ALBERT

Description: The transcription factor signal transducers and activators of transcription 5 (STAT5) is activated by numerous cytokines that orchestrate blood cell development. Multilineage peripheral blood cytopenias were observed in adult mice lacking both isoforms of STAT5 (STAT5A and STAT5B) as well as accelerated rates of apoptosis in the bone marrow. Although the hematopoietic stem cell (HSC) population was preserved in a number of these mice, the post-HSC progenitor populations were diminished and a marked reduction in functional progenitors (spleen colony-forming units) was detected. Competitive bone marrow transplantation studies in vivo revealed a profound impairment of repopulation potential of STAT5-null HSCs, leading to complete lack of contribution to the myeloid, erythroid, and lymphoid lineages. These abnormalities were associated with heightened proliferation activity in the HSC fraction, suggesting the action of homeostatic mechanisms to maintain sufficient levels of diverse blood cell types for viability. Thus, STAT5 normally sustains the robust hematopoietic reserve that contributes to host viability through crucial survival effects on early progenitor cells.

Description: Master transcriptional regulators of development often function through dispersed cis elements at endogenous target genes. While cis-elements are routinely studied in transfection and transgenic reporter assays, it is challenging to ascertain how they function in vivo. To address this problem in the context of the locus encoding the critical hematopoietic transcription factor Gata2, we engineered mice lacking a cluster of GATA motifs 2.8 kb upstream of the Gata2 transcriptional start site. We demonstrate that the −2.8 kb site confers maximal Gata2 expression in hematopoietic stem cells and specific hematopoietic progenitors. By contrast to our previous demonstration that a palindromic GATA motif at the neighboring −1.8 kb site maintains Gata2 repression in terminally differentiating erythroid cells, the −2.8 kb site was not required to initiate or maintain repression. These analyses reveal qualitatively distinct functions of 2 GATA motif-containing regions in vivo.

Description: Abstract 392 DNA methylation is essential for 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 crossed Dnmt1fl/fl mice with Mx1-Cre transgenic mice, and by injection of poly(I)-poly(C) we selectively deleted Dnmt1 in the hematopoietic system (Dnmt1Δ/Δ). In Dnmt1Δ/Δ mice, peripheral blood counts and mature multilineage composition of the bone marrow was found to be normal. Interestingly, specific defects were observed in Dnmt1Δ/Δ HSC self-renewal as assessed by long-term and secondary competitive transplantation, in retention of Dnmt1Δ/Δ HSCs within the bone marrow niche, and in the ability of Dnmt1Δ/Δ HSCs to give rise to multilineage hematopoiesis. Loss of Dnmt1 also had unique impact on myeloid progenitor cells (including common myeloid progenitors, granulocyte-macrophage progenitors, and megakaryocyte-erythrocyte progenitors), regulating their cycling and transcriptional lineage fidelity. To determine the molecular mechanisms underlying these defects, we performed global gene expression microarray analysis and bisulfite sequencing of select loci (IAP, Car1, and Gata1) in purified populations of control and Dnmt1Δ/Δ long-term HSCs, short-term HSCs/multipotent progenitor cells, and myeloid restricted progenitor cells. Through this approach, we demonstrate that loss of Dnmt1 has cell type-specific molecular consequences. For example, demethylation of the Car1 and Gata1 loci in Dnmt1Δ/Δ long-term HSCs is not sufficient to activate gene transcription, whereas demethylation of these genes in Dnmt1Δ/Δ short-term HSCs is associated with activation of transcription. In Dnmt1Δ/Δ myeloid restricted progenitor cells, we observed increases in DNA methylation at specific gene loci such as Car1, indicating that methylation can be established by other methyltransferases in the absence of Dnmt1. Our global gene expression microarray analysis clearly demonstrates that Dnmt1 regulates expression of distinct gene families in these closely related, primitive hematopoietic populations. We were unable to attribute specific functional defects in Dnmt1Δ/Δ hematopoietic stem and progenitor cells to alterations in expression of previously characterized genes, supporting the existence of novel, uncharacterized regulators of HSC and progenitor cell function to be explored from candidates in our data set. We conclude that maintenance methylation induced by Dnmt1 appears to be especially important for HSC and progenitor cell state transitions, such as the stepwise differentiation of long-term HSCs to multipotent progenitors, multipotent progenitors to myeloid restricted progenitors, stem cell mobilization, and regulating cell cycle entry. These findings establish a unique and critical role for Dnmt1 in the primitive hematopoietic compartment. Furthermore, our evidence suggests that epigenetic regulation, at least with respect to DNA methylation, of adult stem cells is distinct from embryonic stem cells and other somatic cell types. Disclosures: No relevant conflicts of interest to declare.