Histone modifications in RNA polymerase II transcription

The genetic blueprint of life occurs in the form of DNA, which is faithfully packaged within the nucleus of each cell in our body.  DNA packaging, and its organization in the nucleus, is regulated by a class of proteins called histones.  These proteins create individual histone-DNA complexes, referred to as nucleosomes, which are further folded into higher-order chromatin structures that are poorly defined (figure at right). It is therefore significant to ask: (i) how do distinct chromosomal domains such as “euchromatin” and “heterochromatin” become established and maintained, and (ii) how is the underlying DNA within this highly compact and repressive chromatin environment made accessible to the protein complexes that utilize it?  Our lab is addressing these questions by examining RNA polymerase II transcription in chromatin.  We seek to understand how this process occurs at the “right place” and at the “right time” in the genome, and the mechanisms by which this mRNA synthesis machine is able to transcribe through a repressive chromatin environment.

One mechanism that has emerged as a major regulator of the organization and function of chromatin are histone post-translational modifications. Surprisingly, a vast number of covalent modifications, such as acetylation, methylation, ubiquitylation and phosphorylation exist on histones.  Numerous studies indicate that these modifications work together in the form of a ‘histone code’ to regulate chromatin-based activities.  This code functions, in part, through the direct recruitment of protein modifiers to the sites of modification, which then alter the organizational state of chromatin and/or facilitate a biological process (e.g. transcription). A famous example is the recruitment of HP1 (heterochromatin protein 1) to histone H3 that is methylated at lysine 9, resulting in the formation of highly compact, transcriptionally inert heterochromatin in cells. (Figure at left)

Of the known histone modifications, histone methylation and ubiquitylation have recently received much attention and are only now becoming understood.  Recent insights indicate that these modifications play a fundamental role in the organization of chromatin and in the activation and repression of genes controlled by RNA polymerase II.  We and others have found, for example, that a variety of enzymes which either methylate (e.g. Set1 and Set2) or ubiquitylate (e.g. Rad6) histones, associate with RNA polymerase II during the elongation cycle of transcription.  While Set2 associates directly with the C-terminal domain of RNA polymerase II, Rad6 and Set1 associate with the polymerase through an interaction with the PAF transcription elongation complex.  This finding indicates that histone modifications play a fundamental role in the transcription process. We seek to understand how these enzymes and their modifications regulate chromatin-based transcription and how defects in these processes result in diseases such as cancer.

A main focus of our lab is the histone methyltransferase Set2.  We have shown that this enzyme “travels” with RNA polymerase II down genes to mediate histone H3 lysine 36 methylation (figure above).  The function of this enzyme remained elusive until recent work showed that the methylation by Set2 recruits a histone deacetylase complex (Rpd3S) to the bodies of genes.  As it turns out, RNA polymerase II transcription involves the recruitment of histone acetyltransferases that function to help disrupt nucleosomes in front of the polymerase.  Set2’s function therefore is to recruit a histone deacetylase enzyme that restores the acetylation levels to their previous state after the passage of the polymerase.  Without this mechanism, acetylation levels remain high in the coding region and cryptic transcription start sites are inappropriately used.  This is just one story to mention, and I would argue that every histone modification has an equally interesting and exciting story to tell (and many of these stories have not yet been told!). 

Finally, I should mention that our lab is using budding yeast as a model organism to determine the functions of the histone modifications mentioned above.  This organism provides a fantastic opportunity to employ both biochemistry and genetics to solve complex problems.  For example, we can easily mutate the histones and chromatin-modifying enzymes we study and examine how their mutation affects chromatin organization, transcription and other biological processes.  We can also insert affinity purification tags on any protein at will to study who they are associated with and where they occur in the genome (and if they get recruited to various ‘marks’).  Thus, this powerful organism affords a unique opportunity to study the fundamentals of chromatin modifications.  Significantly, most, if not all, of the enzymes and modifications we study in yeast are conserved in humans.  Thus, what we learn in yeast is directly applicable to how these enzymes operate in human cells, which is relevant as many of these enzymes have been associated with various human diseases including cancer.

--Brian Strahl