Project description:We have carried out ChIP-seq to study the distribution of Brahma in the genome of S2 cells and to identify the Brahma-bound genes. We have also knocked down Brahma by RNA interference and we have carried out RNA-seq to determine the changes induced by Brahma depletion in the transcriptome of S2 cells. Furthermore, we have carried out RNA-seq experiments to profile S2 cells in which we first deplete Brahma by RNA interference and then express recombinant Brahma (either wild-type or ATPase mutant). These datasets are being used in several projects aimed at gaining understanding on the roles of Brahma in transcription regulation and pre-mRNA processing.
Project description:SWI/SNF chromatin remodeling complexes control gene expression by regulating chromatin structure. However, the full subunit composition of SWI/SNF complexes in plants remains unclear. Here we show that BRAHMA Interacting Protein 1 (BRIP1) and BRIP2 in Arabidopsis thaliana are core subunits of plant SWI/SNF complexes. BRIP1 and 2 are two homolog proteins. brip1 brip2 double mutants exhibit developmental phenotypes and a transcriptome strikingly similar to those of BRAHMA (BRM) mutants. Genetic interaction tests indicated that BRIP1 and 2 act together with BRM to regulate gene expression. Furthermore, BRIP1 and 2 physically interact with BRM-containing SWI/SNF complexes, and extensively co-localize with BRM at endogenous genes. Loss-of-brip1brip2 results in decreased BRM occupancy at almost all BRM target genes and substantially reduced subunits incorporation into the BRM-containing SWI/SNF complexes. Together, our work identifies new core subunits of BRM-containing SWI/SNF complexes in plants, and uncovers the essential role of these subunits in regulating the integrity (assembly) of SWI/SNF complexes in plants.
Project description:BRAHMA (BRM) is a conserved SWI/SNF-type chromatin remodeling ATPase implicated in many key nuclear events. Histone H3 Lysine 27 (H3K27) demethylases specifically remove the repressive histone mark, trimethylation of H3K27 (H3K27me3). Both proteins are thought to play active roles in regulating gene activities at the chromatin level, but their genome-wide coordination remains to be determined. In Arabidopsis thaliana, RELATIVE OF EARLY FLOWERING 6 (REF6, also known as JMJ12) is the first identified plant H3K27 demethylase. Here, genome-wide analyses revealed that REF6 targets to thousands of genes across the Arabidopsis genome and co-localizes with BRM at more than 1,000 genes, many of which are genes involved in response to various stimuli, especially plant hormones. Loss of REF6 activity results in decreased BRM occupancy at hundreds of BRM-REF6 co-targets, indicating that REF6 is required for the recruitment of BRM to chromatin. Further, REF6 targets to genomic loci that contains the CTCTGTTT motif in vivo Examination of BRM occupancy in 14-day-old wt and ref6-1 seedlings. Examination of REF6 occupancy in 14-day-old wt and brm-1 seedlings. Examination of global RNA expression in 14-day-old wt, brm-1, ref6-1 and brm-1 ref6-1 seedlings. Examination of H3K27me3 profiles in 14-day-old wt, brm-1, ref6-1, and brm-1 ref6-1 seedlings.
