The overriding objective of the Druggable Mechanisms Core (DMC) is to provide state-of-the-art technologies in high-throughput chromafin-immunoprecipitafion sequencing (ChlP-seq), RNA sequencing (RNA-seq), and mass spectrometry-based proteomics to each Project for in-depth characterization of transcription factor protein complexes and their respective genetic targets. Data from the DMC will reveal interacting protein partners for Oligl (Project 1), Runxl (Project 2), and NPAS4 (Project 3), characterize associated post-translational modifications, identify downstream genetic targets, and finally quantify the coordinate action of these protein-protein and protein-DNA interactions with respect to transcription of respective target genes. The comprehensive view of molecular interactions provided by the DMC will encompass both direct and surrogate therapeutic entry points for neuropathologies associated with Oligl, Runxl, and NPAS4. Over fime the synergy established between the DMC and Projects 1-3 may provide an experimental paradigm for transcription factor-directed drug discovery.
The specific aims of the DMC are: 1.) Implement innovative mass spectrometry-based proteomics techniques for analysis of protein complexes. We will implement novel proteomics methods to improve dynamic range of protein and pepfide identificafion in conjuncfion with tandem immunoaffinity purificafion of protein complexes. These will include solid-phase capture of cysteine-containing peptides combined with stable isotope labeling for quantitative proteomics, and a multidimension separation strategy based on a combination of reversed phase resins (RP/RP). 2.) Develop ChlP-seq and RNA-seq libraries and optimize sequencing on an Applied Biosystems SOLID genome analyzer. The goal of this aim will be to develop reliable ChlP-seq and RNA-seq library construcfion protocols. Each project will require short read sequencing, which we will perform on our SOLiD genome analyzer. Continuous development of in-house protocols in consultafion with AppliedBiosystems will ensure efficient and rapid sequencing on our SOLiD instrument. 3.) Deploy web-based, bioinformatic tools for integrated analysis of ChIP- an RNA-seq data and for comprehensive, user-driven analysis of proteomics data. The goal of this aim is to deploy a web-based system that will provide personnel in Projects 1-3 with an interactive, comprehensive, and dynamic computational platform for interrogation of their genomic and proteomic data, and extension of their observations to the level of pathways and networks.

Public Health Relevance

The Druggable Mechanisms Core (DMC) will provide systems-level data for the transcripfion factors Oligl, Runxl, and NPAS4. Detailed understanding of the numerous molecular interactions surrounding these transcription factors may reveal direct and surrogate therapeutic targets for various neuropathologies.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Program Projects (P01)
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National Institute of Neurological Disorders and Stroke Initial Review Group (NSD)
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Dana-Farber Cancer Institute
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