CTCF Binding Research Articles

Targeting the 3D genome by anthracyclines for chemotherapeutic effects.

The chromatins are folded into three-dimensional (3D) structures inside cells, which coordinates the regulation of gene transcription by the non-coding regulatory elements. Aberrant chromatin 3D folding has been shown in many diseases, such as acute myeloid leukemia (AML), and may contribute to tumorigenesis. The anthracycline topoisomerase II inhibitors can induce histone eviction and DNA damage. We performed genome-wide high-resolution mapping of the chemotherapeutic effects of various clinically used anthracycline drugs. ATAC-seq was used to profile the histone eviction effects of different anthracyclines. TOP2A ChIP-seq was used to profile the potential DNA damage regions. Integrated analyses show that different anthracyclines have distinct target selectivity on epigenomic regions, based on their respective ATAC-seq and ChIP-seq profiles. We identified the underlying molecular mechanism that unique anthracycline variants selectively target chromatin looping anchors via disrupting CTCF binding, suggesting an additional potential therapeutic effect on the 3D genome. We further performed Hi-C experiments, and data from K562 cells treated with the selective anthracycline drugs indicate that the 3D chromatin organization is disrupted. Furthermore, AML patients receiving anthracycline drugs showed altered chromatin structures around potential looping anchors, which linked to distinct clinical outcomes. Our data indicate that anthracyclines are potent and selective epigenomic targeting drugs and can target the 3D genome for anticancer therapy, which could be used for personalized medicine to treat tumors with aberrant 3D chromatin structures.

Interaction of CTCF and CTCFL in genome regulation through chromatin architecture during the spermatogenesis and carcinogenesis.

The zinc finger protein CTCF is ubiquitously expressed and is integral to the regulation of chromatin architecture through its interaction with cohesin. Conversely, CTCFL expression is predominantly restricted to the adult male testis but is aberrantly expressed in certain cancers. Despite their distinct expression patterns, the cooperative and competitive mechanisms by which CTCF and CTCFL regulate target gene expression in spermatocytes and cancer cells remain inadequately understood. In this review, we comprehensively examine the literature on the divergent amino acid sequences, target sites, expression profiles and functions of CTCF and CTCFL in normal tissues and cancers. We further elucidate the mechanisms by which CTCFL competitively or cooperatively binds to CTCF target sites during spermatogenesis and carcinogenesis to modulate chromatin architecture. We mainly focus on the role of CTCFL in testicular and cancer development, highlighting its interaction with CTCF at CTCF binding sites to regulate target genes. In the testis, CTCF and CTCFL cooperate to regulate the expression of testis-specific genes, essential for proper germ cell progression. In cancers, CTCFL overexpression competes with CTCF for DNA binding, leading to aberrant gene expression, a more relaxed chromatin state, and altered chromatin loops. By uncovering the roles of CTCF and CTCFL in spermatogenesis and carcinogenesis, we can better understand the implications of aberrant CTCFL expression in altering chromatin loops and its contribution to disease pathogenesis.

Dynamic barriers modulate cohesin positioning and genome folding at fixed occupancy.

In mammalian interphase cells, genomes are folded by cohesin loop extrusion limited by directional CTCF barriers. This interplay leads to the enrichment of cohesin at barriers, isolation between neighboring topologically associating domains, and elevated contact frequency between convergent CTCF barriers across the genome. However, recent in vivo measurements present a puzzle: reported residence times for CTCF on chromatin are in the range of a few minutes, while lifetimes for cohesin are much longer. Can the observed features of genome folding result from the action of relatively transient barriers? To address this question, we developed a dynamic barrier model, where CTCF sites switch between bound and unbound states with rates that can be directly compared with biophysical measurements. Using this model, we investigated how barrier dynamics would impact observables for a range of experimental genomic and imaging datasets, including ChIP-seq, Hi-C, and microscopy. We found the interplay of CTCF and cohesin binding timescales influence the strength of each of these features, leaving a signature of barrier dynamics even in the population-averaged snapshots offered by genomic datasets. First, in addition to barrier occupancy, barrier bound times are crucial for instructing features of genome folding. Second, the ratio of boundary to extruder lifetime greatly alters simulated ChIP-seq and simulated Hi-C. Third, large-scale changes in chromosome morphology observed experimentally after increasing extruder lifetime require dynamic barriers. By integrating multiple sources of experimental data, our biophysical model argues that CTCF barrier bound times effectively approach those of cohesin extruder lifetimes. Together, we demonstrate how models that are informed by biophysically measured protein dynamics broaden our understanding of genome folding.

Permeable TAD boundaries and their impact on genome-associated functions.

TAD boundaries are genomic elements that separate biological processes in neighboring domains by blocking DNA loops that are formed through Cohesin-mediated loop extrusion. Most TAD boundaries consist of arrays of binding sites for the CTCF protein, whose interaction with the Cohesin complex blocks loop extrusion. TAD boundaries are not fully impermeable though and allow a limited amount of inter-TAD loop formation. Based on the reanalysis of Nano-C data, a multicontact Chromosome Conformation Capture assay, we propose a model whereby clustered CTCF binding sites promote the successive stalling of Cohesin and subsequent dissociation from the chromatin. A fraction of Cohesin nonetheless achieves boundary read-through. Due to a constant rate of Cohesin dissociation elsewhere in the genome, the maximum length of inter-TAD loops is restricted though. We speculate that the DNA-encoded organization of stalling sites regulates TAD boundary permeability and discuss implications for enhancer-promoter loop formation and other genomic processes.

Fine Mapping Regulatory Variants by Characterizing Native CpG Methylation with Nanopore Long-Read Sequencing.

5-methylcytosine (5mC) is the most common chemical modification occurring on the CpG sites across the human genome. Bisulfite conversion combined with short-read whole genome sequencing can capture and quantify the modification at single nucleotide resolution. However, the PCR amplification process could lead to duplicative methylation patterns and introduce 5mC detection bias. Additionally, the limited read length also restricts co-methylation analysis between distant CpG sites. The bisulfite conversion process presents a significant challenge for detecting variant-specific methylation due to the destruction of allele information in the sequencing reads. To address these issues, we sought to characterize the human methylation profiling with the nanopore long-read sequencing, aiming to demonstrate its potential for long-range co-methylation analysis with native modification call and intact allele information retained. In this regard, we first analyzed the nanopore demo data in the adaptive sampling sequencing run targeting all human CpG islands. We applied the linkage disequilibrium (LD) R2 to calculate the co-methylation in nanopore data, and further identified 27,875, 50,481, 26,542 and 51,189 methylation haplotype blocks (MHB) in COLO829, COLO829BL, HCC1395 and HCC1395BL cell lines, respectively. Interestingly, while we found that majority of the co-methylation were in a short range (≤200bp), a small portion (1~3%) showed long distance (≥1,000bp), suggesting potential remote regulatory mechanisms across the genome. To further characterize the epigenetic changes related to transcription factor binding, we profiled the 5mC percentage changes surrounding various motif sites in JASPAR collection and found that CTCF and KLF5 binding sites showed reduced methylation, while FOXE1 and ZNF354A sites showed increased methylation. To further investigate the allele-specific 5mCG in the prostate genome, we designed a target region covering methylation quantitative trait loci (mQTL) and genome-wide association study (GWAS) risk germline variants and generated long reads with adaptive sampling run in the 22Rv1 cell line. To identify the allele-specific methylation in the 22Rv1 cell line, we performed long-read based phasing and compared the 5mCG signals between the two haplotypes. As a result, we identified 6,390 haplotype-specific methylated regions in the 22Rv1 cell line (p-MWU ≤ 1e-5 and delta ≥ 50%). By examining haplotype-specific methylated regions near the phasing variants, we identified examples of allele-specific methylated regions that showed allelespecific accessibility in the ATAC-seq data. By further integrating the ATAC-seq data of 22Rv1, we found that methylation levels were negatively correlated with chromatin accessibility at the genome-wide scale. Our study has revealed native methylome profiling while preserving haplotype information, offering a novel approach to uncovering the regulatory mechanisms of the human prostate genome.

Clusters of lineage-specific genes are anchored by ZNF274 in repressive perinucleolar compartments.

Long known as the site of ribosome biogenesis, the nucleolus is increasingly recognized for its role in shaping three-dimensional (3D) genome organization. Still, the mechanisms governing the targeting of selected regions of the genome to nucleolus-associated domains (NADs) remain enigmatic. Here, we reveal the essential role of ZNF274, a SCAN-bearing member of the Krüppel-associated box (KRAB)-containing zinc finger protein (KZFP) family, in sequestering lineage-specific gene clusters within NADs. Ablation of ZNF274 triggers transcriptional activation across entire genomic neighborhoods-encompassing, among others, protocadherin and KZFP-encoding genes-with loss of repressive chromatin marks, altered the 3D genome architecture and de novo CTCF binding. Mechanistically, ZNF274 anchors target DNA sequences at the nucleolus and facilitates their compartmentalization via a previously uncharted function of the SCAN domain. Our findings illuminate the mechanisms underlying NAD organization and suggest that perinucleolar entrapment into repressive hubs constrains the activation of tandemly arrayed genes to enable selective expression and modulate cell differentiation programs during development.

Gapped-kmer sequence modeling robustly identifies regulatory vocabularies and distal enhancers conserved between evolutionarily distant mammals

Gene regulatory elements drive complex biological phenomena and their mutations are associated with common human diseases. The impacts of human regulatory variants are often tested using model organisms such as mice. However, mapping human enhancers to conserved elements in mice remains a challenge, due to both rapid enhancer evolution and limitations of current computational methods. We analyze distal enhancers across 45 matched human/mouse cell/tissue pairs from a comprehensive dataset of DNase-seq experiments, and show that while cell-specific regulatory vocabulary is conserved, enhancers evolve more rapidly than promoters and CTCF binding sites. Enhancer conservation rates vary across cell types, in part explainable by tissue specific transposable element activity. We present an improved genome alignment algorithm using gapped-kmer features, called gkm-align, and make genome wide predictions for 1,401,803 orthologous regulatory elements. We show that gkm-align discovers 23,660 novel human/mouse conserved enhancers missed by previous algorithms, with strong evidence of conserved functional activity.

Evolutionarily conserved enhancer-associated features within the MYEOV locus suggest a regulatory role for this non-coding DNA region in cancer.

The myeloma overexpressed gene (MYEOV) has been proposed to be a proto-oncogene due to high RNA transcript levels found in multiple cancers, including myeloma, breast, lung, pancreas and esophageal cancer. The presence of an open reading frame (ORF) in humans and other primates suggests protein-coding potential. Yet, we still lack evidence of a functional MYEOV protein. It remains undetermined how MYEOV overexpression affects cancerous tissues. In this work, we show that MYEOV has likely originated and may still function as an enhancer, regulating CCND1 and LTO1. Firstly, MYEOV 3' enhancer activity was confirmed in humans using publicly available ATAC-STARR-seq data, performed on B-cell-derived GM12878 cells. We detected enhancer histone marks H3K4me1 and H3K27ac overlapping MYEOV in multiple healthy human tissues, which include B cells, liver and lung tissue. The analysis of 3D genome datasets revealed chromatin interactions between a MYEOV-3'-putative enhancer and the proto-oncogene CCND1. BLAST searches and multi-sequence alignment results showed that DNA sequence from this human enhancer element is conserved from the amphibians/amniotes divergence, with a 273bp conserved region also found in all mammals, and even in chickens, where it is consistently located near the corresponding CCND1 orthologues. Furthermore, we observed conservation of an active enhancer state in the MYEOV orthologues of four non-human primates, dogs, rats, and mice. When studying this homologous region in mice, where the ORF of MYEOV is absent, we not only observed an enhancer chromatin state but also found interactions between the mouse enhancer homolog and Ccnd1 using 3D-genome interaction data. This is similar to the interaction observed in humans and, interestingly, coincides with CTCF binding sites in both species. Taken together, this suggests that MYEOV is a primate-specific gene with a de novo ORF that originated at an evolutionarily older enhancer region. This deeply conserved putative enhancer element could regulate CCND1 in both humans and mice, opening the possibility of studying MYEOV regulatory functions in cancer using non-primate animal models.

Evolutionary patterns and functional effects of 3D chromatin structures in butterflies with extensive genome rearrangements

Chromosome rearrangements may distort 3D chromatin architectures and thus change gene regulation, yet how 3D chromatin structures evolve in insects is largely unknown. Here, we obtain chromosome-level genomes for four butterfly species, Graphium cloanthus, Graphium sarpedon, Graphium eurypylus with 2n = 30, 40, and 60, respectively, and Papilio bianor with 2n = 60. Together with large-scale Hi-C data, we find that inter-chromosome rearrangements very rarely disrupted the pre-existing 3D chromatin structure of ancestral chromosomes. However, some intra-chromosome rearrangements changed 3D chromatin structures compared to the ancestral configuration. We find that new TADs and subTADs have emerged across the rearrangement sites where their adjacent compartments exhibit uniform types. Two intra-chromosome rearrangements altered Rel and lft regulation, potentially contributing to wing patterning differentiation and host plant choice. Notably, butterflies exhibited chromatin loops between Hox gene cluster ANT-C and BX-C, unlike Drosophila. Our CRISPR-Cas9 experiments in butterflies confirm that knocking out the CTCF binding site of the loops in BX-C affected the phenotypes regulated by Antp in ANT-C, resulting in legless larva. Our results reveal evolutionary patterns of insect 3D chromatin structures and provide evidence that 3D chromatin structure changes can play important roles in the evolution of traits.

Macrophage Migration Inhibitory Factor (MIF) Upregulates CXCR7 and Contributes to Chemotherapy Resistance in Colorectal Cancer.

Colorectal cancer is one of the most common malignant tumors worldwide, with high incidence and mortality rates making it a focus of research. Chemotherapy is a primary treatment modality for colon cancer, but chemotherapy resistance severely impacts treatment efficacy. MIF has been found to promote tumor progression and resistance in various cancers. This study aims to investigate the role of MIF in chemotherapy resistance in colon cancer and its potential mechanisms, particularly through the upregulation of CXCR7 expression, affecting the metabolism and drug sensitivity of colon cancer cells. The expression levels of MIF in colon cancer tissues and its association with patient prognosis were evaluated by analyzing TCGA and HPA data. Subsequently, the expression levels of MIF in colon cancer cell lines and resistant cell lines were detected by qRT-PCR and immunohistochemistry, and the effect of MIF on oxaliplatin sensitivity was assessed. The impact of MIF on the metabolic activity of colon cancer cells was measured using a cellular energy metabolism analyzer. Further experiments explored the mechanism by which MIF affects the metabolic activity of colon cancer cells through the upregulation of CXCR7 expression, and the role of CTCF in regulating CXCR7 transcription was validated by silencing CTCF. Finally, the effect of MIF on drug sensitivity of colon cancer cells was verified in a mouse xenograft tumor model. In this study, we found that the expression of MIF in colon cancer tissues was significantly higher than in normal tissues, and high MIF expression was associated with poor prognosis in patients. The expression levels of MIF in resistant colon cancer cell lines were significantly higher than in parental cell lines, and MIF overexpression significantly increased the resistance of colon cancer cells to oxaliplatin. Conversely, silencing MIF significantly reduced the IC50 value of resistant cells and increased apoptosis. MIF overexpression significantly increased the ECAR and OCR levels of colon cancer cells, while MIF knockdown significantly reduced these metabolic indicators. Further studies indicated that MIF affects the metabolic activity of colon cancer cells by upregulating CXCR7 expression. CTCF binding peaks at the CXCR7 promoter region and luciferase activity assays indicated that CTCF regulates CXCR7 transcription, and silencing CTCF significantly enhanced the sensitivity of colon cancer cells to oxaliplatin. In vivo experiments in mice showed that MIF silencing combined with oxaliplatin treatment significantly inhibited tumor growth and increased the necrotic area of tumor tissues. In conclusion, this study reveals the crucial role of MIF in chemotherapy resistance in colon cancer through the upregulation of CXCR7 expression, with CTCF playing an important regulatory role in this process. Our findings provide new theoretical insights and potential therapeutic targets for overcoming chemotherapy resistance in colon cancer. Future research should further explore the roles of MIF and CXCR7 in other types of cancers and the potential of MIF and CXCR7 as therapeutic targets.

Deletion of an evolutionarily conserved TAD boundary compromises spermatogenesis in mice.

Spermatogenesis is a complex process that can be disrupted by genetic and epigenetic changes, potentially leading to male infertility. Recent research has rapidly increased the number of protein coding mutations causally linked to impaired spermatogenesis in humans and mice. However, the role of non-coding mutations remains largely unexplored. As a case study to evaluate the effects of non-coding mutations on spermatogenesis, we first identified an evolutionarily conserved topologically associated domain (TAD) boundary near two genes with important roles in mammalian testis function: Dmrtb1 and Lrp8 . We then used CRISPR-Cas9 to generate a mouse line where 26kb of the boundary was removed including a strong and evolutionarily conserved CTCF binding site. ChIP-seq and Hi-C experiments confirmed the removal of the CTCF site and a resulting increase in the DNA-DNA interactions across the domain boundary. Mutant mice displayed significant changes in testis gene expression, abnormal testis histology, a 35% drop in the estimated efficiency of spermatogenesis and a 28% decrease in daily sperm production compared to littermate controls. Despite these quantitative changes in testis function, mutant mice show no significant changes in fertility. This suggests that non-coding deletions affecting testis gene regulation may have smaller effects on fertility compared to coding mutations of the same genes. Our results demonstrate that disruption of a TAD boundary can have a negative impact on sperm production and highlight the importance of considering non-coding mutations in the analysis of patients with male infertility.

Epigenetic alterations affecting hematopoietic regulatory networks as drivers of mixed myeloid/lymphoid leukemia

Leukemias with ambiguous lineage comprise several loosely defined entities, often without a clear mechanistic basis. Here, we extensively profile the epigenome and transcriptome of a subgroup of such leukemias with CpG Island Methylator Phenotype. These leukemias exhibit comparable hybrid myeloid/lymphoid epigenetic landscapes, yet heterogeneous genetic alterations, suggesting they are defined by their shared epigenetic profile rather than common genetic lesions. Gene expression enrichment reveals similarity with early T-cell precursor acute lymphoblastic leukemia and a lymphoid progenitor cell of origin. In line with this, integration of differential DNA methylation and gene expression shows widespread silencing of myeloid transcription factors. Moreover, binding sites for hematopoietic transcription factors, including CEBPA, SPI1 and LEF1, are uniquely inaccessible in these leukemias. Hypermethylation also results in loss of CTCF binding, accompanied by changes in chromatin interactions involving key transcription factors. In conclusion, epigenetic dysregulation, and not genetic lesions, explains the mixed phenotype of this group of leukemias with ambiguous lineage. The data collected here constitute a useful and comprehensive epigenomic reference for subsequent studies of acute myeloid leukemias, T-cell acute lymphoblastic leukemias and mixed-phenotype leukemias.

CTCF mutation at R567 causes developmental disorders via 3D genome rearrangement and abnormal neurodevelopment

The three-dimensional genome structure organized by CTCF is required for development. Clinically identified mutations in CTCF have been linked to adverse developmental outcomes. Nevertheless, the underlying mechanism remains elusive. In this investigation, we explore the regulatory roles of a clinically relevant R567W point mutation, located within the 11th zinc finger of CTCF, by introducing this mutation into both murine models and human embryonic stem cell-derived cortical organoid models. Mice with homozygous CTCFR567W mutation exhibit growth impediments, resulting in postnatal mortality, and deviations in brain, heart, and lung development at the pathological and single-cell transcriptome levels. This mutation induces premature stem-like cell exhaustion, accelerates the maturation of GABAergic neurons, and disrupts neurodevelopmental and synaptic pathways. Additionally, it specifically hinders CTCF binding to peripheral motifs upstream to the core consensus site, causing alterations in local chromatin structure and gene expression, particularly at the clustered protocadherin locus. Comparative analysis using human cortical organoids mirrors the consequences induced by this mutation. In summary, this study elucidates the influence of the CTCFR567W mutation on human neurodevelopmental disorders, paving the way for potential therapeutic interventions.

CCAAT Promoter element regulates transgenerational expression of the MHC class I gene

Transgenerational gene expression depends on both underlying DNA sequences and epigenetic modifications. The latter, which can result in transmission of variegated gene expression patterns across multiple generations without DNA alterations, has been termed epigenetic inheritance and has been documented in plants, worms, flies and mammals. Whereas transcription factors binding to cognate DNA sequence elements regulate gene expression, the molecular basis for epigenetic inheritance has been linked to histone and DNA modifications and non-coding RNA. Here we report that mutation of the CCAAT box promoter element abrogates NF-Y binding and disrupts the stable transgenerational expression of an MHC class I transgene. Transgenic mice with a mutated CCAAT box in the MHC class I transgene display variegated expression of the transgene among littermates and progeny in multiple independently derived transgenic lines. After 4 generations, CCAAT mutant transgenic lines derived from a single founder stably displayed distinct patterns of expression. Histone modifications and RNA polymerase II binding correlate with expression of CCAAT mutant transgenic lines, whereas DNA methylation and nucleosome occupancy do not. Mutation of the CCAAT box also results in changes to CTCF binding and DNA looping patterns across the transgene that correlate with expression status. These studies identify the CCAAT promoter element as a regulator of stable transgenerational gene expression such that mutation of the CCAAT box results in variegated transgenerational inheritance. Considering that the CCAAT box is present in 30% of eukaryotic promoters, this study provides insights into how fidelity of gene expression patterns is maintained through multiple generations.

CTCF regulates global chromatin accessibility and transcription during rod photoreceptor development.

Chromatin architecture facilitates accurate transcription at a number of loci, but it remains unclear how much chromatin architecture is involved in global transcriptional regulation. Previous work has shown that rapid depletion of the architectural protein CTCF in cell culture strongly alters chromatin organization but results in surprisingly limited gene expression changes. This discrepancy has also been observed when other architectural proteins are depleted, and one possible explanation is that full transcriptional changes are masked by cellular heterogeneity. We tested this idea by performing multi-omics analyses with sorted post-mitotic mouse rods, which undergo synchronized development, and identified CTCF-dependent regulation of global chromatin accessibility and gene expression. Depletion of CTCF leads to dysregulation of ∼20% of the entire transcriptome (>3,000 genes) and ∼41% of genome accessibility (>26,000 sites), and these regions are strongly enriched in euchromatin. Importantly, these changes are highly enriched for CTCF occupancy, suggesting direct CTCF binding and transcriptional regulation at these active loci. CTCF mainly promotes chromatin accessibility of these direct binding targets, and a large fraction of these sites correspond to promoters. At these sites, CTCF binding frequently promotes accessibility and inhibits expression, and motifs of transcription repressors are found to be significantly enriched. Our findings provide different and often opposite conclusions from previous studies, emphasizing the need to consider cell heterogeneity and cell type specificity when performing multi-omics analyses. We conclude that the architectural protein CTCF binds chromatin and regulates global chromatin accessibility and transcription during rod development.

Systematic assessment of ISWI subunits shows that NURF creates local accessibility for CTCF

Catalytic activity of the imitation switch (ISWI) family of remodelers is critical for nucleosomal organization and DNA binding of certain transcription factors, including the insulator protein CTCF. Here we define the contribution of individual subcomplexes by deriving a panel of isogenic mouse stem cell lines, each lacking one of six ISWI accessory subunits. Individual deletions of subunits of either CERF, RSF, ACF, WICH or NoRC subcomplexes only moderately affect the chromatin landscape, while removal of the NURF-specific subunit BPTF leads to a strong reduction in chromatin accessibility and SNF2H ATPase localization around CTCF sites. This affects adjacent nucleosome occupancy and CTCF binding. At a group of sites with reduced chromatin accessibility, CTCF binding persists but cohesin occupancy is reduced, resulting in decreased insulation. These results suggest that CTCF binding can be separated from its function as an insulator in nuclear organization and identify a specific role for NURF in mediating SNF2H localization and chromatin opening at bound CTCF sites.

Ectopic Pitx2 expression and sinoatrial node dysfunction in mice recapitulating a novel cardiac syndrome identified in unrelated families with deletions in a gene desert on chromosome 4q25

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch Research Councin/NWO Introduction Several unrelated families presenting with a complex, heterogeneous cardiac syndrome including sinoatrial node dysfunction and atrial fibrillation were found to share overlapping deletions in a 1.5 Megabase pair gene desert on chromosome 4q25. These overlapping regions contain evolutionarily conserved CTCF binding sites that modulate the separation of topologically associating domains containing the transcription factor-encoding gene PITX2 and noncoding RNA genes. Aim To understand mechanistically how the deletion in the gene desert causes sinoatrial node dysfunction and atrial arrhythmias. Methods Mice lacking the orthologue of the minimally overlapping genomic region deleted in affected patients were generated. Electrophysiology, morphology, tissue composition, and transcriptomes were analysed in this mouse model. Results These mice recapitulated major morpho-functional features of the syndrome, including sinoatrial node dysfunction and atrial arrhythmogenesis . Out of the 15 genes flanking the gene desert, only Pitx2 expression was deregulated in the sinoatrial node. During development, cardiac Pitx2c expression is normally restricted to the left side where it drives left sinus venosus and left atrial morphogenesis and prevents the formation of a left-sided sinoatrial node. The PITX2 locus has also been associated with atrial fibrillation in GWAS. Immunohistochemistry together with 3D volume analysis showed that Pitx2c was ectopically expressed in half of the pacemaker cardiomyocytes of the developing and postnatal sinoatrial node while its expression in the left atrium was maintained. Key transcription factors that drive pacemaker cell differentiation, Tbx3, Isl1 and Shox2, were lost where Pitx2c was ectopically expressed. Moreover, a conserved long non-coding RNA (Playrr) previously linked to Pitx2 regulation in the developing dorsal mesentery, was downregulated in the sinoatrial node of homozygous mutants. The downregulation of the pacemaker gene program additionally coincided with the activation of the atrial myocardial gene program in half of the pacemaker cardiomyocytes. Conclusion We generated a mouse model of a novel human cardiac syndrome including sinoatrial node dysfunction associated with the deletion of critical CTCF binding sites in a gene desert on chromosome 4q25. This deletion induced ectopic Pitx2c expression in the sinoatrial node alongside a substantial loss of pacemaker cardiomyocyte identity and a gain in a working atrial myocardium-like phenotype in the sinoatrial node of affected mice.Loss of pacemaker cardiomyocyte identitySinoatrial node dysfunction in mutants

Role of the CTCF Binding Site in Human T-Cell Leukemia Virus-1 Pathogenesis.

During HTLV-1 infection, the virus integrates into the host cell genome as a provirus with a single CCCTC binding protein (CTCF) binding site (vCTCF-BS), which acts as an insulator between transcriptionally active and inactive regions. Previous studies have shown that the vCTCF-BS is important for maintenance of chromatin structure, regulation of viral expression, and DNA and histone methylation. Here, we show that the vCTCF-BS also regulates viral infection and pathogenesis in vivo in a humanized (Hu) mouse model of adult T-cell leukemia/lymphoma. Three cell lines were used to initiate infection of the Hu-mice, i) HTLV-1-WT which carries an intact HTLV-1 provirus genome, ii) HTLV-1-CTCF, which contains a provirus with a mutated vCTCF-BS which abolishes CTCF binding, and a stop codon immediate upstream of the mutated vCTCF-BS which deletes the last 23 amino acids of p12, and iii) HTLV-1-p12stop that contains the intact vCTCF-BS, but retains the same stop codon in p12 as in the HTLV-1-CTCF cell line. Hu-mice were infected with mitomycin treated or irradiated HTLV-1 producing cell lines. There was a delay in pathogenicity when Hu-mice were infected with the CTCF virus compared to mice infected with either p12 stop or WT virus. Proviral load (PVL), spleen weights, and CD4 T cell counts were significantly lower in HTLV-1-CTCF infected mice compared to HTLV-1-p12stop infected mice. Furthermore, we found a direct correlation between the PVL in peripheral blood and death of HTLV-1-CTCF infected mice. In cell lines, we found that the vCTCF-BS regulates Tax expression in a time-dependent manner. The scRNAseq analysis of splenocytes from infected mice suggests that the vCTCF-BS plays an important role in activation and expansion of T lymphocytes in vivo. Overall, these findings indicate that the vCTCF-BS regulates Tax expression, proviral load, and HTLV pathogenicity in vivo.

CTCF and BORIS-mediated autophagy regulation via alternative splicing of BNIP3L in breast cancer

Autophagy is a pivotal regulatory and catabolic process, induced under various stressful conditions, including hypoxia. However, little is known about alternative splicing of autophagy genes in the hypoxic landscape in breast cancer. Our research unravels the hitherto unreported alternative splicing of BNIP3L, a crucial hypoxia-induced autophagic gene. We showed that BNIP3L, under hypoxic condition, forms two isoforms, a full-length isoform (BNIP3L-F) and a shorter isoform lacking exon 1 (BNIP3L-Δ1). The hypoxia-induced BNIP3L-F promotes autophagy, while under normoxia, the BNIP3L-Δ1 inhibits autophagy. We discovered a novel dimension of hypoxia-mediated epigenetic modification that regulates the alternative splicing of BNIP3L. Here, we showed differential DNA methylation of BNIP3L intron 1, causing reciprocal binding of epigenetic factor CTCF and its paralogue BORIS. Additionally, we highlighted the role of CTCF, and BORIS impacting autophagy in breast cancer. The differential binding of CTCF and BORIS results in alternative splicing of BNIP3L forming BNIP3L-F and BNIP3L-Δ1, respectively. The binding of CTCF on unmethylated BNIP3L intron 1 under hypoxia results in RNA Pol-II pause and inclusion of exon 1, promoting BNIP3L-F and autophagy. Interestingly, the binding of BORIS on methylated BNIP3L intron 1 under normoxia also results in RNA Pol-II pause but leads to the exclusion of exon 1 from BNIP3L mRNA. Finally, we reported the critical role of BORIS-mediated RNA Pol-II pause, which subsequently recruits SRSF6, redirecting the proximal splice-site selection, promoting BNIP3L-Δ1, and inhibiting autophagy. Our study provides novel insights into the potential avenues for breast cancer therapy by targeting autophagy regulation, specifically under hypoxic condition.

Differential 3D genome architecture and imprinted gene expression: cause or consequence?

Imprinted genes provide an attractive paradigm to unravel links between transcription and genome architecture. The parental allele-specific expression of these essential genes - which are clustered in chromosomal domains - is mediated by parental methylation imprints at key regulatory DNA sequences. Recent chromatin conformation capture (3C)-based studies show differential organization of topologically associating domains between the parental chromosomes at imprinted domains, in embryonic stem and differentiated cells. At several imprinted domains, differentially methylated regions show allelic binding of the insulator protein CTCF, and linked focal retention of cohesin, at the non-methylated allele only. This generates differential patterns of chromatin looping between the parental chromosomes, already in the early embryo, and thereby facilitates the allelic gene expression. Recent research evokes also the opposite scenario, in which allelic transcription contributes to the differential genome organization, similarly as reported for imprinted X chromosome inactivation. This may occur through epigenetic effects on CTCF binding, through structural effects of RNA Polymerase II, or through imprinted long non-coding RNAs that have chromatin repressive functions. The emerging picture is that epigenetically-controlled differential genome architecture precedes and facilitates imprinted gene expression during development, and that at some domains, conversely, the mono-allelic gene expression also influences genome architecture.