Drosophila architectural protein CTCF is not essential for fly survival and is able to function independently of CP190

SUMMARYLineage-specific transcription factors are critical for long-range enhancer interactions but direct or indirect contributions of architectural proteins such as CTCF to enhancer function remain less clear. The LDB1 complex mediates enhancer-gene interactions at the β-globin locus through LDB1 self-interaction. We find that a novel LDB1-bound enhancer upstream of carbonic anhydrase 2 (Car2) activates its expression by interacting directly with CTCF at the gene promoter. Both LDB1 and CTCF are required for enhancer-Car2 looping and the domain of LDB1 contacted by CTCF is necessary to rescue Car2 transcription in LDB1 deficient cells. Genome wide studies and CRISPR/Cas9 genome editing indicate that LDB1-CTCF enhancer looping underlies activation of a substantial fraction of erythroid genes. Our results provide a mechanism by which long-range interactions of architectural protein CTCF can be tailored to achieve a tissue-restricted pattern of chromatin loops and gene expression.

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RedChIP identifies noncoding RNAs associated with genomic sites occupied by Polycomb and CTCF proteins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2116222119 ◽

2021 ◽

Vol 119 (1) ◽

pp. e2116222119

Author(s):

Alexey A. Gavrilov ◽

Rinat I. Sultanov ◽

Mikhail D. Magnitov ◽

Aleksandra A. Galitsyna ◽

Erdem B. Dashinimaev ◽

...

Keyword(s):

Chromatin Immunoprecipitation ◽

Binding Sites ◽

Wide Spectrum ◽

Noncoding Rnas ◽

Ctcf Binding ◽

Polycomb Repressive Complex 2 ◽

Proximity Ligation ◽

Chromatin Interactions ◽

Genome Wide ◽

Architectural Protein

Nuclear noncoding RNAs (ncRNAs) are key regulators of gene expression and chromatin organization. The progress in studying nuclear ncRNAs depends on the ability to identify the genome-wide spectrum of contacts of ncRNAs with chromatin. To address this question, a panel of RNA–DNA proximity ligation techniques has been developed. However, neither of these techniques examines proteins involved in RNA–chromatin interactions. Here, we introduce RedChIP, a technique combining RNA–DNA proximity ligation and chromatin immunoprecipitation for identifying RNA–chromatin interactions mediated by a particular protein. Using antibodies against architectural protein CTCF and the EZH2 subunit of the Polycomb repressive complex 2, we identify a spectrum of cis- and trans-acting ncRNAs enriched at Polycomb- and CTCF-binding sites in human cells, which may be involved in Polycomb-mediated gene repression and CTCF-dependent chromatin looping. By providing a protein-centric view of RNA–DNA interactions, RedChIP represents an important tool for studies of nuclear ncRNAs.

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Drosophila architectural protein CTCF is not essential for fly survival and is able to function independently of CP190

10.1101/2021.05.31.446447 ◽

2021 ◽

Author(s):

Olga Kyrchanova ◽

Natalia Klimenko ◽

Nikolay Postika ◽

Artem Bonchuk ◽

Nickolay Zolotarev ◽

...

Keyword(s):

Mutational Analysis ◽

Homeotic Genes ◽

Complete Inactivation ◽

Architectural Proteins ◽

Phenotypic Manifestation ◽

Large Clusters ◽

Broad Complex ◽

Cp190 Protein ◽

Architectural Protein

CTCF is the most likely ancestor of proteins that contain large clusters of C2H2 zinc finger domains (C2H2) and is conserved among most bilateral organisms. In mammals, CTCF functions as the main architectural protein involved in the organization of topology-associated domains (TADs). In vertebrates and Drosophila, CTCF is involved in the regulation of homeotic genes. Previously, null mutations in the dCTCF gene were found to result in death during the stage of pharate adults, which failed to eclose from their pupal case. Here, we obtained several new null dCTCF mutations and found that the complete inactivation of dCTCF appears to be limited to phenotypic manifestations of the Abd-B gene and fertility of adult flies. Many modifiers that are not associated with an independent phenotypic manifestation can significantly enhance the expressivity of the null dCTCF mutations, indicating that other architectural proteins are able to functionally compensate for dCTCF inactivation in Drosophila. We also mapped the 715-735 aa region of dCTCF as being essential for the interaction with the BTB (Broad-Complex, Tramtrack, and Bric a brac) and microtubule-targeting (M) domains of the CP190 protein, which binds to many architectural proteins. However, the mutational analysis showed that the interaction with CP190 was not essential for the functional activity of dCTCF in vivo.

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