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    • Genome wide association studies GWASs represent

    2018-10-24

    Genome-wide association studies (GWASs) represent another unbiased approach that has proven particularly successful in humans for gene discovery of numerous disease phenotypes (Hindorff et al., 2015). Encouraged by this success, GWASs also have been proposed in mice using a recently developed panel of classic inbred and RI mouse strains, termed the Hybrid Mouse Diversity Panel (HMDP). Our prior studies with the HMDP have identified key associations for a variety of complex traits that are highly relevant to human diseases, which collectively illustrate the power of this approach for gene discovery in mice (Bennett et al., 2010, 2012; Farber et al., 2011; Ghazalpour et al., 2011, 2012, 2014; Park et al., 2011; Orozco et al., 2012; Davis et al., 2013; Parks et al., 2013; Hartiala et al., 2014). In the present study, we used this mouse platform to carry out a comprehensive genetic screen for HSPC frequency in the adult BM. We identified multiple loci associated with the frequencies of three HSPC populations and functionally validated one positional candidate gene that previously had not been known to play a role in the hematopoietic system.
    Results
    Discussion Using the strain survey data, we also carried out a GWAS analysis and identified multiple distinct and overlapping loci that were significantly or suggestively associated with HSPC sub-populations. At one particular locus on chromosome 5, we provide strong evidence that LSKCD150−CD48− cell frequency and function is regulated by Hopx, a gene that was previously not known to influence HSPC biology. The collective results of the functional validation studies with Hopx−/− mice and competitive transplantation assays demonstrated that Hopx deficiency led to a specific and intrinsic functional defect in the MPP subset of HSCs (LSKCD150−CD48−), possibly related to cell-cycle status, but not in the most primitive long-term subset of igf ir or more committed progenitor populations. These observations were also entirely consistent with the gene expression analyses, showing that Hopx mRNA levels in the BM were positively correlated with LSKCD150−CD48− cells, and the GWAS results, where the Hopx locus was only associated with this HSPC subset and not with LSKs or LSKCD150+CD48− cells. Of direct relevance to our findings, a recent study demonstrated how different HSC populations, and LSKCD150−CD48− cells in particular, play distinct temporal roles in the maintenance of the hematopoietic system (Busch et al., 2015). More specifically, it is the LSKCD150−CD48− sub-population of cells that sustains adult hematopoiesis under homeostatic conditions through multiple rounds of self-renewal. Furthermore, proliferation of this cell population accounts for the differentiation of committed progenitors, while only receiving limited input from the more primitive LSKCD150+CD48− sub-population of cells. Hopx is a homeodomain-only protein that lacks DNA-binding activity yet is predominantly localized to the nucleus (Chen et al., 2002; Shin et al., 2002). There are many different functional roles proposed for this protein depending on the cell type examined. Through direct protein-protein interactions, Hopx was shown to repress the expression of serum response factor (SRF)-dependent genes (Kook and Epstein, 2003), and conditional deletion of Srf in mouse BM leads to an approximate 3-fold increase in the frequency of all HSPC sub-populations (Ragu et al., 2010). Therefore, one plausible mechanism for why Hopx deficiency leads to decreased numbers of LSKCD150−CD48− cells could be as a result of increased Srf-mediated transcription of downstream targets. Alternatively, Hopx may affect HSPC frequency through its effects on cell-cycle status. For example, we demonstrated that Hopx−/− mice have a decreased proportion of LSKCD150−CD48− cells in the quiescent G0 state and increased numbers in the G1 phase. Other groups have shown that Hopx promoter methylation frequently is observed in many malignancies, including esophageal, gastric, pancreatic, and colorectal cancers, where it has been suggested that Hopx functions as a tumor suppressor gene (Yamashita et al., 2013). Since it is known that deficiency of a number of genes in the hematopoietic system leads to increased entry into cell cycle, with a consequent loss of HSPC frequency and function (Orford and Scadden, 2008), it is possible that perturbation of Hopx expression/activity affects the intrinsic homeostasis of HSPCs by altering cellular quiescence and, thus, cell frequency.