CTCF Insulator Research Articles

Tandem CTCF sites function as insulators to balance spatial chromatin contacts and topological enhancer-promoter selection

BackgroundCTCF is a key insulator-binding protein, and mammalian genomes contain numerous CTCF sites, many of which are organized in tandem.ResultsUsing CRISPR DNA-fragment editing, in conjunction with chromosome conformation capture, we find that CTCF sites, if located between enhancers and promoters in the protocadherin (Pcdh) and β-globin clusters, function as an enhancer-blocking insulator by forming distinct directional chromatin loops, regardless whether enhancers contain CTCF sites or not. Moreover, computational simulation in silico and genetic deletions in vivo as well as dCas9 blocking in vitro revealed balanced promoter usage in cell populations and stochastic monoallelic expression in single cells by large arrays of tandem CTCF sites in the Pcdh and immunoglobulin heavy chain (Igh) clusters. Furthermore, CTCF insulators promote, counter-intuitively, long-range chromatin interactions with distal directional CTCF sites, consistent with the cohesin “loop extrusion” model. Finally, gene expression levels are negatively correlated with CTCF insulators located between enhancers and promoters on a genome-wide scale. Thus, single CTCF insulators ensure proper enhancer insulation and promoter activation while tandem CTCF topological insulators determine balanced spatial contacts and promoter choice.ConclusionsThese findings have interesting implications on the role of topological chromatin insulators in 3D genome folding and developmental gene regulation.

Chromosomal Loop Editing Reveals New Epigenomic Landscape in the β-Globin Locus with Implications for Hemoglobinopathy Therapies

Human β-globin locus consists of at least six genes encoding components of the oxygen transport protein hemoglobin and an upstream locus control region (LCR) containing five DNase I hypersensitive sites. In addition, there are at least four conserved CTCF insulator elements surrounding the locus, which form dynamic chromatin interaction patterns across different cell types in the course of hematopoietic stem and progenitor cells (HSPCs) development. Chromatin conformation of the β-globin locus in normal adult CD34+ HSPCs reveals the formation of three topological associated domains (TADs), in which individual TAD boundaries are demarcated by CTCF sites (Figure 1). By comparing chromatin loop interactions between CD34+ HSPCs and its differentiated erythroid progenitors, we identify a chromatin loop that forms in erythroid progenitors and is not evident in the CD34+ HSPCs. Detailed examination of this loop shows that a DNase I hypersensitivity site, also called 3'HS1, overlaps with a CTCF site that forms a loop with another CTCF site adjacent to OR52A5 gene (Figure 2). To investigate the role of this specific chromatin loop in the regulation of hemoglobin gene expression, we knock out the 3'HS1 CTCF motif in adult CD34+ HSPCs under erythroid differentiation medium by CRISPR/Cas9-mediated gene editing. We find that deletion of 3'HS1 CTCF results in a 2-fold decrease of β-globin (HBB) and a 4-fold increase of the fetal hemoglobin gene encoded by γ-globin (HBG1/2) in erythroid colonies of edited CD34+ cells (Figure 3). Elevation of fetal hemoglobin upon 3'HS1 CTCF deletion was also confirmed in human umbilical cord blood-derived erythroid progenitor-2 cells (HUDEP-2), which results in a 12-fold increase of γ-globin expression (Figure 4). These results suggest that the 3'HS1 CTCF plays a crucial role in regulating the expression of fetal hemoglobin gene, however it remains unclear whether changes in chromatin structure is responsible for these changes. CTCF looping interactions have been described to form under convergent directionality. To validate the role of 3'HS1 CTCF in establishing chromatin interactions at the β-globin locus, we aim to invert this binding motif and evaluate how it disrupts chromatin organization and gene expression at this locus. Deciphering the underlying mechanisms will shed light on how three-dimensional chromatin structure is reorganized in differentiating erythroid cells as it undergoes nuclear condensation. Furthermore, elevation of fetal hemoglobin expression can potentially be a new therapeutic gene editing strategy to treat sickle cell disease and some cases of β-hemoglobinopathies. Disclosures No relevant conflicts of interest to declare.

Altered chromosomal topology drives oncogenic programs in SDH-deficient GISTs.

Epigenetic aberrations are widespread in cancer, yet the underlying mechanisms and causality remain poorly understood1-3. A subset of gastrointestinal stromal tumors (GISTs) lack canonical kinase mutations but instead have succinate dehydrogenase (SDH)-deficiency and global DNA hyper-methylation4,5. Here we associate this hyper-methylation with changes in genome topology that activate oncogenic programs. To investigate epigenetic alterations systematically, we mapped DNA methylation, CTCF insulators, enhancers, and chromosome topology in KIT-mutant, PDGFRA-mutant, and SDH-deficient GISTs. Although these respective subtypes shared similar enhancer landscapes, we identified hundreds of putative insulators where DNA methylation replaced CTCF binding in SDH-deficient GISTs. We focused on a disrupted insulator that normally partitions a core GIST super-enhancer from the FGF4 oncogene. Recurrent loss of this insulator alters locus topology in SDH-deficient GISTs, allowing aberrant physical interaction between enhancer and oncogene. CRISPR-mediated excision of the corresponding CTCF motifs in an SDH-intact GIST model disrupted the boundary and strongly up-regulated FGF4 expression. We also identified a second recurrent insulator loss event near the KIT oncogene, which is also highly expressed across SDH-deficient GISTs. Finally, we established a patient-derived xenograft (PDX) from an SDH-deficient GIST that faithfully maintains the epigenetics of the parental tumor, including hyper-methylation and insulator defects. This PDX model is highly sensitive to FGF receptor (FGFR) inhibitor, and more so to combined FGFR and KIT inhibition, validating the functional significance of the underlying epigenetic lesions. Our study reveals how epigenetic alterations can drive oncogenic programs in the absence of canonical kinase mutations, with implications for mechanistic targeting of aberrant pathways in cancers.

Epigenome editing strategies for the functional annotation of CTCF insulators

The human genome is folded into regulatory units termed ‘topologically-associated domains’ (TADs). Genome-wide studies support a global role for the insulator protein CTCF in mediating chromosomal looping and the topological constraint of TAD boundaries. However, the impact of individual insulators on enhancer-gene interactions and transcription remains poorly understood. Here, we investigate epigenome editing strategies for perturbing individual CTCF insulators and evaluating consequent effects on genome topology and transcription. We show that fusions of catalytically-inactive Cas9 (dCas9) to transcriptional repressors (dCas9-KRAB) and DNA methyltransferases (dCas9-DNMT3A, dCas9-DNMT3A3L) can selectively displace CTCF from specific insulators, but only when precisely targeted to the cognate motif. We further demonstrate that stable, partially-heritable insulator disruption can be achieved through combinatorial hit-and-run epigenome editing. Finally, we apply these strategies to simulate an insulator loss mechanism implicated in brain tumorigenesis. Our study provides strategies for stably modifying genome organization and gene activity without altering the underlying DNA sequence.

Identification of Cancer Drivers at CTCF Insulators in 1,962 Whole Genomes.

Recent studies have shown that mutations at non-coding elements, such as promoters and enhancers, can act as cancer drivers. However, an important class of non-coding elements, namely CTCF insulators, has been overlooked in the previous driver analyses. We used insulator annotations from CTCF and cohesin ChIA-PET and analyzed somatic mutations in 1,962 whole genomes from 21 cancer types. Using the heterogeneous patterns of transcription-factor-motif disruption, functional impact, and recurrence of mutations, we developed a computational method that revealed 21 insulators showing signals of positive selection. In particular, mutations in an insulator in multiple cancer types, including 16% of melanoma samples, are associated with TGFB1 up-regulation. Using CRISPR-Cas9, we find that alterations at two of the most frequently mutated regions in this insulator increase cell growth by 40%-50%, supporting the role of this boundary element as a cancer driver. Thus, our study reveals several CTCF insulators as putative cancer drivers.

Trim33 is required for appropriate development of pre-cardiogenic mesoderm

Congenital cardiac malformations are among the most common birth defects in humans. Here we show that Trim33, a member of the Tif1 subfamily of tripartite domain containing transcriptional cofactors, is required for appropriate differentiation of the pre-cardiogenic mesoderm during a narrow time window in late gastrulation. While mesoderm-specific Trim33 mutants did not display noticeable phenotypes, epiblast-specific Trim33 mutant embryos developed ventricular septal defects, showed sparse trabeculation and abnormally thin compact myocardium, and died as a result of cardiac failure during late gestation. Differentiating embryoid bodies deficient in Trim33 showed an enrichment of gene sets associated with cardiac differentiation and contractility, while the total number of cardiac precursor cells was reduced. Concordantly, cardiac progenitor cell proliferation was reduced in Trim33-deficient embryos. ChIP-Seq performed using antibodies against Trim33 in differentiating embryoid bodies revealed more than 4000 peaks, which were significantly enriched close to genes implicated in stem cell maintenance and mesoderm development. Nearly half of the Trim33 peaks overlapped with binding sites of the Ctcf insulator protein. Our results suggest that Trim33 is required for appropriate differentiation of precardiogenic mesoderm during late gastrulation and that it will likely mediate some of its functions via multi-protein complexes, many of which include the chromatin architectural and insulator protein Ctcf.

Crosstalk of Genetic Variants, Allele-Specific DNA Methylation, and Environmental Factors for Complex Disease Risk.

Over the past decades, genome-wide association studies (GWAS) have identified thousands of phenotype-associated DNA sequence variants for potential explanations of inter-individual phenotypic differences and disease susceptibility. However, it remains a challenge for translating the associations into causative mechanisms for complex diseases, partially due to the involved variants in the noncoding regions and the inconvenience of functional studies in human population samples. So far, accumulating evidence has suggested a complex crosstalk among genetic variants, allele-specific binding of transcription factors (ABTF), and allele-specific DNA methylation patterns (ASM), as well as environmental factors for disease risk. This review aims to summarize the current studies regarding the interactions of the aforementioned factors with a focus on epigenetic insights. We present two scenarios of single nucleotide polymorphisms (SNPs) in coding regions and non-coding regions for disease risk, via potentially impacting epigenetic patterns. While a SNP in a coding region may confer disease risk via altering protein functions, a SNP in non-coding region may cause diseases, via SNP-altering ABTF, ASM, and allele-specific gene expression (ASE). The allelic increases or decreases of gene expression are key for disease risk during development. Such ASE can be achieved via either a “SNP-introduced ABTF to ASM” or a “SNP-introduced ASM to ABTF.” Together with our additional in-depth review on insulator CTCF, we are convinced to propose a working model that the small effect of a SNP acts through altered ABTF and/or ASM, for ASE and eventual disease outcome (named as a “SNP intensifier” model). In summary, the significance of complex crosstalk among genetic factors, epigenetic patterns, and environmental factors requires further investigations for disease susceptibility.

Persistent accumulation of unrepaired DNA damage in rat cortical neurons: nuclear organization and ChIP-seq analysis of damaged DNA

Neurons are highly vulnerable to DNA damage induced by genotoxic agents such as topoisomerase activity, oxidative stress, ionizing radiation (IR) and chemotherapeutic drugs. To avert the detrimental effects of DNA lesions in genome stability, transcription and apoptosis, neurons activate robust DNA repair mechanisms. However, defective DNA repair with accumulation of unrepaired DNA are at the basis of brain ageing and several neurodegenerative diseases. Understanding the mechanisms by which neurons tolerate DNA damage accumulation as well as defining the genomic regions that are more vulnerable to DNA damage or refractory to DNA repair and therefore constitute potential targets in neurodegenerative diseases are essential issues in the field. In this work we investigated the nuclear topography and organization together with the genome-wide distribution of unrepaired DNA in rat cortical neurons 15 days upon IR. About 5% of non-irradiated and 55% of irradiated cells accumulate unrepaired DNA within persistent DNA damage foci (PDDF) of chromatin. These PDDF are featured by persistent activation of DNA damage/repair signaling, lack of transcription and localization in repressive nuclear microenvironments. Interestingly, the chromatin insulator CTCF is concentrated at the PDDF boundaries, likely contributing to isolate unrepaired DNA from intact transcriptionally active chromatin. By confining damaged DNA, PDDF would help preserving genomic integrity and preventing the production of aberrant proteins encoded by damaged genes.ChIP-seq analysis of genome-wide γH2AX distribution revealed a number of genomic regions enriched in γH2AX signal in IR-treated cortical neurons. Some of these regions are in close proximity to genes encoding essential proteins for neuronal functions and human neurodegenerative disorders such as epm2a (Lafora disease), serpini1 (familial encephalopathy with neuroserpin inclusion bodies) and il1rpl1 (mental retardation, X-linked 21). Persistent γH2AX signal close to those regions suggests that nearby genes could be either more vulnerable to DNA damage or more refractory to DNA repair.

Depletion of the Insulator Protein CTCF Results in Herpes Simplex Virus 1 Reactivation In Vivo.

Herpes simplex virus 1 (HSV-1) establishes a lifelong latent infection in host peripheral neurons, including the neurons of the trigeminal ganglia (TG). HSV-1 can reactivate from neurons to cause recurrent infection. During latency, the insulator protein CTCF occupies DNA binding sites on the HSV-1 genome, and these sites have been previously characterized as functional enhancer-blocking insulators. Previously, CTCF was found to be dissociated from wild-type virus postreactivation but not in mutants that do not reactivate, indicating that CTCF eviction may also be an important component of reactivation. To further elucidate the role of CTCF in reactivation of HSV-1, we used recombinant adeno-associated virus (rAAV) vectors to deliver a small interfering RNA targeting CTCF to peripheral neurons latent with HSV-1 in rabbit TG. Our data show that CTCF depletion resulted in long-term and persistent shedding of infectious virus in the cornea and increased ICP0 expression in the ganglia, indicating that CTCF depletion facilitates HSV-1 reactivation.IMPORTANCE Increasing evidence has shown that the insulator protein CTCF regulates gene expression of DNA viruses, including the gammaherpesviruses. While CTCF occupation and insulator function control gene expression in DNA viruses, CTCF eviction has been correlated to increased lytic gene expression and the dissolution of transcriptional domains. Our previous data have shown that in the alphaherpesvirus HSV-1, CTCF was found to be dissociated from the HSV-1 genome postreactivation, further indicating a global role for CTCF eviction in the transition from latency to reactivation in HSV-1 genomes. Using an rAAV8, we targeted HSV-1-infected peripheral neurons for CTCF depletion to show that CTCF depletion precedes the shedding of infectious virus and increased lytic gene expression in vivo, providing the first evidence that CTCF depletion facilitates HSV-1 reactivation.

CTCF Binding Sites in the Herpes Simplex Virus 1 Genome Display Site-Specific CTCF Occupation, Protein Recruitment, and Insulator Function.

There are seven conserved CTCF binding domains in the herpes simplex virus 1 (HSV-1) genome. These binding sites individually flank the latency-associated transcript (LAT) and the immediate early (IE) gene regions, suggesting that CTCF insulators differentially control transcriptional domains in HSV-1 latency. In this work, we show that two CTCF binding motifs in HSV-1 display enhancer blocking in a cell-type-specific manner. We found that CTCF binding to the latent HSV-1 genome was LAT dependent and that the quantity of bound CTCF was site specific. Following reactivation, CTCF eviction was dynamic, suggesting that each CTCF site was independently regulated. We explored whether CTCF sites recruit the polycomb-repressive complex 2 (PRC2) to establish repressive domains through a CTCF-Suz12 interaction and found that Suz12 colocalized to the CTCF insulators flanking the ICP0 and ICP4 regions and, conversely, was removed at early times postreactivation. Collectively, these data support the idea that CTCF sites in HSV-1 are independently regulated and may contribute to lytic-latent HSV-1 control in a site-specific manner.IMPORTANCE The role of chromatin insulators in DNA viruses is an area of interest. It has been shown in several beta- and gammaherpesviruses that insulators likely control the lytic transcriptional profile through protein recruitment and through the formation of three-dimensional (3D) chromatin loops. The ability of insulators to regulate alphaherpesviruses has been understudied to date. The alphaherpesvirus HSV-1 has seven conserved insulator binding motifs that flank regions of the genome known to contribute to the establishment of latency. Our work presented here contributes to the understanding of how insulators control transcription of HSV-1.

Loss of Igf2 Gene Imprinting in Murine Prostate Promotes Widespread Neoplastic Growth.

Loss of imprinting (LOI) is an epigenetic event that relaxes an allele-specific restriction on gene expression. One gene that experiences LOI is the paracrine insulin-like growth factor IGF2, which occurs commonly in human prostate tissues during aging and tumorigenesis. However, the relationship between IGF2 LOI and prostate tumorigenesis has not been established functionally. In this study, we created a mouse model with CTCF-binding site mutations at the Igf2-H19 imprint control region that abolishes CTCF insulator activity, resulting in biallelic Igf2 expression that mimics increased levels seen with aging-induced LOI. We found that Igf2 LOI increased the prevalence and severity of prostatic intraepithelial neoplasia (PIN), a premalignant lesion. Engineering Nkx3.1 deficiency into our model increased the frequency of PIN lesions in an additive fashion. Prostates harboring LOI displayed increased MAPK signaling and epithelial proliferation. In human prostate tissue arrays, we documented a positive correlation in benign tissues of IGF2 levels with phospho-ERK and phospho-AKT levels. Overall, our results establish that Igf2 LOI is sufficient on its own to increase rates of neoplastic development in the prostate by upregulating critical cancer-associated signaling pathways. Cancer Res; 77(19); 5236-47. ©2017 AACR.

Inducible CTCF insulator delays the IgH 3′ regulatory region-mediated activation of germline promoters and alters class switching

Class switch recombination (CSR) plays an important role in adaptive immune response by enabling mature B cells to switch from IgM expression to the expression of downstream isotypes. CSR is preceded by inducible germline (GL) transcription of the constant genes and is controlled by the 3' regulatory region (3'RR) in a stimulus-dependent manner. Why the 3'RR-mediated up-regulation of GL transcription is delayed to the mature B-cell stage is presently unknown. Here we show that mice devoid of an inducible CTCF binding element, located in the α constant gene, display a marked isotype-specific increase of GL transcription in developing and resting splenic B cells and altered CSR in activated B cells. Moreover, insertion of a GL promoter downstream of the CTCF insulator led to premature activation of the ectopic promoter. This study provides functional evidence that the 3'RR has a developmentally controlled potential to constitutively activate GL promoters but that this activity is delayed, at least in part, by the CTCF insulator, which borders a transcriptionally active domain established by the 3'RR in developing B cells.

Distinct Roles of Brd2 and Brd4 in Potentiating the Transcriptional Program for Th17 Cell Differentiation

The BET proteins are major transcriptional regulators and have emerged as new drug targets, but their functional distinction has remained elusive. In this study, we report that the BET family members Brd2 and Brd4 exert distinct genomic functions at genes whose transcription they co-regulate during mouse T helper 17 (Th17) cell differentiation. Brd2 is associated with the chromatin insulator CTCF and the cohesin complex to support cis-regulatory enhancer assembly for gene transcriptional activation. In this context, Brd2 binds the transcription factor Stat3 in an acetylation-sensitive manner and facilitates Stat3 recruitment to active enhancers occupied with transcription factors Irf4 and Batf. In parallel, Brd4 temporally controls RNA polymerase II (Pol II) processivity during transcription elongation through cyclin T1 and Cdk9 recruitment and Pol II Ser2 phosphorylation. Collectively, our study uncovers both separate and interdependent Brd2 and Brd4 functions in potentiating the genetic program required for Th17 cell development and adaptive immunity.

GENT-33. EPIGENETIC ALTERATIONS AT INTERGENIC REGIONS ASSOCIATED WITH PROGRESSION IN A SUBSET OF IDH MUTANT GLIOMAS

Malignant gliomas are heterogeneous brain tumors characterized by progression and recurrence with variable patterns. Generally, IDH mutant gliomas are associated with favorable clinical outcome and higher DNA methylation at CpG islands (G-CIMP). However, our group reported a subclassification for G-CIMP (Ceccarelli et al. 2016). We termed these novel subtypes as ‘G-CIMP-low’, with lower DNA methylation profile and significantly worse survival, and ‘G-CIMP-high’, with higher levels of DNA methylation and better clinical outcome. Also, in matched primary and recurrent glioma cases, potential progression from G-CIMP-high to G-CIMP-low was observed. We aimed to understand potential epigenomic mechanisms leading to progression. To evaluate this, we extracted publicly available whole-genome bisulfite sequencing (WGBS) and RNAsequencing/Microarray data for G-CIMP-low (n=1), G-CIMP-high (n=2) and non-tumor brain (n=2) samples. We assess DNA methylation levels at single CpG level (~25 million) and integrated with chromHMM, epigenomic histone modification and transcriptomic data. We observed high concordance between Infinium-CpG-microarray and WGBS data (n=25,516 CpG, p-value<0.05, cor=0.95) suggesting our sample size is sufficient for exploratory analysis. At known intergenic regions and potential regulatory elements, G-CIMP-high and non-tumor brain samples show similar DNA methylation profile whereas G-CIMP-low presents with lower levels of DNA methylation. A significant number of CTCF insulator binding sites (n=24, p-value<0.05) showed DNA methylation differences between G-CIMP-low and G-CIMP-high and deregulated genes near these sites were found to be associated with cell cycle pathways (n=23). Also, there were DNA methylation changes near nuclear lamina domains, usually associated with deregulation of genes at boundaries, suggesting that chromatin accessibility may be affected in G-CIMP-low. Together these results suggest that most of epigenetic alterations in G-CIMP-low are at intergenic regions, specially candidate functional regulatory elements, which may alter 3D structure of chromatin in G-CIMP-low, allowing specific tumor suppressor genes to be deregulated and thereby leading to more aggressive phenotype for G-CIMP-low.

Polymorphism in the TRP8 gene in Kyrgyz population: Putative association with highland adaptation

The human TRPM8 gene encodes a receptor mediating cold sensitivity, and this points to its putative role in cold adaptation. The structural variability of the TRPM8 gene for five single-nucleotide polymorphisms (SNPs) has been studied in the Kyrgyz population. The SNPs are located in the coding regions of the gene, and three of them are confined to a 20-bp segment in exon 7. The frequencies of minor SNP alleles are as follows: rs13004520 G/C = 0.06; rs28901637 A/T = 0.13; rs11562975 G/C = 0.27; rs7593557 G/A = 0.21; rs11563071 C/G = 0.12. The analyzed sample of Kyrgyz population includes 275 individuals living at different altitudes and under drastically different climatic conditions. The frequency of the minor rs11562975 allele in the highlanders (living above 3200 m ASL and higher) is lower by a factor of 1.5 than in the residents of lower regions (760–3000 m ASL; p < 0.01). This result indicates a selective role of rs11562975 in adapting to cold. A comparison of haplotype frequencies in the Kyrgyz population with Europeans, East Asians, and Africans shows a clear narrowing of genotype variation in Europeans in comparison to all the others. Probably, this phenomenon is related to a decline in the population size (bottleneck effect) during evolution. We consider the exon–intron structure of the TRPM8 gene. The epigenetic markers in the vicinity of the gene have been analyzed. Two strong binding sites for insulator ctcf proteins are present there. They are likely to be associated with chromatin conformation and alternative splicing regulation. A structural–functional characterization of genes for the TRP protein family is provided.

50 - Regulatory Mutations of FOXG1 in the Context of Topological Domains

Recent HiC proximity mapping have revealed a 3D-organization of the human genome where cis-regulatory interactions occur within megabase-sized topological associating domains (TADs) ( 1 Dixon J.R. Selvaraj S. Yue F. et al. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature. 2012; 485: 376-380 Crossref PubMed Scopus (3953) Google Scholar ). TADs are separated by boundaries (TDB) which are enriched in Transcriptional Start Sites (TSS), housekeeping genes, binding sites for CTCF insulator factor and SINE elements ( 1 Dixon J.R. Selvaraj S. Yue F. et al. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature. 2012; 485: 376-380 Crossref PubMed Scopus (3953) Google Scholar ). Different structural variations like deletions and translocations may interfere with the TAD organization. Translocations with breakpoints outside of TAD boundaries (the majority) will invariably interfere with TAD organization, creating fusion domains, whereas deletions may or may not, depending on position and size. Although the majority of TADs are identical in human embryonic cells (hESC) and IMR90 fibroblast, some loci display a differential pattern in the two cell lines. This is exemplified by the Forkhead box G1 (FOXG1) gene on 14q12, located within a 2 Mb sized TAD in IMR90 cells which is split into two TADs in hESC. FOXG1 encodes a winged-helix transcriptional repressor critical for early telencephalon development. Coding mutations of FOXG1 causes a congenital variant of the severe neurodevelopmental disorder Rett syndrome2. Balanced chromosomal rearrangements with breakpoints downstream of FOXG1 have been shown to cause the same phenotype as coding mutations ( 2 Alosi D. Klitten L.L. Bak M. et al. Dysregulation of FOXG1 by ring chromosome 14. Mol Cytogenet. 2015; 8: 24 Crossref PubMed Scopus (6) Google Scholar , 3 Shoichet S.A. Kunde S.A. Viertel P. et al. Haploinsufficiency of novel FOXG1B variants in a patient with severe mental retardation and microcephaly. Hum Genet. 2005; 117: 536-544 Crossref PubMed Scopus (79) Google Scholar ). Such long range position effects (LRPE) are presumably caused by the removal of cis-acting regulatory elements. The LRPE critical region at the FOXG1 locus is not defined, and intra-TAD deletions that remove the hESC-specific TAD boundary distal to FOXG1 have been suggested to exemplify enhancer adoption ( 4 Lettice L.A. Daniels S. Sweeney E. et al. Enhancer-adoption as a mechanism of human developmental disease. Hum Mutat. 2011; 32: 1492-1499 Crossref PubMed Scopus (78) Google Scholar ), with the establishment of new deleterious enhancer-target gene interactions ( 5 Ibn-Salem J. Köhler S. Love M.I. et al. Deletions of chromosomal regulatory boundaries are associated with congenital disease. Genome Biol. 2014; 15: 423 Crossref PubMed Scopus (95) Google Scholar ). Herein we have mapped two new LRPE-associated translocations by mate-pair sequencing, which we use together with additional microdeletions distal to FOXG1 to define the critical LRPE-region and the evidence for or against enhancer adoption as a major feature of the FOXG1-locus.

A systematic method to identify modulation of transcriptional regulation via chromatin activity reveals regulatory network during mESC differentiation.

Chromatin regulators (CRs) are crucial for connecting the chromatin level and transcriptome level by modulating chromatin structures, establishing, and maintaining epigenetic modifications. We present a systematic method to identify MOdulation of transcriptional regulation via CHromatin Activity (MOCHA) from gene expression data and demonstrate its advantage in associating CRs to their chromatin localization and understand CRs’ function. We first re-construct the CRs modulation network by integrating the correlation and conditional correlation concepts. Then we quantify the chromatin activity as hidden variable in network by integrating the upstream and downstream information. We applied MOCHA to systematically explore the interplay of CRs, TFs, and target genes in mouse embryonic stem cells (ESC). As a result, MOCHA identified 420 chromatin regulators with modulation preference, including Pou5f1 and Eed. We found that BAF complex, NuRD complex, and polycomb-group proteins, regulate the delicate balance between pluripotency and differentiation by modulating key TFs including Klf4, Tcf3, and Max; NuRD complex members Mbd3 and Hdac1 may modulate Klf4 to achieve its dual functional roles in pluripotent and differentiation stages;Imprinted gene H19 and Igf2 are modulated by DNA methylation, histone acetylation, and insulator CTCF. Finally, we analyzed CR’s combinational modulation pattern by constructing a CR-CR interaction network.

Alterations in expression of imprinted genes from the H19/IGF2 loci in a multigenerational model of intrauterine growth restriction (IUGR)

The H19/IGF2 imprinted loci have attracted recent attention because of their role in cellular differentiation and proliferation, heritable gene regulation, and in utero or early postnatal growth and development. Expression from the imprinted H19/IGF2 locus involves a complex interplay of 3 means of epigenetic regulation: proper establishment of DNA methylation, promoter occupancy of CTCF, and expression of microRNA-675. We have demonstrated previously in a multigenerational rat model of intrauterine growth restriction the epigenetic heritability of adult metabolic syndrome in a F2 generation. We have further demonstrated abrogation of the F2 adult metabolic syndrome phenotype with essential nutrient supplementation of intermediates along the 1-carbon pathway and shown that alterations in the metabolome precede the adult onset of metabolic syndrome. The upstream molecular and epigenomic mediators underlying these observations, however, have yet to be elucidated fully. In the current study, we sought to characterize the impact of the intrauterine growth-restricted lineage and essential nutrient supplementation on both levels and molecular mediators of H19 and IGF2 gene expression in the F2 generation. F2 intrauterine growth-restricted and sham lineages were obtained by exposing P1 (grandmaternal) pregnant dams to bilateral uterine artery ligation or sham surgery at gestational day 19.5. F1 pups were allocated to the essential nutrient supplemented or control diet at postnatal day 21, and bred at 6-7 weeks of age. Hepatic tissues from the resultant F2 offspring at birth and at weaning (day 21) were obtained. Bisulfite modification and sequencing was employed for methylation analysis. H19 and IGF2 expression was measured by quantitative polymerase chain reaction. Promoter occupancy was quantified by the useof chromatin immunoprecipitation, or ChIP, against CTCF insulator proteins. Growth-restricted F2 on control diet demonstrated significant down-regulation in H19 expression compared with sham lineage (0.7831 vs 1.287; P < .05); however, essential nutrient supplementation diet abrogates this difference (4.995 vs 5.100; P > .05). Conversely, Igf2 was up-regulated by essential nutrient supplemented diet on the sham lineage (2.0 fold, P = .01), an effect that was not observed in the growth restricted offspring. A significant differential methylation was observed inthe promoter region of region H19 among the intrauterine growth-restricted lineage (18% vs 25%; P < .05) on a control diet, whereas the essential nutrient supplemented diet was alternately associated with hypermethylation in both lineages (sham: 50%; intrauterine growth restriction: 84%, P < .05). Consistent with essential nutrient supplementation impacting the epigenome, a decrease of CTCF promoter occupancy was observed in CTCF4 of the growth restricted lineage (2.45% vs 0.56%; P < .05) on the control diet, an effect that was repressed with essential nutrient supplementation. Heritable growth restriction is associated with changes in H19 gene expression; these changes are reversible with diet supplementation to favorably impact adult metabolic syndrome.

Autoimmune vitiligo is associated with gain-of-function by a transcriptional regulator that elevates expression of HLA-A*02:01 in vivo

HLA-A is a class I major histocompatibility complex receptor that presents peptide antigens on the surface of most cells. Vitiligo, an autoimmune disease in which skin melanocytes are destroyed by cognate T cells, is associated with variation in the HLA-A gene; specifically HLA-A*02:01, which presents multiple vitiligo melanocyte autoantigens. Refined genetic mapping localizes vitiligo risk in the HLA-A region to an SNP haplotype ∼20-kb downstream, spanning an ENCODE element with many characteristics of a transcriptional enhancer. Convergent CTCF insulator sites flanking the HLA-A gene promoter and the predicted transcriptional regulator, with apparent interaction between these sites, suggests this element regulates the HLA-A promoter. Peripheral blood mononuclear cells from healthy subjects homozygous for the high-risk haplotype expressed 39% more HLA-A RNA than cells from subjects carrying nonhigh-risk haplotypes (P = 0.0048). Similarly, RNAseq analysis of 1,000 Genomes Project data showed more HLA-A mRNA expressed in subjects homozygous for the high-risk allele of lead SNP rs60131261 than subjects homozygous for the low-risk allele (P = 0.006). Reporter plasmid transfection and genomic run-on sequence analyses confirm that the HLA-A transcriptional regulator contains multiple bidirectional promoters, with greatest activity on the high-risk haplotype, although it does not behave as a classic enhancer. Vitiligo risk associated with the MHC class I region thus derives from combined quantitative and qualitative phenomena: a SNP haplotype in a transcriptional regulator that induces gain-of-function, elevating expression of HLA-A RNA in vivo, in strong linkage disequilibrium with an HLA-A allele that confers *02:01 specificity.

Polymorphism in the TRP8 gene in Kyrgyz population: putative association with highland adaptation

The human TRPM8 gene encodes a receptor mediating cold sensitivity, and this fact points to its putative role in cold adaptation. The structural variability of the TRPM8 gene for five single-nucleotide polymorphisms (SNPs) has been studied in Kyrgyz population. The SNPs are located in coding regions of the gene, and three of them are confined to a segment of 20 bp in exon 7. The frequencies of minor SNP alleles are: rs13004520 G/C = 0.06; rs28901637 А/Т = 0.13; rs11562975 G/C = 0.27; rs7593557 G/A = 0.21; rs11563071 С/G = 0.12. The analyzed sample of Kyrgyz population includes 275 individuals living at different altitudes and under drastically different climatic conditions. The frequency of the minor rs11562975 allele in highlanders (living above 2 800 m A. S. L.) is one-third lower than in residents of lower regions (760–2 800 m A. S. L.; p &lt; 0.01). This result presumes a selective role of rs11562975 in cold adaptation. Comparison of haplotype frequencies in Kyrgyz people with Europeans, East Asians, and Africans shows a clear narrowing of genotype variation in Europeans in comparison to all others. Probably, this phenomenon is related to a population size decline (bottleneck effect) during the evolution. We consider the exon– intron structure of the TRPM8 gene. Epigenetic markers in the vicinity of the gene have been analyzed. Two strong binding sites for insulator ctcf proteins are present there. They are likely to be associated with chromatin conformation and alternative splicing regulation. A structure–functional characterization of genes for the TRP protein family is provided.