Promoter nucleosome dynamics regulated by signalling through the CTD code

Abstract

The phosphorylation of the RNA polymerase II C-terminal domain (CTD) plays a key role in delineating transcribed regions within chromatin by recruiting histone methylases and deacetylases. Using genome-wide nucleosome mapping, we show that CTD S2 phosphorylation controls nucleosome dynamics in the promoter of a subset of 324 genes, including the regulators of cell differentiation ste11 and metabolic adaptation inv1. Mechanistic studies on these genes indicate that during gene activation a local increase of phospho-S2 CTD nearby the promoter impairs the phospho-S5 CTD-dependent recruitment of Set1 and the subsequent recruitment of specific HDACs, which leads to nucleosome depletion and efficient transcription. The early increase of phospho-S2 results from the phosphorylation of the CTD S2 kinase Lsk1 by MAP kinase in response to cellular signalling. The artificial tethering of the Lsk1 kinase at the ste11 promoter is sufficient to activate transcription. Therefore, signalling through the CTD code regulates promoter nucleosomes dynamics.The process of activating genes—known as gene expression—involves a number of steps. During the first step, the gene's DNA is copied or ‘transcribed’ to produce a molecule of messenger RNA. However, most of the DNA in a cell is wrapped around proteins called histones to make structures known as nucleosomes, and the DNA has to be unpacked to allow the enzymes that make messenger RNA to access it.Cells regulate how the DNA is packed by attaching chemical groups to the histone proteins. Adding acetyl groups to histones usually causes the nucleosomes to unwrap and creates loosely packed DNA that helps with gene expression. On the other hand, the addition of methyl groups has the opposite effect.RNA polymerase II is the enzyme that carries out transcription of messenger RNAs in all eukaryotic cells—that is, the cells of organisms like plants, animals, and fungi. Like all enzymes, RNA polymerase II is made of smaller building blocks called amino acids. One end of the RNA polymerase II enzyme, called the C-terminal domain (or CTD), contains a unique sequence of amino acids that serves as a scaffold to recruit other proteins involved in transcription and histone modifications. Different amino acids in this region of RNA polymerase II can be modified by the addition of phosphate groups. The pattern of these modifications is often thought of as a code and can influence which other proteins get recruited.It remains poorly understood how RNA polymerase II regulates nucleosomes to allow transcription to occur. Materne, Anandhakumar et al. have now addressed this issue by engineering mutant yeast cells in which phosphate groups cannot be added to specific amino acids in the RNA polymerase II enzyme. Most genes were expressed as normal in these yeast cells, but a few hundred genes were not expressed.Materne, Anandhakumar et al. then used a technique called MNase-Seq to map the position of nucleosomes across the genome and found that there were more nucleosomes near to start of these down-regulated genes. Further experiments showed that the addition of phosphate groups onto the RNA polymerase II is required to deplete the nucleosomes at the start of a gene called ste11, which allows transcription to occur.Materne, Anandhakumar et al. also found that artificially tethering the enzyme that adds phosphate groups to the C-terminal domain to the start of the ste11 gene was sufficient to oust nucleosomes and activate transcription by RNA polymerase II.Future work will address if this newly discovered mechanism is implicated in the activation of specific patterns of gene expression during the development of more complex organisms.