Jef D. Boeke, Ph.D.
The aim of our TCNP technology project is to develop improved methods to identify genetic interactions (synthetic lethality) using microarrays (SLAM) and to define networks and pathways of interacting genes in the brewer's yeast, a relatively simple model for human cells. The special focus of the new technology is on developing methods for essential gene mutants (such as Ts and hypomorphic alleles, which can be systematically generated) or special mutants such as mutations at sites of modification. This complements other work we are doing in which loss of function or null alleles in nonessential genes are examined for genetic interactions. We are also planning to perform, in collaboration with the driving biological projects, systematic high copy screens in Ts mutants in the essential genes involved in lysine modification.
A major effort revolves around systematically mutating all sites at which the four core histones are modified and assessing the phenotypic consequences. Once a complete collection is in hand, we hope to use SLAM technology to build a genetic interaction map for the nucleosome. Joel Bader plays a major role in the resultant network analysis in this project as director of our computational core. His group is also taking responsibility for a histone mutation database, the first of its kind, which will integrate systematic information on mutant phenotypes, sites of modification and other important data on histones. We are also taking step to advance SLAM and related technologies, currently only applicable in yeast, into human systems.
Finally, the lab is collaborating extensively with driving biological projects led by Shelley Berger on histone modifications of various types, and with Cecile Pickart’s group on elucidating networks and pathways of ubiquitylation and SUMOylation. We are also collaborating with Phil Cole on networks and pathways of protein acetylation and Cynthia Wolberger on dissection of deacetylation pathways.
Figure 1. Systematic mutation of yeast histones H3 and H4.
The highly conserved histone proteins are subject to numerous post-translational modifications, especially at lysine residues. Systematic mutagenesis of histones, along with subsequent phenotypic and biochemical characterization provides a "model system" for studying the networks and pathways of lysine modification. To this end, we generated a library of histone H3 and H4 mutants that consists of an alanine scan as well as other systematic residue swaps and tail deletions, totaling 486 mutants, as shown below. To develop a maximally useful and flexible resource, we designed a synthetic cassette with unique features that allow (1) use of a wide range of selectable markers, (2) delivery either as a replication-competent episome or integrated at a native histone locus with high fidelity, and (3) use in complex phenotype analyses too labor intensive for individual mutants. Details of this cassette and initial characterization of these mutants have been published (Dai et al., 2008); mutant phenotypes can also be found in HistoneHits, a database for histone mutations and their phenotypes that has been produced by this TCNP. We are currently constructing similar mutant libraries for histones H2A and H2B.