Abstract
Nucleosome assembly proteins (NAPs) are histone chaperones with an important role in chromatin structure and epigenetic regulation of gene expression. We find that high gene expression levels of mouse Nap1l3 are restricted to haematopoietic stem cells (HSCs) in mice. Importantly, with shRNA or CRISPR-Cas9 mediated loss of function of mouse Nap1l3 and with overexpression of the gene, the number of colony-forming cells and myeloid progenitor cells in vitro are reduced. This manifests as a striking decrease in the number of HSCs, which reduces their reconstituting activities in vivo. Downregulation of human NAP1L3 in umbilical cord blood (UCB) HSCs impairs the maintenance and proliferation of HSCs both in vitro and in vivo. NAP1L3 downregulation in UCB HSCs causes an arrest in the G0 phase of cell cycle progression and induces gene expression signatures that significantly correlate with downregulation of gene sets involved in cell cycle regulation, including E2F and MYC target genes. Moreover, we demonstrate that HOXA3 and HOXA5 genes are markedly upregulated when NAP1L3 is suppressed in UCB HSCs. Taken together, our findings establish an important role for NAP1L3 in HSC homeostasis and haematopoietic differentiation.
Highlights
Nucleosome assembly proteins (NAPs) are histone chaperones with an important role in chromatin structure and epigenetic regulation of gene expression
Given that high levels of Nap1l3 gene expression were restricted to haematopoietic stem cells (HSCs) (Fig. 1b), we investigated the functional importance of Nap1l3 in haematopoietic stem and progenitor cells (HSPCs) differentiation and proliferation of primitive murine bone marrow cells (BM) cells by shRNA-mediated loss of function studies
The shRNA knockdown vectors were introduced by lentiviral transfer to lineage markers (Lin)−Sca+cKit+ (LSK) HSPCs sorted from lineage depleted BM cells. shRNA-mediated downregulation of Nap1l3 resulted in a significant reduction (~85%) in mRNA levels compared to the negative scramble control vector expressing scrambled shRNA (Fig. 1c)
Summary
Nucleosome assembly proteins (NAPs) are histone chaperones with an important role in chromatin structure and epigenetic regulation of gene expression. With shRNA or CRISPR-Cas[9] mediated loss of function of mouse Nap1l3 and with overexpression of the gene, the number of colony-forming cells and myeloid progenitor cells in vitro are reduced This manifests as a striking decrease in the number of HSCs, which reduces their reconstituting activities in vivo. The nucleosome assembly proteins (NAP) represent a family of evolutionarily conserved histone chaperones consisting of five members in mammals, having first been identified in mammalian cells[3] These histone chaperones are thought to facilitate the import of H2A–H2B histone dimers from the cytoplasm to the nucleus[4,5] and to regulate chromatin dynamics by catalysing the assembly or disassembly of nucleosomes[4,6,7,8,9]. We delineate the key transcriptional and signalling pathways underlying the role of NAP1L3 in haematopoiesis
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