2013 — 2014 |
Ruthenburg, Alexander Jackson |
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.) |
Calibrated Chip-Seq: Determining Local Histone Modification Density Genome-Wide
DESCRIPTION (provided by applicant): Post-translational modifications on the histone constituents of nucleosomes are able to transduce changes in local chromatin states that govern the accessibility of underlying DNA, regulating processes that range from transcriptional activation to gene silencing. Yet with present technology, it is impossible to measure the absolute densities of histone modifications in a locus specific manner. Despite serving as the central experimental technique in epigenetics research, chromatin immunoprecipitation coupled to deep sequencing (ChIP-seq) suffers from a number of serious drawbacks: 1.) it is a relative measurement unthered to any external scale in a way that obviates comparison amongst experiments; and 2.) it employs antibody reagents that have differing specificity and affinity for epitopes, which are in turn variable in abundance, yet none of these factors are taken into account in present analysis. Consequently, the peaks of different histone modifications that seem to overlap on certain genomic loci cannot be meaningfully compared. To address these substantial problems, I propose a novel approach to calibrate ChIP-seq data using a panel of nucleosomes derived from recombinant and semisynthetic sources as internal standards (calChIP-seq). To that end, nucleosomes bearing a given mark will be reconstituted with a library of DNAs composed of a constant strong nucleosome positioning sequence that is flanked by a variable barcode that represents each member¿s molar concentration, then spiked into the input of a native ChIP-seq experiment. After immunoprecipitation with modification-specific antibodies followed by sequencing, the tag counts resulting from the exogenous semisynthetic nucleosome DNA series will serve as an internal-standard calibration curve for absolute quantification of mark density with the positional accuracy of ChIP-seq in a genome-wide data set. This basic scheme will be employed in a number of variations to calibrate ChIP-seq in a proof of concept form and critically examine several troublesome sources of experimental error in ChIP measurements. This proposal is centered on developing the calChIP-seq technology, although a number of potential applications that could substantially contribute to understanding how the epigenome contributes to the control of genomic information are presented. The ability to make comparisons of histone modification density on an absolute scale by calChIP-seq will be transformative for our understanding of chromatin states and enable for the first time crucial comparisons between one modification to another, one cell type to another, and from patient to another. I am in a unique position to accomplish this radical and desperately needed improvement to our field¿s most important technology in that I have both expertise in making semisynthetic chromatin and experience with ChIP-sequencing experiments.
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2016 — 2020 |
Ruthenburg, Alexander Jackson |
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. |
Quantitatively Probing Intra-Nucleosomal Chromatin Variation and Function
? DESCRIPTION (provided by applicant): Much epigenetic information is thought to be encoded in the identity and localization of potentially heritable chemical modifications to histone protein that package the genome, to which modification-contingent binding partners bind, thereby transducing downstream functional consequences. The fundamental repeating unit of chromatin, the nucleosome core particle, is a two-fold symmetric octamer of histone proteins enshrouded by two superhelical turns of DNA. This architecture places the two copies of each core histone in defined positions each projecting unstructured tails from the core of the structure to that are subject to dense posttranslational modification. For a given modification, is there meaningful information encoded by having two distinct modifiable sites per fundamental repeating unit of chromatin? There are hints that variation at this level is highly regulated, yet little is known about this scale of chromatin modifications owing to lack of tools that can measure these properties. We have developed a breakthrough calibrated ChIP technology that permits us to query this level of nucleosome sub-structure detail for the first time. In Aim 1 we will directly quantify the symmetry of histone modifications within nucleosomes with our calibrated ChIP method, then probe the function of this newly measurable chromatin property. Given that the unit of recognition for binding partners entire nucleosome and flanking DNA, as opposed to merely the tails, precisely how variation at this level spatially manifests is likely tobe an important element of discrimination. To this end, we have recently developed biochemical evidence that nucleosomal binding partners discriminate as a function of mark-symmetry. We seek to understand the mechanistic properties of this unprecedented level of recognition, both in its biophysical details and its functional consequences for cells and organisms. In Aim 2 we will define the molecular nature of bivalent domains-the seeming apposition of canonically activating and repressive histone modifications decorating critical developmental genes in pluripotent cells-- using calibrated sequential ChIP experiments calibrated with an exhaustive set of internal standards. We will then examine their biogenesis and predictive power as barriers to differentiation. We expect that the results of this study will illuminate the general principlesof sub-nucleosomal mark recognition and function, forming a compelling argument that this relatively un-explored level of chromatin modification is important for genome management.
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2019 — 2021 |
Moskowitz, Ivan Paul [⬀] Ruthenburg, Alexander Jackson |
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. |
Gene Regulatory Non-Coding Rnas in the Human Heart
This collaborative proposal from a cardiac molecular geneticist and a RNA-chromatin biochemist will investigate chromatin associated and transcription factor dependent non-coding RNAs (ncRNAs) as essential components of a cardiac regulatory network. We have applied a novel approach for identification of functional long non-coding RNAs (lncRNAs) and the enhancers that comprise gene regulatory networks. Using transcription factor- dependence, we identified ncRNAs as markers of cis-regulatory elements essential for a mouse cardiac rhythm gene regulatory network. This approach identified exceptionally strong enhancers, and their associated ncRNA are chromatin-bound and required for enhancer function. This proof of principal sets the stage for the investigation of lncRNAs and their associated enhancers in human cardiac gene regulatory networks. We posit that application of our approach to human cardiomyocytes will allow identification of a functional class of human heart enhancer-associated lncRNAs and cis-regulatory elements (CREs) essential for the expression of human cardiac rhythm control genes. In the first specific aim, we will interrogate a set of lncRNAs defined by their TF- dependence and chromatin localization for their requirement for CRE activity and target gene expression. In the second aim, we will utilize novel molecular tools to probe the molecular mechanisms whereby the lncRNAs modulate gene expression. Together these interconnected aims rely on the complementary and non-overlapping expertise of the collaborative investigators to address a highly significant problem in ncRNA biology. These hypotheses are applicable to human genetics, transcriptional regulation, and RNA biology, and therefore may have impact both within cardiovascular genetics and more broadly within human molecular genetics.
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2021 |
Novembre, John (co-PI) [⬀] Rothman-Denes, Lucia B. B (co-PI) [⬀] Ruthenburg, Alexander Jackson Thornton, Joseph W [⬀] |
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. |
Genetic Mechanisms and Evolution
Recent technological advances have transformed genetics research, and social changes have caused major shifts in best practices for graduate education, research training, and mentoring. We propose an innovative interdisciplinary predoctoral T32 program, Genetic Mechanisms and Evolution (GME), which is specifically crafted to meet the challenges and opportunities presented by these changes. The GME program will train a diverse group of world-class Ph.D. scientists in molecular, statistical, and evolutionary genetics research who will serve as the next generation of innovative scientific leaders in genetics. Training will ensure development of multidisciplinary competence across these fields, with a strong foundation in quantitative and computational analysis for every student. The GME training program leverages the world-class strength of the University of Chicago in genetics. Mentors include 56 faculty with extraordinary records of research and graduate training, drawn from 14 departments across the fields of evolutionary, statistical, and molecular genetics. Further, the University?s unique organizational structure brings all areas of genetics into a single division and makes possible the interdisciplinary program we propose. Trainees for 18 funded positions will be selectively drawn from 9 graduate programs across disciplinary areas. The pool of potential trainees is extraordinarily well-qualified and diverse (49% women and 26% URM over the last 5 years). Trainees will be funded in years 2-3 of their studies, but they will participate in training and advising activities from matriculation through graduation. A new interdisciplinary core course and breadth requirements will develop student foundations in molecular, statistical, and evolutionary genetics and build strong skills in programming and statistics. Specialized workshops and an annual hackathon will provide further rigorous training in computational and quantitative analysis of modern genetic data. Formal writing instruction along with workshops in grant-writing and oral presentation skills will train scientists for effective communication and help ameliorate disparities in preparation among students from diverse backgrounds. Individual development plans, mentor-mentee contracts, faculty mentor training, and peer mentoring will facilitate trainee success and allow growth of a mutually supportive community of faculty and students. Participation in a pioneering career development program will support trainees in finding and preparing for a variety of post-PhD career paths. Recruitment and retention of an increasingly diverse group of students will be further strengthened by participating in pipeline and outreach programs, bridge activities for new students, and faculty training to enhance the inclusivity of the training environment and admissions process. All these activities -- building on the strengths of an exceptional cadre of trainees, trainers and institutional support ? will allow us to recruit and train the future leaders of 21st century genetics research.
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