We have recently developed a genetic screening approach, termed E-MAP (Epistatic MiniArray Technology Profiling), that can quantify the strength of systematically generated pair-wise genetic interactions. The method identifies negative double mutant interactions, where combination of mutations causes defects leads to enhancement of growth defects or lethality. Such interactions often specify membership of parallel biochemical pathways. Additionally, E-MAP also identifies positive interactions, where combination of mutants show mutual suppression or lack any additive defects, which are enriched among physically interacting gene products. Using gene deletions of non-essential genes and hypomorphic alleles of essential genes, we have recently generated E-MAPs in S. cerevisiae that have focused on 1) the early secretory pathway and 2) chromosome biology, which includes transcriptional regulation, chromatin remodeling and DNA repair. We now propose the second generation of E-MAP analysis, allowing us to address the next level of complexity, via examination of point mutants of multifunctional and essential genes. Specifically, we will genetically dissect two essential, multisubunit, multifunctional complexes at the heart of gene expression and chromatin structure: RNA polymerase II (RNAPII) and the nucleosome. This approach will allow us to A) map the structural features of these complexes onto their functional roles, and B) characterize the functional relationships between RNAPII and the nucleosome and the wider gene expression apparatus.
In Aim #1, we will use the chromosome biology E-MAP to genetically examine a set of approximately 450 histone H3 and H4 mutants including A) complete alanine (or serine)-scans, B) comprehensive substitution of modifiable residues, and C) semisystematic deletions of the N-terminal tails. The work, which is being done collaboratively with NIH Roadmap TCNP (Technology Center for Networks and Pathways) of Lysine Modification (PI Jef Boeke), will help reveal how histone-histone and histone-DNA contacts and histone modifications influence the steps of transcription and chromatin regulation.
In Aim #2, we will screen approximately 100 distinct and diverse point mutants of several essential RNAPII subunits isolated in collaboration with Craig Kaplan and Roger Kornberg.
In Aim #3, we will subject these data to hierarchical clustering and our recently developed metrics (S- and COP-scoring systems) to help identify functional relationships using the E-MAP data. We will also employ newly developed algorithms that identify functionally related sets of genes (or modules) from large-scale interaction datasets and allows for multi-functional genes to be members of more than one module. We anticipate that a systematic genetic approach described here will provide a more holistic view of chromatin function and transcriptional regulation in eukaryotic cells.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Carter, Anthony D
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of California San Francisco
Schools of Medicine
San Francisco
United States
Zip Code
Nissen, Kelly E; Homer, Christina M; Ryan, Colm J et al. (2017) The histone variant H2A.Z promotes splicing of weak introns. Genes Dev 31:688-701
Janke, Ryan; Kong, Jeremy; Braberg, Hannes et al. (2016) Nonsense-mediated decay regulates key components of homologous recombination. Nucleic Acids Res 44:5218-30
Martin, Humberto; Shales, Michael; Fernandez-Piñar, Pablo et al. (2015) Differential genetic interactions of yeast stress response MAPK pathways. Mol Syst Biol 11:800
Patrick, Kristin L; Ryan, Colm J; Xu, Jiewei et al. (2015) Genetic interaction mapping reveals a role for the SWI/SNF nucleosome remodeler in spliceosome activation in fission yeast. PLoS Genet 11:e1005074
Ding, Li; Paszkowski-Rogacz, Maciej; Winzi, Maria et al. (2015) Systems Analyses Reveal Shared and Diverse Attributes of Oct4 Regulation in Pluripotent Cells. Cell Syst 1:141-51
Sánchez, Arancha; Roguev, Assen; Krogan, Nevan J et al. (2015) Genetic Interaction Landscape Reveals Critical Requirements for Schizosaccharomyces pombe Brc1 in DNA Damage Response Mutants. G3 (Bethesda) 5:953-62
Braberg, Hannes; Alexander, Richard; Shales, Michael et al. (2014) Quantitative analysis of triple-mutant genetic interactions. Nat Protoc 9:1867-81
Kabeche, Ruth; Roguev, Assen; Krogan, Nevan J et al. (2014) A Pil1-Sle1-Syj1-Tax4 functional pathway links eisosomes with PI(4,5)P2 regulation. J Cell Sci 127:1318-26
Braberg, Hannes; Moehle, Erica A; Shales, Michael et al. (2014) Genetic interaction analysis of point mutations enables interrogation of gene function at a residue-level resolution: exploring the applications of high-resolution genetic interaction mapping of point mutations. Bioessays 36:706-13
Moehle, Erica A; Braberg, Hannes; Krogan, Nevan J et al. (2014) Adventures in time and space: splicing efficiency and RNA polymerase II elongation rate. RNA Biol 11:313-9

Showing the most recent 10 out of 55 publications