Foxa2 and H2A.Z Mediate Nucleosome Depletion during Embryonic Stem Cell Differentiation

SummaryNucleosome occupancy is fundamental for establishing chromatin architecture. However, little is known about the relationship between nucleosome dynamics and initial cell lineage specification. Here, we determine the mechanisms that control global nucleosome dynamics during embryonic stem (ES) cell differentiation into endoderm. Both nucleosome depletion and de novo occupation occur during the differentiation process, with higher overall nucleosome density after differentiation. The variant histone H2A.Z and the winged helix transcription factor Foxa2 both act to regulate nucleosome depletion and gene activation, thus promoting ES cell differentiation, whereas DNA methylation promotes nucleosome occupation and suppresses gene expression. Nucleosome depletion during ES cell differentiation is dependent on Nap1l1-coupled SWI/SNF and INO80 chromatin remodeling complexes. Thus, both epigenetic and genetic regulators cooperate to control nucleosome dynamics during ES cell fate decisions

Pluripotent stem cells reveal erythroid-specific activities of the GATA1 N-terminus

Germline GATA1 mutations that result in the production of an amino-truncated protein termed GATA1s (where s indicates short) cause congenital hypoplastic anemia. In patients with trisomy 21, similar somatic GATA1s-producing mutations promote transient myeloproliferative disease and acute megakaryoblastic leukemia. Here, we demonstrate that induced pluripotent stem cells (iPSCs) from patients with GATA1-truncating mutations exhibit impaired erythroid potential, but enhanced megakaryopoiesis and myelopoiesis, recapitulating the major phenotypes of the associated diseases. Similarly, in developmentally arrested GATA1-deficient murine megakaryocyte-erythroid progenitors derived from murine embryonic stem cells (ESCs), expression of GATA1s promoted megakaryopoiesis, but not erythropoiesis. Transcriptome analysis revealed a selective deficiency in the ability of GATA1s to activate erythroid-specific genes within populations of hematopoietic progenitors. Although its DNA-binding domain was intact, chromatin immunoprecipitation studies showed that GATA1s binding at specific erythroid regulatory regions was impaired, while binding at many nonerythroid sites, including megakaryocytic and myeloid target genes, was normal. Together, these observations indicate that lineage-specific GATA1 cofactor associations are essential for normal chromatin occupancy and provide mechanistic insights into how GATA1s mutations cause human disease. More broadly, our studies underscore the value of ESCs and iPSCs to recapitulate and study disease phenotypes12539931005United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; American Society of Hematology Scholar Award; Alex's Lemonade Stand Foundation Springboard Grant; NIH Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD); NIH National Heart Lung & Blood Institute (NHLBI); NIH National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK

Chromatin 3D interaction analysis of the STARD10 locus unveils FCHSD2 as a regulator of insulin secretion

Correction https://doi.org/10.1016/j.celrep.2021.108881Using chromatin conformation capture, we show that an enhancer cluster in the STARD10 type 2 diabetes (T2D) locus forms a defined 3-dimensional (3D) chromatin domain. A 4.1-kb region within this locus, carrying 5 T2D-associated variants, physically interacts with CTCF-binding regions and with an enhancer possessing strong transcriptional activity. Analysis of human islet 3D chromatin interaction maps identifies the FCHSD2 gene as an additional target of the enhancer cluster. CRISPR-Cas9-mediated deletion of the variant region, or of the associated enhancer, from human pancreas-derived EndoC-bH1 cells impairs glucose- stimulated insulin secretion. Expression of both STARD10 and FCHSD2 is reduced in cells harboring CRISPR deletions, and lower expression of STARD10 and FCHSD2 is associated, the latter nominally, with the possession of risk variant alleles in human islets. Finally, CRISPR-Cas9-mediated loss of STARD10 or FCHSD2, but not ARAP1, impairs regulated insulin secretion. Thus, multiple genes at the STARD10 locus influence b cell function.Peer reviewe

The role of non-receptor tyrosine kinases in NK T cell ontogeny and function

NK T cells are a lymphocyte lineage that is selected by CD1d and characterized by the rapid secretion of potent immune regulatory cytokines, such as IFN-γ and IL-4. The signaling mechanisms controlling NK T cell development are poorly characterized. Here, we define the signaling requirements for the Src family kinase members Fyn and Lck as well as the Tec kinase family member Itk, during NK T cell ontogeny. Fyn mutant mice contain a block in NK T cell development, with a five to ten fold decrease in the number of cells. In contrast, conventional T cell development occurs normally. Studies using bone marrow chimeras indicate that the defect behaves in a cell autonomous manner. The few NK T cells present in fyn null mice were analyzed to determine whether Fyn is also required during peripheral NK T cell activation. The fyn−/− NK T cells can respond to ligand and produce cytokines, but have depressed proliferative capacity. Transgenic expression of the NK T cell specific TCR α chain, Vα14Jα18, leads to a complete restoration of NK T cell numbers in fyn−/− mice, suggesting that Fyn may have a role prior to α chain rearrangement. NK T cells are thought to develop from a progenitor expressing the Vα14Jα18 TCR α chain, but which are negative for NK cell markers. Fyn deficient mice have decreased numbers of NK1.1− NK T cell progenitors as well as mature NK1.1+ cells, confirming that Fyn is required early during NK T cell ontogeny. The earliest NK T cell progenitor populations (NK1.1−) could proliferate to IL-7, while mature (NK1.1+) NK T cells did not. The NK1.1 − NK T cell progenitors were capable of upregulating NK1.1 when transferred in vivo. Upon stimulation, the NK1.1− populations secrete IL-4, but little IFN-γ. As the cells mature and upregulate NK1.1, they acquire the ability to secrete IFN-γ. Finally, the Tec family tyrosine kinase Itk is necessary for optimal NK1.1 upregulation and hence final maturation of NK T cells. Itk mutant mice also display a progressive decrease in NK T cells in older mice, suggesting a possible further role in peripheral maintenance

The role of non-receptor tyrosine kinases in NK T cell ontogeny and function

NK T cells are a lymphocyte lineage that is selected by CD1d and characterized by the rapid secretion of potent immune regulatory cytokines, such as IFN-γ and IL-4. The signaling mechanisms controlling NK T cell development are poorly characterized. Here, we define the signaling requirements for the Src family kinase members Fyn and Lck as well as the Tec kinase family member Itk, during NK T cell ontogeny. Fyn mutant mice contain a block in NK T cell development, with a five to ten fold decrease in the number of cells. In contrast, conventional T cell development occurs normally. Studies using bone marrow chimeras indicate that the defect behaves in a cell autonomous manner. The few NK T cells present in fyn null mice were analyzed to determine whether Fyn is also required during peripheral NK T cell activation. The fyn−/− NK T cells can respond to ligand and produce cytokines, but have depressed proliferative capacity. Transgenic expression of the NK T cell specific TCR α chain, Vα14Jα18, leads to a complete restoration of NK T cell numbers in fyn−/− mice, suggesting that Fyn may have a role prior to α chain rearrangement. NK T cells are thought to develop from a progenitor expressing the Vα14Jα18 TCR α chain, but which are negative for NK cell markers. Fyn deficient mice have decreased numbers of NK1.1− NK T cell progenitors as well as mature NK1.1+ cells, confirming that Fyn is required early during NK T cell ontogeny. The earliest NK T cell progenitor populations (NK1.1−) could proliferate to IL-7, while mature (NK1.1+) NK T cells did not. The NK1.1 − NK T cell progenitors were capable of upregulating NK1.1 when transferred in vivo. Upon stimulation, the NK1.1− populations secrete IL-4, but little IFN-γ. As the cells mature and upregulate NK1.1, they acquire the ability to secrete IFN-γ. Finally, the Tec family tyrosine kinase Itk is necessary for optimal NK1.1 upregulation and hence final maturation of NK T cells. Itk mutant mice also display a progressive decrease in NK T cells in older mice, suggesting a possible further role in peripheral maintenance

OCT4 Coordinates with WNT Signaling to Pre-pattern Chromatin at the SOX17 Locus during Human ES Cell Differentiation into Definitive Endoderm

We demonstrate that the pluripotency gene OCT4 has a role in regulating differentiation via Wnt signaling. OCT4 expression levels in human embryonic stem cells increases transiently during the first 24 hr of in vitro differentiation, with OCT4 occupancy increasing at endoderm regulators such as SOX17 and FOXA2. This increased occupancy correlates with loss of the PRC2 complex and the inhibitory histone mark H3K27me3. Knockdown of OCT4 during differentiation inhibits mesendoderm formation and removal of the H3K27me3 mark from the SOX17 promoter, suggesting that OCT4 acts to induce removal of the PRC2 complex. Furthermore, OCT4 and β-catenin can be co-immunoprecipitated upon differentiation, and Wnt stimulation is required for the enhanced OCT4 occupancy and loss of the PRC2 complex from the SOX17 promoter. In conclusion, our study reveals that OCT4, a master regulator of pluripotency, may also collaborate with Wnt signaling to drive endoderm induction by pre-patterning epigenetic markers on endodermal promoters