A distal enhancer at risk locus 11q13.5 promotes suppression of colitis by Treg cells.

Genetic variations underlying susceptibility to complex autoimmune and allergic diseases are concentrated within noncoding regulatory elements termed enhancers1. The functions of a large majority of disease-associated enhancers are unknown, in part owing to their distance from the genes they regulate, a lack of understanding of the cell types in which they operate, and our inability to recapitulate the biology of immune diseases in vitro. Here, using shared synteny to guide loss-of-function analysis of homologues of human enhancers in mice, we show that the prominent autoimmune and allergic disease risk locus at chromosome 11q13.52-7 contains a distal enhancer that is functional in CD4+ regulatory T (Treg) cells and required for Treg-mediated suppression of colitis. The enhancer recruits the transcription factors STAT5 and NF-κB to mediate signal-driven expression of Lrrc32, which encodes the protein glycoprotein A repetitions predominant (GARP). Whereas disruption of the Lrrc32 gene results in early lethality, mice lacking the enhancer are viable but lack GARP expression in Foxp3+ Treg cells, which are unable to control colitis in a cell-transfer model of the disease. In human Treg cells, the enhancer forms conformational interactions with the promoter of LRRC32 and enhancer risk variants are associated with reduced histone acetylation and GARP expression. Finally, functional fine-mapping of 11q13.5 using CRISPR-activation (CRISPRa) identifies a CRISPRa-responsive element in the vicinity of risk variant rs11236797 capable of driving GARP expression. These findings provide a mechanistic basis for association of the 11q13.5 risk locus with immune-mediated diseases and identify GARP as a potential target in their therapy.

SAMHD1-mediated dNTP degradation is required for efficient DNA repair during antibody class switch recombination.

Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1), a dNTP triphosphohydrolase, regulates the levels of cellular dNTPs through their hydrolysis. SAMHD1 protects cells from invading viruses that depend on dNTPs to replicate and is frequently mutated in cancers and Aicardi-Goutières syndrome, a hereditary autoimmune encephalopathy. We discovered that SAMHD1 localizes at the immunoglobulin (Ig) switch region, and serves as a novel DNA repair regulator of Ig class switch recombination (CSR). Depletion of SAMHD1 impaired not only CSR but also IgH/c-Myc translocation. Consistently, we could inhibit these two processes by elevating the cellular nucleotide pool. A high frequency of nucleotide insertion at the break-point junctions is a notable feature in SAMHD1 deficiency during activation-induced cytidine deaminase-mediated genomic instability. Interestingly, CSR induced by staggered but not blunt, double-stranded DNA breaks was impaired by SAMHD1 depletion, which was accompanied by enhanced nucleotide insertions at recombination junctions. We propose that SAMHD1-mediated dNTP balance regulates dNTP-sensitive DNA end-processing enzyme and promotes CSR and aberrant genomic rearrangements by suppressing the insertional DNA repair pathway.

GARP on hepatic stellate cells is essential for the development of liver fibrosis.

BACKGROUND & AIMS: Glycoprotein A repetitions predominant (GARP) is a membrane protein that functions as a latent TGF-ß docking molecule. While the immune regulatory properties of GARP on blood cells have been studied, the function of GARP on tissue stromal cells remains unclear. Here, we investigate the role of GARP expressed on hepatic stellate cells (HSCs) in the development of liver fibrosis. METHODS: The function of GARP on HSCs was explored in toxin-induced and metabolic liver fibrosis models, using conditional GARP-deficient mice or a newly generated inducible system for HSC-specific gene ablation. Primary mouse and human HSCs were isolated to evaluate the contribution of GARP to the activation of latent TGF-ß. Moreover, cell contraction of HSCs in the context of TGF-ß activation was tested in a GARP-dependent fashion. RESULTS: Mice lacking GARP in HSCs were protected from developing liver fibrosis. Therapeutically deleting GARP on HSCs alleviated the fibrotic process in established disease. Furthermore, natural killer T cells exacerbated hepatic fibrosis by inducing GARP expression on HSCs through IL-4 production. Mechanistically, GARP facilitated fibrogenesis by activating TGF-ß and enhancing endothelin-1-mediated HSC contraction. Functional GARP was expressed on human HSCs and significantly upregulated in the livers of patients with fibrosis. Lastly, deletion of GARP on HSCs did not augment inflammation or liver damage. CONCLUSIONS: GARP expressed on HSCs drives the development of liver fibrosis via cell contraction-mediated activation of latent TGF-ß. Considering that systemic blockade of TGF-ß has major side effects, we highlight a therapeutic niche provided by GARP and surface-mediated TGF-ß activation. Thus, our findings suggest an important role of GARP on HSCs as a promising target for the treatment of liver fibrosis. IMPACT AND IMPLICATIONS: Liver fibrosis represents a substantial and increasing public health burden globally, for which specific treatments are not available. Glycoprotein A repetitions predominant (GARP) is a membrane protein that functions as a latent TGF-ß docking molecule. Here, we show that GARP expressed on hepatic stellate cells drives the development of liver fibrosis. Our findings suggest GARP as a novel target for the treatment of fibrotic disease.

Oligoribonucleotide-Mediated Blockade of DNA Extension by Taq DNA Polymerases Increases Specificity and Sensitivity for Detecting Single-Nucleotide Differences.

Blocking PCR is a method that inhibits amplification of DNA possessing a nucleotide sequence complementary to that of a blocker; the method can be used to suppress amplification of target wild-type DNA while amplifying mutated DNA. Previously, we demonstrated that an oligoribonucleotide (ORN) functions as a cost-effective and sequence-specific blocker. This blocking PCR system, named ORN interference-PCR (ORNi-PCR), is compatible with DNA polymerases lacking 5'-3' exonuclease activity but not with those possessing the activity (e.g., Taq DNA polymerase), which can remove a hybridized ORN during DNA extension. Here, we demonstrate that under specific experimental conditions, an intact or phosphorothioated ORN strongly suppresses extension of target DNA by Taq DNA polymerases. This method was applied successfully to real-time ORNi-PCR and one-step real-time reverse transcription-ORNi-PCR using a dual-labeled fluorescent probe to detect a single-nucleotide mutation in DNA and RNA in a sequence-specific manner. The results reaffirm the utility of blocking PCR and provide technical hints for its improvement.

Development of transgenic mice overexpressing mouse carbonyl reductase 1.

BACKGROUND: Carbonyl reductase 1 (CBR1) is a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductase with broad substrate specificity. CBR1 catalyzes the reduction of numerous carbonyl compounds, including quinones, prostaglandins, menadione, and multiple xenobiotics, while also participating in various cellular processes, such as carcinogenesis, apoptosis, signal transduction, and drug resistance. In this study, we aimed to generate transgenic mice overexpressing mouse Cbr1 (mCbr1), characterize the mCbr1 expression in different organs, and identify changes in protein expression patterns. METHODS AND RESULTS: To facilitate a deeper understanding of the functions of CBR1, we generated transgenic mice overexpressing CBR1 throughout the body. These transgenic mice overexpress 3xFLAG-tagged mCbr1 (3xFLAG-mCbr1) under the CAG promoter. Two lines of transgenic mice were generated, one with 3xFLAG-mCbr1 expression in multiple tissues, and the other, with specific expression of 3xFLAG-mCbr1 in the heart. Pathway and network analysis using transgenic mouse hearts identified 73 proteins with levels of expression correlating with mCbr1 overexpression. The expression of voltage-gated anion channels, which may be directly related to calcium ion-related myocardial contraction, was also upregulated. CONCLUSION: mCbr1 transgenic mice may be useful for further in vivo analyses of the molecular mechanisms regulated by Cbr1; such analyses will provide a better understanding of its effects on carcinogenesis and cardiotoxicity of certain cancer drugs.

A stem cell marker KLF5 regulates CCAT1 via three-dimensional genome structure in colorectal cancer cells.

BACKGROUND: KLF5 plays a crucial role in stem cells of colorectum in cooperation with Lgr5 gene. In this study, we aimed to explicate a regulatory mechanism of the KLF5 gene product from a view of three-dimensional genome structure in colorectal cancer (CRC). METHODS: In vitro engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP)-seq method was used to identify the regions that bind to the KLF5 promoter. RESULTS: We revealed that the KLF5 promoter region interacted with the KLF5 enhancer region as well as the transcription start site (TSS) region of the Colon Cancer Associated Transcript 1 (CCAT1) gene. Notably, the heterodeletion mutants of KLF5 enhancer impaired the cancer stem-like properties of CRC cells. The KLF5 protein participated in the core-regulatory circuitry together with co-factors (BRD4, MED1, and RAD21), which constructs the three-dimensional genome structures consisting of KLF5 promoter, enhancer and CCAT1 TSS region. In vitro analysis indicated that KLF5 regulated CCAT1 expression and we found that CCAT1 expression was highly correlated with KLF5 expression in CRC clinical samples. CONCLUSIONS: Our data propose the mechanistic insight that the KLF5 protein constructs the core-regulatory circuitry with co-factors in the three-dimensional genome structure and coordinately regulates KLF5 and CCAT1 expression in CRC.

Circular RNAs hsa_circ_0001438 and hsa_circ_0000417 are downregulated and upregulated, respectively, in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most predominant type of liver cancer and is frequently fatal. Alpha-fetoprotein, alpha-fetoprotein-L3, and protein induced by vitamin K absence or antagonist-II are used as biomarkers to diagnose HCC. However, these biomarkers are not highly specific, especially for early-stage HCC diagnosis; therefore, more specific biomarkers are needed. Recently, circular RNA (circRNA) biomarkers have been used to diagnose several intractable diseases. In this study, we sought to identify circRNA biomarkers for the specific diagnosis of HCC. To this end, we compared the expression levels of circRNAs in primary HCC and normal tissues using publicly available RNA-seq data. Our analysis revealed that the expression levels of eight circRNAs were altered in primary HCC tissues compared with normal tissues. To confirm our findings, we examined the expression levels of selected circRNAs in HCC cell lines and normal hepatocytes. The expression level of hsa_circ_0001438, a circRNA that was downregulated in primary HCC, was lower in poorly and well-differentiated HCC cell lines than in normal hepatocytes. By contrast, the expression level of hsa_circ_0000417, which was increased in primary HCC, was strongly upregulated in a well-differentiated HCC cell line compared with normal hepatocytes. Thus, hsa_circ_0001438 and hsa_circ_0000417 might be potential biomarkers for the specific diagnosis of HCC. The experimental strategy described here, using publicly available RNA-seq data, is a useful and cost-effective method of identifying circRNA biomarkers.

Discrimination of CpG Methylation Status and Nucleotide Differences in Tissue Specimen DNA by Oligoribonucleotide Interference-PCR.

Oligoribonucleotide (ORN) interference-PCR (ORNi-PCR) is a method in which PCR amplification of a target sequence is inhibited in a sequence-specific manner by the hybridization of an ORN with the target sequence. Previously, we reported that ORNi-PCR could detect nucleotide mutations in DNA purified from cultured cancer cell lines or genome-edited cells. In this study, we investigated whether ORNi-PCR can discriminate nucleotide differences and CpG methylation status in damaged DNA, such as tissue specimen DNA and bisulfite-treated DNA. First, we showed that ORNi-PCR could discriminate nucleotide differences in DNA extracted from acetone-fixed paraffin-embedded rat liver specimens or formalin-fixed paraffin-embedded human specimens. Rat whole blood specimens were compatible with ORNi-PCR for the same purpose. Next, we showed that ORNi-PCR could discriminate CpG methylation status in bisulfite-treated DNA. These results demonstrate that ORNi-PCR can discriminate nucleotide differences and CpG methylation status in multiple types of DNA samples. Thus, ORNi-PCR is potentially useful in a wide range of fields, including molecular biology and medical diagnosis.

Transgenic mouse lines expressing the 3xFLAG-dCas9 protein for enChIP analysis.

We developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology to isolate specific genomic regions while retaining their molecular interactions. In enChIP, the locus of interest is tagged with an engineered DNA-binding molecule, such as a modified form of the clustered regularly interspaced short palindromic repeats (CRISPR) system containing a guide RNA (gRNA) and a catalytically inactive form of Cas9 (dCas9). The locus is then affinity-purified to enable identification of associated molecules. In this study, we generated transgenic mice expressing 3xFLAG-tagged Streptococcus pyogenes dCas9 (3xFLAG-dCas9) and retrovirally transduced gRNA into primary CD4+ T cells from these mice for enChIP. Using this approach, we achieved high yields of enChIP at the targeted genomic region. Our novel transgenic mouse lines provide a valuable tool for enChIP analysis in primary mouse cells.

Simultaneous Detection of the T790M and L858R Mutations in the EGFR Gene by Oligoribonucleotide Interference-PCR.

A de novo single-nucleotide mutation in the EGFR gene can cause the development of lung cancer. EGFR tyrosine kinase inhibitors (EGFR-TKIs) are used for clinical treatment of such lung cancers, but acquired resistance often mitigates their efficacy. Accordingly, monitoring of de novo and acquired nucleotide mutations is essential for clinical treatment of lung cancers with EGFR-TKIs. Previously, we reported that oligoribonucleotide interference-PCR (ORNi-PCR) can accurately and cost-effectively detect single-nucleotide mutations. In this study, we applied ORNi-PCR to simultaneous detection of the de novo L858R and acquired T790M mutations in the EGFR gene in lung cancer cells. First, we established optimal experimental conditions for ORNi-PCR to simultaneously detect the two single-nucleotide mutations in genomic DNA from lung cancer cells. The conditions we established could also be used for ORNi-PCR using complementary DNA reverse-transcribed from extracted RNA. We found that ORNi-PCR could detect lung cancer cells possessing both single-nucleotide mutations among a large number of cells harboring wild-type sequences, even when the cancer cells constituted less than ~0.2% of all cells. Our findings demonstrate that ORNi-PCR can simultaneously detect multiple single-nucleotide mutations in a gene of interest and might therefore be useful for simultaneous detection of EGFR mutations in clinical examinations.

Promoter-associated proteins of EPAS1 identified by enChIP-MS - A putative role of HDX as a negative regulator.

Presence of perivascular neuroblastoma cells with high expression of hypoxia inducible factor (HIF)-2α correlates with distant metastasis and aggressive disease. Regulation of HIFs are traditionally considered to occur post-translationally, but we have recently shown that HIF-2α is unconventionally regulated also at the transcriptional level in neuroblastoma cells. Regulatory factors binding directly to EPAS1 (encoding HIF-2α) to promote transcription are yet to be defined. Here, we employ the novel CRISPR/Cas9-based engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) - mass spectrometry (MS) methodology to, in an unbiased fashion, identify proteins that associate with the EPAS1 promoter under normoxic and hypoxic conditions. Our enChIP analysis resulted in 27 proteins binding to the EPAS1 promoter in neuroblastoma cells. In agreement with a general hypoxia-driven downregulation of gene transcription, the majority (24 out of 27) of proteins dissociate from the promoter at hypoxia. Among them were several nucleosome-associated proteins suggesting a general opening of chromatin as one explanation to induced EPAS1 transcription at hypoxia. Of particular interest from the list of released factors at hypoxia was the highly divergent homeobox (HDX) transcription factor, that we show inversely correlates with HIF-2α in neuroblastoma cells. We propose a putative model where HDX negatively regulates EPAS1 expression through a release-of-inhibition mechanism.

Identification of physical interactions between genomic regions by enChIP-Seq.

Physical interactions between genomic regions play critical roles in the regulation of genome functions, including gene expression. Here, we show the feasibility of using engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) in combination with next-generation sequencing (NGS) (enChIP-Seq) to detect such interactions. In enChIP-Seq, the target genomic region is captured by an engineered DNA-binding complex, such as a clustered regularly interspaced short palindromic repeats (CRISPR) system consisting of a catalytically inactive form of Cas9 and a single guide RNA. Subsequently, the genomic regions that physically interact with the target genomic region in the captured complex are sequenced by NGS. Using enChIP-Seq, we found that the 5'HS5 locus, which is involved in the regulation of globin genes expression at the ß-globin locus, interacts with multiple genomic regions upon erythroid differentiation in the human erythroleukemia cell line K562. Genes near the genomic regions inducibly associated with the 5'HS5 locus were transcriptionally up-regulated in the differentiated state, suggesting the existence of a coordinated transcription mechanism mediated by physical interactions between these loci. Thus, enChIP-Seq might be a potentially useful tool for detecting physical interactions between genomic regions in a nonbiased manner, which would facilitate elucidation of the molecular mechanisms underlying regulation of genome functions.

Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins.

The clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for various biological applications, including genome editing. We developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR to isolate target genomic regions from cells for their biochemical characterization. In this study, we developed 'in vitro enChIP' using recombinant CRISPR ribonucleoproteins (RNPs) to isolate target genomic regions. in vitro enChIP has the great advantage over conventional enChIP of not requiring expression of CRISPR complexes in cells. We first showed that in vitro enChIP using recombinant CRISPR RNPs can be used to isolate target DNA from mixtures of purified DNA in a sequence-specific manner. In addition, we showed that this technology can be used to efficiently isolate target genomic regions, while retaining their intracellular molecular interactions, with negligible contamination from irrelevant genomic regions. Thus, in vitro enChIP technology is of potential use for sequence-specific isolation of DNA, as well as for identification of molecules interacting with genomic regions of interest in vivo in combination with downstream analysis.

GARP-TGF-ß complexes negatively regulate regulatory T cell development and maintenance of peripheral CD4+ T cells in vivo.

The role of surface-bound TGF-ß on regulatory T cells (Tregs) and the mechanisms that mediate its functions are not well defined. We recently identified a cell-surface molecule called Glycoprotein A Repetitions Predominant (GARP), which is expressed specifically on activated Tregs and was found to bind latent TGF-ß and mediate a portion of Treg suppressive activity in vitro. In this article, we address the role of GARP in regulating Treg and conventional T cell development and immune suppression in vivo using a transgenic mouse expressing GARP on all T cells. We found that, despite forced expression of GARP on all T cells, stimulation through the TCR was required for efficient localization of GARP to the cell surface. In addition, IL-2 signals enhanced GARP cell surface expression specifically on Tregs. GARP-transgenic CD4(+) T cells and Tregs, especially those expressing higher levels of GARP, were significantly reduced in the periphery. Mature Tregs, but not conventional CD4(+) T cells, were also reduced in the thymus. CD4(+) T cell reduction was more pronounced within the effector/memory subset, especially as the mouse aged. In addition, GARP-overexpressing CD4(+) T cells stimulated through the TCR displayed reduced proliferative capacity, which was restored by inhibiting TGF-ß signaling. Furthermore, inhibiting TGF-ß signals greatly enhanced surface expression of GARP on Tregs and blocked the induction of Foxp3 in activated CD4(+) T cells overexpressing GARP. These findings suggest a role for GARP in natural and induced Treg development through activation of bound latent TGF-ß and signaling, which negatively regulates GARP expression on Tregs.

Regulation of the expression of GARP/latent TGF-ß1 complexes on mouse T cells and their role in regulatory T cell and Th17 differentiation.

GARP/LRRC32 was defined as a marker of activated human regulatory T cells (Tregs) that is responsible for surface localization of latent TGF-ß1. We find that GARP and latent TGF-ß1 are also found on mouse Tregs activated via TCR stimulation; however, in contrast to human Tregs, GARP is also expressed at a low level on resting Tregs. The expression of GARP can be upregulated on mouse Tregs by IL-2 or IL-4 exposure in the absence of TCR signaling. GARP is expressed at a low level on Tregs within the thymus, and Treg precursors from the thymus concomitantly express GARP and Foxp3 upon exposure to IL-2. The expression of GARP is independent of TGF-ß1 and TGF-ß1 loading into GARP and is independent of furin-mediated processing of pro-TGF-ß1 to latent TGF-ß1. Specific deletion of GARP in CD4(+) T cells results in lack of expression of latent TGF-ß1 on activated Tregs. GARP-deficient Tregs develop normally, are present in normal numbers in peripheral tissues, and are fully competent suppressors of the activation of conventional T cells in vitro. Activated Tregs expressing GARP/latent TGF-ß1 complexes are potent inducers of Th17 differentiation in the presence of exogenous IL-6 and inducers of Treg in the presence of IL-2. Induction of both Th17-producing cells and Tregs is caused preferentially by Tregs expressing the latent TGF-ß1/GARP complex on their cell surface rather than by secreted latent TGF-ß1.

Isolation of Specific Genomic Regions and Identification of Their Associated Molecules by Engineered DNA-Binding Molecule-Mediated Chromatin Immunoprecipitation (enChIP) Using the CRISPR System and TAL Proteins.

Comprehensive understanding of genome functions requires identification of molecules (proteins, RNAs, genomic regions, etc.) bound to specific genomic regions of interest in vivo. To perform biochemical and molecular biological analysis of specific genomic regions, we developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) to purify genomic regions of interest. In enChIP, specific genomic regions are tagged for biochemical purification using engineered DNA-binding molecules, such as transcription activator-like (TAL) proteins and a catalytically inactive form of the clustered regularly interspaced short palindromic repeats (CRISPR) system. enChIP is a comprehensive approach that emphasizes non-biased search using next-generation sequencing (NGS), microarrays, mass spectrometry (MS), and other methods. Moreover, this approach is not restricted to cultured cell lines and can be easily extended to organisms. In this review, we discuss applications of enChIP to elucidating the molecular mechanisms underlying genome functions.

Applications of Engineered DNA-Binding Molecules Such as TAL Proteins and the CRISPR/Cas System in Biology Research.

Engineered DNA-binding molecules such as transcription activator-like effector (TAL or TALE) proteins and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) (CRISPR/Cas) system have been used extensively for genome editing in cells of various types and species. The sequence-specific DNA-binding activities of these engineered DNA-binding molecules can also be utilized for other purposes, such as transcriptional activation, transcriptional repression, chromatin modification, visualization of genomic regions, and isolation of chromatin in a locus-specific manner. In this review, we describe applications of these engineered DNA-binding molecules for biological purposes other than genome editing.

Efficient isolation of specific genomic regions retaining molecular interactions by the iChIP system using recombinant exogenous DNA-binding proteins.

BACKGROUND: Comprehensive understanding of mechanisms of genome functions requires identification of molecules interacting with genomic regions of interest in vivo. We previously developed the insertional chromatin immunoprecipitation (iChIP) technology to isolate specific genomic regions retaining molecular interactions and identify their associated molecules. iChIP consists of locus-tagging and affinity purification. The recognition sequences of an exogenous DNA-binding protein such as LexA are inserted into a genomic region of interest in the cell to be analyzed. The exogenous DNA-binding protein fused with a tag(s) is expressed in the cell and the target genomic region is purified with antibody against the tag(s). In this study, we developed the iChIP system using recombinant DNA-binding proteins to make iChIP more straightforward than the conventional iChIP system using expression of the exogenous DNA-binding proteins in the cells to be analyzed. RESULTS: In this system, recombinant 3xFNLDD-D (r3xFNLDD-D) consisting of the 3xFLAG-tag, a nuclear localization signal (NLS), the DNA-binding domain plus the dimerization domain of the LexA protein, and the Dock-tag is used for isolation of specific genomic regions. r3xFNLDD-D was expressed using a silkworm-baculovirus expression system and purified by affinity purification. iChIP using r3xFNLDD-D could efficiently isolate the single-copy chicken Pax5 (cPax5) locus, in which LexA binding elements were inserted, with negligible contamination of other genomic regions. In addition, we could detect RNA associated with the cPax5 locus using this form of the iChIP system combined with RT-PCR. CONCLUSIONS: The iChIP system using r3xFNLDD-D can isolate specific genomic regions retaining molecular interactions without expression of the exogenous DNA-binding protein in the cell to be analyzed. iChIP using r3xFNLDD-D would be more straightforward and useful for analysis of specific genomic regions to elucidate their functions as compared to the previously published iChIP protocol.

iChIP-SILAC analysis identifies epigenetic regulators of CpG methylation of the p16INK4A gene.

Allele-specific epigenetic events regulate the expression of specific genes such as tumor suppressor genes. Methods to biochemically identify epigenetic regulators remain limited. Here, we used insertional chromatin immunoprecipitation (iChIP) to address this issue. iChIP combined with quantitative mass spectrometry identified DNA methyltransferase 1 (DNMT1) and epigenetic regulators as proteins that potentially interact with a region of the p16INK4A gene that is CpG-methylated in one allele in HCT116 cells. Some of the identified proteins are involved in the CpG methylation of this region, and of these, DEAD-box helicase 24 (DDX24) contributes to CpG methylation by regulating the protein levels of DNMT1. Thus, iChIP is a useful method to identify proteins which bind to a target locus of interest.