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Background

Chromatin OrganizationThe 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?

 

(ii) How is the underlying DNA within this highly compact and repressive chromatin environment made accessible to the protein complexes that utilize it?

 

Brian Strahl's lab is addressing these questions by taking advantage of multiple model organisms and through applying a combination of genetic, biochemical and high-throughput proteomic approaches.

 

HP1One mechanism that has emerged as a major regulator of the organization and function of chromatin are histone post-translational modifications (PTMs). 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 above).

Histone modifications in RNA polymerase II transcription

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.

 

The Strahl lab 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 (Figure below).

 

RNAPII 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.

 

Set2 Mechanism

 

One major focus of our lab is the histone methyltransferase Set2. We have shown that this enzyme “travels” with RNA polymerase II during gene transcription 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. Thus, one function of deacetylase activity is to remove transcription-linked histone acetylation that would otherwise maintain a more open chromatin structure in the transcribed region of genes. Without this mechanism, acetylation levels would remain high in the coding regions and cryptic transcription start sites would be inappropriately used. In addition to Rpd3S, other H3K36 methyl-specific effector proteins, including chromatin-remodeling activities, have been recently identified to also function along with Rpd3 in maintaining a suppressed chromatin state in the transcribed region of genes.

 

S. cerevisiaeTo study histone modifictaions in gene regulation, our lab is using budding yeast (Saccharomyces cerevisiae) as a model organism. 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.

High-thoughput proteomics and studies into the ‘histone code’

More recently, the Strahl lab has been engaged in a large-scale, high-throughput proteomics project to uncover the function of histone modifications using peptide arrays. My interest in this stems from the fact that as a postdoc in David Allis’ lab, we proposed the ‘histone code’ hypothesis to explain how distinct covalent histone PTMs might work together to regulate epigenetic inheritance, gene expression and the control of cell growth, differentiation and disease.

Peptide Array Platform schematic

 

However, progress in this area has been slow due to the lack of new tools to study it. Because of this, we set out to develop a novel ‘histone code’ peptide array platform to explore this critical question in chromatin biology (Figure at right).

 

Since initiating this project, we have shown how a number of effector proteins are influenced by combinatorial histone PTMs. We are also collaborating with a wide number of labs across the world to decipher the binding potential of newly identified histone "reading" proteins.

 

One area of particular interest is how adjacent or paired domains in chromatin-associated protein "read" the 'histone code'. Recent studies from our lab have shown that the tandem Tudor (TTD) and adjacent PHD domains of UHRF1, an E3 ubiquitin ligase domain-containing protein, function in unison to bind a specific heterochromatic signature of H3 for its chromatin recruitment and DNA methylation maintenance function (see Rothbart...Strahl, 2013 Genes & Development, 27:1288-98; Figure below). These studies highlight an important feature of how histone-associated proteins engage chromatin, and we note that many other chromatin-associated proteins also contain paired “docking” domains similar to UHRF1. A future goal will be to define the interactions and downstream biological functions of these multi domain-containing proteins.

 

UHRF1.jpg

 

Using epigenetics to fight breast cancer

The Strahl lab has also shifted into the understanding and treatment of breast cancer, as it is becoming increasingly clear that epigenetics plays a central role in breast cancer development.

 

In collaboration with Dr. Pilar Blancafort’s lab at The Univeristy of Western Australia, we have focused on the design and implementation of novel artificial transcription factors that are capable of specifically targeting epigenetic modifications (i.e., DNA methylation) to breast cancer stem cell genes that are important for breast cancer proliferation.

 

We document in two newly published papers in 2012 the use of these transcription factors to shut down breast cancer proliferation in cell culture and in mouse models.

 

--Brian D. Strahl

 

 

Keywords: histones, histone post-translational modifications, epigenetics, gene regulation, Brian Strahl, UNC Chapel Hill