CTCF Gene Research Articles

The N-terminal dimerization domains of human and Drosophila CTCF have similar functionality

BackgroundCTCF is highly likely to be the ancestor of proteins that contain large clusters of C2H2 zinc finger domains, and its conservation is observed across most bilaterian organisms. In mammals, CTCF is the primary architectural protein involved in organizing chromosome topology and mediating enhancer–promoter interactions over long distances. In Drosophila, CTCF (dCTCF) cooperates with other architectural proteins to establish long-range interactions and chromatin boundaries. CTCFs of various organisms contain an unstructured N-terminal dimerization domain (DD) and clusters comprising eleven zinc-finger domains of the C2H2 type. The Drosophila (dCTCF) and human (hCTCF) CTCFs share sequence homology in only five C2H2 domains that specifically bind to a conserved 15 bp motif.ResultsPreviously, we demonstrated that CTCFs from different organisms carry unstructured N-terminal dimerization domains (DDs) that lack sequence homology. Here we used the CTCFattP(mCh) platform to introduce desired changes in the Drosophila CTCF gene and generated a series of transgenic lines expressing dCTCF with different variants of the N-terminal domain. Our findings revealed that the functionality of dCTCF is significantly affected by the deletion of the N-terminal DD. Additionally, we observed a strong impact on the binding of the dCTCF mutant to chromatin upon deletion of the DD. However, chromatin binding was restored in transgenic flies expressing a chimeric CTCF protein with the DD of hCTCF. Although the chimeric protein exhibited lower expression levels than those of the dCTCF variants, it efficiently bound to chromatin similarly to the wild type (wt) protein.ConclusionsOur findings suggest that one of the evolutionarily conserved functions of the unstructured N-terminal dimerization domain is to recruit dCTCF to its genomic sites in vivo.

Ionizing radiation induces vascular smooth muscle cell senescence through activating NF-κB/CTCF/p16 pathway

Radiation injury of blood vessels (RIBV) is a serious long-term complication of radiotherapy, characterized by the development of atherosclerosis. The involvement of vascular smooth muscle cells (VSMCs) senescence in the pathogenesis of radiation-induced atherosclerosis has been implicated, yet the precise mechanisms governing VSMCs senescence remain inadequately comprehended. In this study, the senescence of VSMCs was examined by employing SA-β-gal staining and assessing the expression of p16 and p21, both in vivo and in vitro. Our findings revealed that ionizing radiation (IR) has the potential to augment cellular senescence. In addition, IR significantly activated the NF-κB pathway, as evidenced by increased p65 nuclear translocation, phospho-p65 expression, and enhanced binding ability of p65 (EMSA). Furthermore, a decrease in HMGB2 expression following exposure to IR was observed via Western blot analysis, while CTCF expression remained unchanged. Interestingly, the formation of CTCF spatial clustering was detected under super-resolution fluorescence microscopy. Concurrently, the ChIP technique identified the facilitation of the interaction between CTCF and p16 gene through IR. The inhibition of CTCF or the overexpression of HMGB2 through lentiviruses effectively eliminates the formation of CTCF clusters and the upregulation of p16 and p21 after IR. Inhibition of NF-κB activation induced by IR by PDTC (100 μM) led to a decrease in the staining of SA-β-gal, a reduction in p16 expression, an increase in HMGB2 protein expression and a decrease in CTCF clusters formation. This study provided significant insights into the role and mechanism of IR in VSMCs senescence by regulating NF-κB/CTCF/p16 pathway.

Chromatin architectural factor CTCF is essential for progesterone-dependent uterine maturation.

Receptors for estrogen and progesterone frequently interact, via Cohesin/CTCF loop extrusion, at enhancers distal from regulated genes. Loss-of-function CTCF mutation in >20% of human endometrial tumors indicates its importance in uterine homeostasis. To better understand how CTCF-mediated enhancer-gene interactions impact endometrial development and function, the Ctcf gene was selectively deleted in female reproductive tissues of mice. Prepubertal Ctcfd/d uterine tissue exhibited a marked reduction in the number of uterine glands compared to those without Ctcf deletion (Ctcff/f mice). Post-pubertal Ctcfd/d uteri were hypoplastic with significant reduction in both the amount of the endometrial stroma and number of glands. Transcriptional profiling revealed increased expression of stem cell molecules Lif, EOMES, and Lgr5, and enhanced inflammation pathways following Ctcf deletion. Analysis of the response of the uterus to steroid hormone stimulation showed that CTCF deletion affects a subset of progesterone-responsive genes. This finding indicates (1) Progesterone-mediated signaling remains functional following Ctcf deletion and (2) certain progesterone-regulated genes are sensitive to Ctcf deletion, suggesting they depend on gene-enhancer interactions that require CTCF. The progesterone-responsive genes altered by CTCF ablation included Ihh, Fst, and Errfi1. CTCF-dependent progesterone-responsive uterine genes enhance critical processes including anti-tumorigenesis, which is relevant to the known effectiveness of progesterone in inhibiting progression of early-stage endometrial tumors. Overall, our findings reveal that uterine Ctcf plays a key role in progesterone-dependent expression of uterine genes underlying optimal post-pubertal uterine development.

Clinical characteristics and genetic analysis of two children with Autosomal dominant mental retardation type 21 due to variants of CTCF gene

To explore the clinical and genetic characteristics of two children with developmental delay. Two children who had presented at the Children's Hospital Affiliated to Shandong University on August 18, 2021 were enrolled as the study subjects. Clinical and laboratory examination, chromosomal karyotyping and high-throughput sequencing were carried out for both children. Both children had a 46,XX karyotype. High-throughput sequencing showed that they have respectively carried a c.489delG (p.Q165Rfs*14) and a c.1157_1158delAT (p.Y386Cfs*22) frameshifting variant of the CTCF gene, both had a de novo origin and were unreported previously. The CTCF gene variants probably underlay the development delay in the two children. Above discovery has enriched the mutational spectrum of the CTCF gene and has important implications for revealing the genotype-phenotype correlation for similar patients.

Promising Epigenetic Biomarkers for the Early Detection of Colorectal Cancer: A Systematic Review.

Simple SummaryHigh-performance, non-invasive screening is a requirement in colorectal cancer (CRC) as early detection is a key in reducing disease-related mortality in CRC patients. However, colonoscopy, the actual gold standard in CRC screening, is invasive and often avoided by patients. Conventional screening methods encounter several limitations; therefore, new testing strategies have been considered. DNA methylation is the most prevalent epigenetic alteration that occurs in all stages of carcinogenesis. Our research focused on identifying potential DNA methylation single biomarkers or panels as promising tools in the early detection of CRC; it evaluated methylated genes currently targeted by already approved diagnostic kits. A panel of five CTCF methylated binding sites holds the promise for early-stage specific detection of CRC. CRC screening compliance and accuracy can be enhanced by employing a stool mt-DNA methylation test.In CRC, screening compliance is decreased due to the experienced discomfort associated with colonoscopy, although this method is the gold standard in terms of sensitivity and specificity. Promoter DNA methylation (hypomethylation or hypermethylation) has been linked to all CRC stages. Study objectives: to systematically review the current knowledge on approved biomarkers, reveal new potential ones, and inspect tactics that can improve performance. This research was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines; the risk of bias was evaluated using the revised Quality Assessment of Diagnostic Accuracy Studies criteria (QUADAS-2). The Web of Science® Core Collection, MEDLINE® and Scopus® databases were searched for original articles published in peer-reviewed journals with the specific keywords “colorectal cancer”, “early detection”, “early-stage colorectal cancer”, “epigenetics”, “biomarkers”, “DNA methylation biomarkers”, “stool or blood or tissue or biopsy”, “NDRG4”, “BMP3”, “SEPT9”, and “SDC2”. Based on eligibility criteria, 74 articles were accepted for analysis. mSDC2 and mSEPT9 were frequently assessed in studies, alone or together as part of the ColoDefense panel test—the latter with the greatest performance. mBMP3 may not be an appropriate marker for detecting CRC. A panel of five methylated binding sites of the CTCF gene holds the promise for early-stage specific detection of CRC. CRC screening compliance and accuracy can be enhanced by employing a stool mt-DNA methylation test.

Pericentromeric noncoding RNA changes DNA binding of CTCF and inflammatory gene expression in senescence and cancer

Cellular senescence causes a dramatic alteration of chromatin organization and changes the gene expression profile of proinflammatory factors, thereby contributing to various age-related pathologies through the senescence-associated secretory phenotype (SASP). Chromatin organization and global gene expression are maintained by the CCCTC-binding factor (CTCF); however, the molecular mechanism underlying CTCF regulation and its association with SASP gene expression remains unclear. We discovered that noncoding RNA (ncRNA) derived from normally silenced pericentromeric repetitive sequences directly impairs the DNA binding of CTCF. This CTCF disturbance increases the accessibility of chromatin and activates the transcription of SASP-like inflammatory genes, promoting malignant transformation. Notably, pericentromeric ncRNA was transferred into surrounding cells via small extracellular vesicles acting as a tumorigenic SASP factor. Because CTCF blocks the expression of pericentromeric ncRNA in young cells, the down-regulation of CTCF during cellular senescence triggers the up-regulation of this ncRNA and SASP-related inflammatory gene expression. In this study, we show that pericentromeric ncRNA provokes chromosomal alteration by inhibiting CTCF, leading to a SASP-like inflammatory response in a cell-autonomous and non-cell-autonomous manner and thus may contribute to the risk of tumorigenesis during aging.

CTCF chromatin residence time controls three-dimensional genome organization, gene expression and DNA methylation in pluripotent cells.

The 11 zinc finger (ZF) protein CTCF regulates topologically associating domain formation and transcription through selective binding to thousands of genomic sites. Here, we replaced endogenous CTCF in mouse embryonic stem cells with green-fluorescent-protein-tagged wild-type or mutant proteins lacking individual ZFs to identify additional determinants of CTCF positioning and function. While ZF1 and ZF8-ZF11 are not essential for cell survival, ZF8 deletion strikingly increases the DNA binding off-rate of mutant CTCF, resulting in reduced CTCF chromatin residence time. Loss of ZF8 results in widespread weakening of topologically associating domains, aberrant gene expression and increased genome-wide DNA methylation. Thus, important chromatin-templated processes rely on accurate CTCF chromatin residence time, which we propose depends on local sequence and chromatin context as well as global CTCF protein concentration.

Forebrain glutamatergic neuron-specific Ctcf deletion induces reactive microgliosis and astrogliosis with neuronal loss in adult mouse hippocampus

CCCTC-binding factor (CTCF), a zinc finger protein, is a transcription factor and regulator of chromatin structure. Forebrain excitatory neuron-specific CTCF deficiency contributes to inflammation via enhanced transcription of inflammation-related genes in the cortex and hippocampus. However, little is known about the long-term effect of CTCF deficiency on postnatal neurons, astrocytes, or microglia in the hippocampus of adult mice. To address this, we knocked out the Ctcf gene in forebrain glutamatergic neurons (Ctcf cKO) by crossing Ctcf-floxed mice with Camk2a-Cre mice and examined the hippocampi of 7.5-10-month-old male mice using immunofluorescence microscopy. We found obvious neuronal cell death and reactive gliosis in the hippocampal cornu ammonis (CA)1 in 7.5-10-month-old cKO mice. Prominent rod-shaped microglia that participate in immune surveillance were observed in the stratum pyramidale and radiatum layer, indicating a potential increase in inflammatory mediators released by hippocampal neurons. Although neuronal loss was not observed in CA3, and dentate gyrus (DG) CTCF depletion induced a significant increase in the number of microglia in the stratum oriens of CA3 and reactive microgliosis and astrogliosis in the molecular layer and hilus of the DG in 7.5-10-month-old cKO mice. These results suggest that long-term Ctcf deletion from forebrain excitatory neurons may contribute to reactive gliosis induced by neuronal damage and consequent neuronal loss in the hippocampal CA1, DG, and CA3 in sequence over 7 months of age.

Association between structural brain features and gene expression by weighted gene co-expression network analysis in conversion from MCI to AD

Alzheimer’s disease (AD) is a neurodegenerative disease. Mild cognitive impairment (MCI) represents a state of cognitive function between normal cognition and dementia. Longitudinal studies showed that some MCI patients remained in a state of MCI, and some developed AD. The reason for these different conversions from MCI remains to be investigated. 180 MCI participants were followed for eight years. 143 MCI patients maintained the MCI state (MCI_S), and the remaining thirty-seven MCI patients were re-evaluated as having AD (MCI_AD). We obtained 1,036 structural brain characteristics and 15,481 gene expression values from the 180 MCI participants and applied weighted gene co-expression network analysis (WGCNA) to explore the relationship between structural brain features and gene expression. Regulating mediator effect analysis was employed to explore the relationships among gene expression, brain region measurements and clinical phenotypes. We found that 60 genes from the MCI_S group and 18 genes from the MCI_AD group respectively had the most significant correlations with left paracentral lobule and sulcus (L.PTS) and right subparietal sulcus (R.SubPS) thickness; CTCF, UQCR11 and WDR5B were the mutual genes between the two groups. The expression of CTCF gene and clinical score are completely mediated by L.PTS thickness, and the UQCR11 and WDR5B gene expression levels significantly regulate the mediating effect pathway. In conclusion, the factors affecting the different conversions from MCI are closely related to L.PTS thickness and the CTCF, UQCR11 and WDR5B gene expression levels. Our results add a theoretical foundation of imaging genetics for conversion from MCI to AD.

Autosomal dominant intellectual disability type 21 in a neonate

This is a case report on a 1-day-old male neonate admitted due to a weak cry for 1 day and recurrent circumoral cyanosis for 2 hours. He had unusual facial features at birth, with a single transverse palmar crease on both hands, flat feet, weak cry, feeding difficulties, congenital heart disease, and abnormality on cerebral MRI. Whole exome sequencing showed a de novo mutation, c.778_781delAAAG(p.Lys260ValfsTer2), in exon 3 of the CTCF gene, which was considered a pathogenic mutation by protein function prediction and might damage the function of CTCF protein. He was diagnosed with autosomal dominant intellectual disability type 21 based on the clinical manifestations and genetic analysis results. This case suggests that genetic analysis should be performed as early as possible for neonates with feeding difficulties which cannot be explained by infection or hypoxia, so as to help with early diagnosis and genetic counselling.

Loss of the neuronal genome organizer and transcription factor CTCF induces neuronal death and reactive gliosis in the anterior cingulate cortex.

CCCTC-binding factor (CTCF) is a genome organizer that regulates gene expression through transcription and chromatin structure regulation. CTCF also plays an important role during the developmental and adult stages. Cell-specific CTCF deletion studies have shown that a reduction in CTCF expression leads to the development of distinct clinical features and cognitive disorders. Therefore, we knocked out Ctcf (CTCF cKO) in the excitatory neurons of the forebrain in a Camk2a-Cre mouse strain to examine the role of CTCF in cell death and gliosis in the cortex. CTCF cKO mice were viable, but they demonstrated an age-dependent increase in reactive gliosis of astrocytes and microglia in the anterior cingulate cortex (ACC) from 16 weeks of age prior to neuronal loss observed at over 20 weeks of age. Consistent with these data, qRT-PCR analysis of the CTCF cKO ACC revealed changes in the expression of inflammation-related genes (Hspa1a, Prokr2 and Itga8) linked to gliosis and neuronal death. Our results suggest that prolonged Ctcf gene deficiency in excitatory neurons results in neuronal cell death and gliosis, possibly through functional changes in inflammation-related genes.

Association of mutation and low expression of the CTCF gene with breast cancer progression

BackgroundCTCF encodes 11-zinc finger protein which is implicated in multiple tumors including the carcinoma of the breast. The Present study investigates the association of CTCF mutations and their expression in breast cancer cases. MethodsA total of 155 breast cancer and an equal number of adjacent normal tissue samples from 155 breast cancer patients were examined for CTCF mutation(s) by PCR-SSCP and automated DNA sequencing. Immunohistochemistry (IHC) method was used to analyze CTCF expression. Molecular findings were statistically analyzed with various clinicopathological features to identify associations of clinical relevance. ResultsOf the total, 16.1% (25/155) cases exhibited mutation in the CTCF gene. Missense mutations Gln > His (G > T) in exon 1 and silent mutations Ser > Ser (C > T) in exon 4 of CTCF gene were analyzed. A significant association was observed between CTCF mutations and some clinicopathological parameters namely menopausal status (p = 0.02) tumor stage (p = 0.03) nodal status (p = 0.03) and ER expression (p = 0.04). Protein expression analysis showed 42.58% samples having low or no expression (+), 38.0% with moderate (++) expression and 19.35% having high (+++) expression for CTCF. A significant association was found between CTCF protein expression and clinicopathological parameters include histological grade (p = 0.04), tumor stage (p = 0.04), nodal status (p = 0.03) and ER status (p = 0.04). ConclusionsThe data suggest that CTCF mutations leading to its inactivation significantly contribute to the progression of breast cancer.

Exploring the genetic basis of human population differences in DNA methylation and their causal impact on immune gene regulation

BackgroundDNA methylation is influenced by both environmental and genetic factors and is increasingly thought to affect variation in complex traits and diseases. Yet, the extent of ancestry-related differences in DNA methylation, their genetic determinants, and their respective causal impact on immune gene regulation remain elusive.ResultsWe report extensive population differences in DNA methylation between 156 individuals of African and European descent, detected in primary monocytes that are used as a model of a major innate immunity cell type. Most of these differences (~ 70%) are driven by DNA sequence variants nearby CpG sites, which account for ~ 60% of the variance in DNA methylation. We also identify several master regulators of DNA methylation variation in trans, including a regulatory hub nearby the transcription factor-encoding CTCF gene, which contributes markedly to ancestry-related differences in DNA methylation. Furthermore, we establish that variation in DNA methylation is associated with varying gene expression levels following mostly, but not exclusively, a canonical model of negative associations, particularly in enhancer regions. Specifically, we find that DNA methylation highly correlates with transcriptional activity of 811 and 230 genes, at the basal state and upon immune stimulation, respectively. Finally, using a Bayesian approach, we estimate causal mediation effects of DNA methylation on gene expression in ~ 20% of the studied cases, indicating that DNA methylation can play an active role in immune gene regulation.ConclusionUsing a system-level approach, our study reveals substantial ancestry-related differences in DNA methylation and provides evidence for their causal impact on immune gene regulation.

Abstract 2998: Dynamic 3d chromosomal landscapes in acute leukemia

Abstract T-cell acute lymphoblastic leukemia (T-ALL) accounts for approximately 10-15% of pediatric and 25% of adult ALL cases. Despite improved prognosis driven by the significant advances in the molecular understanding of T-ALL, the outcome of T-ALL patients with primary resistant and relapsed leukemia remains poor. Recent evidences have highlighted a role for changes in 3D chromatin architecture in cancer progression. Herein, for the first time we explored genomewide changes in chromatin organization in primary T-ALL samples relative to T cells from healthy donors. We show how the organization of topologically associated domains (TADs) influences key oncogenic and tumor-suppressive loci by modifying the promoter-enhancer landscape, deregulating the transcriptional program and ultimately causing disease progression. To comprehensively characterize the chromatin landscape of clinically relevant loci in T-ALL, we sought to identify changes in intra-TAD activity and TAD disruptions on a genome-wide scale. We developed and implemented new computational approaches for Hi-C data analysis with a focus on Hi-C data integration with ChIP-Seq and RNA-Seq. We found significant correlations of TAD activity with CTCF occupancy, super-enhancer activity and gene expression of leukemia relevant loci. Furthermore, among the dozens of TAD disruptions we identified, we found a novel TAD “fusion” event around the MYC locus, which allows for chromatin interactions between a recently reported Notch-dependent MYC super-enhancer and promoter to drive overexpression of MYC. This particular TAD fusion is accompanied by a complete CTCF loss in the TAD boundary and increased MYC expression in T-ALL samples. Overall, this study sheds new light into how changes in chromatin architecture influence disease progression by restructuring oncogene and tumor suppressor landscapes. Citation Format: Palaniraja Thandapani, Andreas Kloetgen, Charalampos Lazaris, Xufeng Chen, Panagiotis Ntziachristos, Aristotelis Tsirigos, Iannis Aifantis. Dynamic 3d chromosomal landscapes in acute leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2998.

Multivariate Analysis Identifies Significant Correlations Between Baseline Biomarkers, DNA Demethylation and Clinical Responses in 122 Patients Treated in a Phase 1/2, Study of Guadecitabine (SGI-110), in the Treatment of Relapsed/Refractory Acute Myeloid Leukemia (r/r AML)

Introduction: Guadecitabine (SGI-110) is a novel subcutaneous (SC) next generation hypomethylating agent (HMA) designed as a dinucleotide of decitabine (DAC) and deoxyguanosine that is resistant to degradation by cytidine deaminase (CDA) and results in prolonged in vivo exposure to its active moiety DAC. The differentiated pharmacokinetic profile offers the potential of improved biological and clinical activity and safety over currently available HMAs. In the Phase 1 study, patients with r/r AML were treated at escalating doses of guadecitabine. In the phase 2 study, r/r AML patients were randomized to receive guadecitabine at 60 mg/m2 or 90 mg/m2 SC daily for 5 days (dailyx5). In a separate cohort, patients were treated with 60 mg/m2 SC daily for 10 days (dailyx10: days 1-5 and 8-12) for up to 4 cycles followed by subsequent cycles of the dailyx5 regimen. All regimens were dosed with a 28 day treatment cycle. We have reported the clinical efficacy and safety results from the Phase 1 dose-escalation study in AML and MDS (Issa et al, Lancet Oncology 2015) and the Phase 2 randomized dose-response study in r/r AML at 2 doses (60 and 90 mg/m2) in a 5-day regimen (Kantarjian et al, ASH 2013) and 60 mg/m2 in the 10-day regimen (Griffiths et al, ASCO 2014). Here, we report the associations between clinical responses and global DNA demethylation assessed by LINE1 assay, baseline expression of a panel of 7 genes (CDA, P15, P21, DNMT3B, DNMT3A, DNMT1 and CTCF) assessed by qRT-PCR, and FLT3-ITD/NMP1 mutations.Methods: Samples from 122 patients with r/r AML were analyzed (27 from phase 1 study treated at 36 m/m2/d or higher, 47 from the dailyx5 and 48 from the dailyx10 regimen of the phase 2). Global DNA methylation at baseline and after treatment, were estimated using bisulphite-pyrosequencing on the LINE-1 repetitive sequence for assessing guadecitabine pharmacodynamic (PD) effects. LINE-1 methylation time-course profiles were available in 117 out of 122 analyzed patients. Multivariate analysis was performed using nonlinear ensemble decision tree-based classification algorithms (Random Forests and Gradient Boosting Trees); the algorithms were applied to rank demographic features, treatment schedule features, baseline expression of 7 genes, FLT3-ITD and NPM1 mutations, LINE-1 methylation profiles and baseline hematological features as predictors of clinical response and to measure their directionality. All reported percentages are a measure of the average relative contribution of each feature in predicting response across boosted trees and are normalized across features.Results: In the 122 patients analyzed, the median age was 59.6 (range, 23-86), 75 were males (61.5%). Overall, peak LINE-1 demethylation generally occurred on day 8 after daily x 5 treatment or on day 8 or 15 after daily x 10 treatment. Overall, the maximum peak LINE-1 demethylation was -23.8 % ± 1.24. In 122 r/r AML patients, 28 showed Complete Response (23%, 14 CR and 14 CRi/CRp). High baseline CDA gene expression in peripheral blood was consistently ranked the main predictor of clinical response (mean for responders~1.76 and non-responders~0.12) irrespective of patient gender, dose and dosing cycles, and contributed for 22% of the model accuracy in predicting responses to guadecitabine. Expression of the genes CTCF (11%), DNMT3B (7%) and P21 (6%) were other genes that contributed > 5% in predicting clinical response. Age (8%) was also a significant predictor of response. FLT3-ITD mutations and NPM1 mutations were not significant predictors of response. In an integrated analysis, where methylation levels and gene expression were ranked together on their power to predict clinical response, LINE-1 methylation percent change from baseline on days 8(6%) and 22(19%), age (16%), CTCF (9%), CDA(7%), P21(6.5%) gene expression at baseline had greater than 5% contribution in predicting clinical response. A similar analysis was then performed to rank features on their power to predict day 8 methylation; CDA (28%), DNMT1 (8.5%), P15 (7.5%), and CTCF (7%) gene expression at baseline and age (7%) had greater than 5% predictive power.Conclusions: In conclusion, in r/r AML patients, global DNA demethylation was strongly associated with clinical responses to guadecitabine and analysis of baseline gene expression can identify trends that might enrich for r/r AML patients more likely to respond to HMA therapy with guadecitabine. DisclosuresGottipati:Otsuka Pharmaceutical Development & Commercialization, Inc.: Employment. Rohatagi:Otsuka Pharmaceutical Development & Commercialization, Inc.: Employment. Chung:Astex Pharmaceuticals, Inc.: Research Funding. Taverna:Astex Pharmaceuticals, Inc.: Employment. Kropf:Teva Pharmaceuticals: Consultancy. Azab:Astex Pharmaceuticals, Inc.: Employment. Issa:Astex Pharmaceuticals, Inc.: Consultancy; Janssen: Consultancy.

Quantitative analysis of RNA abondance for CTCF during reprogramming of bovine embryo from oocyte to blastocyst

Abstract. CTCF is a highly conserved protein among eukaryotes and it is involved in many of regulatory functions including, transcriptional repression and activation, chromatin insulation, imprinting, X chromosome inactivation, higher-order chromatin organization, and alternative splicing. Studies performed on mouse embryos indicate that CTCF can be a maternal-effect gene, and is essential for normal development of embryos. CTCF can be used as a molecular effector for the proper epigenetic establishment of embryonic development. The aim of this study was to determine changes in transcript levels of the CTCF gene in bovine preimplantation embryos. RNA was extracted from immature and mature oocytes and embryos at various developmental stages (two-cell, four-cell, eight-cell, and blastocysts). Results showed that the amounts of CTCF transcripts decreased in mature oocyte in comparison with immature oocytes, but this change was not significant. In addition, the amount of CTCF transcript in embryos at two-cell, four-cell, eight-cell, and blastocyst stages significantly increased in comparison with immature oocytes. These data show that CTCF expression in bovine embryo begins at minor embryonic genome activation.

The DNA-binding factor Ctcf critically controls gene expression in macrophages

Macrophages play an important role in immunity and homeostasis. Upon pathogen recognition via specific receptors, they rapidly induce inflammatory responses. This process is tightly controlled at the transcriptional level. The DNA binding zinc-finger protein CCCTC-binding factor (Ctcf) is a crucial regulator of long-range chromatin interactions and coordinates specific communication between transcription factors and gene expression processes. In this study, the Ctcf gene was specifically deleted in myeloid cells by making use of the transgenic Cre-LoxP system. Conditional deletion of the Ctcf gene in myeloid cells induced a mild phenotype in vivo. Ctcf-deficient mice exhibited significantly reduced expression of major histocompatibility complex (MHC) class II in the liver. Ctcf-deficient macrophages demonstrated a normal surface phenotype and phagocytosis capacity. Upon Toll-like receptor (TLR) stimulation, they produced normal levels of the pro-inflammatory cytokines IL-12 and IL-6, but manifested a strongly impaired capacity to produce tumor-necrosis factor (TNF) and IL-10, as well as to express the IL-10 family members IL-19, IL-20 and IL-24. Taken together, our data demonstrate a role of Ctcf that involves fine-tuning of macrophage function.

Expression of chicken CTCF gene in COS-1 cells and partial purification of CTCF protein

The chicken gene for transcription factor CTCF was expressed in COS-1 mammalian cells. The CTCF protein containing polyhistidine tag was partially purified using metallo-affinity and ion-exchange chromatography. The expressed protein localized in the cell nucleus and was shown to be functionally active in the electrophoretic mobility shift assay and specifically interacted with anti-CTCF antibodies.

Expression and Effect of CTCF in Pediatric Acute Lymphoblastic Leukemia

AbstractAbstract 4298Acute lymphoblastic leukemia (ALL) is the most frequent-occurring malignant neoplasm in children, but the pathogenesis of the disease remains unclear. In a microarray assay using samples from 100 Chinese children with ALL, CTCF was found to be up-regulated. DNA-binding nuclear protein CTCF (encoded by CTCF gene) is a highly conserved zinc finger protein involved in multiple cellular processes including transcriptional activation/repression, insulation, imprinting and × chromosome inactivation. It has been shown to be associated with cell apoptosis and differentiation in tumors; however, the biological function of CTCF in pediatric ALL is presently unknown.To investigate the expression features of CTCF in pediatric ALL cells, matched newly diagnosis (ND), complete remission (CR) and relapse (RE) bone marrow samples from 24 patients were collected. Ten ND-CR paired samples (n=20) were selected to detect the mRNA levels of CTCF by Q-PCR. Besides, the protein levels of CTCF at different stages of disease progression were measured by western blot in all patients (20 ND-CR paired samples, n=40; 4 ND-CR-RE matched samples, n=12). To further explore the role of CTCF in the pathogenesis of leukemia, the potential effect of CTCF on the cell apoptosis in lymphoblastic cells was investigated by flow cytometry.We identified significant up-regulation of CTCF in the ND samples. Importantly, the expression of CTCF returned to normal level after CR, but rebounded in the RE samples. Knock-down of CTCF resulted in nearly 3–fold and 15–fold increases in early and late apoptosis of leukemic cells respectively, which indicated that CTCF is an anti-apoptotic factor and plays an anti-apoptotic role in lymphoblastic cells.Our results indicate that CTCF may represent a promising indicator of disease progression as well as reflecting the ongoing therapeutic effects of treatment. Furthermore, CTCF serves as an anti-apoptotic factor and potentially contributes to leukemogenesis in pediatric ALL patients. Disclosures:Zhang:Beijing Health System High-level Technical Personel Plan: Research Funding. Zheng:Beijing Health System High-level Technical Personel Plan: Research Funding.

The DNA-Binding Protein CTCF Limits Proximal Vκ Recombination and Restricts κ Enhancer Interactions to the Immunoglobulin κ Light Chain Locus

Regulation of immunoglobulin (Ig) V(D)J gene rearrangement is dependent on higher-order chromatin organization. Here, we studied the invivo function of the DNA-binding zinc-finger protein CTCF, which regulates interactions between enhancers and promoters. By conditional deletion of the Ctcf gene in the B cell lineage, we demonstrate that loss of CTCF allowed Ig heavy chain recombination, but pre-B cell proliferation and differentiation was severely impaired. In the absence of CTCF, the Igκ light chain locus showed increased proximal and reduced distal Vκ usage. This was associated with enhanced proximal Vκ and reduced Jκ germline transcription. Chromosome conformation capture experiments demonstrated that CTCF limits interactions of the Igκ enhancers with the proximal V(κ) gene region and prevents inappropriate interactions between these strong enhancers and elements outside the Igκ locus. Thus, although Ig gene recombination can occur in the absence of CTCF, it is a critical factor determining Vκ segment choice for recombination.