Conserved role of hnRNPL in alternative splicing of epigenetic modifiers enables B cell activation.

The multifunctional RNA-binding protein hnRNPL is implicated in antibody class switching but its broader function in B cells is unknown. Here, we show that hnRNPL is essential for B cell activation, germinal center formation, and antibody responses. Upon activation, hnRNPL-deficient B cells show proliferation defects and increased apoptosis. Comparative analysis of RNA-seq data from activated B cells and another eight hnRNPL-depleted cell types reveals common effects on MYC and E2F transcriptional programs required for proliferation. Notably, while individual gene expression changes are cell type specific, several alternative splicing events affecting histone modifiers like KDM6A and SIRT1, are conserved across cell types. Moreover, hnRNPL-deficient B cells show global changes in H3K27me3 and H3K9ac. Epigenetic dysregulation after hnRNPL loss could underlie differential gene expression and upregulation of lncRNAs, and explain common and cell type-specific phenotypes, such as dysfunctional mitochondria and ROS overproduction in mouse B cells. Thus, hnRNPL is essential for the resting-to-activated B cell transition by regulating transcriptional programs and metabolism, at least in part through the alternative splicing of several histone modifiers.

GFI1 tethers the NuRD complex to open and transcriptionally active chromatin in myeloid progenitors.

Growth factor indepdendent 1 (GFI1) is a SNAG-domain, DNA binding transcriptional repressor which controls myeloid differentiation through molecular mechanisms and co-factors that still remain to be clearly identified. Here we show that GFI1 associates with the chromodomain helicase DNA binding protein 4 (CHD4) and other components of the Nucleosome remodeling and deacetylase (NuRD) complex. In granulo-monocytic precursors, GFI1, CHD4 or GFI1/CHD4 complexes occupy sites enriched for histone marks associated with active transcription suggesting that GFI1 recruits the NuRD complex to target genes regulated by active or bivalent promoters and enhancers. GFI1 and GFI1/CHD4 complexes occupy promoters that are either enriched for IRF1 or SPI1 consensus binding sites, respectively. During neutrophil differentiation, chromatin closure and depletion of H3K4me2 occurs at different degrees depending on whether GFI1, CHD4 or both are present, indicating that GFI1 is more efficient in depleting of H3K4me2 and -me1 marks when associated with CHD4. Our data suggest that GFI1/CHD4 complexes regulate histone modifications differentially to enable regulation of target genes affecting immune response, nucleosome organization or cellular metabolic processes and that both the target gene specificity and the activity of GFI1 during myeloid differentiation depends on the presence of chromatin remodeling complexes.

Gfi1b regulates the level of Wnt/ß-catenin signaling in hematopoietic stem cells and megakaryocytes.

Gfi1b is a transcriptional repressor expressed in hematopoietic stem cells (HSCs) and megakaryocytes (MKs). Gfi1b deficiency leads to expansion of both cell types and abrogates the ability of MKs to respond to integrin. Here we show that Gfi1b forms complexes with ß-catenin, its co-factors Pontin52, CHD8, TLE3 and CtBP1 and regulates Wnt/ß-catenin-dependent gene expression. In reporter assays, Gfi1b can activate TCF-dependent transcription and Wnt3a treatment enhances this activation. This requires interaction between Gfi1b and LSD1 and suggests that a tripartite ß-catenin/Gfi1b/LSD1 complex exists, which regulates Wnt/ß-catenin target genes. Consistently, numerous canonical Wnt/ß-catenin target genes, co-occupied by Gfi1b, ß-catenin and LSD1, have their expression deregulated in Gfi1b-deficient cells. When Gfi1b-deficient cells are treated with Wnt3a, their normal cellularity is restored and Gfi1b-deficient MKs regained their ability to spread on integrin substrates. This indicates that Gfi1b controls both the cellularity and functional integrity of HSCs and MKs by regulating Wnt/ß-catenin signaling pathway.

Threshold Levels of Gfi1 Maintain E2A Activity for B Cell Commitment via Repression of Id1.

A regulatory circuit that controls myeloid versus B lymphoid cell fate in hematopoietic progenitors has been proposed, in which a network of the transcription factors Egr1/2, Nab, Gfi1 and PU.1 forms the core element. Here we show that a direct link between Gfi1, the transcription factor E2A and its inhibitor Id1 is a critical element of this regulatory circuit. Our data suggest that a certain threshold of Gfi1 is required to gauge E2A activity by adjusting levels of Id1 in multipotent progenitors, which are the first bipotential myeloid/lymphoid-restricted progeny of hematopoietic stem cells. If Gfi1 levels are high, Id1 is repressed enabling E2A to activate a specific set of B lineage genes by binding to regulatory elements for example the IL7 receptor gene. If Gfi1 levels fall below a threshold, Id1 expression increases and renders E2A unable to function, which prevents hematopoietic progenitors from engaging along the B lymphoid lineage.

Heterogeneous Nuclear Ribonucleoprotein L is required for the survival and functional integrity of murine hematopoietic stem cells.

The proliferation and survival of hematopoietic stem cells (HSCs) has to be strictly coordinated to ensure the timely production of all blood cells. Here we report that the splice factor and RNA binding protein hnRNP L (heterogeneous nuclear ribonucleoprotein L) is required for hematopoiesis, since its genetic ablation in mice reduces almost all blood cell lineages and causes premature death of the animals. In agreement with this, we observed that hnRNP L deficient HSCs lack both the ability to self-renew and foster hematopoietic differentiation in transplanted hosts. They also display mitochondrial dysfunction, elevated levels of γH2AX, are Annexin V positive and incorporate propidium iodide indicating that they undergo cell death. Lin(-)c-Kit(+) fetal liver cells from hnRNP L deficient mice show high p53 protein levels and up-regulation of p53 target genes. In addition, cells lacking hnRNP L up-regulated the expression of the death receptors TrailR2 and CD95/Fas and show Caspase-3, Caspase-8 and Parp cleavage. Treatment with the pan-caspase inhibitor Z-VAD-fmk, but not the deletion of p53, restored cell survival in hnRNP L deficient cells. Our data suggest that hnRNP L is critical for the survival and functional integrity of HSCs by restricting the activation of caspase-dependent death receptor pathways.

Distinct mechanisms regulate Cdx2 expression in the blastocyst and in trophoblast stem cells.

The first intercellular differences during mammalian embryogenesis arise in the blastocyst, producing the inner cell mass and the trophectoderm. The trophectoderm is the first extraembryonic tissue and does not contribute to the embryo proper, its differentiation instead forming tissues that sustain embryonic development. Crucial roles in extraembryonic differentiation have been identified for certain transcription factors, but a comprehensive picture of the regulation of this early specification is still lacking. Here, we investigated whether the regulatory mechanisms involved in Cdx2 expression in the blastocyst are also utilized in the postimplantation embryo. We analyzed an enhancer that is regulated through Hippo and Notch in the blastocyst trophectoderm, unexpectedly finding that it is inactive in the extraembryonic structures at postimplantation stages. Further analysis identified other Cdx2 regulatory elements including a stem-cell specific regulatory sequence and an element that drives reporter expression in the trophectoderm, a subset of cells in the extraembryonic region of the postimplantation embryo and in trophoblast stem cells. The cross-comparison in this study of cis-regulatory elements employed in the blastocyst, stem cell populations and the postimplantation embryo provides new insights into early mammalian development and suggests a two-step mechanism in Cdx2 regulation.

Epigenetic therapy as a novel approach for GFI136N-associated murine/human AML.

Epigenetic changes can contribute to development of acute myeloid leukemia (AML), a malignant disease of the bone marrow. A single-nucleotide polymorphism of transcription factor growth factor independence 1 (GFI1) generates a protein with an asparagine at position 36 (GFI1(36N)) instead of a serine at position 36 (GFI1(36S)), which is associated with de novo AML in humans. However, how GFI1(36N) predisposes to AML is poorly understood. To explore the mechanism, we used knock-in mouse strains expressing GFI1(36N) or GFI1(36S). Presence of GFI1(36N) shortened the latency and increased the incidence of AML in different murine models of myelodysplastic syndrome/AML. On a molecular level, GFI1(36N) induced genomewide epigenetic changes, leading to expression of AML-associated genes. On a therapeutic level, use of histone acetyltransferase inhibitors specifically impeded growth of GFI1(36N)-expressing human and murine AML cells in vitro and in vivo. These results establish, as a proof of principle, how epigenetic changes in GFI1(36N)-induced AML can be targeted.

Ring1B and Suv39h1 delineate distinct chromatin states at bivalent genes during early mouse lineage commitment.

Pluripotent cells develop within the inner cell mass of blastocysts, a mosaic of cells surrounded by an extra-embryonic layer, the trophectoderm. We show that a set of somatic lineage regulators (including Hox, Gata and Sox factors) that carry bivalent chromatin enriched in H3K27me3 and H3K4me2 are selectively targeted by Suv39h1-mediated H3K9me3 and de novo DNA methylation in extra-embryonic versus embryonic (pluripotent) lineages, as assessed both in blastocyst-derived stem cells and in vivo. This stably repressed state is linked with a loss of gene priming for transcription through the exclusion of PRC1 (Ring1B) and RNA polymerase II complexes at bivalent, lineage-inappropriate genes upon trophoblast lineage commitment. Collectively, our results suggest a mutually exclusive role for Ring1B and Suv39h1 in regulating distinct chromatin states at key developmental genes and propose a novel mechanism by which lineage specification can be reinforced during early development.

SbcCD regulation and localization in Escherichia coli.

The SbcCD complex and its homologues play important roles in DNA repair and in the maintenance of genome stability. In Escherichia coli, the in vitro functions of SbcCD have been well characterized, but its exact cellular role remains elusive. This work investigates the regulation of the sbcDC operon and the cellular localization of the SbcC and SbcD proteins. Transcription of the sbcDC operon is shown to be dependent on starvation and RpoS protein. Overexpressed SbcC protein forms foci that colocalize with the replication factory, while overexpressed SbcD protein is distributed through the cytoplasm.