Shelley L. Berger, Ph.D.

Hilary Koprowski Professor
Gene Expression and Regulation Program
215-898-3922, Office
berger@wistar.org

Introduction

The laboratory of Shelley L. Berger, Ph.D., focuses on mechanisms that regulate gene expression with a special emphasis on how the DNA-packaging structure of chromatin is manipulated during genomic processes. Their findings inform the study of cancer and other diseases, and ultimately drug discovery.

Research Summary

Eukaryotic genomes are, in general, in a default state of repression, where the vast majority of genes are turned off. This repression is accomplished largely through packaging DNA into tightly coiled packages called chromatin by association with histone proteins. This means that chromatin must be uncoiled before allowing genes to be accessed by proteins that transcribe the genetic code. One mechanism that regulates chromatin structure is the attachment of chemical groups to the histones. It is now becoming clear that a diverse array of enzymes modify histones, and studies by the Berger team explore this range of chemical modification, including acetylation, phosphorylation, methylation, and ubiquitylation. One appealing idea is that the pattern and identity of histone modifications constitute a "code" for specific processes, such as transcription. The researchers use yeast (S. cerevisiae or budding yeast) as a model system to study these enzymes and a potential histone code because yeast research is fast, easy, and inexpensive, allowing for extensive genetic manipulation. This knowledge is then used to examine how these pathways are conserved in human cells. Dysfunction of these pathways underlies cancer and other diseases, so one of the research team's aims is to elucidate normal mechanisms to unravel the basis of many human diseases and to ultimately inform drug discovery.

To study histone modifications, the investigators combine genetics, biochemistry, and structural analyses to understand the mechanism and regulation of histone modification enzymes and the protein complexes in which they reside. Methods include biochemical fractionation, whole genome transcriptional analysis using microarrays, and chromatin immunoprecipitation, combined with an arsenal of more classical genetic and molecular approaches. These approaches are particularly powerful in the model yeast system for framing critical questions in humans.

Recent Scientific Advances

Protein complexes that possess HAT activity: Research on histone modifications began with a study of histone acetyltransferases. For many years the Berger laboratory has studied the transcriptional adaptor, Gcn5, a component of a protein complex that is recruited to promoters through interaction with DNA bound activators. Gcn5 was then identified as the first, and is now the paradigmatic protein found to possess histone acetyltransferase (HAT) activity. Thus, the finding that the Gcn5 component of the bridging adaptor complex is a chromatin modifying enzyme suggests the following powerful model: Particular promoters may be targeted for histone acetylation, and therefore chromatin remodeling, through transcription factor recruitment of the adaptor complex. This model, through work in the Berger laboratory and that of many others, has been confirmed in many respects.

Clearly, the composition and dynamic change in composition of HAT-containing protein complexes determine their physiological role. Hence, one objective is to identify HAT complexes, analyze their composition, and describe how their activity and composition is modulated under various conditions. Only with this knowledge will an understanding of the critical physiological roles of HAT complexes emerge.

Research has identified several native HAT complexes in yeast, and research over the last several years has focused on function of one - termed SAGA - which contains Gcn5 as the HAT enzyme. SAGA, like Gcn5 itself, has become a prototype for the many HAT complexes that have now been identified in yeast and mammalian cells. The research group has been studying the function and composition of two SAGA-related complexes. One appears to be an active form of the complex and the second a repressed form. Thus, it appears that the structure of SAGA is altered through proteolysis and/or addition of subunits, and research concentrates on analyzing how these alterations in composition result in altered activity. Most generally, the Berger team views their studies of SAGA as a paradigm for similar regulation that may occur in numerous other protein complexes that contain histone modifying enzymes.

Patterns of histone modifications: Phosphorylation of histone tails appears to be intimately connected to acetylation. In yeast phosphorylation of histone H3 precedes acetylation by Gcn5 and this pattern is important for transcription. These observations suggest that transcriptional regulation occurs through multiple, linked, covalent modifications of histone tails. Based on these initial results the team searched for kinases in yeast that phosphorylate histone H3. In collaboration with Ramin Shiekhattar's laboratory at Wistar, the Berger laboratory identified a relevant histone H3 kinase yeast as Snf1, a previously known transcription factor that regulates certain Gcn5-dependent genes. Results show that Snf1, functioning as a histone kinase, works together with Gcn5 as a histone acetyltransferase to induce certain genes. The newest studies focus on the recruitment of Snf1 complex to gene promoters and the interplay with recruitment of SAGA.

A connection between acetylation and phosphorylation occurs generally, and in mammalian cells, and studies on H3 in yeast may be a paradigm for these relationships. This was the first histone modification pattern to be identified and has now been confirmed by many other examples of patterns. This line of research indicates that patterns (e.g. acetylation and phosphorylation) provide synergy and increased regulation beyond single histone modifications.

Histone phosphorylation, methylation, and ubiquitylation: The laboratory's focus is expanding to examine a greater range of modifications on the core histone tails. One approach is to use proteomics to test yeast kinases for activity on histones. This approach has led to histone H4 where again a pattern of modifications, in this case between phosphorylation and arginine methylation has been observed. In addition, ubiquitin specific antibodies are being used to determine which histones are marked by this very large peptide modification. Several of the histones are ubiquitylated and the researchers are determining the sites and physiological functions. They are also investigating a potential histone deubiquitylating enzyme in SAGA, and recent studies indicate that deubiquitylation of histone H2B is critical for transcriptional activation. This represents a potentially new paradigm in histone modification, where the cycle of ubiquitylation/deubiquitylation is critical for transcription.

Structure/function analyses of HAT activity: The Berger laboratory has collaborated on a series of studies with Ronen Marmorstein, an X-ray crystallographer at Wistar. In the past several years the two groups have used a combination of functional and structural studies to determine catalytic mechanisms of two HAT families, Gcn5 and the MYST family. The Marmorstein laboratory has also examined the structure of Gcn5 complexed with phosphorylated histone H3, yielding a model that explains the increased acetylation efficiency on the phosphorylated substrate. As before, the Berger laboratory has tested this hypothesis using yeast genetics. The two laboratories are now extending their collaboration to enzymes involved in phosphorylation and ubiquitylation.

p53 modifications and their relationship to histone modifications: p53 is a tumor suppressor and transcriptional trans-activator, which negatively regulates cell growth in mammalian cells. Mutations in p53 are the most common alterations found in human cancer. In collaboration with Thanos Halazonetis' laboratory at Wistar, the Berger team is investigating many aspects of p53 activation function, with regard to the role of modifications. For example, they established that human Gcn5 and CBP acetylate p53 in vivo. More recent work has shown that acetylation of p53 functions to increase interactions with coactivator/HAT proteins, leading to histone acetylation. The interesting general idea is that there may be a modification cascade, where the same enzymes modify both transcription factors and histones. Again, many non-histone transcription factors have emerged as substrates of HAT enzymes and the p53 studies are establishing basic mechanisms in non-histone acetylation.

The Berger team is currently expanding its studies of p53 modification to phosphorylation and have determined that a specific kinase connected to human cancer phosphorylates p53 and one of the histones. Current studies focus on a potential phosphorylation cascade, similar to the acetylation cascade previously identified.

Selected Publications

Huang J, Perez-Burgos L, Placek BJ, Sengupta R, Richter M, Dorsey JA, Kubicek S, Opravil S, Jenuwein T, Berger SL. (2006) Repression of p53 activity by Smyd2-mediated methylation. Nature. 444(7119):629-32.

Krishnamoorthy T, Chen X, Govin J, Cheung WL, Dorsey J, Schindler K, Winter E, Allis CD, Guacci V, Khochbin S, Fuller MT, Berger SL. (2006) Phosphorylation of histone H4 Ser1 regulates sporulation in yeast and is conserved in fly and mouse spermatogenesis. Genes Dev. 20(18):2580-92.

Nathan D, Ingvarsdottir K, Sterner DE, Bylebyl GR, Dokmanovic M, Dorsey JA, Whelan KA, Krsmanovic M, Lane WS, Meluh PB, Johnson ES, Berger SL. (2006) Histone sumoylation is a negative regulator in Saccharomyces cerevisiae and shows dynamic interplay with positive-acting histone modifications. Genes Dev. 20(8):966-76.


Huang J, Kent JR, Placek B, Whelan KA, Hollow CM, Zeng PY, Fraser NW, Berger SL. (2006) Trimethylation of histone H3 lysine 4 by Set1 in the lytic infection of human herpes simplex virus 1. J Virol. 80(12):5740-6.

Emre NC, Ingvarsdottir K, Wyce A, Wood A, Krogan NJ, Henry KW, Li K, Marmorstein R, Greenblatt JF, Shilatifard A, Berger SL. (2005) Maintenance of low histone ubiquitylation by Ubp10 correlates with telomere-proximal Sir2 association and gene silencing. Mol Cell. 17(4):585-94.

Lo WS, Gamache ER, Henry KW, Yang D, Pillus L, Berger SL. (2005) Histone H3 phosphorylation can promote TBP recruitment through distinct promoter-specific mechanisms. EMBO J.24(5):997-1008.