Andrew S. McCallion - US grants

[unreadable] DESCRIPTION (provided by applicant): Although frequently predicted to play critical roles in regulatory control, the nature and identity of functional noncoding sequences require comprehensive investigation. The inability to readily impute the functions of noncoding sequences, or the impact of variation therein significantly hampers attempts to investigate potential associations between noncoding variation and disease susceptibility. The purpose of this grant is to systematically examine the regulatory potential of conserved noncoding sequences at a single human disease locus, RET. RET is a critical gene in the genesis and maintenance of multiple organ systems and a major susceptibility locus in multiple human disorders. This proposal has 3 major aims. First, we will use computational, in vitro and molecular analyses to identify S60 conserved noncoding regulatory sequences at RET, and begin to define critical sequences therein. Second, we will determine the biologic relevance of 3 identified regulatory sequences using plasmid-based transgenesis in mice to examine their in vivo regulatory function. Third, we will specifically examine the disease relevance of a single identified regulatory noncoding sequence. We will delete the selected sequence from a wild-type human BAG encompassing RET and compare the ability of wild-type and mutant transgenic mouse strains to complement the Ret null phenotype in mice. This proposal will yield insights into the nature of functional noncoding sequences at RET and in doing so provide a foundation for increasingly comprehensive investigation of putatively functional sequences at this and other disease loci. The central aim of this proposal is to begin to uncover the nature and identity of regulatory sequences through the systematic implementation of functional genetic analyses at the RET locus. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This proposal builds upon our successes in the first funding cycle, and technological advances in in the wider scientific community. We will expand our understanding of the biology and sequence-based encryption of transcriptional regulatory instructions in clinically pertinent neuronal populations, focusing on tyrosine hydroxylase (Th)-expressing ventral midbrain neurons that are compromised in Parkinson's disease and certain behavioral and neuropsychiatric disorders. In recent years, we have made great strides in characterizing regulatory control at specific neurogenic loci by generating, validating and publicly depositing huge catalogs of neuronal enhancers. We have developed and implemented computational strategies that catalog key motif combinations that identify neuronal enhancers, and developed sequence- based vocabularies (classifiers) for neuroanatomical domains (forebrain, midbrain, hindbrain) among other more homogenous isolated cell populations. By integrating our experiences in functional and computational genomics we have been able to indict several disease-associated variants in pertinent biological processes. We are also beginning to develop the capacity to impute the functional impact of non-coding variation from primary sequence alone. Efforts to understand the architecture of human complex disease through Genome Wide Association Studies have drawn increased attention to potential roles played by regulatory variation. Thus, understanding the connections between regulatory variants and disease risk is very important. We propose detailed characterization of cell-type appropriate genome-wide regulatory sequence catalogs, isolating labeled dopaminergic neurons ex vivo at multiple time points (Aim 1). We will functionally validate the catalogs and define the sequence motifs that specify their function, developing computational classifiers to identify human DA enhancers, and assaying the functional impact of disease-associated variants therein (Aim 2). We will determine the relationship between distal-acting regulatory sequences and their cognate genes using cutting edge chromatin conformation capture (3C)-based strategies to reveal enhancers- promoter interactions. Then we will determine the consequences of deleting selected enhancers using contemporary genome editing strategies (Aim 3). This proposal takes crucial next steps towards a neuronal regulatory lexicon that can inform our observation of disease-associated variation in non-coding, putative regulatory sequence space.

? DESCRIPTION (provided by applicant) This proposal integrates and builds upon our combined experience and strengths, and on technological advances in in the wider scientific community. We will expand our understanding of the biology and sequence-based encryption of transcriptional regulatory instructions in clinically pertinent neuronal populations, focusing on neuronal populations implicated in Schizophrenia (SZ) and related neuropsychiatric disorders. In recent years, we have taken significant strides in characterizing regulatory control at specific neurogenic loci by generating, validating and publicly depositing huge catalogs of neuronal enhancers. We have developed and implemented computational strategies to identify key motif combinations that recognize neuronal enhancers, and used them to develop sequence-based vocabularies (classifiers) for neuroanatomical domains (forebrain, midbrain, hindbrain) and homogenous isolated cell populations. By integrating our experiences in functional and computational genetics and genomics we have already been able to indict several disease-associated variants in pertinent biological processes. Efforts to understand the architecture of human complex disease through Genome Wide Association Studies have drawn increased attention to potential roles played by regulatory variation. Thus, illuminating the connections between regulatory variants and disease risk is an important step towards understanding disease biology. We will systematically assay the regulatory potential of sequences encompassing disease-(SZ)- associated variation, determining the allele-specific activity of identified enhancers and determining their regulatory control by in vitro cellular assays and in whole organisms (Neuron-specific transgenic reporter zebrafish; (Aim 1). Further, to expand neuron-specific annotation to regulatory sequences genome-wide, we propose detailed characterization of cell-type appropriate genome-wide regulatory sequence catalogs, isolating labeled dopaminergic/Glutamatergic/GABAergic neurons ex vivo, functionally validating the catalogs and developing computational classifiers to identify human enhancers with activity in specific neuronal subtypes (Aim 2). Finally, we will determine the relationship between SZ-associated distal-acting regulatory sequences and their cognate genes using cutting edge Chromatin Conformation Capture (3C)-based strategies to reveal putative enhancers-promoter interactions, connect SZ-associated, non-coding loci with genes and determine the regulatory potential of distal sequences identified in this way (Aim 3). This proposal takes significant steps towards understanding neuropsychiatric disease by creating a neuronal regulatory lexicon that can inform our observation of disease-associated variation in non-coding, putative regulatory sequence space and by directly exploring the regulatory roles of SZ associated DNA variants.