PARP1 is an abundant and ubiquitous nuclear enzyme that catalyzes the transfer of poly(ADP-ribose) (pADPr) moiety from NAD to either a protein acceptor or an existing pADPr chain (1
). PARP1 regulates many cellular functions, including stress-induced apoptosis, DNA damage detection and repair (2
), transcriptional regulation and chromatin remodeling (5
), and the control of gene expression by the induction of chromatin loosening at targeted genetic loci (8
). The distribution of PARP1 in chromatin is nonrandom and globally regulates transcription (9
). The dynamic regulation of poly(ADP-ribose) polymerase 1 protein binding to chromatin is mediated by nucleosomal core histones (10
). For example, PARP1 and histone H1 exhibit a reciprocal pattern of chromatin binding at many RNA polymerase II-transcribed promoters (11
). Since PARP1 is involved in the regulation of so many cellular mechanisms, we were inspired to study its genome-wide locations in the human genome in interphase and mitotic cells. Results of this work reveal the true loci of PARP1 in mitotic chromatin, allowing us to further understand the molecular mechanisms of PARP1-dependent processes.
In order to identify PARP1 protein binding sites in the human genome, we applied chromatin immunoprecipitation followed by sequencing (ChIP–seq). In ChIP–seq experiments, the precipitated ChIP-DNA fragments of interest are sequenced directly. In comparison to microarray, ChIP–seq has higher resolution, generates fewer artifacts, and provides greater coverage and a larger dynamic range. ChIP-seq studies have been used to characterize transcription factor binding (12
), genome-wide nucleosome positioning (15
), and to determine epigenetic changes (16
). ChIP-seq technology does not require very long sequencing reads. Large numbers of short reads (35 bp) are sufficient for mapping binding sites in most organisms. Therefore, Illumina/Solexa and ABI/SOLiD have been favored over Roche/454 because they both generate millions of very short reads (about 35 bases/read), whereas Roche/454 generates fewer reads, but longer length (200–300 bases/read). These three main sequencing technologies are utilized on the basis of their applications. As a control, input DNA, consisting of nonimmunoprecipitated, sonicated and cross-linked DNA, has great importance in ChIP-Seq studies, as ChIP DNA samples are normally scored against the input DNA for transcription factor binding site (TFBS) identification (17
). Even after successfully extracting ChIP-seq raw data, determination of binding sites from the data remains a formidable challenge. Therefore, many research groups published different algorithms that allow determining binding sites (18
ChIP-seq can be divided in to the following steps (): 1) ChIP; 2) Library preparation (end repair; addition of an ‘A’ base to the 3′-end of DNA fragments; ligation of adapters to DNA fragments; amplification of adapter-modified DNA fragments and gel purification; pre-sequencing control assays (enrichment check using positive/negative control primers)); and 3) library sequencing (annotation, sequence of DNA and validation by quantitative PCR (qPCR)).
ChIP-seq flow chart. All the steps are same as ChIP up to DNA precipitation; afterward, ChIP-seq steps are followed, adapted from Collas and Dahl (Frontiers in Bioscience) (2008).