CTCF-binding Element Research Articles

Locus folding mechanisms determine modes of antigen receptor gene assembly.

The dynamic folding of genomes regulates numerous biological processes, including antigen receptor (AgR) gene assembly. We show that, unlike other AgR loci, homotypic chromatin interactions and bidirectional chromosome looping both contribute to structuring Tcrb for efficient long-range V(D)J recombination. Inactivation of the CTCF binding element (CBE) or promoter at the most 5'Vβ segment (Trbv1) impaired loop extrusion originating locally and extending to DβJβ CBEs at the opposite end of Tcrb. Promoter or CBE mutation nearly eliminated Trbv1 contacts and decreased RAG endonuclease-mediated Trbv1 recombination. Importantly, Trbv1 rearrangement can proceed independent of substrate orientation, ruling out scanning by DβJβ-bound RAG as the sole mechanism of Vβ recombination, distinguishing it from Igh. Our data indicate that CBE-dependent generation of loops cooperates with promoter-mediated activation of chromatin to juxtapose Vβ and DβJβ segments for recombination through diffusion-based synapsis. Thus, the mechanisms that fold a genomic region can influence molecular processes occurring in that space, which may include recombination, repair, and transcriptional programming.

A de novo transcription-dependent TAD boundary underpins critical multiway interactions during antibody class switch recombination

Interactions between transcription and cohesin-mediated loop extrusion can influence 3D chromatin architecture. However, their relevance in biology is unclear. Here, we report a direct role for such interactions in the mechanism of antibody class switch recombination (CSR) at the murine immunoglobulin heavy chain locus (Igh). Using Tri-C to measure higher-order multiway interactions on single alleles, we find that the juxtaposition (synapsis) of transcriptionally active donor and acceptor Igh switch (S)sequences, an essential step in CSR, occurs via the interaction of loop extrusion complexes with a de novo topologically associating domain (TAD) boundary formed via transcriptional activity across S regions. Surprisingly, synapsis occurs predominantly in proximity to the 3' CTCF-binding element (3'CBE) rather than the Igh super-enhancer, suggesting a two-step mechanism whereby transcription of S regions is not topologically coupled to synapsis, as has been previously proposed. Altogether, these insights advance our understanding of how 3D chromatin architecture regulates CSR.

A CTCF-Binding Element and Histone Deacetylation Cooperatively Maintain Chromatin Loops, Linking to Long-Range Gene Regulation in Cancer Genomes.

BackgroundGenes spanning long chromosomal domains are coordinately regulated in human genome, which contribute to global gene dysregulation and carcinogenesis in cancer. It has been noticed that epigenetic modification and chromatin architecture may participate in the regulation process. However, the regulation patterns and functional elements of long-range gene regulation are unclear.MethodsBased on the clinical transcriptome data from different tumor sets, a novel expressional correlation analysis pipeline was performed to classify the co-regulated regions and subsets of intercorrelated regions. The GLAM2 program was used to predict conserved DNA elements that enriched in regions. Two conserved elements were selected to delete in Ishikawa and HeLa cells by CRISPR-Cas9. SAHA treatment and HDAC knockdown were used to change the histone acetylation status. Using qPCR, MTT, and scratch healing assay, we evaluate the effect on gene expression and cancer cell phenotype. By DNA pull-down and ChIP, the element-binding proteins were testified. 3C and 3D-FISH were performed to depict the alteration in chromatin architecture.ResultsIn multiple cancer genomes, we classified subsets of coordinately regulated regions (sub-CRRs) that possibly shared the same regulatory mechanisms and exhibited similar expression patterns. A new conserved DNA element (CRE30) was enriched in sub-CRRs and associated with cancer patient survival. CRE30 could restrict gene regulation in sub-CRRs and affect cancer cell phenotypes. DNA pull-down showed that multiple proteins including CTCF were recruited on the CRE30 locus, and ChIP assay confirmed the CTCF-binding signals. Subsequent results uncovered that as an essential element, CRE30 maintained chromatin loops and mediated a compact chromatin architecture. Moreover, we found that blocking global histone deacetylation induced chromatin loop disruption and CTCF dropping in the region containing CRE30, linked to promoted gene regulation. Additionally, similar effects were observed with CRE30 deletion in another locus of chromosome 8.ConclusionsOur research clarified a new functional element that recruits CTCF and collaborates with histone deacetylation to maintain high-order chromatin organizations, linking to long-range gene regulation in cancer genomes. The findings highlight a close relationship among conserved DNA element, epigenetic modification, and chromatin architecture in long-range gene regulation process.

CTCF-binding element regulates ESC differentiation via orchestrating long-range chromatin interaction between enhancers and HoxA

Proper expression of Homeobox A cluster genes (HoxA) is essential for embryonic stem cell (ESC) differentiation and individual development. However, mechanisms controlling precise spatiotemporal expression of HoxA during early ESC differentiation remain poorly understood. Herein, we identified a functional CTCF-binding element (CBE+47) closest to the 3'-end of HoxA within the same topologically associated domain (TAD) in ESC. CRISPR-Cas9-mediated deletion of CBE+47 significantly upregulated HoxA expression and enhanced early ESC differentiation induced by retinoic acid (RA) relative to wild-type cells. Mechanistic analysis by chromosome conformation capture assay (Capture-C) revealed that CBE+47 deletion decreased interactions between adjacent enhancers, enabling formation of a relatively loose enhancer–enhancer interaction complex (EEIC), which overall increased interactions between that EEIC and central regions of HoxA chromatin. These findings indicate that CBE+47 organizes chromatin interactions between its adjacent enhancers and HoxA. Furthermore, deletion of those adjacent enhancers synergistically inhibited HoxA activation, suggesting that these enhancers serve as an EEIC required for RA-induced HoxA activation. Collectively, these results provide new insight into RA-induced HoxA expression during early ESC differentiation, also highlight precise regulatory roles of the CTCF-binding element in orchestrating high-order chromatin structure.

Fundamental roles of chromatin loop extrusion in antibody class switching

Antibody class switch recombination (CSR) in B lymphocytes replaces immunoglobulin heavy chain locus (Igh) Cμconstant region exons (CHs) with one of 6 CHs lying 100-200kb downstream1. Each CH is flanked upstream by an I-promoter and long repetitive switch (S) region1. Cytokines/activators induce Activation-Induced Cytidine Deaminase (AID)2 and I-promoter transcription, with 3’IgH regulatory region (3’IgHRR) enhancers controlling the latter via I-promoter competition for long-range 3’IgHRR interactions3-8. Transcription through donor Sμ and an activated downstream acceptor S region targets AID-generated deamination lesions at, potentially, any of 100s of individual S region deamination motifs9-11. General DNA repair pathways convert these lesions to DSBs and join an Sμ upstream DSB-end to an acceptor S region downstream DSB-end for deletional CSR12. AID-initiated DSBs at targets spread across activated S regions routinely participate in such deletional CSR joining11. Here, we report that chromatin loop extrusion underlies the "unprecedented" mechanism11 by which IgH organization in cis promotes deletional CSR. In naive B cells, loop extrusion dynamically juxtaposes 3’IgHRR enhancers with the 200kb upstream Sμ to generate a CSR center ("CSRC"). In CSR-activated primary B cells, I-promoter transcription activates cohesin loading, leading to generation of dynamic sub-domains that directionally align a downstream S region with Sμ for deletional CSR. During constitutive Sα CSR in CH12F3 B lymphoma cells, inversional CSR can be activated by insertion of a CTCF-binding element (“CBE”)-based impediment in the extrusion path. CBE insertion also inactivates upstream S region CSR, while converting adjacent downstream sequences into an ectopic S region by, respectively, inhibiting or promoting their dynamic alignment with Sμ in the CSRC. Our findings suggest that, in a CSRC, dynamically impeded cohesin-mediated loop extrusion juxtaposes proper ends of AID-initiated donor and acceptor S region DSBs for deletional CSR. Such a mechanism might also contribute to pathogenic DSB joining genome-wide.

The fundamental role of chromatin loop extrusion in physiological V(D)J recombination.

RAG endonuclease initiates IgH locus (Igh) V(D)J assembly in progenitor (pro)-B cells by joining Ds to JHs, before joining upstream VHs to DJH intermediates1. In mouse pro-B cells, the CTCF-binding element (CBE)-anchored chromatin loop domain2 at the 3’end of Igh contains an internal sub-domain spanning the 5’CBE anchor (IGCR1)3, the DHs, and a RAG-bound recombination center (RC)4. The RC comprises JH-proximal D (DQ52), 4 JHs, and the intronic enhancer (“iEμ”)5. Robust RAG cleavage is restricted to paired V(D)J segments flanked by complementary recombination signal sequences (12RSSs and 23RSSs)6. Ds are flanked downstream and upstream by 12RSSs that, respectively, mediate deletional joining with convergently-oriented JH-23RSSs and VH-23RSSs6. Despite 12/23 compatibility, inversional D to JH joining via upstream D-12RSSs is rare7,8. Plasmid-based assays attributed lack of inversional D to JH joining to sequence-based preference for downstream D-12RSSs9, as opposed to putative linear scanning mechanisms10,11. Given recent findings that RAG linearly scans convergent CBE-anchored chromatin loops4,12-14, potentially formed by cohesin-mediated loop extrusion15-18, we revisited a scanning role. Here, we report that JH-23RSS chromosomal orientation programs RC-bound RAG to linearly scan upstream chromatin in the 3’Igh sub-domain for convergently-oriented D-12RSSs and, thereby, to mediate deletional joining of all Ds, except RC-based DQ52 that joins by a diffusion-related mechanism. In a DQ52-based RC, formed in the absence of JHs, RAG bound by the downstream DQ52-RSS scans the downstream constant region exon-containing 3’Igh sub-domain in which scanning can be impeded by targeted nuclease-dead Cas9 (dCas9) binding, by transcription through repetitive Igh switch sequences, and by the 3’Igh CBE-based loop anchor. Notably, each scanning impediment focally increases RAG activity on potential substrate sequences within the impeded region. High resolution mapping of RC chromatin interactions reveals that such focal RAG targeting is associated with corresponding impediments to the loop extrusion process that drives chromatin past RC-bound RAG.

Abstract B171: Elucidating the mechanism of RAG tracking and its impacts on V(D)J recombination

Abstract RAG endonuclease initiates V(D)J recombination by binding to the recombination signal sequences (RSSs), introducing DNA breaks between two V(D)J gene segments, and mediating their joining. This process is tightly regulated to generate diverse antigen receptor repertoires and prevent aberrant events that could cause genomic translocations/deletions and lymphoid cancer. RAG can track directionally over chromosomal loop domain in which they reside. Tracking is evidenced by cleavage/joining of short cryptic RSSs lying exclusively in convergent orientation to a bona fide RSS. However, the mechanism of RAG tracking and its impacts remain unknown. Here I established an unbiased assay system by utilizing allele-specific barcode and employing high throughput genome-wide translocation sequencing (HTGTS) to quantitatively evaluate RAG-mediated recombination frequency. I found that insertion of one single bona fide RSS is sufficient to initiate RAG tracking in the c-Myc domain and the recombination between the RSS and its cryptic RSSs seems to follow the 12/23 rule to some degree. When ectopically inserted bona fide RSS pair is 0.5kb apart, convergent configuration of the RSS pair is 4.8-fold more frequent than the tandem configuration. When the distance increases to 267kb, the difference increases to 14-fold, suggesting the contribution of RAG tracking over diffusion-based mechanism increases when the distance between RSS pair increases. In addition, the presence of CTCF-binding element promotes recombination by more than 10-fold. Depletion of Wapl, cohesin remover, to disrupt cohesin turnover reduces V(D)J recombination. The distal RSSs are much preferentially affected. The data suggest that RAG tracking may be driven by cohesin sliding along chromatin. Citation Format: Cheng-Sheng Lee, Jiazhi Hu, Frederick W. Alt. Elucidating the mechanism of RAG tracking and its impacts on V(D)J recombination [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B171.

Diversification of the TCR β Locus Vβ Repertoire by CTCF.

The architectural protein CTCF regulates the conformation and recombination of antigen receptor loci. To study the importance of CTCF in Tcrb locus repertoire formation, we created a conditional knockout mouse line that deletes Ctcf early during thymocyte development. We observed an incomplete block in thymocyte development at the double-negative to double-positive transition, resulting in greatly lowered thymic cellularity. The Tcrb repertoire was altered with a decrease in recombination of Vβ gene segments in close proximity to a CTCF binding element (CBE), resulting in an overall repertoire that was skewed in favor of Vβ gene segments with no nearby CBE. Therefore we show that CTCF functions to diversify the Tcrb repertoire.

Computational and functional characterization of four SNPs in the SOST locus associated with osteoporosis.

The SOST gene encodes sclerostin, a C-terminal cysteine knot-like domain containing key negative regulator of osteoblastic bone formation that inhibits LRP5/6-mediated canonical Wnt signaling. Numerous single nucleotide polymorphisms (SNPs) in the SOST locus are firmly associated with bone mineral density (BMD) and fracture in genome-wide association studies (GWAS) and candidate gene association studies. However, the validation and mechanistic elucidation of causal genetic variants, especially for SNPs located beyond the promoter-proximal region, remain largely unresolved. By employing computational and experimental approaches, here we identify four SNPs rs1230399, rs7220711, rs1107748 and rs75901553 as functional variants which display allelic variation in SOST gene expression. The osteoporosis associated SNP rs1230399 in the SOST distal upstream regulatory region shows FOXA1 binding activity with subsequent transinactivation in a T allele-specific manner. The BMD GWAS lead SNPs rs7220711 and rs1107748 both reside in the 52-kb regulatory element deletion 35-kb downstream of the SOST gene which leads to Van Buchem disease. The rs7220711-A has a higher affinity for the transcriptional repressors MAFF or MAFK homodimers than rs7220711-G, while rs1107748 confers C allele specific transcriptional enhancer activity via a CTCF binding element. The variant rs75901553 C>T located in a conserved site of the SOST 3' UTR abolishes a target binding site for miR-98-5p which is negatively responsive to parathyroid hormone or 17β-estradiol in osteoblastic cell lines. Our findings uncover the biological consequences of four independent genetic variants in the SOST region and their important roles in SOST expression via diverse mechanisms, providing new insights into the genetics and molecular pathogenesis of osteoporosis.

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.

Chromosomal Loop Domains Direct the Recombination of Antigen Receptor Genes

RAG initiates antibody V(D)J recombination in developing lymphocytes by generating "on-target" DNA breaks at matched pairs of bona fide recombination signal sequences (RSSs). We employ bait RAG-generated breaks in endogenous or ectopically inserted RSS pairs to identify huge numbers of RAG "off-target" breaks. Such breaks occur at the simple CAC motif that defines the RSS cleavage site and are largely confined within convergent CTCF-binding element (CBE)-flanked loop domains containing bait RSS pairs. Marked orientation dependence of RAG off-target activity within loops spanning up to 2 megabases implies involvement of linear tracking. In this regard, major RAG off-targets in chromosomal translocations occur as convergent RSS pairs at enhancers within a loop. Finally, deletion of a CBE-based IgH locus element disrupts V(D)J recombination domains and, correspondingly, alters RAG on- and off-target distributions within IgH. Our findings reveal how RAG activity is developmentally focused and implicate mechanisms by which chromatin domains harness biological processes within them.

From the Cover: Loss of imprinting of the insulin-like growth factor II gene occurs by biallelic methylation in a core region of H19-associated CTCF-binding sites in colorectal cancer

We hypothesize that loss of imprinting (LOI) of the insulin-like growth factor II (IGF2) gene is associated with a predisposition to sporadic colorectal cancer. We confirmed a previously known strong correlation between LOI and microsatellite instability and showed that LOI was not a consequence of microsatellite instability or mismatch repair deficiency. LOI of IGF2 correlated strongly with biallelic hypermethylation of a core of five CpG sites in the insulator region of IGF2/H19, which is a known CTCF-binding element. As this methylation-dependent LOI was present in both tumors and normal colonic mucosa, it is possible that hypermethylation creates a field defect predisposing to cancer.