Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H, R, Ca, M, Pm||WB, ChIP-seq, ChIP||Rb||Serum||Polyclonal Antibody|
|Presentation||Rabbit polyclonal antiserum containing 0.05% sodium azide and 30% glycerol.|
|Safety Information according to GHS|
|Material Size||200 µL|
Anti-CTCF Antibody SDS
|Anti-CTCF (rabbit antiserum) - DAM1472197||DAM1472197|
|Anti-CTCF (rabbit antiserum) Polyclonal Antibody||Q2912988|
|Anti-CTCF - 2142232||2142232|
|Anti-CTCF - 2375606||2375606|
|Anti-CTCF - 2452497||2452497|
|Anti-CTCF - 1350637||1350637|
|Anti-CTCF - 1962117||1962117|
|Anti-CTCF - 2016034||2016034|
|Anti-CTCF - 2054523||2054523|
|Reference overview||Application||Species||Pub Med ID|
|Tissue-specific regulation and function of Grb10 during growth and neuronal commitment.|
Plasschaert, RN; Bartolomei, MS
Proceedings of the National Academy of Sciences of the United States of America 112 6841-7 2015
Growth-factor receptor bound protein 10 (Grb10) is a signal adapter protein encoded by an imprinted gene that has roles in growth control, cellular proliferation, and insulin signaling. Additionally, Grb10 is critical for the normal behavior of the adult mouse. These functions are paralleled by Grb10's unique tissue-specific imprinted expression; the paternal copy of Grb10 is expressed in a subset of neurons whereas the maternal copy is expressed in most other adult tissues in the mouse. The mechanism that underlies this switch between maternal and paternal expression is still unclear, as is the role for paternally expressed Grb10 in neurons. Here, we review recent work and present complementary data that contribute to the understanding of Grb10 gene regulation and function, with specific emphasis on growth and neuronal development. Additionally, we show that in vitro differentiation of mouse embryonic stem cells into alpha motor neurons recapitulates the switch from maternal to paternal expression observed during neuronal development in vivo. We postulate that this switch in allele-specific expression is related to the functional role of Grb10 in motor neurons and other neuronal tissues.
|Epigenetic silencing of the XAF1 gene is mediated by the loss of CTCF binding.|
Victoria-Acosta, G; Vazquez-Santillan, K; Jimenez-Hernandez, L; Muñoz-Galindo, L; Maldonado, V; Martinez-Ruiz, GU; Melendez-Zajgla, J
Scientific reports 5 14838 2015
XAF1 is a tumour suppressor gene that compromises cell viability by modulating different cellular events such as mitosis, cell cycle progression and apoptosis. In cancer, the XAF1 gene is commonly silenced by CpG-dinucleotide hypermethylation of its promoter. DNA demethylating agents induce transcriptional reactivation of XAF1, sensitizing cancer cells to therapy. The molecular mechanisms that mediate promoter CpG methylation have not been previously studied. Here, we demonstrate that CTCF interacts with the XAF1 promoter in vivo in a methylation-sensitive manner. By transgene assays, we demonstrate that CTCF mediates the open-chromatin configuration of the XAF1 promoter, inhibiting both CpG-dinucleotide methylation and repressive histone posttranslational modifications. In addition, the absence of CTCF in the XAF1 promoter inhibits transcriptional activation induced by well-known apoptosis activators. We report for the first time that epigenetic silencing of the XAF1 gene is a consequence of the loss of CTCF binding.
|The contribution of cohesin-SA1 to gene expression and chromatin architecture in two murine tissues.|
Cuadrado, A; Remeseiro, S; Graña, O; Pisano, DG; Losada, A
Nucleic acids research 43 3056-67 2015
Cohesin, which in somatic vertebrate cells consists of SMC1, SMC3, RAD21 and either SA1 or SA2, mediates higher-order chromatin organization. To determine how cohesin contributes to the establishment of tissue-specific transcriptional programs, we compared genome-wide cohesin distribution, gene expression and chromatin architecture in cerebral cortex and pancreas from adult mice. More than one third of cohesin binding sites differ between the two tissues and these show reduced overlap with CCCTC-binding factor (CTCF) and are enriched at the regulatory regions of tissue-specific genes. Cohesin/CTCF sites at active enhancers and promoters contain, at least, cohesin-SA1. Analyses of chromatin contacts at the Protocadherin (Pcdh) and Regenerating islet-derived (Reg) gene clusters, mostly expressed in brain and pancreas, respectively, revealed remarkable differences that correlate with the presence of cohesin. We could not detect significant changes in the chromatin contacts at the Pcdh locus when comparing brains from wild-type and SA1 null embryos. In contrast, reduced dosage of SA1 altered the architecture of the Reg locus and decreased the expression of Reg genes in the pancreas of SA1 heterozygous mice. Given the role of Reg proteins in inflammation, such reduction may contribute to the increased incidence of pancreatic cancer observed in these animals.
|T-cell receptor α enhancer is inactivated in αβ T lymphocytes.|
del Blanco, B; Angulo, Ú; Krangel, MS; Hernández-Munain, C
Proceedings of the National Academy of Sciences of the United States of America 112 E1744-53 2015
The Tcra enhancer (Eα) is essential for Tcra locus germ-line transcription and primary Vα-to-Jα recombination during thymocyte development. We found that Eα is inhibited late during thymocyte differentiation and in αβ T lymphocytes, indicating that it is not required to drive transcription of rearranged Tcra genes. Eα inactivation resulted in the disruption of functional long-range enhancer-promoter interactions and was associated with loss of Eα-dependent histone modifications at promoter and enhancer regions, and reduced expression and recruitment of E2A to the Eα enhanceosome in T cells. Enhancer activity could not be recovered by T-cell activation, by forced expression of E2A or by the up-regulation of this and other transcription factors in the context of T helper differentiation. Our results argue that the major function of Eα is to coordinate the formation of a chromatin hub that drives Vα and Jα germ-line transcription and primary rearrangements in thymocytes and imply the existence of an Eα-independent mechanism to activate transcription of the rearranged Tcra locus in αβ T cells.
|Genome-wide targeting of the epigenetic regulatory protein CTCF to gene promoters by the transcription factor TFII-I.|
Peña-Hernández, R; Marques, M; Hilmi, K; Zhao, T; Saad, A; Alaoui-Jamali, MA; del Rincon, SV; Ashworth, T; Roy, AL; Emerson, BM; Witcher, M
Proceedings of the National Academy of Sciences of the United States of America 112 E677-86 2015
CCCTC-binding factor (CTCF) is a key regulator of nuclear chromatin structure and gene regulation. The impact of CTCF on transcriptional output is highly varied, ranging from repression to transcriptional pausing and transactivation. The multifunctional nature of CTCF may be directed solely through remodeling chromatin architecture. However, another hypothesis is that the multifunctional nature of CTCF is mediated, in part, through differential association with protein partners having unique functions. Consistent with this hypothesis, our mass spectrometry analyses of CTCF interacting partners reveal a previously undefined association with the transcription factor general transcription factor II-I (TFII-I). Biochemical fractionation of CTCF indicates that a distinct CTCF complex incorporating TFII-I is assembled on DNA. Unexpectedly, we found that the interaction between CTCF and TFII-I is essential for directing CTCF to the promoter proximal regulatory regions of target genes across the genome, particularly at genes involved in metabolism. At genes coregulated by CTCF and TFII-I, we find knockdown of TFII-I results in diminished CTCF binding, lack of cyclin-dependent kinase 8 (CDK8) recruitment, and an attenuation of RNA polymerase II phosphorylation at serine 5. Phenotypically, knockdown of TFII-I alters the cellular response to metabolic stress. Our data indicate that TFII-I directs CTCF binding to target genes, and in turn the two proteins cooperate to recruit CDK8 and enhance transcription initiation.
|Human papillomaviruses activate and recruit SMC1 cohesin proteins for the differentiation-dependent life cycle through association with CTCF insulators.|
Mehta, K; Gunasekharan, V; Satsuka, A; Laimins, LA
PLoS pathogens 11 e1004763 2015
Human papillomaviruses infect stratified epithelia and link their productive life cycle to the differentiation state of the host cell. Productive viral replication or amplification is restricted to highly differentiated suprabasal cells and is dependent on the activation of the ATM DNA damage pathway. The ATM pathway has three arms that can act independently of one another. One arm is centered on p53, another on CHK2 and a third on SMC1/NBS1 proteins. A role for CHK2 in HPV genome amplification has been demonstrated but it was unclear what other factors provided important activities. The cohesin protein, SMC1, is necessary for sister chromatid association prior to mitosis. In addition the phosphorylated form of SMC1 plays a critical role together with NBS1 in the ATM DNA damage response. In normal cells, SMC1 becomes phosphorylated in response to radiation, however, in HPV positive cells our studies demonstrate that it is constitutively activated. Furthermore, pSMC1 is found localized in distinct nuclear foci in complexes with γ-H2AX, and CHK2 and bound to HPV DNA. Importantly, knockdown of SMC1 blocks differentiation-dependent genome amplification. pSMC1 forms complexes with the insulator transcription factor CTCF and our studies show that these factors bind to conserved sequence motifs in the L2 late region of HPV 31. Similar motifs are found in most HPV types. Knockdown of CTCF with shRNAs blocks genome amplification and mutation of the CTCF binding motifs in the L2 open reading frame inhibits stable maintenance of viral episomes in undifferentiated cells as well as amplification of genomes upon differentiation. These findings suggest a model in which SMC1 factors are constitutively activated in HPV positive cells and recruited to viral genomes through complex formation with CTCF to facilitate genome amplification. Our findings identify both SMC1 and CTCF as critical regulators of the differentiation-dependent life cycle of high-risk human papillomaviruses.
|Genome-wide binding and mechanistic analyses of Smchd1-mediated epigenetic regulation.|
Chen, K; Hu, J; Moore, DL; Liu, R; Kessans, SA; Breslin, K; Lucet, IS; Keniry, A; Leong, HS; Parish, CL; Hilton, DJ; Lemmers, RJ; van der Maarel, SM; Czabotar, PE; Dobson, RC; Ritchie, ME; Kay, GF; Murphy, JM; Blewitt, ME
Proceedings of the National Academy of Sciences of the United States of America 112 E3535-44 2015
Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic repressor with described roles in X inactivation and genomic imprinting, but Smchd1 is also critically involved in the pathogenesis of facioscapulohumeral dystrophy. The underlying molecular mechanism by which Smchd1 functions in these instances remains unknown. Our genome-wide transcriptional and epigenetic analyses show that Smchd1 binds cis-regulatory elements, many of which coincide with CCCTC-binding factor (Ctcf) binding sites, for example, the clustered protocadherin (Pcdh) genes, where we show Smchd1 and Ctcf act in opposing ways. We provide biochemical and biophysical evidence that Smchd1-chromatin interactions are established through the homodimeric hinge domain of Smchd1 and, intriguingly, that the hinge domain also has the capacity to bind DNA and RNA. Our results suggest Smchd1 imparts epigenetic regulation via physical association with chromatin, which may antagonize Ctcf-facilitated chromatin interactions, resulting in coordinated transcriptional control.
|Epigenomic evolution in diffuse large B-cell lymphomas.|
Pan, H; Jiang, Y; Boi, M; Tabbò, F; Redmond, D; Nie, K; Ladetto, M; Chiappella, A; Cerchietti, L; Shaknovich, R; Melnick, AM; Inghirami, GG; Tam, W; Elemento, O
Nature communications 6 6921 2015
The contribution of epigenomic alterations to tumour progression and relapse is not well characterized. Here we characterize an association between disease progression and DNA methylation in diffuse large B-cell lymphoma (DLBCL). By profiling genome-wide DNA methylation at single-base pair resolution in thirteen DLBCL diagnosis-relapse sample pairs, we show that DLBCL patients exhibit heterogeneous evolution of tumour methylomes during relapse. We identify differentially methylated regulatory elements and determine a relapse-associated methylation signature converging on key pathways such as transforming growth factor-β (TGF-β) receptor activity. We also observe decreased intra-tumour methylation heterogeneity from diagnosis to relapsed tumour samples. Relapse-free patients display lower intra-tumour methylation heterogeneity at diagnosis compared with relapsed patients in an independent validation cohort. Furthermore, intra-tumour methylation heterogeneity is predictive of time to relapse. Therefore, we propose that epigenomic heterogeneity may support or drive the relapse phenotype and can be used to predict DLBCL relapse.
|Dynamic estrogen receptor interactomes control estrogen-responsive trefoil Factor (TFF) locus cell-specific activities.|
Quintin, J; Le Péron, C; Palierne, G; Bizot, M; Cunha, S; Sérandour, AA; Avner, S; Henry, C; Percevault, F; Belaud-Rotureau, MA; Huet, S; Watrin, E; Eeckhoute, J; Legagneux, V; Salbert, G; Métivier, R
Molecular and cellular biology 34 2418-36 2014
Estradiol signaling is ideally suited for analyzing the molecular and functional linkages between the different layers of information directing transcriptional regulations: the DNA sequence, chromatin modifications, and the spatial organization of the genome. Hence, the estrogen receptor (ER) can bind at a distance from its target genes and engages timely and spatially coordinated processes to regulate their expression. In the context of the coordinated regulation of colinear genes, identifying which ER binding sites (ERBSs) regulate a given gene still remains a challenge. Here, we investigated the coordination of such regulatory events at a 2-Mb genomic locus containing the estrogen-sensitive trefoil factor (TFF) cluster of genes in breast cancer cells. We demonstrate that this locus exhibits a hormone- and cohesin-dependent reduction in the plasticity of its three-dimensional organization that allows multiple ERBSs to be dynamically brought to the vicinity of estrogen-sensitive genes. Additionally, by using triplex-forming oligonucleotides, we could precisely document the functional links between ER engagement at given ERBSs and the regulation of particular genes. Hence, our data provide evidence of a formerly suggested cooperation of enhancers toward gene regulation and also show that redundancy between ERBSs can occur.
|PAT-ChIP coupled with laser microdissection allows the study of chromatin in selected cell populations from paraffin-embedded patient samples.|
Amatori, S; Ballarini, M; Faversani, A; Belloni, E; Fusar, F; Bosari, S; Pelicci, PG; Minucci, S; Fanelli, M
Epigenetics & chromatin 7 18 2014
The recent introduction of pathology tissue-chromatin immunoprecipitation (PAT-ChIP), a technique allowing chromatin immunoprecipitation from formalin-fixed and paraffin-embedded (FFPE) tissues, has expanded the application potential of epigenetic studies in tissue samples. However, FFPE tissue section analysis is strongly limited by tissue heterogeneity, which hinders linking the observed epigenetic events to the corresponding cellular population. Thus, ideally, to take full advantage of PAT-ChIP approaches, procedures able to increase the purity and homogeneity of cell populations from FFPE tissues are required.In this study, we tested the use of both core needle biopsies (CNBs) and laser microdissection (LMD), evaluating the compatibility of these methods with the PAT-ChIP procedure. Modifications of the original protocols were introduced in order to increase reproducibility and reduce experimental time. We first demonstrated that chromatin can be prepared and effectively immunoprecipitated starting from 0.6-mm-diameter CNBs. Subsequently, in order to assess the applicability of PAT-ChIP to LMD samples, we tested the effects of hematoxylin or eosin staining on chromatin extraction and immunoprecipitation, as well as the reproducibility of our technique when using particularly low quantities of starting material. Finally, we carried out the PAT-ChIP using chromatin extracted from either normal tissue or neoplastic lesions, the latter obtained by LMD from FFPE lung sections derived from mutant K-ras(v12) transgenic mice or from human adeno- or squamous lung carcinoma samples. Well characterized histone post-translational modifications (HPTMs), such as H3K4me3, H3K27me3, H3K27Ac, and H3K9me3, were specifically immunoselected, as well as the CTCF transcription factor and RNA polymerase II (Pol II).Epigenetic profiling can be performed on enriched cell populations obtained from FFPE tissue sections. The improved PAT-ChIP protocol will be used for the discovery and/or validation of novel epigenetic biomarkers in FFPE human samples.
|White Paper - The Message in the Marks: Deciphering Cancer Epigenetics (EMD)|