Microrna-130a Regulates Hematopoietic Stem Cell Self-Renewal By Repressing Chromatin Modifiers and Shaping the Accessible Chromatin Landscape

Background: Residing at the apex of the blood system hierarchy, hematopoietic stem cells (HSCs) are endowed with multi-potency and self-renewal potential. Hematopoietic homeostasis is tightly regulated by controlling the balance between quiescence, self-renewal and lineage-commitment of HSCs. Although many studies have profiled gene expression patterns and epigenomes of HSC and downstream progenitors, post-transcriptional regulation of determinants that control these regulatory networks is largely unknown. MicroRNAs (miRNAs) represent a large class of post-transcriptional regulators that mediate repression of multiple target mRNAs by inhibiting their translation and/or inducing their degradation. A limited number of reports suggest that miRNAs are differentially expressed across the hematopoietic hierarchy and control lineage commitment and cell fate decisions by orchestrating gene regulatory networks, however the mechanisms remain unexplored.

Methods: To identify miRNA(s) that play a functional role in human hematopoiesis, we performed an in vivo competitive repopulation screen in which candidate miRNAs were over-expressed (OE) in human CD34⁺CD38^- umbilical cord blood (CB) cells and subsequently transplanted into immune-deficient mice for 24 weeks. miR-130a was shown to enhance long-term hematopoietic reconstitution and chosen for further investigation.

Results: As miRNAs are negative regulators of gene expression, we studied the functional impact of miR-130a on long-term hematopoietic reconstitution by enforcing its expression in CB cells using lentiviral vector containing orange fluorescent protein (OFP) reporter. At 12 and 24 weeks after transplantation, increased miR-130a expression conferred a statistically significant, competitive advantage to transduced CB cells demonstrated by increased human chimerism and the proportion of OFP⁺/hCD45⁺ cells in the injected femur (IF), bone marrow (BM) and spleen of recipient mice. Xenografts produced by miR-130 O/E showed multi-lineage engraftment with myeloid skewing at the expense of B-lymphoid development and significantly enhanced erythroid output in RF, BM and spleen. In addition, ectopic expression of miR-130a caused splenomegaly in recipient mice. Flow cytometry analysis using several markers expressed during erythroid development revealed accumulation of immature GlyA⁺/CD71⁺/CD36⁺ erythroid progenitors, suggesting an erythroid differentiation block. Enforced expression of miR-130a also perturbed myeloid differentiation shown by the presence of abnormal CD14⁺/CD66b⁺ myeloid cells in the BM. At the primitive and progenitor cell stages, miR-130a O/E caused significant expansion of primitive CD34⁺/CD38^- cells and increased the proportion of immuno-phenotypic HSC. Secondary transplantation involving limited dilution analysis revealed 10-fold increase in HSC frequency, consistent with a role of miR-130a in HSC self-renewal. Analysis of chromatin accessibility surrounding the miR-130a locus across the human hematopoietic hierarchy revealed peaks of accessible chromatin in HSC and downstream progenitors that were absent in mature cells. To ascertain the molecular mechanism of miR-130a function, label-free semi-quantitative proteomics was performed to determine differentially expressed proteins between miR-130a O/E and control-transduced CD34⁺ CB cells. Gene set enrichment analysis (GSEA) identified top miR-130a downregulated gene sets centered on chromatin remodelling. Components of SMRT/N-CoR co-repressor complex and polycomb repressive complex (PRC2) were identified to be among the top downregulated miR-130a targets. We assessed the impact of miR-130a O/E on the global chromatin accessibility landscape by performing ATAC-seq on CD34⁺ CB cells transduced with miR-130a or control lentivirus. Enforced expression of miR-130a resulted in a gain of approximately 450 accessible chromatin peaks. Transcription factor DNA recognition motif analysis revealed significant enrichment of GATA3 motif in accessible sites specific to miR-130a O/E cells.

Conclusion: Together, our data suggests that miR-130a regulates HSC self-renewal and lineage specification. miR-130a mediates repression of several gene networks centered on chromatin remodelling and focally reshapes the accessible chromatin landscape of HSPC.