Chromatin accessibility and cell cycle progression are controlled by the HDAC-associated Sin3B protein in murine hematopoietic stem cells

Blood homeostasis requires the daily production of millions of terminally differentiated effector cells that all originate from hematopoietic stem cells (HSCs). HSCs are rare and exhibit unique self-renewal and multipotent properties, which depend on their ability to maintain quiescence through ill-defined processes. Defective control of cell cycle progression can eventually lead to bone marrow failure or malignancy. In particular, the molecular mechanisms tying cell cell re-entry to cell fate commitment in HSCs remain elusive. Previous studies have identified chromatin coordination as a key regulator of differentiation in embryonic stem cells. Here, we utilized genetic inactivation of the chromatin-associated Sin3B protein to manipulate cell cycle control and found dysregulated chromatin accessibility and cell cycle progression in HSCs. Single cell transcriptional profiling of hematopoietic stem and progenitor cells (HSPCs) inactivated for Sin3B reveals aberrant progression through the G1 phase of the cell cycle, which correlates with the engagement of specific signaling pathways, including aberrant expression of cell adhesion molecules and the interferon signaling program in LT-HSCs. In addition, we uncover the Sin3B-dependent accessibility of genomic elements controlling HSC differentiation, which points to cell cycle progression possibly dictating the priming of HSCs for differentiation. Our findings provide new insights into controlled cell cycle progression as a potential regulator of HSC lineage commitment through the modulation of chromatin features. To investigate the function of SIn3B on murine hematopoiesis, conditional knockouts (cKO) of Sin3B in the hematopoietic system were generated by crossing Sin3B-flox mice with Vav1-iCre mice on a C57BL/6 background. Two cKO mice and two wild type mice were humanely sacrificed as adults, and bone marrow was isolated and subjected to cKit enrichment with magnetic beads. Lineage-cKit+Sca-1+ cells were then sorted by FACS and then samples from each genotype were pooled. Cells were stained with TotalSeq Hashtag Oligonucleotide-antibodies against CD45 (BioLegend). Cells were then washed and the 10X Genomics Chromium Single Cell 3' Kit was used to generate single cell suspension and cDNA synthesis/library preparation. Libraries were sequnced on an Illumina NovaSeq and demultiplexed using 10X Genomics CellRanger software. For stress conditions, two mice of the same genotypes as above were given 5-Fluorauracil (100mg/kg) 9 days before harvest.