William J. Greenleaf, Ph.D. - US grants

DESCRIPTION (provided by applicant): Project summary In a single human cell, two meters of DNA is carefully packaged within a five-micron nucleus in such a way that allows complex biological necessities such as genome replication, DNA repair, and regulated gene expression. This spectacular organizational challenge is overcome through the hierarchical folding of DNA into chromatin. Our understanding of the structure of chromatin, from the level of individual nucleosomes at the ~100 bp level to higher-order, long-range interactions of chromosomes at the megabase level, is undergoing a profound expansion due to high-throughput methods of mapping structural elements to specific locations on the genome, allowing correlation with biological state. We expect the structure of chromatin at an intermediate length scale of ~2 kilobases to play a crucial role in regulating transcription, DNA replication, and DNA repair, but our structural understanding of this secondary structure of chromatin organization continues to lag behind our rapidly developing understanding of both the level of nucleosomes and higher-order, long-range interactions. After decades of work on the intermediate level of chromatin, the topology of this structure - or indeed the very existence of a well-stereotyped structure in vivo - is still holy debated, and almost nothing is known about the variability of these putative structures as a function of genome position. This pilot project builds methods that will develop a clearer picture of this scale of chromatin organization in order to integrate both the physical and biochemical views of the nucleus. We will study chromatin folding both in vivo and in vitro by applying ionizing radiation, which is known to generate correlated nicks to the DNA backbone at spatially proximal locations. The resulting single-stranded DNA fragments, which have ends that were within ~3 nm of each other in the folded chromatin structure, will be analyzed with high-throughput sequencing in order to map these fragments to the genome. This analysis will generate genome-wide pairwise distance constraints on the folded DNA. These data will provide an entirely new window into chromatin compaction and structure at the 30- nm length scale. Our investigations will begin with chromatin fibers assembled in vitro in order to troubleshoot and validate our methodology. Next we will investigate chromatin structure in S. cerevisiae, a model system with a small genome and extremely well characterized, well-positioned nucleosomes. Finally, we will pilot our chromatin structure mapping methodology in primary human fibroblasts and immortalized B-cells. This structural information will be combined with and compared to existing data sets that describe chromatin modifications and nuclease accessibility, laying the groundwork for an integrated physical model of chromatin structure by bridging the gap between our crystallographic understanding of the mononucleosome and our emerging understanding of the higher-order interactions at the megabase scale.

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.

DESCRIPTION: Nucleic-acid-protein interactions are fundamental to diverse biological processes from gene expression to epigenetic control. While the primary sequence of DNA or RNA sets the structural landscape that establishes the biological function of nucleic acids, our ability to predict how perturbations in sequence affect this structure- function relationship eithe at the intra- or inter-molecular interaction level, is limited. Because of the combinatorial complexity of these nucleic acid polymers - especially RNA - obtaining a comprehensive picture of the effects of multiple degrees of sequence perturbation necessarily requires high-throughput methods of assaying nucleic acid species. To this end, we have developed a platform for quantitative biochemistry of tens to hundreds of millions of diverse DNA or RNA molecules on an Illumina sequencing chip. By generating a diverse library of DNA sequences to be probed, we have constructed a post hoc DNA array, using the sequencing data to define the sequences of the clonal clusters (each containing approximately 500 fragments of DNA) on the chip. To probe RNA structures, where the need for combinatorial investigations to probe both structure and function is most acute, we use E. coli RNA polymerase to transcribe the immobilized dsDNA fragments into single stranded RNA, which remains bound to its DNA of origin via a stable, stalled RNAP. Using this RNA array, and custom built fluorescence analysis software, we have demonstrated comprehensive investigations of binding affinities of fluorescently labeled MS2 coat protein, a canonical RNA binding protein. By measuring the equilibrium constants and off-rates for MS2 for all possible single, double, and triple point mutants of the consensus stem-loop sequence, we demonstrate the power of this comprehensive analysis for understanding structure-function relationships in the context of the crystal structure of the interactions, as well as understanding the evolutionary functional constraints of these interactions. By developing three different methods of generating diverse libraries of DNA and RNA on-chip, we will probe the relative affinities of Cas9 and TALEN for target sequences across all near-cognate sequences and across the entire genome. These quantitative investigations will provide detailed biophysical information about the specificity of these protein, as well as their propensity for off-target binding. We will also develop three orthogonal methods for measuring RNA structure on-chip, including FRET-based methods to enable thermodynamic melting measurements. With these methods, we will carry out massive measurements of RNA stability across sequence space, probing all possible short hairpin structures as well as internally mismatched stem loops. These data will multiply the number of thermodynamic measurements of RNA by many orders of magnitude, and will be easily added to current RNA structure prediction suites. Finally we will push the sensitivity of this high-throughput platform o the single molecule level. As proof-of-principle, we will observe the kinetics of folding of divers DNA hairpins, opening the door to single-molecule methods across millions of diverse nucleic acid structures.

Project Summary This application is an administrative supplement request for 5UM1HG009436. The ENCODE project has produced high-resolution, high-quality maps of components of the `regulome' in a set of tissues and cell lines, identifying a collection of putative regulatory elements. Our proposal aims to generate and release data that test the functional relevance of these putative elements with high-throughput, pooled CRISPR screens, as well as a number of other methods that are aimed to understand the relationship between transcription factor levels, global accessibility at regulatory elements, an the effects on gene expression.

In addition to the transcription of protein coding genes in the genome, a large amount of transcription encodes RNA molecules that do not generate mRNA. These noncoding RNAs play important roles in the cell that include regulating dosage compensation, controlling genomic imprinting and regulating transcription. However, human cells transcribe thousands of noncoding RNAs and we have only ascribed functions to a small number. One of the main challenges to understanding the functions of noncoding RNAs is that technologies to rapidly identify and characterize noncoding RNAs are lacking. In this proposal, we develop a novel method that makes it possible to identify, in any cell type, all of the noncoding RNAs that interact with chromosomes and at the same time map the sites where those RNAs bind chromatin. Our approach involves directly linking noncoding RNAs to the underlying DNA by generating a covalent chimera between a chromosome bound RNA and DNA. Using next generation sequencing, we can identify the RNAs in the cell that are likely to regulate chromosome structure or function and define their sites of action on the chromosome. In our first Aim we use Drosophila cells to develop this approach, taking advantage of the fact that established chromosomal RNAs, roX1 and roX2, are known to coat the X chromosome to accomplish dosage compensation in the fly. We then broaden this approach in Aim 2 and identify the RNAs that bind chromatin throughout the human genome and develop a new analytical infrastructure to classify and functionally assign these RNAs. In Aim 3 develop perturbation experiments to test the functions of noncoding RNAs and RNA motifs for their impact on local chromosome accessibility, histone modification state and transcriptional output. We apply a system to redirect noncoding RNAs to new genomic regions to test their functional impact on chromosomes and to regulate different genomic regions through RNA dependent control. By defining the landscape of chromatin associated RNAs in humans and the sites that they regulate in the cell our proposal how these RNAs function as well as the impacts of defects in RNA dependent control that result in cellular dysfunction.

ABSTRACT: Organ Specific Project Beyond nutrient absorption, the gastrointestinal tract has wide-ranging effects on the normal and diseased physiologies of other organ systems, including metabolism, neural function, and the immune system. A high-resolution map of the bowel would be an invaluable resource to understand normal bowel function and the perturbations that lead to disease. We propose to create this map along the length of the small bowel and colon, both of which have nuanced geographic specializations of function. Using single cell RNA-seq and single cell ATAC-seq, we will profile the gene expression and regulatory programs that define the complex cell populations that drive bowel function. We will use CODEX, a highly-multiplexed, antibody-based mapping method, to define the spatial relationships of these cell populations. These investigations will be performed on tissues that are preserved and procured in a manner suitable for human bowel transplantation. Are target milestone is to characterize the small bowel and colon tissues from a total of 22 individuals over the course of this four-year effort. Our protocol to obtain tissues from human organ donors whose families have provided broad, open access consent is in place, and we are actively collecting tissues for other studies.