Association between proteins and DNA is crucial for many vital cellular functions such as gene transcription, DNA replication and recombination, repair, segregation, chromosomal stability, cell cycle progression, and epigenetic silencing. It is important to know the genomic targets of DNA-binding proteins and the mechanisms by which they control and guide gene regulation pathways and cellular proliferation. Chromatin immunoprecipitation (ChIP) is an important technique in the study of protein-gene interactions. Using ChIP, DNA-protein interactions are studied within the context of the cell. The basic steps in this technique are fixation, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. Although ChIP is a very versatile tool, the procedure requires the optimization of reaction conditions. Several modifications to the original ChIP technique have been published to improve the success and to enhance the utility of this procedure. This review addresses the critical parameters and the variants of ChiP as well as the different analytical tools that can be combined with ChIP to enable better understanding of DNA-protein interactions in vivo.
The histone H1t gene is expressed exclusively in testis primary spermatocytes. Previous studies indicate that accumulation of H1t mRNA occurs only in primary spermatocytes in normal rats and in transgenic mice bearing the rat H1t transgene. In this study, DNA sequences of human, monkey, mouse, and rat H1t genes were compared and found to be almost identical in the proximal promoter region extending from the H1/AC box through the TATAA box. In addition to conserved elements common to replication-dependent H1 promoters, the H1t promoter contains a unique TE element, and sequences within this element may contribute to enhanced expression of the gene in primary spermatocytes. Two imperfect inverted repeat sequences designated TE1 and TE2, that are located within the larger TE element, overlap a central GC-rich region and bind specifically to nuclear proteins derived from primary spermatocytes. Protein interactions characterized by methylation interference and UV cross-linking experiments indicate that a complex of proteins with a molecular mass of approximately 180 kDa binds TE1. The GC-rich region in H1t and in some replication dependent histone H1 promoters contains an Sp1 consensus sequence. Although the H1t/TE element that contains the GC-rich region binds nuclear proteins, it does not appear to bind Sp1 obtained from cell populations enriched in primary spermatocytes as determined by electrophoretic mobility supershift assays using polyclonal anti-Sp1 antibodies.
Transcriptional activation of the testis-specific histone H1t gene occurs in pachytene primary spermatocytes during spermatogenesis. Specific binding of testis nuclear proteins to a rat histone H1t promoter sequence, designated the H1t/TE element, correlates with the onset of transcription. This element, located between the H1t/AC box and the H1t/CCAAT box, contains inverted repeats of a shorter element. When the native rat H1t gene along with flanking sequences, including 2453 base pairs (bp) upstream and 3784 bp downstream from the coding region, was microinjected into mouse embryos, the offspring of the resulting transgenic mice transcribed the transgene in a tissue-specific manner and only in primary spermatocytes. In the present study the TE promoter element was deleted and replaced with a heterologous stuffer DNA fragment. When the mutant rat DNA fragment was used to create transgenic mice, offspring of the mice bearing the promoter mutation did not transcribe the rat H1t gene in any tissue. On the other hand, transcription of the rat H4t transgene, which is located approximately 1.5 kilobases downstream from the H1t gene, occurred in these animals. Therefore, these studies support the hypothesis that the TE element is essential for enhanced testis-specific transcription of the H1t gene in primary spermatocytes.
The testis-specific histone Hlt gene is transcribed only in testis. The appearance of testis-specific nuclear proteins that bind to a unique promoter sequence element designated Hlt/TE located between the H1/AC box and the H1/CCAAT box correlates with the onset of transcription of the Hlt gene during the meiotic cell cycle. In order to determine whether sequences flanking the rat Hlt gene are sufficient to confer tissue-specific expression in vivo, a 6859 bp EcoRI restriction fragment of genomic DNA containing the rat histone Hlt gene has been microinjected into mouse embryos. S1 nuclease protection analysis has shown that the descendants of the resulting transgenic mice express the rat gene in the proper tissue and at the proper meiotic cell cycle stage. Furthermore, when populations of mouse testis cells were prepared by centrifugal elutriation, only the fraction enriched in pachytene primary spermatocytes had a significant steady-state level of rat Hlt mRNA. Although the copy-number of the transgene was variable in these animals, rat Hlt mRNA levels in high copy-number animals never exceeded 2.6 times the level in normal rat testes. The appearance of appropriate meiotic cell cycle-specific transcription indicates the importance of the conserved promoter sequence elements between the two species.
The testis-specific histone H1t is synthesized exclusively in late pachytene primary spermatocytes during spermatogenesis. The mechanisms involved in transcriptional repression of the H1t gene during development before the spermatocyte stage and in later stages of germinal cell maturation and in nonexpressing somatic tissues are unknown. To assess the contribution of the upstream DNA sequence to H1t transcriptional silencing in nonexpressing cells, a set of histone H1t-promoted reporter vectors was constructed. Transient transfection of mouse C127I cells with these reporter vectors allowed us to identify a transcriptional silencer located between 948 base pairs (bp) and 780 bp upstream from the H1t transcriptional initiation site. Histone H1t-promoted luciferase activity increased 4-fold when the region between 948 bp and 875 bp upstream from the transcriptional initiation site was eliminated. Addition of a 73-bp rat H1t promoter fragment (-948 to -875, containing the 5' portion of the silencer region) to a site immediately upstream from the histone H1d proximal promoter led to significantly reduced luciferase expression upon transient transfection (56% in C127I cells and 44% in HeLa cells). Nuclear proteins were found to bind to DNA within the H1t silencer region when assayed by in vitro deoxyribonuclease (DNase) I footprinting. Thus, our data suggest that an active transcriptional silencer mechanism involving a specific and autonomous H1t promoter element (nucleotides -948/-875) may be operative to minimize expression of the H1t gene in nontesticular cells.
BackgroundTranscriptional silencing associated with aberrant promoter methylation has been established as an alternate pathway for the development of cancer by inactivating tumor suppressor genes. TMS1 (Target of Methylation induced Silencing), also known as ASC (Apoptosis Speck like protein containing a CARD) is a tumor suppressor gene which encodes for a CARD (caspase recruitment domain) containing regulatory protein and has been shown to promote apoptosis directly and by activation of downstream caspases. This study describes the methylation induced silencing of TMS1/ASC gene in prostate cancer cell lines. We also examined the prevalence of TMS1/ASC gene methylation in prostate cancer tissue samples in an effort to correlate race and clinico-pathological features with TMS1/ASC gene methylation.ResultsLoss of TMS1/ASC gene expression associated with complete methylation of the promoter region was observed in LNCaP cells. Gene expression was restored by a demethylating agent, 5-aza-2'deoxycytidine, but not by a histone deacetylase inhibitor, Trichostatin A. Chromatin Immunoprecipitation (ChIP) assay showed enrichment of MBD3 (methyl binding domain protein 3) to a higher degree than commonly associated MBDs and MeCP2. We evaluated the methylation pattern in 66 prostate cancer and 34 benign prostatic hyperplasia tissue samples. TMS1/ASC gene methylation was more prevalent in prostate cancer cases than controls in White patients (OR 7.6, p 0.002) while no difference between the cases and controls was seen in Black patients (OR 1.1, p 0.91).ConclusionOur study demonstrates that methylation-mediated silencing of TMS1/ASC is a frequent event in prostate cancer, thus identifying a new potential diagnostic and prognostic marker for the treatment of the disease. Racial differences in TMS1/ASC methylation patterns implicate the probable role of molecular markers in determining in susceptibility to prostate cancer in different ethnic groups.
The testis-specific histone H1t gene is expressed only in pachytene primary spermatocytes during spermatogenesis. There is a correlation between the specific binding of testis nuclear proteins to a rat histone H1t promoter sequence, designated the H1t/TE element, and the onset of transcription in primary spermatocytes. Our laboratory has shown that mice bearing the rat gene with a deletion of the TE promoter element and replacement with a heterologous stuffer DNA fragment fail to express the rat H1t transgene in any tissue. In this study we report that five CpGs located within the H1t proximal promoter, including two CpGs located within the essential TE promoter element, contain unmethylated cytosines in vivo in genomic DNA derived from primary spermatocytes where the H1t gene is expressed. All seven CpGs are hypermethylated in vivo in genomic DNA derived from liver cells where gene expression is repressed. Further, in vitro methylation of an H1t promoter-driven reporter plasmid markedly reduced expression in a transient transfection assay system. These results suggest that cytosine methylation may contribute to the transcriptional silencing of the testis-specific histone H1t gene in nonexpressing tissues such as liver.
The recently discovered de novo methyltransferases DNMT3a and DNMT3b have been shown to be critical to embryonic development. However, at a single gene level, little is known about how the methylation pattern is established during development. The avian embryonic -globin gene promoter is completely unmethylated in 4-day-old chicken embryonic erythroid cells, where it is expressed at a high level, and completely methylated in adult erythroid cells, where it is silent. The methylation pattern of the -globin gene promoter, proximal transcribed region, and distal transcribed region on both DNA strands was examined during development in chicken erythroid cells. It was found that de novo methylation targets the CpG-dense proximal transcribed region on the coding (top) strand initially, followed by spreading into the 3 region and into the promoter region. Methylation of the template (bottom) strand lags behind that of the coding strand, and complete methylation of both strands occurs only after the gene has been silenced. The results of the study indicate that establishment of the de novo methylation pattern involves strand-specificity and methylation spreading. IntroductionDNA methylation in eukaryotes involves addition of a methyl group to the carbon 5 position of the cytosine ring. This reaction is catalyzed by DNA methyltransferase in the context of the sequence 5Ј-CG-3Ј, which is also referred to as a CpG dinucleotide. 1 Eukaryotic genomes are not methylated uniformly but contain methylated regions interspersed with unmethylated domains. 2 Approximately 70% to 80% of the CpG residues in most vertebrates are methylated. 3 In contrast to the rest of the genome, smaller regions of DNA called CpG islands are unmethylated and possess the expected CpG frequency. 4,5 During early development a dramatic reduction in methylation levels occurs in the preimplantation embryo. 6 This is followed by a wave of de novo methylation involving most CpG residues. However, CpG island-associated promoter regions are protected from methylation by mechanisms which remain unclear. 7 The methylation profile of genes in the adult is stable over many cell generations. Genomic methylation patterns are conserved after DNA replication by the DNA methyltransferase Dnmt1, which is the major maintenance methyltransferase. 8 Dnmt1 is recruited to replicating DNA to reproduce the methylation pattern of the parental strands in the daughter strands. 9 Inactivation of the mouse Dnmt1 gene by gene targeting resulted in extensive demethylation of all sequences examined. 10,11 However, ES cells completely lacking Dnmt1 were still capable of methylating retroviral DNA de novo. 11 The search for the de novo methyltransferases led to the discovery of Dnmt3a and Dnmt3b. 12 These were found to be essential for de novo methylation and for mouse development. 13 However, it remains unclear how de novo methylation patterns are established during development, over what time intervals these changes occur, or if this process involves any strand-or sequence-specifici...
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