Project description:Chromatin domain insulators are thought to help partition the genome into genetic units called topologically associating domains (TADs). In <i>Drosophila</i>, TADs are often separated by inter-TAD regions containing active housekeeping genes and associated insulator binding proteins. This raises the question of whether insulator binding proteins are involved primarily in chromosomal TAD architecture or gene activation, or if these two activities are linked. The Boundary Element-Associated Factor of 32 kDa (BEAF-32, or BEAF for short) is usually found in inter-TADs. BEAF was discovered based on binding to the scs' insulator, and is important for the insulator activity of scs' and other BEAF binding sites. There are divergent promoters in scs' with a BEAF binding site by each. Here, we dissect the scs' insulator to identify DNA sequences important for insulator and promoter activity, focusing on the half of scs' with a high affinity BEAF binding site. We find that the BEAF binding site is important for both insulator and promoter activity, as is another sequence we refer to as LS4. Aside from that, different sequences play roles in insulator and promoter activity. So while there is overlap and BEAF is important for both, insulator and promoter activity can be separated.
Project description:Data implicate the Drosophila 32 kDa Boundary Element-Associated Factors BEAF-32A and BEAF-32B in both chromatin domain insulator element function and promoter function. They might also function as an epigenetic memory by remaining bound to mitotic chromosomes. Both proteins are made from the same gene. They differ in their N-terminal 80 amino acids, which contain single DNA-binding BED fingers. The remaining 200 amino acids are identical in the two proteins. The structure and function of the middle region of 120 amino acids is unknown, while the C-terminal region of 80 amino acids has a putative leucine zipper and a BESS domain and mediates BEAF-BEAF interactions. Here we report a further characterization of BEAF. We show that the BESS domain alone is sufficient to mediate BEAF-BEAF interactions, although the presence of the putative leucine zipper on at least one protein strengthens the interactions. BEAF-32B is sufficient to rescue a null BEAF mutation in flies. Using mutant BEAF-32B rescue transgenes, we show that the middle region and the BESS domain are essential. In contrast, the last 40 amino acids of the middle region, which is poorly conserved among Drosophila species, is dispensable. Deleting the putative leucine zipper results in a hypomorphic mutant BEAF-32B protein. Finally, we document the dynamics of BEAF-32A-EGFP and BEAF-32B-mRFP during mitosis in embryos. A subpopulation of both proteins appears to remain on mitotic chromosomes and also on the mitotic spindle, while much of the fluorescence is dispersed during mitosis. Differences in the dynamics of the two proteins are observed in syncytial embryos, and both proteins show differences between syncytial and later embryos. This characterization of BEAF lays a foundation for future studies into molecular mechanisms of BEAF function.
Project description:Chromatin insulators are DNA protein complexes that mediate inter and intra chromosomal interactions. Boundary Element Associated Factor- 32 (BEAF-32) is an insulator protein predominantly found near gene promoters and thought to play a role in gene expression. We find that mutations in BEAF-32 are lethal, show loss of epithelial morphology in imaginal discs and cause neoplastic growth defects. To investigate the molecular mechanisms underlying this phenotype, we carried out a genome-wide analysis of BEAF-32 localization in wing imaginal disc cells. Mutation of BEAF-32 results in miss-regulation of 3850 genes by at least 1.5-fold, 647 of which are bound by this protein in wing imaginal cells. Up-regulated genes encode proteins such as Bazooka, a determinant of cell polarity; the Insulin receptor-1 and the p70-S6 kinase, involved in the insulin growth factor pathway that can activate various pathways involved in cell proliferation; and the Unpaired 3 ligand of the Janus kinase (JNK) pathway, and its target gene Socs35, which can also activate cell proliferation. The expression of genes involved in DNA homologous end repair and oxidative stress pathways is also increased in BEAF-32 mutants. Among the down-regulated genes are those encoding components of the Wingless pathway, which is required for cell differentiation, and amino acid metabolism. Miss-regulation of these genes explains the unregulated cell growth and neoplastic phenotypes observed in imaginal tissues of BEAF-32 mutants. Here we examine mapping the genomic binding sites and its distribution of BEAF-32, a Drosophila insulator proteins in wing imaginal tissue. We performed ChIP-seq analysis using BEAF-32B.
Project description: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.
Project description:Boundary Element Associated Factor-32 (BEAF-32) is an insulator protein predominantly found near gene promoters and thought to play a role in gene expression. We find that mutations in BEAF-32 are lethal, show loss of epithelial morphology in imaginal discs and cause neoplastic growth defects. To investigate the molecular mechanisms underlying this phenotype, we carried out a genome-wide analysis of BEAF-32 localization in wing imaginal disc cells. Mutation of BEAF-32 results in miss-regulation of 3850 genes by at least 1.5-fold, 794 of which are bound by this protein in wing imaginal cells. Up-regulated genes encode proteins involved in cell polarity, cell proliferation and cell differentiation. Among the down-regulated genes are those encoding components of the wingless pathway, which is required for cell differentiation. Miss-regulation of these genes explains the unregulated cell growth and neoplastic phenotypes observed in imaginal tissues of BEAF-32 mutants.
Project description:This SuperSeries is composed of the following subset Series: GSE36735: Distribution of Drosophila insulator protein BEAF-32 in Wing imaginal tissue (Wildtype) [ChIP-seq] GSE36736: Genome wide transcriptional profiling of BEAF-32 in wing imaginal tissues of wildtype and mutants [expresion array] Refer to individual Series
Project description:Insulators are considered as chromosome organizers. BEAF, one of the insulator proteins, is highly conserved in Drosophila speies but also limited to Drosophila spcies. BEAF associates with TSS of active genes. Comparative study of BEAF binding landscapes in four Drosophila species reveals BEAF association with gene pairs, and the results suggest the role of gain or loss of BEAF binding during the speciation of Drosophila species. DNA sample from ChIP for BEAF and input are collected for each of four Drosophila species
Project description:Eukaryotic chromosomes are partitioned into topologically associating domains (TADs) that are demarcated by distinct insulator-binding proteins (IBPs) in Drosophila. Whether IBPs regulate specific long-range contacts and how this may impact gene expression remains unclear. Here we identify ‘indirect peaks’ of multiple IBPs, that represent their distant sites of interactions through long-range contacts. Indirect peaks depend on protein-protein interactions among multiple IBPs and their common co-factors, including CP190, as confirmed by high-resolution analyses of long-range contacts. Mutant IBPs unable to interact with CP190 impair long-range contacts as well as the expression of hundreds of distant genes that are specifically flanked by indirect peaks. Regulation of distant genes strongly correlates with RNAPII pausing, highlighting how this key transcriptional stage may trap insulator-based long-range interactions. Our data illustrate how indirect peaks may decipher gene regulatory networks through specific long-range interactions. Binding profiles of Beaf-32 in Drosophila S2 cells by ChIP-Seq (Illumina)