Role and establishment of histone modifications
4214 Molecular Biology Building
Dept. of Biochemistry, Biophysics & Molecular Biology
Iowa State University
Ames, IA 50011
Phone: (515) 294-9015
B.S., Chemistry, Stanford Univ., 1994
Ph.D., Chemistry, California Institute of Technology, 2000
Postdoctoral Fellow, UC San Francisco, 2000-2001
Postdoctoral Fellow, University of Massachussetts, 2001-2005
DNA contains the genetic information that underlies the development and biological functioning of all living organisms. Structurally, DNA is composed of complementary pairs of long nucleic acid strands. However, for organisms with cells that contain a nucleus, from budding yeast to humans, DNA does not exist free in solution, but is instead packaged into a complex structure known as chromatin. The structure of chromatin, and how it changes, plays a central role in how the associated DNA is utilized. My group is focused on achieving a better understanding of chromatin structure, how it is altered, and the functional ramifications of these changes. This knowledge provides a better understanding of how the information contained in DNA is read, propagated, and maintained in normally functioning cells, and offers insight into the way defects in chromatin structure and function lead to human diseases.
Chromatin exhibits multiple levels of structure (Figure 1), but the simplest structural unit of chromatin is the nucleosome. Nucleosomes are composed of 147 base pairs of DNA wrapped around a protein spool known as the histone octamer, which itself contains two copies each of histone proteins H2A, H2B, H3, and H4. The bulk of the histone octamer is contained within the wrappings of the DNA. However, up to a quarter of the histone residues extends past the DNA as largely unstructured histone “tails.” Nucleosomes occur with high frequency in the nucleus, with more than fifty-percent of the genome sequestered as nucleosomes. These nucleosomes can then alone, or with the addition of other structural proteins, adopt complex forms of higher-order chromatin structure. However, the nature and determinants of these higher-order structures are poorly understood.
Figure 1. Model of chromatin structure.
One of the goals in my group is to better understand the higher-order structure that nucleosomes adopt in the absence of additional structural proteins. Using chromatin model systems of repetitive nucleosomes, known as nucleosomal arrays, it has been shown that these molecules can form both a highly intra-array compacted species and an interarray associated species that are believed to reflect higher-order chromatin structure in the nucleus. Formation of both of these species is facilitated especially by the histone H4 tails, and recently we have shown that interarray interactions are mediated in part by direct contacts between H4 tails and a core region at the interface between the histones H2A and H2B. Our studies have also revealed that intra-array compaction requires more than four nucleosomes, while interarray interactions can be formed with as few as two nucleosomes. Additionally, we find that nucleosome stability decreases with decreasing nucleosome array length or saturation, suggesting a potential role of higher-order chromatin structure in regulating nucleosome loss.
Another goal in my group is to better understand the structure of a specialized form of chromatin known as heterochromatin. Heterochromatin has many important biological roles, including insuring proper chromosomal segregation during mitosis and chromosomal end maintenance. Although heterochromatin is believed to be a highly condensed form of chromatin, a long-standing question in the field is what the actual structure of chromatin is. Recently we have been successful in reconstituting a heterochromatin model system with human heterochromatin protein HP1a. Our results suggest that HP1a dimerization allows the molecule to facilitate three types of nucleosome interactions involving histone H3 bridging – within a nucleosome, between nucleosomes within a chromatin strand, and between chromatin strands.
A major way that organisms modulate chromatin structure and function is through chemical modification of the amino acid side-chains of the histone proteins that comprise nucleosomes, especially those contained in the histone tails. Numerous types and sites of histone modifications have been identified to date, and establishment of these modifications is mediated by a number of different chromatin-modifying enzymes.
Our group is actively engaged in trying to understand the action of one of these complexes, the budding yeast SAGA complex, which was one of the first chromatin-modifying enzyme complexes to be identified. The complex is comprised of over 19 different subunits (Figure 2), and this 2 MDa complex is well conserved from yeast to humans. The SAGA family of complexes has a role in facilitating the transcription of inducible genes. These inducible genes involve those that are activated in response to environmental stress. Additionally, in mammals these genes include those that are activated during development, where impaired SAGA function can result in developmental defects and embryonic death. Misregulation of SAGA function has also been shown to be associated with the development of human diseases. For example, SAGA’s ability to induce gene expression can be co-opted by cancerous cells, as well as those expressing viral genes. Thus, an understanding of how SAGA works is important for understanding normal and abnormal gene expression.
Figure 2. Composition and organization of the yeast SAGA complex. A. SAGA subunits. Single stars indicate subunits involved in acetyltransferase activity. B. Low resolution SAGA structure. The proposed sites of selected subunits are indicated. Subunits with only numbers indicate Taf proteins. Localization of Ada2 and Ada3 was not determined, but their direct interactions with Gcn5 have been previously shown. Adapted from Schultz and coworkers, Mol. Cell 2004, 15: 199-208.
One way that SAGA can facilitate gene expression is by catalyzing the acetylation of lysine residues in the histone H3 tails of chromatin (Figure 3). The standard model for this process is that SAGA is recruited to the promoter of induced genes through interactions with transcriptional activators, and this localization results in nucleosome acetylation. While this model is true overall, our recent work suggests that the process is more complicated. We have shown in yeast that in response to environmental stress, the SAGA complex undergoes self-acetylation on one of its subunits, Ada3. This autoacetylation is then recognized by a domain in another subunit, the bromodomain of Gcn5, allowing multimerization of the complex. This multimerization has not previously been observed for any other chromatin-modifying complex, and provides a novel mechanism of potentially regulating SAGA activity. Indeed, we find that one implication of SAGA multimerization is that it allows the complex to cooperatively acetylate nucleosomes, where binding of one nucleosomal substrate facilitates the binding and acetylation of other distal nucleosomes. We believe that this cooperative acetylation reflects a potential ability of acetylated SAGA complex to mediate long-range bridging of nucleosomes within a gene, and even between genes. Interestingly, cooperative nucleosome acetylation by the SAGA complex requires that both H3 tail within a nucleosome be present and unacetylated, suggesting the binding interactions between H3 tails within a nucleosome somehow lead to cooperative behavior.
Figure 3. SAGA-mediated Nucleosome acetylation. A. Acetyl transfer reaction. B. Histone H3 lysine acetylation sites.
The complexity and heterogeneity of isolated nuclear chromatin makes it difficult to characterize its properties in a test tube. A common theme in a number of projects in my group is the development and use of tools to generate and study well-defined chromatin model systems. To supplement previously developed mononucleosome and nucleosomal array systems, we have established methods to incorporate specific, homogeneous combinations of post-translational modifications into the histone H3 and H4 histone tails. We have developed a strategy to generate nucleosomes that within the histone octamer vary in composition between the two copies of a given histones. We have also generated a system of mononucleosomes that can undergo DNA ligation to give nucleosomal arrays up to four nucleosomes long, with the potential to vary the composition of each nucleosome in a desired manner. Additionally, we have adapted a cysteine-mediated cross-linking strategy to directly trap cross-strand array interactions. These nucleosomal systems have been used as substrates for enzyme assays by the SAGA acetyltransferase complex and various ATP-dependent remodeling complexes in order to better understand how their activity toward chromatin is modulated by the presence of pre-existing post-translational histone modifications. Also, these systems have allowed us to better understand the determinants of higher-order chromatin, including the role that specific post-translational histone modifications play in modulating these structures.
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