Postdoctoral, University of Washington, Zoology, 1980.
Ph.D., Harvard University, Biochemistry and Molecular Biology, 1977.
There has been extraordinary progress in molecular biology during the 50-year span that began with the discovery of the DNA double helix and culminated with the nearly complete specification of our genetic inheritance. In contrast, the inheritance of differences between cells and tissues is poorly understood. To better understand inheritance that does not depend on DNA sequence, we apply genomic tools to the study of epigenetic markers, such as histone variants and DNA methylation.
The bulk of the eukaryotic genome is packaged into nucleosome particles, each of which comprises an octamer of four core histones--H2A, H2B, H3, and H4--which wrap nearly two turns of DNA. Nucleosomes can be differentiated both by numerous post-translational histone modifications and by incorporation of a few histone variants. H3 variants fall into three categories: canonical H3, which is deposited during replication; H3.3, which is the general constitutive form; and CenH3 (CENP-A in mammals), which is deposited exclusively at centromeres. We wondered whether the presence of CenH3 might result in a profoundly different nucleosome, and so we biochemically characterized CenH3 nucleosomes and directly visualized them in their native form. We found that, in stark contrast to octameric bulk nucleosomes, centromeric nucleosomes are stable heterotypic tetramers (hemisomes) with one copy of CenH3, H2A, H2B, and H4 each, wrapping only one turn of DNA.
To study the process whereby these unusual nucleosomes are deposited, we purified the Drosophila CenH3 assembly complex, and showed that its only non-histone component, the RbAp48 histone chaperone, can assemble CenH3 nucleosomes in vitro. We discovered that CenH3 nucleosomes induce positive DNA supercoils, implying a right-handed wrap. This is the opposite direction of wrapping of conventional nucleosomes. We then confirmed this finding in vivo for functional centromeres in budding yeast using centromere minichromosomes and conditional mutants. The right-handed wrapping of DNA around the histone core implied by positive supercoiling means that interaction surfaces between histones that prevent the nucleosome core from springing apart would be facing away from one another, consistent with hemisomes but not with octameric particles. The mutual incompatibility of nucleosomes with opposite topologies can potentially explain how centromeres are efficiently maintained as a unique loci on chromosomes.
We had previously shown that the H3.3 histone replacement variant marks sites of active chromatin. Because nucleosomes must be unraveled to replace H3/H4 with H3.3/H4, these findings suggested that the mapping of H3.3 patterns would provide insights into nucleosome dynamics. Indeed, we found striking patterns of replacement with H3.3 over active genes and transposons. Active genes were found to be depleted of histones at promoters but enriched in H3.3 from upstream to downstream of transcription units. Homeotic gene clusters displayed conspicuous peaks of histone replacement at boundaries of cis-regulatory domains superimposed over broad regions of low replacement. Peaks of histone replacement closely corresponded to binding sites for Polycomb and trithorax group proteins, and sites of nucleosome depletion. Our results suggested the existence of a continuous process that disrupts nucleosomes and maintains accessibility of cis-regulatory elements.
To better understand this disruptive process, we have applied epigenomic profiling to the classical salt fractionation method. Salt competes for interactions between the highly basic histone core and highly acidic DNA, and so salt-solubility measures a nucleosomal physical property. Chromatin fractions extracted with low salt after micrococcal nuclease digestion contain predominantly mononucleosomes and represent classical 'active' chromatin. We found that profiles of these low-salt soluble fractions displayed phased nucleosomes over transcriptionally active genes and correspond closely to profiles of the H2A replacement histone, H2A.Z.
We have also introduced a metabolic labeling strategy to obtain a more direct measure of nucleosome disruption genome-wide. Newly synthesized proteins are labeled with an amino acid analog, derivatized with a biotin moiety, nucleosome core particles are selectively extracted and affinity purified with streptavidin, and DNA is extracted for genome-wide profiling. We have successfully obtained genome-wide nucleosome turnover profiles for Drosophila cultured cells, and we have used these data to address the relationship between histone turnover and fundamental processes, including transcriptional initiation and elongation, epigenetic regulation and replication origin activity.
Another technology that we have introduced addresses the requirement for abundant cell-type-specific chromatin from tissues for epigenomic studies. A nuclear envelope protein is expressed under control of a cell-type-specific promoter, and in vivo biotin labeling is followed by affinity isolation of labeled nuclei to rapidly obtain large quantities of pure nuclei. We have applied this method to measure gene expression and chromatin features of the hair and non-hair cell types of the Arabidopsis root epidermis. We identified hundreds of genes that are preferentially expressed in each cell type and found that genes with the largest expression differences between hair and non-hair cells also show differences between cell types in H3K4me3 and H3K27me3. Our method should be applicable to any organism that is amenable to transformation.
DNA methylation represents another mode of epigenetic inheritance. To gain a better understanding of how DNA methylation is targeted and how it silences imprinted genes, we have used Arabidopsis as a model system, which combines powerful genetic tools and a small well-annotated gene-rich genome. Our mapping of DNA methylation genome-wide revealed the existence of gene-body CG methylation and its surprising interplay with transcription. We also profiled Arabidopsis DNA methylation genome-wide in the embryo and endosperm, and found that large-scale methylation changes accompany endosperm development and endosperm-specific gene expression. Transposable element fragments are extensively demethylated in the endosperm. We discovered several new imprinted genes by identifying candidates associated with differentially methylated regions. Our data suggest that imprinting in plants evolved from genome defense against transposable elements.
In comparing patterns of Arabidopsis DNA methylation with patterns of H2A.Z, we discovered that regions of DNA methylation are quantitatively deficient in H2A.Z. Exclusion of H2A.Z was seen at sites of DNA methylation in the bodies of actively transcribed genes and in methylated transposons. Mutation of the maintenance DNA methyltransferase, which causes both losses and gains of DNA methylation, engenders opposite changes in H2A.Z deposition, while mutation of the catalytic subunit of the SWR1 complex that deposits H2A.Z leads to genome-wide hypermethylation. Our findings indicate that DNA methylation can influence chromatin structure and effect gene silencing by excluding H2A.Z, and that H2A.Z protects genes from DNA methylation. This first documented example of a causal relationship between a DNA modification and a protein structural component of chromatin, led us to propose that exclusion of H2A.Z is a general mechanism for gene silencing by DNA methylation.
We also continue to develop computational and genomics tools. One is SIFT (for Sorting Intolerant From Tolerant), which uses evolutionary information to estimate the probability that a mutational change will be damaging to protein function. The SIFT server has become an important tool for predicting the effect of human non-synonymous polymorphisms. Our general reverse genetics strategy, called TILLING (Targeting Induced Local Lesions IN Genomes), uses chemical mutagenesis followed by screening for point mutations. The resulting allelic series can be used to determine gene function in the context of a whole organism. In our TILLING technique, heteroduplexes formed from denatured and annealed PCR products are cleaved at single-base mismatches and the resulting fragments detected on electrophoretic gels. We have established TILLING services for several organisms, and maintain public services for the Arabidopsis and Drosophila communities.
Honors and Awards
2005, National Academy of Sciences
Nucleosomes Are Context-Specific, H2A.Z-Modulated Barriers to RNA Polymerase.. Molecular cell. 53(5):819-30.. 2014.
CENP-A octamers do not confer a reduction in nucleosome height by AFM.. Nature structural & molecular biology. 21(1):4-5.. 2014.
Doxorubicin, DNA torsion, and chromatin dynamics.. Biochimica et biophysica acta. 1845(1):84-9.. 2014.
Transcription-generated torsional stress destabilizes nucleosomes.. Nature structural & molecular biology. 21(1):88-94.. 2014.
High-resolution mapping of transcription factor binding sites on native chromatin.. Nature methods. 11(2):203-9.. 2014.
Phylogeny as the basis for naming histones.. Trends in genetics : TIG. 29(9):499-500.. 2013.
Doxorubicin Enhances Nucleosome Turnover around Promoters.. Current biology : CB. 23(9):782-7.. 2013.
Regulation of nucleosome dynamics by histone modifications.. Nature structural & molecular biology. 20(3):259-66.. 2013.
Histone variants in pluripotency and disease.. Development (Cambridge, England). 140(12):2513-24.. 2013.
The heat shock response: A case study of chromatin dynamics in gene regulation.. Biochemistry and cell biology = Biochimie et biologie cellulaire. 91(1):42-8.. 2013.
ISWI and CHD Chromatin Remodelers Bind Promoters but Act in Gene Bodies.. PLoS genetics. 9(2):e1003317.. 2013.
The CentO satellite confers translational and rotational phasing on cenH3 nucleosomes in rice centromeres.. Proceedings of the National Academy of Sciences of the United States of America. 110(50):E4875-83.. 2013.
Mot1 Redistributes TBP from TATA-Containing to TATA-Less Promoters.. Molecular and cellular biology. 33(24):4996-5004.. 2013.
Sixty years of genome biology.. Genome biology. 14(4):113.. 2013.
Reconstitution of hemisomes on budding yeast centromeric DNA.. Nucleic acids research.. 2013.
Epigenetics & chromatin: interactions and processes.. Epigenetics & chromatin. 6(1):2.. 2013.
TILLING and Ecotilling for Rice.. Methods in molecular biology (Clifton, N.J.). 956:39-56.. 2013.
Surveying the epigenomic landscape, one base at a time.. Genome biology. 13(10):250.. 2012.
SIFT web server: predicting effects of amino acid substitutions on proteins.. Nucleic acids research. 40(Web Server issue):W452-7.. 2012.
Tripartite organization of centromeric chromatin in budding yeast.. Proceedings of the National Academy of Sciences of the United States of America. 109(1):243-8.. 2012.
Chromatin processes, epigenetic inheritance, centromere structure and function and evolution.. Current biology : CB. 22(4):R106-7.. 2012.