Our laboratory is interested in epigenetic control of genome organization. In eukaryotic cells, genomic DNA is folded with histone and non-histone proteins in the form of chromatin. The building block of chromatin is the nucleosome, which contains 146 bp of DNA wrapped around an octamer of histones. Factors involved in covalent modifications of histones, together with chromatin-remodeling activities and DNA modifications, are components of intricate epigenetic mechanisms that help organize genomes into discrete domains that play important regulatory roles in almost every aspect of DNA metabolism. These epigenetic mechanisms gradually restrict the developmental potential of stem cells during differentiation and also constitute memories of gene activity that ensure faithful inheritance of cell identity. Defects in epigenetic regulation have been extensively demonstrated to have causal roles in numerous developmental disorders and cancers.
Recently, extensive efforts have been undertaken to identify new epigenetic histone modifications and the enzymes that catalyze these modifications. However, how histone-modifying activities are targeted to specific locations and how their activities are regulated is poorly understood. In addition, apart from a few well-known examples, how the cellular machinery interprets these modifications to achieve diverse epigenetic states is not clear. The fission yeast is a great model organism to study epigenetic regulation due to its highly conserved histone modifications, simple genome organization, and amenability to genetic and biochemical manipulations. Our laboratory use system biology approaches such as high throughput genetic screens, genomics, and proteomics combined with traditional genetic and biochemical analyses to study the role of epigenetic mechanisms in regulating the functions of the genome, which is essential to our understanding of stem cell and cancer biology.
1. Mechanisms of higher-order chromatin assembly
Eukaryotic genomes contain large amounts of repetitive DNA sequences, which are preferred sites for the assembly of heterochromatin structures. The formation of heterochromatin results in highly condensed chromosomal domains that limit the access of the transcription and recombination machinery. Consequently, heterochromatin is critical for regulating gene expression as well as maintaining genome integrity by rendering repetitive structures recombinationally inert and by prohibiting potentially mutagenic transposition events. Heterochromatin is also crucial for functional organization of vital chromosomal structures such as centromeres and telomeres.
Heterochromatin formation has been a paradigm for studying the role of histone modifications in regulating chromatin structures. The establishment of heterochromatin involves the concerted recruitment of diverse histone-modifying activities, culminating in the methylation of histone H3 lysine 9 (H3K9me) and the recruitment of HP1 proteins. One of the most intriguing aspects of heterochromatin assembly is that the underlying DNA repeats are transcribed, and the transcription process is required for the generation of RNAs for heterochromatin assembly through the RNA interference (RNAi) pathway. We are investigating how histone modifying enzymes and non-coding RNAs function together to organize heterochromatin domains.
2. Mechanisms of 3D genome organization
Eukaryotic chromosomes are arranged in distinct spatial and temporal patterns rather than being randomly scattered within the nucleus. It is increasingly recognized that spatial and temporal genome organization is essential for gene expression, DNA replication, and maintenance of genome stability. The inner nuclear membrane proteins provide anchoring points for the association of chromatin with the nuclear envelope and are critical for three-dimensional genome organization. Mutations of these proteins are associated with human diseases termed "nuclear envelopathies."
One of the most striking examples of genome organization is the "Rabl-like" configuration of chromosomes in the interphase nuclei, in which centromeres are clustered at the nuclear periphery at one side of the nucleus and telomeres are clustered on the opposite side of the nuclear envelope. This configuration of chromosomes has been seen in diverse cell types ranging from yeast to plants, flies, and mammals. We have identified a nuclear membrane associated protein complex that is directly links the centromeres to the nuclear envelope (Hou et al, 2012). We will further examine how centromere clustering regulates the 3D organization of genome and investigate the role of this organization in controlling the DNA damage response.
3. Using fission yeast to model human disease
Mutations of many epigenetic regulators have been causally linked to human diseases. Since the processes involved in chromatin metabolism in fission yeast are highly conserved with those of mammals, introducing human disease-associated mutations into yeast can recapitulate disease phenotypes. We will then use high throughput genetic screens to identify pathways required for the survival of these cells and design new approaches to inactivate these pathways to kill cells with disease-causing mutations.
As a test of principle, we used fission yeast cells containing extra chromosomes to model aneuploidy, which refers to the abnormal number of chromosomes. Aneuploidy is a common feature of most tumor cells and contributes to tumor progression. We performed a screen with the fission yeast deletion library and found that mutations in a number of genes cause sensitivity to the presence of additional chromosomes. Therefore, these gene products represent new targets for drug design to selectively kill aneuploid cancer cells. We will apply similar approaches to study other human diseases caused by mutations in epigenetic regulators.