The School of Molecular and Cellular Biology at the University of Illinois at Urbana-Champaign

In eukaryotic cells, genomic DNA is associated with proteins to form the complex referred to as chromatin. This includes abundant architectural proteins such as histones about which DNA is wrapped to form the repeating structural subunit of chromatin, nucleosomes, and a diverse array of markedly less abundant transcription factors and enzymes. The accessibility of DNA for gene transcription, replication, recombination and repair within chromatin is determined at the nucleotide level by the binding of histones and at higher levels of chromatin organization by the extent to which nucleosomal filaments are folded upon themselves. The compaction of DNA achieved through histone:DNA interactions and higher order folding of chromatin are required to fit genomes within nuclei but must be altered locally for DNA-templated activities such as gene transcription to occur. Biologists have long recognized that post-translational modifications of histones and other nuclear proteins such as acetylation, methylation, phosphorylation, poly(ADP-ribosylation) and ubiquitination, are associated with gene activation, mitosis and meiosis, but elucidating how these events are linked has been complicated by the low abundance and lability of modified forms of histones and other nuclear proteins and the even lower abundance of the enzymes that add or remove these modifications.

Our work strives to elucidate the signaling pathways responsible for specific nuclear protein modifications and the molecular mechanisms by which these modifications affect chromatin structure and DNA-templated processes. We use a variety of biochemical and molecular biology techniques in model systems ranging from yeast to mammalian cells. The experimental approaches include:

1) Biochemical purification in conjunction with mass spectrometry and other proteomics techniques are used to characterize the repertoire of modified chromosomal proteins found in cells under different physiological conditions. Similar approaches are also employed to identify enzymes responsible for specific modifications and characterize their interactions with other proteins in the signaling pathways that regulate them.

2) Raising antibodies specific for modified forms of proteins and using these in immunocytochemistry and immunofractionations (e.g. chromatin immunoprecipitation) to further determine “when and where” modifications occur within cells.

3) Engineering cells to express proteins in which modification sites have been mutated to determine the roles of specific modifications in vivo.

This work contributes to our understanding of cell biology in general and the mechanisms underlying the regulation of growth, development and senescence of all eukaryotic organisms. Aberrant modification of nuclear proteins is increasingly implicated in the etiology of human diseases including cancer and we hope that our work will enable the development of new therapeutic strategies.