Genomic approaches to chromatin structure and function, and to epigenetic inheritance.

Public data sets:
http://users.umassmed.edu/Oliver.Rando/

Organisms sharing identical genomes may nonetheless exhibit heritable variation in traits that are now called “epigenetic” traits. Information carriers for epigenetic traits include prion conformation and cytosine methylation, and it is widely believed that the packaging of eukaryotic genomes into chromatin provides another carrier of epigenetic information. Epigenetics is of great interest for researchers interested in fields ranging from systems biology to evolution to human disease.

Our lab is broadly interested in epigenetic inheritance, but most of our research focuses on one putative carrier of epigenetic information – the nucleoprotein complex known as chromatin. We utilize “genomics” tools such as DNA microarrays and high-throughput sequencing to measure chromatin structure over entire genomes at single-nucleosome resolution, with the eventual goal of determining how chromatin states are established and maintained.

We consider the primary sequence of chromatin to consist of three major features – the positioning of nucleosomes relative to underlying genomic sequence, the covalent modification pattern of each nucleosome, and the histone variants comprising each histone octamer. To date, we have measured these three features in actively growing yeast cultures, and have also measured the exchange rates of histone H3 in G1-arrested yeast. A number of interesting features emerge from these studies:

Most interesting, we have identified what may be considered “motifs” in chromatin structure in yeast. For example, yeast promoters are characterized by a nucleosome-free region (NFR) of about 140 bp flanked by two well-positioned nucleosomes that lack a number of modifications such as H4K16ac, and that are rapidly replaced throughout the cell cycle. The +1 nucleosome also tends to carry Htz1 in place of H2A, while the -1 nucleosome carries Htz1 at a subset of promoters.

Another interesting feature of yeast chromatin is that covalent modifications tend to occur in a small number of highly-correlated groups, suggesting that histone modification patterns do not encode complex “messages.” Nonetheless, the abundance of covalent modifications (over 100 have been described!!!) raises the question of why so many exist.

By measuring histone replacement dynamics, we have found that coding regions are surprisingly “cold”, meaning the passage of RNA polymerase does not result in nucleosome replacement except at extremely high transcription rates. Heterochromatin is also cold, while promoter and tRNA genes are associated with hot nucleosomes. Chromatin boundaries – sequences whose presence prevents the lateral spread of silencing complexes from a nucleating element – are also associated with rapidly-exchanged nucleosomes, suggesting that “scrubbing” or chromatin by rapid replacement serves as the mechanism to limit this lateral spreading.

Our lab is interested in the following questions:

1) What are the rules by which chromatin motifs are generated? How do genomic sequences, and factors such as RNA polymerase passage, result in the common chromatin patterns seen at so many genes?

2) What are the mechanisms resulting in replication-independent histone replacement?

3) What are there so many histone modifications? What are the “systems” features that result from histone modification crosstalk?

4) More generally, how does chromatin act as a signal filter in cells, given its location between upstream signaling pathways and downstream transcriptional outcomes?

5) What happens to nucleosomes during genomic replication, and how do old nucleosomes influence the states of newly-incorporated nucleosomes? What is the machinery required to maintain a chromatin state?

6) How does chromatin structure change over evolution, and how do chromatin regulators contribute to phenotypic divergence in closely-related species?

7) What phenotypes are epigenetically heritable, and under what conditions is a selective advantage conferred by stochastic switching as opposed to plastic responsiveness to the environment?

8) How is the genome packaged in sperm and embryonic stem cells, and how do histone dynamics change during differentiation?