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1.
Mapping DNase I hypersensitive sites is an accurate method of identifying the location of gene regulatory elements, including promoters, enhancers, silencers and locus control regions. Although Southern blots are the traditional method of identifying DNase I hypersensitive sites, the conventional manual method is not readily scalable to studying large chromosomal regions, much less the entire genome. Here we describe DNase-chip, an approach that can rapidly identify DNase I hypersensitive sites for any region of interest, or potentially for the entire genome, by using tiled microarrays. We used DNase-chip to identify DNase I hypersensitive sites accurately from a representative 1% of the human genome in both primary and immortalized cell types. We found that although most DNase I hypersensitive sites were present in both cell types studied, some of them were cell-type specific. This method can be applied globally or in a targeted fashion to any tissue from any species with a sequenced genome.  相似文献   

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We have developed a new high resolution method for screening 400-600 base pairs of DNA in chromatin for DNase I hypersensitive sites and protein-DNA interactions. By separating the DNA isolated from nuclease-digested nuclei in small, native polyacrylamide gels prior to electroblotting onto nylon membranes, we increased the resolution by greater than 3-fold as compared with the traditional approach whereby the nuclease-digested DNA is fractionated electrophoretically in agarose gels (11). In addition, our native genomic blotting method has the advantage of combining the ability of the traditional agarose approach to detect DNase I hypersensitive sites, with the genomic sequencing method (2), where individual protein-DNA contacts can be observed. Native genomic blotting therefore permits for the first time the display of DNase I hypersensitive sites and protein-DNA interactions at high resolution on the same autoradiograph. This method allows us to investigate a new level of chromatin structure and to therefore obtain better insight into levels of gene structure, organization and gene regulation.  相似文献   

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YM Wang  P Zhou  LY Wang  ZH Li  YN Zhang  YX Zhang 《PloS one》2012,7(8):e42414
Mapping DNase I hypersensitive sites (DHSs) within nuclear chromatin is a traditional and powerful method of identifying genetic regulatory elements. DHSs have been mapped by capturing the ends of long DNase I-cut fragments (>100,000 bp), or 100-1200 bp DNase I-double cleavage fragments (also called double-hit fragments). But next generation sequencing requires a DNA library containing DNA fragments of 100-500 bp. Therefore, we used short DNA fragments released by DNase I digestion to generate DNA libraries for next generation sequencing. The short segments are 100-300 bp and can be directly cloned and used for high-throughput sequencing. We identified 83,897 DHSs in 2,343,479 tags across the human genome. Our results indicate that the DHSs identified by this DHS assay are consistent with those identified by longer fragments in previous studies. We also found: (1) the distribution of DHSs in promoter and other gene regions of similarly expressed genes differs among different chromosomes; (2) silenced genes had a more open chromatin structure than previously thought; (3) DHSs in 3'untranslated regions (3'UTRs) are negatively correlated with level of gene expression.  相似文献   

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The role of local sequence information in establishing the chromatin structure of the human c-myc upstream region (MUR) was investigated. Adeno-associated virus (AAV)-mediated gene transduction was used to introduce an additional unrearranged copy of the 2.4 kb HindIII-XhoI fragment of the MUR into a novel location in the genome in each of two cloned HeLa cell lines. The AAV-based rep- cap- viral vector SKMA used to transduce the MUR retained only 1.4 kb (24%) of the AAV genome and could accommodate inserts as large as 2.4 kb. SKMA was capable of infecting HeLa cells and integrating into the host genome at single copy number. Integration may have occurred at a preferred site in the HeLa genome, but this site was apparently distinct from the previously identified preferred AAV integration site on human chromosome 19. Indirect end-labelling was used to map DNase I and micrococcal nuclease (MNase) cleavage sites over the transduced c-myc sequences and the endogenous c-myc loci in infected HeLa cells. A similarly ordered chromatin domain, extending 5' from c-myc promoter P0, was found to exist at the transduced c-myc locus in each clone. The position and relative sensitivity of 13 MNase cleavage sites and five DNase I hypersensitive sites, originally identified at the endogenous MUR in non-transduced cells, were shown to be conserved when this DNA was moved to a new chromosome site. A conserved DNase I hypersensitive site also was mapped to the region between the left AAV terminal repeat and AAV promoter P5. These results suggest that the information required to establish the particular chromatin structure of the MUR resides within the local DNA sequence of that region.  相似文献   

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Binding of nogalamycin and adriamycin with Sarcoma-180 ascites tumor cell chromatin was studied by a spectrofluorometric method. There was significant reduction in the number of available drug binding sites per nucleotide when the chromatin was digested with DNase I for a period which releases only 7% of the chromosomal DNA. Results indicate preferential binding of these drugs with DNase I hypersensitive sites of chromatin. The DNase-I hypersensitive sites of chromatin were shown to correlate to the sequences required for gene expression. Further digestion with DNase I increases availability of drug binding sites, probably due to relaxation of the compact chromatin.  相似文献   

11.
To analyse the relationship between DNA undermethylation at some sites in the ovalbumin and conalbumin gene regions (1) and the expression of these genes in chick oviduct, digestions with HhaI, which differentiates between methylated and unmethylated HhaI restriction sites, was performed on DNA isolated from chicken erythrocyte or oviduct chromatin treated with DNase I which degrades preferentially "active" chromatin. This was followed by analysis with ovalbumin- and conalbumin-specific hybridization probes. We conclude that the residual DNA methylation found at some sites of the ovalbumin and conalbumin gene regions is derived from the fraction of cells in which the chromatin of these genes is not in an "active" form. On the other hand, the ovalbumin and conalbumin sites which are partially unmethylated in erythrocyte DNA correspond to chromatin regions which are not DNase I-senitive. We have also detected a site about 1 kb downstream from the 3' end of the conalbumin gene that is hypersensitive to DNase I in all tissues tested.  相似文献   

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The DNase I sensitivity of chromosomal DNA regions carrying integrated proviral genomes of Moloney (M-MuLV) and AKR Murine Leukemia Virus (AKR-MuLV), and the cellular homologue of the mos-gene (c-mos) of Moloney Sarcoma Virus (MSV) were studied in tumor tissues of leukemic mice. The genetically transmitted sequences of M-MuLV, AKR-MuLV, and the c-mos gene are all in DNase I resistant chromatin conformations in M-MuLV-induced tumors. Each M-MuLV-induced tumor contained at least one somatically acquired integrated recombinant MuLV genome that displayed two main characteristic features of active chromatin: a) a configuration hypersensitive to DNase I, and b) extensive hypomethylation. DNase I hypersensitive sites were mapped at the junction of cellular sequences and the 5'-viral large terminal repeat (LTR). Expression of a recombinant MuLV seems therefore to be a necessary feature to maintain the transformed state.  相似文献   

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Structure of transcriptionally active chromatin   总被引:18,自引:0,他引:18  
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染色质转座酶可及性测序研究进展   总被引:1,自引:0,他引:1  
吴杰  全建平  叶勇  吴珍芳  杨杰  杨明  郑恩琴 《遗传》2020,(4):333-346
染色质转座酶可及性测序(assay for transposase-accessible chromatin with high-throughput sequencing,ATAC-seq)诞生于2013年,具有比脱氧核糖核酸酶I超敏感位点测序(deoxyribonuclease I hypersensitive site sequencing, DNase-seq)和微球菌核酸酶敏感位点测序(micrococcal nuclease sequencing, MNase-seq)更快速、灵敏、简便的优点,是目前分析全基因组范围染色质开放区域的热点技术。通过该技术能获得染色质开放区域的相关信息,从而映射出转录因子等调控蛋白的结合区域和核小体定位等信息,对于研究表观遗传分子机制具有重要意义。本文比较了5种获取染色质开放区域技术的优缺点,重点介绍了ATAC-seq的原理和主要流程,描述了利用ATAC-seq技术研究染色质开放区域的发展概况以及ATAC-seq的相关应用,期望对真核生物全基因组水平的染色质开放区域研究、顺式调控元件鉴定以及遗传调控网络的解析等提供借鉴。  相似文献   

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DNA topoisomerase II-β (TOP2B) is fundamental to remove topological problems linked to DNA metabolism and 3D chromatin architecture, but its cut-and-reseal catalytic mechanism can accidentally cause DNA double-strand breaks (DSBs) that can seriously compromise genome integrity. Understanding the factors that determine the genome-wide distribution of TOP2B is therefore not only essential for a complete knowledge of genome dynamics and organization, but also for the implications of TOP2-induced DSBs in the origin of oncogenic translocations and other types of chromosomal rearrangements. Here, we conduct a machine-learning approach for the prediction of TOP2B binding using publicly available sequencing data. We achieve highly accurate predictions, with accessible chromatin and architectural factors being the most informative features. Strikingly, TOP2B is sufficiently explained by only three features: DNase I hypersensitivity, CTCF and cohesin binding, for which genome-wide data are widely available. Based on this, we develop a predictive model for TOP2B genome-wide binding that can be used across cell lines and species, and generate virtual probability tracks that accurately mirror experimental ChIP-seq data. Our results deepen our knowledge on how the accessibility and 3D organization of chromatin determine TOP2B function, and constitute a proof of principle regarding the in silico prediction of sequence-independent chromatin-binding factors.  相似文献   

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A Almer  W H?rz 《The EMBO journal》1986,5(10):2681-2687
The chromatin structure of two tandemly linked acid phosphatase genes (PHO5 and PHO3) from Saccharomyces cerevisiae was analyzed under conditions at which the strongly regulated PHO5 gene is repressed. Digestion experiments with DNase I, DNase II, micrococcal nuclease and restriction nucleases reveal the presence of five hypersensitive sites at the PHO5/PHO3 locus, two of them upstream of PHO5 at distances of 920 and 370 bp, one in between the two genes and two downstream of PHO3. Specifically positioned nucleosomes are located next to these hypersensitive sites as shown by indirect end-labeling experiments. The positions deduced from these experiments could be verified by monitoring the accessibility of various restriction sites to the respective nucleases. Sites within putative linker regions were about 50-60% susceptible, whereas sites located within nucleosome cores were resistant. Hybridizing micrococcal nuclease digests to a probe from in between the two upstream hypersensitive sites leads to an interruption of an otherwise regular nucleosomal DNA pattern. This shows directly that these hypersensitive sites represent gaps within ordered nucleosomal arrays.  相似文献   

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