Parallel functional annotation of cancer-associated missense mutations in histone methyltransferases.

Using exome sequencing for biomarker discovery and precision medicine requires connecting nucleotide-level variation with functional changes in encoded proteins. However, for functionally annotating the thousands of cancer-associated missense mutations, or variants of uncertain significance (VUS), purifying variant proteins for biochemical and functional analysis is cost-prohibitive and inefficient. We describe parallel functional annotation (PFA) of large numbers of VUS using small cultures and crude extracts in 96-well plates. Using members of a histone methyltransferase family, we demonstrate high-throughput structural and functional annotation of cancer-associated mutations. By combining functional annotation of paralogs, we discovered two phylogenetic and clustering parameters that improve the accuracy of sequence-based functional predictions to over 90%. Our results demonstrate the value of PFA for defining oncogenic/tumor suppressor functions of histone methyltransferases as well as enhancing the accuracy of sequence-based algorithms in predicting the effects of cancer-associated mutations.

Biochemical reconstitution and phylogenetic comparison of human SET1 family core complexes involved in histone methylation.

Mixed lineage leukemia protein-1 (MLL1) is a member of the SET1 family of histone H3 lysine 4 (H3K4) methyltransferases that are required for metazoan development. MLL1 is the best characterized human SET1 family member, which includes MLL1-4 and SETd1A/B. MLL1 assembles with WDR5, RBBP5, ASH2L, DPY-30 (WRAD) to form the MLL1 core complex, which is required for H3K4 dimethylation and transcriptional activation. Because all SET1 family proteins interact with WRAD in vivo, it is hypothesized they are regulated by similar mechanisms. However, recent evidence suggests differences among family members that may reflect unique regulatory inputs in the cell. Missing is an understanding of the intrinsic enzymatic activities of different SET1 family complexes under standard conditions. In this investigation, we reconstituted each human SET1 family core complex and compared subunit assembly and enzymatic activities. We found that in the absence of WRAD, all but one SET domain catalyzes at least weak H3K4 monomethylation. In the presence of WRAD, all SET1 family members showed stimulated monomethyltransferase activity but differed in their di- and trimethylation activities. We found that these differences are correlated with evolutionary lineage, suggesting these enzyme complexes have evolved to accomplish unique tasks within metazoan genomes. To understand the structural basis for these differences, we employed a "phylogenetic scanning mutagenesis" assay and identified a cluster of amino acid substitutions that confer a WRAD-dependent gain-of-function dimethylation activity on complexes assembled with the MLL3 or Drosophila trithorax proteins. These results form the basis for understanding how WRAD differentially regulates SET1 family complexes in vivo.

Linking genetics to structural biology: complex heterozygosity screening with actin alanine scan alleles identifies functionally related surfaces on yeast actin.

Previous genome-level genetic interaction screens with the single essential actin gene of yeast identified 238 nonessential genes that upon deletion result in deleterious, digenic complex haploinsufficiences with an actin null allele. Deletion alleles of these 238 genes were tested for complex heterozygous interactions with 32 actin alanine scan alleles, which target clusters of residues on the surface of actin. A total of 891 deleterious digenic combinations were identified with 203 of the 238 genes. Two-dimensional hierarchical cluster analysis of the interactions identified nine distinct groups, and the alleles within clusters tended to affect localized regions on the surface of actin. The mutants in one cluster all affect electrostatic interactions between stacked subunits in the long pitch helix of the actin filament. A second cluster that contains the most highly interactive alleles may disrupt the tropomyosin/myosin system, as one of the mutants in that cluster cannot support Type V myosin-dependent movement of secretory vesicles in haploids and causes processivity defects in heterozygous diploids. These examples suggest the clusters represent mutations with shared protein-protein interaction defects. These results show that complex heterozygous interaction screens have benefit for detecting actin-related genes and suggest that having actin filaments of mixed composition, containing both mutant and wild-type subunits, presents unique challenges to the cell.

Automethylation activities within the mixed lineage leukemia-1 (MLL1) core complex reveal evidence supporting a "two-active site" model for multiple histone H3 lysine 4 methylation.

The mixed lineage leukemia-1 (MLL1) core complex predominantly catalyzes mono- and dimethylation of histone H3 at lysine 4 (H3K4) and is frequently altered in aggressive acute leukemias. The molecular mechanisms that account for conversion of mono- to dimethyl H3K4 (H3K4me1,2) are not well understood. In this investigation, we report that the suppressor of variegation, enhancer of zeste, trithorax (SET) domains from human MLL1 and Drosophila Trithorax undergo robust intramolecular automethylation reactions at an evolutionarily conserved cysteine residue in the active site, which is inhibited by unmodified histone H3. The location of the automethylation in the SET-I subdomain indicates that the MLL1 SET domain possesses significantly more conformational plasticity in solution than suggested by its crystal structure. We also report that MLL1 methylates Ash2L in the absence of histone H3, but only when assembled within a complex with WDR5 and RbBP5, suggesting a restraint for the architectural arrangement of subunits within the complex. Using MLL1 and Ash2L automethylation reactions as probes for histone binding, we observed that both automethylation reactions are significantly inhibited by stoichiometric amounts of unmethylated histone H3, but not by histones previously mono-, di-, or trimethylated at H3K4. These results suggest that the H3K4me1 intermediate does not significantly bind to the MLL1 SET domain during the dimethylation reaction. Consistent with this hypothesis, we demonstrate that the MLL1 core complex assembled with a catalytically inactive SET domain variant preferentially catalyzes H3K4 dimethylation using the H3K4me1 substrate. Taken together, these results are consistent with a "two-active site" model for multiple H3K4 methylation by the MLL1 core complex.

Chemical suppression of defects in mitotic spindle assembly, redox control, and sterol biosynthesis by hydroxyurea.

We describe the results of a systematic search for a class of hitherto-overlooked chemical-genetic interactions in the Saccharomyces cerevisiae genome, which exists between a detrimental genetic mutation and a chemical/drug that can ameliorate, rather than exacerbate, that detriment. We refer to this type of interaction as "chemical suppression." Our work was driven by the hypothesis that genome instability in a certain class of mutants could be alleviated by mild replication inhibition using chemicals/drugs. We queried a collection of conditionally lethal, i.e., temperature-sensitive, alleles representing 40% of the yeast essential genes for those mutants whose growth defect can be suppressed by hydroxyurea (HU), known as a potent DNA replication inhibitor, at the restrictive temperature. Unexpectedly, we identified a number of mutants defective in diverse cellular pathways other than DNA replication. Here we report that HU suppresses selected mutants defective in the kinetochore-microtubule attachment pathway during mitotic chromosome segregation. HU also suppresses an ero1-1 mutant defective for a thiol oxidase of the endoplasmic reticulum by providing oxidation equivalents. Finally, we report that HU suppresses an erg26-1 mutant defective for a C-3 sterol dehydrogenase through regulating iron homeostasis and in turn impacting ergosterol biosynthesis. We further demonstrate that cells carrying the erg26-1 mutation show an increased rate of mitochondrial DNA loss and delayed G1 to S phase transition. We conclude that systematic gathering of a compendium of "chemical suppression" of yeast mutants by genotoxic drugs will not only enable the identification of novel functions of both chemicals and genes, but also have profound implications in cautionary measures of anticancer intervention in humans.

Actin dosage lethality screening in yeast mediated by selective ploidy ablation reveals links to urmylation/wobble codon recognition and chromosome stability.

The actin cytoskeleton exists in a dynamic equilibrium with monomeric and filamentous states of its subunit protein actin. The spatial and temporal regulation of actin dynamics is critical to the many functions of actin. Actin levels are remarkably constant, suggesting that cells have evolved to function within a narrow range of actin concentrations. Here we report the results of screens in which we have increased actin levels in strains deleted for the ~4800 nonessential yeast genes using a technical advance called selective ploidy ablation. We detected 83 synthetic dosage interactions with actin, 78 resulted in reduced growth, whereas in 5 cases overexpression of actin suppressed the growth defects caused by the deleted genes. The genes were highly enriched in several classes, including transfer RNA wobble uridine modification, chromosome stability and segregation, cell growth, and cell division. We show that actin overexpression sequesters a limited pool of eEF1A, a bifunctional protein involved in aminoacyl-transfer RNA recruitment to the ribosome and actin filament cross-linking. Surprisingly, the largest class of genes is involved in chromosome stability and segregation. We show that actin mutants have chromosome segregation defects, suggesting a possible role in chromosome structure and function. Monomeric actin is a core component of the INO80 and SWR chromatin remodeling complexes and the NuA4 histone modification complex, and our results suggest these complexes may be sensitive to actin stoichiometry. We propose that the resulting effects on chromatin structure can lead to synergistic effects on chromosome stability in strains lacking genes important for chromosome maintenance.

Novel interactions between actin and the proteasome revealed by complex haploinsufficiency.

Saccharomyces cerevisiae has been a powerful model for uncovering the landscape of binary gene interactions through whole-genome screening. Complex heterozygous interactions are potentially important to human genetic disease as loss-of-function alleles are common in human genomes. We have been using complex haploinsufficiency (CHI) screening with the actin gene to identify genes related to actin function and as a model to determine the prevalence of CHI interactions in eukaryotic genomes. Previous CHI screening between actin and null alleles for non-essential genes uncovered ∼240 deleterious CHI interactions. In this report, we have extended CHI screening to null alleles for essential genes by mating a query strain to sporulations of heterozygous knock-out strains. Using an act1Δ query, knock-outs of 60 essential genes were found to be CHI with actin. Enriched in this collection were functional categories found in the previous screen against non-essential genes, including genes involved in cytoskeleton function and chaperone complexes that fold actin and tubulin. Novel to this screen was the identification of genes for components of the TFIID transcription complex and for the proteasome. We investigated a potential role for the proteasome in regulating the actin cytoskeleton and found that the proteasome physically associates with actin filaments in vitro and that some conditional mutations in proteasome genes have gross defects in actin organization. Whole-genome screening with actin as a query has confirmed that CHI interactions are important phenotypic drivers. Furthermore, CHI screening is another genetic tool to uncover novel functional connections. Here we report a previously unappreciated role for the proteasome in affecting actin organization and function.

Synthetic genetic array analysis in Saccharomyces cerevisiae provides evidence for an interaction between RAT8/DBP5 and genes encoding P-body components.

Coordination of the multiple steps of mRNA biogenesis helps to ensure proper regulation of gene expression. The Saccharomyces cerevisiae DEAD-box protein Rat8p/Dbp5p is an essential mRNA export factor that functions at the nuclear pore complex (NPC) where it is thought to remodel mRNA/protein complexes during mRNA export. Rat8p also functions in translation termination and has been implicated in functioning during early transcription. We conducted a synthetic genetic array analysis (SGA) using a strain harboring the temperature-sensitive rat8-2 allele. Although RAT8 had been shown to interact genetically with >15 other genes, we identified >40 additional genes whose disruption in a rat8-2 background causes synthetic lethality or dramatically reduced growth. Included were five that encode components of P-bodies, sites of cytoplasmic mRNA turnover and storage. Wild-type Rat8p localizes to NPCs and diffusely throughout the cell but rat8-2p localized to cytoplasmic granules at nonpermissive temperature that are distinct from P-bodies. In some genetic backgrounds, these granules also contain poly(A)-binding protein, Pab1p, and additional mRNA export factors. Although these foci are distinct from P-bodies, the two merge under heat-stress conditions. We suggest that these granules reflect defective mRNP remodeling during mRNA export and during cytoplasmic mRNA metabolism.

Modeling complex genetic interactions in a simple eukaryotic genome: actin displays a rich spectrum of complex haploinsufficiencies.

Multigenic influences are major contributors to human genetic disorders. Since humans are highly polymorphic, there are a high number of possible detrimental, multiallelic gene pairs. The actin cytoskeleton of yeast was used to determine the potential for deleterious bigenic interactions; approximately 4800 complex hemizygote strains were constructed between an actin-null allele and the nonessential gene deletion collection. We found 208 genes that have deleterious complex haploinsufficient (CHI) interactions with actin. This set is enriched for genes with gene ontology terms shared with actin, including several actin-binding protein genes, and nearly half of the CHI genes have defects in actin organization when deleted. Interactions were frequently seen with genes for multiple components of a complex or with genes involved in the same function. For example, many of the genes for the large ribosomal subunit (RPLs) were CHI with act1Delta and had actin organization defects when deleted. This was generally true of only one RPL paralog of apparently duplicate genes, suggesting functional specialization between ribosomal genes. In many cases, CHI interactions could be attributed to localized defects on the actin protein. Spatial congruence in these data suggest that the loss of binding to specific actin-binding proteins causes subsets of CHI interactions.

A genetic dissection of Aip1p's interactions leads to a model for Aip1p-cofilin cooperative activities.

Actin interacting protein 1 (Aip1p) and cofilin cooperate to disassemble actin filaments in vitro and are thought to promote rapid turnover of actin networks in vivo. The precise method by which Aip1p participates in these activities has not been defined, although severing and barbed-end capping of actin filaments have been proposed. To better describe the mechanisms and biological consequences of Aip1p activities, we undertook an extensive mutagenesis of AIP1 aimed at disrupting and mapping Aip1p interactions. Site-directed mutagenesis suggested that Aip1p has two actin binding sites, the primary actin binding site lies on the edge of its N-terminal beta-propeller and a secondary actin binding site lies in a comparable location on its C-terminal beta-propeller. Random mutagenesis followed by screening for separation of function mutants led to the identification of several mutants specifically defective for interacting with cofilin but still able to interact with actin. These mutants suggested that cofilin binds across the cleft between the two propeller domains, leaving the actin binding sites exposed and flanking the cofilin binding site. Biochemical, genetic, and cell biological analyses confirmed that the actin binding- and cofilin binding-specific mutants are functionally defective, whereas the genetic analyses further suggested a role for Aip1p in an early, internalization step of endocytosis. A complementary, unbiased molecular modeling approach was used to derive putative structures for the Aip1p-cofilin complex, the most stable of which is completely consistent with the mutagenesis data. We theorize that Aip1p-severing activity may involve simultaneous binding to two actin subunits with cofilin wedged between the two actin binding sites of the N- and C-terminal propeller domains.