Lucio Comai - US grants

DESCRIPTION (adapted from the applicant's abstract): Simian virus 40 (SV40) large T antigen is a multifunctional regulatory protein that plays a key role in the viral life cycle. In addition, large T antigen immortalizes primary cells, and induces cell transformation and tumor formation in animals. To accomplish these functions, large T antigen has to alter the cellular mechanisms that control the expression of genes involved in cell cycle progression and cell proliferation. Ribosomal RNA synthesis by RNA polymerase I (pol I) is tightly associated with cell growth and proliferation, and previous studies demonstrated that large T antigen up-regulates RNA pol I transcription in SV40-infected cells. To better understand how large T antigen stimulates RNA pol I transcription, he has established an in vitro transcription system that responds to large T antigen. During these studies, his laboratory has discovered that the transcription factor SL1 is one of the cellular components of the pol I transcriptional machinery targeted by large T antigen. In addition, these studies suggested that, in part, the phosphorylation of UBF by a still unidentified T antigen-associated kinase, may also contribute to the overall stimulatory process. In this grant proposal, using a biochemical approach, the PI proposed to dissect the mechanism of regulation by RNA pol I transcription by large T antigen. The first goal is to understand the role of large T antigen-SL1 interaction in the stimulatory process. For this purpose, he will analyze the effects of deletion and single point mutations in large T antigen in a variety of in vivo and in vitro protein-protein interaction assays and trancription assays, and correlate the ability to bind to SL1 with the transcriptional stimulation. Second, he proposes to use footprinting and EMSA assays to test whether these protein-protein interactions may potentiate functional interactions between the transcription factors and facilitate the formation of the transcription initiation complex. Interestingly, his preliminary studies indicate that the region of large T antigen necessary for pol I stimulation overlaps with the domain involved in the binding and inactivation of p53. Thus, he has designed a set of experiments to determine the potential functional link between the tumor suppressor p53 and large T antigen stimulation of pol I transcription. Finally, he will address the significance and the role of UBF phosphorylation in the T antigen-mediated activation process and characterize the associated-protein kinase activity. These studies will provide new insights on the molecular mechanisms of mammalian gene regulation and will unravel some of the strategies adopted by small DNA viruses such as SV40 to alter the regulation of key cellular processes.

DESCRIPTION (provided by applicant): Werner syndrome (WS) is an autosomal recessive disorder leading to premature onset aging and aging-related diseases including cancer and atherosclerosis. WS results from the loss of function of the WRN gene. The WRN gene encodes a RecQ helicase protein with a unique exonuclease activity (WRN) whose cellular function is poorly understood. Cells from WS patients demonstrate premature senescence and sensitivity to DNA damaging agents such as camptothecin (CPT). Importantly, we have shown that WRN binds to Ku70/80, a heterodimeric complex known to play a critical role in the repair of DNA damage. This observation strongly supports the idea that WRN functions in a DNA damage response pathway. We therefore hypothesize that WRN is required for S-phase checkpoint activation or is directly involved in the repair of DNA lesions following exposure of cells to CPT. Specifically, in Aim I we will test whether loss of WRN leads to defective S-phase checkpoint controls in CPT-treated cells. Experiments proposed in Aim 2 will study the dynamics of the recruitment of WRN and its associated factors to chromatin in response to CPT-induced DNA damage. In Aim 3, we will test the hypothesis that loss of WRN results in genetic instability at the rDNA locus leading to aberrant ribosomal RNAs biosynthesis upon DNA damage. In Aim 4, biochemical insights into these processes will be obtained by studying the response to CPT-induced DNA damage of cells expressing mutant WRN proteins deficient in exonuclease or helicase activity, or lacking conserved structural domains. Taken together, these experiments should provide important mechanistic insights into the process of human aging.

DESCRIPTION (provided by applicant): The broad objective of this project is to analyze at the molecular level the regulatory mechanisms of altered RNA splicing that are controlled by the formation of pathological MBNL1 mega-complexes in myotonic dystrophy 1 (DM1) patient cells. The genetic defect in DM1 results in the production of mutant RNAs encoding expanded CUG tracts. Abnormally expanded CUG tracts have been shown to form aberrant mega-complexes that contain the alternative splice factor, MBNL1, within the nucleus. Several lines of evidence implicate the formation of these high molecular weight complexes in altered splicing of a subset of physiologically important RNAs and in the subsequent development of DM1 pathology in vivo. To determine the mechanism whereby formation of the MBNL1 mega-complexes alters the splice code in DM1 we propose to purify both normal MBNL1 complexes and the aberrant MBNL1 mega-complexes that develop in DM1 myoblasts. In complementary experiments the role of these complexes in dictating RNA splice site choice will be defined. The Aims of this application are: 1. Purification and functional characterization of normal MBNL1 complexes in spliceosome assembly and RNA catalysis. 2. Purification of MBNL1 mega-complexes from DM1 myoblasts and definition of the mechanics of mega-complex formation in vivo. 3. Elucidation of the mechanisms by which formation of MBNL1 mega-complexes alters the splice code in DM1 myoblasts.

DESCRIPTION (provided by applicant): The goal of this study is to gain mechanistic insights on the function of the Werner syndrome protein (WRN) complex in the processes that maintain telomere integrity and prevent the formation of extrachromosomal telomeric circles. WRN is a nuclear protein with helicase and exonuclease activities, whose loss-of-function mutations are associated with the premature aging and cancer prone disease Werner syndrome (WS). Genetic and biochemical evidence implicate WRN in telomere metabolism and suggest that abnormal telomere length homeostasis contributes to the pathology of WS. Our biochemical studies have shown that WRN operates as a functional unit with the Ku70/80 heterodimer and have further demonstrated that loss of WRN function causes the production of telomeric circles in fibroblasts expressing telomerase. To determine the mechanism whereby the WRN complex regulates telomere homeostasis, we propose to mechanistically characterize the functional interplay between WRN, Ku and telomere-specific factors found at chromosome ends and define the process activated by loss of WRN function responsible for the formation of telomeric circles. To accomplish these objectives we propose the following three aims. In Aim 1, we will characterize the role of WRN and its interacting partner Ku70/80 in the regulation of telomeric termini in human cells. In Aim 2, we will dissect the molecular mechanisms leading to the formation of extrachromosomal t- circles resulting from loss of WRN function. In Aim 3, we will characterize the biochemical properties of WRN in the context of model telomeric substrates in vitro. The studies proposed in this application will elucidate the role of WRN at telomeres and provide valuable information for understanding how loss of WRN function promotes genome instability, premature aging and the early onset of diseases such as cancer and cardiovascular disease. It is recognized that the incidence of these diseases increases progressively with age. Therefore, the changes at the molecular, cellular and physiologic levels that occur during aging profoundly influence the development and progression of these diseases. Functional analysis of WRN will provide a clearer understanding of these changes and will help in the development of therapeutic agents aimed at preventing the early onset of age-associated diseases.

DESCRIPTION (provided by applicant): Myotonic dystrophy (DM1) is the most common adult onset muscular dystrophy in humans. Currently, there is no cure or an FDA approved drug for DM1 and related diseases. DM1 is an autosomal dominant disorder resulting from the expansion of a CTG-repeat sequence in the 3'untranslated region of the DMPK gene. This defect results in the expression of mutant DMPK RNAs encoding expanded CUG repeats (CUGexp) that form large intra nuclear RNA-protein complexes or foci. Expression of CUGexp RNAs leads to abnormal RNA splicing, which in turn has been linked to the development of key features of DM1 pathology. We hypothesize that small molecules that degrade or disperse CUGexp RNAs in DM1 cells can re-establish normal splice patterns and reverse DM1 pathology. To test this hypothesis, we have developed a primary HTS and a secondary hit validation assay to identify small-molecules that selectively alter the biology of DMPK CUGexp RNAs without affecting the normal DMPK transcripts. Our in house library was developed using a robust machine learning chemoinformatics platform and consists of 40,000 highly diverse small-molecules representing a library of several million compounds. Preliminary results obtained from a screen of 2,500 compounds demonstrate that our strategy allows the rapid identification of potent molecules that successfully reverse DM1 pathology in both patient cells and DM1 mouse models. In a concerted effort to identify a set of potent lead compounds that can be developed as a therapeutic cocktail for DM1 we propose the following Aims: Aim 1. Implement primary HTS and the secondary hit validation assay to screen 20,000 molecules from our in-house library. Aim 2. Test hits in tertiary DM1 patient cell-based assays to identify highly potent leads that reverse five key cellular DM1 phenotypes. Selectivity, toxicity and synergy of leads will be measured in parallel. Aim 3. The chemical structure of lead compounds will be reiteratively refined to optimize pharmacological properties and establish structure-activity relationships. PUBLIC HEALTH RELEVANCE: We have developed a HTS screen to identify compounds that alter the biology of toxic CUGexp RNAs in myotonic dystrophy 1 (DM1). This screen will be used to identify therapeutic compounds that can be used to treat DM1.

Large expansion of CTG trinucleotide repeats in the 3? untranslated region of the DMPK gene cause myotonic dystrophy type 1 (DM1), the most common form of adult muscular dystrophy. Toxic CUG repeats-containing RNAs transcribed from the DMPK gene sequester and disable RNA binding proteins that are critical for cell function. At present, there is no therapeutic approach to reduce disease severity or delay disease onset. Several labs are considering approaches aimed at neutralizing the toxic RNA. These could provide short-term effects, unless continuously applied. A strategy that induces the contraction or deletion of the expanded CTG repeats could provide an alternative approach to permanently eliminate the production of the toxic RNA. Expanded CTG repeats have the potential to form hairpins structures in vitro and a mechanism that involves the failure to resolve secondary structures that form sporadically during lagging strand synthesis has been proposed to explain trinucleotide instability. Several lines of evidence suggest that specialized helicases can either facilitate or antagonize expansion of repeat sequences. However, to what extent and under what circumstances specific helicases contribute to repeat stability in vivo is not known. The WRN protein is a member of the RecQ family of helicases that has been shown to resolve secondary structures formed by repetitive G-rich sequences, and in vitro studies from our and other labs have indicated that WRN prevents stalling of the replicative polymerase at these repetitive sequences. To determine whether WRN influences the stability of expanded CTG repeats in vivo, we crossed Wrn knock-out mice with transgenic mice bearing expanded CTG repeats that recapitulate DM1-specific skeletal muscle pathologies. Our preliminary data indicate that Wrn deficiency results in the stochastic loss of the expanded repeats, suggesting that this helicase contributes to the maintenance of long pathogenic CTG repeats in vivo. To gain critical insights on the relationship between WRN and expanded CTG repeats stability and explore the therapeutic potential of WRN inhibition for DM1, we propose to characterize CTG instability in Wrn deficient mice, assess the effects of Wrn deficiency on the skeletal muscle phenotype of DM1 mice, and gain insights on factors that influence CTG repeat instability resulting from WRN deficiency. These studies will reveal the potential value of approaches that target WRN for developing novel therapeutic strategies for DM1.

Nucleotide repeat expansion mutations cause a variety of genetic disorders including myotonic dystrophy (DM) types 1 and 2, and a large fraction of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). We developed a cell-based screen and secondary validation assays to identify small molecules for Dm1. We screened a diverse collection of small molecules and identified 60 novel hit compounds (myotonic dystrophy inhibitors-MDIs) that in secondary assays selectively disperse expanded CUG repeat- containing RNAs prone to form nuclear aggregates and sequester critical RNA-binding proteins in DM1 patient?s derived cells. Significantly, we showed that these compounds also dissipate expanded CCUG repeat-containing RNA foci in DM2 patient?s derived cells, suggesting that they may hold therapeutic potential across distinct GC-rich repeat expansion diseases. Building on the knowledge gained in our DM1 project, we have designed a screening strategy to identify potential small molecule therapeutics for FTD/ALS caused by an hexanucleotide expansion in the C9orf72 gene (C9FTD/ALS). In this project, wepropose to develop, optimize and validate a cell-based assay platform to measure critical molecular parameters of C9FTD/ALS disease. Next, we will use this platform to test whether compounds that affect the stability of toxic CUG RNA foci can disperse expanded G4C2 repeat RNA foci and decrease dipeptide proteins accumulation, two causes of toxicity in C9FTD/ALS patient?s derived cells. Lastly, we will perform a proof-of-concept screen of a diverse custom set of small-molecule compounds to best assess the value and efficacy of our screening platform to identify potential therapeutic agents for C9FTD/ALS. As there is no effective treatment for FTD or ALS, the identification of small molecules of potential therapeutic value offers hope for individuals with these debilitating and fatal diseases.