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We observed that microRNAs (miRNAs) that regulate differentiation in a variety of simpler systems also regulate differentiation of human multipotent stromal cells (hMSCs) from bone marrow. Differentiation of hMSCs into osteoblasts and adipocytes was inhibited by using lentiviruses expressing shRNAs to decrease expression of Dicer and Drosha, two enzymes that process early transcripts to miRNA. Expression analysis of miRNAs during hMSC differentiation identified 19 miRNAs that were up-regulated during osteogenic differentiation and 20 during adipogenic differentiation, 11 of which were commonly up-regulated in both osteogenic and adipogenic differentiation. In silico models predicted that five of the up-regulated miRNAs targeted leukemia inhibitory factor (LIF) expression. The prediction was confirmed for two of the miRNAs, hsa-mir 199a and hsa-mir346, in that over-expression of the miRNAs decreased LIF secretion by hMSCs. The results demonstrate that differentiation of hMSCs is regulated by miRNAs and that several of these miRNAs target LIF. hsa-mir199a | hsa-mir346 | stem cells | plasticity

Analysis of the carboxyl-terminal region of E2F-1 activation domain reveals the over expression of retinoblastoma (RB), which inhibits GAL4/E2F-1 activity in vivo, and the RB mutant, RBd22, is not over expressed. The carboxyl-terminal region of E2F1 supports the transcriptional activation when linked to the DNA binding domain of yeast transcription factor GAL4. An 18-residue retinoblastoma (RB)-binding sequence is found in this region. The E2F-1 mediated transcriptional activation through direct physical association is suppressed by the RB.

Cyclosporin A (CsA) and FK506 are immunosuppressants which upon binding to their respective receptor proteins, inhibit the transcriptional induction of several genes involved in the activation of T cells. An investigation was conducted to further elucidate the regulatory roles of CsA and FK506. The results showed that both CsA and FK506 are able to block the induction of tumor necrosis factor-alpha gene transcription by anti-Ig in B cells. Therefore, CsA and FK506 may be used as probes to elucidate the transcriptional pathway followed during activation of B cells.

Regulation of gene expression in mammals through methylation of cytosine residues at CpG dinucleotides is involved in the development and progression of tumors. Because many genes that are involved in the control of cell proliferation are regulated by members of the E2F family of transcription factors and because some E2F DNA-binding sites are methylated in vivo, we have investigated whether CpG methylation can regulate E2F functions. We show here that methylation of E2F elements derived from the dihydrofolate reductase, E2F1, and cdc2 promoters prevents the binding of all E2F family members tested (E2F1 through E2F5). In contrast, methylation of the E2F elements derived from the c-myc and c-myb promoters minimally affects the binding of E2F2, E2F3, E2F4, and E2F5 but significantly inhibits the binding of E2F1. Consistent with these studies, E2F3, but not E2F1, activates transcription through methylated E2F sites derived from the c-myb and c-myc genes whereas both E2F1 and E2F3 fail to transactivate a reporter gene that is under the control of a methylated dihydrofolate reductase E2F site. Together, these data illustrate a means through which E2F activity can be controlled.