Homeobox (HOX) genes play a definitive role in determination of cell fate during embryogenesis and hematopoiesis. MLL-related leukemia is coincident with increased expression of a subset of HOX genes, including HOXA9. MLL functions to maintain, rather than initiate, expression of its target genes. However, the mechanism of MLL maintenance of target gene expression is not understood. Here, we demonstrate that Mll binds to specific clusters of CpG residues within the Hoxa9 locus and regulates expression of multiple transcripts. The presence of Mll at these clusters provides protection from DNA methylation. shRNA knock-down of Mll reverses the methylation protection status at the previously protected CpG clusters; methylation at these CpG residues is similar to that observed in Mll null cells. Furthermore, reconstituting MLL expression in Mll null cells can reverse DNA methylation of the same CpG residues, demonstrating a dominant effect of MLL in protecting this specific region from DNA methylation. Intriguingly, an oncogenic MLL-AF4 fusion can also reverse DNA methylation, but only for a subset of these CpGs. This method of transcriptional regulation suggests a mechanism that explains the role of Mll in transcriptional maintenance, but it may extend to other CpG DNA binding proteins. Protection from methylation may be an important mechanism of epigenetic inheritance by regulating the function of both de novo and maintenance DNA methyltransferases.homeodomain ͉ leukemia ͉ maintenance
To evaluate the role of the erythropoietin (Epo) receptor (R) in erythropoiesis in more primitive stem cells, we assessed the influence of retrovirus-mediated gene transfer of human (h) EpoR complementary DNA (cDNA) into murine embryonic stem (ES) cells on erythroid differentiation of these cells. The hEpoR cDNA was efficiently transduced into ES cells, forming hEpoR that stably expressed ES (ES-hEpoR) cells. Expression of hEpoR cDNA was confirmed in ES-hEpoR cells by reverse transcriptase-polymerase chain reaction and Northern blot analysis. Colony assays demonstrated that definitive erythroid and primitive erythroid colonies were significantly increased from ES-hEpoR cells, when compared with mock virus-transduced ES (ES-Neo) cells, during the time course of differentiation induced by withdrawal of leukemia inhibitory factor, in either the presence or the absence of Epo. Multipotential colony-forming units (CFU-Mix) were also increased in ES-hEpoR cells at different stages of differentiation, but no changes were detected for CFU-granulocyte-macrophage colonies (CFU-GM). Time course studies by Northern blot analysis demonstrated elevated levels of expression of beta-H1 and beta-Major globin genes in embryoid bodies derived from ES-hEpoR cells stimulated with Epo, when compared with similar expression from ES-Neo cells. Expression of the GATA-1 gene was enhanced in ES-hEpoR cells, when compared with ES-Neo cells, beginning immediately after initiation of the cultures until 8 days of differentiation. These data indicate that primitive and definitive erythropoiesis in differentiating embryoid bodies can be enhanced by retrovirus-mediated gene transfer of an hEpoR gene.
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