The common understanding of the function of RecD, as derived predominantly from studies in Escherichia coli, is that RecD is one of three enzymes in the RecBCD double-stranded break repair DNA recombination complex. However, comparative genomics has revealed that many organisms possess a recD gene even though the other members of the complex, recB and recC, are not present. Further, bioinformatic analyses have shown that there is substantial sequence dissimilarity between recD genes associated with recB and recC (recD1), and those that are not associated with recBC (recD2). Deinococcus radiodurans, known for its extraordinary DNA repair capability, is one such organism that does not possess either recB or recC, and yet does possess a recD gene. The recD of D. radiodurans was deleted and this mutant was shown to have a capacity to repair double-stranded DNA breaks equivalent to wild-type. The phylogenetic history of recD was studied using a dataset of 120 recD genes from 91 fully sequenced species. The analysis focused upon the role of gene duplication and functional genomic context in the evolution of recD2, which appears to have undergone numerous independent events resulting in duplicate recD2 genes. The role of RecD as part of the RecBCD complex appears to have a divergence from an earlier ancestral RecD function still preserved in many species including D. radiodurans.

Histone deacetylases (HDACs) catalyze deacetylation of histones that results in altered transcriptional activity. Inhibitors of HDACs have been shown to induce transcriptional changes that contribute positively to reprogramming somatic cells either by nuclear transfer or inducing a pluripotent state. However, the exact molecular mechanisms whereby HDAC inhibitors function and the specificity of the HDAC isoforms in cell reprogramming are not yet fully understood. Herein, we report the ability of individual isoform-specific HDACs to modulate endogenous expression of pluripotency-associated genes in bovine somatic cells. This in vitro study showed that a transient selective depletion of HDACs resulted in elevated mRNA levels of Oct-4, Sox2, and Nanog. In particular, we found that inhibition of specific HDAC isoforms using small interfering (si) RNA significantly increased expression of Nanog, a key factor required for totipotency induced by somatic cell nuclear transfer and for maintaining pluripotency in embryonic and induced pluripotent stem cells. Our study suggests that this gene might be the most susceptible to HDAC activity inhibition. Moreover, a regulatory role of the class III HDAC, SIRT3, on an Oct4-Sox2-Nanog transcriptional network was revealed. We observed the upregulation of pluripotency-related genes by depletion of SIRT3. SIRT3 is localized to mitochondria and is associated with energy metabolism processes, suggesting metabolic changes may be linked to reprogramming in bovine fibroblasts. In conclusion, we show that targeting selective HDACs can potentially be useful to enhance reprogramming and that sirtuins may play a pivotal role in somatic cell reprogramming by upregulating an Oct4-Sox2-Nanog transcriptional network. Dedifferentiating donor somatic cells by upregulating developmentally important genes through specific knockdown of epigenetic targets, in particular HDACs, may provide a path to improving livestock cloning and the in vitro production of pluripotent cells.

Human somatic cells can be directly reprogrammed to induced pluripotent stem (iPS) cells by forced expression of the transcription factors Oct4, Sox2, and either Klf4 and cMyc or Nanog and Lin28, using virus-based systems. However, low reprogramming efficiency and the potential for deleterious virus-induced genomic modification limit the clinical potential of this technology. Recent reports indicate, however, that the generation of iPS cells can be enhanced by the addition of synthetic small molecules, including epigenetic modulators. In this report, we demonstrate that the epigenetic modifiers Valproic Acid (VPA) and 5-azacytidine activate the reciprocal transcriptional regulation of endogenous pluripotency transcription factor genes in human dermal fibroblasts and that VPA alone can directly activate endogenous Oct4 in the absence of transgenes. Moreover, using human adipose cells, we demonstrate that histone deacetylase inhibition, prior to reprogramming factor transfection, increases embryonic stem (ES) cell-like colony formation ~2 - 3 fold. In addition, DNA methyltransferase (DNMT) inhibition during human ES cell culture promotes maturation of reprogrammed somatic cells, increasing the yield ~4 fold. These data provide proof of principle that reprogramming efficiency can be improved by inhibiting specific repressive epigenetic regulatory components at the levels of ES cell-like colony formation and maturation. In addition, these studies raise the interesting possibility that a more efficient small molecule-based reprogramming system may provide a superior alternative to current virus-based approaches

The identification of a single, early marker for full developmental potential of induced pluripotent stem (iPS) cells has proven elusive. Recently, however, activation of the imprinted gene cluster, Dlk1-Dio3 has emerged as a viable candidate in the mouse. To explore the relationship between Dlk1-Dio3 expression and developmental potential more fully, we used murine ear mesenchymal stem cells (mEMSC) for iPS cell induction. Mouse EMSC are easily obtained and share functional characteristics with embryonic stem (ES) cells and therefore, may be a reliable non-embryonic source for iPS cell production. We report that mEMSC express high levels of Gtl2, a maternally expressed gene within the Dlk1-Dio3 imprinted cluster. Moreover, mEMSC produce Gtl2 expressing (Gtl2 on ) iPSC clones that share functional characteristics with ES cell clones. The production of Gtl2 on iPS cell clones from mEMSC provides a new model with which to investigate the regulation of Dlk1-Dio3 cluster activity during direct cell reprogramming.

Small molecules will need to be identified and/or developed that target protein classes limiting reprogramming efficiency. A specific class of proteins includes epigenetic regulators that silence, or minimize expression, of pluripotency genes in differentiated cells. To better understand the role of specific epigenetic modulators in reprogramming, we have used shRNA delivered by lentivirus to assess the significance of individual epi-proteins in reprogramming pluripotent gene expression.