Identifying multi-locus chromatin contacts in human cells using tethered multiple 3C.

BACKGROUND: Several recently developed experimental methods, each an extension of the chromatin conformation capture (3C) assay, have enabled the genome-wide profiling of chromatin contacts between pairs of genomic loci in 3D. Especially in complex eukaryotes, data generated by these methods, coupled with other genome-wide datasets, demonstrated that non-random chromatin folding correlates strongly with cellular processes such as gene expression and DNA replication. RESULTS: We describe a genome architecture assay, tethered multiple 3C (TM3C), that maps genome-wide chromatin contacts via a simple protocol of restriction enzyme digestion and religation of fragments upon agarose gel beads followed by paired-end sequencing. In addition to identifying contacts between pairs of loci, TM3C enables identification of contacts among more than two loci simultaneously. We use TM3C to assay the genome architectures of two human cell lines: KBM7, a near-haploid chronic leukemia cell line, and NHEK, a normal diploid human epidermal keratinocyte cell line. We confirm that the contact frequency maps produced by TM3C exhibit features characteristic of existing genome architecture datasets, including the expected scaling of contact probabilities with genomic distance, megabase scale chromosomal compartments and sub-megabase scale topological domains. We also confirm that TM3C captures several known cell type-specific contacts, ploidy shifts and translocations, such as Philadelphia chromosome formation (Ph+) in KBM7. We confirm a subset of the triple contacts involving the IGF2-H19 imprinting control region (ICR) using PCR analysis for KBM7 cells. Our genome-wide analysis of pairwise and triple contacts demonstrates their preference for linking open chromatin regions to each other and for linking regions with higher numbers of DNase hypersensitive sites (DHSs) to each other. For near-haploid KBM7 cells, we infer whole genome 3D models that exhibit clustering of small chromosomes with each other and large chromosomes with each other, consistent with previous studies of the genome architectures of other human cell lines. CONCLUSION: TM3C is a simple protocol for ascertaining genome architecture and can be used to identify simultaneous contacts among three or four loci. Application of TM3C to a near-haploid human cell line revealed large-scale features of chromosomal organization and multi-way chromatin contacts that preferentially link regions of open chromatin.

Loss of IGF2 imprinting is associated with abrogation of long-range intrachromosomal interactions in human cancer cells.

Nuclear architecture and chromatin geography are important factors in the regulation of gene expression, as these components may play a vital epigenetic role both in normal physiology as well as in the initiation and progression of malignancies. Using a modification of the chromosome conformation capture (3C) technique, we examined long-range chromatin interactions of the imprinted human IGF2 gene. We demonstrate that numerous intrachromosomal interactions occur along both parental alleles in normal tissues, where the IGF2 is paternally expressed, as well as in normal liver where gene expression is biallelic. Long-range and allele-specific interactions occur between the IGF2/H19 imprinting control region-1 (ICR1) and ICR2, a region which regulates an imprinted gene cluster nearly a megabase distant from IGF2. Loss of genomic imprinting is a common epigenetic event in cancer, and long-range interactions have not been examined in malignant cells. In cancer cell lines in which IGF2 imprinting is maintained (MOI), essentially all of the 3C interactions seen in normal cells were preserved. However, in cells in which IGF2 imprinting was lost (LOI), nearly all of the long-range chromatin interactions involving IGF2 were abrogated. A three-dimensional computer model depicts the physical interactions between the IGF2 promoter and ICR1 in MOI cells, while the model of LOI lung cancer cells is flattened with few long-range interactions. This dramatic change in the three-dimension configuration of the chromatin at the IGF2 locus in LOI cancer cells suggests that the loss of imprinting may lead to a variety of changes in gene expression in addition to changes in IGF2 transcription.

A complex deoxyribonucleic acid looping configuration associated with the silencing of the maternal Igf2 allele.

Alternate interactions between the H19 imprinting control region (ICR) and one of the two Igf2 differentially methylated regions has been proposed as a model regulating the reciprocal imprinting of Igf2 and H19. To study the conformation of this imprint switch, we performed a systematic structural analysis across the 140 kb of the mouse Igf2-H19 region, which includes enhancers located both between the two genes as well as downstream of H19, by using a scanning chromosome conformation capture (3C) technique. Our results suggest that on the active paternal Igf2 allele, the various enhancers have direct access to the Igf2 promoters, whereas the imprinted silent maternal Igf2 allele assumes a complex three-dimensional knotted loop that keeps the enhancers away from the Igf2 promoters and allows them to interact with the H19 promoter. This complex DNA looping of the maternal allele is formed by interactions involving differentially methylated region 1, the ICR, and enhancers. Binding of CTC-binding factor to the maternal, unmethylated ICR in conjunction with the presence of multicomplex components including interchromosomal interactions, create a barrier blocking the access of all enhancers to Igf2, thereby silencing the maternal Igf2. This silencing configuration exists in newborn liver, mouse embryonic fibroblast, and embryonic stem cells and persists during mitosis, conferring a mechanism for epigenetic memory.

Loss of imprinting of IGF2 sense and antisense transcripts in Wilms' tumor.

Human insulin-like growth factor II gene (IGF2) is overexpressed, and its imprinting is disrupted in many tumors, including Wilms' tumor. A transcript that is antisense to IGF2, IGF2-antisense (IGF2-AS), is transcribed from within IGF2 in a reverse orientation. This transcript is also maternally imprinted and overexpressed in Wilms' tumor. IGF2-AS was detected as a 2.2 kb mRNA in Hep 3B cells by Northern blotting, and it encodes a putative 168 amino acid peptide. An alternative splicing mRNA observed predominantly in adult liver encodes an additional putative 199 amino acid peptide. We have examined the expression of IGF2 and IGF2-AS in normal tissue, breast and ovarian tumors, and 25 informative, well-characterized Wilms' tumors and determined the relationship between IGF2 and IGF2-AS imprinting. IGF2-AS was expressed at levels comparable with IGF2 sense expression derived from promoters P1 and P2 in normal tissue and in breast, ovarian, and Wilms' tumor tissues. In Wilms' tumors that demonstrate maintenance of imprinting of IGF2, IGF2-AS was imprinted. In contrast, in tumors which demonstrate LOI of IGF2, only two of six tumors showed loss of imprinting of IGF2-AS, whereas four of six tumors demonstrated maintenance of imprinting for IGF2-AS. The discrepancy between IGF2 and IGF2-AS loss of imprinting in some tumors demonstrates the control complexity of the imprinting status of the various transcripts derived from the IGF2 gene.

The histone code regulating expression of the imprinted mouse Igf2r gene.

The mouse IGF-II receptor (Igf2r) and its antisense transcript Air are reciprocally imprinted in most normal tissues. Several mechanisms have been hypothesized to explain Igf2r-Air imprinting, including Igf2r silencing by Air, and transcriptional repression of Igf2r-Air by two differentially methylated regions (DMR1 and DMR2). We employed Mus musculus x Mus spretus interspecific mice and chromatin immunoprecipitation (ChIP) to investigate allele-specific histone modifications in the two DMRs. We show that, in both DMRs, the active alleles of both Igf2r, and Air are associated with acetylated histones (H3, and H4), acetyl lysine 9 of histone H3 (H3 K9-Ac), and methyl lysine 4 of histone H3 (H3 K4-Me). The silenced alleles are associated with methylated DNA, deacetylated H3 K9, and unmethylated H3 K4. Allele-specific histone modifications are present in the DMR2 that is established in the gametes and represents the DNA gametic-imprint of the Igf2r. In the DMR2 from liver, kidney, and central nervous system tissues, H3 K9 methylation is associated exclusively with the silenced allele, and H3 S10 phosphorylation with the active alleles. Treatment of fibroblast cells with 5-aza-deoxycytidine and/or Trichostatin A led to partial reactivation of the silenced allele, which correlates with biallelic histone acetylation. In central nervous system, despite the presence of imprinted Air transcripts, biallelic expression of Igf2r occurs. The tissue-specific relaxation of Igf2r imprinting correlates with biallelic histone acetylation, and biallelic H3 K4 methylation in the promoter region of Igf2r (DMR1). We propose a model of the histone code for Igf2r, and Air imprinting that defines histone modifications specific for the putative gametic imprint DMR2, and explains the tissue-specific imprinting of Igf2r in the mouse and the absence of IGF2R imprinting in human.

CTCF binding at the insulin-like growth factor-II (IGF2)/H19 imprinting control region is insufficient to regulate IGF2/H19 expression in human tissues.

The adjacent IGF2 and H19 genes are imprinted in most normal mouse and human tissues, but imprinting is often lost in tumors. Mouse models suggest that parental-allele specific CCCTC-binding factor (CTCF) binding at the IGF2/H19 imprinting control region (ICR) regulates the expression of these two genes. Using chromatin immunoprecipitation and PCR, we show that in several normal and neoplastic human tissues, CTCF consistently binds unmethylated ICR elements, but CTCF binding does not result in predictable gene expression. In the fetal brain, CTCF binding is monoallelic and specific for the unmethylated ICR, yet IGF2/H19 expression is biallelic. In osteosarcoma tumors, aberrant methylation of the IGF2/H19 ICR results in equally aberrant CTCF binding, yet expression of these genes does not correlate with CTCF binding. This is the first description of chromatin immunoprecipitation for CTCF binding at the human IGF2/H19 ICR, and the results demonstrate that CTCF binding at the IGF2/H19 ICR is insufficient to regulate the expression of IGF2/H19 in many human tissues.

Epigenetic regulation of the taxol resistance-associated gene TRAG-3 in human tumors.

TRAG-3, originally identified as a taxol resistance-associated gene from an ovarian carcinoma cell line, is upregulated in many human tumors. Like many tumor antigens, TRAG-3 mRNA is not detectable or is expressed at very low levels in normal fetal and adult human tissues except for testis, where TRAG-3 mRNA transcripts are detected abundantly. TRAG-3 mRNA is frequently overexpressed in tumors but is rarely detected in adjacent normal tissues. To delineate the transcriptional regulation of this tumor antigen, we cloned and sequenced the TRAG-3 promoter. A 539-base pair fragment upstream of the initiation site, which contains two unusual CT repeat stretches, was sufficient to drive the maximum activity of a luciferase reporter gene. Sodium bisulfite sequencing of genomic DNA revealed that the amount of DNA methylation in exon 2 and in the promoter regions is inversely correlated with gene expression. In normal tissues, TRAG-3 is hypermethylated and is thus transcriptionally silenced. In those tumors where TRAG-3 is actively transcribed, the TRAG-3 promoter and exon 2 are hypomethylated. Treatment of a TRAG-3-silenced cell line H23 with the demethylating reagent 5-aza-cytosine reduced DNA methylation and induced TRAG-3 expression in a dose-dependent manner. These results indicate that DNA demethylation is an important epigenetic mechanism that regulates the TRAG-3 tumor antigen in human tumors.

CTCF regulates allelic expression of Igf2 by orchestrating a promoter-polycomb repressive complex 2 intrachromosomal loop.

CTCF is a zinc finger DNA-binding protein that regulates the epigenetic states of numerous target genes. Using allelic regulation of mouse insulin-like growth factor II (Igf2) as a model, we demonstrate that CTCF binds to the unmethylated maternal allele of the imprinting control region (ICR) in the Igf2/H19 imprinting domain and forms a long-range intrachromosomal loop to interact with the three clustered Igf2 promoters. Polycomb repressive complex 2 is recruited through the interaction of CTCF with Suz12, leading to allele-specific methylation at lysine 27 of histone H3 (H3-K27) and to suppression of the maternal Igf2 promoters. Targeted mutation or deletion of the maternal ICR abolishes this chromatin loop, decreases allelic H3-K27 methylation, and causes loss of Igf2 imprinting. RNA interference knockdown of Suz12 also leads to reactivation of the maternal Igf2 allele and biallelic Igf2 expression. CTCF and Suz12 are coprecipitated from nuclear extracts with antibodies specific for either protein, and they interact with each other in a two-hybrid system. These findings offer insight into general epigenetic mechanisms by which CTCF governs gene expression by orchestrating chromatin loop structures and by serving as a DNA-binding protein scaffold to recruit and bind polycomb repressive complexes.

CTCF mediates interchromosomal colocalization between Igf2/H19 and Wsb1/Nf1.

Gene transcription may be regulated by remote enhancer or insulator regions through chromosome looping. Using a modification of chromosome conformation capture (3C) and fluorescence in situ hybridization, we found that one allele of the insulin-like growth factor 2 (Igf2)/H19 imprinting control region (ICR) on chromosome 7 colocalized with one allele of Wsb1/Nf1 on chromosome 11. Omission of CCCTC-binding factor (CTCF) or deletion of the maternal ICR abrogated this association and altered Wsb1/Nf1 gene expression. These findings demonstrate that CTCF mediates an interchromosomal association, perhaps by directing distant DNA segments to a common transcription factory, and the data provide a model for long-range allele-specific associations between gene regions on different chromosomes that suggest a framework for DNA recombination and RNA trans-splicing.

Correction of aberrant imprinting of IGF2 in human tumors by nuclear transfer-induced epigenetic reprogramming.

Loss of genomic imprinting of insulin-like growth factor II (IGF2) is a hallmark of many human neoplasms. We attempted to correct this aberrant epigenotype by transferring nuclei from human tumor cells that showed loss of IGF2 imprinting into enucleated mouse and human fibroblasts that had maintained normal IGF2 imprinting. After nuclear transfer, the abnormal biallelic expression of IGF2 in tumor nuclei transiently converted to normal monoallelic imprinted expression in the reconstructed diploid cells. In tetraploid hybrid cells, however, normal IGF2 imprinting was permanently restored in the tumor genome. Inhibition of the synthesis of putative trans imprinting factors with cycloheximide led to loss of IGF2 imprinting in normal cultured fibroblasts, suggesting that normal cells produce proteins that act in trans to induce or maintain genomic imprinting. These data demonstrate that an abnormal tumor epigenotype can be corrected by in vitro reprogramming, and suggest that loss of imprinting is associated with the loss of activity of non-CTCF trans imprinting factor(s) that are either inactivated or mutated in tumors.

IVF results in de novo DNA methylation and histone methylation at an Igf2-H19 imprinting epigenetic switch.

Recent studies suggest that IVF and assisted reproduction technologies (ART) may result in abnormal genomic imprinting, leading to an increased frequency of Angelman syndrome (AS) and Beckwith-Weidemann syndrome (BWS) in IVF children. To learn how ART might alter the epigenome, we examined morulas and blastocysts derived from C57BL/6J X M. spretus F1 mice conceived in vivo and in vitro and determined the allelic expression of four imprinted genes: Igf2, H19, Cdkn1c and Slc221L. IVF-derived mouse embryos that were cultured in human tubal fluid (HTF) (Quinn's advantage) media displayed a high frequency of aberrant H19 imprinting, whereas in vivo and IVF embryos showed normal maternal expression of Cdkn1c and normal biallelic expression of Igf2 and Slc221L. Embryonic stem (ES) cells derived from IVF blastocysts also showed abnormal Igf2/H19 imprinting. Allele-specific bisulphite PCR reveals abnormal DNA methylation at a CCCTC-binding factor (CTCF) site in the imprinting control region (ICR), as the normally unmethylated maternal allele acquired a paternal methylation pattern. Chromatin immunoprecipitation (ChIP) assays indicate an increase of lysine 4 methylation (dimethyl Lys4-H3) on the paternal chromatin and a gain in lysine 9 methylation (trimethyl Lys9-H3) on the maternal chromatin at the same CTCF-binding site. Our results indicate that de novo DNA methylation on the maternal allele and allele-specific acquisition of histone methylation lead to aberrant Igf2/H19 imprinting in IVF-derived ES cells. We suggest that ART, which includes IVF and various culture media, might cause imprinting errors that involve both aberrant DNA methylation and histone methylation at an epigenetic switch of the Igf2-H19 gene region.

Promoter-restricted histone code, not the differentially methylated DNA regions or antisense transcripts, marks the imprinting status of IGF2R in human and mouse.

Imprinting of the mouse Igf2r depends upon an intronic differentially methylated DNA region (DMR) and the presence of the Air antisense transcript. However, biallelic expression of mouse Igf2r in brain occurs despite the presence of Air, and biallelic expression of human IGF2R in peripheral tissues occurs despite the presence of an intronic DMR. We examined histone modifications throughout the mouse and human Igf2r/IGF2R using chromatin immuno-precipitation (ChIP) assays in combination with quantitative real time PCR. Methylation of Lys4 and Lys9 of histone H3 in the promoter regions marks the active and silenced alleles, respectively. We measured di- and tri-methyl Lys4 and Lys9 across the Igf2r and Air promoters. While both di- and tri-methyl Lys4 marked the active Igf2r and the active Air allele, tri-methyl Lys9, but not di-methyl Lys9, marked the suppressed Air allele. We show here that enrichment of parental allele-specific histone modifications in the promoter region, rather than the presence of DNA methylation or antisense transcription, correctly identifies the tissue- and species- specific imprinting status of Igf2r/IGF2R. We discuss these findings in light of recent progress in identifying specific components of the epigenetic marks in imprinted genes.

Epigenetic regulation of Igf2/H19 imprinting at CTCF insulator binding sites.

The mouse insulin-like growth factor II (Igf2) and H19 genes are located adjacent to each other on chromosome 7q11-13 and are reciprocally imprinted. It is believed that the allelic expression of these two genes is regulated by the binding of CTCF insulators to four parent-specific DNA methylation sites in an imprinting control center (ICR) located between these two genes. Although monoallelically expressed in peripheral tissues, Igf2 is biallelically transcribed in the CNS. In this study, we examined the allelic DNA methylation and CTCF binding in the Igf2/H19 imprinting center in CNS, hypothesizing that the aberrant CTCF binding as one of the mechanisms leads to biallelic expression of Igf2 in CNS. Using hybrid F1 mice (M. spretus males x C57BL/6 females), we showed that in CNS, CTCF binding sites in the ICR were methylated exclusively on the paternal allele, and CTCF bound only to the unmethylated maternal allele, showing no differences from the imprinted peripheral tissues. Among three other epigenetic modifications examined, histone H3 lysine 9 methylation correlated well with Igf2 allelic expression in CNS. These results suggest that CTCF binding to the ICR alone is not sufficient to insulate the Igf2 maternal promoter and to regulate the allelic expression of the gene in the CNS, thus challenging the aberrant CTCF binding as a common mechanism for lack of Igf2 imprinting in CNS. Further studies should be focused on the identification of factors that are involved in histone methylation and CTCF-associated factors that may be needed to coordinate Igf2 imprinting.

Activating and silencing histone modifications form independent allelic switch regions in the imprinted Gnas gene.

Activation and suppression of gene transcription is tightly controlled by epigenetic modifications. The imprinted Gnas1 gene region contains closely juxtaposed maternally expressed (Nesp) and paternally expressed (Nespas, Gnasxl, Exon 1A) transcripts, providing a unique opportunity to study how epigenetic modifications change in nucleosomes from active to silenced promoters. Using 30 polymorphic sites across the Gnas1 gene region in (C57BL/6JxMus spretus) F(1) mice and chromatin immunoprecipitation (ChIP) assays we identified two allelic switch regions (ASRs) that mark boundaries of epigenetic information. We show that activating signals (histone acetylation and methylation of H3 Lys4) and silencing signals (histone methylation of H3 Lys9 and DNA methylation) segregate independently across the ASRs and suggest that these ASRs allow the transcriptional elongation to proceed through the silenced domain of nearby imprinted promoters. We discuss these findings in light of recent progress in the conceptualization of nucleosome remodeling during transcriptional elongation and in the development of histone code.

Loss of imprinting of IGF2 and H19 in osteosarcoma is accompanied by reciprocal methylation changes of a CTCF-binding site.

The adjacent insulin-like growth factor 2 (IGF2) and H19 genes are imprinted in most normal human tissues, but imprinting is often lost in tumors. The mechanisms involved in maintenance of imprinting (MOI) and loss of imprinting (LOI) are unresolved. We show here that osteosarcoma (OS) tumors with IGF2/H19 MOI exhibit allele-specific differential methylation of a CTCF-binding site upstream of H19. LOI of IGF2 or H19 in OS occurs in a mutually exclusive manner, and occurs with monoallelic expression of the other gene. Bisulfite sequencing reveals IGF2 LOI occurs with biallelic CpG methylation of the CTCF-binding site, while H19 LOI occurs with biallelic hypomethylation of this site. Our data demonstrate that IGF2 LOI and H19 LOI are accompanied by reciprocal methylation changes at a critical CTCF-binding site. We propose a model in which incomplete gain or loss of methylation at this CTCF-binding site during tumorigenesis explains the complex and often conflicting expression patterns of IGF2 and H19 in tumors.

An imprinted PEG1/MEST antisense expressed predominantly in human testis and in mature spermatozoa.

PEG1 (or MEST) is an imprinted gene located on human chromosome 7q32 that is expressed predominantly from the paternal allele. In the mouse, Peg1/Mest is associated with embryonic growth and maternal behavior. Human PEG1 is transcribed from two promoters; the transcript from promoter P1 is derived from both parental alleles, and the transcript from P2 is exclusively from the paternal allele. We characterized the P1 and P2 transcripts in various normal and neoplastic tissues. In the normal tissues, PEG1 was transcribed from both promoters P1 and P2, whereas in six of eight neoplastic tissues, PEG1 was transcribed exclusively from promoter P1. Bisulfite sequencing demonstrated high levels of CpG methylation in the P2 region of DNA from a lung tumor. In the region between P1 and P2, we identified a novel transcript, PEG1-AS, in an antisense orientation to PEG1. PEG1-AS is a spliced transcript and was detected as a strong 2.4-kilobase band on a Northern blot. PEG1-AS and PEG1 P2-sense transcript were expressed exclusively from the paternal allele. Fragments of DNA from within the 1.5-kilobase region between PEG1-AS and the P2 exon were ligated to a pGL3 luciferase reporter vector and transfected into NCI H23 cells. This DNA exhibited strong promoter activity in both the sense and antisense directions, indicating that PEG1-AS and P2 exon share a common promoter region. Treatment of the transfected DNA fragments with CpG methylase abolished the promoter activity. Of interest, PEG1-AS was expressed predominantly in testis and in mature motile spermatozoa, indicating a possible role for this transcript in human sperm physiology and fertilization.