RNF12 initiates X-chromosome inactivation by targeting REX1 for degradation.
Nature. 2012;485(7398):386-90
Authors: Gontan C, Achame EM, Demmers J, Barakat TS, Rentmeester E, van Ijcken W, Grootegoed JA, Gribnau J
Abstract
Evolution of the mammalian sex chromosomes has resulted in a heterologous X and Y pair, where the Y chromosome has lost most of its genes. Hence, there is a need for X-linked gene dosage compensation between XY males and XX females. In placental mammals, this is achieved by random inactivation of one X chromosome in all female somatic cells. Upregulation of Xist transcription on the future inactive X chromosome acts against Tsix antisense transcription, and spreading of Xist RNA in cis triggers epigenetic changes leading to X-chromosome inactivation. Previously, we have shown that the X-encoded E3 ubiquitin ligase RNF12 is upregulated in differentiating mouse embryonic stem cells and activates Xist transcription and X-chromosome inactivation. Here we identify the pluripotency factor REX1 as a key target of RNF12 in the mechanism of X-chromosome inactivation. RNF12 causes ubiquitination and proteasomal degradation of REX1, and Rnf12 knockout embryonic stem cells show an increased level of REX1. Using chromatin immunoprecipitation sequencing, REX1 binding sites were detected in Xist and Tsix regulatory regions. Overexpression of REX1 in female embryonic stem cells was found to inhibit Xist transcription and X-chromosome inactivation, whereas male Rex1(+/-) embryonic stem cells showed ectopic X-chromosome inactivation. From this, we propose that RNF12 causes REX1 breakdown through dose-dependent catalysis, thereby representing an important pathway to initiate X-chromosome inactivation. Rex1 and Xist are present only in placental mammals, which points to co-evolution of these two genes and X-chromosome inactivation.
PMID: 22596162 [PubMed - in process]
Genome-wide analysis of p63 binding sites identifies AP-2 factors as co-regulators of epidermal differentiation.
Nucleic Acids Res. 2012 May 9;
Authors: McDade SS, Henry AE, Pivato GP, Kozarewa I, Mitsopoulos C, Fenwick K, Assiotis I, Hakas J, Zvelebil M, Orr N, Lord CJ, Patel D, Ashworth A, McCance DJ
Abstract
The p63 transcription factor (TP63) is critical in development, growth and differentiation of stratifying epithelia. This is highlighted by the severity of congenital abnormalities caused by TP63 mutations in humans, the dramatic phenotypes in knockout mice and de-regulation of TP63 expression in neoplasia altering the tumour suppressive roles of the TP53 family. In order to define the normal role played by TP63 and provide the basis for better understanding how this network is perturbed in disease, we used chromatin immunoprecipitation combined with massively parallel sequencing (ChIP-seq) to identify >7500 high-confidence TP63-binding regions across the entire genome, in primary human neonatal foreskin keratinocytes (HFKs). Using integrative strategies, we demonstrate that only a subset of these sites are bound by TP53 in response to DNA damage. We identify a role for TP63 in transcriptional regulation of multiple genes genetically linked to cleft palate and identify AP-2alpha (TFAP2A) as a co-regulator of a subset of these genes. We further demonstrate that AP-2gamma (TFAP2C) can bind a subset of these regions and that acute depletion of either TFAP2A or TFAP2C alone is sufficient to reduce terminal differentiation of organotypic epidermal skin equivalents, indicating overlapping physiological functions with TP63.
PMID: 22573176 [PubMed - as supplied by publisher]
Identification of novel NRF2-regulated genes by ChIP-Seq: influence on retinoid X receptor alpha.
Nucleic Acids Res. 2012 May 11;
Authors: Chorley BN, Campbell MR, Wang X, Karaca M, Sambandan D, Bangura F, Xue P, Pi J, Kleeberger SR, Bell DA
Abstract
Cellular oxidative and electrophilic stress triggers a protective response in mammals regulated by NRF2 (nuclear factor (erythroid-derived) 2-like; NFE2L2) binding to deoxyribonucleic acid-regulatory sequences near stress-responsive genes. Studies using Nrf2-deficient mice suggest that hundreds of genes may be regulated by NRF2. To identify human NRF2-regulated genes, we conducted chromatin immunoprecipitation (ChIP)-sequencing experiments in lymphoid cells treated with the dietary isothiocyanate, sulforaphane (SFN) and carried out follow-up biological experiments on candidates. We found 242 high confidence, NRF2-bound genomic regions and 96% of these regions contained NRF2-regulatory sequence motifs. The majority of binding sites were near potential novel members of the NRF2 pathway. Validation of selected candidate genes using parallel ChIP techniques and in NRF2-silenced cell lines indicated that the expression of about two-thirds of the candidates are likely to be directly NRF2-dependent including retinoid X receptor alpha (RXRA). NRF2 regulation of RXRA has implications for response to retinoid treatments and adipogenesis. In mouse, 3T3-L1 cells' SFN treatment affected Rxra expression early in adipogenesis, and knockdown of Nrf2-delayed Rxra expression, both leading to impaired adipogenesis.
PMID: 22581777 [PubMed - as supplied by publisher]
Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel.
Proc Natl Acad Sci U S A. 2012 May 14;
Authors: Cipriany BR, Murphy PJ, Hagarman JA, Cerf A, Latulippe D, Levy SL, Benítez JJ, Tan CP, Topolancik J, Soloway PD, Craighead HG
Abstract
Epigenetic modifications, such as DNA and histone methylation, are responsible for regulatory pathways that affect disease. Current epigenetic analyses use bisulfite conversion to identify DNA methylation and chromatin immunoprecipitation to collect molecules bearing a specific histone modification. In this work, we present a proof-of-principle demonstration for a new method using a nanofluidic device that combines real-time detection and automated sorting of individual molecules based on their epigenetic state. This device evaluates the fluorescence from labeled epigenetic modifications to actuate sorting. This technology has demonstrated up to 98% accuracy in molecule sorting and has achieved postsorting sample recovery on femtogram quantities of genetic material. We have applied it to sort methylated DNA molecules using simultaneous, multicolor fluorescence to identify methyl binding domain protein-1 (MBD1) bound to full-duplex DNA. The functionality enabled by this nanofluidic platform now provides a workflow for color-multiplexed detection, sorting, and recovery of single molecules toward subsequent DNA sequencing.
PMID: 22586076 [PubMed - as supplied by publisher]
Functional Genomic Methods to Study Estrogen Receptor Activity.
J Mammary Gland Biol Neoplasia. 2012 May 16;
Authors: Gilfillan S, Fiorito E, Hurtado A
Abstract
Estrogen Receptor (ER) is a nuclear receptor that mediates the actions of estrogen and tamoxifen. ER is expressed in a major fraction of human breast cancers. Recently, genomic maps for estrogen- and tamoxifen-ER have been published. Interestingly, estrogen and tamoxifen induce similar genomic interactions and both ligands have been shown to use co-operating factors. The interactions of these co-operating factors within ER regions have impact both on ER-DNA interactions and gene expression regulated by estrogen and tamoxifen. Moreover, the study of chromatin changes induced by these factors has also provided significant insight into our understanding of ER transcriptional regulation. This methods review describes some functional genomic methods to study the influence of both ER ligands and ER co-operating factors. The analysis of protein-DNA interactions and chromatin changes can be explored by using classical and novel methods such as Chromatin Immunoprecipitation (ChIP) or Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE). This review also explores the properties of each of these methods and the advantages of combining them with high throughput sequencing.
PMID: 22588661 [PubMed - as supplied by publisher]
Chromatin immunoprecipitation identifies genes under direct VraSR regulation in Staphylococcus aureus.
Can J Microbiol. 2012 May 9;
Authors: Sengupta M, Jain V, Wilkinson BJ, Jayaswal RK
Abstract
Transcriptional profiling of Staphylococcus aureus treated with cell wall-active antibiotics identified the 2-component system, VraSR, as one of the key players in response to antibiotic stress. Although it has been shown that a number of genes are regulated by the VraSR system, it has not been shown which genes are under direct VraSR regulation and which genes are not. In this study, chromatin immunoprecipitation techniques were used to identify the genes which are regulated by the direct interaction of VraR with their promoter regions. The results showed for the first time, that the VraSR mediated regulation of cell wall biosynthesis-associated genes, pbp2, murZ, and sgtB are facilitated by the direct binding of VraR to their respective promoters. Conversely, fmtA, indicated previously to be under VraSR regulation did not exhibit direct regulation by the binding of VraR to its promoter. The VraSR system plays a very important role in antibiotic resistance against cell wall-active antibiotics, and hence, it is essential to understand its complete regulatory mechanism.
PMID: 22571705 [PubMed - as supplied by publisher]
Genome-Wide Identification of Transcription Factor-Binding Sites in Plants Using Chromatin Immunoprecipitation Followed by Microarray (ChIP-chip) or Sequencing (ChIP-seq).
Methods Mol Biol. 2012;876:173-88
Authors: Zhu JY, Sun Y, Wang ZY
Abstract
Nearly all signal transduction pathways lead to regulation of gene expression by controlling specific transcription factors (TFs). Chromatin immunoprecipitation (ChIP) is a powerful method for studying TF-DNA interactions in vivo. To identify all binding sites of a TF in the genome, the DNA obtained in ChIP experiments needs to be analyzed by hybridization to genome-tiling microarrays (ChIP-chip) or by next-generation sequencing (ChIP-seq). Here, we provide detailed protocols of ChIP for two model plant species Arabidopsis and rice, procedures of DNA sample preparation for ChIP-chip or ChIP-seq, and a general guide for computational data analysis. We have used these protocols to successfully identify direct target genes of the BZR1 TF of the brassinosteroid signaling pathway in both Arabidopsis and rice.
PMID: 22576095 [PubMed - in process]
Vitamin D-mediated regulation of CYP21A2 transcription - A novel mechanism for vitamin D action.
Biochim Biophys Acta. 2012 Apr 27;
Authors: Lundqvist J, Wikvall K, Norlin M
Abstract
BACKGROUND: 1α,25-Dihydroxyvitamin D(3) has recently been reported to decrease expression and activity of CYP21A2. In this paper, we have studied the mechanisms for the 1α,25-dihydroxyvitamin D(3)-mediated effect on CYP21A2 transcriptional rate. METHODS: We have studied the effects of 1α,25-dihydroxyvitamin D(3) using luciferase reporter constructs containing different lengths of the CYP21A2 promoter. These constructs were transfected into cell lines derived from human and mouse adrenal cortex. The mechanism for the effects of vitamin D on the CYP21A2 promoter was studied using chromatin immunoprecipitation assay, mutagenesis and gene silencing by siRNA. RESULTS: 1α,25-Dihydroxyvitamin D(3) was found to alter the promoter activity via a VDR-mediated mechanism, including the comodulators VDR interacting repressor (VDIR) and Williams syndrome transcription factor (WSTF). The involvement of comodulator VDIR was confirmed by gene silencing. We identified a vitamin D response element in the CYP21A2 promoter. Interaction between this novel response element and VDR, WSTF and VDIR was shown by chromatin immunoprecipitation assay. When this sequence was deleted, the effect of 1α,25-dihydroxyvitamin D(3) was abolished, indicating that this sequence in the CYP21A2 promoter functions as a vitamin D response element. Interestingly, an altered balance between nuclear receptors and comodulators reversed the suppressing effect of vitamin D to a stimulatory effect. GENERAL SIGNIFICANCE: This paper reports data important for the understanding of the mechanisms for vitamin D-mediated suppression of gene expression as well as for the vitamin D-mediated effects on CYP21A2. We report a novel mechanism for effects of 1α,25-dihydroxyvitamin D(3).
PMID: 22561756 [PubMed - as supplied by publisher]
Involvement of multiple elements in FXR-mediated transcriptional activation of FGF19.
J Steroid Biochem Mol Biol. 2012 May 2;
Authors: Miyata M, Hata T, Yamakawa H, Kagawa T, Yoshinari K, Yamazoe Y
Abstract
The intestinal endocrine hormone human fibroblast growth factor 19 (FGF19) is involved in the regulation of not only hepatic bile acid metabolism but also carbohydrate and lipid metabolism. In the present study, bile acid/farnesoid X receptor (FXR) responsiveness in the FGF19 promoter region was investigated by a reporter assay using the human colon carcinoma cell line LS174T. The assay revealed the presence of bile acid/FXR-responsive elements in the 5'-flanking region up to 8.8kb of FGF19. Deletion analysis indicated that regions from -1866 to -1833, from -1427 to -1353, and from -75 to +262 were involved in FXR responsiveness. Four, four, and two consecutive half-sites of nuclear receptors were observed in the three regions, respectively. An electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay revealed FXR/retinoid X receptor α (RXRα) heterodimer binding in these three regions. EMSA and reporter assays using mutated constructs indicated that the nuclear receptor IR1, ER2, and DR8 motifs in the 5'-flanking region were involved in FXR responsiveness of FGF19. Lithocholic acid (LCA) (10μM), chenodeoxycholic acid (CDCA) (10μM), or GW4064 (0.1μM) treatment increased reporter activity in a construct including the three motifs under FXR-expressing conditions whereas LCA and not CDCA or GW4064 treatment increased the reporter activity under pregnane X receptor (PXR)-expressing conditions. These results suggest that FGF19 is transcriptionally activated through multiple FXR-responsive elements in the promoter region.
PMID: 22561792 [PubMed - as supplied by publisher]
Bloom's Syndrome and PICH Helicases Cooperate with Topoisomerase IIα in Centromere Disjunction before Anaphase.
PLoS One. 2012;7(4):e33905
Authors: Rouzeau S, Cordelières FP, Buhagiar-Labarchède G, Hurbain I, Onclercq-Delic R, Gemble S, Magnaghi-Jaulin L, Jaulin C, Amor-Guéret M
Abstract
Centromeres are specialized chromosome domains that control chromosome segregation during mitosis, but little is known about the mechanisms underlying the maintenance of their integrity. Centromeric ultrafine anaphase bridges are physiological DNA structures thought to contain unresolved DNA catenations between the centromeres separating during anaphase. BLM and PICH helicases colocalize at these ultrafine anaphase bridges and promote their resolution. As PICH is detectable at centromeres from prometaphase onwards, we hypothesized that BLM might also be located at centromeres and that the two proteins might cooperate to resolve DNA catenations before the onset of anaphase. Using immunofluorescence analyses, we demonstrated the recruitment of BLM to centromeres from G2 phase to mitosis. With a combination of fluorescence in situ hybridization, electron microscopy, RNA interference, chromosome spreads and chromatin immunoprecipitation, we showed that both BLM-deficient and PICH-deficient prometaphase cells displayed changes in centromere structure. These cells also had a higher frequency of centromeric non disjunction in the absence of cohesin, suggesting the persistence of catenations. Both proteins were required for the correct recruitment to the centromere of active topoisomerase IIα, an enzyme specialized in the catenation/decatenation process. These observations reveal the existence of a functional relationship between BLM, PICH and topoisomerase IIα in the centromere decatenation process. They indicate that the higher frequency of centromeric ultrafine anaphase bridges in BLM-deficient cells and in cells treated with topoisomerase IIα inhibitors is probably due not only to unresolved physiological ultrafine anaphase bridges, but also to newly formed ultrafine anaphase bridges. We suggest that BLM and PICH cooperate in rendering centromeric catenates accessible to topoisomerase IIα, thereby facilitating correct centromere disjunction and preventing the formation of supernumerary centromeric ultrafine anaphase bridges.
PMID: 22563370 [PubMed - in process]