EBF1-deficient bone marrow stroma elicits persistent changes in HSC potential.

Crosstalk between mesenchymal stromal cells (MSCs) and hematopoietic stem cells (HSCs) is essential for hematopoietic homeostasis and lineage output. Here, we investigate how transcriptional changes in bone marrow (BM) MSCs result in long-lasting effects on HSCs. Single-cell analysis of Cxcl12-abundant reticular (CAR) cells and PDGFRα+Sca1+ (PαS) cells revealed an extensive cellular heterogeneity but uniform expression of the transcription factor gene Ebf1. Conditional deletion of Ebf1 in these MSCs altered their cellular composition, chromatin structure and gene expression profiles, including the reduced expression of adhesion-related genes. Functionally, the stromal-specific Ebf1 inactivation results in impaired adhesion of HSCs, leading to reduced quiescence and diminished myeloid output. Most notably, HSCs residing in the Ebf1-deficient niche underwent changes in their cellular composition and chromatin structure that persist in serial transplantations. Thus, genetic alterations in the BM niche lead to long-term functional changes of HSCs.

Tnpo3 enables EBF1 function in conditions of antagonistic Notch signaling.

Transcription factor EBF1 (early B cell factor 1) acts as a key regulator of B cell specification. The transcriptional network in which EBF1 operates has been extensively studied; however, the regulation of EBF1 function remains poorly defined. By mass spectrometric analysis of proteins associated with endogenous EBF1 in pro-B cells, we identified the nuclear import receptor Transportin-3 (Tnpo3) and found that it interacts with the immunoglobulin-like fold domain of EBF1. We delineated glutamic acid 271 of EBF1 as a critical residue for the association with Tnpo3. EBF1E271A showed normal nuclear localization; however, it had an impaired B cell programming ability in conditions of Notch signaling, as determined by retroviral transduction of Ebf1 -/- progenitors. By RNA-seq analysis of EBF1E271A-expressing progenitors, we found an up-regulation of T lineage determinants and down-regulation of early B genes, although similar chromatin binding of EBF1E271A and EBF1wt was detected in pro-B cells expressing activated Notch1. B lineage-specific inactivation of Tnpo3 in mice resulted in a block of early B cell differentiation, accompanied by a down-regulation of B lineage genes and up-regulation of T and NK lineage genes. Taken together, our observations suggest that Tnpo3 ensures B cell programming by EBF1 in nonpermissive conditions.

Repetitive Elements Trigger RIG-I-like Receptor Signaling that Regulates the Emergence of Hematopoietic Stem and Progenitor Cells.

Inflammatory signaling is required for hematopoietic stem and progenitor cell (HSPC) development. Here, we studied the involvement of RIG-I-like receptors (RLRs) in HSPC formation. Rig-I or Mda5 deficiency impaired, while Lgp2 deficiency enhanced, HSPC emergence in zebrafish embryos. Rig-I or Mda5 deficiency reduced HSPC numbers by inhibiting inflammatory signals that were in turn enhanced in Lgp2 deficient embryos. Simultaneous reduction of Lgp2 and either Rig-I or Mda5 rescued inflammatory signals and HSPC numbers. Modulating the expression of the signaling mediator Traf6 in RLR deficient embryos restored HSPC numbers. Repetitive element transcripts could be detected in hemogenic endothelial cells and HSPCs, suggesting a role as RLR ligands. Indeed, ectopic expression of repetitive elements enhanced HSPC formation in wild-type, but not in Rig-I or Mda5 deficient embryos. Manipulation of RLR expression in mouse fetal liver HSPCs indicated functional conservation among species. Thus, repetitive elements transcribed during development drive RLR-mediated inflammatory signals that regulate HSPC formation.

ZFP451-mediated SUMOylation of SATB2 drives embryonic stem cell differentiation.

The establishment of cell fates involves alterations of transcription factor repertoires and repurposing of transcription factors by post-translational modifications. In embryonic stem cells (ESCs), the chromatin organizers SATB2 and SATB1 balance pluripotency and differentiation by activating and repressing pluripotency genes, respectively. Here, we show that conditional Satb2 gene inactivation weakens ESC pluripotency, and we identify SUMO2 modification of SATB2 by the E3 ligase ZFP451 as a potential driver of ESC differentiation. Mutations of two SUMO-acceptor lysines of Satb2 (Satb2K →R ) or knockout of Zfp451 impair the ability of ESCs to silence pluripotency genes and activate differentiation-associated genes in response to retinoic acid (RA) treatment. Notably, the forced expression of a SUMO2-SATB2 fusion protein in either Satb2K →R or Zfp451-/- ESCs rescues, in part, their impaired differentiation potential and enhances the down-regulation of Nanog The differentiation defect of Satb2K →R ESCs correlates with altered higher-order chromatin interactions relative to Satb2wt ESCs. Upon RA treatment of Satb2wt ESCs, SATB2 interacts with ZFP451 and the LSD1/CoREST complex and gains binding at differentiation genes, which is not observed in RA-treated Satb2K →R cells. Thus, SATB2 SUMOylation may contribute to the rewiring of transcriptional networks and the chromatin interactome of ESCs in the transition of pluripotency to differentiation.

Dynamic EBF1 occupancy directs sequential epigenetic and transcriptional events in B-cell programming.

B-cell fate determination requires the action of transcription factors that operate in a regulatory network to activate B-lineage genes and repress lineage-inappropriate genes. However, the dynamics and hierarchy of events in B-cell programming remain obscure. To uncouple the dynamics of transcription factor expression from functional consequences, we generated induction systems in developmentally arrested Ebf1-/- pre-pro-B cells to allow precise experimental control of EBF1 expression in the genomic context of progenitor cells. Consistent with the described role of EBF1 as a pioneer transcription factor, we show in a time-resolved analysis that EBF1 occupancy coincides with EBF1 expression and precedes the formation of chromatin accessibility. We observed dynamic patterns of EBF1 target gene expression and sequential up-regulation of transcription factors that expand the regulatory network at the pro-B-cell stage. A continuous EBF1 function was found to be required for Cd79a promoter activity and for the maintenance of an accessible chromatin domain that is permissive for binding of other transcription factors. Notably, transient EBF1 occupancy was detected at lineage-inappropriate genes prior to their silencing in pro-B cells. Thus, persistent and transient functions of EBF1 allow for an ordered sequence of epigenetic and transcriptional events in B-cell programming.

Interactions between lineage-associated transcription factors govern haematopoietic progenitor states.

Recent advances in molecular profiling provide descriptive datasets of complex differentiation landscapes including the haematopoietic system, but the molecular mechanisms defining progenitor states and lineage choice remain ill-defined. Here, we employed a cellular model of murine multipotent haematopoietic progenitors (Hoxb8-FL) to knock out 39 transcription factors (TFs) followed by RNA-Seq analysis, to functionally define a regulatory network of 16,992 regulator/target gene links. Focussed analysis of the subnetworks regulated by the B-lymphoid TF Ebf1 and T-lymphoid TF Gata3 revealed a surprising role in common activation of an early myeloid programme. Moreover, Gata3-mediated repression of Pax5 emerges as a mechanism to prevent precocious B-lymphoid differentiation, while Hox-mediated activation of Meis1 suppresses myeloid differentiation. To aid interpretation of large transcriptomics datasets, we also report a new method that visualises likely transitions that a progenitor will undergo following regulatory network perturbations. Taken together, this study reveals how molecular network wiring helps to establish a multipotent progenitor state, with experimental approaches and analysis tools applicable to dissecting a broad range of both normal and perturbed cellular differentiation landscapes.

Epigenetic regulator genes direct lineage switching in MLL/AF4 leukemia.

The fusion gene MLL/AF4 defines a high-risk subtype of pro-B acute lymphoblastic leukemia. Relapse can be associated with a lineage switch from acute lymphoblastic to acute myeloid leukemia, resulting in poor clinical outcomes caused by resistance to chemotherapies and immunotherapies. In this study, the myeloid relapses shared oncogene fusion breakpoints with their matched lymphoid presentations and originated from various differentiation stages from immature progenitors through to committed B-cell precursors. Lineage switching is linked to substantial changes in chromatin accessibility and rewiring of transcriptional programs, including alternative splicing. These findings indicate that the execution and maintenance of lymphoid lineage differentiation is impaired. The relapsed myeloid phenotype is recurrently associated with the altered expression, splicing, or mutation of chromatin modifiers, including CHD4 coding for the ATPase/helicase of the nucleosome remodelling and deacetylation complex. Perturbation of CHD4 alone or in combination with other mutated epigenetic modifiers induces myeloid gene expression in MLL/AF4+ cell models, indicating that lineage switching in MLL/AF4 leukemia is driven and maintained by disrupted epigenetic regulation.

ERBB2b mRNA isoform encodes a nuclear variant of the ERBB2 oncogene in breast cancer.

The presence of nuclear ERBB2 receptor-type tyrosine kinase is one of the causes of the resistance to membrane ERBB2-targeted therapy in breast cancers. It has been previously reported that this nuclear location arises through at least two different mechanisms: proteolytic shedding of the extracellular domain of the full-length receptor and translation of the messenger RNA (mRNA)-encoding ERBB2 from internal initiation codons. Here, we report a new mechanism and function where a significant portion of nuclear ERBB2 results from the translation of the variant ERBB2 mRNA under the transcriptional control of a distal promoter that is actively used in breast cancer cells. We show that both membrane ERBB2a and nuclear ERBB2b isoforms are prevalently expressed in breast cancer cell lines and carcinoma samples. The ERBB2b isoform, which is translated from mRNA variant 2, can directly translocate into the nucleus due to the lack of the signal peptide which is required for an intermediate membrane location. Small interfering RNA-mediated gene silencing showed that ERBB2b can repress ERBB2a expression, encoded by variant 1, whereas ERBB2a activates ERBB2b. Nuclear ERBB2 binding to its own promoter was revealed by chromatin immunoprecipitation assay. Altogether, our results provide new insights into the origin and function of nuclear ERBB2 where it can participate at the same time in a positive or a negative feedback autoregulatory loop, dependent on which of its promoters this bona fide transcription factor is acting. They also provide a new understanding for the resistance to therapies targeting the membrane-anchored ERBB2 in breast cancer.

Inducible chromatin priming is associated with the establishment of immunological memory in T cells.

Immunological memory is a defining feature of vertebrate physiology, allowing rapid responses to repeat infections. However, the molecular mechanisms required for its establishment and maintenance remain poorly understood. Here, we demonstrated that the first steps in the acquisition of T-cell memory occurred during the initial activation phase of naïve T cells by an antigenic stimulus. This event initiated extensive chromatin remodeling that reprogrammed immune response genes toward a stably maintained primed state, prior to terminal differentiation. Activation induced the transcription factors NFAT and AP-1 which created thousands of new DNase I-hypersensitive sites (DHSs), enabling ETS-1 and RUNX1 recruitment to previously inaccessible sites. Significantly, these DHSs remained stable long after activation ceased, were preserved following replication, and were maintained in memory-phenotype cells. We show that primed DHSs maintain regions of active chromatin in the vicinity of inducible genes and enhancers that regulate immune responses. We suggest that this priming mechanism may contribute to immunological memory in T cells by facilitating the induction of nearby inducible regulatory elements in previously activated T cells.

Cooperative binding of AP-1 and TEAD4 modulates the balance between vascular smooth muscle and hemogenic cell fate.

The transmission of extracellular signals into the nucleus involves inducible transcription factors, but how different signalling pathways act in a cell type-specific fashion is poorly understood. Here, we studied the regulatory role of the AP-1 transcription factor family in blood development using embryonic stem cell differentiation coupled with genome-wide transcription factor binding and gene expression analyses. AP-1 factors respond to MAP kinase signalling and comprise dimers of FOS, ATF and JUN proteins. To examine genes regulated by AP-1 and to examine how it interacts with other inducible transcription factors, we abrogated its global DNA-binding activity using a dominant-negative FOS peptide. We show that FOS and JUN bind to and activate a specific set of vascular genes and that AP-1 inhibition shifts the balance between smooth muscle and hematopoietic differentiation towards blood. Furthermore, AP-1 is required for de novo binding of TEAD4, a transcription factor connected to Hippo signalling. Our bottom-up approach demonstrates that AP-1- and TEAD4-associated cis-regulatory elements form hubs for multiple signalling-responsive transcription factors and define the cistrome that regulates vascular and hematopoietic development by extrinsic signals.

Integration of Kinase and Calcium Signaling at the Level of Chromatin Underlies Inducible Gene Activation in T Cells.

TCR signaling pathways cooperate to activate the inducible transcription factors NF-κB, NFAT, and AP-1. In this study, using the calcium ionophore ionomycin and/or PMA on Jurkat T cells, we show that the gene expression program associated with activation of TCR signaling is closely related to specific chromatin landscapes. We find that calcium and kinase signaling cooperate to induce chromatin remodeling at ∼2100 chromatin regions, which demonstrate enriched binding motifs for inducible factors and correlate with target gene expression. We found that these regions typically function as inducible enhancers. Many of these elements contain composite NFAT/AP-1 sites, which typically support cooperative binding, thus further reinforcing the need for cooperation between calcium and kinase signaling in the activation of genes in T cells. In contrast, treatment with PMA or ionomycin alone induces chromatin remodeling at far fewer regions (∼600 and ∼350, respectively), which mostly represent a subset of those induced by costimulation. This suggests that the integration of TCR signaling largely occurs at the level of chromatin, which we propose plays a crucial role in regulating T cell activation.

Chromatin priming elements establish immunological memory in T cells without activating transcription: T cell memory is maintained by DNA elements which stably prime inducible genes without activating steady state transcription.

We have identified a simple epigenetic mechanism underlying the establishment and maintenance of immunological memory in T cells. By studying the transcriptional regulation of inducible genes we found that a single cycle of activation of inducible factors is sufficient to initiate stable binding of pre-existing transcription factors to thousands of newly activated distal regulatory elements within inducible genes. These events lead to the creation of islands of active chromatin encompassing nearby enhancers, thereby supporting the accelerated activation of inducible genes, without changing steady state levels of transcription in memory T cells. These studies also highlighted the need for more sophisticated definitions of gene regulatory elements. The chromatin priming elements defined here are distinct from classical enhancers because they function by maintaining chromatin accessibility rather than directly activating transcription. We propose that these priming elements are members of a wider class of genomic elements that support correct developmentally regulated gene expression.

LMO2 is required for TAL1 DNA binding activity and initiation of definitive haematopoiesis at the haemangioblast stage.

LMO2 is a bridging factor within a DNA binding complex and is required for definitive haematopoiesis to occur. The developmental stage of the block in haematopoietic specification is not known. We show that Lmo2-/- mouse embryonic stem cells differentiated to Flk-1+ haemangioblasts, but less efficiently to haemogenic endothelium, which only produced primitive haematopoietic progenitors. Genome-wide approaches indicated that LMO2 is required at the haemangioblast stage to position the TAL1/LMO2/LDB1 complex to regulatory elements that are important for the establishment of the haematopoietic developmental program. In the absence of LMO2, the target site recognition of TAL1 is impaired. The lack of LMO2 resulted in altered gene expression levels already at the haemangioblast stage, with transcription factor genes accounting for ∼15% of affected genes. Comparison of Lmo2-/- with Tal1-/- Flk-1+ cells further showed that TAL1 was required to initiate or sustain Lmo2 expression.

Dynamic recruitment of Ets1 to both nucleosome-occupied and -depleted enhancer regions mediates a transcriptional program switch during early T-cell differentiation.

Ets1 is a sequence-specific transcription factor that plays an important role during hematopoiesis, and is essential for the transition of CD4(-)/CD8(-) double negative (DN) to CD4(+)/CD8(+) double positive (DP) thymocytes. Using genome-wide and functional approaches, we investigated the binding properties, transcriptional role and chromatin environment of Ets1 during this transition. We found that while Ets1 binding at distal sites was associated with active genes at both DN and DP stages, its enhancer activity was attained at the DP stage, as reflected by levels of the core transcriptional hallmarks H3K4me1/3, RNA Polymerase II and eRNA. This dual, stage-specific ability reflected a switch from non-T hematopoietic toward T-cell specific gene expression programs during the DN-to-DP transition, as indicated by transcriptome analyses of Ets1(-/-) thymic cells. Coincidentally, Ets1 associates more specifically with Runx1 in DN and with TCF1 in DP cells. We also provide evidence that Ets1 predominantly binds distal nucleosome-occupied regions in DN and nucleosome-depleted regions in DP. Finally and importantly, we demonstrate that Ets1 induces chromatin remodeling by displacing H3K4me1-marked nucleosomes. Our results thus provide an original model whereby the ability of a transcription factor to bind nucleosomal DNA changes during differentiation with consequences on its cognate enhancer activity.

Pasha: a versatile R package for piling chromatin HTS data.

UNLABELLED: We describe an R package designed for processing aligned reads from chromatin-oriented high-throughput sequencing experiments. Pasha (preprocessing of aligned sequences from HTS analyses) allows easy manipulation of aligned reads from short-read sequencing technologies (ChIP-seq, FAIRE-seq, MNase-Seq, …) and offers innovative approaches such as ChIP-seq reads elongation, nucleosome midpoint piling strategy for positioning analyses, or the ability to subset paired-end reads by groups of insert size that can contain biologically relevant information. AVAILABILITY AND IMPLEMENTATION: Pasha is a multi-platform R package, available on CRAN repositories under GPL-3 license (https://cran.r-project.org/web/packages/Pasha/). CONTACTS: rfenouil@gmail.com or jean-christophe.andrau@igmm.cnrs.fr SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Transcription-dependent generation of a specialized chromatin structure at the TCRß locus.

V(D)J recombination assembles Ag receptor genes during lymphocyte development. Enhancers at AR loci are known to control V(D)J recombination at associated alleles, in part by increasing chromatin accessibility of the locus, to allow the recombination machinery to gain access to its chromosomal substrates. However, whether there is a specific mechanism to induce chromatin accessibility at AR loci is still unclear. In this article, we highlight a specialized epigenetic marking characterized by high and extended H3K4me3 levels throughout the Dß-Jß-Cß gene segments. We show that extended H3K4 trimethylation at the Tcrb locus depends on RNA polymerase II (Pol II)-mediated transcription. Furthermore, we found that the genomic regions encompassing the two DJCß clusters are highly enriched for Ser(5)-phosphorylated Pol II and short-RNA transcripts, two hallmarks of transcription initiation and early transcription. Of interest, these features are shared with few other tissue-specific genes. We propose that the entire DJCß regions behave as transcription "initiation" platforms, therefore linking a specialized mechanism of Pol II transcription with extended H3K4 trimethylation and highly accessible Dß and Jß gene segments.

Mapping of transcription factor motifs in active chromatin identifies IRF5 as key regulator in classical Hodgkin lymphoma.

Deregulated transcription factor (TF) activities are commonly observed in hematopoietic malignancies. Understanding tumorigenesis therefore requires determining the function and hierarchical role of individual TFs. To identify TFs central to lymphomagenesis, we identified lymphoma type-specific accessible chromatin by global mapping of DNaseI hypersensitive sites and analyzed enriched TF-binding motifs in these regions. Applying this unbiased approach to classical Hodgkin lymphoma (HL), a common B-cell-derived lymphoma with a complex pattern of deregulated TFs, we discovered interferon regulatory factor (IRF) sites among the top enriched motifs. High-level expression of the proinflammatory TF IRF5 was specific to HL cells and crucial for their survival. Furthermore, IRF5 initiated a regulatory cascade in human non-Hodgkin B-cell lines and primary murine B cells by inducing the TF AP-1 and cooperating with NF-κB to activate essential characteristic features of HL. Our strategy efficiently identified a lymphoma type-specific key regulator and uncovered a tumor promoting role of IRF5.

Wellington-bootstrap: differential DNase-seq footprinting identifies cell-type determining transcription factors.

BACKGROUND: The analysis of differential gene expression is a fundamental tool to relate gene regulation with specific biological processes. Differential binding of transcription factors (TFs) can drive differential gene expression. While DNase-seq data can provide global snapshots of TF binding, tools for detecting differential binding from pairs of DNase-seq data sets are lacking. RESULTS: In order to link expression changes with changes in TF binding we introduce the concept of differential footprinting alongside a computational tool. We demonstrate that differential footprinting is associated with differential gene expression and can be used to define cell types by their specific TF occupancy patterns. CONCLUSIONS: Our new tool, Wellington-bootstrap, will enable the detection of differential TF binding facilitating the study of gene regulatory systems.

CpG islands and GC content dictate nucleosome depletion in a transcription-independent manner at mammalian promoters.

One clear hallmark of mammalian promoters is the presence of CpG islands (CGIs) at more than two-thirds of genes, whereas TATA boxes are only present at a minority of promoters. Using genome-wide approaches, we show that GC content and CGIs are major promoter elements in mammalian cells, able to govern open chromatin conformation and support paused transcription. First, we define three classes of promoters with distinct transcriptional directionality and pausing properties that correlate with their GC content. We further analyze the direct influence of GC content on nucleosome positioning and depletion and show that CpG content and CGI width correlate with nucleosome depletion both in vivo and in vitro. We also show that transcription is not essential for nucleosome exclusion but influences both a weak +1 and a well-positioned nucleosome at CGI borders. Altogether our data support the idea that CGIs have become an essential feature of promoter structure defining novel regulatory properties in mammals.

Wellington: a novel method for the accurate identification of digital genomic footprints from DNase-seq data.

The expression of eukaryotic genes is regulated by cis-regulatory elements such as promoters and enhancers, which bind sequence-specific DNA-binding proteins. One of the great challenges in the gene regulation field is to characterise these elements. This involves the identification of transcription factor (TF) binding sites within regulatory elements that are occupied in a defined regulatory context. Digestion with DNase and the subsequent analysis of regions protected from cleavage (DNase footprinting) has for many years been used to identify specific binding sites occupied by TFs at individual cis-elements with high resolution. This methodology has recently been adapted for high-throughput sequencing (DNase-seq). In this study, we describe an imbalance in the DNA strand-specific alignment information of DNase-seq data surrounding protein-DNA interactions that allows accurate prediction of occupied TF binding sites. Our study introduces a novel algorithm, Wellington, which considers the imbalance in this strand-specific information to efficiently identify DNA footprints. This algorithm significantly enhances specificity by reducing the proportion of false positives and requires significantly fewer predictions than previously reported methods to recapitulate an equal amount of ChIP-seq data. We also provide an open-source software package, pyDNase, which implements the Wellington algorithm to interface with DNase-seq data and expedite analyses.