Having shown that nucleosomes are precisely positioned at the 5’ ends of most genes, and is positioned by chromatin remodelers, our next objective is to reconstitute genome-wide nucleosome organization with purified proteins. This will allow us to dissect specific mechanisms. We now achieved this in 2016. Our next step is to explore the assembly of the transcription machinery in the context of properly positioned nucleosomes, as this reflects the in vivo
There are nearly a thousand different gene and chromatin regulatory proteins which need to work together to regulate the genome. We want to know their precise positional organization relative to each other, within several bp of resolution. This will be achieved using our ChIP-exo assay applied to a thousand yeast TAP-tagged ORF strains. Such an all-encompassing view will help us better understand genome regulation. This will set the stage for an equivalent endeavor in human systems.
What is the mechanism by which transcription is regulated?
Ultimately sequence-specific transcription factors need to recruit the transcription machinery to promoters. The machinery then locks in on the DNA, wherein RNA polymerase II initiates transcription. Hundreds of different proteins regulate this process through opposing activities that respond to environmental signaling. We tackle each of these activities (e.g. how histone acetylation affects PIC assembly) in great mechanistic detail. This is being accomplished through genome-wide binding and transcription assays to obtain positional organization, and though use of factor depletion to explore what happens when a regulatory protein is unable to do its job.