CCCTC-binding factor (CTCF) is a DNA-binding protein that has various, often seemingly contradictory, roles in gene regulation. This Review describes these disparate functions and how the context-dependent looping of DNA regions by CTCF is emerging as a potential unifying mechanism that underpins these diverse roles.

CTCF: an architectural protein bridging genome topology and function

https://www.cell.com/cell/pdf/S0092-8674(14)01179-9.pdf

Control of Cell Identity Genes Occurs in Insulated Neighborhoods in Mammalian Chromosomes

Chromatin Domains: The Unit of Chromosome Organization

The pluripotent state of embryonic stem cells (ESCs) is produced by active transcription of genes that control cell identity and repression of genes encoding lineage-specifying developmental regulators. Here, we use ESC cohesin ChIA-PET data to identify the local chromosomal structures at both active and repressed genes across the genome. The results produce a map of enhancer-promoter interactions and reveal that super-enhancer-driven genes generally occur within chromosome structures that are formed by the looping of two interacting CTCF sites co-occupied by cohesin. These looped structures form insulated neighborhoods whose integrity is important for proper expression of local genes. We also find that repressed genes encoding lineage-specifying developmental regulators occur within insulated neighborhoods. These results provide insights into the relationship between transcriptional control of cell identity genes and control of local chromosome structure.

Despite an abundance of new studies about topologically associating domains (TADs), the role of genetic information in TAD formation is still not fully understood. Here we use our software, HiCExplorer (hicexplorer.readthedocs.io) to annotate >2800 high-resolution (570 bp) TAD boundaries in Drosophila melanogaster. We identify eight DNA motifs enriched at boundaries, including a motif bound by the M1BP protein, and two new boundary motifs. In contrast to mammals, the CTCF motif is only enriched on a small fraction of boundaries flanking inactive chromatin while most active boundaries contain the motifs bound by the M1BP or Beaf-32 proteins. We demonstrate that boundaries can be accurately predicted using only the motif sequences at open chromatin sites. We propose that DNA sequence guides the genome architecture by allocation of boundary proteins in the genome. Finally, we present an interactive online database to access and explore the spatial organization of fly, mouse and human genomes, available at http://chorogenome.ie-freiburg.mpg.de
 . Although topologically associating domains (TADs) have been extensively investigated, it is not clear to what extent DNA sequence contributes to their formation. Here the authors develop software to identify high-resolution TAD boundaries and reveal their relationship to underlying DNA motifs.

/pdf/high-resolution-tads-reveal-dna-sequences-underlying-genome-2ho3i29at7.pdf

High-resolution TADs reveal DNA sequences underlying genome organization in flies.

Several multiprotein DNA complexes capable of insulator activity have been identified in Drosophila melanogaster, yet only CTCF, a highly conserved zinc finger protein, and the transcription factor TFIIIC have been shown to function in mammals. CTCF is involved in diverse nuclear activities, and recent studies suggest that the proteins with which it associates and the DNA sequences that it targets may underlie these various roles. Here we show that the Drosophila homolog of CTCF (dCTCF) aligns in the genome with other Drosophila insulator proteins such as Suppressor of Hairy wing [SU(HW)] and Boundary Element Associated Factor of 32 kDa (BEAF-32) at the borders of H3K27me3 domains, which are also enriched for associated insulator proteins and additional cofactors. RNAi depletion of dCTCF and combinatorial knockdown of gene expression for other Drosophila insulator proteins leads to a reduction in H3K27me3 levels within repressed domains, suggesting that insulators are important for the maintenance of appropriate repressive chromatin structure in Polycomb (Pc) domains. These results shed new insights into the roles of insulators in chromatin domain organization and support recent models suggesting that insulators underlie interactions important for Pc-mediated repression. We reveal an important relationship between dCTCF and other Drosophila insulator proteins and speculate that vertebrate CTCF may also align with other nuclear proteins to accomplish similar functions.

/pdf/drosophila-ctcf-tandemly-aligns-with-other-insulator-473v1vmvjs.pdf

Drosophila CTCF tandemly aligns with other insulator proteins at the borders of H3K27me3 domains

Insulators, long-range interactions, and genome function.

Chromatin insulators are DNA–protein complexes with broad functions in nuclear biology. Based on the ability of insulator proteins to interact with each other, it was originally found that insulators form loops that bring together distant regions of the genome. Data from genome-wide localization studies indicate that insulator proteins can be present in intergenic regions as well as at the 5′, introns or 3′ of genes, suggesting a variety of roles for insulator loops in chromosome biology. Recent results suggest that insulators mediate intra- and interchromosomal interactions to affect transcription, imprinting, and recombination. Cells have developed mechanisms to control insulator activity by recruiting specialized proteins or by covalent modification of core components. It is then possible that insulator-mediated interactions set up cell-specific blueprints of nuclear organization that may contribute to the establishment of different patterns of gene expression during cell differentiation and development. As a consequence, disruption of insulator activity could result in the development of cancer or other disease states.

Chromatin Insulators: A Role in Nuclear Organization and Gene Expression

Myc has been characterized as a transcription factor that activates expression of genes involved in pluripotency and cancer, and as a component of the replication complex. Here we find that Myc is present at promoters and enhancers of Drosophila melanogaster genes during interphase. Myc colocalizes with Orc2, which is part of the prereplication complex, during G1. As is the case in mammals, Myc associates preferentially with paused genes, suggesting that it may also be involved in the release of RNA polymerase II from the promoter-proximal pausing in Drosophila. Interestingly, about 40% of Myc sites present in interphase persists during mitosis. None of the Myc mitotic sites correspond to enhancers, and only some correspond to promoters. The rest of the mitotic Myc sites overlap with binding sites for multiple insulator proteins that are also maintained in mitosis. These results suggest alternative mechanisms to explain the role of Myc in pluripotency and cancer.

/pdf/a-subset-of-drosophila-myc-sites-remain-associated-with-hrg3p9uooc.pdf

A subset of Drosophila Myc sites remain associated with mitotic chromosomes colocalized with insulator proteins.

Understanding the relationship between genome organization and expression is central to understanding genome function. Closely apposed genes in a head-to-head orientation share the same upstream region and are likely to be coregulated. Here we identify the Drosophila BEAF-32 insulator as a cis regulatory element separating close head-to-head genes with different transcription regulation modes. We then compare the binding landscapes of the BEAF-32 insulator protein in four different Drosophila genomes and highlight the evolutionarily conserved presence of this protein between close adjacent genes. We find that changes in binding of BEAF-32 to sites in the genome of different Drosophila species correlate with alterations in genome organization caused by DNA rearrangements or genome size expansion. The cross-talk between BEAF-32 genomic distribution and genome organization contributes to new gene-expression profiles, which in turn translate into specific and distinct phenotypes. The results suggest a mechanism for the establishment of differences in transcription patterns during evolution.

/pdf/the-beaf-32-insulator-coordinates-genome-organization-and-4gbzx1d3qt.pdf

Jingping Yang

Papers

Drosophila CTCF tandemly aligns with other insulator proteins at the borders of H3K27me3 domains

Insulators, long-range interactions, and genome function.

Chromatin Insulators: A Role in Nuclear Organization and Gene Expression

A subset of Drosophila Myc sites remain associated with mitotic chromosomes colocalized with insulator proteins.

The BEAF-32 insulator coordinates genome organization and function during the evolution of Drosophila species