DNA damage is emerging to be a key mediator of age-associated chromatin reorganization, promoting many of the epigenetic changes that correlate with, or mimic, transcriptional deregulation observed with age11. The (histone) deacetylase SIRT1 was shown to be critically involved in several aspects of the DNA damage response as well as in the transcriptional regulation of a variety of key developmental regulators, placing it at the crossroads of DNA damage and epigenetic gene regulation. Consistent with this notion, we have shown previously that SIRT1 redistributes on chromatin from certain promoters and repetitive DNA to sites of DNA breaks, resulting in gene expression changes at ostensibly undamaged genomic loci that are normally regulated by SIRT112. While the resulting chromatin reorganization may at least in part explain age-associated gene deregulation, functional consequences of this process remain to be indentified. To study the role of SIRT1 in epigenomic maintenance, we thus turned to a physiological system that displays a well-established link between DNA damage and epigenomic changes, the hematopoietic stem and precursor cell (HSPC) compartment. Work from Weisman and colleagues showed that various types of DNA damage could severely impair stem cell maintenance, predominantly by reducing self-renewal capacity. Moreover, HSPCs accumulate DNA damage during normal aging and harbor characteristic gene expression changes that may ultimately affect HSPC function. However, it is unclear how DNA damage triggers HSPC exhaustion and if the observed gene expression changes are functionally related. Supporting a role for SIRT1 in the epigenetic (de)regulation of stem cell fate, SIRT1 was shown to be part of a novel polycomb repressive complex, PRC4, which has distinctive chromatin modifying activity and is expressed specifically in undifferentiated cells including embryonic stem cells. Polycomb Group (PcG)-mediated gene regulation is known to play a critical role in HSPC maintenance as loss of the PcG component Bmi-1 ablates HSPC development. Together these observations call for an in depth investigation of SIRT1 function in hematopoietic stem cells. RESULTS AND FUTURE DIRCTIONS: To test the hypothesis that SIRT1 is a critical (epigenetic) mediator of HSPC maintenance and function, we generated a mouse model that allows for the inducible ablation of SIRT1 gene expression in adult animals, thereby avoiding perinatal lethality and/or possible compensation observed in SIRT1-deficient mice. We found that, upon inactivation of SIRT1, the HSPC compartment undergoes an initial phase of proliferative expansion, followed by increased DNA damage and an eventual dramatic depletion in the long-term hematopoietic stem cell compartment. These data demonstrate an important role for SIRT1 in the maintenance of HSPC quiescence as well as the regulation of DNA damage accumulation. We then sought to investigate the mechanism by which SIRT1 mediates these effects, focusing on SIRT1-dependent changes in maintenance of genomic integrity and gene expression. Using microarray analysis in sorted HSPCs, we found that, out of a group of 11 Gene Ontology annotated genes associated with definitive hematopoiesis, a single candidate gene was strongly upregulated in the absence of SIRT1. This gene was previously shown to promote HSPC expansion and has further been linked to acute myelogenous leukemia (AML), a cancer derived from aberrantly proliferating hematopoietic progenitor cells. Using chromatin immunoprecipitation (ChIP) we found that Sirt1 binds to the this gene at the DNA level and that Sirt1 enrichment is correlated with the polycomb-group (PcG) repressive mark H3K27 (3Me), provinding the first evidence that Sirt1 may modulate PcG associated developmental genes to promote the maintenance of stem cells. Future work will dissect the molecular basis for this observation using depletion of this SIRT1 target gene by RNAi as well as a detailed analysis of theo role of PRC4 in this process. To address the functional consequences of HSPC expansion/exhaustion associated with loss of SIRT1, we are currently performing both competitive and (serial) non-competitive bone marrow reconstitution experiments. IMPLICATIONS: Our preliminary data suggest an important role for SIRT1 in HSPC maintenance, where loss of SIRT1 promotes HSPC expansion and eventually exhaustion corroborating the importance of SIRT1 as a regulator of developmental programs. The tight regulation of HSPC proliferation is a critical aspect of mammalian development and does not only affect the generation of normal hematopoietic cells but is closely linked to tumors of the hematopoietic lineage. Both acute and chronic myeloid leukemia arise from aberrant proliferation of HSPCs and a better understanding of the underlying regulators is critical to design targeted therapy.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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