The tumor suppressor p53 regulates cell-cycle progression and apoptosis in response to genotoxic stress, and inactivation of p53 is a common feature of cancer cells. The levels and activity of p53 are tightly regulated by posttranslational modifications, including phosphorylation, ubiquitination, and acetylation. Here, we demonstrate that the transcription factor Yin Yang 1 (YY1) interacts with p53 and inhibits its transcriptional activity. We show that YY1 disrupts the interaction between p53 and the coactivator p300 and that expression of YY1 blocks p300-dependent acetylation and stabilization of p53. Furthermore, expression of YY1 inhibits the accumulation of p53 and the induction of p53 target genes in response to genotoxic stress. YY1 also interacts with Mdm2 and the expression of YY1 promotes the assembly of the p53-Mdm2 complex. Consequently, YY1 enhances Mdm2-mediated ubiquitination of p53. Inactivation of endogenous YY1 enhances the accumulation of p53 as well as the expression of p53 target genes in response to DNA damage, and it sensitizes cells to DNA damageinduced apoptosis. Hence, our results demonstrate that YY1 regulates the transcriptional activity, acetylation, ubiquitination, and stability of p53 by inhibiting its interaction with the coactivator p300 and by enhancing its interaction with the negative regulator Mdm2. YY1 may, therefore, be an important negative regulator of the p53 tumor suppressor in response to genotoxic stress. p300 ͉ acetylation ͉ ubiquitin ͉ Mdm2 ͉ transcription

Friedreich ataxia is a degenerative disease caused by deficiency of the protein frataxin (FXN). An intronic expansion of GAA triplets in the FXN-encoding gene, FXN, causes gene silencing and thus reduced FXN protein levels. Although it is widely assumed that GAA repeats block transcription via the assembly of an inaccessible chromatin structure marked by methylated H3K9, direct proof for this is lacking. In this study, we analysed different histone modification patterns along the human FXN gene in FRDA patient-derived lymphoblastoid cell lines. We show that FXN mRNA synthesis, but not turnover rates are affected by an expanded GAA repeat tract. Importantly, rather than preventing transcription initiation, long GAA repeat tracts affect transcription at the elongation step and this can occur independently of H3K9 methylation. Our data demonstrate that finding novel strategies to overcome the transcription elongation problem may develop into promising new treatments for FRDA.

Members of the sterol regulatory element-binding protein (SREBP) family of transcription factors control cholesterol and lipid metabolism and play critical roles during adipocyte differentiation. The transcription factor SREBP1 is degraded by the ubiquitin-proteasome system following phosphorylation of Thr 426 and Ser 430 in its phosphodegron. We now demonstrate that the glycogen synthase kinase (GSK)-3␤-dependent phosphorylation of these residues in SREBP1 is enhanced in response to specific DNA binding. DNA binding enhances the direct interaction between the C-terminal domain of SREBP1 and GSK-3␤. Accordingly, we demonstrate that GSK-3␤ is recruited to the promoters of SREBP target genes in vivo. As a result of the phosphorylation of Thr 426 and Ser 430 , the ubiquitin ligase Fbw7 is recruited to SREBP molecules associated with target promoters. Using a reconstituted ubiquitination system, we demonstrate that Fbw7-mediated ubiquitination of SREBP1 is dependent on its DNA binding activity. Thus, DNA binding could provide a mechanistic link between the phosphorylation, ubiquitination, and degradation of active transcription factors.Members of the sterol regulatory element-binding protein (SREBP) 3 family of transcription factors control cholesterol and lipid metabolism and play critical roles during adipocyte differentiation and insulin signaling (1, 2). The SREBP family of transcription factors consists of three different SREBP proteins; SREBP1a, SREBP1c, and SREBP2. The SREBPs are synthesized as large precursor proteins that are inserted into the nuclear and endoplasmic reticulum membranes and are transcriptionally inactive (3). In sterol-depleted cells, SREBPs are processed sequentially by two membrane-associated proteases that release the mature form of the proteins (4, 5). These transcriptionally active fragments of the SREBPs are translocated to the nucleus and bind to the promoters of SREBP target genes (6). It has been demonstrated that the mature forms of SREBPs are modified by phosphorylation (7-9), acetylation (10), sumoylation (11), and ubiquitination (8, 12), and it has been shown that these modifications regulate their stability and/or transcriptional activity.Many transcription factors, particularly those involved in the control of cell growth, are unstable proteins targeted for degradation by the ubiquitin-proteasome system (13). Numerous observations have pointed to the existence of a close connection between the ubiquitin-proteasome system and transcriptional activation (14,15). It has been demonstrated that the sequences that specify proteolysis of some activators overlap with their transcriptional activation domains and that components of the proteasome can be recruited to promoters through interactions with transcriptional regulators (16 -21). It has also been demonstrated that the activity of certain transcription factors can be enhanced by ubiquitination or through interactions with specific ubiquitin ligases (22-25). However, the mechanistic link between the transcriptional activity of...

In the fission yeast S. pombe, the RNA interference (RNAi) pathway is required to generate small interfering RNAs (siRNAs) that mediate heterochromatic silencing of centromeric repeats. Here, we demonstrate that RNAi also functions to repress genomic elements other than constitutive heterochromatin. Using DNA adenine methyltransferase identification (DamID), we show that the RNAi proteins Dcr1 and Rdp1 physically associate with some euchromatic genes, noncoding RNA genes and retrotransposon long terminal repeats, and that this association is independent of the Clr4 histone methyltransferase. Physical association of RNAi with chromatin is sufficient to trigger a silencing response but not to assemble heterochromatin. The mode of silencing at the newly identified RNAi targets is consistent with a co-transcriptional gene silencing model, as proposed earlier, and functions with trace amounts of siRNAs. We anticipate that similar mechanisms could also be operational in other eukaryotes.

ObjectiveMyasthenia gravis (MG) is a chronic autoimmune disorder where autoantibodies target the nicotinic acetylcholine receptors (AChR+) in about 85% of cases, in which the thymus is considered to play a pathogenic role. As there are no reliable biomarkers to monitor disease status in MG, we analyzed circulating miRNAs in sera of MG patients to find disease-specific miRNAs.MethodsOverall, 168 miRNAs were analyzed in serum samples from four AChR+ MG patients and four healthy controls using Exiqon Focus miRNA polymerase chain reaction (PCR) Panel I + II. Specific accumulation pattern of 13 miRNAs from the discovery set was subsequently investigated in the sera of 16 AChR+ MG patients and 16 healthy controls. All patients were without immunosuppressive treatment. Selected specific miRNAs were further analyzed in the serum of nine MG patients before and after thymectomy to assess the effect of thymus removal on the accumulation of the candidate miRNAs in patient sera.ResultsThree miRNAs were specifically dysregulated in AChR+ MG patient sera samples. Hsa-miR150-5p, which induces T-cell differentiation, as well as hsa-miR21-5p, a regulator of Th1 versus Th2 cell responses, were specifically elevated in MG sera. Additionally, hsa-miR27a-3p, involved in natural killer (NK) cell cytotoxicity, was decreased in MG. Hsa-miR150-5p levels had the highest association with MG and were significantly reduced after thymus removal in correlation with disease improvement.InterpretationWe propose that the validated miRNAs: hsa-miR150-5p, hsa-miR21-5p, and hsa-miR27a-3p can serve as novel serum biomarkers in AChR+ MG. Hsa-miR-150-5p could be a helpful marker to monitor disease severity.

The sterol regulatory element-binding protein (SREBP) family of transcription factors controls the biosynthesis of cholesterol and other lipids, and lipid synthesis is critical for cell growth and proliferation. We were, therefore, interested in the expression and activity of SREBPs during the cell cycle. We found that the expression of a number of SREBP-responsive promoter-reporter genes were induced in a SREBP-dependent manner in cells arrested in G 2͞M. In addition, the mature forms of SREBP1a and SREBP1c were hyperphosphorylated in mitotic cells, giving rise to a phosphoepitope recognized by the mitotic protein monoclonal-2 (MPM-2) antibody. In contrast, SREBP2 was not hyperphosphorylated in mitotic cells and was not recognized by the MPM-2 antibody. The MPM-2 epitope was mapped to the C terminus of mature SREBP1, and the mitosis-specific hyperphosphorylation of SREBP1 depended on this domain of the protein. The transcriptional and DNA-binding activity of SREBP1 was enhanced in cells arrested in G 2͞M, and these effects depended on the C-terminal domain of the protein. In part, these effects could be explained by our observation that mature SREBP1 was stabilized in G 2͞M. In agreement with these observations, we found that the synthesis of cholesterol was increased in G 2͞M-arrested cells. Thus, our results demonstrate that the activity of mature SREBP1 is regulated by phosphorylation during the cell cycle, suggesting that SREBP1 may provide a link between lipid synthesis, proliferation, and cell growth.cell cycle ͉ cholesterol ͉ phosphorylation ͉ proliferation M embers of the sterol regulatory element-binding protein (SREBP) family of transcription factors control cholesterol and lipid metabolism and play critical roles in insulin signaling and during adipocyte differentiation (1). Two genes, srebf1 and srebf2, encode three different SREBPs (SREBP1a, SREBP1c, and SREBP2) (2). SREBPs are synthesized as large precursor proteins that are inserted into the nuclear and endoplasmic reticulum (ER) membranes and are transcriptionally inactive. In sterol-depleted cells, SREBP cleavage-activating protein (SCAP) escorts SREBP to the Golgi apparatus where SREBP is processed sequentially by two membrane-associated proteases, resulting in the release of the mature form of the protein (3, 4). This transcriptionally active fragment of SREBP is translocated to the nucleus and binds to the promoters of SREBP target genes. Most of these genes are involved in the control of lipid biosynthesis and metabolism (5). In sterolloaded cells, the SCAP͞SREBP complex is trapped in the ER as a result of sterol-induced binding of SCAP to Insigs, which are resident proteins of the ER membrane (6, 7).It is well established that the synthesis of membrane lipids is critical for cell growth and proliferation (8, 9). Here, we report that the expression of a number of SREBP-responsive promoterreporter genes are induced in a SREBP-dependent manner in cells arrested in G 2 ͞M. In addition, the mature forms of SREBP1a and SREBP1c are hyperphosphory...

Human adenoviruses (HAds) encode for one or two highly abundant virus-associated RNAs, designated VA RNAI and VA RNAII, which fold into stable hairpin structures resembling miRNA precursors. Here we show that the terminal stem of the VA RNAs originating from Ad4, Ad5, Ad11 and Ad37, all undergo Dicer dependent processing into virus-specific miRNAs (so-called mivaRNAs). We further show that the mivaRNA duplex is subjected to a highly asymmetric RISC loading with the 3′-strand from all VA RNAs being the favored strand, except for the Ad37 VA RNAII, where the 5′-mivaRNAII strand was preferentially assembled into RISC. Although the mivaRNA seed sequences are not fully conserved between the HAds a bioinformatics prediction approach suggests that a large fraction of the VA RNAII-, but not the VA RNAI-derived mivaRNAs still are able to target the same cellular genes. Using small RNA deep sequencing we demonstrate that the Dicer processing event in the terminal stem of the VA RNAs is not unique and generates 3′-mivaRNAs with a slight variation of the position of the 5′ terminal nucleotide in the RISC loaded guide strand. Also, we show that all analyzed VA RNAs, except Ad37 VA RNAI and Ad5 VA RNAII, utilize an alternative upstream A start site in addition to the classical +1 G start site. Further, the 5′-mivaRNAs with an A start appears to be preferentially incorporated into RISC. Although the majority of mivaRNA research has been done using Ad5 as the model system our analysis demonstrates that the mivaRNAs expressed in Ad11- and Ad37-infected cells are the most abundant mivaRNAs associated with Ago2-containing RISC. Collectively, our results show an unexpected variability in Dicer processing of the VA RNAs and a serotype-specific loading of mivaRNAs into Ago2-based RISC.

The immuno-microRNAs miR-150-5p and miR-21-5p show a disease specific signature, which suggests these microRNAs as possible biological autoimmune markers of MG.