Project description:The formation of hematopoietic cells relies on the chromatin remodeling activities of ISWI ATPase SMARCA5 (SNF2H) and its complexes. The Smarca5 null and conditional alleles have been used to study its functions in embryonic and organ development in mice. These mouse model phenotypes vary from embryonic lethality of constitutive knockout to less severe phenotypes observed in tissue-specific Smarca5 deletions, e.g., in the hematopoietic system. Here we show that, in a gene dosage-dependent manner, the hypomorphic allele of SMARCA5 (S5tg) can rescue not only the developmental arrest in hematopoiesis in the hCD2iCre model but also the lethal phenotypes associated with constitutive Smarca5 deletion or Vav1iCre-driven conditional knockout in hematopoietic progenitor cells. Interestingly, the latter model also provided evidence for the role of SMARCA5 expression level in hematopoietic stem cells, as the Vav1iCre S5tg animals accumulate stem and progenitor cells. Furthermore, their hematopoietic stem cells exhibited impaired lymphoid lineage entry and differentiation. This observation contrasts with the myeloid lineage which is developing without significant disturbances. Our findings indicate that animals with low expression of SMARCA5 exhibit normal embryonic development with altered lymphoid entry within the hematopoietic stem cell compartment.
Project description:In the mouse embryo during midgestation, hematopoietic stem cells (HSCs) are generated from aortic endothelium through the transdifferentiation process known as endothelial-to-hematopoietic transition, or EHT. During EHT, flat-shaped hemogenic endothelium transform into round-shaped hematopoietic cells, which subsequently form cell aggregates, called hematopoietic clusters. To understand HSC specification during ontogeny and to search for nascent HSC markers, we performed single-cell microarray analysis of developing HSC populations.
Project description:Cullin proteins are scaffolds that coordinate assembly of cullin-RING E3 ubiquitin (Ub) ligases (CRL), complexes that control post-translational ubiquitin modification and degradation of cellular proteins. Cullin-5 (Cul5) coordinates assembly of CRL complexes containing the RING E3 ligase Rbx1/2, the adapter proteins Elongins B and C, and a Suppressor of Cytokine Signalling (SOCS) box-containing substrate recognition protein. To explore potential roles for Cul5, we generated mice lacking Cul5 in the in hematopoietic system. Analyses included biological and molecular studies including proteomic analysis of differential expression of proteins in primary purified hematopoietic stem/progenitor (LSK) cells lacking Cul5.
Project description:DNA methylation is essential for mammalian development and plays crucial roles in a variety of biological processes. The DNA methyltransferase Dnmt1 serves to maintain parental cell methylation patterns on daughter DNA strands in mitotic cells, however, the precise role of Dnmt1 in regulation of quiescent adult stem cells is not known. To examine the role of Dnmt1 in adult hematopoietic stem cells (HSCs), we conditionally disrupted Dnmt1 in the hematopoietic system. We used microarrays to profile the global gene expression program in hematopoietic stem and progenitor cells following deletion of Dnmt1.
Project description:Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) characterized by hyper-proliferation of the erythroid, megakaryocytic and granulocytic lineages and the presence of an activating mutation in JAK2. To elucidate mechanisms that regulate PV stem cells, we applied a newly developed data-independent acquisition (DIA) mass spectrometry (MS) technology to purified hematopoietic stem and progenitor cell (HSPC) subpopulations of patients with chronic and progressed PV. Proteomic analyses were supplemented by RNA-sequencing (RNA-seq) and identified targets validated by flow cytometry and functional in vitro assays.