CTCF shapes chromatin by multiple mechanisms: the impact of 20 years of CTCF research on understanding the workings of chromatin

More than 109 base pairs of the genome in higher eucaryotes are positioned in the interphase nucleus such that gene activation, gene repression, remote gene regulation by enhancer elements, and reading as well as adjusting epigenetic marks are possible. One important structural and functional component of chromatin organization is the zinc finger factor CTCF. Two decades of research has advanced the understanding of the fundamental role that CTCF plays in regulating such a vast expanse of DNA

Insulators and domains of gene expression

The genomic organization into active and inactive chromatin domains imposes specific requirements for having domain boundaries to prohibit interference between the opposing activities of neighbouring domains. These boundaries provide an insulator function by binding architectural proteins that mediate long-range interactions. Among these, CTCF plays a prominent role in establishing chromatin loops (between pairs of CTCF binding sites) through recruiting cohesin. CTCF-mediated long-range interactions are integral for a multitude of topological features of interphase chromatin, such as the formation of topologically associated domains, domain insulation, enhancer blocking and even enhancer function

Hand is a direct target of the forkhead transcription factor Biniou during Drosophila visceral mesoderm differentiation

<p>Abstract</p> <p>Background</p> <p>The visceral trunk mesoderm in <it>Drosophila melanogaster </it>develops under inductive signals from the ectoderm. This leads to the activation of the key regulators Tinman, Bagpipe and Biniou that are crucial for specification of the circular visceral muscles. How further differentiation is regulated is widely unknown, therefore it seems to be essential to identify downstream target genes of the early key regulators. In our report we focus on the analysis of the transcriptional control of the highly conserved transcription factor Hand in circular visceral muscle cells, providing evidence that the <it>hand </it>gene is a direct target of Biniou.</p> <p>Results</p> <p>Herein we describe the identification of a regulatory region in the <it>hand </it>gene essential and sufficient for the expression in the visceral mesoderm during embryogenesis. We found that <it>hand </it>expression in the circular visceral mesoderm is abolished in embryos mutant for the FoxF domain containing transcription factor Biniou. Furthermore we demonstrate that Biniou regulates <it>hand </it>expression by direct binding to a 300 bp sequence element, located within the 3^rdintron of the <it>hand </it>gene. This regulatory element is highly conserved in different <it>Drosophila </it>species. In addition, we provide evidence that Hand is dispensable for the initial differentiation of the embryonic visceral mesoderm.</p> <p>Conclusion</p> <p>In the present report we show that cross species sequence comparison of non-coding sequences between orthologous genes is a powerful tool to identify conserved regulatory elements. Combining functional dissection experiments <it>in vivo </it>and protein/DNA binding studies we identified <it>hand </it>as a direct target of Biniou in the circular visceral muscles.</p

CTCF genomic binding sites in Drosophila and the organisation of the bithorax complex.

Insulator or enhancer-blocking elements are proposed to play an important role in the regulation of transcription by preventing inappropriate enhancer/promoter interaction. The zinc-finger protein CTCF is well studied in vertebrates as an enhancer blocking factor, but Drosophila CTCF has only been characterised recently. To date only one endogenous binding location for CTCF has been identified in the Drosophila genome, the Fab-8 insulator in the Abdominal-B locus in the Bithorax complex (BX-C). We carried out chromatin immunopurification coupled with genomic microarray analysis to identify CTCF binding sites within representative regions of the Drosophila genome, including the 3-Mb Adh region, the BX-C, and the Antennapedia complex. Location of in vivo CTCF binding within these regions enabled us to construct a robust CTCF binding-site consensus sequence. CTCF binding sites identified in the BX-C map precisely to the known insulator elements Mcp, Fab-6, and Fab-8. Other CTCF binding sites correlate with boundaries of regulatory domains allowing us to locate three additional presumptive insulator elements; "Fab-2," "Fab-3," and "Fab-4." With the exception of Fab-7, our data indicate that CTCF is directly associated with all known or predicted insulators in the BX-C, suggesting that the functioning of these insulators involves a common CTCF-dependent mechanism. Comparison of the locations of the CTCF sites with characterised Polycomb target sites and histone modification provides support for the domain model of BX-C regulation

Choice of binding sites for CTCFL compared to CTCF is driven by chromatin and by sequence preference

The two paralogous zinc finger factors CTCF and CTCFL differ in expression such that CTCF is ubiquitously expressed, whereas CTCFL is found during spermatogenesis and in some cancer types in addition to other cell types. Both factors share the highly conserved DNA binding domain and are bound to DNA sequences with an identical consensus. In contrast, both factors differ substantially in the number of bound sites in the genome. Here, we addressed the molecular features for this binding specificity. In contrast to CTCF we found CTCFL highly enriched at 'open' chromatin marked by H3K27 acetylation, H3K4 di- and trimethylation, H3K79 dimethylation and H3K9 acetylation plus the histone variant H2A.Z. CTCFL is enriched at transcriptional start sites and regions bound by transcription factors. Consequently, genes deregulated by CTCFL are highly cell specific. In addition to a chromatin-driven choice of binding sites, we determined nucleotide positions critical for DNA binding by CTCFL, but not by CTCF

Heterodimerization of AML1/ETO with CBFβ is required for leukemogenesis but not for myeloproliferation

The AML1/Runx1 transcription factor and its heterodimerization partner CBFβ are essential regulators of myeloid differentiation. The chromosomal translocation t(8;21), fusing the DNA binding domain of AML1 to the corepressor eight-twenty-one (ETO), is frequently associated with acute myeloid leukemia and generates the AML1/ETO (AE) fusion protein. AE represses target genes usually activated by AML1 and also affects the endogenous repressive function of ETO at Notch target genes. In order to analyze the contribution of CBFβ in AE-mediated leukemogenesis and deregulation of Notch target genes, we introduced two point mutations in a leukemia-initiating version of AE in mice, called AE9a, that disrupt the AML1/CBFβ interaction (AE9aNT). We report that the AE9a/CBFβ interaction is not required for the AE9a-mediated aberrant expression of AML1 target genes, while upregulation/derepression of Notch target genes does require the interaction with CBFβ. Using retroviral transduction to express AE9a in murine adult bone marrow-derived hematopoietic progenitors, we observed that both AE9a and AE9aNT lead to increased myeloproliferation in vivo. However, both development of leukemia and long-term replating capacity are only observed with AE9a but not with AE9aNT. Thus, deregulation of both AML1 and Notch target genes is required for the development of AE9a-driven leukemia

PWWP2A binds distinct chromatin moieties and interacts with an MTA1-specific core NuRD complex.

Chromatin structure and function is regulated by reader proteins recognizing histone modifications and/or histone variants. We recently identified that PWWP2A tightly binds to H2A.Z-containing nucleosomes and is involved in mitotic progression and cranial-facial development. Here, using in vitro assays, we show that distinct domains of PWWP2A mediate binding to free linker DNA as well as H3K36me3 nucleosomes. In vivo, PWWP2A strongly recognizes H2A.Z-containing regulatory regions and weakly binds H3K36me3-containing gene bodies. Further, PWWP2A binds to an MTA1-specific subcomplex of the NuRD complex (M1HR), which consists solely of MTA1, HDAC1, and RBBP4/7, and excludes CHD, GATAD2 and MBD proteins. Depletion of PWWP2A leads to an increase of acetylation levels on H3K27 as well as H2A.Z, presumably by impaired chromatin recruitment of M1HR. Thus, this study identifies PWWP2A as a complex chromatin-binding protein that serves to direct the deacetylase complex M1HR to H2A.Z-containing chromatin, thereby promoting changes in histone acetylation levels

The male germ cell gene regulator CTCFL is functionally different from CTCF and binds CTCF-like consensus sites in a nucleosome composition-dependent manner.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: CTCF is a highly conserved and essential zinc finger protein expressed in virtually all cell types. In conjunction with cohesin, it organizes chromatin into loops, thereby regulating gene expression and epigenetic events. The function of CTCFL or BORIS, the testis-specific paralog of CTCF, is less clear. RESULTS: Using immunohistochemistry on testis sections and fluorescence-based microscopy on intact live seminiferous tubules, we show that CTCFL is only transiently present during spermatogenesis, prior to the onset of meiosis, when the protein co-localizes in nuclei with ubiquitously expressed CTCF. CTCFL distribution overlaps completely with that of Stra8, a retinoic acid-inducible protein essential for the propagation of meiosis. We find that absence of CTCFL in mice causes sub-fertility because of a partially penetrant testicular atrophy. CTCFL deficiency affects the expression of a number of testis-specific genes, including Gal3st1 and Prss50. Combined, these data indicate that CTCFL has a unique role in spermatogenesis. Genome-wide RNA expression studies in ES cells expressing a V5- and GFP-tagged form of CTCFL show that genes that are downregulated in CTCFL-deficient testis are upregulated in ES cells. These data indicate that CTCFL is a male germ cell gene regulator. Furthermore, genome-wide DNA-binding analysis shows that CTCFL binds a consensus sequence that is very similar to that of CTCF. However, only ~3,700 out of the ~5,700 CTCFL- and ~31,000 CTCF-binding sites overlap. CTCFL binds promoters with loosely assembled nucleosomes, whereas CTCF favors consensus sites surrounded by phased nucleosomes. Finally, an ES cell-based rescue assay shows that CTCFL is functionally different from CTCF. CONCLUSIONS: Our data suggest that nucleosome composition specifies the genome-wide binding of CTCFL and CTCF. We propose that the transient expression of CTCFL in spermatogonia and preleptotene spermatocytes serves to occupy a subset of promoters and maintain the expression of male germ cell genes

A functional insulator screen identifies NURF and dREAM components to be required for enhancer-blocking

Chromatin insulators of higher eukaryotes functionally divide the genome into active and inactive domains. Furthermore, insulators regulate enhancer/promoter communication, which is evident from the Drosophila bithorax locus in which a multitude of regulatory elements control segment specific gene activity. Centrosomal protein 190 (CP190) is targeted to insulators by CTCF or other insulator DNA-binding factors. Chromatin analyses revealed that insulators are characterized by open and nucleosome depleted regions. Here, we wanted to identify chromatin modification and remodelling factors required for an enhancer blocking function. We used the well-studied Fab-8 insulator of the bithorax locus to apply a genome-wide RNAi screen for factors that contribute to the enhancer blocking function of CTCF and CP190. Among 78 genes required for optimal Fab-8 mediated enhancer blocking, all four components of the NURF complex as well as several subunits of the dREAM complex were most evident. Mass spectrometric analyses of CTCF or CP190 bound proteins as well as immune precipitation confirmed NURF and dREAM binding. Both co-localise with most CP190 binding sites in the genome and chromatin immune precipitation showed that CP190 recruits NURF and dREAM. Nucleosome occupancy and histone H3 binding analyses revealed that CP190 mediated NURF binding results in nucleosomal depletion at CP190 binding sites. Thus, we conclude that CP190 binding to CTCF or to other DNA binding insulator factors mediates recruitment of NURF and dREAM. Furthermore, the enhancer blocking function of insulators is associated with nucleosomal depletion and requires NURF and dREAM

Distinct IL-1α-responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

How cytokine-driven changes in chromatin topology are converted into gene regulatory circuits during inflammation still remains unclear. Here, we show that interleukin (IL)-1α induces acute and widespread changes in chromatin accessibility via the TAK1 kinase and NF-κB at regions that are highly enriched for inflammatory disease-relevant SNPs. Two enhancers in the extended chemokine locus on human chromosome 4 regulate the IL-1α-inducible IL8 and CXCL1-3 genes. Both enhancers engage in dynamic spatial interactions with gene promoters in an IL-1α/TAK1-inducible manner. Microdeletions of p65-binding sites in either of the two enhancers impair NF-κB recruitment, suppress activation and biallelic transcription of the IL8/CXCL2 genes, and reshuffle higher-order chromatin interactions as judged by i4C interactome profiles. Notably, these findings support a dominant role of the IL8 “master” enhancer in the regulation of sustained IL-1α signaling, as well as for IL-8 and IL-6 secretion. CRISPR-guided transactivation of the IL8 locus or cross-TAD regulation by TNFα-responsive enhancers in a different model locus supports the existence of complex enhancer hierarchies in response to cytokine stimulation that prime and orchestrate proinflammatory chromatin responses downstream of NF-κB