Cohesin complexes mediate sister-chromatid cohesion in dividing cells but may also contribute to gene regulation in postmitotic cells. How cohesin regulates gene expression is not known. Here we describe cohesin-binding sites in the human genome and show that most of these are associated with the CCCTC-binding factor (CTCF), a zinc-finger protein required for transcriptional insulation. CTCF is dispensable for cohesin loading onto DNA, but is needed to enrich cohesin at specific binding sites. Cohesin enables CTCF to insulate promoters from distant enhancers and controls transcription at the H19/IGF2 (insulin-like growth factor 2) locus. This role of cohesin seems to be independent of its role in cohesion. We propose that cohesin functions as a transcriptional insulator, and speculate that subtle deficiencies in this function contribute to 'cohesinopathies' such as Cornelia de Lange syndrome.

Cohesin mediates transcriptional insulation by CCCTC-binding factor

http://rulai.cshl.edu/reprints/CTCFBindingSites.pdf

Analysis of the Vertebrate Insulator Protein CTCF-Binding Sites in the Human Genome

https://www.cell.com/cell/pdf/S0092-8674(11)01507-8.pdf

Waves of Retrotransposon Expansion Remodel Genome Organization and CTCF Binding in Multiple Mammalian Lineages.

We gather together: insulators and genome organization.

Cohesins, which mediate sister chromatin cohesion, and CTCF, which functions at chromatin boundaries, play key roles in the structural and functional organization of chromosomes. We examined the binding of these two factors on the Kaposi's sarcoma-associated herpesvirus (KSHV) episome during latent infection and found a striking colocalization within the control region of the major latency transcript responsible for expressing LANA (ORF73), vCyclin (ORF72), vFLIP (ORF71), and vmiRNAs. Deletion of the CTCF-binding site from the viral genome disrupted cohesin binding, and crippled colony formation in 293 cells. Clonal instability correlated with elevated expression of lytic cycle gene products, notably the neighbouring promoter for K14 and vGPCR (ORF74). siRNA depletion of RAD21 from latently infected cells caused an increase in K14 and ORF74, and lytic inducers caused a rapid dissociation of RAD21 from the viral genome. RAD21 and SMC1 also associate with the cellular CTCF sites at mammalian c-myc promoter and H19/Igf2 imprinting control region. We conclude that cohesin subunits associate with viral and cellular CTCF sites involved in complex gene regulation and chromatin organization.

/pdf/cohesins-localize-with-ctcf-at-the-kshv-latency-control-16mozxyy90.pdf

Cohesins localize with CTCF at the KSHV latency control region and at cellular c-myc and H19/Igf2 insulators

Eukaryotic transcriptional regulation often involves regulatory elements separated from the cognate genes by long distances, whereas appropriately positioned insulator or enhancer-blocking elements shield promoters from illegitimate enhancer action. Four proteins have been identified in Drosophila mediating enhancer blocking—Su(Hw), Zw5, BEAF32 and GAGA factor. In vertebrates, the single protein CTCF, with 11 highly conserved zinc fingers, confers enhancer blocking in all known chromatin insulators. Here, we characterize an orthologous CTCF factor in Drosophila with a similar domain structure, binding site specificity and transcriptional repression activity as in vertebrates. In addition, we demonstrate that one of the insulators (Fab-8) in the Drosophila Abdominal-B locus mediates enhancer blocking by dCTCF. Therefore, the enhancer-blocking protein CTCF and, most probably, the mechanism of enhancer blocking mediated by this remarkably versatile factor are conserved from Drosophila to humans.

/pdf/ctcf-is-conserved-from-drosophila-to-humans-and-confers-2wmtsw37nz.pdf

CTCF is conserved from Drosophila to humans and confers enhancer blocking of the Fab-8 insulator

Cells latently infected with herpes simplex virus (HSV) contain nucleosomal DNA similar to that of host cell chromatin. Recent studies have demonstrated that histones in the latency-associated transcript (LAT) promoter and intron regions contain histone modifications permissive for transcription. However, those histones associated with the lytic-specific ICP0 gene, which lies only 5 kb away, contain modifications typical of silenced chromatin. How this active chromatin is kept separate from the repressed chromatin in the nearby ICP0 region remains crucial to the understanding of the HSV lytic cycle. In this study, we show that the LAT intron region contains an insulator. Specifically, we show that an 800-bp region from the LAT intron can block enhancers in both tissue culture cells and Drosophila melanogaster embryos. Importantly, the 800-bp HSV insulator protects a LAT transgene from positional effects in Drosophila eye tissue. The 800-bp region contains nine copies of 16-bp repeats. In vitro electrophoretic mobility shift assay revealed that CTCF interacts with the CTCCC sequence within the repeats. In vivo chromatin immunoprecipitation assay demonstrated that CTCF interacts with these repeats in latently infected trigeminal ganglion neurons. The deletion of these repeats impaired insulator activity in human K562 cells and Drosophila embryos. Finally, double-spaced RNA knockdown of CTCF disrupts enhancer-blocking activity of the LAT insulator in transfected Drosophila S3 cells. These results strongly support the hypothesis that the 800-bp DNA in the LAT intron region works as a chromatin boundary during latency to separate active chromatin associated with the LAT promoter region from repressed chromatin in the ICP0 gene.

CTCF-Dependent Chromatin Boundary Element between the Latency-Associated Transcript and ICP0 Promoters in the Herpes Simplex Virus Type 1 Genome

Multiple Promoter Targeting Sequences exist in Abdominal-B to regulate long-range gene activation

Large gene complexes frequently use "specialized" DNA elements to ensure proper regulation of gene activities. The Promoter Targeting Sequence (PTS) from the Abdominal-B locus of the Drosophila Bithorax complex overcomes an insulator, and facilitates, yet restricts, distant enhancers to a single promoter. We found that this promoter-targeting activity is independent of an enhancer's tissue or temporal specificity, and can be remembered in all somatic cells in the absence of promoter activation. It requires an insulator for its establishment, but can be maintained by the PTS in the absence of an insulator. More importantly, the promoter-targeting activity can be remembered after the transgene is translocated to new chromosomal locations. These results suggest that promoter targeting is established independent of enhancer activity, and is maintained epigenetically throughout development and subsequent generations.

/pdf/the-promoter-targeting-sequence-mediates-epigenetically-1lj4ueshah.pdf

The Promoter Targeting Sequence mediates epigenetically heritable transcription memory

Gene transcription can be regulated by promoter competition when multiple promoters are available for a single enhancer. The converse, however, is largely undescribed. Here we report an occurrence of several enhancers competing for one promoter (enhancer interference) and propose its underlying mechanism. The promoter targeting sequence from the Abdominal-B locus of the Drosophila bithorax complex overcomes the enhancer-blocking activity of insulators in transgenic embryos. It may facilitate the long-range enhancer–promoter communication in Abdominal-B by bypassing insulator elements such as Frontabdominal-7 and Frontabdominal-8. In transgenic embryos, the anti-insulator activity allowed both an insulator-blocked enhancer and a nonblocked enhancer to contact the same promoter. We found that these two enhancers exhibited mutual inhibition or mutual exclusion in cells where both are transcriptionally active. This enhancer interference occurred at the level of enhancer–promoter communication. It occurred only between enhancers on opposite sides of an insulator and depended on enhancer activity. We hypothesize that enhancer interference limits the interaction of the Abdominal-B promoter to the enhancer(s) from only one regulatory domain in a specific abdominal segment.

https://www.pnas.org/content/pnas/104/9/3237.full.pdf

Qi Chen

Papers

CTCF is conserved from Drosophila to humans and confers enhancer blocking of the Fab-8 insulator

CTCF-Dependent Chromatin Boundary Element between the Latency-Associated Transcript and ICP0 Promoters in the Herpes Simplex Virus Type 1 Genome

Multiple Promoter Targeting Sequences exist in Abdominal-B to regulate long-range gene activation

The Promoter Targeting Sequence mediates epigenetically heritable transcription memory

Promoter targeting sequence mediates enhancer interference in the Drosophila embryo