FACT Complex Recruitment and the Role of H2A.Z in Transcriptional Regulation [ChIP-seq]
The FACT complex (FAcilitates Chromatin Transcription) plays a key role in chromatin remodeling during transcription, enhancing DNA accessibility. It is believed to alter chromatin by temporarily displacing histones, enabling RNA polymerase II (Pol II) to engage, and then reinstating the original chromatin structure after transcription is complete. In this study, we show that FACT complex subunit ssrp1 undergoes neddylation in vivo, which might be important for its subcellular localization and its interaction with Pol II. This, in turn, promotes the recruitment of the FACT complex to actively transcribed chromatin, establishing a positive feedback loop. In addition, the FACT complex mediates H3K4me3 while not influencing the transcriptional rate. Lastly, in contrast to its canonical form, ubiquitinated H2A.Z does not impede FACT's binding to chromatin and may alleviate the transcriptional repression associated with canonical H2A ubiquitination. Overall design: ChIP-seq of NRPB1 in Columbia-0(WT) and nrpb2-3, ssrp1-2 mutant background;ChIP-seq of H3K4me3 in Columbia-0(WT) and nrpb2-3 ssrp1-2 spt16-2 mutant.Flag ChIP used Flag antibody, and HA ChIP used beads In all cases, seeds were stratified for 4 days at 4°C, and seedlings were harvested 12 days after germination.