Michiel Vermeulen: Proteomics and Chromatin Biology
Tel.: 088 75 50829/68086
Fax: 088 75 68101
Email: M.Vermeulen-3 - at - umcutrecht.nl
Vermeulen Group pictures 2009
In a eukaryotic nucleus, DNA is packed in a structural polymer called chromatin. Chromatin serves to store genetic material, but also plays an active role in regulating processes such as DNA repair, replication and transcription. The nucleosome, an octamer of four different histone proteins around which the DNA is wrapped, represents the basic repeating unit within chromatin. Nucleosomes pose a barrier for reading the stored DNA-sequence information. Recently, a large number of transcription factors have been identified and characterized that are able to alter the structure of chromatin and by doing so are able to regulate the accessibility and transcriptional activity of genes. Of particular interest are proteins and protein complexes that post-translationally modify histones (so-called chromatin “writers”). These modifications include acetylation, phosphorylation, methylation and ubiquitination which are thought to provide an epigenetic “barcode” that partially determines which genes are expressed in a given cell type at any particular time point.
Emerging evidence causally links epigenetic alterations of chromatin to a disturbed proliferation - differentiation balance implicated in many diseases, including cancer. These epigenetic alterations, unlike genetic mutations, are in pinciple all reversible. Chromatin regulators are therefore seen as promising targets for the development of so-called epidrugs. To gain insights into the physiological and pathological versions of these enzymes it is not only essential to determine their genomic targets but also to decipher their downstream effects. One of these effects is thought to be the recruitment of stabilization of protein complexes that subsequently can exert their function at the site of recruitment.
We apply high-accuracy quantitative mass spectrometry to study chromatin structure and function in general and histone and DNA modifications in particular. Our long term goal is to understand the mechanisms via which histone and DNA modifications contribute to regulate processes in the nucleus such as transcription and DNA repair. Our current main interests are:
- To decipher and functionally characterize the histone methyl lysine interactome using a SILAC-based peptide pull-down approach (see figure).
- To study interactions with methylated and hydroxymethylated DNA during embryonic stem cell differentiation.
- The identification and functional characterization of novel protein complexes involved in chromatin structure and function and their dynamics during stem cell differentiation.
- Establishment of technology that allows the identification and quantitation of proteins that are brought down in chromatin immunoprecipitation (ChIP) experiments.
Mass spectrometry-based quantitative proteomics forms the basis of many of our experiments. The department recently acquired an LTQ-Orbitrap-Velos mass spectrometer. In addition, we apply classic biochemical techniques such as recombinant protein expression and purification, protein tagging in mammalian cells, ChIP-sequencing and functional knock-down approaches.
Figure: a histone methyl lysine interactome.
A schematic overview of data that was obtained in a large scale quantitative proteomics screening to identify novel protein complexes binding to histone lysine trimethylations on histone H3 and H4. Proteins interacting with the five trimethyl lysine marks that were studied are indicated. Encircled are proteins that were additionally identified in GFP pull-down experiments; baits in these pull-downs are underlined. For proteins that are color coded red in vivo verification by ChIP-seq was also provided. The results that were obtained during this study may serve as a start point for novel therapeutic strategies aimed at disrupting interactions between histone lysine methylations and their readers in tumors that are characterized by aberrant histone methylation patterns. For more information refer to Vermeulen et al., Cell. 2010 Sep 17;142(6):967-80.