Research Areas:Molecular Biology and Biochemistry, Chromatin and Epigenetics
Research Interests and Goals
Regulation of gene expression is a fundamental biological process that determines growth, differentiation, identity and function of all cells. Our laboratory uses a combination of molecular biology and biochemistry techniques to dissect the mechanisms that regulate mammalian gene expression through histone proteins and post-translational modifications of chromatin.
In particular, we focus on histone phosphorylation, methylation and ubiquitylation, processes that act as molecular switches to both regulate and fine-tune gene transcription.
Our goals are to dissect the molecular details of how these modifications on various histones and histone variants work individually, as well as how they work together in combinations to diversify the regulatory outcomes on different cell functions.
Achieving these goals will not only yield greater insights into basic cell biology principles, but will also impact on our understanding of cancer and other diseases caused by dysregulation of gene expression.
The Cancer-Chromatin Connection
Cancer is often caused by dysregulated expression of oncogenes or tumour suppressor genes. Post-translational modifications of histone proteins is one of the fundamental mechanisms that regulates gene expression.
In the context of the human genome, DNA is complexed with histone proteins to form nucleosomes and chromatin (Figure 1). This functional organization of the genome is tightly regulated to ensure the appropriate gene expression patterns.
Active genes are located in euchromatin. Euchromatin has an open structure, which allows transcription factors to bind the promoters of these genes. Conversely, inactive genes are sequestered into compact structures called heterochromatin where they are silenced.
Disruption of this balance between euchromatin and heterochromatin can have disastrous effects on normal cell functions, and can lead to unrestricted cell growth, resulting in the development of cancer.
Our lab utilizes a combination of molecular biology and biochemistry techniques to dissect the mechanisms of how histones and histone modifications regulate gene functions.
We have two main questions:
- How do signal transduction pathways converge onto histones to regulate gene functions?
- How do histone variants function in the epigenetic regulation of gene expression?
To address how signal transduction pathways converge onto histones to regulate gene expression we are are studying the role of H3 phohorylation in the activation of immediate-early genes in mammalian cells.
The Role of Histone H3 Phosphorylation in Gene Activation
Upon growth factor stimulation, the MAP kinase pathway is activated and ultimately converges onto chromatin resulting in the phosphorylatin of H3 at two specific serine resides (S10 and S28). These phosphorylation events are very rapid and transient, and correlate with the transcriptional activation of genes such as c-fos and c-jun (Figure 1).
The mechanism of transcriptional activation by phosphorylated S10 and/or S28 remains unclear. Currently, we are testing the significance of phosphorylation at these two sites and how they interact with the transcriptional machinary to regulate immediate-early gene expression.
Our previous work (Cheung et al., 2000) showed that H3 phosphorylation promotes acetylation on the same histone and that these modifications function together to activate gene expression (Figure 2). This idea that histone modifications can work in combinations was formally described as the Histone Code Hypothesis (Strahl and Allis, 2000).
In addition, we are also developing new techniques and assays to dissect the intricate interplay between histone modifications in vivo.
To study the role of histone variants in the epigenetic regulation of gene expression, we are studying the H2A variant, H2A.Z.
Histone Variant H2A.Z and Epigenetic Regulation of Gene Expression
H2A.Z is the only histone variant that is essential for cell viability. Knocking down H2A.Z expression by RNAi in mammalian cells leads to cell death or senescence.
Cumulative evidence suggests that this H2A variant has both positive and negative regulatory effects on gene expression. H2A.Z localizes to the transcription start sites of genes and we have found that a fraction of H2A.Z is modified by a single ubiquitin group. Monoubiquitylated H2A.Z is associated with transcriptionally silenced heterochromatin, and is linked to polycomb silencing.
Currently, we are testing how addition or removal of the ubiquitin modification on H2A.Z regulates transcription and gene expression.
- Histone phosphorylation: chromatin modifications that link cell signaling pathways to nuclear function regulation
Lau, P. and Cheung, P. (Book Chapter) The Handbook of Cell Signaling. In press
- Monoubiquitylation of H2A.Z distinguishes its association with euchromatin or facultative heterochromatin
- Chromatin immunoprecipitation assay for mammalian tissues
Turner, F.B., Cheung, W.L., Cheung, P. Methods in Molecular Biology: Nuclear Programming. 2005 325: 261-272.
- The enhancement of histone H4 and H2A serine 1 phosphorylation during mitosis and S-phase is evolutionaily conserved
- Generation and characterization of antibodies directed against di-modified histones, and comments on antibody and epitope recognition
Cheung, P. Methods Enzmol. 2003 376: 221-234.
- Differentially methylated forms of histone H3 show unique association patterns with inactive human X chromosomes
- Follicle-stimulating hormone stimulates protein kinase A-mediated histone H3 phosphorylation and acetylation leading to select gene activation in ovarian granulosa cells
- Synergistic coupling of histone H3 phosphorylation and acetylation in response to epidermal growth factor stimulation
- Herpes simplex virus ICP27 induces cytoplasmic accumulation of unspliced polyadenylated alpha-globin pre-mRNA in infected HeLa cells
- Herpes simplex virus immediate-early proteins ICP0 and ICP4 activate the endogenous human alpha-globin gene in non-erythroid cells