Publications

2024:2

1. Drug Resist. Updates

   ABCC4 Impacts Megakaryopoiesis and Protects Megakaryocytes against 6-Mercaptopurine Induced Cytotoxicity

   Ranjit Sabina, Wang Yao, Zhu Jingwen, Cheepala Satish B., Schuetz Erin G., Cho Woo Jung, Xu Beisi, Robinson Camenzind G., Wu Gang, Naren Anjaparavanda. P., Schuetz John D.

   Drug Resist. Updates 2024

   Abstract DOI PMID GEO:Human.Mouse.RNA

   The role of ABCC4, an ATP-binding cassette transporter, in the process of platelet formation, megakaryopoiesis, is unknown. Here, we show that ABCC4 is highly expressed in megakaryocytes (MKs). Mining of public genomic data (ATAC-seq and genome wide chromatin interactions, Hi-C) revealed that key megakaryopoiesis transcription factors (TFs) interacted with ABCC4 regulatory elements and likely accounted for high ABCC4 expression in MKs. Importantly these genomic interactions for ABCC4 ranked higher than for genes with known roles in megakaryopoiesis suggesting a role for ABCC4 in megakaryopoiesis. We then demonstrate that ABCC4 is required for optimal platelet formation as in vitro differentiation of fetal liver derived MKs from Abcc4-/- mice exhibited impaired proplatelet formation and polyploidization, features required for optimal megakaryopoiesis. Likewise, a human megakaryoblastic cell line, MEG-01 showed that acute ABCC4 inhibition markedly suppressed key processes in megakaryopoiesis and that these effects were related to reduced cAMP export and enhanced dissociation of a negative regulator of megakaryopoiesis, protein kinase A (PKA) from ABCC4. PKA activity concomitantly increased after ABCC4 inhibition which was coupled with significantly reduced GATA-1 expression, a TF needed for optimal megakaryopoiesis. Further, ABCC4 protected MKs from 6-mercaptopurine (6-MP) as Abcc4-/- mice show a profound reduction in MKs after 6-MP treatment. In total, our studies show that ABCC4 not only protects the MKs but is also required for maximal platelet production from MKs, suggesting modulation of ABCC4 function might be a potential therapeutic strategy to regulate platelet production.

2. Nucleic Acids Res.

   THUMPD2 Catalyzes the N2-methylation of U6 snRNA of the Spliceosome Catalytic Center and Regulates Pre-mRNA Splicing and Retinal Degeneration

   Yang Wen-Qing*, Ge Jian-Yang*, Zhang Xiaofeng, Zhu Wen-Yu, Lin Lin, Shi Yigong, Xu Beisi#, Liu Ru-Juan#

   Nucleic Acids Res. 2024

   Abstract DOI GEO:Human.RNA

   The mechanisms by which the relatively conserved spliceosome manages the enormously large number of splicing events that occur in humans (∼200 000 versus ∼300 in yeast) are poorly understood. Here, we show deposition of one RNA modification-N2-methylguanosine (m2G) on the G72 of U6 snRNA (the catalytic center of the spliceosome) promotes efficient pre-mRNA splicing activity in human cells. This modification was identified to be conserved among vertebrates. Further, THUMPD2 was demonstrated as the methyltransferase responsible for U6 m2G72 by explicitly recognizing the U6-specific sequences and structural elements. The knock-out of THUMPD2 eliminated U6 m2G72 and impaired the pre-mRNA splicing activity, resulting in thousands of changed alternative splicing events of endogenous pre-mRNAs in human cells. Notably, the aberrantly spliced pre-mRNA population elicited the nonsense-mediated mRNA decay pathway. We further show that THUMPD2 was associated with age-related macular degeneration and retinal function. Our study thus demonstrates how an RNA epigenetic modification of the major spliceosome regulates global pre-mRNA splicing and impacts physiology and disease.

2023:12

1. Nat Commun

   PAX3-FOXO1 Dictates Myogenic Reprogramming and Rhabdomyosarcoma Identity in Endothelial Progenitors

   Searcy Madeline B., Larsen Randolph K., Stevens Bradley T., Zhang Yang, Jin Hongjian, Drummond Catherine J., Langdon Casey G., Gadek Katherine E., Vuong Kyna, Reed Kristin B., Garcia Matthew R., Xu Beisi, Kimbrough Darden W., Adkins Grace E., Djekidel Nadhir, Porter Shaina N., Schreiner Patrick A., Pruett-Miller Shondra M., Abraham Brian J., Rehg Jerold E., Hatley Mark E.

   Nat Commun 2023

   Abstract DOI PMC PMID GEO:Human.RNA.CutRun.Mouse.HiC.scRNA.RNA.CutRun

   Fusion-positive rhabdomyosarcoma (FP-RMS) driven by the expression of the PAX3-FOXO1 (P3F) fusion oncoprotein is an aggressive subtype of pediatric rhabdomyosarcoma. FP-RMS histologically resembles developing muscle yet occurs throughout the body in areas devoid of skeletal muscle highlighting that FP-RMS is not derived from an exclusively myogenic cell of origin. Here we demonstrate that P3F reprograms mouse and human endothelial progenitors to FP-RMS. We show that P3F expression in aP2-Cre expressing cells reprograms endothelial progenitors to functional myogenic stem cells capable of regenerating injured muscle fibers. Further, we describe a FP-RMS mouse model driven by P3F expression and Cdkn2a loss in endothelial cells. Additionally, we show that P3F expression in TP53-null human iPSCs blocks endothelial-directed differentiation and guides cells to become myogenic cells that form FP-RMS tumors in immunocompromised mice. Together these findings demonstrate that FP-RMS can originate from aberrant development of non-myogenic cells driven by P3F.

2. Blood

   Acute Myeloid Leukemias with UBTF Tandem Duplications Are Sensitive to Menin Inhibitors

   Barajas Juan Martin, Rasouli Milad, Umeda Masayuki, Hiltenbrand Ryan Lea, Abdelhamed Sherif, Mohnani Rebecca, Arthur Bright, Westover Tamara, Thomas Melvin E., Ashtiani Minoo, Janke Laura J, Xu Beisi, Chang Ti-Cheng, Rosikiewicz Wojciech, Xiong Emily, Rolle Chandra, Low Jonathan, Krishnan Reethu, Song Guangchun, Walsh Michael P., Ma Jing J., Rubnitz Jeffrey E., Iacobucci Ilaria, Chen Taosheng, Krippner-Heidenreich Anja, Zwaan Christian Michel, Heidenreich Olaf, Klco Jeffery M

   Blood 2023

   Abstract DOI PMID GEO:Human.RNA GEO:Human.CutRun

   UBTF tandem duplications (UBTF-TDs) have recently emerged as a recurrent alteration in pediatric and adult acute myeloid leukemia (AML). UBTF-TD leukemias are characterized by a poor response to conventional chemotherapy and a transcriptional signature that mirrors NUP98-rearranged and NPM1-mutant AMLs, including HOX gene dysregulation. However, the mechanism of how UBTF-TD drives leukemogenesis remains unknown. In this study, we investigated the genomic occupancy of UBTF-TD in transformed cord-blood CD34+ (cbCD34+) cells and patient-derived xenograft models. We found that UBTF-TD protein maintained genomic occupancy at ribosomal DNA (rDNA) loci while also occupying genomic targets commonly dysregulated in UBTF-TD myeloid malignancies, such as the HOXA/HOXB gene clusters and MEIS1. These data suggest that UBTF-TD is a gain-of-function alteration that results in mislocalization to genomic loci dysregulated in UBTF-TD leukemias. UBTF-TD also co-occupies key genomic loci with KMT2A and Menin, which are known to be key partners involved in HOX-dysregulated leukemias. Using a protein degradation system, we showed that stemness, proliferation, and transcriptional signatures are dependent on sustained UBTF-TD localization to chromatin. Finally, we demonstrate that primary cells from UBTF-TD leukemias are sensitive to the Menin inhibitor SNDX-5613, resulting in markedly reduced in vitro and in vivo tumor growth, myeloid differentiation, and abrogation of the UBTF-TD leukemic expression signature. These findings provide a viable therapeutic strategy for patients with this high-risk AML subtype.

3. Nat Commun

   SNIP1 and PRC2 Coordinate Cell Fates of Neural Progenitors during Brain Development

   Matsui Yurika, Djekidel Mohamed Nadhir, Lindsay Katherine, Samir Parimal, Connolly Nina, Wu Gang, Yang Xiaoyang, Fan Yiping, Xu Beisi, Peng Jamy C.

   Nat Commun 2023

   Abstract DOI PMC PMID GEO:Mouse.RNA.CutRun

   Stem cell survival versus death is a developmentally programmed process essential for morphogenesis, sizing, and quality control of genome integrity and cell fates. Cell death is pervasive during development, but its programming is little known. Here, we report that Smad nuclear interacting protein 1 (SNIP1) promotes neural progenitor cell survival and neurogenesis and is, therefore, integral to brain development. The SNIP1-depleted brain exhibits dysplasia with robust induction of caspase 9-dependent apoptosis. Mechanistically, SNIP1 regulates target genes that promote cell survival and neurogenesis, and its activities are influenced by TGFβ and NFκB signaling pathways. Further, SNIP1 facilitates the genomic occupancy of Polycomb complex PRC2 and instructs H3K27me3 turnover at target genes. Depletion of PRC2 is sufficient to reduce apoptosis and brain dysplasia and to partially restore genetic programs in the SNIP1-depleted brain in vivo. These findings suggest a loci-specific regulation of PRC2 and H3K27 marks to toggle cell survival and death in the developing brain.

4. Cancer Res

   PP2Ac Deficiency Enhances Tumor Immunogenicity by Activating STING-Type I Interferon Signaling in Glioblastoma

   Mondal Isha*, Das Oishika*, Sun Raymond, Gao Jian, Yu Bohyeon, Diaz Aaron, Behnan Jinan, Dubey Abhishek, Meng Zhipeng, Eskandar Emad, Xu Beisi#, Lu Rongze Olivia.#, Ho Winson S.#

   Cancer Res 2023

   Abstract DOI PMC PMID Browser:WES GEO:Mouse.scRNA.RNA

   Glioblastoma (GBM) is an immunologically “cold” tumor that does not respond to current immunotherapy. Here, we demonstrate a fundamental role for the α-isoform of the catalytic subunit of protein phosphatase-2A (PP2Ac) in regulating glioma immunogenicity. Genetic ablation of PP2Ac in glioma cells enhanced double stranded DNA (dsDNA) production and cGAS-type I interferon (IFN) signaling, MHC-I expression, and tumor mutational burden. In co-culture experiments, PP2Ac deficiency in glioma cells promoted dendritic cell (DC) cross presentation and clonal expansion of CD8+ T cells. In vivo, PP2Ac depletion sensitized tumors to immune checkpoint blockade and radiotherapy treatment. Single cell analysis demonstrated that PP2Ac deficiency increased CD8+ T cell, NK cell, and DC accumulation and reduced immunosuppressive tumor associated macrophages. Furthermore, loss of PP2Ac increased IFN signaling in myeloid and tumor cells and reduced expression of a tumor gene signature associated with worse patient survival in TCGA. Collectively, this study establishes a novel role for PP2Ac in inhibiting dsDNA-cGAS-STING signaling to suppress anti-tumor immunity in glioma.

5. Proceedings of the N

   Interrogating Bromodomain Inhibitor Resistance in KMT2A-rearranged Leukemia through Combinatorial CRISPR Screens

   Wright Shaela, Hu Jianzhong, Wang Hong, Hyle Judith, Zhang Yang, Du Guoqing, Konopleva Marina Y., Kornblau Steven M., Djekidel Mohamed Nadhir, Rosikiewicz Wojciech, Xu Beisi, Lu Rui, Yang Jun J., Li Chunliang

   Proceedings of the National Academy of Sciences 2023

   Abstract DOI PMC PMID GEO:Human.RNA

   Bromo- and extra-terminal domain inhibitors (BETi) have exhibited therapeutic activities in many cancers. However, the mechanisms controlling BETi response and resistance are not well understood. We conducted genome-wide loss-of-function CRISPR screens using BETi-treated KMT2A-rearranged (KMT2A-r) cell lines. We revealed that Speckle-type POZ protein (SPOP) gene (Speckle Type BTB/POZ Protein) deficiency caused significant BETi resistance, which was further validated in cell lines and xenograft models. Proteomics analysis and a kinase-vulnerability CRISPR screen indicated that cells treated with BETi are sensitive to GSK3 perturbation. Pharmaceutical inhibition of GSK3 reversed the BETi-resistance phenotype. Based on this observation, a combination therapy regimen inhibiting both BET and GSK3 was developed to impede KMT2A-r leukemia progression in patient-derived xenografts in vivo. Our results revealed molecular mechanisms underlying BETi resistance and a promising combination treatment regimen of ABBV-744 and CHIR-98014 by utilizing unique ex vivo and in vivo KMT2A-r PDX models.

6. Viruses

   How Estrogen, Testosterone, and Sex Differences Influence Serum Immunoglobulin Isotype Patterns in Mice and Humans

   Surman Sherri L., Jones Bart G., Penkert Rhiannon R., Sealy Robert E., Marion Tony, Thomas Paul G., Neale Geoffrey, Xu Beisi, Hurwitz Julia L.

   Viruses 2023

   Abstract DOI PMC PMID

   Females often exhibit superior immune responses compared to males toward vaccines and pathogens such as influenza viruses and SARS-CoV-2. To help explain these differences, we first studied serum immunoglobulin isotype patterns in C57BL/6 male and female mice. We focused on IgG2b, an isotype that lends to virus control and that has been previously shown to be elevated in murine females compared to males. Improvements in IgG2b serum levels, and/or IgG2b ratios with other non-IgM isotypes, were observed when: (i) wildtype (WT) female mice were compared to estrogen receptor knockout mice (IgG2b, IgG2b/IgG3, IgG2b/IgG1, and IgG2b/IgA were all higher in WT mice), (ii) unmanipulated female mice were compared to ovariectomized mice (IgG2b/IgA was higher in unmanipulated animals), (iii) female mice were supplemented with estrogen in the context of an inflammatory insult (IgG2b and IgG2b/IgG3 were improved by estrogen supplementation), and (iv) male mice were supplemented with testosterone, a hormone that can convert to estrogen in vivo (IgG2b, IgG2b/IgG3, IgG2b/IgG1, and IgG2b/IgA were all improved by supplementation). We next examined data from three sets of previously described male and female human blood samples. In each case, there were higher IgG2 levels, and/or ratios of IgG2 with non-IgM isotypes, in human females compared to males. The effects of sex and sex hormones in the mouse and human studies were subtle, but frequent, suggesting that sex hormones represent only a fraction of the factors that influence isotype patterns. Examination of the gene loci suggested that upregulation of murine IgG2b or human IgG2 could be mediated by estrogen receptor binding to estrogen response elements and cytosine-adenine (CA) repeats upstream of respective Cγ genes. Given that murine IgG2b and human IgG2 lend to virus control, the isotype biases in females may be sufficient to improve outcomes following vaccination or infection. Future attention to sex hormone levels, and consequent immunoglobulin isotype patterns, in clinical trials are encouraged to support the optimization of vaccine and drug products for male and female hosts.

7. Nat Commun

   Proteasome Inhibition Targets the KMT2A Transcriptional Complex in Acute Lymphoblastic Leukemia

   Kamens Jennifer L., Nance Stephanie, Koss Cary, Xu Beisi, Cotton Anitria, Lam Jeannie W., Garfinkle Elizabeth A. R., Nallagatla Pratima, Smith Amelia M. R., Mitchell Sharnise, Ma Jing, Currier Duane, Wright Charlie, Kavdia Kanisha, Pagala Vishwajeeth R., Kim Wonil, Wallace LaShanale M., Cho Ji-Hoon, Fan Yiping, Seth Aman, Twarog Nathaniel, Choi John K., Obeng Esther A., Hatley Mark E., Metzger Monika L., Inaba Hiroto, Jeha Sima, Rubnitz Jeffrey E., Peng Junmin, Chen Taosheng, Shelat Anang A., Guy R. Kiplin, Gruber Tanja A.

   Nat Commun 2023

   Abstract DOI PMC PMID More:Proteome.PRIDE GEO:Human.ChIP.RNA

   Rearrangments in Histone-lysine-N-methyltransferase 2A (KMT2Ar) are associated with pediatric, adult and therapy-induced acute leukemias. Infants with KMT2Ar acute lymphoblastic leukemia (ALL) have a poor prognosis with an event-free-survival of 38%. Herein we evaluate 1116 FDA approved compounds in primary KMT2Ar infant ALL specimens and identify a sensitivity to proteasome inhibition. Upon exposure to this class of agents, cells demonstrate a depletion of histone H2B monoubiquitination (H2Bub1) and histone H3 lysine 79 dimethylation (H3K79me2) at KMT2A target genes in addition to a downregulation of the KMT2A gene expression signature, providing evidence that it targets the KMT2A transcriptional complex and alters the epigenome. A cohort of relapsed/refractory KMT2Ar patients treated with this approach on a compassionate basis had an overall response rate of 90%. In conclusion, we report on a high throughput drug screen in primary pediatric leukemia specimens whose results translate into clinically meaningful responses. This innovative treatment approach is now being evaluated in a multi-institutional upfront trial for infants with newly diagnosed ALL.

8. J Clin Invest

   PP2Ac/STRN4 Negatively Regulates STING-Type I Interferon Signaling in Tumor Associated Macrophages

   Ho Winson S.*, Mondal Isha*, Xu Beisi*, Das Oishika*, Sun Raymond C., Chiou Pochin, Cai Xiaomin, Tahmasebinia Foozhan, Wu Caren Yu-Ju, Wu Zhihao, Matsui William, Lim Michael, Meng Zhipeng, Lu Rongze Olivia

   J Clin Invest 2023

   Abstract DOI PMC PMID GEO:Mouse.scRNA.RNA

   Stimulator of IFN genes type I (STING-Type I) IFN signaling in myeloid cells plays a critical role in effective antitumor immune responses, but STING agonists as monotherapy have shown limited efficacy in clinical trials. The mechanisms that downregulate STING signaling are not fully understood. Here, we report that protein phosphatase 2A (PP2A), with its specific B regulatory subunit Striatin 4 (STRN4), negatively regulated STING-Type I IFN in macrophages. Mice with macrophage PP2A deficiency exhibited reduced tumor progression. The tumor microenvironment showed decreased immunosuppressive and increased IFN-activated macrophages and CD8+ T cells. Mechanistically, we demonstrated that Hippo kinase MST1/2 was required for STING activation. STING agonists induced dissociation of PP2A from MST1/2 in normal macrophages, but not in tumor conditioned macrophages. Furthermore, our data showed that STRN4 mediated PP2A binding to and dephosphorylation of Hippo kinase MST1/2, resulting in stabilization of YAP/TAZ to antagonize STING activation. In human patients with glioblastoma (GBM), YAP/TAZ was highly expressed in tumor-associated macrophages but not in nontumor macrophages. We also demonstrated that PP2A/STRN4 deficiency in macrophages reduced YAP/TAZ expression and sensitized tumor-conditioned macrophages to STING stimulation. In summary, we demonstrated that PP2A/STRN4-YAP/TAZ has, in our opinion, been an unappreciated mechanism that mediates immunosuppression in tumor-associated macrophages, and targeting the PP2A/STRN4-YAP/TAZ axis can sensitize tumors to immunotherapy.

9. Oncogene

   Spatiotemporal Regulation of Cholangiocarcinoma Growth and Dissemination by Peritumoral Myofibroblasts in a Vcam1-dependent Manner

   Tian Cheng, Li Liyuan, Pan Qingfei, Xu Beisi, Li Yizhen, Fan Li, Brown Anthony, Morrison Michelle, Dey Kaushik, Yang Jun J., Yu Jiyang, Glazer Evan S., Zhu Liqin

   Oncogene 2023

   Abstract DOI PMC PMID GEO:Mouse.RNA

   Intrahepatic cholangiocarcinoma (iCCA) is characterized by its highly desmoplastic stroma. Myofibroblasts (MFs) are present both within the tumor mass (intratumoral MFs, iMFs) and at the tumor border (peritumoral MFs, pMFs). Using a spheroid-based coculture system, we show that the initial iCCA-pMF contact is growth suppressive to the tumor cells. However, prolonged iCCA-pMF interaction elicits significant tumor cell invasion and dissemination. We find that vascular cell adhesion molecule-1 (Vcam1) level is elevated in tumor cells in contact with pMFs but low in disseminated tumor cells both in vitro and in vivo. A gene regulatory network analysis of mouse and patient iCCA tumors and Vcam1 knockout (Vcam1KO) demonstrate a heavy involvement of Vcam1 in epithelial-to-mesenchymal transition. While Vcam1KO has only a limited impact on tumor cell growth in their monoculture, Vcam1KO spheroids exhibit instant dissemination and a severe growth defect when cocultured with pMFs. When transplanted into the liver, Vcam1KO iCCA cells show a similar increase in dissemination but a significant defect in establishing primary and metastatic tumors. Incomplete blocking of Vcam1 in vivo reduces the size but increase the number of metastatic lesions. Overall, our study shows a spatiotemporal regulation of iCCA growth and dissemination by pMFs in a Vcam1-dependent manner.

10. Genome Biol.

    Auxin-Inducible Degron 2 System Deciphers Functions of CTCF Domains in Transcriptional Regulation

    Hyle Judith*, Djekidel Mohamed Nadhir*, Williams Justin, Wright Shaela, Shao Ying, Xu Beisi#, Li Chunliang#

    Genome Biol. 2023

    Abstract DOI PMC PMID GEO:Human.HiCHIP.SLAM.ChIP.RNA

    CTCF is a well-established chromatin architectural protein that also plays various roles in transcriptional regulation. While CTCF biology has been extensively studied, how the domains of CTCF function to regulate transcription remains unknown. Additionally, the original auxin-inducible degron 1 (AID1) system has limitations in investigating the function of CTCF.

11. Cell Reports

    Modulation of Protease Expression by the Transcription Factor Ptx1/PITX Regulates Protein Quality Control during Aging

    Jiao Jianqin, Curley Michelle, Graca Flavia A., Robles-Murguia Maricela, Shirinifard Abbas, Finkelstein David, Xu Beisi, Fan Yiping, Demontis Fabio

    Cell Reports 2023

    Abstract DOI GEO:FruitFly.RNA GEO:FruitFly.RNA

    Protein quality control is important for healthy aging and is dysregulated in age-related diseases. The autophagy-lysosome and ubiquitin-proteasome are key for proteostasis, but it remains largely unknown whether other proteolytic systems also contribute to maintain proteostasis during aging. Here, we find that expression of proteolytic enzymes (proteases/peptidases) distinct from the autophagy-lysosome and ubiquitin-proteasome systems declines during skeletal muscle aging in Drosophila. Age-dependent protease downregulation undermines proteostasis, as demonstrated by the increase in detergent-insoluble poly-ubiquitinated proteins and pathogenic huntingtin-polyQ levels in response to protease knockdown. Computational analyses identify the transcription factor Ptx1 (homologous to human PITX1/2/3) as a regulator of protease expression. Consistent with this model, Ptx1 protein levels increase with aging, and Ptx1 RNAi counteracts the age-associated downregulation of protease expression. Moreover, Ptx1 RNAi improves muscle protein quality control in a protease-dependent manner and extends lifespan. These findings indicate that proteases and their transcriptional modulator Ptx1 ensure proteostasis during aging.

12. Science Advances

    ZBTB18 Restricts Chromatin Accessibility and Prevents Transcriptional Adaptations That Drive Metastasis

    Wang Ruishan, Bhatt Akshita B., Minden-Birkenmaier Benjamin A., Travis Olivia K., Tiwari Srishti, Jia Hong, Rosikiewicz Wojciech, Martinot Ophelie, Childs Eleanor, Loesch Robin, Tossou Guenole, Jamieson Sophie, Finkelstein David, Xu Beisi, Labelle Myriam

    Science Advances 2023

    Abstract DOI PMC PMID GEO:Mouse.RNA.ATAC

    Metastases arise from rare cancer cells that successfully adapt to the diverse microenvironments encountered during dissemination through the bloodstream and colonization of distant tissues. How cancer cells acquire the ability to appropriately respond to microenvironmental stimuli remains largely unexplored. Here, we report an epigenetic pliancy mechanism that allows cancer cells to successfully metastasize. We find that a decline in the activity of the transcriptional repressor ZBTB18 defines metastasis-competent cancer cells in mouse models. Restoration of ZBTB18 activity reduces chromatin accessibility at the promoters of genes that drive metastasis, such as Tgfbr2, and this prevents TGFβ1 pathway activation and consequently reduces cell migration and invasion. Besides repressing the expression of metastatic genes, ZBTB18 also induces widespread chromatin closing, a global epigenetic adaptation previously linked to reduced phenotypic flexibility. Thus, ZBTB18 is a potent chromatin regulator, and the loss of its activity enhances chromatin accessibility and transcriptional adaptations that promote the phenotypic changes required for metastasis.

2022:8

1. Nat Commun

   Epigenetic Activation of the FLT3 Gene by ZNF384 Fusion Confers a Therapeutic Susceptibility in Acute Lymphoblastic Leukemia

   Zhao Xujie, Wang Ping, Diedrich Jonathan D., Smart Brandon, Reyes Noemi, Yoshimura Satoshi, Zhang Jingliao, Yang Wentao, Barnett Kelly, Xu Beisi, Li Zhenhua, Huang Xin, Yu Jiyang, Crews Kristine, Yeoh Allen Eng Juh, Konopleva Marina, Wei Chia-Lin, Pui Ching-Hon, Savic Daniel, Yang Jun J.

   Nat Commun 2022

   Abstract DOI PMC PMID GEO:Human.ChIP.CutTag.ChIAPET.ATAC

   FLT3 is an attractive therapeutic target in acute lymphoblastic leukemia (ALL) but the mechanism for its activation in this cancer is incompletely understood. Profiling global gene expression in large ALL cohorts, we identify over-expression of FLT3 in ZNF384-rearranged ALL, consistently across cases harboring different fusion partners with ZNF384. Mechanistically, we discover an intergenic enhancer element at the FLT3 locus that is exclusively activated in ZNF384-rearranged ALL, with the enhancer-promoter looping directly mediated by the fusion protein. There is also a global enrichment of active enhancers within ZNF384 binding sites across the genome in ZNF384-rearranged ALL cells. Downregulation of ZNF384 blunts FLT3 activation and decreases ALL cell sensitivity to FLT3 inhibitor gilteritinib in vitro. In patient-derived xenograft models of ZNF384-rearranged ALL, gilteritinib exhibits significant anti-leukemia efficacy as a monotherapy in vivo. Collectively, our results provide insights into FLT3 regulation in ALL and point to potential genomics-guided targeted therapy for this patient population.

2. Science Translationa

   Targeting KDM4 for Treating PAX3-FOXO1–Driven Alveolar Rhabdomyosarcoma

   Singh Shivendra*, Abu-Zaid Ahmed*, Jin Hongjian*, Fang Jie, Wu Qiong, Wang Tingting, Feng Helin, Quarni Waise, Shao Ying, Maxham Lily, Abdolvahabi Alireza, Yun Mi-Kyung, Vaithiyalingam Sivaraja, Tan Haiyan, Bowling John, Honnell Victoria, Young Brandon, Guo Yian, Bajpai Richa, Pruett-Miller Shondra M., Grosveld Gerard C., Hatley Mark, Xu Beisi, Fan Yiping, Wu Gang, Chen Eleanor Y., Chen Taosheng, Lewis Peter W., Rankovic Zoran, Li Yimei, Murphy Andrew J., Easton John, Peng Junmin, Chen Xiang, Wang Ruoning, White Stephen W., Davidoff Andrew M., Yang Jun

   Science Translational Medicine 2022

   Abstract DOI PMC PMID

   Chimeric transcription factors drive lineage-specific oncogenesis but are notoriously difficult to target. Alveolar rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue sarcoma transformed by the pathognomonic Paired Box 3–Forkhead Box O1 (PAX3-FOXO1) fusion protein, which governs a core regulatory circuitry transcription factor network. Here, we show that the histone lysine demethylase 4B (KDM4B) is a therapeutic vulnerability for PAX3-FOXO1+ RMS. Genetic and pharmacologic inhibition of KDM4B substantially delayed tumor growth. Suppression of KDM4 proteins inhibited the expression of core oncogenic transcription factors and caused epigenetic alterations of PAX3-FOXO1–governed superenhancers. Combining KDM4 inhibition with cytotoxic chemotherapy led to tumor regression in preclinical PAX3-FOXO1+ RMS subcutaneous xenograft models. In summary, we identified a targetable mechanism required for maintenance of the PAX3-FOXO1–related transcription factor network, which may translate to a therapeutic approach for fusion-positive RMS.

3. Blood Cancer Discov.

   ZNF384 Fusion Oncoproteins Drive Lineage Aberrancy in Acute Leukemia

   Dickerson Kirsten M., Qu Chunxu, Gao Qingsong, Iacobucci Ilaria, Gu Zhaohui, Yoshihara Hiroki, Backhaus Emily A., Chang Yunchao, Janke Laura J., Xu Beisi, Wu Gang, Papachristou Evangelia K., D’Santos Clive S., Roberts Kathryn G., Mullighan Charles G.

   Blood Cancer Discovery 2022

   Abstract DOI PMID More:Proteome.PRIDE More:WES.SRA GEO:Human.HiChIP.CHIP.RNA.scRNA

   ZNF384-rearranged fusion oncoproteins (FO) define a subset of lineage ambiguous leukemias, but their mechanistic role in leukemogenesis and lineage ambiguity is poorly understood. Using viral expression in mouse and human hematopoietic stem and progenitor cells (HSPC) and a Ep300::Znf384 knockin mouse model, we show that ZNF384 FO promote hematopoietic expansion, myeloid lineage skewing, and self-renewal. In mouse HSPCs, concomitant lesions, such as NRASG12D, were required for fully penetrant leukemia, whereas in human HSPCs, expression of ZNF384 FO drove B/myeloid leukemia, with sensitivity of a ZNF384-rearranged xenograft to FLT3 inhibition in vivo. Mechanistically, ZNF384 FO occupy a subset of predominantly intragenic/enhancer regions with increased histone 3 lysine acetylation and deregulate expression of hematopoietic stem cell transcription factors. These data define a paradigm for FO-driven lineage ambiguous leukemia, in which expression in HSPCs results in deregulation of lineage-specific genes and hematopoietic skewing, progressing to full leukemia in the context of proliferative stress.Expression of ZNF384 FO early in hematopoiesis results in binding and deregulation of key hematopoietic regulators, skewing of hematopoiesis, and priming for leukemic transformation. These results reveal the interplay between cell of origin and expression of ZNF384 FO to mediate lineage ambiguity and leukemia development.This article is highlighted in the In This Issue feature, p. 171

4. JNCI: J. Natl. Cancer Inst.

   Molecular Mechanisms of ARID5B-Mediated Genetic Susceptibility to Acute Lymphoblastic Leukemia

   Zhao Xujie*, Qian Maoxiang*, Goodings Charnise*, Zhang Yang, Yang Wenjian, Wang Ping, Xu Beisi, Tian Cheng, Pui Ching-Hon, Hunger Stephen P, Raetz Elizabeth A, Devidas Meenakshi, Relling Mary V, Loh Mignon L, Savic Daniel, Li Chunliang, Yang Jun J

   JNCI: Journal of the National Cancer Institute 2022

   Abstract DOI PMID GEO:Human

   There is growing evidence for the inherited basis of susceptibility to childhood acute lymphoblastic leukemia (ALL). Genome-wide association studies have identified non-coding ALL risk variants at the ARID5B gene locus, but their exact functional effects and the molecular mechanism linking ARID5B to B-cell ALL leukemogenesis remain largely unknown.We performed targeted sequencing of ARID5B in germline DNA of 5008 children with ALL. Variants were evaluated for association with ALL susceptibility using 3644 patients from the UK10K cohort as non-ALL controls, under an additive model. Cis-regulatory elements in ARID5B were systematically identified using dCas9-KRAB–mediated enhancer interference system enhancer screen in ALL cells. Disruption of transcription factor binding by ARID5B variant was predicted informatically and then confirmed using chromatin immunoprecipitation and coimmunoprecipitation. ARID5B variant association with hematological traits was examined using UK Biobank dataset. All statistical tests were 2-sided.We identified 54 common variants in ARID5B statistically significantly associated with leukemia risk, all of which were noncoding. Six cis-regulatory elements at the ARID5B locus were discovered using CRISPR-based high-throughput enhancer screening. Strikingly, the top ALL risk variant (rs7090445, P\,= 5.57\,\texttimes 10–45) is located precisely within the strongest enhancer element, which is also distally tethered to the ARID5B promoter. The variant allele disrupts the MEF2C binding motif sequence, resulting in reduced MEF2C affinity and decreased local chromosome accessibility. MEF2C influences ARID5B expression in ALL, likely via a transcription factor complex with RUNX1. Using the UK Biobank dataset (n\,= 349\mkern1mu861), we showed that rs7090445 was also associated with lymphocyte percentage and count in the general population (P\,= 8.6\,\texttimes 10–22 and 2.1\,\texttimes 10–18, respectively).Our results indicate that ALL risk variants in ARID5B function by modulating cis-regulatory elements at this locus.

5. Mol. Cell

   NSD1 Mediates Antagonism between SWI/SNF and Polycomb Complexes and Is Required for Transcriptional Activation upon EZH2 Inhibition

   Drosos Yiannis, Myers Jacquelyn A., Xu Beisi, Mathias Kaeli M., Beane Emma C., Radko-Juettner Sandi, Mobley Robert J., Larsen Margaret E., Piccioni Federica, Ma Xiaotu, Low Jonathan, Hansen Baranda S., Peters Samuel T., Bhanu Natarajan V., Dhanda Sandeep K., Chen Taosheng, Upadhyaya Santhosh A., Pruett-Miller Shondra M., Root David E., Garcia Benjamin A., Partridge Janet F., Roberts Charles W. M.

   Molecular Cell 2022

   Abstract DOI PMID GEO:Human.CHIP.RNA GEO:Pub.CHIP GEO:Pub.RNA

   Disruption of antagonism between SWI/SNF chromatin remodelers and polycomb repressor complexes drives the formation of numerous cancer types. Recently, an inhibitor of the polycomb protein EZH2 was approved for the treatment of a sarcoma mutant in the SWI/SNF subunit SMARCB1, but resistance occurs. Here, we performed CRISPR screens in SMARCB1-mutant rhabdoid tumor cells to identify genetic contributors to SWI/SNF-polycomb antagonism and potential resistance mechanisms. We found that loss of the H3K36 methyltransferase NSD1 caused resistance to EZH2 inhibition. We show that NSD1 antagonizes polycomb via cooperation with SWI/SNF and identify co-occurrence of NSD1 inactivation in SWI/SNF-defective cancers, indicating in vivo relevance. We demonstrate that H3K36me2 itself has an essential role in the activation of polycomb target genes as inhibition of the H3K36me2 demethylase KDM2A restores the efficacy of EZH2 inhibition in SWI/SNF-deficient cells lacking NSD1. Together our data expand the mechanistic understanding of SWI/SNF and polycomb interplay and identify NSD1 as the key for coordinating this transcriptional control.

6. Nat Commun

   The Myokine Fibcd1 Is an Endogenous Determinant of Myofiber Size and Mitigates Cancer-Induced Myofiber Atrophy

   Graca Flavia A., Rai Mamta, Hunt Liam C., Stephan Anna, Wang Yong-Dong, Gordon Brittney, Wang Ruishan, Quarato Giovanni, Xu Beisi, Fan Yiping, Labelle Myriam, Demontis Fabio

   Nat Commun 2022

   Abstract DOI PMC PMID GEO:Mouse.RNA.FIBCD1 GEO:Mouse.RNA.CACMEL GEO:Mouse.RNA.Dia

   Decline in skeletal muscle cell size (myofiber atrophy) is a key feature of cancer-induced wasting (cachexia). In particular, atrophy of the diaphragm, the major muscle responsible for breathing, is an important determinant of cancer-associated mortality. However, therapeutic options are limited. Here, we have used Drosophila transgenic screening to identify muscle-secreted factors (myokines) that act as paracrine regulators of myofiber growth. Subsequent testing in mouse myotubes revealed that mouse Fibcd1 is an evolutionary-conserved myokine that preserves myofiber size via ERK signaling. Local administration of recombinant Fibcd1 (rFibcd1) ameliorates cachexia-induced myofiber atrophy in the diaphragm of mice bearing patient-derived melanoma xenografts and LLC carcinomas. Moreover, rFibcd1 impedes cachexia-associated transcriptional changes in the diaphragm. Fibcd1-induced signaling appears to be muscle selective because rFibcd1 increases ERK activity in myotubes but not in several cancer cell lines tested. We propose that rFibcd1 may help reinstate myofiber size in the diaphragm of patients with cancer cachexia.

7. J Pathol

   Genomic Profiling Identifies Genes and Pathways Dysregulated by HEY1-NCOA2 Fusion and Shines a Light on Mesenchymal Chondrosarcoma Tumorigenesis

   Qi Wenqing*, Rosikiewicz Wojciech*, Yin Zhaohong, Xu Beisi, Jiang Huihong, Wan Shibiao, Fan Yiping, Wu Gang, Wang Lu

   J Pathol 2022

   Abstract DOI PMID GEO:Human.ChIP.RNA

   Mesenchymal chondrosarcoma is a rare, high-grade, primitive mesenchymal tumor. It accounts for around 2-10% of all chondrosarcomas and mainly affects adolescents and young adults. We previously described the HEY1-NCOA2 as a recurrent gene fusion in mesenchymal chondrosarcoma, an important breakthrough for characterizing this disease; however, little study had been done to characterize the fusion protein functionally, in large part due to a lack of suitable models for evaluating the impact of HEY1-NCOA2 expression in the appropriate cellular context. We used iPSC-derived mesenchymal stem cells (iPSC-MSCs), which can differentiate into chondrocytes, and generated stable transduced iPSC-MSCs with inducible expression of HEY1-NCOA2 fusion protein, wildtype HEY1 or wildtype NCOA2. We next comprehensively analyzed both the DNA binding properties and transcriptional impact of HEY1-NCOA2 expression by integrating genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) and expression profiling (RNA-seq). We demonstrated that HEY1-NCOA2 fusion protein preferentially binds to promoter regions of canonical HEY1 targets, resulting in transactivation of HEY1 targets, and significantly enhances cell proliferation. Intriguingly, we identified that both PDGFB and PDGFRA were directly targeted and upregulated by HEY1-NCOA2; and the fusion protein, but not wildtype HEY1 or NCOA2, dramatically increased the level of phospho-AKT (Ser473). Our findings provide a rationale for exploring PDGF/PI3K/AKT inhibition in treating mesenchymal chondrosarcoma. \textcopyright 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

8. Life Sci. Alliance

   PROSER1 Mediates TET2 O-GlcNAcylation to Regulate DNA Demethylation on UTX-Dependent Enhancers and CpG Islands

   Wang Xiaokang, Rosikiewicz Wojciech, Sedkov Yurii, Martinez Tanner, Hansen Baranda S., Schreiner Patrick, Christensen Jesper, Xu Beisi, Pruett-Miller Shondra M., Helin Kristian, Herz Hans-Martin

   Life Science Alliance 2022

   Abstract DOI PMID GEO:Human.CHIP.RNA.WGBS.hMeDIP

   DNA methylation at enhancers and CpG islands usually leads to gene repression, which is counteracted by DNA demethylation through the TET protein family. However, how TET enzymes are recruited and regulated at these genomic loci is not fully understood. Here, we identify TET2, the glycosyltransferase OGT and a previously undescribed proline and serine rich protein, PROSER1 as interactors of UTX, a component of the enhancer-associated MLL3/4 complexes. We find that PROSER1 mediates the interaction between OGT and TET2, thus promoting TET2 O-GlcNAcylation and protein stability. In addition, PROSER1, UTX, TET1/2, and OGT colocalize on many genomic elements genome-wide. Loss of PROSER1 results in lower enrichment of UTX, TET1/2, and OGT at enhancers and CpG islands, with a concomitant increase in DNA methylation and transcriptional down-regulation of associated target genes and increased DNA hypermethylation encroachment at H3K4me1-predisposed CpG islands. Furthermore, we provide evidence that PROSER1 acts as a more general regulator of OGT activity by controlling O-GlcNAcylation of multiple other chromatin signaling pathways. Taken together, this study describes for the first time a regulator of TET2 O-GlcNAcylation and its implications in mediating DNA demethylation at UTX-dependent enhancers and CpG islands and supports an important role for PROSER1 in regulating the function of various chromatin-associated proteins via OGT-mediated O-GlcNAcylation.

2021:9

1. Cell. Rep.

   Control of Foxp3 Induction and Maintenance by Sequential Histone Acetylation and DNA Demethylation

   Li Jun*, Xu Beisi*, He Minghong, Zong Xinying, Cunningham Trevor, Sha Cher, Fan Yiping, Cross Richard, Hanna Jacob H., Feng Yongqiang

   Cell Report 2021

   Abstract DOI PMID GEO:Mouse.ATAC.WGBS.CHIP.CutRun.SLAM

   Regulatory T (Treg) cells play crucial roles in suppressing deleterious immune response. Here, we investigate how Treg cells are mechanistically induced in vitro (iTreg) and stabilized via transcriptional regulation of Treg lineage-specifying factor Foxp3. We find that acetylation of histone tails at the Foxp3 promoter is required for inducing Foxp3 transcription. Upon induction, histone acetylation signals via bromodomain-containing proteins, particularly targets of inhibitor JQ1, and sustains Foxp3 transcription via a global or trans effect. Subsequently, Tet-mediated DNA demethylation of Foxp3 cis-regulatory elements, mainly enhancer CNS2, increases chromatin accessibility and protein binding, stabilizing Foxp3 transcription and obviating the need for the histone acetylation signal. These processes transform stochastic iTreg induction into a stable cell fate, with the former sensitive and the latter resistant to genetic and environmental perturbations. Thus, sequential histone acetylation and DNA demethylation in Foxp3 induction and maintenance reflect stepwise mechanical switches governing iTreg cell lineage specification.

2. EMBO J.

   A Dual Role of Human tRNA Methyltransferase hTrmt13 in Regulating Translation and Transcription

   Li Hao*, Dong Han*, Xu Beisi*, Xiong Qing-Ping, Li Cai-Tao, Yang Wen-Qing, Li Jing, Huang Zhi-Xuan, Zeng Qi-Yu, Wang En-Duo, Liu Ru-Juan

   EMBO J 2021

   Abstract DOI PMID GEO:Human.CHIP.RNA.CLIP

   Since numerous RNAs and RBPs prevalently localize to active chromatin regions, many RNA-binding proteins (RBPs) may be potential transcriptional regulators. RBPs are generally thought to regulate transcription via noncoding RNAs. Here, we describe a distinct, dual mechanism of transcriptional regulation by the previously uncharacterized tRNA-modifying enzyme, hTrmt13. On one hand, hTrmt13 acts in the cytoplasm to catalyze 2’-O-methylation of tRNAs, thus regulating translation in a manner depending on its tRNA-modification activity. On the other hand, nucleus-localized hTrmt13 directly binds DNA as a transcriptional co-activator of key epithelial-mesenchymal transition factors, thereby promoting cell migration independent of tRNA-modification activity. These dual functions of hTrmt13 are mutually exclusive, as it can bind either DNA or tRNA through its CHHC zinc finger domain. Finally, we find that hTrmt13 expression is tightly associated with poor prognosis and survival in diverse cancer patients. Our discovery of the noncatalytic roles of an RNA-modifying enzyme provides a new perspective for understanding epitranscriptomic regulation.

3. Nat. Commun.

   KDM6B Promotes Activation of the Oncogenic CDK4/6-pRB-E2F Pathway by Maintaining Enhancer Activity in MYCN-amplified Neuroblastoma

   D’Oto Alexandra, Fang Jie, Jin Hongjian, Xu Beisi, Singh Shivendra, Mullasseril Anoushka, Jones Victoria, Abu-Zaid Ahmed, von Buttlar Xinyu, Cooke Bailey, Hu Dongli, Shohet Jason, Murphy Andrew J., Davidoff Andrew M., Yang Jun

   Nature Communication 2021

   Abstract DOI PMID GEO:Human.CutTag.RNA.ATAC GEO:Human.Microarray

   The H3K27me2/me3 histone demethylase KDM6B is essential to neuroblastoma cell survival. However, the mechanism of KDM6B action remains poorly defined. We demonstrate that inhibition of KDM6B activity 1) reduces the chromatin accessibility of E2F target genes and MYCN, 2) selectively leads to an increase of H3K27me3 but a decrease of the enhancer mark H3K4me1 at the CTCF and BORIS binding sites, which may, consequently, disrupt the long-range chromatin interaction of MYCN and E2F target genes, and 3) phenocopies the transcriptome induced by the specific CDK4/6 inhibitor palbociclib. Overexpression of CDK4/6 or Rb1 knockout confers neuroblastoma cell resistance to both palbociclib and the KDM6 inhibitor GSK-J4. These data indicate that KDM6B promotes an oncogenic CDK4/6-pRB-E2F pathway in neuroblastoma cells via H3K27me3-dependent enhancer-promoter interactions, providing a rationale to target KDM6B for high-risk neuroblastoma.

4. Genome Biol.

   Acute Depletion of CTCF Rewires Genome-Wide Chromatin Accessibility

   Xu Beisi*#, Wang Hong*, Wright Shaela, Hyle Judith, Zhang Yang, Shao Ying, Niu Mingming, Fan Yiping, Rosikiewicz Wojciech, Djekidel Mohamed Nadhir, Peng Junmin, Lu Rui, Li Chunliang#

   Genome Biology 2021

   Abstract DOI PMC PMID More:Proteome.PRIDE GEO:All.Human.ATAC.WGBS GEO:RNA GEO:CutRun GEO:HiC

   Background The transcription factor CTCF appears indispensable in defining topologically associated domain boundaries and maintaining chromatin loop structures within these domains, supported by numerous functional studies. However, acute depletion of CTCF globally reduces chromatin interactions but does not significantly alter transcription. Results Here, we systematically integrate multi-omics data including ATAC-seq, RNA-seq, WGBS, Hi-C, Cut&Run, and CRISPR-Cas9 survival dropout screens, and time-solved deep proteomic and phosphoproteomic analyses in cells carrying auxin-induced degron at endogenous CTCF locus. Acute CTCF protein degradation markedly rewires genome-wide chromatin accessibility. Increased accessible chromatin regions are frequently located adjacent to CTCF-binding sites at promoter regions and insulator sites associated with enhanced transcription of nearby genes. In addition, we use CTCF-associated multi-omics data to establish a combinatorial data analysis pipeline to discover CTCF co-regulatory partners. We successfully identify 40 candidates, including multiple established partners. Interestingly, many CTCF co-regulators that have alterations of their respective downstream gene expression do not show changes of their own expression levels across the multi-omics measurements upon acute CTCF loss, highlighting the strength of our system to discover hidden co-regulatory partners associated with CTCF-mediated transcription. Conclusions This study highlights that CTCF loss rewires genome-wide chromatin accessibility, which plays a critical role in transcriptional regulation.

5. Cancer Discov.

   Enhancer Hijacking Drives Oncogenic BCL11B Expression in Lineage Ambiguous Stem Cell Leukemia

   Montefiori Lindsey E., Bendig Sonja, Gu Zhaohui, Chen Xiaolong, Polonen Petri, Ma Xiaotu, Murison Alex, Zeng Andy, Garcia-Prat Laura, Dickerson Kirsten, Iacobucci Ilaria, Abdelhamed Sherif, Hiltenbrand Ryan, Mead Paul E., Mehr Cyrus M., Xu Beisi, Cheng Zhongshan, Chang Ti-Cheng, Westover Tamara, Ma Jing, Stengel Anna, Kimura Shunsuke, Qu Chunxu, Valentine Marcus B., Rashkovan Marissa, Luger Selina, Litzow Mark R., Rowe Jacob M., Boer Monique L., Wang Victoria, Yin Jun, Kornblau Steven M., Hunger Stephen P., Loh Mignon L., Pui Ching-Hon, Yang Wenjian, Crews Kristine R., Roberts Kathryn G., Yang Jun J., Relling Mary V., Evans William E., Stock Wendy, Paietta Elisabeth M., Ferrando Adolfo A., Zhang Jinghui, Kern Wolfgang, Haferlach Torsten, Wu Gang, Dick John E., Klco Jeffery M., Haferlach Claudia, Mullighan Charles G.

   Cancer Discovery 2021

   Abstract DOI PMID GEO:Human_RNA.CHIP.HiCHIP.scRNA.scATAC EGA:Human.RNA.WGS.PacBio

   Lineage ambiguous leukemias are high-risk malignancies of poorly understood genetic basis. Here, we describe a distinct subgroup of acute leukemia with expression of myeloid, T lymphoid and stem cell markers driven by aberrant allele-specific deregulation of BCL11B, a master transcription factor responsible for thymic T-lineage commitment and specification. Mechanistically, this deregulation was driven by chromosomal rearrangements that juxtapose BCL11B to super-enhancers active in hematopoietic progenitors, or focal amplifications that generate a super-enhancer from a non-coding element distal to BCL11B. Chromatin conformation analyses demonstrate long range interactions of rearranged enhancers with the expressed BCL11B allele, and association of BCL11B with activated hematopoietic progenitor cell cis-regulatory elements, suggesting BCL11B is aberrantly co-opted into a gene regulatory network that drives transformation by maintaining a progenitor state. These data support a role for ectopic BCL11B expression in primitive hematopoietic cells mediated by enhancer hijacking as an oncogenic driver of human lineage ambiguous leukemia.

6. J. Exp. Med.

   Foxp3 Enhancers Synergize to Maximize Regulatory T Cell Suppressive Capacity

   Zong Xinying, Hao Xiaolei, Xu Beisi, Crawford Jeremy Chase, Wright Shaela, Li Jun, Zhang Yang, Bai Lu, He Minghong, Jiang Menglin, Fan Yiping, Connelly Jon P., Pruett-Miller Shondra M., Berns Hartmut, Janke Laura, Li Chunliang, Feng Yongqiang

   Journal of Experimental Medicine 2021

   Abstract DOI PMC PMID GEO:Mouse_WGBS.CutRun.ATAC.TCR.CaptureC

   T reg cells bearing a diverse antigen receptor repertoire suppress pathogenic T cells and maintain immune homeostasis during their long lifespan. How their robust function is determined genetically remains elusive. Here, we investigate the regulatory space of the cis-regulatory elements of T reg lineage–specifying factor Foxp3. Foxp3 enhancers are known as distinct readers of environmental cues controlling T reg cell induction or lineage stability. However, their single deficiencies cause mild, if any, immune dysregulation, leaving the key transcriptional mechanisms determining Foxp3 expression and thereby T reg cell suppressive capacity uncertain. We examined the collective activities of Foxp3 enhancers and found that they coordinate to maximize T reg cell induction, Foxp3 expression level, or lineage stability through distinct modes and that ablation of synergistic enhancers leads to lethal autoimmunity in young mice. Thus, the induction and maintenance of a diverse, stable T reg cell repertoire rely on combinatorial Foxp3 enhancers, suggesting broad, stage-specific, synergistic activities of cell-intrinsic factors and cell-extrinsic cues in determining T reg cell suppressive capacity.

7. Immunity

   A Distal Foxp3 Enhancer Enables Interleukin-2 Dependent Thymic Treg Cell Lineage Commitment for Robust Immune Tolerance

   Dikiy Stanislav, Li Jun, Bai Lu, Jiang Menglin, Janke Laura, Zong Xinying, Hao Xiaolei, Hoyos Beatrice, Wang Zhong-Min, Xu Beisi, Fan Yiping, Rudensky Alexander Y., Feng Yongqiang

   Immunity 2021

   Abstract DOI PMC PMID GEO:Mouse_RNA.ATAC.CHIP.TCR

   Activation of the STAT5 transcription factor downstream of the Interleukin-2 receptor (IL-2R) induces expression of Foxp3, a critical step in the differentiation of regulatory T (Treg) cells. Due to the pleiotropic effects of IL-2R signaling, it is unclear how STAT5 acts directly on the Foxp3 locus to promote its expression. Here, we report that IL-2 – STAT5 signaling converged on an enhancer (CNS0) during Foxp3 induction. CNS0 facilitated the IL-2 dependent CD25+Foxp3– precursor to Treg cell transition in the thymus. Its deficiency resulted in impaired Treg cell generation in neonates, which was partially mitigated with age. While the thymic Treg cell paucity caused by CNS0 deficiency did not result in autoimmunity on its own, it exacerbated autoimmune manifestations caused by disruption of the Aire gene. Thus, CNS0 enhancer activity ensures robust Treg cell differentiation early in postnatal life and cooperatively with other tolerance mechanisms minimizes autoimmunity.

8. Cell Metab.

   Proteasome Stress in Skeletal Muscle Mounts a Long-Range Protective Response That Delays Retinal and Brain Aging

   Rai Mamta, Coleman Zane, Curley Michelle, Nityanandam Anjana, Platt Anna, Robles-Murguia Maricela, Jiao Jianqin, Finkelstein David, Wang Yong-Dong, Xu Beisi, Fan Yiping, Demontis Fabio

   Cell Metabolism 2021

   Abstract DOI PMC PMID GEO:RNA_Human_FruitFly

   Neurodegeneration in the central nervous system (CNS) is a defining feature of organismal aging that is influenced by peripheral tissues. Clinical observations indicate that skeletal muscle influences CNS aging, but the underlying muscle-to-brain signaling remains unexplored. In Drosophila, we find that moderate perturbation of the proteasome in skeletal muscle induces compensatory preservation of CNS proteostasis during aging. Such long-range stress signaling depends on muscle-secreted Amyrel amylase. Mimicking stress-induced Amyrel upregulation in muscle reduces age-related accumulation of poly-ubiquitinated proteins in the brain and retina via chaperones. Preservation of proteostasis stems from the disaccharide maltose, which is produced via Amyrel amylase activity. Correspondingly, RNAi for SLC45 maltose transporters reduces expression of Amyrel-induced chaperones and worsens brain proteostasis during aging. Moreover, maltose preserves proteostasis and neuronal activity in human brain organoids challenged by thermal stress. Thus, proteasome stress in skeletal muscle hinders retinal and brain aging by mounting an adaptive response via amylase/maltose.

9. Genome Biol.

   MethylationToActivity: A Deep-Learning Framework That Reveals Promoter Activity Landscapes from DNA Methylomes in Individual Tumors

   Williams Justin, Xu Beisi, Putnam Daniel, Thrasher Andrew, Li Chunliang, Yang Jun, Chen Xiang

   Genome Biology 2021

   Abstract DOI PMC PMID More:STJUDE.CLOUD.workflow More:Code

   Although genome-wide DNA methylomes have demonstrated their clinical value as reliable biomarkers for tumor detection, subtyping, and classification, their direct biological impacts at the individual gene level remain elusive. Here we present MethylationToActivity (M2A), a machine learning framework that uses convolutional neural networks to infer promoter activities based on H3K4me3 and H3K27ac enrichment, from DNA methylation patterns for individual genes. Using publicly available datasets in real-world test scenarios, we demonstrate that M2A is highly accurate and robust in revealing promoter activity landscapes in various pediatric and adult cancers, including both solid and hematologic malignant neoplasms.

2020:8

1. Blood

   FBXO11-Mediated Proteolysis of BAHD1 Relieves PRC2-Dependent Transcriptional Repression in Erythropoiesis

   Xu Peng, Scott Daniel C., Xu Beisi, Yao Yu, Feng Ruopeng, Cheng Li, Mayberry Kalin, Bi Wenjian, Palmer Lance E, King Moeko T., Wang Hong, Li Yuxin, Fan Yiping, Alpi Arno F., Li Chunliang, Peng Junmin, Papizan James, Pruett-Miller Shondra M, Spallek Ria, Bassermann Florian, Cheng Yong, Schulman Brenda A., Weiss Mitchell J.

   Blood 2020

   Abstract DOI PMC PMID GEO:Human.CHIP.RNA

   The histone mark H3K27me3 and its reader/writer Polycomb repressive complex 2 (PRC2) mediate widespread transcriptional repression in stem and progenitor cells. Mechanisms that regulate this activity are critical for hematopoietic development but poorly understood. Here we show that the E3 ubiquitin ligase FBXO11 relieves PRC2-mediated repression during erythroid maturation by targeting its newly identified substrate BAHD1, an H3K27me3 reader that recruits transcriptional co-repressors. Erythroblasts lacking FBXO11 are developmentally delayed, with reduced expression of maturation-associated genes, most of which harbor bivalent histone marks (activating H3K4me3 and repressive H3K27me3), bind BAHD1, and fail to recruit the erythroid transcription factor GATA1. The BAHD1 complex interacts physically with PRC2 and depletion of either component restores FBXO11-deficient erythroid gene expression. Our studies identify BAHD1 as a novel effector of PRC2-mediated repression and reveal how a single E3 ubiquitin ligase eliminates PRC2 repression at developmentally poised bivalent genes during erythropoiesis.

2. eLife

   Functional Interrogation of HOXA9 Regulome in MLLr Leukemia via Reporter-Based CRISPR/Cas9 Screen

   Zhang Hao, Zhang Yang, Zhou Xinyue, Wright Shaela, Hyle Judith, Zhao Lianzhong, An Jie, Zhao Xujie, Shao Ying, Xu Beisi, Lee Hyeong-Min, Chen Taosheng, Zhou Yang, Chen Xiang, Lu Rui, Li Chunliang

   eLife 2020

   Abstract DOI PMC PMID GEO:Human.CutRun

   Aberrant HOXA9 expression is a hallmark of most aggressive acute leukemias, notably those with KMT2A (MLL) gene rearrangements. HOXA9 overexpression not only predicts poor diagnosis and outcome but also plays a critical role in leukemia transformation and maintenance. However, our current understanding of HOXA9 regulation in leukemia is limited, hindering development of therapeutic strategies. Here, we generated the HOXA9-mCherry knock-in reporter cell lines to dissect HOXA9 regulation. By utilizing the reporter and CRISPR/Cas9 screens, we identified transcription factors controlling HOXA9 expression, including a novel regulator, USF2, whose depletion significantly down-regulated HOXA9 expression and impaired MLLr leukemia cell proliferation. Ectopic expression of Hoxa9 rescued impaired leukemia cell proliferation upon USF2 loss. Cut and Run analysis revealed the direct occupancy of USF2 at HOXA9 promoter in MLLr leukemia cells. Collectively, the HOXA9 reporter facilitated the functional interrogation of the HOXA9 regulome and has advanced our understanding of the molecular regulation network in HOXA9-driven leukemia.

3. Epigenet. Chromatin

   UTX/KDM6A Suppresses AP-1 and a Gliogenesis Program during Neural Differentiation of Human Pluripotent Stem Cells

   Xu Beisi*, Mulvey Brett*, Salie Muneeb, Yang Xiaoyang, Matsui Yurika, Nityanandam Anjana, Fan Yiping, Peng Jamy C.

   Epigenetics & Chromatin 2020

   Abstract DOI PMC PMID GEO:Human.CHIP.RNA.ATAC

   Background UTX/KDM6A is known to interact and influence multiple different chromatin modifiers to promote an open chromatin environment to facilitate gene activation, but its molecular activities in developmental gene regulation remain unclear. Results We report that in human neural stem cells, UTX binding correlates with both promotion and suppression of gene expression. These activities enable UTX to modulate neural stem cell self-renewal, promote neurogenesis, and suppress gliogenesis. In neural stem cells, UTX has a less influence over histone H3 lysine 27 and lysine 4 methylation but more predominantly affects histone H3 lysine 27 acetylation and chromatin accessibility. Furthermore, UTX suppresses components of AP-1 and, in turn, a gliogenesis program. Conclusions Our findings revealed that UTX coordinates dualistic gene regulation to govern neural stem cell properties and neurogenesis–gliogenesis switch.

4. Nat. Commun.

   Ybx1 Fine-Tunes PRC2 Activities to Control Embryonic Brain Development

   Evans Myron K., Matsui Yurika*, Xu Beisi*, Willis Catherine, Loome Jennifer, Milburn Luis, Fan Yiping, Pagala Vishwajeeth, Peng Jamy C.

   Nature Communications 2020

   Abstract DOI PMC PMID GEO:Mouse.CHIP.RNA

   Chromatin modifiers affect spatiotemporal gene expression programs that underlie organismal development. The Polycomb repressive complex 2 (PRC2) is a crucial chromatin modifier in executing neurodevelopmental programs. Here, we find that PRC2 interacts with the nucleic acid–binding protein Ybx1. In the mouse embryo in vivo, Ybx1 is required for forebrain specification and restricting mid-hindbrain growth. In neural progenitor cells (NPCs), Ybx1 controls self-renewal and neuronal differentiation. Mechanistically, Ybx1 highly overlaps PRC2 binding genome-wide, controls PRC2 distribution, and inhibits H3K27me3 levels. These functions are consistent with Ybx1-mediated promotion of genes involved in forebrain specification, cell proliferation, or neuronal differentiation. In Ybx1-knockout NPCs, H3K27me3 reduction by PRC2 enzymatic inhibitor or genetic depletion partially rescues gene expression and NPC functions. Our findings suggest that Ybx1 fine-tunes PRC2 activities to regulate spatiotemporal gene expression in embryonic neural development and uncover a crucial epigenetic mechanism balancing forebrain–hindbrain lineages and self-renewal–differentiation choices in NPCs.

5. Viral Immunol.

   From Influenza Virus Infections to Lupus: Synchronous Estrogen Receptor α and RNA Polymerase II Binding Within the Immunoglobulin Heavy Chain Locus

   Jones Bart G., Sealy Robert E., Penkert Rhiannon R., Surman Sherri L., Birshtein Barbara K., Xu Beisi, Neale Geoffrey, Maul Robert W., Gearhart Patricia J., Hurwitz Julia L.

   Viral Immunology 2020

   Abstract DOI PMC PMID

   Males and females respond to pathogens differently and exhibit significantly different frequencies of autoimmune disease. For example, vaccinated adult females control influenza virus better than males, but females suffer systemic lupus erythematosus at a 9:1 frequency compared to males. Numerous explanations have been offered for these sex differences, but most have involved indirect mechanisms by which estrogen, a nuclear hormone, modifies cell barriers or immunity. In search of a direct mechanism, we examined the binding of estrogen receptor α (ERα), a class I nuclear hormone receptor, to the immunoglobulin heavy chain locus. Here, we show that in purified murine B cells, ERα and RNA polymerase II (RNA Pol II) exhibit extraordinarily similar DNA binding patterns. We further demonstrate that ERα preferentially binds adenosine–cytidine (AC)-repeats in the immunoglobulin heavy chain locus when supplemental estrogen is added to purified, lipopolysaccharide-activated B cells. Based on these and previous data, we hypothesize that (i) estrogen guides the binding of ERα and its RNA Pol II partner within the locus, which in turn instructs sterile transcription and class switch recombination (CSR), (ii) ERα binding to AC-repeats modifies the DNA architecture and loops associated with CSR, and (iii) by these mechanisms, estrogen instructs antibody expression. By targeting ERα-DNA interactions in the immunoglobulin heavy chain locus, clinicians may ultimately enhance antibody responses in the context of infectious diseases and reduce antibody responses in the context of allergic or autoimmune reactions.

6. Nat. Commun.

   MYCN Amplification and ATRX Mutations Are Incompatible in Neuroblastoma

   Zeineldin Maged, Federico Sara, Chen Xiang, Fan Yiping, Xu Beisi, Stewart Elizabeth, Zhou Xin, Jeon Jongrye, Griffiths Lyra, Nguyen Rosa, Norrie Jackie, Easton John, Mulder Heather, Yergeau Donald, Liu Yanling, Wu Jianrong, Van Ryn Collin, Naranjo Arlene, Hogarty Michael D., Kamiński Marcin M., Valentine Marc, Pruett-Miller Shondra M., Pappo Alberto, Zhang Jinghui, Clay Michael R., Bahrami Armita, Vogel Peter, Lee Seungjae, Shelat Anang, Sarthy Jay F., Meers Michael P., George Rani E., Mardis Elaine R., Wilson Richard K., Henikoff Steven, Downing James R., Dyer Michael A.

   Nature Communications 2020

   Abstract DOI PMC PMID Browser:Human_hg19 EGA:WGS EGA:CHIP

   Aggressive cancers often have activating mutations in growth-controlling oncogenes and inactivating mutations in tumor-suppressor genes. In neuroblastoma, amplification of the MYCN oncogene and inactivation of the ATRX tumor-suppressor gene correlate with high-risk disease and poor prognosis. Here we show that ATRX mutations and MYCN amplification are mutually exclusive across all ages and stages in neuroblastoma. Using human cell lines and mouse models, we found that elevated MYCN expression and ATRX mutations are incompatible. Elevated MYCN levels promote metabolic reprogramming, mitochondrial dysfunction, reactive-oxygen species generation, and DNA-replicative stress. The combination of replicative stress caused by defects in the ATRX–histone chaperone complex, and that induced by MYCN-mediated metabolic reprogramming, leads to synthetic lethality. Therefore, ATRX and MYCN represent an unusual example, where inactivation of a tumor-suppressor gene and activation of an oncogene are incompatible. This synthetic lethality may eventually be exploited to improve outcomes for patients with high-risk neuroblastoma.

7. Cancer Res.

   An ABC Transporter Drives Medulloblastoma Pathogenesis by Regulating Sonic Hedgehog Signaling

   Wijaya Juwina, Vo BaoHan T., Liu Jingjing, Xu Beisi, Wu Gang, Wang Yao, Peng Junmin, Zhang Jin, Janke Laura J., Orr Brent A., Yu Jiyang, Roussel Martine F., Schuetz John D.

   Cancer Research 2020

   Abstract DOI PMC PMID

   Mutations in Sonic hedgehog (SHH) signaling promote aberrant proliferation and tumor growth. SHH medulloblastoma (MB) is among the most frequent brain tumors in children less than three years of age. While key components of the SHH pathway are well-known, we hypothesized that new disease-modifying targets of SHH MB might be identified from large-scale bioinformatics and systems biology analyses. Using a data-driven systems biology approach, we built a medulloblastoma-specific interactome. The ATP-binding cassette transporter ABCC4 was identified as a modulator of SHH-MB. Accordingly, increased ABCC4 expression correlated with poor overall survival in SHH MB patients. Knockdown of ABCC4 expression markedly blunted the constitutive activation of the SHH pathway secondary to Ptch1 or Sufu insufficiency. In human tumor cell lines, ABCC4 knockdown and inhibition reduced full-length GLI3 levels. In a clinically relevant murine SHH MB model, targeted ablation of Abcc4 in primary tumors significantly reduced tumor burden and extended the lifespan of tumor-bearing mice. These studies reveal ABCC4 as a potent SHH pathway regulator and a new candidate to target with the potential to improve SHH MB therapy.

8. Genes Dev.

   Muscle-Derived Dpp Regulates Feeding Initiation via Endocrine Modulation of Brain Dopamine Biosynthesis

   Robles-Murguia Maricela, Rao Deepti, Finkelstein David, Xu Beisi, Fan Yiping, Demontis Fabio

   Genes & Development 2020

   Abstract DOI PMC PMID GEO:FruitFly.RNA

   In animals, the brain regulates feeding behavior in response to local energy demands of peripheral tissues, which secrete orexigenic and anorexigenic hormones. Although skeletal muscle is a key peripheral tissue, it remains unknown whether muscle-secreted hormones regulate feeding. In Drosophila, we found that decapentaplegic (dpp), the homolog of human bone morphogenetic proteins BMP2 and BMP4, is a muscle-secreted factor (a myokine) that is induced by nutrient sensing and that circulates and signals to the brain. Muscle-restricted dpp RNAi promotes foraging and feeding initiation, whereas dpp overexpression reduces it. This regulation of feeding by muscle-derived Dpp stems from modulation of brain tyrosine hydroxylase (TH) expression and dopamine biosynthesis. Consistently, Dpp receptor signaling in dopaminergic neurons regulates TH expression and feeding initiation via the downstream transcriptional repressor Schnurri. Moreover, pharmacologic modulation of TH activity rescues the changes in feeding initiation due to modulation of dpp expression in muscle. These findings indicate that muscle-to-brain endocrine signaling mediated by the myokine Dpp regulates feeding behavior.

2019:10

1. Nature

   Targeting REGNASE-1 Programs Long-Lived Effector T Cells for Cancer Therapy

   Wei Jun, Long Lingyun, Zheng Wenting, Dhungana Yogesh, Lim Seon Ah, Guy Cliff, Wang Yanyan, Wang Yong-Dong, Qian Chenxi, Xu Beisi, Kc Anil, Saravia Jordy, Huang Hongling, Yu Jiyang, Doench John G., Geiger Terrence L., Chi Hongbo

   Nature 2019

   Abstract DOI PMC PMID GEO:Mouse.ATAC.scRNA.RNA.Microarray

   Adoptive cell therapy represents a new paradigm in cancer immunotherapy, but it can be limited by the poor persistence and function of transferred T cells1. Here we use an in vivo pooled CRISPR–Cas9 mutagenesis screening approach to demonstrate that, by targeting REGNASE-1, CD8+ T cells are reprogrammed to long-lived effector cells with extensive accumulation, better persistence and robust effector function in tumours. REGNASE-1-deficient CD8+ T cells show markedly improved therapeutic efficacy against mouse models of melanoma and leukaemia. By using a secondary genome-scale CRISPR–Cas9 screening, we identify BATF as the key target of REGNASE-1 and as a rheostat that shapes antitumour responses. Loss of BATF suppresses the increased accumulation and mitochondrial fitness of REGNASE-1-deficient CD8+ T cells. By contrast, the targeting of additional signalling factors—including PTPN2 and SOCS1—improves the therapeutic efficacy of REGNASE-1-deficient CD8+ T cells. Our findings suggest that T cell persistence and effector function can be coordinated in tumour immunity and point to avenues for improving the efficacy of adoptive cell therapy for cancer.

2. Cell. Immunol.

   Matters of Life and Death: How Estrogen and Estrogen Receptor Binding to the Immunoglobulin Heavy Chain Locus May Influence Outcomes of Infection, Allergy, and Autoimmune Disease

   Jones Bart G., Penkert Rhiannon R., Surman Sherri L., Sealy Robert E., Pelletier Stephane, Xu Beisi, Neale Geoff, Maul Robert W., Gearhart Patricia J., Hurwitz J. L.

   Cellular Immunology 2019

   Abstract DOI PMC PMID

   Sex hormones are best known for their influences on reproduction, but they also have profound influences on the immune response. Examples of sex-specific differences include: (i) the relatively poor control of influenza virus infections in males compared to females, (ii) allergic asthma, an IgE-associated hypersensitivity reaction that is exacerbated in adolescent females compared to males, and (iii) systemic lupus erythematosus, a life-threatening autoimmune disease with a 9:1 female:male bias. Here we consider how estrogen and estrogen receptor α (ERα) may influence the immune response by modifying class switch recombination (CSR) and immunoglobulin expression patterns. We focus on ERα binding to enhancers (Eμ and the 3’ regulatory region) and switch sites (S\textmu and Sε) in the immunoglobulin heavy chain locus. Our preliminary data from ChIP-seq analyses of purified, activated B cells show estrogen-mediated changes in the positioning of ERα binding within and near S\textmu and Sε. In the presence of estrogen, ERα is bound not only to estrogen response elements (ERE), but also to adenosine-cytidine (AC)-repeats and poly adenosine (poly A) sequences, in some cases within constant region gene introns. We propose that by binding these sites, estrogen and ERα directly participate in the DNA loop formation required for CSR. We further suggest that estrogen regulates immunoglobulin expression patterns and can thereby influence life-and-death outcomes of infection, hypersensitivity, and autoimmune disease.

3. Neuron

   Nucleome Dynamics during Retinal Development

   Norrie Jackie L.*, Lupo Marybeth S.*, Xu Beisi*, Al Diri Issam*, Valentine Marc*, Putnam Daniel*, Griffiths Lyra, Zhang Jiakun, Johnson Dianna, Easton John, Shao Ying, Honnell Victoria, Frase Sharon, Miller Shondra, Stewart Valerie, Zhou Xin, Chen Xiang, Dyer Michael A.

   Neuron 2019

   Abstract DOI PMC PMID Browser:Mouse_mm9 Browser:Human_hg19 GEO:GSE87064

   Summary More than 8,000 genes are turned on or off as progenitor cells produce the 7 classes of retinal cell types during development. Thousands of enhancers are also active in the developing retinae, many having features of cell- and developmental stage-specific activity. We studied dynamic changes in the 3D chromatin landscape important for precisely orchestrated changes in gene expression during retinal development by ultra-deep in situ Hi-C analysis on murine retinae. We identified developmental-stage-specific changes in chromatin compartments and enhancer-promoter interactions. We developed a machine learning-based algorithm to map euchromatin and heterochromatin domains genome-wide and overlaid it with chromatin compartments identified by Hi-C. Single-cell ATAC-seq and RNA-seq were integrated with our Hi-C and previous ChIP-seq data to identify cell- and developmental-stage-specific super-enhancers (SEs). We identified a bipolar neuron-specific core regulatory circuit SE upstream of Vsx2, whose deletion in mice led to the loss of bipolar neurons.

4. Nucleic Acids Res.

   Acute Depletion of CTCF Directly Affects MYC Regulation through Loss of Enhancer–Promoter Looping

   Hyle Judith, Zhang Yang, Wright Shaela, Xu Beisi, Shao Ying, Easton John, Tian Liqing, Feng Ruopeng, Xu Peng, Li Chunliang

   Nucleic Acids Research 2019

   Abstract DOI PMC PMID GEO:Human.RNA GEO:Human.CaptureC GEO:GSE126619

   Numerous pieces of evidence support the complex, 3D spatial organization of the genome dictates gene expression. CTCF is essential to define topologically associated domain boundaries and to facilitate the formation of insulated chromatin loop structures. To understand CTCF’s direct role in global transcriptional regulation, we integrated the miniAID-mClover3 cassette to the endogenous CTCF locus in a human pediatric B-ALL cell line, SEM, and an immortal erythroid precursor cell line, HUDEP-2, to allow for acute depletion of CTCF protein by the auxin-inducible degron system. In SEM cells, CTCF loss notably disrupted intra-TAD loops and TAD integrity in concurrence with a reduction in CTCF-binding affinity, while showing no perturbation to nuclear compartment integrity. Strikingly, the overall effect of CTCF’s loss on transcription was minimal. Whole transcriptome analysis showed hundreds of genes differentially expressed in CTCF-depleted cells, among which MYC and a number of MYC target genes were specifically downregulated. Mechanically, acute depletion of CTCF disrupted the direct interaction between the MYC promoter and its distal enhancer cluster residing ∼1.8 Mb downstream. Notably, MYC expression was not profoundly affected upon CTCF loss in HUDEP-2 cells suggesting that CTCF could play a B-ALL cell line specific role in maintaining MYC expression.

5. Nat. Commun.

   Long-Read Sequencing Unveils IGH-DUX4 Translocation into the Silenced IGH Allele in B-Cell Acute Lymphoblastic Leukemia

   Tian Liqing, Shao Ying, Nance Stephanie, Dang Jinjun, Xu Beisi, Ma Xiaotu, Li Yongjin, Ju Bensheng, Dong Li, Newman Scott, Zhou Xin, Schreiner Patrick, Tseng Elizabeth, Hon Ting, Ashby Meredith, Li Chunliang, Easton John, Gruber Tanja A., Zhang Jinghui

   Nature Communications 2019

   Abstract DOI PMC PMID GEO:Human.ChIP.Hi-C.HiChIP.CaptureC.ATAC SRA:Human.PacBio.RRBS.10XChromiumWGS

   IGH@ proto-oncogene translocation is a common oncogenic event in lymphoid lineage cancers such as B-ALL, lymphoma and multiple myeloma. Here, to investigate the interplay between IGH@ proto-oncogene translocation and IGH allelic exclusion, we perform long-read whole-genome and transcriptome sequencing along with epigenetic and 3D genome profiling of Nalm6, an IGH-DUX4 positive B-ALL cell line. We detect significant allelic imbalance on the wild-type over the IGH-DUX4 haplotype in expression and epigenetic data, showing IGH-DUX4 translocation occurs on the silenced IGH allele. In vitro, this reduces the oncogenic stress of DUX4 high-level expression. Moreover, patient samples of IGH-DUX4 B-ALL have similar expression profile and IGH breakpoints as Nalm6, suggesting a common mechanism to allow optimal dosage of non-toxic DUX4 expression.

6. Acta Neuropathol.

   H3.3 K27M Depletion Increases Differentiation and Extends Latency of Diffuse Intrinsic Pontine Glioma Growth in Vivo

   Silveira André B., Kasper Lawryn H., Fan Yiping, Jin Hongjian, Wu Gang, Shaw Timothy I., Zhu Xiaoyan, Larson Jon D., Easton John, Shao Ying, Yergeau Donald A., Rosencrance Celeste, Boggs Kristy, Rusch Michael C., Ding Liang, Zhang Junyuan, Finkelstein David, Noyes Rachel M., Russell Brent L., Xu Beisi, Broniscer Alberto, Wetmore Cynthia, Pounds Stanley B., Ellison David W., Zhang Jinghui, Baker Suzanne J.

   Acta Neuropathologica 2019

   Abstract DOI PMC PMID GEO:Human.CHIP.RNA

   Histone H3 K27M mutation is the defining molecular feature of the devastating pediatric brain tumor, diffuse intrinsic pontine glioma (DIPG). The prevalence of histone H3 K27M mutations indicates a critical role in DIPGs, but the contribution of the mutation to disease pathogenesis remains unclear. We show that knockdown of this mutation in DIPG xenografts restores K27M-dependent loss of H3K27me3 and delays tumor growth. Comparisons of matched DIPG xenografts with and without K27M knockdown allowed identification of mutation-specific effects on the transcriptome and epigenome. The resulting transcriptional changes recapitulate expression signatures from K27M primary DIPG tumors and are strongly enriched for genes associated with nervous system development. Integrated analysis of ChIP-seq and expression data showed that genes upregulated by the mutation are overrepresented in apparently bivalent promoters. Many of these targets are associated with more immature differentiation states. Expression profiles indicate K27M knockdown decreases proliferation and increases differentiation within lineages represented in DIPG. These data suggest that K27M-mediated loss of H3K27me3 directly regulates a subset of genes by releasing poised promoters, and contributes to tumor phenotype and growth by limiting differentiation. The delayed tumor growth associated with knockdown of H3 K27M provides evidence that this highly recurrent mutation is a relevant therapeutic target.

7. Int. Immunol.

   Complex Sex-Biased Antibody Responses: Estrogen Receptors Bind Estrogen Response Elements Centered within Immunoglobulin Heavy Chain Gene Enhancers

   Jones Bart G, Sealy Robert E, Penkert Rhiannon R, Surman Sherri L, Maul Robert W, Neale Geoff, Xu Beisi, Gearhart Patricia J, Hurwitz Julia L

   International Immunology 2019

   Abstract DOI PMC PMID

   Nuclear hormone receptors including the estrogen receptor (ERα) and the retinoic acid receptor regulate a plethora of biological functions including reproduction, circulation and immunity. To understand how estrogen and other nuclear hormones influence antibody production, we characterized total serum antibody isotypes in female and male mice of C57BL/6J, BALB/cJ and C3H/HeJ mouse strains. Antibody levels were higher in females compared to males in all strains and there was a female preference for IgG2b production. Sex-biased patterns were influenced by vitamin levels, and by antigen specificity toward influenza virus or pneumococcus antigens. To help explain sex biases, we examined the direct effects of estrogen on immunoglobulin heavy chain sterile transcript production among purified, lipopolysaccharide-stimulated B cells. Supplemental estrogen in B-cell cultures significantly increased immunoglobulin heavy chain sterile transcripts. Chromatin immunoprecipitation analyses of activated B cells identified significant ERα binding to estrogen response elements (EREs) centered within enhancer elements of the immunoglobulin heavy chain locus, including the E\textmu enhancer and hypersensitive site 1,2 (HS1,2) in the 3’ regulatory region. The ERE in HS1,2 was conserved across animal species, and in humans marked a site of polymorphism associated with the estrogen-augmented autoimmune disease, lupus. Taken together, the results highlight: (i) the important targets of ERα in regulatory regions of the immunoglobulin heavy chain locus that influence antibody production, and (ii) the complexity of mechanisms by which estrogen instructs sex-biased antibody production profiles.

8. Nat. Neurosci.

   Differentiation of Human Pluripotent Stem Cells into Neurons or Cortical Organoids Requires Transcriptional Co-Regulation by UTX and 53BP1

   Yang Xiaoyang*, Xu Beisi*, Mulvey Brett, Evans Myron, Jordan Samuel, Wang Yong-Dong, Pagala Vishwajeeth, Peng Junmin, Fan Yiping, Patel Arishna, Peng Jamy C.

   Nature Neuroscience 2019

   Abstract DOI PMC PMID GEO:Human.CHIP.RNA.Mouse.RNA

   UTX is a chromatin modifier required for development and neural lineage specification, but how it controls these biological processes is unclear. To determine the molecular mechanisms of UTX, we identified novel UTX protein interaction partners. Here we show that UTX and 53BP1 directly interact and co-occupy promoters in human embryonic stem cells and differentiating neural progenitor cells. Human 53BP1 contains a UTX-binding site that diverges from its mouse homolog by 41%, and disruption of the 53BP1–UTX interaction abrogated human, but not mouse, neurogenesis in vitro. The 53BP1–UTX interaction is required to upregulate key neurodevelopmental genes during the differentiation of human embryonic stem cells into neurons or into cortical organoids. 53BP1 promotes UTX chromatin binding, and in turn H3K27 modifications and gene activation, at a subset of genomic regions, including neurogenic genes. Overall, our data suggest that the 53BP1–UTX interaction supports the activation of key genes required for human neurodevelopment.

9. Nature

   Metabolic Heterogeneity Underlies Reciprocal Fates of T H 17 Cell Stemness and Plasticity

   Karmaus Peer W. F., Chen Xiang, Lim Seon Ah, Herrada Andrés A., Nguyen Thanh-Long M., Xu Beisi, Dhungana Yogesh, Rankin Sherri, Chen Wenan, Rosencrance Celeste, Yang Kai, Fan Yiping, Cheng Yong, Easton John, Neale Geoffrey, Vogel Peter, Chi Hongbo

   Nature 2019

   Abstract DOI PMC PMID GEO:Mouse.Microarray

   A defining feature of adaptive immunity is the development of long-lived memory T cells to curtail infection. Recent studies have identified a unique stem-like T-cell subset amongst exhausted CD8-positive T cells in chronic infection1–3, but it remains unclear whether CD4-positive T-cell subsets with similar features exist in chronic inflammatory conditions. Amongst helper T cells, TH17 cells have prominent roles in autoimmunity and tissue inflammation and are characterized by inherent plasticity4–7, although how such plasticity is regulated is poorly understood. Here we demonstrate that TH17 cells in a mouse model of autoimmune disease are functionally and metabolically heterogeneous; they contain a subset with stemness-associated features but lower anabolic metabolism, and a reciprocal subset with higher metabolic activity that supports transdifferentiation into TH1-like cells. These two TH17-cell subsets are defined by selective expression of the transcription factors TCF-1 and T-bet, and by discrete levels of CD27 expression. We also identify signalling via the kinase complex mTORC1 as a central regulator of TH17-cell fate decisions by coordinating metabolic and transcriptional programmes. TH17 cells with disrupted mTORC1 signalling or anabolic metabolism fail to induce autoimmune neuroinflammation or to develop into TH1-like cells, but instead upregulate TCF-1 expression and acquire stemness-associated features. Single-cell RNA sequencing and experimental validation reveal heterogeneity in fate-mapped TH17 cells, and a developmental arrest in the TH1 transdifferentiation trajectory upon loss of mTORC1 activity or metabolic perturbation. Our results establish that the dichotomy of stemness and effector function underlies the heterogeneous TH17 responses and autoimmune pathogenesis, and point to previously unappreciated metabolic control of plasticity in helper T cells.

10. Cancer Cell

    Histone H3.3 K27M Accelerates Spontaneous Brainstem Glioma and Drives Restricted Changes in Bivalent Gene Expression

    Larson Jon D., Kasper Lawryn H., Paugh Barbara S., Jin Hongjian, Wu Gang, Kwon Chang-Hyuk, Fan Yiping, Shaw Timothy I., Silveira André B., Qu Chunxu, Xu Raymond, Zhu Xiaoyan, Zhang Junyuan, Russell Helen R., Peters Jennifer L., Finkelstein David, Xu Beisi, Lin Tong, Tinkle Christopher L., Patay Zoltan, Onar-Thomas Arzu, Pounds Stanley B., McKinnon Peter J., Ellison David W., Zhang Jinghui, Baker Suzanne J.

    Cancer Cell 2019

    Abstract DOI PMC PMID GEO:Mouse.CHIP.RNA

    Summary Diffuse intrinsic pontine gliomas (DIPGs) are incurable childhood brainstem tumors with frequent histone H3 K27M mutations and recurrent alterations in PDGFRA and TP53. We generated genetically engineered inducible mice and showed that H3.3 K27M enhanced neural stem cell self-renewal while preserving regional identity. Neonatal induction of H3.3 K27M cooperated with activating platelet-derived growth factor receptor α (PDGFRα) mutant and Trp53 loss to accelerate development of diffuse brainstem gliomas that recapitulated human DIPG gene expression signatures and showed global changes in H3K27 posttranslational modifications, but relatively restricted gene expression changes. Genes upregulated in H3.3 K27M tumors were enriched for those associated with neural development where H3K27me3 loss released the poised state of apparently bivalent promoters, whereas downregulated genes were enriched for those encoding homeodomain transcription factors.

2018:6

1. Dev. Cell

   The Hippo Pathway Prevents YAP/TAZ-Driven Hypertranscription and Controls Neural Progenitor Number

   Lavado Alfonso, Park Jun Young, Paré Joshua, Finkelstein David, Pan Haitao, Xu Beisi, Fan Yiping, Kumar Ram Parikshan, Neale Geoffrey, Kwak Young Don, McKinnon Peter J., Johnson Randy L., Cao Xinwei

   Developmental Cell 2018

   Abstract DOI PMC PMID GEO:Mouse.CHIP.RNA.Microarray

   Summary The Hippo pathway controls the activity of YAP/TAZ transcriptional coactivators through a kinase cascade. Despite the critical role of this pathway in tissue growth and tumorigenesis, it remains unclear how YAP/TAZ-mediated transcription drives proliferation. By analyzing the effects of inactivating LATS1/2 kinases, the direct upstream inhibitors of YAP/TAZ, on mouse brain development and applying cell-number-normalized transcriptome analyses, we discovered that YAP/TAZ activation causes a global increase in transcription activity, known as hypertranscription, and upregulates many genes associated with cell growth and proliferation. In contrast, conventional read-depth-normalized RNA-sequencing analysis failed to detect the scope of the transcriptome shift and missed most relevant gene ontologies. Following a transient increase in proliferation, however, hypertranscription in neural progenitors triggers replication stress, DNA damage, and p53 activation, resulting in massive apoptosis. Our findings reveal a significant impact of YAP/TAZ activation on global transcription activity and have important implications for understanding YAP/TAZ function.

2. Nature

   The Genetic Basis and Cell of Origin of Mixed Phenotype Acute Leukaemia

   Alexander Thomas B., Gu Zhaohui, Iacobucci Ilaria, Dickerson Kirsten, Choi John K., Xu Beisi, Payne-Turner Debbie, Yoshihara Hiroki, Loh Mignon L., Horan John, Buldini Barbara, Basso Giuseppe, Elitzur Sarah, de Haas Valerie, Zwaan C. Michel, Yeoh Allen, Reinhardt Dirk, Tomizawa Daisuke, Kiyokawa Nobutaka, Lammens Tim, De Moerloose Barbara, Catchpoole Daniel, Hori Hiroki, Moorman Anthony, Moore Andrew S., Hrusak Ondrej, Meshinchi Soheil, Orgel Etan, Devidas Meenakshi, Borowitz Michael, Wood Brent, Heerema Nyla A., Carrol Andrew, Yang Yung-Li, Smith Malcolm A., Davidsen Tanja M., Hermida Leandro C., Gesuwan Patee, Marra Marco A., Ma Yussanne, Mungall Andrew J., Moore Richard A., Jones Steven J. M., Valentine Marcus, Janke Laura J., Rubnitz Jeffrey E., Pui Ching-Hon, Ding Liang, Liu Yu, Zhang Jinghui, Nichols Kim E., Downing James R., Cao Xueyuan, Shi Lei, Pounds Stanley, Newman Scott, Pei Deqing, Guidry Auvil Jaime M., Gerhard Daniela S., Hunger Stephen P., Inaba Hiroto, Mullighan Charles G.

   Nature 2018

   Abstract DOI PMC PMID Browser:MPAL.Oncoprint GEO:Mouse.CHIP.RNA

   Mixed phenotype acute leukaemia (MPAL) is a high-risk subtype of leukaemia with myeloid and lymphoid features, limited genetic characterization, and a lack of consensus regarding appropriate therapy. Here we show that the two principal subtypes of MPAL, T/myeloid (T/M) and B/myeloid (B/M), are genetically distinct. Rearrangement of ZNF384 is common in B/M MPAL, and biallelic WT1 alterations are common in T/M MPAL, which shares genomic features with early T-cell precursor acute lymphoblastic leukaemia. We show that the intratumoral immunophenotypic heterogeneity characteristic of MPAL is independent of somatic genetic variation, that founding lesions arise in primitive haematopoietic progenitors, and that individual phenotypic subpopulations can reconstitute the immunophenotypic diversity in vivo. These findings indicate that the cell of origin and founding lesions, rather than an accumulation of distinct genomic alterations, prime tumour cells for lineage promiscuity. Moreover, these findings position MPAL in the spectrum of immature leukaemias and provide a genetically informed framework for future clinical trials of potential treatments for MPAL.

3. Cancer Cell

   Identification of Therapeutic Targets in Rhabdomyosarcoma through Integrated Genomic, Epigenomic, and Proteomic Analyses

   Stewart Elizabeth, McEvoy Justina, Wang Hong, Chen Xiang, Honnell Victoria, Ocarz Monica, Gordon Brittney, Dapper Jason, Blankenship Kaley, Yang Yanling, Li Yuxin, Shaw Timothy I., Cho Ji-Hoon, Wang Xusheng, Xu Beisi, Gupta Pankaj, Fan Yiping, Liu Yu, Rusch Michael, Griffiths Lyra, Jeon Jongrye, Freeman Burgess B., Clay Michael R., Pappo Alberto, Easton John, Shurtleff Sheila, Shelat Anang, Zhou Xin, Boggs Kristy, Mulder Heather, Yergeau Donald, Bahrami Armita, Mardis Elaine R., Wilson Richard K., Zhang Jinghui, Peng Junmin, Downing James R., Dyer Michael A.

   Cancer Cell 2018

   Abstract DOI PMC PMID Browser:Human_hg19 EGA:Link

   Summary Personalized cancer therapy targeting somatic mutations in patient tumors is increasingly being incorporated into practice. Other therapeutic vulnerabilities resulting from changes in gene expression due to tumor specific epigenetic perturbations are progressively being recognized. These genomic and epigenomic changes are ultimately manifest in the tumor proteome and phosphoproteome. We integrated transcriptomic, epigenomic, and proteomic/phosphoproteomic data to elucidate the cellular origins and therapeutic vulnerabilities of rhabdomyosarcoma (RMS). We discovered that alveolar RMS occurs further along the developmental program than embryonal RMS. We also identified deregulation of the RAS/MEK/ERK/CDK4/6, G2/M, and unfolded protein response pathways through our integrated analysis. Comprehensive preclinical testing revealed that targeting the WEE1 kinase in the G2/M pathway is the most effective approach in vivo for high-risk RMS.

4. Sci. Rep.

   Mouse Medulloblastoma Driven by CRISPR Activation of Cellular Myc

   Vo BaoHan T., Kwon Jin Ah, Li Chunliang, Finkelstein David, Xu Beisi, Orr Brent A., Sherr Charles J., Roussel Martine F.

   Scientific Reports 2018

   Abstract DOI PMC PMID GEO:Mouse.Microarray GEO:Mouse.CHIP

   MYC-driven Group 3 (G3) medulloblastoma (MB) is the most aggressive of four molecular subgroups classified by transcriptome, genomic landscape and clinical outcomes. Mouse models that recapitulate human G3 MB all rely on retroviral vector-induced Myc expression driven by viral regulatory elements (Retro-Myc tumors). We used nuclease-deficient CRISPR/dCas9-based gene activation with combinatorial single guide RNAs (sgRNAs) to enforce transcription of endogenous Myc in Trp53-null neurospheres that were orthotopically transplanted into the brains of naïve animals. Three combined sgRNAs linked to dCas9-VP160 induced cellular Myc expression and large cell anaplastic MBs (CRISPR-Myc tumors) which recapitulated the molecular characteristics of mouse and human G3 MBs. The BET inhibitor JQ1 suppressed MYC expression in a human G3 MB cell line (HD-MB03) and CRISPR-Myc, but not in Retro-Myc MBs. This G3 MB mouse model in which Myc expression is regulated by its own promoter will facilitate pre-clinical studies with drugs that regulate Myc transcription.

5. Cancer Discov.

   MYC Drives a Subset of High-Risk Pediatric Neuroblastomas and Is Activated through Mechanisms Including Enhancer Hijacking and Focal Enhancer Amplification

   Zimmerman Mark W., Liu Yu, He Shuning, Durbin Adam D., Abraham Brian J., Easton John, Shao Ying, Xu Beisi, Zhu Shizhen, Zhang Xiaoling, Li Zhaodong, Weichert-Leahey Nina, Young Richard A., Zhang Jinghui, Look A. Thomas

   Cancer Discovery 2018

   Abstract DOI PMC PMID

   The amplified MYCN gene serves as an oncogenic driver in approximately 20% of high-risk pediatric neuroblastomas. Here, we show that the family member MYC is a potent transforming gene in a separate subset of high-risk neuroblastoma cases (∼10%), based on (i) its upregulation by focal enhancer amplification or genomic rearrangements leading to enhancer hijacking, and (ii) its ability to transform neuroblastoma precursor cells in a transgenic animal model. The aberrant regulatory elements associated with oncogenic MYC activation include focally amplified distal enhancers and translocation of highly active enhancers from other genes to within topologically associating domains containing the MYC gene locus. The clinical outcome for patients with high levels of MYC expression is virtually identical to that of patients with amplification of the MYCN gene, a known high-risk feature of this disease. Together, these findings establish MYC as a bona fide oncogene in a clinically significant group of high-risk childhood neuroblastomas. Significance: Amplification of the MYCN oncogene is a recognized hallmark of high-risk pediatric neuroblastoma. Here, we demonstrate that MYC is also activated as a potent oncogene in a distinct subset of neuroblastoma cases through either focal amplification of distal enhancers or enhancer hijacking mediated by chromosomal translocation. Cancer Discov; 8(3); 320–35. \textcopyright 2017 AACR. This article is highlighted in the In This Issue feature, p. 253

6. Cell Rep.

   Retinal Cell Type DNA Methylation and Histone Modifications Predict Reprogramming Efficiency and Retinogenesis in 3D Organoid Cultures

   Wang Lu*, Hiler Daniel*, Xu Beisi*, AlDiri Issam, Chen Xiang, Zhou Xin, Griffiths Lyra, Valentine Marc, Shirinifard Abbas, Sablauer András, Thiagarajan Suresh, Barabas Marie-Elizabeth, Zhang Jiakun, Johnson Dianna, Frase Sharon, Dyer Michael A.

   Cell Reports 2018

   Abstract DOI PMC PMID Browser:Mouse_mm9.iPSC Browser:Mouse_mm9_All Browser:Human_hg19 GEO:GSE87064

   Summary Diverse cell types can be reprogrammed into pluripotent stem cells by ectopic expression of Oct4 (Pou5f1), Klf4, Sox2, and Myc. Many of these induced pluripotent stem cells (iPSCs) retain memory, in terms of DNA methylation and histone modifications (epigenetic memory), of their cellular origins, and this may bias subsequent differentiation. Neurons are difficult to reprogram, and there has not been a systematic side-by-side characterization of reprogramming efficiency or epigenetic memory across different neuronal subtypes. Here, we compare reprogramming efficiency of five different retinal cell types at two different stages of development. Retinal differentiation from each iPSC line was measured using a quantitative standardized scoring system called STEM-RET and compared to the epigenetic memory. Neurons with the lowest reprogramming efficiency produced iPSC lines with the best retinal differentiation and were more likely to retain epigenetic memory of their cellular origins. In addition, we identified biomarkers of iPSCs that are predictive of retinal differentiation.

2017:2

1. Nature

   Orthotopic Patient-Derived Xenografts of Paediatric Solid Tumours

   Stewart Elizabeth, Federico Sara M., Chen Xiang, Shelat Anang A., Bradley Cori, Gordon Brittney, Karlstrom Asa, Twarog Nathaniel R., Clay Michael R., Bahrami Armita, Iii Burgess B. Freeman, Xu Beisi, Zhou Xin, Wu Jianrong, Honnell Victoria, Ocarz Monica, Blankenship Kaley, Dapper Jason, Mardis Elaine R., Wilson Richard K., Downing James, Zhang Jinghui, Easton John, Pappo Alberto, Dyer Michael A.

   Nature 2017

   Abstract DOI PMC PMID Browser:Human_hg19 EGA:Link

   Preclinical models of paediatric solid tumours that could help identify predictive biomarkers of a patient’s sensitivity to therapy have been lacking. Over five years, the authors have developed an open access collection of orthotopic xenografts of 12 types of paediatric tumour. Genomic and epigenetic characterization reveals that xenografts retain characteristics of the tumour of origin. A high-throughput drug screen provides a resource for the community to identify potentially efficacious drug combinations.

2. Neuron

   The Dynamic Epigenetic Landscape of the Retina During Development, Reprogramming, and Tumorigenesis

   Aldiri Issam*, Xu Beisi*, Wang Lu, Chen Xiang, Hiler Daniel, Griffiths Lyra, Valentine Marc, Shirinifard Abbas, Thiagarajan Suresh, Sablauer Andras, Barabas Marie-Elizabeth, Zhang Jiakun, Johnson Dianna, Frase Sharon, Zhou Xin, Easton John, Zhang Jinghui, Mardis Elaine R., Wilson Richard K., Downing James R., Dyer Michael A.

   Neuron 2017

   Abstract DOI PMC PMID Browser:Mouse_mm9 Browser:Human_hg19 GEO:Mouse.Human.CHIP.RNA.WGBS.ATAC

   In the developing retina, multipotent neural progenitors undergo unidirectional differentiation in a precise spatiotemporal order. Here we profile the epigenetic and transcriptional changes that occur during retinogenesis in mice and humans. Although some progenitor genes and cell cycle genes were epigenetically silenced during retinogenesis, the most dramatic change was derepression of cell-type-specific differentiation programs. We identified developmental-stage-specific super-enhancers and showed that most epigenetic changes are conserved in humans and mice. To determine how the epigenome changes during tumorigenesis and reprogramming, we performed integrated epigenetic analysis of murine and human retinoblastomas and induced pluripotent stem cells (iPSCs) derived from murine rod photoreceptors. The retinoblastoma epigenome mapped to the developmental stage when retinal progenitors switch from neurogenic to terminal patterns of cell division. The epigenome of retinoblastomas was more similar to that of the normal retina than that of retina-derived iPSCs, and we identified retina-specific epigenetic memory.

2016:3

1. Nat. Genet.

   Deregulation of DUX4 and ERG in Acute Lymphoblastic Leukemia

   Zhang Jinghui, McCastlain Kelly, Yoshihara Hiroki, Xu Beisi, Chang Yunchao, Churchman Michelle L., Wu Gang, Li Yongjin, Wei Lei, Iacobucci Ilaria, Liu Yu, Qu Chunxu, Wen Ji, Edmonson Michael, Payne-Turner Debbie, Kaufmann Kerstin B., Takayanagi Shin-ichiro, Wienholds Erno, Waanders Esmé, Ntziachristos Panagiotis, Bakogianni Sofia, Wang Jingjing, Aifantis Iannis, Roberts Kathryn G., Ma Jing, Song Guangchun, Easton John, Mulder Heather L., Chen Xiang, Newman Scott, Ma Xiaotu, Rusch Michael, Gupta Pankaj, Boggs Kristy, Vadodaria Bhavin, Dalton James, Liu Yanling, Valentine Marcus L., Ding Li, Lu Charles, Fulton Robert S., Fulton Lucinda, Tabib Yashodhan, Ochoa Kerri, Devidas Meenakshi, Pei Deqing, Cheng Cheng, Yang Jun, Evans William E., Relling Mary V., Pui Ching-Hon, Jeha Sima, Harvey Richard C., Chen I.-Ming L., Willman Cheryl L., Marcucci Guido, Bloomfield Clara D., Kohlschmidt Jessica, Mrózek Krzysztof, Paietta Elisabeth, Tallman Martin S., Stock Wendy, Foster Matthew C., Racevskis Janis, Rowe Jacob M., Luger Selina, Kornblau Steven M., Shurtleff Sheila A., Raimondi Susana C., Mardis Elaine R., Wilson Richard K., Dick John E., Hunger Stephen P., Loh Mignon L., Downing James R., Mullighan Charles G., Project the St Jude Children’s Research Hospital-Washington University Pediatric Cancer Genome

   Nature Genetics 2016

   Abstract DOI PMC PMID Browser:BALL.Oncoprint EGA:Link EGA:Link EGA:Link

   Chromosomal rearrangements deregulating hematopoietic transcription factors are common in acute lymphoblastic leukemia (ALL). Here we show that deregulation of the homeobox transcription factor gene DUX4 and the ETS transcription factor gene ERG is a hallmark of a subtype of B-progenitor ALL that comprises up to 7% of B-ALL. DUX4 rearrangement and overexpression was present in all cases and was accompanied by transcriptional deregulation of ERG, expression of a novel ERG isoform, ERGalt, and frequent ERG deletion. ERGalt uses a non-canonical first exon whose transcription was initiated by DUX4 binding. ERGalt retains the DNA-binding and transactivation domains of ERG, but it inhibits wild-type ERG transcriptional activity and is transforming. These results illustrate a unique paradigm of transcription factor deregulation in leukemia in which DUX4 deregulation results in loss of function of ERG, either by deletion or induced expression of an isoform that is a dominant-negative inhibitor of wild-type ERG function.

2. Mol. Immunol.

   Binding of Estrogen Receptors to Switch Sites and Regulatory Elements in the Immunoglobulin Heavy Chain Locus of Activated B Cells Suggests a Direct Influence of Estrogen on Antibody Expression

   Jones Bart G., Penkert Rhiannon R., Xu Beisi, Fan Yiping, Neale Geoff, Gearhart Patricia J., Hurwitz Julia L.

   Molecular Immunology 2016

   Abstract DOI PMC PMID

   Females and males differ in antibody isotype expression patterns and in immune responses to foreign- and self-antigens. For example, systemic lupus erythematosus is a condition that associates with the production of isotype-skewed anti-self antibodies, and exhibits a 9:1 female:male disease ratio. To explain differences between B cell responses in males and females, we sought to identify direct interactions of the estrogen receptor (ER) with the immunoglobulin heavy chain locus. This effort was encouraged by our previous identification of estrogen response elements (ERE) in heavy chain switch (S) regions. We conducted a full-genome chromatin immunoprecipitation analysis (ChIP-seq) using DNA from LPS-activated B cells and an ERα-specific antibody. Results revealed ER binding to a wide region of DNA, spanning sequences from the JH cluster to Cδ, with peaks in Eμ and Sμ sites. Additional peaks of ERα binding were coincident with hs1,2 and hs4 sites in the 3’ regulatory region (3’RR) of the heavy chain locus. This first demonstration of direct binding of ER to key regulatory elements in the immunoglobulin locus supports our hypothesis that estrogen and other nuclear hormone receptors and ligands may directly influence antibody expression and class switch recombination (CSR). Our hypothesis encourages the conduct of new experiments to evaluate the consequences of ER binding. A better understanding of ER:DNA interactions in the immunoglobulin heavy chain locus, and respective mechanisms, may ultimately translate to better control of antibody expression, better protection against pathogens, and prevention of pathologies caused by auto-immune disease.

3. Viral Immunol.

   Hotspots for Vitamin–Steroid–Thyroid Hormone Response Elements Within Switch Regions of Immunoglobulin Heavy Chain Loci Predict a Direct Influence of Vitamins and Hormones on B Cell Class Switch Recombination

   Hurwitz Julia L., Penkert Rhiannon R., Xu Beisi, Fan Yiping, Partridge Janet F., Maul Robert W., Gearhart Patricia J.

   Viral Immunology 2016

   Abstract DOI PMC PMID

   Vitamin A deficiencies are common throughout the world and have a significant negative influence on immune protection against viral infections. Mouse models demonstrate that the production of IgA, a first line of defense against viruses at mucosal sites, is inhibited in the context of vitamin A deficiency. In vitro, the addition of vitamin A to activated B cells can enhance IgA expression, but downregulate IgE. Previous reports have demonstrated that vitamin A modifies cytokine patterns, and in so doing may influence antibody isotype expression by an indirect mechanism. However, we have now discovered hundreds of potential response elements among Sμ, S\varepsilon, and Sα switch sites within immunoglobulin heavy chain loci. These hotspots appear in both mouse and human loci and include targets for vitamin receptors and related proteins (e.g., estrogen receptors) in the nuclear receptor superfamily. Full response elements with direct repeats are relatively infrequent or absent in Sγ regions although half-sites are present. Based on these results, we pose a hypothesis that nuclear receptors have a direct effect on the immunoglobulin heavy chain class switch recombination event. We propose that vitamin A may alter S site accessibility to activation-induced deaminase and nonhomologous end-joining machinery, thereby influencing the isotype switch, antibody production, and protection against viral infections at mucosal sites.

2015:3

1. Genes Dev.

   The Glucose-Sensing Transcription Factor MLX Promotes Myogenesis via Myokine Signaling

   Hunt Liam C., Xu Beisi, Finkelstein David, Fan Yiping, Carroll Patrick A., Cheng Pei-Feng, Eisenman Robert N., Demontis Fabio

   Genes & Development 2015

   Abstract DOI PMC PMID

   Metabolic stress and changes in nutrient levels modulate many aspects of skeletal muscle function during aging and disease. Growth factors and cytokines secreted by skeletal muscle, known as myokines, are important signaling factors, but it is largely unknown whether they modulate muscle growth and differentiation in response to nutrients. Here, we found that changes in glucose levels increase the activity of the glucose-responsive transcription factor MLX (Max-like protein X), which promotes and is necessary for myoblast fusion. MLX promotes myogenesis not via an adjustment of glucose metabolism but rather by inducing the expression of several myokines, including insulin-like growth factor 2 (IGF2), whereas RNAi and dominant-negative MLX reduce IGF2 expression and block myogenesis. This phenotype is rescued by conditioned medium from control muscle cells and by recombinant IGF2, which activates the myogenic kinase Akt. Importantly, MLX-null mice display decreased IGF2 induction and diminished muscle regeneration in response to injury, indicating that the myogenic function of MLX is manifested in vivo. Thus, glucose is a signaling molecule that regulates myogenesis and muscle regeneration via MLX/IGF2/Akt signaling.

2. Development

   Brg1 Coordinates Multiple Processes during Retinogenesis and Is a Tumor Suppressor in Retinoblastoma

   Aldiri Issam, Ajioka Itsuki, Xu Beisi, Zhang Jiakun, Chen Xiang, Benavente Claudia, Finkelstein David, Johnson Dianna, Akiyama Jennifer, Pennacchio Len A., Dyer Michael A.

   Development 2015

   Abstract DOI PMC PMID GEO:Microarray GEO:CHIP

   Skip to Next Section Retinal development requires precise temporal and spatial coordination of cell cycle exit, cell fate specification, cell migration and differentiation. When this process is disrupted, retinoblastoma, a developmental tumor of the retina, can form. Epigenetic modulators are central to precisely coordinating developmental events, and many epigenetic processes have been implicated in cancer. Studying epigenetic mechanisms in development is challenging because they often regulate multiple cellular processes; therefore, elucidating the primary molecular mechanisms involved can be difficult. Here we explore the role of Brg1 (Smarca4) in retinal development and retinoblastoma in mice using molecular and cellular approaches. Brg1 was found to regulate retinal size by controlling cell cycle length, cell cycle exit and cell survival during development. Brg1 was not required for cell fate specification but was required for photoreceptor differentiation and cell adhesion/polarity programs that contribute to proper retinal lamination during development. The combination of defective cell differentiation and lamination led to retinal degeneration in Brg1-deficient retinae. Despite the hypocellularity, premature cell cycle exit, increased cell death and extended cell cycle length, retinal progenitor cells persisted in Brg1-deficient retinae, making them more susceptible to retinoblastoma. ChIP-Seq analysis suggests that Brg1 might regulate gene expression through multiple mechanisms.

3. Cell Stem Cell

   Quantification of Retinogenesis in 3D Cultures Reveals Epigenetic Memory and Higher Efficiency in iPSCs Derived from Rod Photoreceptors

   Hiler Daniel, Chen Xiang, Hazen Jennifer, Kupriyanov Sergey, Carroll Patrick A., Qu Chunxu, Xu Beisi, Johnson Dianna, Griffiths Lyra, Frase Sharon, Rodriguez Alberto R., Martin Greg, Zhang Jiakun, Jeon Jongrye, Fan Yiping, Finkelstein David, Eisenman Robert N., Baldwin Kristin, Dyer Michael A.

   Cell Stem Cell 2015

   Abstract DOI PMC PMID EGA:Link

   Summary Cell-based therapies to treat retinal degeneration are now being tested in clinical trials. However, it is not known whether the source of stem cells is important for the production of differentiated cells suitable for transplantation. To test this, we generated induced pluripotent stem cells (iPSCs) from murine rod photoreceptors (r-iPSCs) and scored their ability to make retinae by using a standardized quantitative protocol called STEM-RET. We discovered that r-iPSCs more efficiently produced differentiated retinae than did embryonic stem cells (ESCs) or fibroblast-derived iPSCs (f-iPSCs). Retinae derived from f-iPSCs had fewer amacrine cells and other inner nuclear layer cells. Integrated epigenetic analysis showed that DNA methylation contributes to the defects in f-iPSC retinogenesis and that rod-specific CTCF insulator protein-binding sites may promote r-iPSC retinogenesis. Together, our data suggest that the source of stem cells is important for producing retinal neurons in three-dimensional (3D) organ cultures.