miR-21 Is a Transcriptional Target of Growth Factor Independent -1; Implications for Transcriptional Regulation of microRNA.

Hematopoiesis is a tightly regulated multistage process, wherein pluripotent self-renewing stem cells gives rise to all blood cell lineages. Growth factor independent -1 (Gfi1) is a transcription factor that relies on corepressor proteins such as G9a to repress transcription of target genes. Gfi1 is required for hematopoietic stem cell self-renewal and subsequent granulocytic lineage development. Recent studies have identified microRNAs (miRs) as 21–23 nucleotide non-coding RNA that regulate gene expression. It is currently unclear which microRNAs control hematopoiesis. The deregulated Gfi1 null environment provides a unique setting to screen for miRNA that may control hematopoiesis. Therefore, we performed microRNA gene expression array analyses on RNA extracted from total bone marrow cells of Gfi1 null and wild type littermate mice. Our results demonstrate that several miR genes are differentially expressed in Gfi1 knock-out mice; however, we have focused further analyses on miR-21, which was consistently upregulated in Gfi1 null cells. Thus, miR-21 provides a potential direct target for Gfi1. Moreover, miR-21 was previously shown to regulate apoptosis in neuronal cell lines. As demonstrated by both expression array and Taqman, the relative expression of the mature miR-21 in Gfi1 null bone marrow cells is more than 7 fold higher than wild type Gfi1 littermate bone marrow. Because loss of Gfi1 alters hematopoiesis, we normalized the cells under analysis by lineage marker depletion. Still, in comparison to similar Lin-populations from wild type littermates, the expression of miR-21 was consistently higher in Gfi1 null Lin-bone marrow cells. We next attempted to determine whether miR-21 is directly regulated by Gfi1; however, bioinformatic analyses of microRNA regulatory sequences are currently underdeveloped. We therefore adopted a systematic gene-centric pipeline approach and adapted a web-accessible database resource termed Genome TraFac (http://genometrafac.cchmc.org) to microRNA orthologs that are conserved between human and murine genomes. The new “miRBase” analyzed for the occurrence of conserved individual cis -elements and compositionally similar cis- culsters to identify validated transcription factor targets within promoter and distal genomic regulatory regions of microRNA genes. Interestingly, many microRNA genes contain conserved regulatory sequences within transcribed precursor sequences, not in the predicted promoter regions. Within the miR-21 gene, two putative Gfi1 binding sites were identified within transcribed precursor sequences. To determine the accuracy of our bioinformatic analyses, we performed chromatin immunoprecipitation (ChIP) with anti-Gfi1 and an anti-G9A antisera. Our data reveal Gfi1, along with its co-factor G9A, is bound to the predicted region (but not control regions lacking predicted Gfi1 binding sites). Notably, electrophoretic mobility shift assay (EMSA) indicated that in vitro transcribed and translated Gfi1 binds strongly to one of the two putative Gfi1 binding sites in the miR-21 gene. These data suggest that the level of miR-21 is regulated directly by Gfi1, and that microRNA genes are commonly regulated by conserved cis-elements within transcribed precursor sequences. The role of miR-21 in controlling differentiation/proliferation in hematopoietic stem cells is currently under investigation.