Functional sub-division of the Drosophila genome via chromatin looping

Ahanger, Sajad Hamid; Shouche, Yogesh S.; Mishra, Rakesh K.

doi:10.4161/nucl.23389

Cited by 26 publications

(29 citation statements)

References 81 publications

(123 reference statements)

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“…Identification of Pita and ZIPIC as partners of the CP190 protein CP190 plays a central role in Drosophila boundary and domain formation (for review, see Ahanger et al 2013) To identify unknown DNA binding factors that might be associated with a chromatin domain function of CP190, we searched for zincfinger proteins that can interact with CP190. We purified CP190 from an extract prepared from S2 cells stably expressing a tagged FLAG-CP190 transgene.…”

Section: Resultsmentioning

confidence: 99%

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Two new insulator proteins, Pita and ZIPIC, target CP190 to chromatin

et al. 2014

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Insulators are multiprotein-DNA complexes that regulate the nuclear architecture. The Drosophila CP190 protein is a cofactor for the DNA-binding insulator proteins Su(Hw), CTCF, and BEAF-32. The fact that CP190 has been found at genomic sites devoid of either of the known insulator factors has until now been unexplained. We have identified two DNA-binding zinc-finger proteins, Pita, and a new factor named ZIPIC, that interact with CP190 in vivo and in vitro at specific interaction domains. Genomic binding sites for these proteins are clustered with CP190 as well as with CTCF and BEAF-32. Model binding sites for Pita or ZIPIC demonstrate a partial enhancer-blocking activity and protect gene expression from PRE-mediated silencing. The function of the CTCF-bound MCP insulator sequence requires binding of Pita. These results identify two new insulator proteins and emphasize the unifying function of CP190, which can be recruited by many DNA-binding insulator proteins.

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Section: Resultsmentioning

confidence: 99%

“…CP190 (1096 amino acids) contains an N-terminal BTB/POZ domain, an aspartic-acid-rich D-region, four C2H2 zinc-finger motifs, and a C-terminal E-rich domain (Oliver et al 2010;Ahanger et al 2013). The BTB domain of CP190 forms stable homodimers that may be involved in protein-protein interactions (Oliver et al 2010;Bonchuk et al 2011).…”

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confidence: 99%

Two new insulator proteins, Pita and ZIPIC, target CP190 to chromatin

et al. 2014

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“…Vertebrates possess only one known insulator protein, CTCF, while in Drosophila different proteins, dCTCF, Su(Hw), BEAF and GAGA, bind to the different insulators characterized. In addition, CP190 protein seems to be common to all of them (Gurudatta & Corces, ; Ahanger et al , ). Several studies indicate that insulator elements and the proteins they bind play a fundamental role in development and disease.…”

Section: Introductionmentioning

confidence: 99%

Ibf1 and Ibf2 are novel CP190-interacting proteins required for insulator function

et al. 2014

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Insulators are DNA-protein complexes that play a central role in chromatin organization and regulation of gene expression. In Drosophila different proteins, dCTCF, Su(Hw), and BEAF bind to specific subsets of insulators most of them having in common CP190. It has been shown that there are a number of CP190-binding sites that are not shared with any other known insulator protein, suggesting that other proteins could cooperate with CP190 to regulate insulator activity. Here we report on the identification of two previously uncharacterized proteins as CP190-interacting proteins, that we have named Ibf1 and Ibf2. These proteins localize at insulator bodies and associate with chromatin at CP190-binding sites throughout the genome. We also show that Ibf1 and Ibf2 are DNA-binding proteins that form hetero-oligomers that mediate CP190 binding to chromatin. Moreover, Ibf1 and Ibf2 are necessary for insulator activity in enhancer-blocking assays and Ibf2 null mutation cause a homeotic phenotype. Taken together our data reveal a novel pathway of CP190 recruitment to chromatin that is required for insulator activity.

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“…Overall, there are about 6,000 CP190 sites in the Drosophila genome with 80% of them being bound by at least one of the five DNA-binding insulator factors. This suggests that CP190 may confer an important role in insulator function [13]. In fact, when comparing CTCF sites devoid of CP190 binding with those that are bound by both factors, nucleosomal occupancy at these sites is strikingly different [12].…”

Section: Introductionmentioning

confidence: 99%

A Functional Insulator Screen Identifies NURF and dREAM Components to Be Required for Enhancer-Blocking

et al. 2014

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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.

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Functional sub-division of the Drosophila genome via chromatin looping

Cited by 26 publications

References 81 publications

Two new insulator proteins, Pita and ZIPIC, target CP190 to chromatin

Two new insulator proteins, Pita and ZIPIC, target CP190 to chromatin

Ibf1 and Ibf2 are novel CP190-interacting proteins required for insulator function

A Functional Insulator Screen Identifies NURF and dREAM Components to Be Required for Enhancer-Blocking

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