Lorraine Pillus - US grants

Dr. Lorraine Pillus has provided general support for her studies of positional control of gene expression in yeast. Her research program is directed toward understanding the molecular basis for control of gene expression and cell identity. She is using the combined techniques of biochemistry, cell biology and genetics to examine the assembly and inheritance of transcriptionally repressed states of cell type information. In addition, she is attempting to identify functional correlates of the yeast genes in vertebrates using a yeast assay system, as well as identifying signals and targets in yeast controlling the germination of yeast spores. %%% The ultimate goal of this research is to understand molecular mechanisms for establishing and maintaining transcriptionally inactive regions in the chromosomes of dividing cells. Because the position effect that is the focus of these experiments involves a heterochromatic structure of chromatin, the results of these studies will bear on the genetic control of imprinting and on the structure of eukaryotic chromosomes.

The proposed project's goals are to understand the function of the newly identified SAS gene family in chromatin-mediated transcriptional regulation and genomic silencing. A sequence signature shared by Sas proteins and enzymes known to have acetyltransferase activity suggests that the Sas proteins function by acetylating key chromatin components. SAS genes were discovered in the yeast Saccharomyces, but sequence homologs including human MOZ and Tip60, have significant implications for human health. MOZ is the common 5' partner in recurrent 8p11 translocations that lead to the M4/M5 subtype of acute myeloid leukemia. Tip60 is a human gene associated with HIV-Tat that facilitates increased levels of Tat-dependent transcription. Understanding the most conserved elements of SAS function may thus lead to increased understanding of leukemia and HIV related disease including AIDS and AIDS-related malignancies. Analysis of yeast SAS function revealed that SAS2 or SAS3 mutants are defective in transcriptional silencing. The third yeast gene, ESA1, is essential for viability. Because ESA1 is most closely related to the human homologs, much of the proposal focuses on its analysis. Conditional alleles, including mutations in the putative acetyltransferase domain, will identify critical regions of ESA 1. Analysis of conditional alleles will facilitate the proposed genetic and cell biological dissection of ESA 1. It will be determined if ESA 1 is required at a single or multiple points in the cell cycle and if loss of ESA1 function leads to silencing defects, thereby potentially identifying genetic loci whose repression is essential for normal mitotic growth. Biochemical characterization will test directly the hypothesis that SAS genes function through chromatin acetylation. Identification of relevant substrates and regulators of activity will be sought through biochemical and genetic approaches. Tests to determine limits of functional conservation between yeast and human SAS genes will be performed by determining if human genes can suppress yeast mutant phenotypes. Experiments will be performed to test the hypothesis that mis-localization of SAS activity may lead to disease by altering locally defined patterns of transcriptional regulation. Results from these diverse experimental approaches should establish mechanisms of SAS gene function and suggest how disruption of this function leads to alterations in genomic silencing and activation.

This grant will provide partial support for the sixth consecutive FASEB Summer Research Conference on "Chromatin and Transcription," to be held on July 7-12, 2001, at the Conference Center in Snowmass Village, Colorado. The goal of this highly successful series of meetings is to foster interactions between researchers who study DNA-templated processes with individuals who study chromatin structure and organization. These disciplines are moving together at a rapid pace and are providing much new information for the basic biological sciences. The meeting's central topic is the relationship between chromatin and transcription. The regulation of chromatin impacts other critical biological areas; for instance, there are important links between chromatin and nuclear structure, chromosome organization, and the cell cycle, particularly relating to recent discoveries about histone modification. Moreover, questions of genomic stability and instability, which are relevant to genetic disease, will be addressed in a session on developmental/epigenetic regulation. The timing and location of the 2001 conference are ideal for productive and interactive discussions about data in these rapidly evolving areas. NSF support will be used to help new and young investigators to attend the meeting and present their results in an oral format.

DESCRIPTION (provided by applicant): Transcriptional regulatory mechanisms are critical for normal growth control and development. In recent years, key elements of transcription have been recognized to be mediated by chromatin modification, often through large macromolecular complexes which combine modifying enzymes with the basic transcriptional machinery. One major form of chromatin modification is histone acetylation. Disruption of regulated chromatin acetylation and deacetylation has been increasingly correlated with loss of normal metabolism, growth control, and development of cancer. Recognition that the SIR2/HST gene family encodes a chromatin acetylation modifying activity places these genes even more centrally in regulatory circuits for genomic control. The SIR2/HST family of genes is perhaps the most broadly conserved family regulating chromatin and gene expression and includes members in the Archaea, the eubacteria, and in all eukaryotes examined. Progress toward understanding the mechanism of function of Sir2p and its homologues came from the recent discovery that these proteins have intrinsic NAD-dependent protein deacetylase (NAD-DAC) activity. This activity provides important insight into chromatin and chromosomal control, however, it also raises a number of questions, the answers to which are critical for understanding the biological function, specificity, and regulation of this newly defined class of enzymes. Experiments to address these questions are the foundation for the proposed research. The long-term goal of our research is to understand the in vivo regulation, targeting and specificity of the SJR2/HST family of NAD-DACs. This goal will be approached through three specific aims: 1. To define the enzymatic activity of Sir2 and the Hst proteins. 2. To establish a molecular definition of the macromolecular complexes through which the enzymes act. 3. To evaluate the functional significance of the NAD-DAC complexes and their genomic targeting. These aims will be accomplished through a combination of molecular genetic, cell biological and biochemical approaches that will be extended with emerging microarray technologies.

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of this research is to understand mechanisms by which chromatin modifications regulate gene expression and chromosomal structure and function. The project centers on the MYST family of acetyltransferases that is implicated in normal growth and development and in conditions as diverse as leukemia, HIV-infection, and Alzheimer's disease. The proposed experiments build on results demonstrating that MYST proteins function in both transcriptional activation and silencing and have critical roles in DNA damage repair, cell cycle control, stationary phase survival, and chromosomal and subnuclear architecture. Previous studies focused on the three MYST genes, ESA1, SAS2 and SAS3. Planned studies will test the hypothesis that distinct roles for the MYST enzymes are specified through genomic targeting, mediated by interacting proteins and coordination with other chromatin modifying proteins. Transcriptional assays, affinity assays, mutational analysis, chromatin immunoprecipitation and microarray experiments will be used in these tests. Genetic and physical interactions that contribute to the distinct functions of SAS3 and ESA1 will be identified. Mechanisms of cell cycle control and DNA damage for SAS3 and ESA1 will be pursued to evaluate transcriptional vs. structural effects. These experiments will monitor checkpoint functions and will validate and extend microarray data. Cross-complementation experiments will be performed to address the specificity and extent of functional conservation of human MYST proteins. Preferred histone substrates have been identified for the yeast MYST enzymes, yet non-histone substrates are likely to be key to diverse MYST functions. Proteomic approaches will identify non-histone substrates, the significance of which will be evaluated through combined biochemical and genetic approaches. [unreadable] [unreadable]

Project Summary (ABSTRACT) Chromosomal functions are regulated by dynamic modifications of their structural proteins. Acetylation and deacetylation are well-established modifications that affect how histones and other chromosomal proteins influence transcription, recombination, replication and repair of damage. A distinct, newly defined activity is SUMO-targeted ubiquitin ligation (STUbL), catalyzed by proteins previously identified for their roles in genome stability and in response to DNA. The goals of the research are to define the mechanisms by which chromatin deacetylation and STUbL together yield optimal transcriptional silencing, genome stability and growth regulation. The proposed research builds on the lab's recent discovery that the Sir2 deacetylase is physically and functionally linked to a STUbL activity catalyzed by the Slx5-Slx8 complex. The project will be accomplished through three aims. In the first aim, genetic and biochemical studies will test the hypothesis that a second predicted STUbL protein functions in parallel to SLX5-SLX8 to promote optimal structure and function through STUbL activity. Transcriptional silencing defects of mutants will be characterized through molecular and genetic approaches, including genetic analysis and chromatin immunoprecipitation (ChIP). In the second aim, the genomic and subnuclear localization of STUbL components will be evaluated. Genomic and cell biological experiments will test if STUbLs occupy silent chromatin and define a distinct genomic binding pattern and subnuclear compartment(s). The third aim will define chromatin-specific substrates of STUbL activity through biochemical approaches. A combination of candidate substrate and proteomic analyses will be used. Potential substrates will be independently validated through additional biochemical and molecular genetic approaches. It will be determined if Sir2 deacetylase activity influences STUbL activity or substrate specificity. Together, the results from these three aims will establish the key mechanisms, substrates, and genomic targets of STUbL that are critical for chromatin function.

DESCRIPTION (provided by applicant): The goal of this project is to develop platform technology that measures how the epigenome influences transcription factor binding and gene activation at a controlled promoter sequence as the chromosome location is varied. This platform is built around the idea that the value of the existing yeast deletion libraries (Yeast Knock Outs, YKO) can be used for position-effect experiments to measure epigenetic regulation on a genome- wide scale. The availability of thousands of isogenic strains, each of which differs only in the position of a single gene cassette at a distinct chromosomal locus, provides a controlled genetic marker that can be assayed to understand how epigenetic features at different positions influence transcription. YKO libraries will be used in conjunction with molecular barcode microarrays, ChIP-chip, and pooled transformation technologies to measure position effects on transcription factor binding, gene expression, and homologous recombination. The NIH has previously funded each of these individual technologies for functional genomics research. Here, we propose to give them new value by leveraging them for epigenetic studies. Three technologies are proposed: 1) A molecular barcode immunoprecipitation on microchip (BIP-chip) assay that measures genome-wide position effects on binding between a DNA-binding protein and a controlled promoter sequence. Development will involve combining elements of traditional and histone ChIP-chip with elements of quantitative barcode microarrays. 2) Quantitative RT-PCR of the expression level of the kanMX gene as its chromosomal location is varied. Reverse transcription and quantitative PCR will be used to quantify the expression level of kanMX in individual yeast strains in the YKO library covering all of yeast chromosome I. 3) En masse integrative transformation of custom promoter sequences into pooled yeast cultures. Methods for performing pooled transformations developed for diploid-based Synthetic Lethal Analysis by Microarray (dSLAM) will be adapted to investigate position effects on homologous recombination and to enable the use of BIP-chip for evaluating epigenetic effects on any transcription factor (TF). Together, the development of these technologies will enable future research to directly measure the effects of epigenetic regulation on DNA binding and gene activation for any TF-promoter combination of interest. PUBLIC HEALTH RELEVANCE: Epigenetics contribute to critical cellular functions and pathologies ranging from gene silencing and DNA repair to tissue-specific gene expression, cell differentiation, carcinogenesis, and aging. Throughout development, differentiating cells accumulate epigenetic instructions that ultimately determine fully differentiated patterns of expression. Many developmental syndromes, and specific disease phenotypes, including cancer, stem from fundamental epigenetic changes that inactivate critical genes or activate disruptive genes. Identifying disease- causing epigenetic changes and finding ways to mitigate, alter, or reverse deleterious ones will be the subject of biomedical research for the foreseeable future. Combining reference epigenome maps with TF-epigenome interaction measurements, as proposed in this study, will enable researchers to specifically pinpoint epigenetic changes that induce altered TF binding behavior and contribute to developmental defects and disease.

The 23 pairs of chromosomes that carry human genetic information contain both DNA and proteins that package the genome to protect it and regulate its use within cells. This protein-DNA material, known as chromatin, and the complex linear chromosomes, are very different than genetic packaging in simpler organisms like bacteria, which ordinarily carry their genomes on a single, circular molecule. It has become increasingly clear that chromatin-based processes are deeply tied to ancient metabolic activities. Understanding these links, including defining novel, unsuspected functions of metabolic enzymes, holds the promise of revealing critical aspects of the evolution of genomic processes and stability. Major impact of the project comes from the training opportunity provided for the students and fellows performing the research. They will participate in frontline interdisciplinary research to foster both experimental and computational skills. Importantly, the students and fellows will contribute to the lab's work to promote diversity in science through mentoring high school and undergraduate students identified through outreach programs, including students transferring to UCSD from the community college system, a population with a significant component of first-generation and under-represented college students. The participation of these students in a mentored and meaningful research program will contribute to the development of a more highly trained and diverse scientific community for the 21st century.


The long-term goal of the proposed research is to define the chromatin-directed functions of metabolic enzymes with previously unsuspected functions in epigenetic transcriptional silencing and DNA damage repair. These studies build upon progress with the model eukaryote, Saccharomyces cerevisiae in which, in proof of principle studies, the investigators discovered previously unsuspected nuclear roles for two enzymes with earlier defined functions in amino acid metabolism. The objectives for the research are to define chromatin-directed functions of a panel of newly identified metabolic enzymes with nuclear roles, using the strategies that the investigators have successfully developed. By probing long known but incompletely understood metabolic enzymes, the progress made in this research will be of fundamental significance defining proteins that have contributed to the molecular and cellular evolution of nuclear processes in the course of genomic evolution. Significantly, study of these enzymes will establish a framework defining the interplay between metabolism and chromatin functions that are critical for gene regulation, response to DNA damage, and maintenance of genome stability. The project employs a combination of classical functional studies, genome-scale analysis and newly developed microfluidics and single-cell imaging technologies that enable visualization of the silencing dynamics in single cells.

Project Summary Cellular aging is a complex biological process, associated with many diseases, such as cancer, diabetes, and neurodegenerative diseases. New therapeutic approaches to slow aging hold promise for reducing global healthcare burdens of chronic diseases. However, the development of these approaches requires a deep understanding of the mechanisms of aging, which remains a challenging goal. Static population-based studies are insufficient to reveal sophisticated molecular mechanisms that underlie the aging process, because genetically identical cells have various intrinsic causes of aging and widely different rates of aging. Furthermore, although many single genes have profound effects on lifespan, how they interact and function within gene regulatory networks to regulate aging and how these interactions change dynamically during aging remain largely unknown. To overcome these challenges, we have developed high-throughput microfluidic technologies to track the dynamics of molecular processes throughout the replicative lifespans of single S.cerevisiae cells. In the proposed research, these dynamic measurement technologies will be integrated with computational modeling to systematically characterize and quantify the collective dynamic behaviors of aging-related molecular networks. In Aim 1, we will quantitatively characterize the phenotypic changes associated with distinct causes of cell aging and, based on these data, construct a phenomenological model of the aging process, upon which we will build mechanistic models of the conserved Sir2 and protein kinase A (PKA)-regulated molecular networks, both of which are deeply connected to aging. In particular, in Aim 2, we will develop a mechanistic model of the Sir2-regulated molecular network to predict its dynamics and regulatory roles during aging. High-throughout single-cell analysis will be performed to track the dynamics of Sir2-regulated genes and test the model predictions. In Aim 3, we will systematically characterize the PKA- regulated stress response during aging and develop a mechanistic model to quantify and predict the effects of environmental cues on aging. We will systematically examine the dynamics and contribution of stress response genes under various environmental perturbations. These experimental measurements will be used to test the predictions, refine the model, and more importantly, provide insight into the basic mechanisms underlying the environmental control of aging. To accomplish these aims, we have assembled a strong interdisciplinary team of investigators with complementary expertise, who will work synergistically to tackle fundamental questions in the biology of aging from a systems biology perspective. !