Epigenetic regulation of hematopoietic stem cell (HSC) and progenitor cell fate decisions
It is becoming increasingly clear that epigenetic regulation (through DNA methylation, histone modifications, and chromatin remodeling) is an important component dictating HSC and progenitor cell fate decisions. In the hematopoietic system, many lineage-restricted promoters are associated with specific, combinatorial histone modification patterns, which may determine their selective priming of gene expression during lineage commitment. We are using functional screening approaches, in vitro and in vivo, to identify key epigenetic regulators that drive lineage commitment steps during early hematopoietic differentiation. Validated regulators will be pursued by complete in vivo characterization using mouse conditional knockout or overexpression models, and molecular characterization using ChIP-Seq, RNA-Seq, bisulfite sequencing, and other emerging technologies to interrogate epigenetic changes within rare cell populations. In addition, we have begun to study how these mechanisms influence hematopoietic lineage commitment during aging.
DNA methylation patterning of HSC and leukemia stem cell (LSC) self-renewal
DNA methylation, one of the most common epigenetic modifications highly deregulated in leukemias and other cancers, is established and maintained by three major DNA methyltransferase enzymes (Dnmt1, Dnmt3a, and Dnmt3b). Our recent work has demonstrated that HSCs and LSCs depend on distinct levels of DNA methylation mediated by Dnmt1. Conditional knockout of Dnmt1 in the hematopoietic system in mice significantly impaired the ability of HSCs to self-renew and differentiate (Trowbridge et al., Cell Stem Cell 2010). In LSCs, however, loss of only one copy of Dnmt1 was sufficient to impair leukemia formation in a mouse model of MLL-AF9-induced acute myeloid leukemia (AML) (Trowbridge et al., PNAS 2012). Normal HSCs were not impaired by loss of one copy of Dnmt1, indicating that there is an increased requirement for Dnmt1 in LSCs and leukemia cells. We have extended these findings to a second model of hematopoietic malignancy, adult peripheral T cell lymphoma, demonstrating that loss of only one copy of Dnmt1 is sufficient to impair lymphomagenesis. Together, this work suggests that DNA methylation mediated by Dnmt1 is a relevant mechanism regulating hematopoietic malignancy.
To further investigate the cellular and molecular basis underlying the differential requirement for Dnmt1 expression in HSCs and LSCs, we are characterizing the changes that occur in both global DNA methylation patterns and gene expression patterns of HSCs and LSCs upon loss or heterozygosity of Dnmt1. Global DNA methylation patterns are characterized by a state-of-the-art technique termed reduced representation bisulfite sequencing (RRBS). The elucidation of both global methylation patterns and gene expression patterns rely upon next-generation high-throughput sequencing technology. In addition, we have performed a large-scale co-immunoprecipitation and mass spectrometry-based screen to identify novel interacting proteins of Dnmt1 in leukemia cells. Our top candidates will be further probed using conditional knockout mouse models or knockdown strategies to evaluate their importance in HSC and LSC function. Using this strategy, we strive to understand how Dnmt1 may be uniquely targeted to DNA, and discover the breadth of cellular mechanisms that may be regulated by Dnmt1 in HSCs and LSCs.
Influence of the microenvironment on epigenetic patterns in HSCs
The local microenvironment or “niche” within bone marrow is of critical importance to supporting proper function of HSCs. While a significant amount of effort has been devoted to defining the specific cell types and growth factors that comprise the HSC niche, not much is known about how the signals received by HSCs are integrated at the chromatin level to generate an appropriate transcriptional response. It is becoming increasingly clear that epigenetic modifications of chromatin and DNA are important components of signal integration and cellular “memory”. We are currently focused on defining the epigenetic changes that occur in HSCs in response to activation of developmentally important signaling pathways, such as Wnt, Notch and Hedgehog. To determine the type of epigenetic changes that are critical determinants of HSC self-renewal in response to activation of these pathways, we are performing screens using small molecule inhibitors of the enzymes or “writers” that establish various epigenetic modifications, including histone lysine methylation, acetylation, and DNA methylation. Specifically, we are evaluating whether HSC self-renewal or differentiation, as would normally occur in response to activation of these signaling pathways, is altered when the epigenetic machinery is not able to appropriately respond to these environmental signals. This experimental approach will provide novel insight into our understanding of how HSCs integrate and respond to extracellular signals, and how these signals establish cellular memory that is critical for life-long HSC self-renewal and sustained hematopoiesis.