MYSM1 maintains ribosomal protein gene expression in hematopoietic stem cells to prevent hematopoietic dysfunction II.

MYSM1 is a transcriptional regulator essential for HSC function and hematopoiesis. We established that HSC dysfunction in Mysm1-deficiency is driven by p53 stress response, however, the molecular function of MYSM1 as a transcriptional activator and its essential role in p53 stress response repression remain difficult to reconcile. Here, we performed genome-wide analyses of MYSM1-regulated genes in hematopoietic stem and progenitor cells (HSPCs). This included RNA-Seq of sorted Mysm1-deficient mouse HSCs and MPPs, and ChIP-Seq mapping of MYSM1 DNA-binding sites in hematopoietic progenitor cell lines. We demonstrate a direct role for MYSM1 in the regulation of genes encoding protein components of the ribosome (RP-genes) and other regulators of translation. Mechanistically, the dysregulation of RP-genes in Mysm1-deficiency was upstream of p53-activation and associated with reduced HSCs protein synthesis rates and p53-dependent anemia. We performed RNA-Seq transcriptional profiling of FACS-sorted primary HSC, MPP1, and MPP2 cells. Briefly, bone marrow cells were harvested from Mysm1 fl/fl CreERT2 mice following tamoxifen-induced Mysm1-deletion, and from tamoxifen-treated Mysm1 fl/+ CreERT2 and corn-oil vehicle-treated Mysm1 fl animals. The cells were sorted using FACS into primary HSC, MPP1, and MPP2 cells, gating on Lin-cKit+Sca1+CD150+Flt3-, and CD34-CD48- for HSCs, CD34+CD48- for MPP1, and CD34+CD48+ for MPP2 cells. RNA was isolated from the FACS-sorted cells using the MagMAX total RNA kit (Ambion, Life Technologies), and quality assessed using Bioanalyzer RNA Pico chips (Agilent). rRNA depletion and library preparation were performed using the SMARTer Stranded RNA-Seq kit (Takara Clontech). The libraries were sequenced on an Illumina HiSeq 2000 sequencer in paired-end 50bp configuration. Cells from multiple mice were pooled to achieve yields of >1ng of RNA per sample, and three independent samples were analyzed per genotype for each cell-type for tamoxifen-induced mice and two independent samples for vehicle-treated mice.