CTCF Binding Sites Research Articles

CBIO-05. SOX2 CHROMATIN ARCHITECTURE AND ENHANCER ACTIVITY IN THE MAINTENANCE AND DEVELOPMENT OF NEURAL STEM CELLS

Abstract Gain of function mutations in isocitrate dehydrogenase I (IDH1) result in the formation of the oncometabolite 2-hydroxyglutarate (2HG) in adult lower grade gliomas. To gain insight into mechanisms of gliomagenesis, our lab previously created a tractable human cellular model of low grade astrocytoma (LGA) using the putative cell-of-origin, human neural stem cells (NSCs), engineered to express mutant IDH1 and knockdown constructs against TP53 and ATRX, the two other genetic changes that accompany the IDH mutation in these tumors. We found that transcription factor (sex determining region Y)-box 2 SOX2, which is essential to NSC multipotency, the ability to differentiate to neuroglial lineages, behaves as a tumor suppressor during glioma initiation. In this context, we showed SOX2 is transcriptionally downregulated to impair NSC multipotency, thus locking NSCs in an undifferentiated state to initiate gliomagenesis. This downregulation occurs secondary to dynamic reorganization of the topologically associating domain (TAD) of SOX2 and the loss of contact with several genomic loci with histone modifications and chromatin accessibility suggestive of being enhancers. Here we show that those putative enhancers acquire enhancer-like features simultaneous to tje TAD organizing in a way that facilitates interaction with the SOX2 promoter during the process of pluripotent stem cell differentiation into neuroectodermal lineages, suggesting a developmental role. Preliminary data suggests that disruption of the SOX2 TAD by preventing binding of the genome organizer CTCF downregulates SOX2 expression in NSCs. Targeted silencing of several regions of a putative enhancer with CRISPRi also downregulates SOX2. In human embryonic stem cells (hESCs), interfering with these CTCF binding sites biases their differentiation away from the neuroectoderm. We are currently performing CRISPRi screen against all putative enhancer loci, teratoma formation assays on hESCs lacking relevant CTCF binding, and CRISPR mediated deletion of putative enhancers. Understanding this developmental process may reveal underlying vulnerabilities in LGA.

Myeloid Leukemia Dependencies at CTCF-Enriched Long Noncoding RNA Loci

The noncoding genome presents a largely untapped source of biological insights, including tens of thousands of long noncoding RNA (lncRNA) loci. While some produce bona fide lncRNAs, others exert transcript-independent cis-regulatory effects, and the lack of predictive features renders their mechanistic dissection highly challenging. Here, we describe CTCF-enriched lncRNA loci (C-LNC) as a putative new subclass of functional genetic elements exemplified by MYNRL15 - myeloid leukemia noncoding regulatory locus on chromosome 15.Initially identified by an expression-guided CRISPRi screen of hematopoietic stem and progenitor (HSPC) / acute myeloid leukemia (AML) lncRNA signatures (480 genes, 1545 sgRNAs), we found MYNRL15 dependency in myeloid leukemia cells of diverse genetic backgrounds. Interestingly, cis and trans perturbation approaches revealed both the MYNRL15 transcript and its flanking protein-coding genes to be dispensable. High density CRISPR tiling of a 15 kb area centered on MYNRL15 (1613 sgRNAs) instead uncovered two crucial, candidate cis-regulatory DNA elements in the locus, which drive the MYNRL15 perturbation phenotype. To determine the molecular basis of MYNRL15 dependence, we performed transcriptome, chromatin conformation, chromatin accessibility, and CTCF profiling. RNA-sequencing established MYNRL15's involvement in maintaining key cancer dependency pathways (e.g. cell cycle, ribosome, spliceosome). Further, MYNRL15 perturbation associated with the coordinated dysregulation of several chromosome 15 neighbourhoods, and formation of a long-range chromatin interaction between the locus and the base of a distal loop, as detected via next-generation Capture-C. The gained interaction was accompanied by diffuse gains in chromatin accessibility across the distal interaction sites (ATAC-seq) as well as reduced CTCF occupancy at the MYRNL15 locus (CTCF CUT&RUN), altogether indicating the 3D re-organization of chromosome 15 following MYNRL15 perturbation. Integrative analysis of the chromatin conformation and transcriptome data, combined with a small CRISPR-Cas9 knockout screen of protein-coding genes from the gained interaction region (29 genes, 149 sgRNAs), pinpointed two potent cancer dependency genes that are located in the region and downregulated following MYNRL15 perturbation: namely, WDR61 and IMP3. Individual knockout of both genes robustly depleted myeloid leukemia cells, recapitulating the MYNRL15 perturbation phenotype and positioning WDR61 and IMP3 as its regulatory targets. Importantly, in primary cells, MYNRL15 perturbation eradicated AML blasts while sparing 50-60% of CD34 + HSPCs in vitro, and reduced patient-derived AML xenografts up to 10-fold in vivo, indicating a potential therapeutic window.Having implicated MYNRL15 in 3D genome organization and demonstrated its role in myeloid leukemia cells, we explored whether MYNRL15 may belong to a sub-category of biologically relevant lncRNA loci that have thus far been overlooked due to their lack of transcript-specific functions. Remarkably, elevated CTCF density (e.g. number of CTCF binding sites per kb of gene length) distinguishes MYNRL15 and 531 other lncRNA loci in K562 cells, of which 43-54% associate with genetic subgroups and/or survival in AML patient cohorts, and 18.4% are functionally required for leukemia maintenance as determined by CRISPR-Cas9 screening. The latter hit identification rate represents a substantial improvement over typical lncRNA essentiality screens (which range from 2-6%) - illustrating the effectiveness of CTCF density metrics in refining functional lncRNA candidate lists, and underlining the relevance such loci hold for AML and cancer pathophysiology in general. Curated C-LNC catalogs in other cell types will facilitate the search for noncoding oncogenic vulnerabilities in AML and other malignancies. [Display omitted] DisclosuresReinhardt: Celgene Corporation: Consultancy; Novartis: Consultancy; Bluebird Bio: Consultancy; Janssen: Consultancy; CLS Behring: Research Funding; Roche: Research Funding. Klusmann: Bluebird Bio: Consultancy; Novartis: Consultancy; Roche: Consultancy; Jazz Pharmaceuticals: Consultancy.

CTCF puts a new twist on UV damage and repair in skin cancer

ABSTRACT Somatic mutations in skin cancers are highly enriched at binding sites for CCCTC-binding factor (CTCF). We have discovered that CTCF binding alters the DNA structure to render it more susceptible to UV damage. Elevated UV damage formation at CTCF binding sites, in conjunction with subsequent repair inhibition, promotes UV mutagenesis.

CHD4 Conceals Aberrant CTCF-Binding Sites at TAD Interiors by Regulating Chromatin Accessibility in Mouse Embryonic Stem Cells.

CCCTC-binding factor (CTCF) critically contributes to 3D chromatin organization by determining topologically associated domain (TAD) borders. Although CTCF primarily binds at TAD borders, there also exist putative CTCF-binding sites within TADs, which are spread throughout the genome by retrotransposition. However, the detailed mechanism responsible for masking the putative CTCF-binding sites remains largely elusive. Here, we show that the ATP-dependent chromatin remodeler, chromodomain helicase DNA-binding 4 (CHD4), regulates chromatin accessibility to conceal aberrant CTCF-binding sites embedded in H3K9me3-enriched heterochromatic B2 short interspersed nuclear elements (SINEs) in mouse embryonic stem cells (mESCs). Upon CHD4 depletion, these aberrant CTCF-binding sites become accessible and aberrant CTCF recruitment occurs within TADs, resulting in disorganization of local TADs. RNA-binding intrinsically disordered domains (IDRs) of CHD4 are required to prevent this aberrant CTCF binding, and CHD4 is critical for the repression of B2 SINE transcripts. These results collectively reveal that a CHD4-mediated mechanism ensures appropriate CTCF binding and associated TAD organization in mESCs.

Dissecting CTCF site function in a tense HoxD locus.

In this issue of Genes & Development, Amândio and colleagues (pp. 1490-1509) dissect the function of a cluster of several CTCF binding sites in the HoxD cluster by iterative deletions in mice. They found additive functions for some, and intriguingly found that some sites function as insulators, while others function as anchors for enhancer-promoter interactions. These functions vary depending on developmental context. The work provides new insights into the roles played by CTCF in regulating developmental patterns and 3D chromatin organization.

Sequential in cis mutagenesis in vivo reveals various functions for CTCF sites at the mouse HoxD cluster.

Mammalian Hox gene clusters contain a range of CTCF binding sites. In addition to their importance in organizing a TAD border, which isolates the most posterior genes from the rest of the cluster, the positions and orientations of these sites suggest that CTCF may be instrumental in the selection of various subsets of contiguous genes, which are targets of distinct remote enhancers located in the flanking regulatory landscapes. We examined this possibility by producing an allelic series of cumulative in cis mutations in these sites, up to the abrogation of CTCF binding in the five sites located on one side of the TAD border. In the most impactful alleles, the global chromatin architecture of the locus was modified, yet not drastically, illustrating that CTCF sites located on one side of a strong TAD border are sufficient to organize at least part of this insulation. Spatial colinearity in the expression of these genes along the major body axis was nevertheless maintained, despite abnormal expression boundaries. In contrast, strong effects were scored in the selection of target genes responding to particular enhancers, leading to the misregulation of Hoxd genes in specific structures. Altogether, while most enhancer-promoter interactions can occur in the absence of this series of CTCF sites, the binding of CTCF in the Hox cluster is required to properly transform a rather unprecise process into a highly discriminative mechanism of interactions, which is translated into various patterns of transcription accompanied by the distinctive chromatin topology found at this locus. Our allelic series also allowed us to reveal the distinct functional contributions for CTCF sites within this Hox cluster, some acting as insulator elements, others being necessary to anchor or stabilize enhancer-promoter interactions, and some doing both, whereas they all together contribute to the formation of a TAD border. This variety of tasks may explain the amazing evolutionary conservation in the distribution of these sites among paralogous Hox clusters or between various vertebrates.

CGGBP1-dependent CTCF-binding sites restrict ectopic transcription

ABSTRACT Binding sites of the chromatin regulator protein CTCF function as important landmarks in the human genome. The recently characterized CTCF-binding sites at LINE-1 repeats depend on another repeat-regulatory protein CGGBP1. These CGGBP1-dependent CTCF-binding sites serve as potential barrier elements for epigenetic marks such as H3K9me3. Such CTCF-binding sites are associated with asymmetric H3K9me3 levels as well as RNA levels in their flanks. The functions of these CGGBP1-dependent CTCF-binding sites remain unknown. By performing targeted studies on candidate CGGBP1-dependent CTCF-binding sites cloned in an SV40 promoter-enhancer episomal system we show that these regions act as inhibitors of ectopic transcription from the SV40 promoter. CGGBP1-dependent CTCF-binding sites that recapitulate their genomic function of loss of CTCF binding upon CGGBP1 depletion and H3K9me3 asymmetry in immediate flanks are also the ones that show the strongest inhibition of ectopic transcription. By performing a series of strand-specific reverse transcription PCRs we demonstrate that this ectopic transcription results in the synthesis of RNA from the SV40 promoter in a direction opposite to the downstream reporter gene in a strand-specific manner. The unleashing of the bidirectionality of the SV40 promoter activity and a breach of the transcription barrier seems to depend on depletion of CGGBP1 and loss of CTCF binding proximal to the SV40 promoter. RNA-sequencing reveals that CGGBP1-regulated CTCF-binding sites act as barriers to transcription at multiple locations genome-wide. These findings suggest a role of CGGBP1-dependent binding sites in restricting ectopic transcription.

3'HS1 CTCF binding site in human β-globin locus regulates fetal hemoglobin expression.

Mutations in the adult β-globin gene can lead to a variety of hemoglobinopathies, including sickle cell disease and β-thalassemia. An increase in fetal hemoglobin expression throughout adulthood, a condition named hereditary persistence of fetal hemoglobin (HPFH), has been found to ameliorate hemoglobinopathies. Deletional HPFH occurs through the excision of a significant portion of the 3' end of the β-globin locus, including a CTCF binding site termed 3'HS1. Here, we show that the deletion of this CTCF site alone induces fetal hemoglobin expression in both adult CD34+ hematopoietic stem and progenitor cells and HUDEP-2 erythroid progenitor cells. This induction is driven by the ectopic access of a previously postulated distal enhancer located in the OR52A1 gene downstream of the locus, which can also be insulated by the inversion of the 3'HS1 CTCF site. This suggests that genetic editing of this binding site can have therapeutic implications to treat hemoglobinopathies.

The leukemic oncogene EVI1 hijacks a MYC super-enhancer by CTCF-facilitated loops

Chromosomal rearrangements are a frequent cause of oncogene deregulation in human malignancies. Overexpression of EVI1 is found in a subgroup of acute myeloid leukemia (AML) with 3q26 chromosomal rearrangements, which is often therapy resistant. In AMLs harboring a t(3;8)(q26;q24), we observed the translocation of a MYC super-enhancer (MYC SE) to the EVI1 locus. We generated an in vitro model mimicking a patient-based t(3;8)(q26;q24) using CRISPR-Cas9 technology and demonstrated hyperactivation of EVI1 by the hijacked MYC SE. This MYC SE contains multiple enhancer modules, of which only one recruits transcription factors active in early hematopoiesis. This enhancer module is critical for EVI1 overexpression as well as enhancer-promoter interaction. Multiple CTCF binding regions in the MYC SE facilitate this enhancer-promoter interaction, which also involves a CTCF binding site upstream of the EVI1 promoter. We hypothesize that this CTCF site acts as an enhancer-docking site in t(3;8) AML. Genomic analyses of other 3q26-rearranged AML patient cells point to a common mechanism by which EVI1 uses this docking site to hijack enhancers active in early hematopoiesis.

Research progress of CTCF in mediating 3D genome formation and regulating gene expression.

In interphase eukaryotic nuclei, chromatin is folded to form a higher-order topological structure. The spatial organization of such chromatin domain has an important impact on the regulation of gene expression. As a key architectural structural protein, CTCF (CCCTC-binding factor) plays an important role in the formation of chromatin three-dimensional chromatin structure. CTCF can also bind to many insulator elements in the genome and insulate enhancers from activating target genes via modulating remote chromatin interactions. A recent study by Dr. Chunliang Li and his team at St. Jude Children's Research Hospital in the United States showed that when CTCF was acutely degraded, significant changes were found in the three-dimensional structure of chromatin. The mechanism by which CTCF binding sites function as insulator elements was investigated by Prof. Qiang Wu's team at Institute of Systems Biomedicine and Shanghai Jiao Tong University in China and Prof. Bing Ren's team at Ludwig Institute for Cancer Research in the United States. Here we mainly review and discuss some of these latest progresses.

DNA 5-hydroxymethylcytosine in pediatric central nervous system tumors may impact tumor classification and is a positive prognostic marker

BackgroundNucleotide-specific 5-hydroxymethylcytosine (5hmC) remains understudied in pediatric central nervous system (CNS) tumors. 5hmC is abundant in the brain, and alterations to 5hmC in adult CNS tumors have been reported. However, traditional approaches to measure DNA methylation do not distinguish between 5-methylcytosine (5mC) and its oxidized counterpart 5hmC, including those used to build CNS tumor DNA methylation classification systems. We measured 5hmC and 5mC epigenome-wide at nucleotide resolution in glioma, ependymoma, and embryonal tumors from children, as well as control pediatric brain tissues using tandem bisulfite and oxidative bisulfite treatments followed by hybridization to the Illumina Methylation EPIC Array that interrogates over 860,000 CpG loci.ResultsLinear mixed effects models adjusted for age and sex tested the CpG-specific differences in 5hmC between tumor and non-tumor samples, as well as between tumor subtypes. Results from model-based clustering of tumors was used to test the relation of cluster membership with patient survival through multivariable Cox proportional hazards regression. We also assessed the robustness of multiple epigenetic CNS tumor classification methods to 5mC-specific data in both pediatric and adult CNS tumors. Compared to non-tumor samples, tumors were hypohydroxymethylated across the epigenome and tumor 5hmC localized to regulatory elements crucial to cell identity, including transcription factor binding sites and super-enhancers. Differentially hydroxymethylated loci among tumor subtypes tended to be hypermethylated and disproportionally found in CTCF binding sites and genes related to posttranscriptional RNA regulation, such as DICER1. Model-based clustering results indicated that patients with low 5hmC patterns have poorer overall survival and increased risk of recurrence. Our results suggest 5mC-specific data from OxBS-treated samples impacts methylation-based tumor classification systems giving new opportunities for further refinement of classifiers for both pediatric and adult tumors.ConclusionsWe identified that 5hmC localizes to super-enhancers, and genes commonly implicated in pediatric CNS tumors were differentially hypohydroxymethylated. We demonstrated that distinguishing methylation and hydroxymethylation is critical in identifying tumor-related epigenetic changes. These results have implications for patient prognostication, considerations of epigenetic therapy in CNS tumors, and for emerging molecular neuropathology classification approaches.

CTCF binding modulates UV damage formation to promote mutation hot spots in melanoma.

Somatic mutations in DNA-binding sites for CCCTC-binding factor (CTCF) are significantly elevated in many cancers. Prior analysis has suggested that elevated mutation rates at CTCF-binding sites in skin cancers are a consequence of the CTCF-cohesin complex inhibiting repair of UV damage. Here, we show that CTCF binding modulates the formation of UV damage to induce mutation hot spots. Analysis of genome-wide CPD-seq data in UV-irradiated human cells indicates that formation of UV-induced cyclobutane pyrimidine dimers (CPDs) is primarily suppressed by CTCF binding but elevated at specific locations within the CTCF motif. Locations of CPD hot spots in the CTCF-binding motif coincide with mutation hot spots in melanoma. A similar pattern of damage formation is observed at CTCF-binding sites invitro, indicating that UV damage modulation is a direct consequence of CTCF binding. We show that CTCF interacts with binding sites containing UV damage and inhibits repair by a model repair enzyme invitro. Structural analysis and molecular dynamic simulations reveal the molecular mechanism for how CTCF binding modulates CPD formation.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Acute depletion of CTCF rewires genome-wide chromatin accessibility

BackgroundThe transcription factor CTCF appears indispensable in defining topologically associated domain boundaries and maintaining chromatin loop structures within these domains, supported by numerous functional studies. However, acute depletion of CTCF globally reduces chromatin interactions but does not significantly alter transcription.ResultsHere, we systematically integrate multi-omics data including ATAC-seq, RNA-seq, WGBS, Hi-C, Cut&Run, and CRISPR-Cas9 survival dropout screens, and time-solved deep proteomic and phosphoproteomic analyses in cells carrying auxin-induced degron at endogenous CTCF locus. Acute CTCF protein degradation markedly rewires genome-wide chromatin accessibility. Increased accessible chromatin regions are frequently located adjacent to CTCF-binding sites at promoter regions and insulator sites associated with enhanced transcription of nearby genes. In addition, we use CTCF-associated multi-omics data to establish a combinatorial data analysis pipeline to discover CTCF co-regulatory partners. We successfully identify 40 candidates, including multiple established partners. Interestingly, many CTCF co-regulators that have alterations of their respective downstream gene expression do not show changes of their own expression levels across the multi-omics measurements upon acute CTCF loss, highlighting the strength of our system to discover hidden co-regulatory partners associated with CTCF-mediated transcription.ConclusionsThis study highlights that CTCF loss rewires genome-wide chromatin accessibility, which plays a critical role in transcriptional regulation.

Serial deletions of tandem reverse CTCF sites reveal balanced HOXD regulatory landscape of enhancers

The genome architectural protein CTCF (CCCTC-binding factor) not only mediates long-distance chromatin interactions between distal enhancers and target promoters, but also functions as an important insulator-binding factor to block improper enhancer activation of non-target promoters, and is thus of great significance to transcriptional regulation of developmental genes. The Hox (Homeobox) gene family plays an important role in the development of the brain, bones, and limbs. The spatiotemporal colinear expression of the HOXD cluster along the proximal-distal axis of limbs is regulated by two clusters of enhancers known as super-enhancers located in the flanking regulatory regions. We focused on the HOXD cluster to explore the architectural role of CTCF in transcriptional regulation of developmental genes. The HOXD cluster contains 9 paralogous genes intermixed with a series of CBS (CTCF-binding site) elements. Using the CRISPR DNA-fragment editing system, we generated a series of single-cell HEK293T clones with deletion of increasing numbers of reverse CBS elements. RNA-seq experiments revealed decreased levels of HOXD gene expression. In addition, chromosome conformation capture experiments revealed increased long-distance chromatin interactions between HOXD and the upstream enhancer cluster and corresponding decreased interactions between HOXD and the downstream enhancer cluster. Thus, tandem reverse CTCF sites function as insulators to maintain HOXD regulatory balance between the upstream and downstream enhancer clusters. This study has interesting implications on the precise gene expression control of the Hox family during animal development.

Combinatorial CRISPR inversions of CTCF sites in HOXD cluster reveal complex insulator function

CTCF (CCCTC-binding factor) is a zinc-finger protein which plays a vital role in the three-dimensional (3D) genome architecture. A pair of forward-reverse convergent CTCF binding sites (CBS elements) mediates long-distance DNA interactions to form chromatin loops with the assistance of the cohesin complex, while CBS elements at the chromatin domain boundaries show reverse-forward divergent patterns and function as insulators to discriminate against DNA interaction between chromatin domains. However, there are still many unresolved problems regarding CTCF-mediated insulation function. In order to study the connections between chromatin loops and the insulation function of CBS elements, we combinatorically inverted CBS elements at the HOXD locus by using CRISPR/Cas9 DNA-fragment editing methods in the HEK293T cell line and obtained five different kinds of single-cell CRISPR clones. By performing quantitative high-resolution chromosome conformation capture copy (QHR-4C) experiments, we found that boundary CBS inversions abolish original chromatin loops and establish new loops from the opposite direction, thus shifting the insulator boundary to the new divergent CTCF sites. Furthermore, tandem CBS elements block cohesin permeated from the opposite orientation to function as insulators. RNA-seq experiments showed that alterations of local three-dimensional genome architecture would further influence gene expression of the HOXD cluster. In conclusion, a pair of divergent CBS elements function as insulators by forming chromatin loops within chromatin domains to block cohesin sliding.

CTCF is a barrier for 2C-like reprogramming

Totipotent cells have the ability to generate embryonic and extra-embryonic tissues. Interestingly, a rare population of cells with totipotent-like potential, known as 2 cell (2C)-like cells, has been identified within ESC cultures. They arise from ESC and display similar features to those found in the 2C embryo. However, the molecular determinants of 2C-like conversion have not been completely elucidated. Here, we show that the CCCTC-binding factor (CTCF) is a barrier for 2C-like reprogramming. Indeed, forced conversion to a 2C-like state by the transcription factor DUX is associated with DNA damage at a subset of CTCF binding sites. Depletion of CTCF in ESC efficiently promotes spontaneous and asynchronous conversion to a 2C-like state and is reversible upon restoration of CTCF levels. This phenotypic reprogramming is specific to pluripotent cells as neural progenitor cells do not show 2C-like conversion upon CTCF-depletion. Furthermore, we show that transcriptional activation of the ZSCAN4 cluster is necessary for successful 2C-like reprogramming. In summary, we reveal an unexpected relationship between CTCF and 2C-like reprogramming.

EBNA2 driven enhancer switching at the CIITA-DEXI locus suppresses HLA class II gene expression during EBV infection of B-lymphocytes.

Viruses suppress immune recognition through diverse mechanisms. Epstein-Barr Virus (EBV) establishes latent infection in memory B-lymphocytes and B-cell malignancies where it impacts B-cell immune function. We show here that EBV primary infection of naïve B-cells results in a robust down-regulation of HLA genes. We found that the viral encoded transcriptional regulatory factor EBNA2 bound to multiple regulatory regions in the HLA locus. Conditional expression of EBNA2 correlated with the down regulation of HLA class II transcription. EBNA2 down-regulation of HLA transcription was found to be dependent on CIITA, the major transcriptional activator of HLA class II gene transcription. We identified a major EBNA2 binding site downstream of the CIITA gene and upstream of DEXI, a dexamethasone inducible gene that is oriented head-to-head with CIITA gene transcripts. CRISPR/Cas9 deletion of the EBNA2 site upstream of DEXI attenuated CIITA transcriptional repression. EBNA2 caused an increase in DEXI transcription and a graded change in histone modifications with activation mark H3K27ac near the DEXI locus, and a loss of activation marks at the CIITA locus. A prominent CTCF binding site between CIITA and DEXI enhancers was mutated and further diminished the effects of EBNA2 on CIITA. Analysis of HiC data indicate that DEXI and CIITA enhancers are situated in different chromosome topological associated domains (TADs). These findings suggest that EBNA2 down regulates HLA-II genes through the down regulation of CIITA, and that this down regulation is an indirect consequence of EBNA2 enhancer formation at a neighboring TAD. We propose that enhancer competition between these neighboring chromosome domains represents a novel mechanism for gene regulation demonstrated by EBNA2.

Differential CTCF binding in motor neurons and lymphocytes

Background: Diversity is critical to lymphocyte roles in the immune system and to neurons, which form complex network structures in the brain. Emerging evidence suggests that an increasing number of molecules associated with the immune system are also expressed in various central nervous system (CNS) regions and play crucial roles in brain development. Examination of shared molecular mechanisms underlying neurogenesis and lymphocyte differentiation may clarify relevant pathways, and suggests additional biomarkers in lymphocytes for neurological disorders. These results can contribute to find biomarkers that can be monitored through patient lymphocyte populations. Methods: We analysed similarities and conserved regions in several genes regulated by CCCTC-binding factor (CTCF) during lymphocyte and neuronal developmental stages. We performed epigenetic analyses of CTCF binding Trak1, Gabpa, Gabpb1, Gabpb2, Gfi1, Gfi1b gene loci in T and B lymphocytes at different developmental stages, as well as in neural progenitor cells and motor neurons. Results: Common and shared CTCF binding events at Trak1 suggest additional transcriptional regulatory factors that control Trak1 gene expression levels differ in neurons and lymphocytes. Gabpb1 includes a common CTCF binding site shared with neurons and lymphocytes. Correlation of CTCF binding analysis and gene expression profile suggests that CTCF binding plays a role in epigenetic regulation of Gabpb1 gene. While Gfi1a is phylogenetically well-conserved and CTCF sites are occupied in lymphocytes, there are no CTCF binding occupied in neurons and neural progenitor cells. Although Gfi1b is highly homologous to Gfi1, differences in expression levels suggest that Gfi1b is critical for both lymphogenesis and neurogenesis. Neurons and lymphocytes have multiple common CTCF binding sites in the Gfi1b gene. Conclusions: The partial overlap in CTCF regulatory sites for some genes in neurons and lymphocytes suggests that there may be markers which can exhibit parallel changes in these cells and serve as biomarkers.

Multiple CTCF sites cooperate with each other to maintain a TAD for enhancer-promoter interaction in the β-globin locus.

Insulators are cis-regulatory elements that block enhancer activity and prevent heterochromatin spreading. The binding of CCCTC-binding factor (CTCF) protein is essential for insulators to play the roles in a chromatin context. The β-globin locus, consisting of multiple genes and enhancers, is flanked by two insulators 3'HS1 and HS5. However, it has been reported that the absence of these insulators did not affect the β-globin transcription. To explain the unexpected finding, we have deleted a CTCF motif at 3'HS1 or HS5 in the human β-globin locus and analyzed chromatin interactions around the locus. It was found that a topologically associating domain (TAD) containing the β-globin locus is maintained by neighboring CTCF sites in the CTCF motif-deleted loci. The additional deletions of neighboring CTCF motifs disrupted the β-globin TAD, resulting in decrease of the β-globin transcription. Chromatin interactions of the β-globin enhancers with gene promoter were weakened in the multiple CTCF motifs-deleted loci, even though the enhancers have still active chromatin features such as histone H3K27ac and histone H3 depletion. Genome-wide analysis using public CTCF ChIA-PET and ChIP-seq data showed that chromatin domains possessing multiple CTCF binding sites tend to contain super-enhancers like the β-globin enhancers. Taken together, our results show that multiple CTCF sites surrounding the β-globin locus cooperate with each other to maintain a TAD. The β-globin TAD appears to provide a compact spatial environment that enables enhancers to interact with promoter.