br Experimental Procedures br Author Contributions br Acknow
Acknowledgments We wish to thank Nicole Varain and Donna Colon for their excellent technical support assisting with the fetal sheep transplants. This work was supported by NIH, NHLBIR01HL097623, and the Intramural Pilot Program of Wake Forest School of Medicine.
Introduction All myeloid and lymphoid blood cell lineages are continually replenished throughout adult life from a reservoir of rare multipotent hematopoietic stem l-name manufacturer (HSC) residing in the bone marrow. Studies in both humans and mice have shown that HSC are not constant throughout life (Chambers et al., 2007; Dykstra et al., 2011; Flach et al., 2014; Kuranda et al., 2011; Lescale et al., 2010; Pang et al., 2011; Rossi et al., 2005). There is an increase in the number of phenotypically defined HSC with age, but the stem cells that accumulate exhibit a diminished long-term reconstitution potential as well as a cell-intrinsic reduction in their capacity to generate immune-competent B lymphocytes, leading to a myeloid-biased differentiation output. This age-associated skewing of HSC differentiation potential from lymphoid to myeloid lineages, and the resultant decreased output of naive B cells, leads to a decline in antibody diversity and is believed to contribute to the general depletion of immune function observed in the elderly (reviewed in Denkinger et al., 2015). HSC aging is driven by both cell-extrinsic alterations in the stem cell niche and systemic signals, as well as changes intrinsic to the stem cells themselves (reviewed in Garrick et al., 2015; Geiger et al., 2013), including widespread changes in gene-expression patterns (Chambers et al., 2007; Flach et al., 2014; Pang et al., 2011; Rossi et al., 2005; Sun et al., 2014). While the molecular triggers for these transcriptomic and functional changes are still incompletely understood, recent studies in mouse HSC have demonstrated that aging is associated with alterations in the DNA methylation and histone modification profiles (Beerman et al., 2013; Sun et al., 2014), suggesting that disruption of the normal epigenetic state is an important factor in the aging HSC phenotype. One key component of the epigenetic landscape is the formation of domains of heterochromatin. These regions of compacted and transcriptionally repressive chromatin are critical for diverse aspects of nuclear biology, including the regulation of gene-expression patterns, the transcriptional silencing of genomic repeats, and the maintenance of genome stability, as well as normal centromere and telomere function (Bulut-Karslioglu et al., 2014; Grewal and Jia, 2007; Peters et al., 2001; Schoeftner and Blasco, 2009). One of the principal enzymes involved in the formation of heterochromatin is SUV39H, a family of two histone methyltransferases (SUV39H1/KMT1A and SUV39H2/KMT1B) that catalyze tri-methylation of lysine 9 of histone H3 (H3K9me3) (Peters et al., 2001). The H3K9me3 histone modification is recognized and bound by members of the heterochromatin protein 1 (HP1) family (Lachner et al., 2001), critical adaptor proteins that coordinate chromatin compaction by undergoing self-association as well as recruiting histone deacetylases, DNA methyltransferases, and structural RNAs (reviewed in Maison and Almouzni, 2004). Consistent with a crucial role for heterochromatin during differentiation and development, it has been shown that SUV39H1-mediated H3K9me3 regulates lineage commitment during early mouse development by repressing lineage-inappropriate genes (Alder et al., 2010) and that depletion of SUV39H gives rise to pre- and postnatal developmental defects and lethality in mice (Peters et al., 2001). Accumulating evidence suggests that SUV39H may also regulate various aspects of hematopoiesis. The SUV39H1/HP1 regulatory axis is important to maintain cellular fate following commitment to the T helper 2 (TH2) lymphocyte lineage (Allan et al., 2012). Further, deletion of Suv39h in mice leads to the development of late-onset B cell lymphomas (Peters et al., 2001), while overexpression of SUV39H leads to impaired erythroid differentiation (Czvitkovich et al., 2001). However, at present the role of SUV39H and heterochromatin structure in HSC aging and the regulation of differentiation potential has not been investigated directly.