Alexander E. Urban, Ph.D. - US grants

DESCRIPTION (Provided by the applicant) Abstract: I propose to study through which molecular mechanisms of genetic control it is that large genomic Copy Number Variants (CNVs) exert their effects on gene regulation, dynamically over cellular differentiation and cellular functioning. We aim to establish a novel research program and paradigm in our laboratory that combines the use of induced Pluripotent Stem Cells (iPSC) and multi-level, comprehensive and integrated analysis of genomic and epigenomic layers of control and activity. In the process we will be testing several novel hypotheses on the molecular mechanisms through which CNVs affect gene regulation and thus cellular and eventually organismal phenotypes. This will also serve as a model for smaller CNVs and their potentially subtle and cumulative or combinatorial yet potentially substantive effects on phenotype in health and disease. Copy Number Variation (CNV) is common in the genome of healthy humans and is associated with phenotypic variation. CNV is also frequently and strongly associated with major disease phenotypes, especially in neuropsychiatric diseases that involve an aberrant development of the brain such as schizophrenia and autism. There are now several prominent examples for CNVs that are strongly associated with neuropsychiatric disorders such as schizophrenia, autism, mental retardation and epilepsy (22q11.2 deletions, 1q21.1 deletions and duplications, 15q13.3 deletions, 16p11.2 deletions and duplications, 3q29 microduplications). CNVs (and sometimes the same CNVs as mentioned above) are also strongly associated with malformation diseases of the heart as well as in phenotypes that involve the functioning of the immune system. CNVs are therefore an important phenomenon to study both in its own right as a strong predisposing factor for disease as well as an enticing point of entry for the better understanding of the molecular etiology of complex diseases with a strong and complex genetic and genomic component. A major barrier to a better understanding of how the molecular mechanisms through which CNV affect phenotype is the lack of access to relevant human tissues that carry a given disease associated CNV, for example neuronal tissue cultures with a large genomic deletion that is strongly associated with schizophrenia or autism. The use of iPSC lines from probands with a given CNV promises to overcome this barrier and will allow us to observe the effects of CNVs on a molecular level in the relevant tissue as it progresses along a developmental trajectory and then settles into the neuronal phenotype. The complete and multilevel high-resolution genomics and epigenomics analyses based on massively parallel DNA next-generation-sequencing will include comprehensive transcriptome analyses by RNA-Seq, meDNA- Seq to study DNA methylation patterns and ChIP-Seq to map regulatory histone modifications and transcription factor networks. Public Health Relevance: Neurodevelopmental, neuropsychiatric disorders such as schizophrenia and autism spectrum disorders are a major public health concern. Copy Number Variations are amongst the candidate loci with the strongest association with such diseases but they typically affect multiple genes and only very little basic knowledge about their molecular mechanisms of action exists. Our project will create a new research paradigm into the effects of CNV during neuronal development by combining iPSC techniques with comprehensive nextgeneration genomics analyses.

DESCRIPTION (provided by applicant): Despite the rapidly increasing capacity to sequence human genomes, our incomplete ability to read and interpret the information content in genomes and epigenomes remain a central challenge. A comprehensive set of regulatory events across a genome - the regulome - is needed to make full use of genomic information, but is currently out of reach for practically all clinical applications and many biological systems The proposed Center will develop technologies that greatly increase the sensitivity, speed, and comprehensiveness of understanding genome regulation. We will develop new technologies to interrogate the transactions between the genome and regulatory factors, such as proteins and noncoding RNAs, and integrate variations in DNA sequences and chromatin states over time and across individuals. Novel molecular engineering and biosensor strategies are deployed to encapsulate the desired complex DNA transformations into the probe system, such that the probe system can be directly used on very small human clinical samples and capture genome-wide information in one or two steps. These technologies will be applied to clinical samples and workflows in real time to exercise their robustness and reveal for the first time epigenomic dynamics of human diseases during progression and treatment. These technologies will be broadly applicable to many biomedical investigations, and the Center will disseminate the technologies via training and diverse means.

Project Summary/Abstract After a century of debate about the fundamental nature of neuropsychiatric disorders, we know that genetics lie at their core, yet do not fully understand the critical underlying mechanisms of their disease-causing pathology. The overall goal of our proposal is the creation of comprehensive and integrated maps of chromatin accessibility, chromosome folding and transcriptional patterns, delineating regulatory regions in the genomes of key disease relevant anatomical regions of adult and fetal brains, in brains from patients with Schizophrenia, Autism Spectrum Disorder, Bipolar Affective Disorder and matched controls, and those with known CNVs (Copy-Number Variants) that may unmask regional or long-range targets of epigenomic regulatory interactions that may also be of great relevance in patients with the same clinical phenotype. We will use comprehensive and highly-resolving epigenomics assays, that were recently developed by us, and novel ways to integrate the data for the first time in neuropsychiatrically relevant brain tissues. We will generate comprehensive maps of the spectrum of organization and function of regulatory regions by integrating complementary techniques: single-cell ATAC-seq (scATAC-seq) to characterize chromatin openness and HiChIP to characterize long- range folding interactions of sorted neuronal and non-neuronal cells, both of which are coupled to single-cell RNA-seq and long-range RNA-seq for expression information, further complimented by information about transcription factors through proteomic analysis of nuclear fractions. These maps will then be combined with coding or non-coding/regulatory variants in the genomic sequence in the candidate regions and integrated into the overall PsychENCODE database, which will allow us to create and validate reference maps for epigenomic marks and interactions, determine aberrations to the reference state in patient tissue, and connect such aberrations to genetic disease loci as well as assemble such loci into disease pathways. This project will not only greatly expand our understanding of regulatory information encoded in the human genome and its impact on human brain development and neuropsychiatric disorders, but also produce the bioinformatics tools necessary to analyze the complex data being generated in PsychENCODE.