Project description:Genome organization is driven by forces affecting transcriptional state, but the relationship between transcription and genome architecture remains unclear. Here, we identified the Drosophila transcription factor Motif 1 Binding Protein (M1BP) in physical association with the gypsy chromatin insulator core complex, including the universal insulator protein CP190. M1BP is required for enhancer-blocking and barrier activities of the gypsy insulator as well as its proper nuclear localization. Genome-wide, M1BP specifically colocalizes with CP190 at Motif 1-containing promoters, which are enriched at topologically associating domain (TAD) borders. M1BP facilitates CP190 chromatin binding at many shared sites and vice versa. Both factors promote Motif 1-dependent gene expression and transcription near TAD borders genome-wide. Finally, loss of M1BP reduces chromatin accessibility and increases both inter- and intra-TAD local genome compaction. Our results reveal physical and functional interaction between CP190 and M1BP to activate transcription at TAD borders and mediate chromatin insulator-dependent genome organization.
Project description:CTCF and Cp190 proteins are implicated at many insulator elements throughout Drosophila genome. Here we compared Hi-C maps, transcriptomes and binding of multiple insulator proteins in cultured Drosophila cells derived from CTCF-KO, Cp190-KO and control embryos.
Project description:CTCF and Cp190 proteins are implicated at many insulator elements throughout Drosophila genome. Here we compared Hi-C maps, transcriptomes and binding of multiple insulator proteins in cultured Drosophila cells derived from CTCF-KO, Cp190-KO and control embryos.
Project description:CTCF and Cp190 proteins are implicated at many insulator elements throughout Drosophila genome. Here we compared Hi-C maps, transcriptomes and binding of multiple insulator proteins in cultured Drosophila cells derived from CTCF-KO, Cp190-KO and control embryos.
Project description:Chromatin insulators and Polycomb group (PcG) complexes control nuclear organization to effect changes in gene expression. In Drosophila, RNA silencing pathways influence long range interactions mediated by PcG proteins and nuclear localization of the gypsy insulator; however, the underlying mechanisms are unknown. Here, we identify a singular requirement for Argonaute2 (AGO2) for the activity of the CCCTC-binding factor (CTCF)/Centrosomal protein 190 (CP190) dependent Fab-8 insulator. AGO2 and CP190 interact physically, and genome wide localization of AGO2 by chromatin immunoprecipitation and sequencing (ChIP-seq) reveals extensive colocalization of AGO2 with insulators and Polycomb Response Elements (PREs) but minimal overlap with regions of endogenous small interfering RNA (endo-siRNA) production. Finally, depletion of either CTCF or CP190 results in loss of AGO2 association with insulators, PREs, and other cis-regulatory regions. Our findings suggest that Dicer-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome. ChIP-seq of AGO2 in two Drosophila cell types (S2 and S3)
Project description:Genome organization is driven by forces affecting transcriptional state, but the relationship between transcription and genome architecture remains unclear. Here, we identified the Drosophila transcription factor Motif 1 Binding Protein (M1BP) in physical association with the gypsy chromatin insulator core complex, including the universal insulator protein CP190. M1BP is required for enhancer-blocking and barrier activities of the gypsy insulator as well as its proper nuclear localization. Genome-wide, M1BP specifically colocalizes with CP190 at Motif 1-containing promoters, which are enriched at topologically associating domain (TAD) borders. M1BP is required for CP190 chromatin binding at many shared sites, and CP190 also affects M1BP chromatin association. Both factors are required for Motif 1-dependent gene expression and transcription near TAD borders genome-wide. Finally, loss of M1BP alters local genome compaction. Our results reveal physical and functional interaction between CP190 and M1BP to activate transcription at TAD borders and mediate chromatin insulator-dependent genome organization.
Project description:Chromatin insulators are DNA-protein complexes that can prevent the spread of repressive chromatin and block communication between enhancers and promoters to regulate gene expression. In Drosophila, the gypsy chromatin insulator complex consists of three core proteins: CP190, Su(Hw), and Mod(mdg4)67.2. These factors concentrate at nuclear foci termed insulator bodies, and their normal localization is correlated with proper insulator function. Here, we identified NURF301/E(bx), a nucleosome remodeling factor, as a novel regulator of gypsy insulator body localization through a high-throughput RNAi imaging screen. NURF301 promotes gypsy-dependent insulator barrier activity and physically interacts with gypsy insulator proteins. Using ChIP-seq, we found that NURF301 co-localizes with insulator proteins genome-wide, and NURF301 promotes chromatin association of Su(Hw) and CP190 at gypsy insulator binding sites. These effects correlate with NURF301-dependent nucleosome repositioning. At the same time, CP190 and Su(Hw) are also required for recruitment of NURF301 to chromatin. Finally, Oligopaint FISH combined with immunofluorescence revealed that NURF301 promotes 3D contact between insulator bodies and gypsy binding site DNA, and NURF301 is required for proper nuclear positioning of gypsy binding sites. Our data provide new insights into how a nucleosome remodeling factor and insulator proteins cooperatively contribute to nuclear organization.
Project description:Chromatin insulators shield promoters and chromatin domains from neighbouring enhancers or chromatin regions with opposing activities. Insulator binding proteins and their cofactors mediate the boundary function. In general, covalent modification of proteins by the Small Ubiqutin-like Modifier (SUMO) is an important mechanism to control the interaction of proteins within complexes. Here we addressed the impact of SUMO in respect to insulator function, chromatin binding of insulator factors and formation of insulator speckles. SUMOylation augments the enhancer blocking function of four different insulator sequences and increases the genome-wide binding of the insulator cofactor CP190. A model is discussed with enhanced chromatin binding of SUMOylated CP190 causing fusion of insulator speckles, which may allow for more efficient insulation.
Project description:Chromatin insulators and Polycomb group (PcG) complexes control nuclear organization to effect changes in gene expression. In Drosophila, RNA silencing pathways influence long range interactions mediated by PcG proteins and nuclear localization of the gypsy insulator; however, the underlying mechanisms are unknown. Here, we identify a singular requirement for Argonaute2 (AGO2) for the activity of the CCCTC-binding factor (CTCF)/Centrosomal protein 190 (CP190) dependent Fab-8 insulator. AGO2 and CP190 interact physically, and genome wide localization of AGO2 by chromatin immunoprecipitation and sequencing (ChIP-seq) reveals extensive colocalization of AGO2 with insulators and Polycomb Response Elements (PREs) but minimal overlap with regions of endogenous small interfering RNA (endo-siRNA) production. Finally, depletion of either CTCF or CP190 results in loss of AGO2 association with insulators, PREs, and other cis-regulatory regions. Our findings suggest that Dicer-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome.