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Novel in vivo targets of DeltaNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach.

ABSTRACT: BACKGROUND: p63 is a transcription factor that plays an important role in skin epidermal development and differentiation. The p63 gene encodes for two major protein isoforms, those containing an amino-terminal trans-activation domain (TAp63) and those lacking this domain (DeltaNp63). Both the TA and DeltaN transcripts are also alternatively spliced at the 3' end producing proteins with unique C-termini that are designated as alpha, beta and gamma isoforms. Recent research has suggested that DeltaNp63 is the predominant isoform expressed and active in keratinocytes. RESULTS: To better elucidate the biological role of p63 in regulating gene expression in keratinocytes we performed chromatin immunoprecipitation (ChIP) experiments with DeltaNp63-specific antibodies. We included an additional step in the ChIP procedure to enrich for DeltaNp63 targets by screening the library of immunoprecipitated DNA for its ability to bind recombinant GST-DeltaNp63. Cloning of DeltaNp63-ChIP-derived DNA fragments identified more than 60 potential DeltaNp63 target loci that were located close to or embedded within known or predicted genes. Identity of these target genes suggests that they may participate in a myriad of cellular processes including transcriptional regulation, signaling and metabolism. Here we confirm the binding of DeltaNp63 to several of these genomic loci both by EMSA and replicate ChIP assays. Finally we show that the expression of many of these target genes is altered when DeltaNp63 levels in keratinocytes are reduced by siRNA, further confirming that these are bona fide targets. CONCLUSION: This unbiased genomic approach has allowed us to uncover functional targets of DeltaNp63 and serves as the initial step in further analysis of the transcriptional regulatory mechanisms that are governed by p63 in keratinocytes.

SUBMITTER: Birkaya B

PROVIDER: S-EPMC1890296 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Novel in vivo targets of DeltaNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach.

Birkaya Barbara B Ortt Kori K Sinha Satrajit S

BMC molecular biology 20070523

<h4>Background</h4>p63 is a transcription factor that plays an important role in skin epidermal development and differentiation. The p63 gene encodes for two major protein isoforms, those containing an amino-terminal trans-activation domain (TAp63) and those lacking this domain (DeltaNp63). Both the TA and DeltaN transcripts are also alternatively spliced at the 3' end producing proteins with unique C-termini that are designated as alpha, beta and gamma isoforms. Recent research has suggested th ...[more]

PMID: 17521434

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Project description:The hepatitis B-X (HBx) protein is strongly associated with hepatocellular carcinoma. It is implicated not to directly cause cancer but to play a role in hepatocellular carcinoma as a co-factor. The oncogenic potential of HBx primarily lies in its interaction with transcriptional regulators resulting in aberrant gene expression and deregulated cellular pathways. Utilizing ultraviolet irradiation to simulate a tumor-initiating event, we integrated chip-based chromatin immunoprecipitation (ChIP-chip) with expression microarray profiling and identified 184 gene targets directly deregulated by HBx. One-hundred forty-four transcription factors interacting with HBx were computationally inferred. We experimentally validated that HBx interacts with some of the predicted transcription factors (pTF) as well as the promoters of the deregulated target genes of these pTFs. Significantly, we demonstrated that the pTF interacts with the promoters of the deregulated HBx target genes and that deregulation by HBx of these HBx target genes carrying the pTF consensus sequences can be reversed using pTF small interfering RNAs. The roles of these deregulated direct HBx target genes and their relevance in cancer was inferred via querying against biogroup/cancer-related microarray databases using web-based NextBio(TM) software. Six pathways, including the Jak-STAT pathway, were predicted to be significantly deregulated when HBx binds indirectly to direct target gene promoters. In conclusion, this study represents the first ever demonstration of the utilization of ChIP-chip to identify deregulated direct gene targets from indirect protein-DNA binding as well as transcriptional factors directly interacting with HBx. Increased knowledge of the gene/transcriptional factor targets of HBx will enhance our understanding of the role of HBx in hepatocellular carcinogenesis and facilitate the design of better strategies in combating hepatitis B virus-associated hepatocellular carcinoma.

Project description:Study questionCan novel meiotic RNA targets of DAZL (deleted in azoospermia-like) be identified in the human foetal ovary?Summary answerSYCP1 (synaptonemal complex protein-1), TEX11 (testis expressed 11) and SMC1B (structural maintenance of chromosomes 1B) are novel DAZL targets in the human foetal ovary, thus DAZL may have previously unrecognised roles in the translational regulation of RNAs involved in chromosome cohesion and DNA recombination in the oocyte from the time of initiation of meiosis.What is known alreadyThe phenotype of Dazl deficiency in mouse is infertility in both sexes and DAZL has also been linked to infertility in humans. Few studies have explored targets of this RNA-binding protein. The majority of these investigations have been carried out in mouse, and have focussed on the male thus the basis for its central function in regulating female fertility is largely unknown.Study design size, durationWe carried out RNA sequencing after immunoprecipitation of endogenous DAZL from human foetal ovarian tissue (17 weeks of gestation, obtained after elective termination of pregnancy) to identify novel DAZL targets involved in meiosis (n = 3 biological replicates).Participants/materials, setting, methodsUsing quantitative RT-PCR, we examined the expression of selected RNAs identified by our immunoprecipitation across gestation, and visualised the expression of potential target SMC1B in relation to DAZL, with a combination of in situ hybridisation and immunohistochemistry. 3' untranslated region (3'UTR)-luciferase reporter assays and polysome profile analysis were used to investigate the regulation of three RNA targets by DAZL, representing key functionalities: SYCP1, TEX11 and SMC1B.Main results and the role of chanceWe identified 764 potential RNA targets of DAZL in the human foetal ovary (false discovery rate 0.05 and log-fold change ≥ 2), with functions in synaptonemal complex formation (SYCP1, SYCP3), cohesin formation (SMC1B, RAD21), spindle assembly checkpoint (MAD2L1, TRIP13) and recombination and DNA repair (HORMAD1, TRIP13, TEX11, RAD18, RAD51). We demonstrated that the translation of novel targets SYCP1 (P = 0.004), TEX11 (P = 0.004) and SMC1B (P = 0.002) is stimulated by the presence of DAZL but not by a mutant DAZL with impaired RNA-binding activity.Large scale dataThe raw data are available at GEO using the study ID: GSE81524.Limitations, reasons for cautionThis analysis is based on identification of DAZL targets at the time when meiosis starts in the ovary: it may have other targets at other stages of oocyte development, and in the testis. Representative targets were validated, but detailed analysis was not performed on the majority of putative targets.Wider implications of the findingsThese data indicate roles for DAZL in the regulation of several key functions in human oocytes. Through the translational regulation of novel RNA targets SMC1B and TEX11, DAZL may have a key role in regulating chromosome cohesion and DNA recombination; two processes fundamental in determining oocyte quality and whose establishment in foetal life may support lifelong fertility.Study funding and competing interest(s)This study was supported by the UK Medical Research Council (grant no G1100357 to R.A.A. and an intramural MRC programme grant to I.R.A.). The authors declare no competing interests.

Dataset Information

Novel in vivo targets of DeltaNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach.

Publications

Novel in vivo targets of DeltaNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach.

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