Repression of p53-target gene Bbc3/puma by MYSM1 is essential for the survival of hematopoietic multipotent progenitors and contributes to stem cell maintenance

MYSM1 is a transcriptional regulator essential for HSC function and hematopoiesis. We established that p53 activation is a common mechanism mediating HSC dysfunction, MPP depletion, and lymphopenia in Mysm1-deficiency, however, the specific p53-induced effectors that trigger dysfunction in Mysm1-deficient HSPCs remain unknown. Here, we performed RNA-Seq of Lin-cKit+Sca1+CD150+ and CD150- HSPCs from Mysm1-/-Puma-/-, Mysm1-/-Puma+/-, Puma-/-, and wild-type mice; (Mysm1-/-Puma+/- phenocopies Mysm1-/-). We showed that Bbc3/PUMA is the primary non-redundant mediator of MPP depletion in Mysm1-deficiency and contributes to HSC dysfunction, whereas depletion of lymphoid-lineage cells involves PUMA-independent p53 activities. We also identified a broad downregulation of genes encoding protein components of the ribosome (RP-genes) and other regulators of translation in Mysm1-deficiency, and the downregulation persisted in Mysm1-/-Puma-/-. We performed RNA-Seq transcriptional profiling of FACS-sorted primary hematopoietic stem and progenitor cells. Briefly, bone marrow cells were harvested from Mysm1-/-Puma-/-, Mysm1-/-Puma+/-, Puma-/-, and wild-type mice; (Mysm1-/-Puma+/- phenocopies Mysm1-/-), and sorted using FACS into primary HSPCs, gating on Lin-cKit+Sca1+CD150+ and CD150-. 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). RNA (10 ng) was rRNA-depleted and used for library preparation with the SMARTer Stranded RNA-Seq kit (Takara Clontech, Mountain View, CA, USA) designed for low input. The RNA-seq libraries were sequenced on an Illumina HiSeq 2000 sequencer in a paired-end 50-bp configuration.