The goals of our research are to understand how chromatin states are established and maintained and to understand how chromatin regulates transcription. We use a combination of biochemical, molecular biological, and genomics approaches.
The nucleosome is the basic repeating unit of chromatin. Nucleosomes are composed of approximately 150 bp of DNA that is wrapped around an octameric core of histone proteins. Nucleosomes cover the eukaryotic genome such that, depending on the organism or cell type, between 60 and 90 percent of the DNA is bound by histones. Because nucleosomes can restrict access to the DNA, the precise positioning of nucleosomes on DNA can have a profound impact on many basic biological processes.
The core histone proteins within the nucleosome mostly interact with the phosphate backbone of DNA, which allows nucleosomes to occupy almost any DNA sequence. Nevertheless, certain DNA sequences, those that are better able to adopt the severe bend and twist imposed by the histone octamer, are more readily occupied than others. Indeed, it is now apparent that regions of genomes have evolved, at least in part, to favor or disfavor nucleosomes.
Histones are decorated by many post-translational modifications. Distinct modification patterns are often correlated with basic DNA transactions such as gene transcription, DNA repair, and DNA replication and recombination. The function of many modifications is not well understood; however, some are known to facilitate open chromatin states by altering the charge of histones, whereas others serve as recruitment sites for trans-acting factors.
Nucleosomes and transcription
Figure 1: Chromatin structure at the 5’ end of a typical gene
Mapping of nucleosome positions genome-wide has shown that regulatory elements, such as gene promoters and enhancers, contain a stereotypical arrangement of nucleosomes (Figure 1). Directly upstream of the transcription start site lies a region of longer linker DNA termed the nucleosome depleted region (NDR). Nucleosomes surrounding the NDR are extensively modified and frequently contain the histone variant H2A.Z. The precise positioning (and composition) of NDR-proximal nucleosomes is controlled by a number of different ATP-dependent chromatin remodeling enzymes, which in some cases act in collaboration with transcription factors. The NDR is bound by many proteins and is very likely the site for the assembly of the RNA polymerase II preinitiation complex. Therefore, the establishment and maintenance of the NDR is of key importance for gene transcription.
Epigenetics and chromatin replication
The term epigenetics is commonly used to describe differences in programs of gene expression that result in different cell types, tissues, and organs, but are not based on changes in the DNA sequence. Underlying such phenomena are mechanisms that allow a particular gene expression pattern to be maintained through many cell divisions.
In our understanding, an epigenetic process has three key requirements: (i) the ability to regulate gene expression, (ii) maintenance through the cell cycle, and (iii) the ability to template its own duplication.
If histones (and their modifications) can be stably inherited, then the transcriptional outcomes they help specify may also persist. Therefore, particular chromatin structures and patterns of histone modification could facilitate epigenetic inheritance.
DNA replication and chromatin
During S-phase a complex and coordinated series of events must occur to ensure that chromatin is duplicated along with the DNA (Figure 2). First, directly ahead of the replication fork, histone-DNA interactions from preexisting (old) nucleosomes are disrupted. As the fork progresses the old histones are displaced from the DNA and a significant quantity are transferred onto the nascent DNA. During this process, the H2A/B dimers are thought to separate from the H3/H4 tetramer. Importantly, the tetramer itself likely remains intact and is not disseminated into H3/H4 dimers or free histones.
Figure 2: Replicating chromatin
Despite decades of investigation the mechanism of octamer segregation is far from clear, but some evidence suggests that the old tetramers (and the H2A/B dimers) segregate randomly to the daughter strands. Accompanying the recycling of the old histones, newly synthesized histones produced during S-phase are deposited to ensure that both daughters have a full complement of nucleosomes. The finding that old octamers are recycled into the new chromatin on the daughter DNA potentially provides a mechanism that explains how chromatin states and post-translational modifications can be inherited through generations: If subunits of the old parental octamers (either H2A/B dimers or H3/4 tetramers) are randomly segregated to the daughters, each daughter duplex could inherit approximately 50 percent of the parental histones. The parental histones could then recruit enzyme complexes that recognize their own modifications and catalyze the same modification on nearby nascent histones. In this way old parental histone modifications template their duplication.
Epigenetic expression patterns mediated by the inheritance of histones have received considerable attention and post-translational modifications of histones are typically referred to as “epigenetic” modifications. However, to date there is very little data that describes how histones and their modifications can be propagated through a cell cycle. Furthermore, we have only rudimentary models that explain how chromatin structures are duplicated during S-phase.