Publications by authors named "Jane A Skok"

61 Publications

The Ig heavy chain protein but not its message controls early B cell development.

Proc Natl Acad Sci U S A 2020 12 23;117(49):31343-31352. Epub 2020 Nov 23.

Division of Tumor Biology and Immunology, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands;

Development of progenitor B cells (ProB cells) into precursor B cells (PreB cells) is dictated by immunoglobulin heavy chain checkpoint (IgHCC), where the IgHC encoded by a productively rearranged allele assembles into a PreB cell receptor complex (PreBCR) to generate signals to initiate this transition and suppressing antigen receptor gene recombination, ensuring that only one productive allele is expressed, a phenomenon known as allelic exclusion. In contrast to a productively rearranged allele, the messenger RNA (mRNA) () from a nonproductively rearranged allele is degraded by nonsense-mediated decay (NMD). This fact prohibited firm conclusions regarding the contribution of stable to the molecular and developmental changes associated with the IgHCC. This point was addressed by generating the mouse model from mice having a premature termination codon at position +5 in leader exon of allele. This prohibited NMD, and the lack of a transmembrane region (∆TM) prevented the formation of any signaling-competent PreBCR complexes that may arise as a result of read-through translation across premature Ter5 stop codon. A highly sensitive sandwich Western blot revealed read-through translation of message, indicating that previous conclusions regarding a role of in establishing allelic exclusion requires further exploration. As determined by RNA sequencing (RNA-Seq), this low amount of IgHC sufficed to initiate PreB cell markers normally associated with PreBCR signaling. In contrast, the knock-in allele, which generated stable but no detectable IgHC, failed to induce PreB development. Our data indicate that the IgHCC is controlled at the level of IgHC and not expression.
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http://dx.doi.org/10.1073/pnas.2004810117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7733823PMC
December 2020

Context-Dependent Requirement of Euchromatic Histone Methyltransferase Activity during Reprogramming to Pluripotency.

Stem Cell Reports 2020 12 24;15(6):1233-1245. Epub 2020 Sep 24.

Skirball Institute of Biomolecular Medicine, Department of Cell Biology, NYU Langone Medical Center, New York, NY 10016, USA; Helen L. and Martin S. Kimmel Center for Biology and Medicine, NYU Langone Medical Center, New York, NY 10016, USA; Laura and Isaac Perlmutter Cancer Center, NYU Langone Medical Center, New York, NY 10016, USA; Sanford I. Weill Department of Medicine, Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, New York, NY 10021, USA. Electronic address:

Methylation of histone 3 at lysine 9 (H3K9) constitutes a roadblock for cellular reprogramming. Interference with methyltransferases or activation of demethylases by the cofactor ascorbic acid (AA) facilitates the derivation of induced pluripotent stem cells (iPSCs), but possible interactions between specific methyltransferases and AA treatment remain insufficiently explored. We show that chemical inhibition of the methyltransferases EHMT1 and EHMT2 counteracts iPSC formation in an enhanced reprogramming system in the presence of AA, an effect that is dependent on EHMT1. EHMT inhibition during enhanced reprogramming is associated with rapid loss of H3K9 dimethylation, inefficient downregulation of somatic genes, and failed mesenchymal-to-epithelial transition. Furthermore, transient EHMT inhibition during reprogramming yields iPSCs that fail to efficiently give rise to viable mice upon blastocyst injection. Our observations establish novel functions of H3K9 methyltransferases and suggest that a functional balance between AA-stimulated enzymes and EHMTs supports efficient and less error-prone iPSC reprogramming to pluripotency.
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http://dx.doi.org/10.1016/j.stemcr.2020.08.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724475PMC
December 2020

Author Correction: Defining the relative and combined contribution of CTCF and CTCFL to genomic regulation.

Genome Biol 2020 Jun 2;21(1):133. Epub 2020 Jun 2.

Department of Pathology, New York University Langone Health, New York, NY, 10016, USA.

An amendment to this paper has been published and can be accessed via the original article.
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http://dx.doi.org/10.1186/s13059-020-02056-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265643PMC
June 2020

Defining the relative and combined contribution of CTCF and CTCFL to genomic regulation.

Genome Biol 2020 05 11;21(1):108. Epub 2020 May 11.

Department of Pathology, New York University Langone Health, New York, NY, 10016, USA.

Background: Ubiquitously expressed CTCF is involved in numerous cellular functions, such as organizing chromatin into TAD structures. In contrast, its paralog, CTCFL, is normally only present in the testis. However, it is also aberrantly expressed in many cancers. While it is known that shared and unique zinc finger sequences in CTCF and CTCFL enable CTCFL to bind competitively to a subset of CTCF binding sites as well as its own unique locations, the impact of CTCFL on chromosome organization and gene expression has not been comprehensively analyzed in the context of CTCF function. Using an inducible complementation system, we analyze the impact of expressing CTCFL and CTCF-CTCFL chimeric proteins in the presence or absence of endogenous CTCF to clarify the relative and combined contribution of CTCF and CTCFL to chromosome organization and transcription.

Results: We demonstrate that the N terminus of CTCF interacts with cohesin which explains the requirement for convergent CTCF binding sites in loop formation. By analyzing CTCF and CTCFL binding in tandem, we identify phenotypically distinct sites with respect to motifs, targeting to promoter/intronic intergenic regions and chromatin folding. Finally, we reveal that the N, C, and zinc finger terminal domains play unique roles in targeting each paralog to distinct binding sites to regulate transcription, chromatin looping, and insulation.

Conclusion: This study clarifies the unique and combined contribution of CTCF and CTCFL to chromosome organization and transcription, with direct implications for understanding how their co-expression deregulates transcription in cancer.
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http://dx.doi.org/10.1186/s13059-020-02024-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212617PMC
May 2020

CTCF and CTCFL in cancer.

Curr Opin Genet Dev 2020 04 22;61:44-52. Epub 2020 Apr 22.

Dept. of Pathology, New York University Langone Health, New York, NY 10016, USA; Laura and Isaac Perlmutter Cancer Center, NYU School of Medicine, New York, NY 10016, USA. Electronic address:

CTCF plays a key role in organizing chromatin into TAD structures but it can also function as a transcription factor. CTCFL (CTCF-like), the paralog of CTCF, is normally transiently expressed in pre-meiotic male germ cells together with ubiquitously expressed CTCF. It plays a unique role in spermatogenesis by regulating expression of testis-specific genes. Genetic alterations in CTCF and its paralog CTCFL have both been found in numerous cancers, but it remains unknown to what extent CTCFL deregulates transcription on its own or by opposing CTCF. Here, we discuss some of the potential mechanisms by which these two proteins could alter gene regulation and contribute to oncogenic transcriptional programs.
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http://dx.doi.org/10.1016/j.gde.2020.02.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7893514PMC
April 2020

DNA methylation disruption reshapes the hematopoietic differentiation landscape.

Nat Genet 2020 04 23;52(4):378-387. Epub 2020 Mar 23.

New York Genome Center, New York, NY, USA.

Mutations in genes involved in DNA methylation (DNAme; for example, TET2 and DNMT3A) are frequently observed in hematological malignancies and clonal hematopoiesis. Applying single-cell sequencing to murine hematopoietic stem and progenitor cells, we observed that these mutations disrupt hematopoietic differentiation, causing opposite shifts in the frequencies of erythroid versus myelomonocytic progenitors following Tet2 or Dnmt3a loss. Notably, these shifts trace back to transcriptional priming skews in uncommitted hematopoietic stem cells. To reconcile genome-wide DNAme changes with specific erythroid versus myelomonocytic skews, we provide evidence in support of differential sensitivity of transcription factors due to biases in CpG enrichment in their binding motif. Single-cell transcriptomes with targeted genotyping showed similar skews in transcriptional priming of DNMT3A-mutated human clonal hematopoiesis bone marrow progenitors. These data show that DNAme shapes the topography of hematopoietic differentiation, and support a model in which genome-wide methylation changes are transduced to differentiation skews through biases in CpG enrichment of the transcription factor binding motif.
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http://dx.doi.org/10.1038/s41588-020-0595-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7216752PMC
April 2020

NSD2 overexpression drives clustered chromatin and transcriptional changes in a subset of insulated domains.

Nat Commun 2019 10 24;10(1):4843. Epub 2019 Oct 24.

Department of Pathology, New York University Langone Health, New York, NY, 10016, USA.

CTCF and cohesin play a key role in organizing chromatin into topologically associating domain (TAD) structures. Disruption of a single CTCF binding site is sufficient to change chromosomal interactions leading to alterations in chromatin modifications and gene regulation. However, the extent to which alterations in chromatin modifications can disrupt 3D chromosome organization leading to transcriptional changes is unknown. In multiple myeloma, a 4;14 translocation induces overexpression of the histone methyltransferase, NSD2, resulting in expansion of H3K36me2 and shrinkage of antagonistic H3K27me3 domains. Using isogenic cell lines producing high and low levels of NSD2, here we find oncogene activation is linked to alterations in H3K27ac and CTCF within H3K36me2 enriched chromatin. A logistic regression model reveals that differentially expressed genes are significantly enriched within the same insulated domain as altered H3K27ac and CTCF peaks. These results identify a bidirectional relationship between 2D chromatin and 3D genome organization in gene regulation.
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http://dx.doi.org/10.1038/s41467-019-12811-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6813313PMC
October 2019

B-1a cells acquire their unique characteristics by bypassing the pre-BCR selection stage.

Nat Commun 2019 10 18;10(1):4768. Epub 2019 Oct 18.

Department of Pathology, New York University School of Medicine, New York University, New York, NY, USA.

B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells, B-1a cells have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues, the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver versus bone marrow environment, reduced IL-7R/STAT5 levels promote immunoglobulin kappa gene recombination at the early pro-B cell stage. As a result, differentiating B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain. This 'alternate pathway' of development enables the production of B cells with self-reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing lineage and selection models of B-1a cell development and explain how these cells acquire their unique properties.
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http://dx.doi.org/10.1038/s41467-019-12824-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6802180PMC
October 2019

Impaired Expression of Rearranged Immunoglobulin Genes and Premature p53 Activation Block B Cell Development in BMI1 Null Mice.

Cell Rep 2019 01;26(1):108-118.e4

Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016, USA; Department of Urology, New York University School of Medicine, New York, NY 10016, USA; Perlmutter Cancer Center, New York University School of Medicine, New York, NY 10016, USA. Electronic address:

B cell development is a highly regulated process that requires stepwise rearrangement of immunoglobulin genes to generate a functional B cell receptor (BCR). The polycomb group protein BMI1 is required for B cell development, but its function in developing B cells remains poorly defined. We demonstrate that BMI1 functions in a cell-autonomous manner at two stages during early B cell development. First, loss of BMI1 results in a differentiation block at the pro-B cell to pre-B cell transition due to the inability of BMI1-deficient cells to transcribe newly rearranged Igh genes. Accordingly, introduction of a pre-rearranged Igh allele partially restored B cell development in Bmi1 mice. In addition, BMI1 is required to prevent premature p53 signaling, and as a consequence, Bmi1 large pre-B cells fail to properly proliferate. Altogether, our results clarify the role of BMI1 in early B cell development and uncover an unexpected function of BMI1 during VDJ recombination.
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http://dx.doi.org/10.1016/j.celrep.2018.12.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6362848PMC
January 2019

Analysis of 3D genomic interactions identifies candidate host genes that transposable elements potentially regulate.

Genome Biol 2018 12 13;19(1):216. Epub 2018 Dec 13.

Department of Pathology, New York University School of Medicine, New York, NY, 10016, USA.

Background: The organization of chromatin in the nucleus plays an essential role in gene regulation. About half of the mammalian genome comprises transposable elements. Given their repetitive nature, reads associated with these elements are generally discarded or randomly distributed among elements of the same type in genome-wide analyses. Thus, it is challenging to identify the activities and properties of individual transposons. As a result, we only have a partial understanding of how transposons contribute to chromatin folding and how they impact gene regulation.

Results: Using PCR and Capture-based chromosome conformation capture (3C) approaches, collectively called 4Tran, we take advantage of the repetitive nature of transposons to capture interactions from multiple copies of endogenous retrovirus (ERVs) in the human and mouse genomes. With 4Tran-PCR, reads are selectively mapped to unique regions in the genome. This enables the identification of transposable element interaction profiles for individual ERV families and integration events specific to particular genomes. With this approach, we demonstrate that transposons engage in long-range intra-chromosomal interactions guided by the separation of chromosomes into A and B compartments as well as topologically associated domains (TADs). In contrast to 4Tran-PCR, Capture-4Tran can uniquely identify both ends of an interaction that involve retroviral repeat sequences, providing a powerful tool for uncovering the individual transposable element insertions that interact with and potentially regulate target genes.

Conclusions: 4Tran provides new insight into the manner in which transposons contribute to chromosome architecture and identifies target genes that transposable elements can potentially control.
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http://dx.doi.org/10.1186/s13059-018-1598-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6292174PMC
December 2018

Stage-specific epigenetic regulation of CD4 expression by coordinated enhancer elements during T cell development.

Nat Commun 2018 09 5;9(1):3594. Epub 2018 Sep 5.

The Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, New York, NY, 10016, USA.

The inheritance of gene expression patterns is dependent on epigenetic regulation, but the establishment and maintenance of epigenetic landscapes during T cell differentiation are incompletely understood. Here we show that two stage-specific Cd4 cis-elements, the previously characterized enhancer E4p and a novel enhancer E4m, coordinately promote Cd4 transcription in mature thymic MHC-II-specific T cells, in part through the canonical Wnt pathway. Specifically, E4p licenses E4m to orchestrate DNA demethylation by TET1 and TET3, which in turn poises the Cd4 locus for transcription in peripheral T cells. Cd4 locus demethylation is important for subsequent Cd4 transcription in activated peripheral T cells wherein these cis-elements become dispensable. By contrast, in developing thymocytes the loss of TET1/3 does not affect Cd4 transcription, highlighting an uncoupled event between transcription and epigenetic modifications. Together our findings reveal an important function for thymic cis-elements in governing gene expression in the periphery via a heritable epigenetic mechanism.
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http://dx.doi.org/10.1038/s41467-018-05834-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6125341PMC
September 2018

Capturing the Onset of PRC2-Mediated Repressive Domain Formation.

Mol Cell 2018 06;70(6):1149-1162.e5

Howard Hughes Medical Institute, New York University School of Medicine, New York, NY 10016, USA; Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016, USA. Electronic address:

Polycomb repressive complex 2 (PRC2) maintains gene silencing by catalyzing methylation of histone H3 at lysine 27 (H3K27me2/3) within chromatin. By designing a system whereby PRC2-mediated repressive domains were collapsed and then reconstructed in an inducible fashion in vivo, a two-step mechanism of H3K27me2/3 domain formation became evident. First, PRC2 is stably recruited by the actions of JARID2 and MTF2 to a limited number of spatially interacting "nucleation sites," creating H3K27me3-forming Polycomb foci within the nucleus. Second, PRC2 is allosterically activated via its binding to H3K27me3 and rapidly spreads H3K27me2/3 both in cis and in far-cis via long-range contacts. As PRC2 proceeds further from the nucleation sites, its stability on chromatin decreases such that domains of H3K27me3 remain proximal, and those of H3K27me2 distal, to the nucleation sites. This study demonstrates the principles of de novo establishment of PRC2-mediated repressive domains across the genome.
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http://dx.doi.org/10.1016/j.molcel.2018.05.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7700016PMC
June 2018

Enhancer talk.

Epigenomics 2018 04 27;10(4):483-498. Epub 2018 Mar 27.

Department of Pathology, New York University School of Medicine, 550 First Avenue, MSB 599, New York, NY 10016, USA.

Enhancers are short noncoding segments of DNA (100-1000 bp) that control the temporal and spatial activity of genes in an orientation-independent manner. They can be separated from their target genes by large distances and are thus known as distal regulatory elements. One consequence of the variability in the distance separating enhancers and their target promoters is that it is difficult to determine which elements are involved in the regulation of a particular gene. Moreover, enhancers can be found in clusters in which multiple regulatory elements control expression of the same target gene. However, little is known about how the individual elements contribute to gene expression. Here, we describe how chromatin conformation promotes and constraints enhancer activity. Further, we discuss enhancer clusters and what is known about the contribution of individual elements to the regulation of target genes. Finally, we examine the reliability of different methods used to identify enhancers.
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http://dx.doi.org/10.2217/epi-2017-0157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5925435PMC
April 2018

Control of B-1a cell development by instructive BCR signaling.

Curr Opin Immunol 2018 04 3;51:24-31. Epub 2018 Feb 3.

Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Campus-Vienna-Biocenter 1, A-1030 Vienna, Austria. Electronic address:

B-1a cells remain one of the most enigmatic lymphocyte subsets. In this review, we discuss recent advances in our understanding of the development of these cells and their regulation by the transcription factors Bhlhe41 and Arid3a as well as by the RNA-binding protein Lin28b. A large body of literature supports an instructive role of BCR signaling in B-1a cell development and lineage commitment, which is initiated only after signaling from an autoreactive BCR. While both fetal and adult hematopoiesis can generate B-1a cells, the contribution of adult hematopoiesis to the B-1a cell compartment is low under physiological conditions. We discuss several models that can reconcile the instructive role of BCR signaling with this fetal bias in B-1a cell development.
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http://dx.doi.org/10.1016/j.coi.2018.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5943138PMC
April 2018

Low-Grade Astrocytoma Mutations in IDH1, P53, and ATRX Cooperate to Block Differentiation of Human Neural Stem Cells via Repression of SOX2.

Cell Rep 2017 Oct;21(5):1267-1280

Department of Neurosurgery, NYU School of Medicine, New York, NY 10016, USA; Kimmel Center for Stem Cell Biology, NYU School of Medicine, New York, NY 10016, USA; Laura and Isaac Perlmutter Cancer Center, NYU School of Medicine, New York, NY 10016, USA; Brain Tumor Center, NYU School of Medicine, New York, NY 10016, USA; Neuroscience Institute, NYU School of Medicine, New York, NY 10016, USA. Electronic address:

Low-grade astrocytomas (LGAs) carry neomorphic mutations in isocitrate dehydrogenase (IDH) concurrently with P53 and ATRX loss. To model LGA formation, we introduced R132H IDH1, P53 shRNA, and ATRX shRNA into human neural stem cells (NSCs). These oncogenic hits blocked NSC differentiation, increased invasiveness in vivo, and led to a DNA methylation and transcriptional profile resembling IDH1 mutant human LGAs. The differentiation block was caused by transcriptional silencing of the transcription factor SOX2 secondary to disassociation of its promoter from a putative enhancer. This occurred because of reduced binding of the chromatin organizer CTCF to its DNA motifs and disrupted chromatin looping. Our human model of IDH mutant LGA formation implicates impaired NSC differentiation because of repression of SOX2 as an early driver of gliomagenesis.
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http://dx.doi.org/10.1016/j.celrep.2017.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5687844PMC
October 2017

The Conserved ATM Kinase RAG2-S365 Phosphorylation Site Limits Cleavage Events in Individual Cells Independent of Any Repair Defect.

Cell Rep 2017 Oct;21(4):979-993

Department of Pathology, New York University School of Medicine, New York, NY 10016, USA. Electronic address:

Many DNA lesions associated with lymphoid malignancies are linked to off-target cleavage by the RAG1/2 recombinase. However, off-target cleavage has mostly been analyzed in the context of DNA repair defects, confounding any mechanistic understanding of cleavage deregulation. We identified a conserved SQ phosphorylation site on RAG2 365 to 366 that is involved in feedback control of RAG cleavage. Mutation of serine 365 to a non-phosphorylatable alanine permits bi-allelic and bi-locus RAG-mediated breaks in the same cell, leading to reciprocal translocations. This phenomenon is analogous to the phenotype we described for ATM kinase inactivation. Here, we establish deregulated cleavage itself as a driver of chromosomal instability without the associated repair defect. Intriguingly, a RAG2-S365E phosphomimetic rescues the deregulated cleavage of ATM inactivation, reducing the incidence of reciprocal translocations. These data support a model in which feedback control of cleavage and maintenance of genome stability involves ATM-mediated phosphorylation of RAG2.
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http://dx.doi.org/10.1016/j.celrep.2017.09.084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5662208PMC
October 2017

Wolf-Hirschhorn Syndrome Candidate 1 Is Necessary for Correct Hematopoietic and B Cell Development.

Cell Rep 2017 05;19(8):1586-1601

Department of Cell Biology and Immunology, Centro de Biologia Molecular Severo Ochoa (CBMSO), CSIC/UAM, Madrid 28049, Spain. Electronic address:

Immunodeficiency is one of the most important causes of mortality associated with Wolf-Hirschhorn syndrome (WHS), a severe rare disease originated by a deletion in chromosome 4p. The WHS candidate 1 (WHSC1) gene has been proposed as one of the main genes responsible for many of the alterations in WHS, but its mechanism of action is still unknown. Here, we present in vivo genetic evidence showing that Whsc1 plays an important role at several points of hematopoietic development. Particularly, our results demonstrate that both differentiation and function of Whsc1-deficient B cells are impaired at several key developmental stages due to profound molecular defects affecting B cell lineage specification, commitment, fitness, and proliferation, demonstrating a causal role for WHSC1 in the immunodeficiency of WHS patients.
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http://dx.doi.org/10.1016/j.celrep.2017.04.069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5510986PMC
May 2017

miRNAs Are Essential for the Regulation of the PI3K/AKT/FOXO Pathway and Receptor Editing during B Cell Maturation.

Cell Rep 2016 11;17(9):2271-2285

Department of Pathology, New York University School of Medicine, New York, NY 10016, USA. Electronic address:

B cell development is a tightly regulated process dependent on sequential rearrangements of immunoglobulin loci that encode the antigen receptor. To elucidate the role of microRNAs (miRNAs) in the orchestration of B cell development, we ablated all miRNAs at the earliest stage of B cell development by conditionally targeting the enzymes critical for RNAi in early B cell precursors. Absence of any one of these enzymes led to a block at the pro- to pre-B cell transition due to increased apoptosis and a failure of pre-B cells to proliferate. Expression of a Bcl2 transgene allowed for partial rescue of B cell development, however, the majority of the rescued B cells had low surface immunoglobulin expression with evidence of ongoing light chain editing. Our analysis revealed that miRNAs are critical for the regulation of the PTEN-AKT-FOXO1 pathway that in turn controls Rag expression during B cell development.
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http://dx.doi.org/10.1016/j.celrep.2016.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5679080PMC
November 2016

MED12 Regulates HSC-Specific Enhancers Independently of Mediator Kinase Activity to Control Hematopoiesis.

Cell Stem Cell 2016 12 25;19(6):784-799. Epub 2016 Aug 25.

Department of Pathology, Laura & Isaac Perlmutter Cancer Center, and The Helen L. and Martin S. Kimmel Center for Stem Cell Biology, NYU School of Medicine, New York, NY 10016, USA. Electronic address:

Hematopoietic-specific transcription factors require coactivators to communicate with the general transcription machinery and establish transcriptional programs that maintain hematopoietic stem cell (HSC) self-renewal, promote differentiation, and prevent malignant transformation. Mediator is a large coactivator complex that bridges enhancer-localized transcription factors with promoters, but little is known about Mediator function in adult stem cell self-renewal and differentiation. We show that MED12, a member of the Mediator kinase module, is an essential regulator of HSC homeostasis, as in vivo deletion of Med12 causes rapid bone marrow aplasia leading to acute lethality. Deleting other members of the Mediator kinase module does not affect HSC function, suggesting kinase-independent roles of MED12. MED12 deletion destabilizes P300 binding at lineage-specific enhancers, resulting in H3K27Ac depletion, enhancer de-activation, and consequent loss of HSC stemness signatures. As MED12 mutations have been described recently in blood malignancies, alterations in MED12-dependent enhancer regulation may control both physiological and malignant hematopoiesis.
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http://dx.doi.org/10.1016/j.stem.2016.08.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5268820PMC
December 2016

Identification of multi-loci hubs from 4C-seq demonstrates the functional importance of simultaneous interactions.

Nucleic Acids Res 2016 Oct 20;44(18):8714-8725. Epub 2016 Jul 20.

Interdepartmental Program in Computational Biology and Bioinformatics, Yale University, New Haven, CT 06511, USA Department of Pathology and Yale Cancer Center, Yale University School of Medicine, New Haven, CT, USA Program of Applied Mathematics, Yale university, New Haven, CT, USA

Use of low resolution single cell DNA FISH and population based high resolution chromosome conformation capture techniques have highlighted the importance of pairwise chromatin interactions in gene regulation. However, it is unlikely that associations involving regulatory elements act in isolation of other interacting partners that also influence their impact. Indeed, the influence of multi-loci interactions remains something of an enigma as beyond low-resolution DNA FISH we do not have the appropriate tools to analyze these. Here we present a method that uses standard 4C-seq data to identify multi-loci interactions from the same cell. We demonstrate the feasibility of our method using 4C-seq data sets that identify known pairwise and novel tri-loci interactions involving the Tcrb and Igk antigen receptor enhancers. We further show that the three Igk enhancers, MiEκ, 3'Eκ and Edκ, interact simultaneously in this super-enhancer cluster, which add to our previous findings showing that loss of one element decreases interactions between all three elements as well as reducing their transcriptional output. These findings underscore the functional importance of simultaneous interactions and provide new insight into the relationship between enhancer elements. Our method opens the door for studying multi-loci interactions and their impact on gene regulation in other biological settings.
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http://dx.doi.org/10.1093/nar/gkw568DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5062970PMC
October 2016

A Damage-Independent Role for 53BP1 that Impacts Break Order and Igh Architecture during Class Switch Recombination.

Cell Rep 2016 06 16;16(1):48-55. Epub 2016 Jun 16.

Department of Pathology, New York University School of Medicine, New York, NY 10016, USA. Electronic address:

During class switch recombination (CSR), B cells replace the Igh Cμ or δ exons with another downstream constant region exon (CH), altering the antibody isotype. CSR occurs through the introduction of AID-mediated double-strand breaks (DSBs) in switch regions and subsequent ligation of broken ends. Here, we developed an assay to investigate the dynamics of DSB formation in individual cells. We demonstrate that the upstream switch region Sμ is first targeted during recombination and that the mechanism underlying this control relies on 53BP1. Surprisingly, regulation of break order occurs through residual binding of 53BP1 to chromatin before the introduction of damage and independent of its established role in DNA repair. Using chromosome conformation capture, we show that 53BP1 mediates changes in chromatin architecture that affect break order. Finally, our results explain how changes in Igh architecture in the absence of 53BP1 could promote inversional rearrangements that compromise CSR.
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http://dx.doi.org/10.1016/j.celrep.2016.05.073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4927351PMC
June 2016

Active and Inactive Enhancers Cooperate to Exert Localized and Long-Range Control of Gene Regulation.

Cell Rep 2016 06 26;15(10):2159-2169. Epub 2016 May 26.

Department of Pathology, NYU School of Medicine, New York, NY 10016, USA. Electronic address:

V(D)J recombination relies on the presence of proximal enhancers that activate the antigen receptor (AgR) loci in a lineage- and stage-specific manner. Unexpectedly, we find that both active and inactive AgR enhancers cooperate to disseminate their effects in a localized and long-range manner. Here, we demonstrate the importance of short-range contacts between active enhancers that constitute an Igk super-enhancer in B cells. Deletion of one element reduces the interaction frequency between other enhancers in the hub, which compromises the transcriptional output of each component. Furthermore, we establish that, in T cells, long-range contact and cooperation between the inactive Igk enhancer MiEκ and the active Tcrb enhancer Eβ alters enrichment of CBFβ binding in a manner that impacts Tcrb recombination. These findings underline the complexities of enhancer regulation and point to a role for localized and long-range enhancer-sharing between active and inactive elements in lineage- and stage-specific control.
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http://dx.doi.org/10.1016/j.celrep.2016.04.087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4899175PMC
June 2016

CRISPR-dCas9 and sgRNA scaffolds enable dual-colour live imaging of satellite sequences and repeat-enriched individual loci.

Nat Commun 2016 05 25;7:11707. Epub 2016 May 25.

Department of Pathology, New York University School of Medicine, 550 First Avenue, Sml 311, New York, New York 10016, USA.

Imaging systems that allow visualization of specific loci and nuclear structures are highly relevant for investigating how organizational changes within the nucleus play a role in regulating gene expression and other cellular processes. Here we present a live imaging system for targeted detection of genomic regions. Our approach involves generating chimaeric transcripts of viral RNAs (MS2 and PP7) and single-guide RNAs (sgRNAs), which when co-expressed with a cleavage-deficient Cas9 can recruit fluorescently tagged viral RNA-binding proteins (MCP and PCP) to specific genomic sites. This allows for rapid, stable, low-background visualization of target loci. We demonstrate the efficiency and flexibility of our method by simultaneously labelling major and minor satellite regions as well as two individual loci on mouse chromosome 12. This system provides a tool for dual-colour labelling, which is important for tracking the dynamics of chromatin interactions and for validating epigenetic processes identified in fixed cells.
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http://dx.doi.org/10.1038/ncomms11707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4894952PMC
May 2016

4C-ker: A Method to Reproducibly Identify Genome-Wide Interactions Captured by 4C-Seq Experiments.

PLoS Comput Biol 2016 Mar 3;12(3):e1004780. Epub 2016 Mar 3.

Department of Pathology, New York University School of Medicine, New York, New York, United States of America.

4C-Seq has proven to be a powerful technique to identify genome-wide interactions with a single locus of interest (or "bait") that can be important for gene regulation. However, analysis of 4C-Seq data is complicated by the many biases inherent to the technique. An important consideration when dealing with 4C-Seq data is the differences in resolution of signal across the genome that result from differences in 3D distance separation from the bait. This leads to the highest signal in the region immediately surrounding the bait and increasingly lower signals in far-cis and trans. Another important aspect of 4C-Seq experiments is the resolution, which is greatly influenced by the choice of restriction enzyme and the frequency at which it can cut the genome. Thus, it is important that a 4C-Seq analysis method is flexible enough to analyze data generated using different enzymes and to identify interactions across the entire genome. Current methods for 4C-Seq analysis only identify interactions in regions near the bait or in regions located in far-cis and trans, but no method comprehensively analyzes 4C signals of different length scales. In addition, some methods also fail in experiments where chromatin fragments are generated using frequent cutter restriction enzymes. Here, we describe 4C-ker, a Hidden-Markov Model based pipeline that identifies regions throughout the genome that interact with the 4C bait locus. In addition, we incorporate methods for the identification of differential interactions in multiple 4C-seq datasets collected from different genotypes or experimental conditions. Adaptive window sizes are used to correct for differences in signal coverage in near-bait regions, far-cis and trans chromosomes. Using several datasets, we demonstrate that 4C-ker outperforms all existing 4C-Seq pipelines in its ability to reproducibly identify interaction domains at all genomic ranges with different resolution enzymes.
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http://dx.doi.org/10.1371/journal.pcbi.1004780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4777514PMC
March 2016

Mediator facilitates transcriptional activation and dynamic long-range contacts at the IgH locus during class switch recombination.

J Exp Med 2016 Mar 22;213(3):303-12. Epub 2016 Feb 22.

Institut de Génétique et de Biologie Moléculaire et Cellulaire, 67400 Illkirch, France Institut National de la Santé et de la Recherche Médicale, Unité 964, 67404 Illkirch, France Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7104, 67404 Illkirch, France Université de Strasbourg, 67400 Illkirch, France

Immunoglobulin (Ig) class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation-induced cytidine deaminase (AID) to Ig switch regions (S regions). During CSR, the IgH locus undergoes dynamic three-dimensional structural changes in which promoters, enhancers, and S regions are brought to close proximity. Nevertheless, little is known about the underlying mechanisms. In this study, we show that Med1 and Med12, two subunits of the mediator complex implicated in transcription initiation and long-range enhancer/promoter loop formation, are dynamically recruited to the IgH locus enhancers and the acceptor regions during CSR and that their knockdown in CH12 cells results in impaired CSR. Furthermore, we show that conditional inactivation of Med1 in B cells results in defective CSR and reduced acceptor S region transcription. Finally, we show that in B cells undergoing CSR, the dynamic long-range contacts between the IgH enhancers and the acceptor regions correlate with Med1 and Med12 binding and that they happen at a reduced frequency in Med1-deficient B cells. Our results implicate the mediator complex in the mechanism of CSR and are consistent with a model in which mediator facilitates the long-range contacts between S regions and the IgH locus enhancers during CSR and their transcriptional activation.
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http://dx.doi.org/10.1084/jem.20141967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4813673PMC
March 2016

RAG Off-Target Activity Is in the Loop.

Trends Mol Med 2015 Dec 18;21(12):733-735. Epub 2015 Nov 18.

Department of Pathology, New York University School of Medicine, New York, NY 10016, USA. Electronic address:

The 'off-target' activity of RAG recombinases contributes to mutations and cancer. Recent studies show that the influence of DNA regulatory elements is largely constrained by the formation of chromatin loops and interaction frequencies. Now, the Alt lab has identified major RAG off-target activity bearing similar limitations, with joining events restricted to convergent paired RSS elements in loop domains.
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http://dx.doi.org/10.1016/j.molmed.2015.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4679453PMC
December 2015

Long-Range Regulation of V(D)J Recombination.

Adv Immunol 2015 20;128:123-82. Epub 2015 Aug 20.

Department of Pathology, New York University School of Medicine, New York, USA. Electronic address:

Given their essential role in adaptive immunity, antigen receptor loci have been the focus of analysis for many years and are among a handful of the most well-studied genes in the genome. Their investigation led initially to a detailed knowledge of linear structure and characterization of regulatory elements that confer control of their rearrangement and expression. However, advances in DNA FISH and imaging combined with new molecular approaches that interrogate chromosome conformation have led to a growing appreciation that linear structure is only one aspect of gene regulation and in more recent years, the focus has switched to analyzing the impact of locus conformation and nuclear organization on control of recombination. Despite decades of work and intense effort from numerous labs, we are still left with an incomplete picture of how the assembly of antigen receptor loci is regulated. This chapter summarizes our advances to date and points to areas that need further investigation.
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http://dx.doi.org/10.1016/bs.ai.2015.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4660984PMC
February 2016

Cohesin loss alters adult hematopoietic stem cell homeostasis, leading to myeloproliferative neoplasms.

J Exp Med 2015 Oct 5;212(11):1833-50. Epub 2015 Oct 5.

Howard Hughes Medical Institute, Department of Pathology, and Center for Health Informatics and Bioinformatics, School of Medicine and Department of Biology, Center for Genomics and Systems Biology, New York University, New York, NY 10016 Howard Hughes Medical Institute, Department of Pathology, and Center for Health Informatics and Bioinformatics, School of Medicine and Department of Biology, Center for Genomics and Systems Biology, New York University, New York, NY 10016

The cohesin complex (consisting of Rad21, Smc1a, Smc3, and Stag2 proteins) is critically important for proper sister chromatid separation during mitosis. Mutations in the cohesin complex were recently identified in a variety of human malignancies including acute myeloid leukemia (AML). To address the potential tumor-suppressive function of cohesin in vivo, we generated a series of shRNA mouse models in which endogenous cohesin can be silenced inducibly. Notably, silencing of cohesin complex members did not have a deleterious effect on cell viability. Furthermore, knockdown of cohesin led to gain of replating capacity of mouse hematopoietic progenitor cells. However, cohesin silencing in vivo rapidly altered stem cells homeostasis and myelopoiesis. Likewise, we found widespread changes in chromatin accessibility and expression of genes involved in myelomonocytic maturation and differentiation. Finally, aged cohesin knockdown mice developed a clinical picture closely resembling myeloproliferative disorders/neoplasms (MPNs), including varying degrees of extramedullary hematopoiesis (myeloid metaplasia) and splenomegaly. Our results represent the first successful demonstration of a tumor suppressor function for the cohesin complex, while also confirming that cohesin mutations occur as an early event in leukemogenesis, facilitating the potential development of a myeloid malignancy.
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http://dx.doi.org/10.1084/jem.20151323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4612095PMC
October 2015

Breaking TADs: insights into hierarchical genome organization.

Epigenomics 2015 ;7(4):523-6

Department of Pathology, New York University School of Medicine, New York, NY 10016, USA.

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http://dx.doi.org/10.2217/epi.15.25DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624204PMC
June 2016

CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation.

Science 2015 Feb;347(6225):1017-21

Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA. Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016, USA.

Polycomb and Trithorax group proteins encode the epigenetic memory of cellular positional identity by establishing inheritable domains of repressive and active chromatin within the Hox clusters. Here we demonstrate that the CCCTC-binding factor (CTCF) functions to insulate these adjacent yet antagonistic chromatin domains during embryonic stem cell differentiation into cervical motor neurons. Deletion of CTCF binding sites within the Hox clusters results in the expansion of active chromatin into the repressive domain. CTCF functions as an insulator by organizing Hox clusters into spatially disjoint domains. Ablation of CTCF binding disrupts topological boundaries such that caudal Hox genes leave the repressed domain and become subject to transcriptional activation. Hence, CTCF is required to insulate facultative heterochromatin from impinging euchromatin to produce discrete positional identities.
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http://dx.doi.org/10.1126/science.1262088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4428148PMC
February 2015