Combined chromatin immunoprecipitation and next-generation sequencing (ChIP-seq) has enabled genome-wide epigenetic profiling of numerous cell lines and tissue types. A major limitation of ChIP-seq, however, is the large number of cells required to generate high-quality data sets, precluding the study of rare cell populations. Here, we present an ultra-low-input micrococcal nuclease-based native ChIP (ULI-NChIP) and sequencing method to generate genome-wide histone mark profiles with high resolution from as few as 10 3 cells. We demonstrate that ULI-NChIP-seq generates high-quality maps of covalent histone marks from 10 3 to 10 6 embryonic stem cells. Subsequently, we show that ULI-NChIP-seq H3K27me3 profiles generated from E13.5 primordial germ cells isolated from single male and female embryos show high similarity to recent data sets generated using 50-180 Â more material. Finally, we identify sexually dimorphic H3K27me3 enrichment at specific genic promoters, thereby illustrating the utility of this method for generating high-quality and -complexity libraries from rare cell populations.

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Transcription of endogenous retroviruses (ERVs) is inhibited by de novo DNA methylation during gametogenesis, a process initiated after birth in oocytes and at approximately embryonic day 15.5 (E15.5) in prospermatogonia. Earlier in germline development, the genome, including most retrotransposons, is progressively demethylated. Young ERVK and ERV1 elements, however, retain intermediate methylation levels. As DNA methylation reaches a low point in E13.5 primordial germ cells (PGCs) of both sexes, we determined whether retrotransposons are marked by H3K9me3 and H3K27me3 using a recently developed low-input ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing) method. Although these repressive histone modifications are found predominantly on distinct genomic regions in E13.5 PGCs, they concurrently mark partially methylated long terminal repeats (LTRs) and LINE1 elements. Germline-specific conditional knockout of the H3K9 methyltransferase SETDB1 yields a decrease of both marks and DNA methylation at H3K9me3-enriched retrotransposon families. Strikingly, Setdb1 knockout E13.5 PGCs show concomitant derepression of many marked ERVs, including intracisternal A particle (IAP), ETn, and ERVK10C elements, and ERV-proximal genes, a subset in a sex-dependent manner. Furthermore, Setdb1 deficiency is associated with a reduced number of male E13.5 PGCs and postnatal hypogonadism in both sexes. Taken together, these observations reveal that SETDB1 is an essential guardian against proviral expression prior to the onset of de novo DNA methylation in the germline.

Among the earliest responses of mammalian cells to DNA damage is catalytic activation of a nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1). Activated PARP-1 forms the polymers of ADPribose (pADPr or PAR) that posttranslationally modify its target proteins, such as PARP-1 and DNA repair-related proteins. Although this metabolism is known to be implicated in other repair pathways, here we show its role in the versatile nucleotide excision repair pathway (NER) that removes a variety of DNA damages including those induced by UV. We show that PARP inhibition or specific depletion of PARP-1 decreases the efficiency of removal of UV-induced DNA damage from human skin fibroblasts or mouse epidermis. Using NER-proficient and -deficient cells and in vitro PARP-1 assays, we show that damaged DNA-binding protein 2 (DDB2), a key lesion recognition protein of the global genomic subpathway of NER (GG-NER), associates with PARP-1 in the vicinity of UV-damaged chromatin, stimulates its catalytic activity, and is modified by pADPr. PARP inhibition abolishes UV-induced interaction of DDB2 with PARP-1 or xeroderma pigmentosum group C (XPC) and also decreases localization of XPC to UV-damaged DNA, which is a key step that leads to downstream events in GG-NER. Thus, PARP-1 collaborates with DDB2 to increase the efficiency of the lesion recognition step of GG-NER.M ammalian cells respond very rapidly to different types of DNA damage by activation of an abundant and ubiquitous nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1). The activated PARP-1 uses NAD + to form polymers of ADP-ribose (pADPr or PAR) that modify PARP-1 itself and selected target proteins, such as histones and DNA repair proteins (1). This posttranslational modification, i.e., PARylation, has been implicated in cellular responses ranging from DNA repair to cell death. Among mammalian DNA repair pathways, PARP-1 has been implicated in base excision repair, homologous recombination, and nonhomologous end-joining pathways (2, 3), but we do not know its role in the most versatile nucleotide excision repair (NER) pathway that removes a wide variety of DNA lesions, including UV-induced thymine dimers (T-T) and other cyclobutane pyrimidine dimers (CPD), as well as 6-4 photoproducts (6-4PP) (4).The core mammalian NER pathway uses more than 30 proteins to recognize the damaged site on DNA, remove 24-to 32-nucleotide-long single-stranded DNA containing the lesion, fill the gap using the nondamaged strand as a template, and finally ligate the nick (4). There are two subpathways of NER: the transcription-coupled NER (TC-NER) removes lesions from the actively transcribed strands of the genes and the global genomic NER (GG-NER) repairs lesions from the entire genome. These two pathways differ in the initial step of lesion recognition: TC-NER is initiated when elongating RNA polymerase II stalls at the lesion, whereas GG-NER is initiated when the lesion is recognized in the chromatin context by DDB2 (XPE), which through its participation in the UV-DDB-E3 ligase complex ub...