Chromatin, or the epigenome, as it is sometimes called, is the complex of DNA, histone proteins and associated macromolecules that package and organize eukaryotic genomes within the confines of the cell nucleus and facilitate cell- and tissue-specific programs of gene expression. Studies of chromatin, from the earliest stages of embryonic development to senescence, are important for understanding the basis of normal gene expression in health, and the causes of gene dysregulation in human disease.
The eukaryotic chromatin fiber is constituted by an array of nucleosomes—fundamental particles of DNA compaction—each containing 147 bp of DNA wound over an octameric histone core. Many thousands of nucleosomes connected by short DNA linkers are arrayed from one end of each chromosome to the other. At regulatory DNA elements, the placement, repositioning or depletion of nucleosomes control gene-specific expression while suppressing indiscriminate transcription genome-wide. Additional condensation and folding of nucleosome arrays, by mechanisms not fully understood, further compress the genome into the familiar mitotic chromosome structures visible by light microscopy.
Historically, our studies began with the application of nuclease digestion techniques to probe the chromatin structure of specific genes. We found that DNA sequences near transcription start sites are hypersensitive to DNase I digestion, providing initial evidence for structural changes in chromatin organization linked to early steps of gene expression. DNase I hypersensitive site mapping has since been widely employed to discover promoters, enhancers and locus control regions, and extended to powerful genome-wide approaches to nucleosome organization. By investigating the underlying causes of DNase I hypersensitivity, we discovered in 1994 the existence of ATP-dependent chromatin remodeling enzymes, the structure and function of which remain an abiding interest of the laboratory.
Principles of Chromatin Remodeling
Advances in the field over the past several decades have defined the main principles of nucleosome organization genome-wide. Anti-nucleosomal DNA sequences such as stretches of poly-A and poly-G nucleotides influence nucleosome exclusion at gene promoters and enhancers. These intrinsic properties of DNA work in combination with sequence-specific DNA binding protein factors, ATP-dependent chromatin remodeling enzymes, and numerous covalent histone modifying enzymes to create chromatin landscapes that are permissive or non-permissive for the assembly of the transcription machinery. The conserved NURF enzyme complex studied in our laboratory is a prototypical ATP-dependent chromatin remodeler that is essential for embryonic and post-embryonic development, affecting transcription of several hundred genes in the mouse and fly. Upon recruitment by sequence-specific transcription factors and histone methyl- and acetyl-lysine marks on chromatin, NURF acts by 'sliding' nucleosomes to cover or uncover regulatory DNA elements.
We are currently focused on yeast SWR1, a conserved 14-subunit ATP-dependent chromatin remodeling complex essential for viability in metazoans. SWR1 is dedicated to catalyzing the replacement of histone H2A in conventional nucleosomes with the histone variant H2A.Z. This histone variant is implicated in transcription, in part through altered nucleosome stability, and is universally localized to eukaryotic promoters and enhancers. Our studies of the replacement mechanism reveal step-wise eviction of an H2A-H2B dimer coupled to deposition of H2A.Z-H2B, in a reaction dependent on activation of the SWR1 ATPase by its two natural substrates. We are dissecting the histone replacement reaction by multiple approaches, including a long-term collaboration with Andres Leschziner's group at Harvard to elucidate the structure of SWR1. In related work, we are developing new methods to map the epigenomic landscape in the course of cell division cycles.
CenH3, also called CENP-A, is another variant of histone H3 that assembles into nucleosomes at chromosome centromeres. CenH3-containing nucleosomes provide the foundation of the kinetochore, a multi-protein superstructure that connects daughter chromosomes to microtubules of the mitotic and meiotic spindle, enabling chromosome segregation to opposite poles during cell division. We are studying the assembly, composition and function of this highly specialized nucleosome in budding yeast.
At Janelia, we interact with colleagues in optical physics, chemistry, and engineering at the cutting edge of fluorescence and electron microscopy, and with members of the Transcription Imaging Consortium. The technologies being developed and a highly collaborative environment provide fresh and unique opportunities to study the local dynamics of chromatin architecture in live cells. We are also beginning to explore the large scale, three-dimensional folding of chromatin fibers—a major, longstanding problem in chromosome biology.
As of August 13, 2012