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Mechanisms of Epigenetic Inheritance
Summary: Steven Henikoff performs experimental research on epigenetic inheritance, chromosome structure, and centromere evolution, and develops tools for sequence comparison, in vivo mapping, and functional genomics.
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 and evolutionary 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.
CenH3 nucleosomes wrap only a minute fraction of the genomic landscape and have received little attention relative to bulk nucleosomes. 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. We are investigating the implications of our surprising finding for centromere maintenance, function, and evolution.
CenH3 deposition does not appear to depend on DNA sequence, which implies that centromeres are faithfully inherited by an epigenetic process. To better understand the basis for CenH3 localization, we have purified soluble Drosophila CenH3 to identify the protein complex that assembles it into nucleosomes. The complex consists of CenH3, H4, and a single chaperone protein, RbAp48, which is also found in H3 and H3.3 assembly complexes. The simple generic composition of the machinery that deposits CenH3 might be an evolutionary adaptation to centromeric DNA, which consists of highly repetitive satellite sequences that are rapidly evolving in most plants and animals. Remarkably, CenH3s are also rapidly evolving, in contrast to H3 and H3.3, which are virtually invariant. We have found that CenH3s and CENP-C, which is another essential centromere foundation protein, are evolving by positive selection in plants and animals. What process underlies the rapid and adaptive evolution of the centromere, which is essential for the segregation of every chromosome each time a cell divides?
We have proposed that centromeres act selfishly during meiosis, and that foundation proteins, such as CenH3 and CENP-C, evolve to suppress meiotic drive. During female meiosis of plants and animals, only one of the four meiotic products succeeds, and the other three degenerate. A competition between ootids to achieve a favored orientation could result in meiotic drive, because a centromere that ends up in a favorable position at meiosis I is more likely to be included in the oocyte nucleus, leading to rapid fixation. A new satellite DNA expansion near an existing centromere would allow it to outcompete its homologous centromere for a favorable orientation, accounting for recurrent and rapid satellite evolution. Centromere foundation proteins would evolve to counter drive, and a perpetual arms race ensues. Centromere drive and suppression by foundation proteins might explain why centromeres of organisms with female meiosis are almost always composed of variable lengths of highly repetitive DNA.
Our studies of H3.3 have revealed that it is deposited by a distinct nucleosome assembly pathway. Whereas canonical H3 is incorporated strictly during DNA replication, amino acid changes toward H3.3 allow replication-independent (RI) deposition. Active genes are the predominant sites of RI deposition, and thus H3.3 is a marker for active chromatin. This paradigm is general, and we find that H3.3 marks active chromatin in the germline and during the early cleavage divisions of Caenorhabditis elegans embryos.
Replacement of H3 with H3.3 requires unraveling of the nucleosome, so that mapping of this chromatin repair process over the genome will identify sites at which chromatin is disrupted. To map histone replacement patterns genome-wide, we introduced an efficient profiling strategy: H3.3 and H3 are tagged with biotin in vivo, which allows for high-efficiency pull-down of tagged nucleosomes by streptavidin-coated beads. Pulled-down DNA is extracted and hybridized to genome-scale tiling microarrays to assay histone replacement at subnucleosomal resolution. Using this method, we found striking patterns of histone replacement over active Drosophila genes and transposons. Replacement occurs prominently at sites of abundant RNA polymerase II and active histone modifications throughout the Drosophila genome. Active genes are depleted of histones at promoters and are enriched in H3.3 from upstream to downstream of transcription units. Homeotic gene clusters display conspicuous peaks of histone replacement at boundaries of cis-regulatory domains superimposed over broad regions of low replacement. Peaks of histone replacement closely correspond to nuclease-hypersensitive sites, binding sites for Polycomb and trithorax group proteins, and sites of nucleosome depletion. These findings suggest the existence of a continuous process that disrupts nucleosomes and maintains accessibility of cis-regulatory elements.
Another mode of epigenetic inheritance is DNA methylation. We have introduced a method for profiling DNA methylation in Arabidopsis using microarrays, and have provided strong support for the role of DNA methylation in silencing transposons. In addition, we have discovered that a large fraction of Arabidopsis genes are methylated. Unlike transposon methylation, which occurs on nearly all cytosines, genic methylation is exclusively found at CG dinucleotides and is present in short dense clusters. Using tiling microarrays, we have mapped DNA methylation over the entire Arabidopsis genome at high resolution. DNA methylation covers transposons and is present within a large fraction of genes. Methylation within genes is conspicuously biased away from gene ends, and is strongly influenced by transcription: moderately transcribed genes are most likely to be methylated, while genes at either extreme are least likely. Transcription is in turn influenced by methylation: short methylated genes are poorly expressed, and loss of methylation in the body of a gene leads to enhanced transcription. Our results indicate that genic transcription and DNA methylation are closely interwoven processes.
In addition to our studies of chromatin inheritance, DNA methylation, and centromere evolution, we continue to develop tools to facilitate our own research and that of others. These have included computational tools for interpreting sequence information, such as SIFT (sorting intolerant from tolerant) for predicting deleterious mutations, and the DamID method for mapping in vivo binding targets of chromatin proteins and transcription factors.
We have also introduced a general reverse genetics strategy, called TILLING (targeting induced local lesions in genomes), whereby chemical mutagenesis is 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 (polymerase chain reaction) products are cleaved at single-base mismatches and the resulting fragments detected on electrophoretic gels. We have established TILLING services for the Arabidopsis, maize, and Drosophila communities. We have also applied the basic method to the discovery of rare polymorphisms, and have used this "Ecotilling" method to discover rare SNPs (single-nucleotide polymorphisms) in human DNA and new mutations in cancer.
Last updated: February 11, 2008
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