Yin Shen - US grants

Mutations in gene regulatory elements are a major cause of human disease. The ENCODE, Epigenome Roadmap and other projects have identified millions of putative regulatory elements across more than one hundred cell types and tissues. While these maps have significantly expanded our knowledge of regulatory sequences, they are only descriptive and further high-throughput functional assays are needed in order to understand the biology of these elements. In addition, with whole-genomes on the verge of being commonly available, there is a pressing need to develop high-throughput assays that can rapidly analyze the functional effect of the thousands of variants detected in these genomes. Massively parallel reporter assays (MPRAs) provide such a technique, enabling the testing of thousands of sequences and their variants for reporter activity. However, they currently have several caveats. These include amongst others the inability to test long DNA sequences and the majority of MPRAs testing sequences in an episomal manner and alongside a minimal promoter instead of the target gene promoter. Here, we will develop a novel MPRA technology that will address all of these caveats. Using capture Hi-C technology that allows for the hybridization of regulatory elements to their target promoter, we will generate an MPRA library that encompasses long regulatory sequences cloned in front of their target promoters. Using a lentivirus based MPRA (lentiMPRA) method that we developed, we will test these sequences in a genome integrated manner. To functionally validate that there are indeed differences between a minimal promoter versus a target promoter based MPRA library, we will compare similar sequences in both contexts. Finally, we will also dissect enhancer-promoter interactions to pinpoint important sequences driving these interactions. The technology that we will develop will enable the testing of thousands of regulatory elements and their variants alongside their target promoter. As such, it will increase our understanding of regulatory element function and how gene regulatory elements communicate with their target promoter.

Project Summary/Abstract Alzheimer's disease (AD) is a devastating complex neurological degenerative disorder affecting 10% of people over 65 with no cure. The overarching goal of the proposed study is to identify and functionally characterize AD-associated SNPs utilizing novel functional genomic approaches and iPSC-derived cellular models. Our plans include: (1) Determine the functional significance of candidate SNPs in three iPSC-drived 2D AD relevant cell types. (2) Identify genes regulated by distal non-coding SNPs in three iPSC-drived 2D AD relevant cell types. (3) Test the biological consequences of high confidence AD rSNPs from (1) and (2) in isogenic iPSC- derived 2D cell cultures and 3D minibrain organoids. The designed study will be very first comprehensive investigation of AD associated SNPs, thus will shed light on how non-coding genetic variations contribute to AD. Obtaining knowledge for the fundamental genetic mechanisms of AD will expand our horizons to develop improved preventative and diagnostic methods, and also yield targets for novel therapeutic interventions, ultimately leading to a cure for AD.

Project Summary The overarching goal of the proposed study is to functionally characterize a large number of candidate functional elements in the mammalian genome. The ENCODE projects have revealed millions of putative regulatory elements across more than one hundred cell types and tissues. While these maps have significantly expanded our knowledge of non-coding sequences, there are still large gaps between having descriptive maps of functional elements and understanding the biology of these elements underlying gene regulation. These include: (a) few candidate functional elements predicted by the ENCODE experiments are functionally validated; (b) Epigenomic studies have not given/revealed information on the target genes of candidate functional elements. Therefore, it is still a challenge to interpret the biological functions of non-coding DNA sequences. To address these issues, the objective of this UM1 application is to perform large scale functional characterization of candidate functional elements in their native chromatin context. We will first identify candidate regulatory elements utilizing ENCODE data and generate reporter tagged genes of interest in cell lines utilizing a high throughput, automated platform. Second, we will interrogate candidate functional elements in their native chromatin contexts utilizing two complementary high throughput CRIPSR/Cas9 mediated genome editing approaches. We anticipate these analyses will significantly advance our knowledge of the biological functions of candidate regulatory regions and gene regulation in mammalian cells.

Abstract The overarching goal of this study is to understand how BEST1 expression is modulated by cis-regulatory elements in the retinal pigment epithelium (RPE). The variability of retinal phenotypes and age of onset of visual loss, even in individuals who carry the same causative mutation, is one of the biggest mysteries for BEST1 related diseases. Genetic variations in non-coding regulatory elements can serve as an attractive model to provide the molecular basis for the observed various onsets of BEST1 vitelliform macular dystrophy (BVMD) in patients. Therefore understanding the transcriptional control mediated by cis-regulatory elements in the RPE will give us insight into novel target regions for therapeutic editing of the enhancer elements identified by this study. We are utilizing human induced pluripotent stem cells (iPSCs)-derived RPEs as a model to understand cell type-specific transcriptional controls of BEST1. We are also utilizing integrative, unbiased, and high throughput epigenomic and genetic tools to achieve the following aims: (1) We will use a high resolution 4C-seq analysis to identify potential regulatory elements that interact with the BEST1 promoter in human primary RPE. (2) In Aim 2, we will generate BEST1 dual allele reporter lines and use these lines to interrogate cis-regulatory elements of BEST1 in their native chromatin contexts utilizing high throughput CRIPSR/Cas9 mediated genome editing approach. (3) Finally, we will test the utilities of allelic specific enhancer deletion to modulate allelic expression of BEST1. We expect these analyses will significantly advance our knowledge of the precise control of BEST1 transcription in RPEs.

Project Summary Cis-regulatory elements control Cell-type-specific gene regulation via looping with their targeting genes. Therefore, mapping the 3D chromatin interactions between promoters and cis-regulatory elements will be pivotal to understand the functions of regulatory regions. Besides, many genetic variants associated with neuropsychiatric diseases reside in the putative cis-regulatory elements. They may contribute to disease by affecting regulatory sequences function, but the exact mechanisms of how they contribute to diseases via gene regulation remain unknown. Here, we aim to make substantial advances in understanding how the 3D epigenome contributes to brain development and diseases by mapping and analyzing the dynamic changes during the human prefrontal cortex development. We will first map transcriptome, chromatin accessibility, and 3D chromatin loops in six distinct cell types from the developing prefrontal cortex and perform an integrative analysis to interrogate how chromatin interaction control gene expression and development. Second, we will integrate the 3D epigenomic datasets with medical genetics resources to gain insights into cell types, genomic loci, and biological pathways that are causal to diseases, link GWAS SNPs with their target genes. Third, to establish cell-type-specific functional links between chromatin loops and target gene expression, we will test the biological consequences of distal regulatory regions interacting with promoters in iPSC models and primary cells. Our project will reveal new insight into the biological functions of the 3D epigenome in brain development and diseases.