2009 — 2013 |
Ahituv, Nadav Bejerano, Gill (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Characterization of Regulatory Elements Leading to Human Limb Malformations @ University of California, San Francisco
DESCRIPTION (provided by applicant): Characterization of regulatory elements leading to human limb malformations ABSTRACT Limb malformations are the second most common human congenital abnormality with a prevalence of 1 for every 500 births. Although several mutations in genes have been identified that explain syndromic forms (associated with other symptoms) of limb malformations, the characterization of mutations causing non- syndromic/isolated limb malformations has been less successful. A variety of molecular and clinical data suggests that mutations responsible for non-syndromic limb malformations may reside in distant noncoding regulatory sequences such as enhancers (sequences that regulate gene promoters). These data are based on position effects (chromosomal rearrangements that leave the gene intact but remove its regulatory elements) that lead to limb malformations, the observed modular nature of enhancers, and the recent example of a non- syndromic preaxial polydactyly in humans that has been linked to a long distance enhancer of the Sonic Hedgehog (SHH) gene. Long distance regulatory enhancers have traditionally been difficult to identify and very few of them have been characterized for limb regulatory expression thus far. In preliminary studies for this proposal, we have discovered 43 novel human limb enhancers using a mouse enhancer transgenic assay and verified several of them for pectoral fin expression in zebrafish. In order to discover additional human limb enhancers we are using advanced computational tools to dissect the unique sequence signatures in both the novel limb enhancers we discovered and previously reported ones. These signatures allow us to predict novel limb enhancers surrounding known limb-associated genes and throughout the human genome. These predicted limb enhancers will initially be tested in a high-throughput manner in zebrafish for fin expression. Positive fin enhancers will then be reverified in mice for limb expression. All characterized enhancers both in zebrafish and mouse will be available to the biomedical community through a web accessible browser. In addition, we have collected numerous DNA samples of patients with non-syndromic limb malformations and are in the process of collecting numerous more. We will conduct mutation analysis of these DNA samples within limb enhancers, and potential causative nucleotide changes will be tested for their effect on limb formation using the mouse as our model. The identification of causative sequences leading to non-syndromic limb malformations will result in improved patient counseling, the development of molecular testing including prenatal genetic testing, and an increased knowledge about the pathogenesis of human limb malformations and limb development. PUBLIC HEALTH RELEVANCE: Characterization of regulatory elements leading to human limb malformations Mutations in genes leading to limb malformations, the second most common human congenital abnormality with a prevalence of 1 for every 500 births, have been discovered on the majority in a syndromic form (associated with other symptoms). There is a variety of molecular and clinical data to suggest that non- syndromic (isolated) limb malformations can be caused by mutations in distant regulatory noncoding sequences (DNA switches that tell the genes when and where to turn on or off), but only a small number of limb regulatory noncoding sequences have been discovered thus far and only one of these sequences has been linked to human non-syndromic limb malformations. In this proposal we have discovered 43 novel human limb regulatory noncoding sequences and have collected numerous DNA samples from patients with non- syndromic limb malformations enabling us not only to screen these 43 DNA sequences for mutations in non- syndromic patients, but to also identify in a high-throughput manner using advanced computational tools and zebrafish and mouse assays, novel limb regulatory sequences throughout the human genome, which will make for additional limb malformation mutation candidates.
|
1 |
2009 — 2011 |
Ahituv, Nadav Bejerano, Gill [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Computational &Functional Annotation of the Zebrafish Genome Regulatory Toolbox
DESCRIPTION (provided by applicant): Zebrafish with its growing arsenal of tools that allow the generation of transgenics, gene knockdowns and knockouts, and mutant resources coupled with its high-throughput and cost efficiency is quickly becoming the major animal model for drug screens and gene related studies. However, as with other vertebrate genomes, the majority of the zebrafish genome (97%) is made up of non-genic sequences whose functional necessity remains largely unknown. One vital function that is clearly embedded in these regions is gene regulation, instructing genes when and where to turn on or off. However, unlike genes where we know their genomic location, their code, and the consequences of nucleotide changes within them, in gene regulatory sequences we don't have that knowledge. This knowledge is extremely vital, with a wide variety of clinical and molecular data supporting these sequences to be an important driver for development, evolution, diversity, and disease. In this proposal, we will combine advanced computational tools with high-throughput zebrafish functional studies to annotate this noncoding terrain. Using and refining multiple vertebrate genome alignments we have generated an unprecedented set of 166,693 zebrafish conserved noncoding elements (CNEs), with at least 8,805 regions having a direct ortholog in the human genome. Preliminary studies for a portion of these sequences using a zebrafish transgenic enhancer assay, find 41% of these sequences to function as enhancers at 24 to 48 hours post fertilization. Taking advantage of this transgenic assay we aim to screen 200 sequences a year for enhancer activity. These sequences will be selected from our large CNE set, sequences whose enhancer activity and tissue-timepoint specificity will be predicted using sophisticated computational tools, and community requested sequences. This characterization will not only allow the functional annotation of these sequences, but will also generate a novel and extremely important toolkit of gene regulatory elements that can drive expression of any gene of interest at precise locations and precise developmental time points. In addition, we will also use the annotated regulatory landscape to discover novel genes with potential important developmental function. This will be carried out by analyzing the expression patterns and functional consequences due to knockdown of less characterized genes that lie in rich regulatory regions, a common sign for the existence of important developmental gene regulators. Additional computational techniques will be used to discover genes under tight regulation in novel tissue contexts, as well as pathways which are currently not studied in the context we find them enriched in. All the data generated in this proposal, both computational and functional, will be made available to the community through a dedicated web browser (http://zebrafish.stanford.edu/) as well as integration into ZFIN, Ensemble, and the UCSC genome browser. Combined, our work will advance zebrafish as the major animal model for annotating and characterizing the noncoding portion of the vertebrate genome. PUBLIC HEALTH RELEVANCE: Computational &Functional Annotation of the Zebrafish Genome Regulatory Toolbox While genes make up less than 3% of our DNA, within the remaining 97% lie other numerous extremely important sequences such as gene regulatory elements, that instruct the genes when and where to turn on or off. Mutations in these gene regulatory elements can have a great impact on human disease, yet their location and code still remains on the majority unknown. In this proposal we will take advantage of the unique properties of the zebrafish model organism to couple advanced computational tools with rapid functional zebrafish assays to annotate these sequences and obtain a better understanding of the vertebrate gene regulatory code, which will be of extreme importance to our comprehension of the genetic cause for numerous human diseases.
|
0.954 |
2010 — 2014 |
Ahituv, Nadav |
U19Activity Code Description: To support a research program of multiple projects directed toward a specific major objective, basic theme or program goal, requiring a broadly based, multidisciplinary and often long-term approach. A cooperative agreement research program generally involves the organized efforts of large groups, members of which are conducting research projects designed to elucidate the various aspects of a specific objective. Substantial Federal programmatic staff involvement is intended to assist investigators during performance of the research activities, as defined in the terms and conditions of award. The investigators have primary authorities and responsibilities to define research objectives and approaches, and to plan, conduct, analyze, and publish results, interpretations and conclusions of their studies. Each research project is usually under the leadership of an established investigator in an area representing his/her special interest and competencies. Each project supported through this mechanism should contribute to or be directly related to the common theme of the total research effort. The award can provide support for certain basic shared resources, including clinical components, which facilitate the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence. |
Functional Genomics @ University of California, San Francisco
The Functional Genomics Research Team brings together a world-class group of experts with diverse backgrounds in membrane transporters and in transcriptional and regulatory genomics. The UCSF investigators with expertise in transporter biology include Kathleen Giacomini, Deanna Kroetz and Leslie Benet who participated in the last funding cycle. They made significant contributions in characterizing the function of amino acid variants and basal promoter regions of ABC and SLC membrane transporters. In this funding cycle, these investigators will continue their active role and expertise to direct the studies of characterizing the function of non-coding region (promoter, UTRs region and enhancer region) variants of membrane transporters in cells and mouse models. In planning the renewal application, the group felt that they needed to phenotypically characterize more variants and to extend their studies beyond amino acid variants and basal promoter region variants. To this end, Nadav Ahituv was recruited in the forth funding year of the last funding cycle to the project to complement and extend the existing expertise in transcriptional and gene regulation. His expertise and knowledge in comparative genomic strategies and regulatory element analysis have led to discoveries of regulatory sequence variation in the non-coding regions of genes associated with human congenital abnormalities and other clinically relevant phenotypes. Collectively, the Functional Genomics Research Team provides outstanding diverse and complementary expertise in experimental methods to characterize and to map the functional regions and the sequence variants of these regions of the membrane transporters in the upcoming five years. In addition to the investigators mentioned above, Hobart Harris and Xin Chen will support our pharmacogenomics studies with their individual expertise in initiating and setting standard protocols to accrue and to bank kidney and liver tissues. These unique and ethnically diverse kidney and liver tissue samples are valuable for determining transporter expressions and also in identifying and supporting the findings from our functional studies. This team will also work closely with the computational genomics team to identify key regulatory and functional regions in the transporter genes.
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1 |
2012 — 2015 |
Ahituv, Nadav |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Characterization of Neuronal Gene Regulatory Elements Associated With Epilepsy @ University of California, San Francisco
DESCRIPTION (provided by applicant): Epilepsy is one of the most common neurological disorders. It is a complex and heterogeneous disease which makes it difficult to precisely diagnose and provide effective treatment. A major and underexplored cause of complex disorders such as epilepsy could be mutations in gene regulatory elements. For example, disruption of these elements and subsequently the gene regulatory networks that are involved in brain development can lead to epilepsy subtypes such as infantile spasms (IS). However, the regulatory elements of brain expressed genes involved in IS are unknown. Using chromatin immunoprecipitation followed by deep sequencing (ChIP- Seq) with active enhancer chromatin marks (H3K4me, H3K27ac, p300), we will identify potential enhancers in the mouse embryonic day 16.5 (E16.5) developing forebrain. In order to determine which genes physically interact with these potential enhancers, we will carry out chromatin interaction analysis followed by paired-end tag sequencing (ChIA-PET) on E16. 5 mouse forebrains. Candidate enhancers of genes associated with IS will be tested for forebrain enhancer expression using zebrafish and mouse transgenic enhancer assays. IS patients from two different cohorts will be screened for coding and copy number variant (CNV) mutations. Potential forebrain enhancers that are found within IS-associated CNVs will be assayed for their enhancer activity in mice. IS, a patient without IS-associated CNVs and coding mutations will be screened for mutations in our characterized enhancers? Potential causative enhancer mutations will be functionally assessed for their enhancer expression in mice compared to the wild type allele and for differential binding affinity to transcription factors. Combined, these results will generate a regulatory landscape of the developing mouse forebrain, identify and functionally characterize potential IS-associated gene regulatory elements; screens IS patients for mutations in these elements and provide novel functional noncoding DNA sequences for the genetic diagnosis of epilepsy. In addition, this study will serve as a model for the functional characterization of gene regulatory elements involved in other complex human diseases.
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1 |
2012 — 2014 |
Ahituv, Nadav Shendure, Jay Ashok [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Massively Parallel, in Vivo Functional Testing of Regulatory Elements @ University of Washington
DESCRIPTION (provided by applicant): The overall aim of the ENCODE project is to comprehensively identify functional elements in the human genome. Currently applicable high-throughput technologies, such as RNA-Seq, ChIP-Seq, and DNase-Seq, exploit patterns of marks to infer the role of specific sequences, but generally fall short of functionally interrogatig and thereby validating these predictions. To address this gap, we propose a novel paradigm for the massively parallel functional testing of candidate regulatory elements. In preliminary work, we have developed a system whereby sequence-based transcribed barcodes enable the extensive multiplexing of classic reporter assays, in vitro or in vivo. Here, we propose to adapt this approach for testing tens-of- thousands of human regulatory elements in single assays, and furthermore to shift these assays from an episomal to a chromosomal context. Our specific aims are: (1) To develop high-throughput methods to clone, by capture or by synthesis, large numbers of candidate regulatory elements and to link them to transcribed, synthetic barcodes within complex populations of reporter vectors. (2) To test in parallel tens-of-thousands of candidate regulatory elements nominated by liver ChIP-Seq for in vitro and in vivo activity using HepG2 transfections and the hydrodynamic tail vein assay, with RNA-Seq of the synthetic barcodes serving as a single readout for the differential activity of distinct candidate regulatory elements. (3) To develop a similarly multiplexed lentiviral assay for regulatory element analysis that is chromosomally based and generically applicable to diverse cell and tissue types. We anticipate that these methods can be scaled for the efficient, in vivo functional testing of large numbers of candidate regulatory elements nominated by other technologies. Furthermore, our approach can easily be adopted by other researchers and used for many related goals, such as testing which regulatory elements work together, dissecting the fine-scale architecture of individual regulatory elements, and evaluating the performance of synthetic regulatory elements.
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0.955 |
2012 — 2015 |
Ahituv, Nadav Vaisse, Christian [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Identification & Functional Characterization of Sim1 Obesity-Associated Variants @ University of California, San Francisco
DESCRIPTION (provided by applicant): Project Summary/Abstract Identification & functional characterization of SIM1 obesity-associated variants Obesity leads to an increased risk for type 2 diabetes, heart attack, many types of cancer, hypertension, stroke, and is estimated to soon be the leading cause of death in the US. Through twin and family studies, obesity has been found to have a 40-70% heritability rate, pointing to a strong genetic etiology. In a large-scale human resequencing project of obese and lean individuals we have discovered that rare coding variants in the Single Minded 1 (SIM1) gene could have a large effect on obesity predisposition. Haploinsuficiency of SIM1 in humans and in heterozygous null Sim1 mice was shown to lead to severe obesity, and a common non- synonymous haplotype predisposes to obesity, suggesting that both altered function and altered expression of SIM1 can lead to obesity susceptibility. In this proposal, we will take advantage of comparative genomics coupled with zebrafish and mouse enhancer assays to identify SIM1 regulatory elements. Using this approach we have already uncovered five hypothalamus enhancers in the SIM1 region. These functional regulatory elements in addition to the SIM1 coding region will be sequenced in several large cohorts of obese and lean individuals in order to uncover obesity-associated variants. Obesity-associated coding variants will be assessed for their effect on the protein function using an in vitro functional assay that we generated for this project. Enhancer variants will be assayed for differential enhancer activity in mice compared to the reference allele. Future assays, such as removal of an obesity-associated enhancer in mice and further sequencing of SIM1 obesity-associated variants in the NIDDK Longitudinal Assessment of Bariatric Surgery (LABS) cohort (a cohort of adults that have undergone bariatric surgery and that is being analyzed for their subsequent outcome) will be considered as a follow-up to this proposal. Identifying and functionally characterizing SIM1 obesity-associated nucleotide variants will increase our understanding of the different genetic contributions of SIM1 to this phenotype. In addition, this study will serve as a model to functionally characterize the effect of noncoding regulatory elements on human disease.
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1 |
2012 |
Ahituv, Nadav Bejerano, Gill [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Computational & Functional Annotation of the Zebrafish Genome Regulatory Toolbox
Computational & Functional Annotation of the Zebrafish Genome Regulatory Toolbox Zebrafish with its growing arsenal of tools that allow the generation of transgenics, gene knockdowns and knockouts, and mutant resources coupled with its high-throughput and cost efficiency is quickly becoming the major animal model for drug screens and gene related studies. However, as with other vertebrate genomes, the majority of the zebrafish genome (97%) is made up of non-genic sequences whose functional necessity remains largely unknown. One vital function that is clearly embedded in these regions is gene regulation, instructing genes when and where to turn on or off. However, unlike genes where we know their genomic location, their code, and the consequences of nucleotide changes within them, in gene regulatory sequences we don't have that knowledge. This knowledge is extremely vital, with a wide variety of clinical and molecular data supporting these sequences to be an important driver for development, evolution, diversity, and disease. In this proposal, we will combine advanced computational tools with high-throughput zebrafish functional studies to annotate this noncoding terrain. Using and refining multiple vertebrate genome alignments we have generated an unprecedented set of 166,693 zebrafish conserved noncoding elements (CNEs), with at least 8,805 regions having a direct ortholog in the human genome. Preliminary studies for a portion of these sequences using a zebrafish transgenic enhancer assay, find 41% of these sequences to function as enhancers at 24 to 48 hours post fertilization. Taking advantage of this transgenic assay we aim to screen 200 sequences a year for enhancer activity. These sequences will be selected from our large CNE set, sequences whose enhancer activity and tissue-timepoint specificity will be predicted using sophisticated computational tools, and community requested sequences. This characterization will not only allow the functional annotation of these sequences, but will also generate a novel and extremely important toolkit of gene regulatory elements that can drive expression of any gene of interest at precise locations and precise developmental time points. In addition, we will also use the annotated regulatory landscape to discover novel genes with potential important developmental function. This will be carried out by analyzing the expression patterns and functional consequences due to knockdown of less characterized genes that lie in rich regulatory regions, a common sign for the existence of important developmental gene regulators. Additional computational techniques will be used to discover genes under tight regulation in novel tissue contexts, as well as pathways which are currently not studied in the context we find them enriched in. All the data generated in this proposal, both computational and functional, will be made available to the community through a dedicated web browser (http://zebrafish.stanford.edu/) as well as integration into ZFIN, Ensembl, and the UCSC genome browser. Combined, our work will advance zebrafish as the major animal model for annotating and characterizing the noncoding portion of the vertebrate genome.
|
0.954 |
2016 — 2020 |
Ahituv, Nadav Solnicakrezel, Lilianna Wise, Carol A |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Developmental Mechanisms of Human Idiopathic Scoliosis @ Ut Southwestern Medical Center
Project Summary/Abstract Adolescent idiopathic scoliosis (AIS) is a twisting condition of the spine and is the most common pediatric musculoskeletal disorder, affecting 3% of children worldwide. Children with AIS risk severe disfigurement, back pain, and pulmonary dysfunction later in life. Girls requiring treatment for AIS outnumber boys by more than five-fold, for reasons that are unknown. AIS is treated symptomatically rather than preventively because the underlying etiology is unknown. Hospital charges for AIS surpass one billion dollars annually in the U.S. and are rising significantly faster than for other pediatric procedures. Our overall purpose is to understand the biologic causes of AIS as a means to early diagnosis, prevention and non-invasive biologic treatment. Adolescent idiopathic scoliosis is a complex genetic disease. Genome wide association studies (GWAS) of common non-coding variants by our group and others have identified AIS-associated haplotypes, but the mechanistic basis of these associations remains to be defined. Furthermore these findings explain less than 5% of overall heritability due in part to the fact that the AIS exome has yet to be fully interrogated. Another barrier to understanding the pathogenesis of AIS in humans has been the lack of appropriate, genetically- defined animal models that are essential for defining spatiotemporal involvement in the disease. Finally the developmental regulation of postnatal spinal development generally, and the specific tissue of origin in AIS specifically, are poorly understood. To address these issues we have established an innovative collaborative approach combining unbiased gene discovery in humans, modeling and gene discovery in zebrafish, and genomic analysis of postnatal spine development. Specifically, the component activities of our proposed Program will synergize to yield the tools and fundamental knowledge that the field of AIS research has lacked, addressing the following goals: (1) We will define the genetic architecture conveying AIS susceptibility as identified in human populations; (2) We will develop the first genetically tractable vertebrate system for modeling AIS and studying the functional consequences of AIS mutations identified in humans; (3) We will define cis-and trans-regulation of AIS causal genes; (4) We will pilot a large-scale genomics platform to begin to characterize the molecular mechanisms controlling spinal development; (5) We will identify and characterize causal mutations in patients that may identify gene-based AIS subtypes; (6) By filling gaps in fundamental knowledge of the disease we will drive innovative efforts to develop new therapies for AIS. Our discoveries will spur the field toward much-needed hypothesis-driven research aimed at early molecular diagnosis, prevention and therapies. We also expect that these studies will enlighten other structural defects of humans.
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0.903 |
2016 — 2020 |
Ahituv, Nadav Pollard, Katherine S. [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Massively Parallel Dissection of Psychiatric Regulatory Networks @ J. David Gladstone Institutes
? DESCRIPTION (provided by applicant): Abnormal neuronal development can lead to a wide array of mental disorders. Genes important for neurodevelopment have been combed for coding mutations leading to psychiatric disease with limited success, suggesting that other regions in the genome could be causative. A variety of molecular and clinical data indicates that mutations associated with psychiatric disease can reside in gene regulatory sequences such as enhancers. However, only a few enhancers have been definitively linked with these disorders to date. This is primarily because regulatory mutations are challenging to functionally characterize and link to specific genes and phenotypes. To address this challenge, we will use functional genomics data, sequence motifs, and evolutionary signatures to train EnhancerFinder, software that we developed that predicts functional enhancers at high success rates, to now specifically identify active neurodevelopmental enhancers. Over 12,000 candidate neurodevelopmental enhancers will then be cloned and assayed en masse for their enhancer activity using massively parallel reporter assays (MPRAs) in three human embryonic stem cell (hESC) derived neuronal lines: early initiation, neural progenitor cell stage that produces only neurons upon further differentiation, and astrocytes. In addition, we will link enhancers to their target genes using a novel chromatin structure-based prediction approach, called TargetFinder, thereby establishing a network connecting regulatory regions to neurodevelopmental genes. By overlaying reproducible psychiatric disease associated loci with this network, we will identify and prioritize non-coding mutations that are likely to affect expression of neurodevelopmental genes with roles in psychiatric disease. These predictions will be validated using genome- editing techniques to knock out regulatory elements and then assay changes in chromatin interactions and gene expression in developing neurons. The key innovations of our approach are: (i) accurate, quantitative measurements of activity for thousands of psychiatric disease associated enhancer candidates in parallel, (ii) chromatin based inference of gene regulatory networks linking enhancer mutations to genes and pathways, and (iii) a well-characterized stem cell based system to apply these techniques in a high-throughput manner to developing human neurons. We will rapidly disseminate software, reagents, protocols, and datasets to enable follow-up functional studies in the labs of our mental health collaborators and many others. Our long-term aim is to pinpoint causative regulatory variants in the many genomic loci associated with psychiatric disease where an obvious coding mutation is lacking. This approach could easily be adapted to functionally characterize gene regulatory elements involved in other complex human diseases.
|
0.904 |
2016 — 2020 |
Ahituv, Nadav |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Non-Coding/Epigenetic Regulation @ Ut Southwestern Medical Center
Adolescent idiopathic scoliosis (AIS) affects ~3% of the population worldwide and is estimated to cost several billion dollars annually in surgeries alone in the US. The causes of AIS remain largely unknown. While mutations in genes leading to syndromic scoliosis (associated with other symptoms) have been discovered, the identification of mutations causing non-syndromic/isolated AIS (only AIS without any other symptoms), have been less successful. Several genome-wide association studies (GWAS) have identified AIS-associated single nucleotide polymorphisms (SNPs) in noncoding regions adjacent to promising candidate genes, suggesting a role for gene regulatory sequences, such as enhancers, in AIS. In our preliminary results for example, we show that the activity of a somitic muscle and spinal cord enhancer near PAX1, a gene known to be involved in spinal development, is abolished by AIS-associated SNPs identified by GWAS. Here, we plan to take advantage of functional enhancer assays in zebrafish and mice combined with mouse CRISPR/Cas9 knockouts to characterize gene regulatory sequences that are associated with AIS. These sequences will be selected from GWAS studies, both from the literature and Project 1 (Human) and near genes shown to cause AIS in zebrafish from our Project 2 (Zebrafish). In addition, using RNA-seq and ChIP-seq for a repressive mark (H3K27me3), active marks (H3K27ac, H3K4me1, RNAPII) and candidate transcription factors on AIS- associated tissues, we will identify gene regulatory elements that could be associated with AIS, thus revealing additional sequence candidates whose alteration could lead to AIS. While there is no specific tissue whose aberration is widely known to cause AIS, there is now ample evidence linking chondrocytes to AIS. Our collaborators in Project 2 have shown that chondrocyte-specific deletion of the Gpr126 gene (a AIS GWAS gene) in mice, leads to scoliosis beginning at 20 days of age. We thus plan to initially carry out RNA-seq and ChIP-seq on mouse chondrocytes at early time points. Later on in the project we plan to use RNA-seq and ChIP-seq to characterize additional cell types/tissues where AIS candidate genes identified in Project 1 (Human) and Project 2 (Zebrafish) are expressed. Combined, our work will allow for the functional characterization of regulatory regions that are important in AIS pathogenesis, and begin to provide a genomic encyclopedia of regulatory elements that could be associated with this disease, thus providing additional candidate regions for mutation screening in individuals with AIS. In addition, our work will serve as a model for the functional characterization of gene regulatory elements involved in additional subtypes of scoliosis, musculoskeletal and other human disease.
|
0.903 |
2017 — 2021 |
Ahituv, Nadav Shendure, Jay Ashok |
UM1Activity Code Description: To support cooperative agreements involving large-scale research activities with complicated structures that cannot be appropriately categorized into an available single component activity code, e.g. clinical networks, research programs or consortium. The components represent a variety of supporting functions and are not independent of each component. Substantial federal programmatic staff involvement is intended to assist investigators during performance of the research activities, as defined in the terms and conditions of the award. The performance period may extend up to seven years but only through the established deviation request process. ICs desiring to use this activity code for programs greater than 5 years must receive OPERA prior approval through the deviation request process. |
Massively Parallel Reporter Assays and Genome Editing of Encode Predicted Regulatory Elements @ University of California, San Francisco
PROJECT SUMMARY Since its inception in 2003, the Encyclopedia of DNA Elements (ENCODE) Consortium has made remarkable progress towards the identification of all functional elements in the human genome. However, major limitations of the current catalog are that the vast majority of elements have not been functionally characterized, the impact of genetic variation on their function is poorly defined, the precise levels of activation or repression that they confer remain unmeasured, and the specific gene(s) that they regulate are not definitively known. To address these gaps, we will implement `in genome' massively parallel functional assays to characterize over 100,000 ENCODE-based candidate regulatory elements, to confirm and quantify their activities as well as to link many of them to their target genes. In a systematic comparison of episomal vs. genomic massively parallel reporter assays (MPRA), we show that episomal assays fail to accurately capture the full patterns of regulatory activity that are observed in the context of chromatin. We therefore focus exclusively on methods that test candidate regulatory elements in an integrated, `in genome' context. First, using lentivirus-based massively parallel reporter assays, we will characterize at least 100,000 ENCODE-based regulatory elements for their promoter/enhancer activity while integrated into the genome (lentiMPRA; Aim 1a). Importantly, lentiMPRA can be carried out in almost every cell type and leverages ongoing developments in lentivirus technology. Early results will be used to iteratively develop models that make better selections for subsequent rounds of functional characterization. Second, we will use CRISPR/Cas9 and multiplex homology directed repair to integrate a subset of candidate enhancers to the 3' UTR of transcriptionally inactive genes, allowing us to further validate and characterize their ability to activate transcription in a natural genomic context (`in genome' STARR-seq; Aim 1b). Finally, we will implement a new paradigm involving CRISPR/Cas9-based multiplex genome editing followed by RNA-seq/ATAC-seq molecular profiling to characterize a genome-wide subset of candidate regulatory elements in their native genomic context for the functional consequences of mutations on them, while also determining the target gene(s) that they regulate (massively parallel genome editing; Aim 2). Although we will initially focus our efforts on K562 and HepG2 cells, we will also perform work in other cell lines as appropriate for the needs of the ENCODE Consortium, with 25% of our capacity dedicated to a common set of elements. Combined with the efforts of the other functional characterization centers, our work will provide unprecedented `in genome' validation and characterization of ENCODE-defined candidate regulatory elements, while also facilitating insights into our understanding of the basic biology of gene regulation and how regulatory variants contribute to human disease risk.
|
1 |
2018 — 2019 |
Ahituv, Nadav Shen, Yin [⬀] |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Development of Massively Parallel Reporter Assays That Use Cognate Promoters @ University of California, San Francisco
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.
|
1 |
2018 — 2020 |
Ahituv, Nadav Jorgenson, Eric |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Genetic Etiology of Abdominal Hernia Susceptibility @ Kaiser Foundation Research Institute
Abdominal hernias are some of the most frequently diagnosed conditions in clinical practice, with more than twenty million hernia repair surgeries performed annually around the world. Many patients experience serious post-surgical complications, including chronic pain (6%) and hernia recurrence (10%). Delaying treatment carries the risk of bowel incarceration, which requires emergency hernia repair surgery and is associated with a substantial risk of mortality. Because the risks associated with hernias must be balanced against the risks associated with their treatment, there is a clear need for a better understanding of hernia etiology and improved treatment options. We conducted the first large-scale genetic study of hernia risk and identified noncoding variants at four novel genetic loci underlying the risk of inguinal hernia?the most common type of hernia?and showed that four genes in these loci (EFEMP1, WT1, EBF2, and ADAMTS6) are expressed in mouse connective tissue. Here, we will extend our findings by identifying genetic risk loci underlying additional abdominal hernia subtypes, locating and characterizing regulatory elements within these loci, and demonstrating, using both in vitro and in vivo assays, how nucleotide variation within these elements can lead to their altered regulation and hernia susceptibility. By linking specific genetic variants in hernia risk loci to their functional effect on gene regulation, we can begin to understand the biological mechanisms that lead to hernia susceptibility. Our study will fill an important gap in the literature by identifying genetic loci underlying hernia subtypes and provide insights into the specific biological mechanisms that lead to hernia development. An improved understanding of the mechanisms through which hernias develop can guide a modern `precision medicine' approach for hernia treatment that will lead to preventative non-surgical treatments.
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0.903 |
2018 — 2020 |
Ahituv, Nadav Pollard, Katherine S. (co-PI) [⬀] |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Massively Parallel Characterization of Psychiatric Disease Associated Regulatory Elements in Defined Cell Types @ University of California, San Francisco
Project Summary / Abstract Abnormal neuronal development can lead to a wide array of psychiatric disorders. Mutations disrupting protein coding genes have been found to cause some of these disorders but a large number of them still remain unsolved. A variety of molecular and clinical data suggests that mutations in gene regulatory sequences could be a major contributor to these disorders. However, only a few causal regulatory mutations have been found to date. This is primarily because functional regulatory elements are difficult to identify, particularly in mixed cell populations such as the developing brain. In addition, these elements are difficult to functionally characterize in a high-throughput manner in these cell types. To address these challenges, we propose to use novel single-cell genomic technologies along with massively parallel reporter assays (MPRAs) in human primary cells and organoids to characterize thousands of brain development associated genes, regulatory elements and pathways. First, using single cell RNA-seq (scRNA-seq) and ATAC-seq (sci-ATAC-seq) across multiple cortical areas and subcortical regions of developing human brain at three development stages, we will generate a comprehensive map of genes, regulatory elements and networks involved in human brain development (Aim 1). Next, we will use similar techniques (scRNA-seq and sci-ATAC-seq) on human cerebral organoid cultures derived from induced pluripotent stem cells (iPSCs). We will compare regulatory programs in organoid cells to cells present during normal human brain development. To assess the contribution of key transcription factors involved in psychiatric disorders to gene regulatory pathways in the developing brain, we will use genome editing on the same genetic background to create heterozygous loss-of-function mutations in key transcription factors involved in psychiatric disorders and assess their effects on gene expression (scRNA-seq) and gene regulation (sci- ATAC-seq) (Aim 2). Finally, we will functionally characterize over 37,500 candidate enhancers and nucleotide variants within them using a lentiviral-based MPRA (lentiMPRA) in disease-relevant cell types purified from human primary cells and organoids. Several of these sequences will also be assayed in organoids lacking key transcription factors deleted in Aim 2 to test the importance of these genes to regulatory activity and to identify interactions with regulatory variants (Aim 3). Data from all aims will be used to build predictive models of gene expression and enhancer activity as a function of regulatory sequences, which will be used to design lentiMPRA libraries and iteratively improve models using results from initial libraries. Combined our project will use cutting- edge techniques such as scRNA-seq, sci-ATAC-seq and MPRA coupled with advanced computational analyses to significantly increase the number of functionally characterized human brain developmental regulatory elements and how their activity changes in the presence of disease associated mutations to shed light on the genetic basis for psychiatric disorders.
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1 |
2018 |
Ahituv, Nadav Shendure, Jay Ashok |
UM1Activity Code Description: To support cooperative agreements involving large-scale research activities with complicated structures that cannot be appropriately categorized into an available single component activity code, e.g. clinical networks, research programs or consortium. The components represent a variety of supporting functions and are not independent of each component. Substantial federal programmatic staff involvement is intended to assist investigators during performance of the research activities, as defined in the terms and conditions of the award. The performance period may extend up to seven years but only through the established deviation request process. ICs desiring to use this activity code for programs greater than 5 years must receive OPERA prior approval through the deviation request process. |
Ahituv Nih Um1 Regalado Uw Diversity Supplement P0531415 @ University of California, San Francisco
PROJECT SUMMARY Since its inception in 2003, the Encyclopedia of DNA Elements (ENCODE) Consortium has made remarkable progress towards the identification of all functional elements in the human genome. However, major limitations of the current catalog are that the vast majority of elements have not been functionally characterized, the impact of genetic variation on their function is poorly defined, the precise levels of activation or repression that they confer remain unmeasured, and the specific gene(s) that they regulate are not definitively known. To address these gaps, we will implement `in genome' massively parallel functional assays to characterize over 100,000 ENCODE-based candidate regulatory elements, to confirm and quantify their activities as well as to link many of them to their target genes. In a systematic comparison of episomal vs. genomic massively parallel reporter assays (MPRA), we show that episomal assays fail to accurately capture the full patterns of regulatory activity that are observed in the context of chromatin. We therefore focus exclusively on methods that test candidate regulatory elements in an integrated, `in genome' context. First, using lentivirus-based massively parallel reporter assays, we will characterize at least 100,000 ENCODE-based regulatory elements for their promoter/enhancer activity while integrated into the genome (lentiMPRA; Aim 1a). Importantly, lentiMPRA can be carried out in almost every cell type and leverages ongoing developments in lentivirus technology. Early results will be used to iteratively develop models that make better selections for subsequent rounds of functional characterization. Second, we will use CRISPR/Cas9 and multiplex homology directed repair to integrate a subset of candidate enhancers to the 3' UTR of transcriptionally inactive genes, allowing us to further validate and characterize their ability to activate transcription in a natural genomic context (`in genome' STARR-seq; Aim 1b). Finally, we will implement a new paradigm involving CRISPR/Cas9-based multiplex genome editing followed by RNA-seq/ATAC-seq molecular profiling to characterize a genome-wide subset of candidate regulatory elements in their native genomic context for the functional consequences of mutations on them, while also determining the target gene(s) that they regulate (massively parallel genome editing; Aim 2). Although we will initially focus our efforts on K562 and HepG2 cells, we will also perform work in other cell lines as appropriate for the needs of the ENCODE Consortium, with 25% of our capacity dedicated to a common set of elements. Combined with the efforts of the other functional characterization centers, our work will provide unprecedented `in genome' validation and characterization of ENCODE-defined candidate regulatory elements, while also facilitating insights into our understanding of the basic biology of gene regulation and how regulatory variants contribute to human disease risk.
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1 |
2019 — 2020 |
Ahituv, Nadav |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Technologies For Simultaneous Characterization of Regulatory Activity and Protein Binding @ University of California, San Francisco
Project Summary Mutations in gene regulatory elements are a major cause of human disease. Large-scale genomic assays, such as ChIP-seq and ATAC-seq, have identified millions of putative regulatory elements across many different cell types and tissues. Furthermore, massively parallel reporter assays (MPRAs), have allowed us to test thousands of regulatory sequences and their variants for their functional activity in a high-throughput manner. In addition, lentivirus-based MPRAs (lentiMPRAs) have enabled the testing of candidate sequences for regulatory activity with high reproducibility in hard to transfect cells and in chromatin context via genomic integration. While these assays have significantly expanded our knowledge of regulatory elements, technologies that can simultaneously analyze both the regulatory function of a specific sequence and the transcription factors, cofactors and epigenomic modifications that determine it do not exist. Here, we will develop a novel technology, crMPRA (CUT&RUN MPRA), that combines two separate techniques, lentiMPRA and cleavage under targets and release using nuclease (CUT&RUN) to simultaneously analyze in a high-throughput manner the regulatory activity, protein binding and epigenetic modification of thousands of sequences. We will take advantage of lentiMPRA both for testing thousands of candidate sequences for their regulatory activity, but also to enrich the genome with thousands of integrations of a specific sequence, such that it could be assayed for protein binding and epigenetic modifications via CUT&RUN. In Aim 1, we will develop crMPRA, by taking advantage of sequences that were previously characterized via lentiMPRA (regulatory activity) and ChIP-seq (protein binding and epigenetic marks) in hepatocellular carcinoma HepG2 cells. We will use these sequences to build an MPRA library and characterize them for their regulatory activity via lentiMPRA. We will also simultaneously carry out CUT&RUN on specific TFs (e.g. EP300, FOXA2, HNF4A) and epigenetic marks (e.g. H3K27ac, H3K27me3). For Aim 2, we will further test a transcription factor binding site perturbation library via crMPRA how these perturbations affect regulatory activity, protein binding and epigenetic modification, and analyze the functional correlation or independency between these states. As such, this novel technology will allow us to increase our understanding of the regulatory code at several levels including TF binding, histone modification, and transcriptional activation, and how its alteration can lead to human disease.
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1 |
2019 — 2021 |
Ahituv, Nadav Burchard, Esteban Gonzalez [⬀] Seibold, Max (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Airway Functional Genomics of Bronchodilator Drug Response in Minority Children With Asthma @ University of California, San Francisco
ABSTRACT Asthma is the most common chronic disease among children. Asthma prevalence, mortality, and drug response vary by race/ethnicity and genetic ancestry. In the U.S., asthma prevalence is highest among Puerto Ricans (36.5%), intermediate among African Americans (13.0%) and whites (12.1%), and lowest in Mexicans (7.5%). These disparities extend to asthma mortality, which is four-fold higher in Puerto Ricans and African Americans compared to Mexican Americans. Albuterol is the most commonly prescribed asthma medication in the world and is the mainstay of acute asthma management. Among low income and minority populations in the U.S., albuterol is often the only medication used regardless of asthma severity. Poor drug response contributes to racial/ethnic disparities in asthma morbidity and mortality. Disturbingly, Americans with the highest asthma prevalence and death rate also have the lowest drug response. Chronic albuterol use can decrease acute airway smooth muscle response to albuterol and increase airway inflammation through beta-agonist signaling in the airway epithelium, suggesting that chronic albuterol use may alter acute response through genomic and epigenomic modification of airway cells. Furthermore, acute bronchodilator drug response (BDR) to albuterol is a complex phenotype with an estimated heritability of 28.5%, indicating genetic factors contribute to BDR variability. Genome-wide and whole genome association analyses have revealed population-specific common and rare variants in non-coding regions of the genome associated with the extremes of BDR. The roles of genomic regulatory regions and population-specific variants in BDR have yet to be fully investigated. To this end, we have created an investigative system involving airway-specific cell types, patient-derived cells, and detailed clinical data to generate an encyclopedia of genes, regulatory regions, and pathways involved in BDR to albuterol. We will integrate RNA-seq, ChIP-seq, ATAC-seq, and whole genome sequencing data with detailed clinical data to identify trans-ethnic and population-specific variants contributing to differential expression and chromatin structure patterns in response to albuterol exposure. Furthermore, we will functionally characterize the regulatory regions that underlie acute and chronic albuterol BDR in multi-ethnic children with asthma using CRISPR-Cas9 activation/inhibition assays. These analyses will allow us to determine on a genomic scale the functional consequences of acute and chronic albuterol treatment on airway cells, and provide insight into potential targetable genes, regulatory elements, and pathways for improved asthma therapies in at-risk populations.
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1 |
2020 — 2021 |
Ahituv, Nadav Vaisse, Christian [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
From Obesity Gwas to Therapeutic Targets @ University of California, San Francisco
Project Summary/Abstract Obesity leads to an increased risk for type 2 diabetes, heart attack, many types of cancer, hypertension, stroke, and is estimated to soon be the leading cause of death in the US. Through twin and family studies, obesity has been found to have a 40-70% heritability rate, pointing to a strong genetic etiology. The long-term objective of our studies is to determine how genetic variation predisposes humans to obesity and what the therapeutic implications are for this condition. To find common genetic variants associated with obesity, numerous genome-wide association studies (GWAS) have been performed. Over 500 loci have been found to associate with increased body weight index (BMI), all of which reside in noncoding regions of the genome . However, little progress has been made in outlining the causal SNPs and understanding the mechanisms by which they actually cause obesity. In this proposal, we explore the hypothesis that obesity-associated SNPs affect regulatory regions that are active in neuronal sub-population implicated in the regulation of food intake and body weight. Using state-of-the-art approaches that we have recently pioneered through collaborations between the Ahituv and Vaisse laboratories we propose to: - Extend and refine the regulatory landscape of hypothalamic neuronal subpopulations implicated in body weight regulation to identify candidate regulatory elements overlapping obesity GWAS single nucleotide polymorphisms (SNPs). - Use CRISPR inactivation (CRISPRi) in mice to directly test the functional role of regulatory elements that encompass obesity-associated SNPs. - Use CRISPR activation (CRISPRa) in mice to test the therapeutic potential of activity modulation of identified target regulatory regions. Combined, our work will not only provide a regulatory map of neuronal subtypes associated obesity, but also functionally characterize these regions and show their therapeutic potential.
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1 |
2020 — 2021 |
Ahituv, Nadav Kroetz, Deanna L [⬀] |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Pharmaceutical Sciences and Pharmacogenomics @ University of California, San Francisco
The Pharmaceutical Sciences and Pharmacogenomics (PSPG) Graduate Program is a unique, dynamic, contemporary program in pharmaceutical sciences and pharmacogenomics at the University of California San Francisco that attracts diverse faculty and students who share a common interest in applying basic sciences to challenging research topics in drug development and precision drug therapy. The graduate program reflects exciting scientific developments in the area of genomics, quantitative and systems pharmacology, and computation that have far-reaching implications to the pharmaceutical and pharmacological sciences. The goal of the PSPG graduate program is to educate and train Ph.D. students to conceptualize, design and execute innovative scientific research in the interdisciplinary scientific areas encompassed by modern pharmaceutical sciences. The program brings together 59 well- funded faculty members spanning 23 departments. This multidisciplinary and unique graduate program has a dual focus: 1) pharmaceutical sciences and drug development, including molecular and systems pharmacology, drug delivery and therapeutic bioengineering, and pharmacokinetics/pharmacodynamics and modeling; and 2) pharmacogenomics, the application of genetics and genomics for the development of novel therapeutics and the optimal use of drugs in individual patients for precision medicine. The training program includes a series of core courses providing an in-depth understanding of the principles of pharmaceutical sciences and pharmacogenomics, including an innovative new core course in systems pharmacology. Core courses are complemented by electives covering advanced drug delivery and pharmacokinetic principles, principles of genetics and cell biology, bioinformatics, tissue and organ biology, and computer programming. Students also participate in laboratory rotations that expose them to the diversity of potential projects available for their dissertation research and a university-wide course on responsible conduct of research. The program immerses trainees in the culture of science through a journal club with students across four basic science graduate programs that are focused on quantitative approaches to studying biology, a seminar program which brings in leading academic, regulatory and industrial scientists, student research presentations, and an annual retreat. The program goal is to recruit 8-10 outstanding Ph.D. students per year, plus at least one student for a combined Pharm.D./Ph.D. degree. Underrepresented minority students are actively recruited through a number of faculty activities and represent 16% of our students; 16% of our students also come from disadvantaged backgrounds. Upon graduation, the new PSPG Ph.D. scientist will have the ethics, knowledge and tools necessary to become independent researchers, and also the passion and enthusiasm to make impactful contributions to the pharmaceutical sciences and pharmacogenomics field throughout their career.
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1 |
2021 |
Ahituv, Nadav Kircher, Martin (co-PI) [⬀] Shendure, Jay Ashok [⬀] |
UM1Activity Code Description: To support cooperative agreements involving large-scale research activities with complicated structures that cannot be appropriately categorized into an available single component activity code, e.g. clinical networks, research programs or consortium. The components represent a variety of supporting functions and are not independent of each component. Substantial federal programmatic staff involvement is intended to assist investigators during performance of the research activities, as defined in the terms and conditions of the award. The performance period may extend up to seven years but only through the established deviation request process. ICs desiring to use this activity code for programs greater than 5 years must receive OPERA prior approval through the deviation request process. |
Massively Parallel Characterization of Variants and Elements Impacting Transcriptional Regulation in Dynamic Cellular Systems @ University of Washington
SUMMARY / ABSTRACT A major fraction of heritability for common diseases, as well as for the penetrance and expressivity of rare diseases, partitions to distal regulatory elements in the human genome, overwhelmingly cell type-specific enhancers. However, a rate-limiting challenge for the field has been how to identify the specific variants, elements and regulated genes that mediate these effects on disease liability. Towards the overall goals of the Impact of Genomic Variation on Function (IGVF) Consortium, we propose to test over one million human regulatory elements or variants for their functional effects on transcriptional regulation, as well as to query over 100,000 distal regulatory elements for the gene(s) that they regulate. A first theme of our proposal is the diversity of multiplex technologies that we will employ to these ends, including massively parallel reporter assays (MPRAs), crisprQTL, saturation genome editing, multiplex prime editing and single cell combinatorial indexing, many of which we pioneered. A second theme is a focus on dynamic cellular systems that enable a given library of variants and/or elements to be tested across a broad range of cell types and states within a single experiment; these will include ESC-derived neuronal progenitors, cardiomyocytes, embryoid bodies, gastruloids and organoids, and in select cases, mice. A third theme involves leveraging our experience (e.g. CADD, a widely used, genome-wide catalog of variant effect predictions) to support the overarching goals of IGVF. Specifically, we envision using functional measurements generated by us and others to produce well-calibrated predictions of enhancer activity and variant effects that are continuous along the branching trajectories that comprise human development. Our specific aims are as follows: (1) To perform massively parallel validation and functional characterization of candidate human enhancers in a broad range of cell type contexts. (2) To perform massively parallel characterization of human genetic variants with potential roles in human disease. (3) To contribute to a comprehensive variant-element-phenotype catalog while taking a leadership role in synergistic interactions within IGVF, in the dissemination of methods, data and predictions, and in the overarching goals of the consortium.
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0.955 |