Anja Ebert

CTCF-binding elements mediate control of V(D)J recombination

Immunoglobulin heavy chain (IgH) variable region exons are assembled from VH, D and JH gene segments in developing B lymphocytes. Within the 2.7-megabase mouse Igh locus, V(D)J recombination is regulated to ensure specific and diverse antibody repertoires. Here we report in mice a key Igh V(D)J recombination regulatory region, termed intergenic control region 1 (IGCR1), which lies between the VH and D clusters. Functionally, IGCR1 uses CTCF looping/insulator factor-binding elements and, correspondingly, mediates Igh loops containing distant enhancers. IGCR1 promotes normal B-cell development and balances antibody repertoires by inhibiting transcription and rearrangement of DH-proximal VH gene segments and promoting rearrangement of distal VH segments. IGCR1 maintains ordered and lineage-specific VH(D)JH recombination by suppressing VH joining to D segments not joined to JH segments, and VH to DJH joins in thymocytes, respectively. IGCR1 is also required for feedback regulation and allelic exclusion of proximal VH-to-DJH recombination. Our studies elucidate a long-sought Igh V(D)J recombination control region and indicate a new role for the generally expressed CTCF protein.

The Distal VH Gene Cluster of the Igh Locus Contains Distinct Regulatory Elements with Pax5 Transcription Factor-Dependent Activity in Pro-B Cells

V(H)-DJ(H) recombination of the immunoglobulin heavy chain (Igh) locus is temporally and spatially controlled during early B cell development, and yet no regulatory elements other than the V(H) gene promoters have been identified throughout the entire V(H) gene cluster. Here, we discovered regulatory sequences that are interspersed in the distal V(H) gene region. These conserved repeat elements were characterized by the presence of Pax5 transcription factor-dependent active chromatin by binding of the regulators Pax5, E2A, CTCF, and Rad21, as well as by Pax5-dependent antisense transcription in pro-B cells. The Pax5-activated intergenic repeat (PAIR) elements were no longer bound by Pax5 in pre-B and B cells consistent with the loss of antisense transcription, whereas E2A and CTCF interacted with PAIR elements throughout early B cell development. The pro-B cell-specific and Pax5-dependent activity of the PAIR elements suggests that they are involved in the regulation of distal V(H)-DJ(H) recombination at the Igh locus.

The B‐cell identity factor Pax5 regulates distinct transcriptional programmes in early and late B lymphopoiesis

Pax5 controls the identity and development of B cells by repressing lineage‐inappropriate genes and activating B‐cell‐specific genes. Here, we used genome‐wide approaches to identify Pax5 target genes in pro‐B and mature B cells. In these cell types, Pax5 bound to 40% of the cis‐regulatory elements defined by mapping DNase I hypersensitive (DHS) sites, transcription start sites and histone modifications. Although Pax5 bound to 8000 target genes, it regulated only 4% of them in pro‐B and mature B cells by inducing enhancers at activated genes and eliminating DHS sites at repressed genes. Pax5‐regulated genes in pro‐B cells account for 23% of all expression changes occurring between common lymphoid progenitors and committed pro‐B cells, which identifies Pax5 as an important regulator of this developmental transition. Regulated Pax5 target genes minimally overlap in pro‐B and mature B cells, which reflects massive expression changes between these cell types. Hence, Pax5 controls B‐cell identity and function by regulating distinct target genes in early and late B lymphopoiesis.

Opposing roles of polycomb repressive complexes in hematopoietic stem and progenitor cells.

Polycomb group (PcG) proteins are transcriptional repressors with a central role in the establishment and maintenance of gene expression patterns during development. We have investigated the role of polycomb repressive complexes (PRCs) in hematopoietic stem cells (HSCs) and progenitor populations. We show that mice with loss of function mutations in PRC2 components display enhanced HSC/progenitor population activity, whereas mutations that disrupt PRC1 or pleiohomeotic repressive complex are associated with HSC/progenitor cell defects. Because the hierarchical model of PRC action would predict synergistic effects of PRC1 and PRC2 mutation, these opposing effects suggest this model does not hold true in HSC/progenitor cells. To investigate the molecular targets of each complex in HSC/progenitor cells, we measured genome-wide expression changes associated with PRC deficiency, and identified transcriptional networks that are differentially regulated by PRC1 and PRC2. These studies provide new insights into the mechanistic interplay between distinct PRCs and have important implications for approaching PcG proteins as therapeutic targets.

The transcription factor Pax5 regulates its target genes by recruiting chromatin-modifying proteins in committed B cells

Pax5 is a critical regulator of B‐cell commitment. Here, we identified direct Pax5 target genes by streptavidin‐mediated ChIP‐chip analysis of pro‐B cells expressing in vivo biotinylated Pax5. By binding to promoters and enhancers, Pax5 directly regulates the expression of multiple transcription factor, cell surface receptor and signal transducer genes. One of the newly identified enhancers was shown by transgenic analysis to confer Pax5‐dependent B‐cell‐specific activity to the Nedd9 gene controlling B‐cell trafficking. Profiling of histone modifications in Pax5‐deficient and wild‐type pro‐B cells demonstrated that Pax5 induces active chromatin at activated target genes, while eliminating active chromatin at repressed genes in committed pro‐B cells. Pax5 rapidly induces these chromatin and transcription changes by recruiting chromatin‐remodelling, histone‐modifying and basal transcription factor complexes to its target genes. These data provide novel insight into the regulatory network and epigenetic regulation, by which Pax5 controls B‐cell commitment.

PU.1 cooperates with IRF4 and IRF8 to suppress pre-B-cell leukemia

The Ets family transcription factor PU.1 and the interferon regulatory factor (IRF)4 and IRF8 regulate gene expression by binding to composite DNA sequences known as Ets/interferon consensus elements. Although all three factors are expressed from the onset of B-cell development, single deficiency of these factors in B-cell progenitors only mildly impacts on bone marrow B lymphopoiesis. Here we tested whether PU.1 cooperates with IRF factors in regulating early B-cell development. Lack of PU.1 and IRF4 resulted in a partial block in development the pre-B-cell stage. The combined deletion of PU.1 and IRF8 reduced recirculating B-cell numbers. Strikingly, all PU.1/IRF4 and ~50% of PU.1/IRF8 double deficient mice developed pre-B-cell acute lymphoblastic leukemia (B-ALL) associated with reduced expression of the established B-lineage tumor suppressor genes, Ikaros and Spi-B. These genes are directly regulated by PU.1/IRF4/IRF8, and restoration of Ikaros or Spi-B expression inhibited leukemic cell growth. In summary, we demonstrate that PU.1, IRF4 and IRF8 cooperate to regulate early B-cell development and to prevent pre-B-ALL formation.

SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis

Profiling transcription—a SLAM dunk Identification of the direct target genes of transcription factors could shed light on how healthy cells become malignant. Muhar et al. applied a modified version of a transcript-mapping method called SLAM-seq to identify the target genes of two transcriptional regulators of major interest in cancer research (see the Perspective by Sabò and Amati). The MYC oncoprotein selectively activates transcription of just a few genes, primarily those involved in basic cell metabolism. In contrast, BRD4, a bromodomain-containing protein that is being targeted for cancer therapy, activates transcription of many genes. Science, this issue p. 800; see also p. 713 An mRNA mapping method identifies the direct gene targets of two transcriptional regulators implicated in cancer. Defining direct targets of transcription factors and regulatory pathways is key to understanding their roles in physiology and disease. We combined SLAM-seq [thiol(SH)–linked alkylation for the metabolic sequencing of RNA], a method for direct quantification of newly synthesized messenger RNAs (mRNAs), with pharmacological and chemical-genetic perturbation in order to define regulatory functions of two transcriptional hubs in cancer, BRD4 and MYC, and to interrogate direct responses to BET bromodomain inhibitors (BETis). We found that BRD4 acts as general coactivator of RNA polymerase II–dependent transcription, which is broadly repressed upon high-dose BETi treatment. At doses triggering selective effects in leukemia, BETis deregulate a small set of hypersensitive targets including MYC. In contrast to BRD4, MYC primarily acts as a selective transcriptional activator controlling metabolic processes such as ribosome biogenesis and de novo purine synthesis. Our study establishes a simple and scalable strategy to identify direct transcriptional targets of any gene or pathway.

Retrotransposon derepression leads to activation of the unfolded protein response and apoptosis in pro-B cells

ABSTRACT The H3K9me3-specific histone methyltransferase Setdb1 impacts on transcriptional regulation by repressing both developmental genes and retrotransposons. How impaired retrotransposon silencing may lead to developmental phenotypes is currently unclear. Here, we show that loss of Setdb1 in pro-B cells completely abrogates B cell development. In pro-B cells, Setdb1 is dispensable for silencing of lineage-inappropriate developmental genes. Instead, we detect strong derepression of endogenous murine leukemia virus (MLV) copies. This activation coincides with an unusual change in chromatin structure, with only partial loss of H3K9me3 and unchanged DNA methylation, but strongly increased H3K4me3. Production of MLV proteins leads to activation of the unfolded protein response pathway and apoptosis. Thus, our data demonstrate that B cell development depends on the proper repression of retrotransposon sequences through Setdb1. Summary: Setdb1-deficient pro-B cells display derepression of endogenous murine leukemia virus (MLV) elements. This induces the unfolded protein response and apoptosis, blocking B cell development.

Polycomb repression complex 2 is required for the maintenance of retinal progenitor cells and balanced retinal differentiation

Polycomb repressive complexes maintain transcriptional repression of genes encoding crucial developmental regulators through chromatin modification. Here we investigated the role of Polycomb repressive complex 2 (PRC2) in retinal development by inactivating its key components Eed and Ezh2. Conditional deletion of Ezh2 resulted in a partial loss of PRC2 function and accelerated differentiation of Müller glial cells. In contrast, inactivation of Eed led to the ablation of PRC2 function at early postnatal stage. Cell proliferation was reduced and retinal progenitor cells were significantly decreased in this mutant, which subsequently caused depletion of Müller glia, bipolar, and rod photoreceptor cells, primarily generated from postnatal retinal progenitor cells. Interestingly, the proportion of amacrine cells was dramatically increased at postnatal stages in the Eed-deficient retina. In accordance, multiple transcription factors controlling amacrine cell differentiation were upregulated. Furthermore, ChIP-seq analysis showed that these deregulated genes contained bivalent chromatin (H3K27me3+ H3K4me3+). Our results suggest that PRC2 is required for proliferation in order to maintain the retinal progenitor cells at postnatal stages and for retinal differentiation by controlling amacrine cell generation.

Epigenetic regulation of brain region-specific microglia clearance activity

The rapid elimination of dying neurons and nonfunctional synapses in the brain is carried out by microglia, the resident myeloid cells of the brain. Here we show that microglia clearance activity in the adult brain is regionally regulated and depends on the rate of neuronal attrition. Cerebellar, but not striatal or cortical, microglia exhibited high levels of basal clearance activity, which correlated with an elevated degree of cerebellar neuronal attrition. Exposing forebrain microglia to apoptotic cells activated gene-expression programs supporting clearance activity. We provide evidence that the polycomb repressive complex 2 (PRC2) epigenetically restricts the expression of genes that support clearance activity in striatal and cortical microglia. Loss of PRC2 leads to aberrant activation of a microglia clearance phenotype, which triggers changes in neuronal morphology and behavior. Our data highlight a key role of epigenetic mechanisms in preventing microglia-induced neuronal alterations that are frequently associated with neurodegenerative and psychiatric diseases.Microglia clearance activity in adult brain is regulated epigenetically and region-specifically to match neuronal attrition rates. Uncoupling this activity from neural apoptosis leads to aberrant microglia activation & neurodegenerative-like changes.