Project description:Age-related functional decline of hematopoietic stem cells (HSC) contributes to the decline of the hematopoietic system. Growth Arrest and DNA Damage-inducible proteins (Gadd45a, Gadd45b, and Gadd45g) are robustly expressed early in HSC activation and are known to be important for DNA damage response. Gadd45b has also been shown to promote removal of 5-methylcytosine at specific loci. Given that both DNA methylation (DNAm) alterations and DNA damage have been associated with HSC aging, we sought to examine the role of Gadd45b in HSCs. To examine if Gadd45b is critical for regulating DNAm in HSCs, we performed whole genome bisulfite sequence and RNA sequencing on wild-type (WT) and Gadd45b knock-out (KO) HSCs isolated from young and old mice. Global DNAm of young KO showed increased DNAm compared to young WT, and the DNAm profiles were more similar to old HSCs, from either WT and KO, than to young WT. We examined the DNAm changes common between the Gadd45b KO (YKOvsYWT) or WT aging (OWTvsYWT). 52.5% of differentially methylated cytosines (DMCs) in YKO were also seen in normal aging, and analysis of genes with promoters near these overlapping DMCs are enriched for pathways related to immune response and development processes. We compared the expression of genes with DMCs in the promoter regions, but DMCs did not strongly correspond with transcriptional alterations. To test if these overlapping DNAm changes in young KO mice primed HSCs towards aging phenotypes, we evaluated the composition of peripheral blood and bone marrow in KO, and performed functional evaluation experiments. We found no significant differences between young KO and WT HSCs suggesting these overlapping DMCs do not contribute to aging phenotypes, reducing the potential DNAm changes contributing to aging HSC phenotypes.
Project description:Recent work from others and us revealed interactions between the Sin3/HDAC complex, the H3K4me3 demethylase KDM5A, GATAD1 and EMSY. Here, we characterize the EMSY/KDM5A/SIN3B complex in detail by quantitative interaction proteomics and ChIP-sequencing. We identify a novel substoichiometric interactor of the complex, ZNF131, which recruits EMSY to a large number of active, H3K4me3 marked promoters. Interestingly, using an EMSY knock-out line and subsequent rescue experiments, we show that EMSY is in most cases positively correlated with transcriptional activity of its target genes and stimulates cell proliferation. Finally, by immunohistochemical staining of primary breast tissue microarrays we find that EMSY/KDM5A/SIN3B complex subunits are frequently overexpressed in primary breast cancer cases in a correlative manner. Taken together, these data open venues for exploring the possibility that sporadic breast cancer patients with EMSY amplification might benefit from epigenetic combination therapy targeting both the KDM5A demethylase and histone deacetylases.
Project description: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.
Project description: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.