Brd2 is a member of the bromodomain extra terminal (BET) protein family, which consists of four chromatin-interacting proteins that regulate gene expression. Each BET protein contains two N-terminal bromodomains, which recognize acetylated histones, and the C-terminal protein-protein interaction domain. Using a genome-wide screen, we identify 1450 genes whose transcription is regulated by Brd2. In addition, almost 290 genes change their alternative splicing pattern upon Brd2 depletion. Brd2 is specifically localized at promoters of target genes, and our data show that Brd2 interaction with chromatin cannot be explained solely by histone acetylation. Using coimmunoprecipitation and live-cell imaging, we show that the C-terminal part is crucial for Brd2 association with chromatin. Live-cell microscopy also allows us to map the average binding time of Brd2 to chromatin and quantify the contributions of individual Brd2 domains to the interaction with chromatin. Finally, we show that bromodomains and the C-terminal domain are equally important for transcription and splicing regulation, which correlates with the role of these domains in Brd2 binding to chromatin.

• MeSH
• alternativní sestřih MeSH
• chromatin metabolismus   MeSH
• genetická transkripce MeSH
• genom lidský * MeSH
• HeLa buňky MeSH
• histony genetika   metabolismus   MeSH
• lidé MeSH
• promotorové oblasti (genetika) MeSH
• protein-serin-threoninkinasy genetika   metabolismus   MeSH
• regulace genové exprese * MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• signální transdukce MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• videomikroskopie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Precursor messenger RNA (pre-mRNA) splicing is catalyzed by the spliceosome, a large ribonucleoprotein (RNP) complex composed of five small nuclear RNP particles (snRNPs) and additional proteins. Using live cell imaging of GFP-tagged snRNP components expressed at endogenous levels, we examined how the spliceosome assembles in vivo. A comprehensive analysis of snRNP dynamics in the cell nucleus enabled us to determine snRNP diffusion throughout the nucleoplasm as well as the interaction rates of individual snRNPs with pre-mRNA. Core components of the spliceosome, U2 and U5 snRNPs, associated with pre-mRNA for 15-30 s, indicating that splicing is accomplished within this time period. Additionally, binding of U1 and U4/U6 snRNPs with pre-mRNA occurred within seconds, indicating that the interaction of individual snRNPs with pre-mRNA is distinct. These results are consistent with the predictions of the step-wise model of spliceosome assembly and provide an estimate on the rate of splicing in human cells.

• MeSH
• buněčné jádro metabolismus   MeSH
• buněčné linie MeSH
• fluorescenční spektrometrie MeSH
• FRAP MeSH
• HeLa buňky MeSH
• kinetika MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• prekurzory RNA metabolismus   MeSH
• ribonukleoproteiny malé jaderné metabolismus   fyziologie   MeSH
• sestřih RNA fyziologie   MeSH
• spliceozomy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH