EZH2-dependent chromatin looping controls INK4a and INK4b, but not ARF, during human progenitor cell differentiation and cellular senescence

<p>Abstract</p> <p>Background</p> <p>The <it>INK4b-ARF-INK4a </it>tumour suppressor locus controls the balance between progenitor cell renewal and cancer. In this study, we investigated how higher-order chromatin structure modulates differential expression of the human <it>INK4b-ARF-INK4a </it>locus during progenitor cell differentiation, cellular ageing and senescence of cancer cells.</p> <p>Results</p> <p>We found that <it>INK4b </it>and <it>INK4a</it>, but not <it>ARF</it>, are upregulated following the differentiation of haematopoietic progenitor cells, in ageing fibroblasts and in senescing malignant rhabdoid tumour cells. To investigate the underlying molecular mechanism we analysed binding of polycomb group (PcG) repressive complexes (PRCs) and the spatial organization of the <it>INK4b-ARF-INK4a </it>locus. In agreement with differential derepression, PcG protein binding across the locus is discontinuous. As we described earlier, PcG repressors bind the INK4a promoter, but not ARF. Here, we identified a second peak of PcG binding that is located ~3 kb upstream of the <it>INK4b </it>promoter. During progenitor cell differentiation and ageing, PcG silencer EZH2 attenuates, causing loss of PRC binding and transcriptional activation of <it>INK4b </it>and <it>INK4a</it>. The expression pattern of the locus is reflected by its organization in space. In the repressed state, the PRC-binding regions are in close proximity, while the intervening chromatin harbouring <it>ARF </it>loops out. Down regulation of EZH2 causes release of the ~35 kb repressive chromatin loop and induction of both <it>INK4a </it>and <it>INK4b</it>, whereas <it>ARF </it>expression remains unaltered.</p> <p>Conclusion</p> <p>PcG silencers bind and coordinately regulate <it>INK4b </it>and <it>INK4a</it>, but not <it>ARF</it>, during a variety of physiological processes. Developmentally regulated EZH2 levels are one of the factors that can determine the higher order chromatin structure and expression pattern of the <it>INK4b-ARF-INK4a </it>locus, coupling human progenitor cell differentiation to proliferation control. Our results revealed a chromatin looping mechanism of long-range control and argue against models involving homogeneous spreading of PcG silencers across the <it>INK4b-ARF-INK4a </it>locus.</p

Whole-transcriptome analysis of endothelial to hematopoietic stem cell transition reveals a requirement for Gpr56 in HSC generation

10.1084/jem.20140767Journal of Experimental Medicine212193-10

In vivo single cell analysis reveals Gata2 dynamics in cells transitioning to hematopoietic fate

Cell fate is established through coordinated gene expression programs in individual cells. Regulatory networks that include the Gata2 transcription factor play central roles in hematopoietic fate establishment. Although Gata2 is essential to the embryonic development and function of hematopoietic stem cells that form the adult hierarchy, little is known about the in vivo expression dynamics of Gata2 in single cells. Here, we examine Gata2 expression in single aortic cells as they establish hematopoietic fate in Gata2Venus mouse embryos. Time-lapse imaging reveals rapid pulsatile level changes in Gata2 reporter expression in cells undergoing endothelial-to-hematopoietic transition. Moreover, Gata2 reporter pulsatile expression is dramatically altered in Gata2+/- aortic cells, which undergo fewer transitions and are reduced in hematopoietic potential. Our novel finding of dynamic pulsatile expression of Gata2 suggests a highly unstable genetic state in single cells concomitant with their transition to hematopoietic fate. This reinforces the notion that threshold levels of Gata2 influence fate establishment and has implications for transcription factor-related hematologic dysfunctions

Functional and molecular characterization of mouse Gata2-independent hematopoietic progenitors

The Gata2 transcription factor is a pivotal regulator of hematopoietic cell development and maintenance, highlighted by the fact that Gata2 haploinsufficiency has been identified as the cause of some familial cases of acute myelogenous leukemia/myelodysplastic syndrome and in MonoMac syndrome. Genetic deletion in mice has shown that Gata2 is pivotal to the embryonic generation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). It functions in the embryo during endothelial cell to hematopoietic cell transition to affect hematopoietic cluster, HPC, and HSC formation. Gata2 conditional deletion and overexpression studies show the importance of Gata2 levels in hematopoiesis, during all developmental stages. Although previous studies of cell populations phenotypically enriched in HPCs and HSCs show expression of Gata2, therehasbeen nodirect study of Gata2 expressing cells during normal hematopoiesis. In this study, we generate a Gata2Venus reporter mouse model with unperturbed Gata2 expression to examine the hematopoietic function and transcriptome of Gata2 expressing and nonexpressing cells. We show that all the HSCs are Gata2 expressing. However, not allHPCs in the aorta, vitellineand umbilical arteries, and fetal liver require or express Gata2. These Gata2-independent HPCs exhibit a different functional output and genetic program, including Ras and cyclic AMP response element-binding protein pathways and other Gata factors, compared with Gata2-dependent HPCs. Our results, indicating that Gata2 is of major importance in programming toward HSC fate but not in all cells with HPC fate, have implications for current reprogramming strategies

HIF1α is a regulator of hematopoietic progenitor and stem cell development in hypoxic sites of the mouse embryo

Hypoxia affects many physiologic processes during early stages of mammalian ontogeny, particularly placental and vascular development. In the adult, the hypoxic bone marrow microenvironment plays a role in regulating hematopoietic stem cell (HSC) function. HSCs are generated from the major vasculature of the embryo, but whether the hypoxic response affects the generation of these HSCs is as yet unknown. Here we examined whether Hypoxia Inducible Factor1-alpha (HIF1α), a key modulator of the response to hypoxia, is essential for HSC development. We found hypoxic cells in embryonic tissues that generate and expand hematopoietic cells (aorta, placenta and fetal liver), and specifically aortic endothelial and hematopoietic cluster cells. A Cre/loxP conditional knockout (cKO) approach was taken to delete HIF1α in Vascular Endothelial-Cadherin expressing endothelial cells, the precursors to definitive hematopoietic cells. Functional assays show that HSC and hematopoietic progenitor cells (HPCs) are significantly reduced in cKO aorta and placenta. Moreover, decreases in phenotypic aortic hematopoietic cluster cells in cKO embryos indicate that HIF1α is necessary for generation and/or expansion of HPCs and HSCs. cKO adult BM HSCs are also affected under transplantation conditions. Thus, HIF1α is a regulator of HSC generation and function beginning at the earliest embryonic stages

BMP and Hedgehog Regulate Distinct AGM Hematopoietic Stem Cells Ex Vivo

Hematopoietic stem cells (HSC), the self-renewing cells of the adult blood differentiation hierarchy, are generated during embryonic stages. The first HSCs are produced in the aorta-gonad-mesonephros (AGM) region of the embryo through endothelial to a hematopoietic transition. BMP4 and Hedgehog affect their production and expansion, but it is unknown whether they act to affect the same HSCs. In this study using the BRE GFP reporter mouse strain that identifies BMP/Smad-activated cells, we find that the AGM harbors two types of adult-repopulating HSCs upon explant culture: One type is BMP-activated and the other is a non-BMP-activated HSC type that is indirectly controlled by Hedgehog signaling through the VEGF pathway. Transcriptomic analyses demonstrate that the two HSC types express distinct but overlapping genetic programs. These results revealing the bifurcation in HSC types at early embryonic stages in the AGM explant model suggest that their development is dependent upon the signaling molecules in the microenvironment