Who is william greenleaf

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View all. Associate Professor of Genetics, Stanford University. Verified email at stanford. Articles Cited by Public access. Title Sort Sort by citations Sort by year Sort by title. Current protocols in molecular biology 1 , The American Journal of Human Genetics 93 5 , , Articles 1—20 Show more. On a visit to St. It is not only the beginning of a great work, capable of indefinite extension, but each step in its progress and the first step in its commencement involve the sacrifice both of time and money.

During his chancellorship, the first women graduated, the first African-American was admitted, the School of Fine Arts was established, and the university celebrated its silver anniversary. William Greenleaf Eliot, scholar, literary figure, civic leader, social reformer and educator, died on January 23, , after a lengthy illness.

After calibrating RICC-seq signal to three-dimensional distances, we show that compact two-start helical fibre structures with stacked alternating nucleosomes are consistent with RICC-seq fragmentation patterns from H3K9me3-marked chromatin, while non-compact structures and solenoid structures are consistent with open chromatin.

Our data support a model of chromatin architecture in intact interphase nuclei consistent with variable longitudinal compaction of two-start helical fibres.

The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer-promoter interactions, specifically at regions of shared DNA accessibility.

These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets.

Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases.

We identify three dominant patterns of transcription factor TF activation that drive leukemia regulomes, as well as TF deactivations that alter host T cells in CTCL patients. Clinical response to histone deacetylase inhibitors HDACi is strongly associated with a concurrent gain in chromatin accessibility.

These results provide a foundational framework to study personal regulomes in human cancer and epigenetic therapy. We present Omni-ATAC, an improved ATAC-seq protocol for chromatin accessibility profiling that works across multiple applications with substantial improvement of signal-to-background ratio and information content.

The Omni-ATAC protocol enables the interrogation of personal regulomes in tissue context and translational studies. The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular cues.

Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption, but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells.

Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals.

Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper TH17 cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs. Most disease variants lie within noncoding genomic regions, making their functional interpretation challenging. Because chromatin openness strongly influences transcriptional activity, we hypothesized that cell-type-specific open chromatin regions OCRs might highlight disease-relevant noncoding sequences.

To investigate, we mapped global OCRs in neurons differentiating from hiPSCs, a cellular model for studying neurodevelopmental disorders such as schizophrenia SZ. Our study shows that noncoding disease variants in OCRs can affect neurodevelopment, and that analysis of open chromatin regions can help prioritize functionally relevant noncoding variants identified by GWAS.

Chromosome conformation is an important feature of metazoan gene regulation; however, enhancer-promoter contact remodeling during cellular differentiation remains poorly understood. To address this, genome-wide promoter capture Hi-C CHi-C was performed during epidermal differentiation.

Two classes of enhancer-promoter contacts associated with differentiation-induced genes were identified. The first class 'gained' increased in contact strength during differentiation in concert with enhancer acquisition of the H3K27ac activation mark. The second class 'stable' were pre-established in undifferentiated cells, with enhancers constitutively marked by H3K27ac. The stable class was associated with the canonical conformation regulator cohesin, whereas the gained class was not, implying distinct mechanisms of contact formation and regulation.

Analysis of stable enhancers identified a new, essential role for a constitutively expressed, lineage-restricted ETS-family transcription factor, EHF, in epidermal differentiation. Furthermore, neither class of contacts was observed in pluripotent cells, suggesting that lineage-specific chromatin structure is established in tissue progenitor cells and is further remodeled in terminal differentiation. Genome conformation is central to gene control but challenging to interrogate.

Here we present HiChIP, a protein-centric chromatin conformation method. HiChIP of cohesin reveals multiscale genome architecture with greater signal-to-background ratios than those of in situ Hi-C. Spatial organization of the genome plays a central role in gene expression, DNA replication, and repair.

But current epigenomic approaches largely map DNA regulatory elements outside of the native context of the nucleus. Here we report assay of transposase-accessible chromatin with visualization ATAC-see , a transposase-mediated imaging technology that employs direct imaging of the accessible genome in situ, cell sorting, and deep sequencing to reveal the identity of the imaged elements.

ATAC-see revealed the cell-type-specific spatial organization of the accessible genome and the coordinated process of neutrophil chromatin extrusion, termed NETosis. Integration of ATAC-see with flow cytometry enables automated quantitation and prospective cell isolation as a function of chromatin accessibility, and it reveals a cell-cycle dependence of chromatin accessibility that is especially dynamic in G1 phase.

The integration of imaging and epigenomics provides a general and scalable approach for deciphering the spatiotemporal architecture of gene control. We define the chromatin accessibility and transcriptional landscapes in 13 human primary blood cell types that span the hematopoietic hierarchy.

Exploiting the finding that the enhancer landscape better reflects cell identity than mRNA levels, we enable 'enhancer cytometry' for enumeration of pure cell types from complex populations. We identify regulators governing hematopoietic differentiation and further show the lineage ontogeny of genetic elements linked to diverse human diseases. In acute myeloid leukemia AML , chromatin accessibility uncovers unique regulatory evolution in cancer cells with a progressively increasing mutation burden.

Single AML cells exhibit distinctive mixed regulome profiles corresponding to disparate developmental stages.

A method to account for this regulatory heterogeneity identified cancer-specific deviations and implicated HOX factors as key regulators of preleukemic hematopoietic stem cell characteristics. Thus, regulome dynamics can provide diverse insights into hematopoietic development and disease. Metastases are the main cause of cancer deaths, but the mechanisms underlying metastatic progression remain poorly understood. We isolated pure populations of cancer cells from primary tumors and metastases from a genetically engineered mouse model of human small cell lung cancer SCLC to investigate the mechanisms that drive the metastatic spread of this lethal cancer.

Genome-wide characterization of chromatin accessibility revealed the opening of large numbers of distal regulatory elements across the genome during metastatic progression. These changes correlate with copy number amplification of the Nfib locus, and differentially accessible sites were highly enriched for Nfib transcription factor binding sites. Nfib is necessary and sufficient to increase chromatin accessibility at a large subset of the intergenic regions.

Nfib promotes pro-metastatic neuronal gene expression programs and drives the metastatic ability of SCLC cells. The identification of widespread chromatin changes during SCLC progression reveals an unexpected global reprogramming during metastatic progression. Cancer sequencing studies have primarily identified cancer driver genes by the accumulation of protein-altering mutations. An improved method would be annotation independent, sensitive to unknown distributions of functions within proteins and inclusive of noncoding drivers.

We employed density-based clustering methods in 21 tumor types to detect variably sized significantly mutated regions SMRs. SMRs demonstrate spatial clustering of alterations in molecular domains and at interfaces, often with associated changes in signaling. Mutation frequencies in SMRs demonstrate that distinct protein regions are differentially mutated across tumor types, as exemplified by a linker region of PIK3CA in which biophysical simulations suggest that mutations affect regulatory interactions.

The functional diversity of SMRs underscores both the varied mechanisms of oncogenic misregulation and the advantage of functionally agnostic driver identification.

Here we survey variation and dynamics of active regulatory elements genome-wide using longitudinal samples from human individuals. Over 4, predicted regulatory elements 7. Gender was the most significant attributable source of variation. ATAC-seq revealed previously undescribed elements that escape X chromosome inactivation and predicted gender-specific gene regulatory networks across autosomes, which coordinately affect genes with immune function.

Noisy regulatory elements with personal variation in accessibility are significantly enriched for autoimmune disease loci. Over one third of regulome variation lacked genetic variation in cis, suggesting contributions from environmental or epigenetic factors.

These results refine concepts of human individuality and provide a foundational reference for comparing disease-associated regulomes. Cell-to-cell variation is a universal feature of life that affects a wide range of biological phenomena, from developmental plasticity to tumour heterogeneity. Although recent advances have improved our ability to document cellular phenotypic variation, the fundamental mechanisms that generate variability from identical DNA sequences remain elusive.

Here we reveal the landscape and principles of mammalian DNA regulatory variation by developing a robust method for mapping the accessible genome of individual cells by assay for transposase-accessible chromatin using sequencing ATAC-seq integrated into a programmable microfluidics platform.

Single-cell ATAC-seq scATAC-seq maps from hundreds of single cells in aggregate closely resemble accessibility profiles from tens of millions of cells and provide insights into cell-to-cell variation. Accessibility variance is systematically associated with specific trans-factors and cis-elements, and we discover combinations of trans-factors associated with either induction or suppression of cell-to-cell variability. We further identify sets of trans-factors associated with cell-type-specific accessibility variance across eight cell types.

Targeted perturbations of cell cycle or transcription factor signalling evoke stimulus-specific changes in this observed variability. The pattern of accessibility variation in cis across the genome recapitulates chromosome compartments de novo, linking single-cell accessibility variation to three-dimensional genome organization. Single-cell analysis of DNA accessibility provides new insight into cellular variation of the 'regulome'. A decade of rapid method development has begun to yield exciting insights into the 3D architecture of the metazoan genome and the roles it may play in regulating transcription.

Here we review core methods and new tools in the modern genomicist's toolbox at three length scales, ranging from single base pairs to megabase-scale chromosomal domains, and discuss the emerging picture of the 3D genome that these tools have revealed. Blind spots remain, especially at intermediate length scales spanning a few nucleosomes, but thanks in part to new technologies that permit targeted alteration of chromatin states and time-resolved studies, the next decade holds great promise for hypothesis-driven research into the mechanisms that drive genome architecture and transcriptional regulation.

Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell iPSC reprogramming factors Oct4, Sox2, Klf4 and c-Myc OSKM followed by cell expansion in media that promote lineage differentiation.

Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency.

We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced 'transdifferentiation' pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by various methods.

Spectacular advances in the throughput of DNA sequencing have allowed genome-wide analysis of epigenetic features such as methylation, nucleosome position and post-translational modification, chromatin accessibility and connectivity, and transcription factor binding. However, for rare or precious biological samples, input requirements of many of these methods limit their application. In this review we discuss recent advances for low-input genome-wide analysis of chromatin immunoprecipitation, methylation, DNA accessibility, and chromatin conformation.

This unit describes Assay for Transposase-Accessible Chromatin with high-throughput sequencing ATAC-seq , a method for mapping chromatin accessibility genome-wide.

This method probes DNA accessibility with hyperactive Tn5 transposase, which inserts sequencing adapters into accessible regions of chromatin. Sequencing reads can then be used to infer regions of increased accessibility, as well as to map regions of transcription-factor binding and nucleosome position. The method is a fast and sensitive alternative to DNase-seq for assaying chromatin accessibility genome-wide, or to MNase-seq for assaying nucleosome positions in accessible regions of the genome.

Paired-end sequencing has enabled a variety of new methods for high-throughput interrogation of both genome structure and chromatin architecture. Here, we discuss how the paired-end paradigm can be used to interpret sequencing data as biophysical measurements of in vivo chromatin structure that report on single molecules in single cells. Conditional gene deletion in mice has contributed immensely to our understanding of many biological and biomedical processes.

Despite an increasing awareness of nonprotein-coding functional elements within protein-coding transcripts, current gene-targeting approaches typically involve simultaneous ablation of noncoding elements within targeted protein-coding genes. The potential for protein-coding genes to have additional noncoding functions necessitates the development of novel genetic tools capable of precisely interrogating individual functional elements.

Importantly, cassette inversion maintains physiologic transcript structure, thereby ensuring proper microRNA-mediated regulation of the GFP reporter, as well as maintaining expression of nonprotein-coding elements.

To test this potentially generalizable strategy, we generated and analyzed mice with this conditional knockin reporter targeted to the Hmga2 locus. RNA-protein interactions drive fundamental biological processes and are targets for molecular engineering, yet quantitative and comprehensive understanding of the sequence determinants of affinity remains limited.

Studying the MS2 coat protein, we decompose the binding energy contributions from primary and secondary RNA structure, and observe that differences in affinity are often driven by sequence-specific changes in both association and dissociation rates. By analyzing the biophysical constraints and modeling mutational paths describing the molecular evolution of MS2 from low- to high-affinity hairpins, we quantify widespread molecular epistasis and a long-hypothesized, structure-dependent preference for G:U base pairs over C:A intermediates in evolutionary trajectories.

Our results suggest that quantitative analysis of RNA on a massively parallel array RNA-MaP provides generalizable insight into the biophysical basis and evolutionary consequences of sequence-function relationships.

Although individual pauses have been studied in vitro, the determinants of pauses in vivo and their distribution throughout the bacterial genome remain unknown. In vitro single-molecule and ensemble analyses demonstrate that these pauses result from RNAP-nucleic acid interactions that inhibit next-nucleotide addition. The consensus sequence also leads to pausing by RNAPs from diverse lineages and is enriched at translation start sites in both E. Our results thus reveal a conserved mechanism unifying known and newly identified pause events.

Limb-girdle muscular dystrophy primarily affects the muscles of the hips and shoulders the "limb-girdle" muscles , although it is a heterogeneous disorder that can present with varying symptoms. There is currently no cure. We sought to identify the genetic basis of limb-girdle muscular dystrophy type 1 in an American family of Northern European descent using exome sequencing. Exome sequencing was performed on DNA samples from two affected siblings and one unaffected sibling and resulted in the identification of eleven candidate mutations that co-segregated with the disease.

Phe89Ile, which was recently identified as a cause of limb-girdle muscular dystrophy type 1D. Phe89Ile mutation likely arose independently of the previously reported mutation. Exome sequencing provided an unbiased and effective method for identifying the genetic etiology of limb-girdle muscular dystrophy type 1 in a previously genetically uncharacterized family. We describe an assay for transposase-accessible chromatin using sequencing ATAC-seq , based on direct in vitro transposition of sequencing adaptors into native chromatin, as a rapid and sensitive method for integrative epigenomic analysis.

ATAC-seq captures open chromatin sites using a simple two-step protocol with , cells and reveals the interplay between genomic locations of open chromatin, DNA-binding proteins, individual nucleosomes and chromatin compaction at nucleotide resolution. We discovered classes of DNA-binding factors that strictly avoided, could tolerate or tended to overlap with nucleosomes. Most ancient specimens contain very low levels of endogenous DNA, precluding the shotgun sequencing of many interesting samples because of cost.

We developed a simple, compact microfluidic device to perform high dynamic-range digital polymerase chain reaction dPCR in an array of isolated femtoliter microreactors. This device, made from polydimethylsiloxane PDMS , allows the samples to be loaded into all microreactors simultaneously. The microreactors are completely sealed through the deformation of a PDMS membrane. The small volume of the microreactors ensures a compact device with high reaction efficiency and low reagent and sample consumption.

Future potential applications of this platform include multicolor dPCR and massively parallel dPCR for next generation sequencing library preparation. We developed a multiplex sequencing-by-synthesis method combining terminal phosphate-labeled fluorogenic nucleotides TPLFNs and resealable polydimethylsiloxane PDMS microreactors.

In the presence of phosphatase, primer extension by DNA polymerase using nonfluorescent TPLFNs generates fluorophores, which are confined in the microreactors and detected. We immobilized primed DNA templates in the microreactors, then sequentially introduced one of the four identically labeled TPLFNs, sealed the microreactors and recorded a fluorescence image after template-directed primer extension.

With cycle times of View details for DOI We present details of the design, construction, and testing of a single-beam optical tweezers apparatus capable of measuring and exerting torque, as well as force, on microfabricated, optically anisotropic particles an "optical torque wrench". The control of angular orientation is achieved by rotating the linear polarization of a trapping laser with an electro-optic modulator EOM , which affords improved performance over previous designs.

The torque imparted to the trapped particle is assessed by measuring the difference between left- and right-circular components of the transmitted light, and constant torque is maintained by feeding this difference signal back into a custom-designed electronic servo loop.

In addition, we developed particles suitable for rotation in this apparatus using microfabrication techniques. Altogether, the system allows for the simultaneous application of forces approximately 0. As a proof of principle, we demonstrate how our instrument can be used to study the supercoiling of single DNA molecules.

We used single-molecule techniques to investigate the mechanism by which three representative terminators his, t, and tR2 destabilize the elongation complex EC. For his and tR2 terminators, loads exerted to bias translocation did not affect termination efficiency TE.

However, the force-dependent kinetics of release and the force-dependent TE of a mutant imply a forward translocation mechanism for the t terminator. We deduce that different mechanisms, involving hypertranslocation or shearing, operate at terminators with different U-tracts.

Tension applied to RNA at terminators suggests that closure of the final hairpin bases destabilizes the hybrid and that competing RNA structures modulate TE. We propose a quantitative, energetic model that predicts the behavior for these terminators and mutant variants. Riboswitches regulate genes through structural changes in ligand-binding RNA aptamers.

With the use of an optical-trapping assay based on in situ transcription by a molecule of RNA polymerase, single nascent RNAs containing pbuE adenine riboswitch aptamers were unfolded and refolded. Multiple folding states were characterized by means of both force-extension curves and folding trajectories under constant force by measuring the molecular contour length, kinetics, and energetics with and without adenine.

Distinct folding steps correlated with the formation of key secondary or tertiary structures and with ligand binding. Adenine-induced stabilization of the weakest helix in the aptamer, the mechanical switch underlying regulatory action, was observed directly.

These results provide an integrated view of hierarchical folding in an aptamer, demonstrating how complex folding can be resolved into constituent parts, and supply further insights into tertiary structure formation. A new arsenal of approaches, including single-molecule fluorescence, atomic-force microscopy, magnetic tweezers, and optical traps OTs have been employed to probe the many facets of the transcription cycle.

These approaches supply fresh insights into the means by which RNAP identifies a promoter, initiates transcription, translocates and pauses along the DNA template, proofreads errors, and ultimately terminates transcription. Results from single-molecule experiments complement the knowledge gained from biochemical and genetic assays by facilitating the observation of states that are otherwise obscured by ensemble averaging, such as those resulting from heterogeneity in molecular structure, elongation rate, or pause propensity.

Most studies to date have been performed with bacterial RNAP, but work is also being carried out with eukaryotic polymerase Pol II and single-subunit polymerases from bacteriophages. We discuss recent progress achieved by single-molecule studies, highlighting some of the unresolved questions and ongoing debates. Interdisciplinary work in the life sciences at the boundaries of biology, chemistry and physics is making enormous strides.