Np95 interacts with de novo DNA methyltransferases, Dnmt3a and Dnmt3b, and mediates epigenetic silencing of the viral CMV promoter in embryonic stem cells

Abstract

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation without specific permission.Recent studies have indicated that nuclear protein of 95 kDa (Np95) is essential for maintaining genomic methylation by recruiting DNA methyltransferase (Dnmt) 1 to hemi-methylated sites. Here, we show that Np95 interacts more strongly with regulatory domains of the de novo methyltransferases Dnmt3a and Dnmt3b. To investigate possible functions, we developed an epigenetic silencing assay using fluorescent reporters in embryonic stem cells (ESCs). Interestingly, silencing of the cytomegalovirus promoter in ESCs preceded DNA methylation and was strictly dependent on the presence of either Np95, histone H3 methyltransferase G9a or Dnmt3a and Dnmt3b. Our results indicate a regulatory role for Np95, Dnmt3a and Dnmt3b in mediating epigenetic silencing through histone modification followed by DNA methylation.

UHRF1 coordinates peroxisome proliferator activated receptor gamma (PPARG) epigenetic silencing and mediates colorectal cancer progression

“…We have already shown that H3K27me3 is present on this promoter along with its specific methyltransferase EZH2 (Pancione et al, 2010b); here, we provide evidence that H3K9me2 and H3K9me3 are engaged along with Suv39H1 to silence PPARG in CRC cells, in line with recent reports showing that CRC progression correlates with higher levels of Suv39H1 and the presence of these repressive histone marks on promoter sequences (Fullgrabe et al, 2011;Varier and Timmers, 2011). Consistently, UHRF1 depletion is associated with PPARG re-expression through DNA demethylation at the promoter region and H3K9 and H3K27 demethylation, likely due to displacement of EZH2, MeCP2 and Suv39H1, confirming previous data on their physical interactions with UHRF1 (Unoki et al, 2004;Meilinger and Fellinger, 2009;Alhosin et al, 2011). Our data support that PPARG epigenetic deregulation is coordinated by UHRF1 through both DNA and histone methylation.…”

“…High UHRF1 levels are associated with a more advanced tumor grade (grades 3 and 4), as recently shown in lung, bladder and prostate cancers (Meilinger and Fellinger, 2009;Phe et al, 2010;Alhosin et al, 2011). High UHRF1 expression is also associated with a shorter survival only in advanced stages (stages III-IV).…”

“…UHRF1 was engaged at very low levels in HT29 cells, more in HCT116 and even more in RKO cells, confirming the inverse relationship with PPARG expression (Figure 3a). In line with the notion that UHRF1 interacts and recruits DNMTs to silence transcription of target genes (Meilinger and Fellinger, 2009;Alhosin et al, 2011), we analyzed DNMT1, DNMT3a and 3b. DNMT1 and DNMT3a were detected at low, if any, levels on the PPARG promoter in all three cell lines (Figure 3a), whereas DNMT3b was engaged only slightly in HT29, more in HCT116 and even more in RKO cells.…”

Histone tails regulate DNA methylation by allosterically activating de novo methyltransferase

“…In this model, the methyltransferase is recruited to a specific genomic site by directly recognizing nucleosomal DNA or histones [13-15, 18, 25], or alternatively through other adaptor proteins such as Dnmt3L [13,23], G9a [26][27][28], Np95 [29] and PML-RAR [30]. The localized Dnmt3 is then allosterically activated to methylate nearby cytosines upon the binding of an unmodified H3K4 N-terminus to the PHD domain.…”

Reprogramming Adipose Tissue-Derived Mesenchymal Stem Cells into Pluripotent Stem Cells by a Mutant Adeno-Associated Viral Vector

“…Such retroviral transgene silencing may result from the activation of potent repressor factors or the reduction of certain activating factors after directly reprogramming somatic cells to pluripotent states (Hotta and Ellis, 2008). In addition, CMV promoter activity has been shown to be progressively silenced in pluripotent stem cells (Xia et al, 2007;Meilinger et al, 2009). In rAAV2.3m-mediated cell reprogramming, whether transgene silencing resulted from the activation of certain endogenous potent repressor or from the reduction of CMV promoter activity in rAAV-iPS cells remains elusive.…”