We previously identified a hematopoietic stem and progenitor cell (HSPC)-specific silencer element (the methylation-determining region [G1MDR]) that recruits DNA methyltransferase 1 (Dnmt1) and provokes methylation of the gene enhancer
We previously identified a hematopoietic stem and progenitor cell (HSPC)-specific silencer element (the methylation-determining region [G1MDR]) that recruits DNA methyltransferase 1 (Dnmt1) and provokes methylation of the gene enhancer. apoptosis of HSPCs. Furthermore, genetic deletion of in HSPCs activated expression and depleted HSPCs, thus recapitulating the HSC phenotype associated with GATA1 gain of function. These results demonstrate that this G1MDR holds the key to HSPC maintenance and suggest that release from this suppressive mechanism is usually a fundamental requirement for subsequent initiation of erythroid differentiation. gene regulation, hematopoietic stem and progenitor cell, erythropoiesis INTRODUCTION The transcription factor GATA1 plays a key role in erythroid and megakaryocytic cell differentiation and in the differentiation of basophilic, eosinophilic, and mast cell lineages (1,C8). For erythroid differentiation, GATA1 expression increases in stages, from common myeloid progenitors (CMPs) to megakaryocyte-erythroid progenitors (MEPs), and reaches a peak at the proerythroblast (ProEB) stage (9, 10). Earlier studies identified a series of gene that participate in the elaborate transcriptional regulation of the gene (11, 12). Based on both and studies, gene expression during erythropoiesis has been reported to require promoter-proximal CACCC motifs, a palindromic double-GATA motif (dbG) approximately 650 bp upstream of the transcription start site (TSS), and a GATA motif in the hematopoietic enhancer (G1HE), 3.7 kb upstream of the TSS (10, 13,C15). GATA2 is usually abundantly expressed in hematopoietic stem and progenitor cells (HSPCs) (16,C22). During commitment to the erythroid-megakaryocytic lineages, GATA2 acts to induce gene expression, while at the same time, the enhanced level of GATA1 represses gene expression. This exchange of GATA2 for GATA1 has been referred to as GATA factor switching (23,C28). One intriguing observation is usually that while GATA2 is usually abundantly expressed in HSPCs, the gene is usually expressed only at a low level. This observation suggests that a certain predominantly repressive mechanism controls the transcription of the gene in HSPCs. In this regard, we previously identified a gene repression in HSPCs. We also found that DNA methyltransferase 1 (Dnmt1) is usually recruited to the element and participates in gene methylation (29). We refer to this silencer element as the methylation-determining region (G1MDR). While the mechanisms maintaining the high level of expression in maturing erythroid lineage cells have been studied extensively, the initial activation of the gene in early hematopoietic Rabbit polyclonal to ZNF248 progenitors Syncytial Virus Inhibitor-1 remains to be fully understood. As mentioned above, in HSPCs, expression is usually repressed to a very low level, even in the presence of abundant GATA2. In light of the GATA factor switching mechanism, we surmise that certain cues that release the gene from the silencing machinery may serve as the central mechanism that initiates gene activation. In this regard, several lines of evidence are worthy of attention. First, Dnmt1 is usually strongly recruited to the G1MDR to maintain DNA methylation of the locus in HSPCs, and deletion of the G1MDR selectively abrogates gene repression in HSPCs, which is usually associated with an increase of GATA2 occupancy in the gene enhancer (29). Second, demethylation of the enhancer and promoter around the G1MDR is usually associated with the enhancement of gene expression during erythropoiesis (29). Based on these observations, we hypothesized that derepression Syncytial Virus Inhibitor-1 of Dnmt1-G1MDR-mediated repression is the key molecular mechanism that triggers the initial activation of gene expression in HSPCs, which subsequently leads HSPCs to differentiate toward the erythroid lineage. To address this hypothesis, we generated three transgenic mouse lines that carry modified bacterial artificial chromosome (BAC) DNAs in which the G1MDR was deleted to express either wild-type GATA1, a GATA1-estrogen receptor fusion protein (G1ERT2), or a Cre-estrogen receptor fusion protein (CreERT2) in HSPCs. The mouse lines harboring these constructs individually are referred to as MG-G1, MG-G1ERT2, and MG-CreERT2, for minigene-gene expression in HSPCs, in a GATA2-dependent manner. The release of from repressive regulation by means of this strategy provoked skewed hematopoiesis toward erythroid differentiation and concomitantly induced HSPC apoptosis. Importantly, the genetic deletion of in HSPCs in MG-CreERT2 mice activated gene expression and recapitulated the hematopoietic phenotype of MG-G1 and MG-G1ERT2 transgenic mice. Taken together, these results demonstrate that this Dnmt1-G1MDR complex plays a key role in the repression of the gene for HSPC maintenance and suggest that release from this repression is critical for subsequent gene activation for erythroid commitment and differentiation. RESULTS Syncytial Virus Inhibitor-1 G1MDR-mediated repression is crucial for hematopoietic homeostasis and perinatal survival. To address the consequences of release from G1MDR-mediated gene repression in HSPCs on subsequent.