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Short tandem DNA repeats are drivers of genome instability. To identify suppressors of break-induced mutagenesis human cells, unbiased genetic screens were conducted using a lentiviral shRNA library. The recipient cells possessed fragile non-B DNA that could induce DNA double-strand breaks (DSBs), integrated at an ectopic chromosomal site adjacent to a thymidine kinase marker gene. Mutagenesis of the thymidine kinase gene rendered cells resistant to the nucleoside analog ganciclovir (GCV). The screen identified genes that have established roles in DNA replication and repair, chromatin modification, responses to ionizing radiation, and genes encoding proteins enriched at replication forks. Novel loci implicated in BIR included olfactory receptors, the G0S2 oncogene/tumor suppressor axis, the EIF3H-METTL3 translational regulator, and the SUDS3 subunit of the Sin3A corepressor. Consistent with a role in suppressing BIR, siRNA knockdown of selected candidates increased the frequency of the GCVr phenotype and increased DNA rearrangements near the ectopic non-B DNA. Inverse PCR and DNA sequence analyses showed that hits identified in the screen increased genome instability. Further analysis quantitated repeat-induced hypermutagenesis at the ectopic site and showed that knockdown of a primary hit, COPS2, induced mutagenic hotspots, remodeled the replication fork, and increased nonallelic chromosome template switches.

Liver X receptor alpha (LXRα) is crucial for the maintenance of lipid and cholesterol homeostasis. Ligand binding and dimerization with retinoid X receptor (RXR) or peroxisome proliferator-activated receptor (PPAR) is required for forming active DNA binding complexes leading to gene regulation. Structure based prediction and solvent accessibility of LXRα LBD shows that residues H383, E387, H390, L414, and R415 which are located in helices 9 and 10 may be critical for mediating protein-protein interactions. In this study, LXRα interface residues were individually mutated to determine their effects on ligand binding, protein-protein association, subcellular localization, and transactivation activity. LXRα L414R and R415A lacked binding to T-0901317, but retained binding to 25-Hydroxycholesterol. assay and a cell based assay demonstrated that LXRα L414R was specifically impaired for interactions with RXRα but not PPARα suggesting that charge reversal at the interface provides selectivity to LXRα dimerization. Furthermore, binding of LXRα L414R or R415A with PPARα exhibited minimal conformational changes in the dimer secondary structure. Interestingly, all LXRα mutants exhibited lower levels of ligand dependent luciferase activity driven by the SREBP-1c or ApoA1 promoter. Taken together, our data demonstrates that intact hydrophobic interactions and salt bridges at the interface mediate efficient ligand-dependent transactivation activities.

A seed marks the transition between two developmental states; a plant is an embryo during seed formation, whereas it is a seedling after emergence from the seed. Two factors have been identified in Arabidopsis that play a role in establishment of repression of the embryonic state: PKL (PICKLE), which codes for a putative CHD3 chromatin remodeling factor, and gibberellin (GA), a plant growth regulator. Previous observations have also suggested that PKL mediates some aspects of GA responsiveness in the adult plant. To investigate possible mechanisms by which PKL and GA might act to repress the embryonic state, we further characterized the ability of PKL and GA to repress embryonic traits and reexamined the role of PKL in mediating GA-dependent responses. We found that PKL acts throughout the seedling to repress expression of embryonic traits. Although the ability of pkl seedlings to express embryonic traits is strongly induced by inhibiting GA biosynthesis, it is only marginally responsive to abscisic acid and SPY (SPINDLY), factors that have previously been demonstrated to inhibit GA-dependent responses during germination. We also observed that pkl plants exhibit the phenotypic hallmarks of a mutation in a positive regulator of a GA response pathway including reduced GA responsiveness and increased synthesis of bioactive GAs. These observations indicate that PKL may mediate a subset of GA-dependent responses during shoot development.

Embryos express several unique differentiation characteristics, including the accumulation of a number of metabolites that are generally considered to be unique to seeds. PICKLE (PKL) codes for a CHD3-chromatin remodeling factor that is necessary for repression of embryonic traits in seedlings of Arabidopsis thaliana (L.) Heynh. In pkl mutants, primary roots are capable of expressing many embryonic traits after germination and are referred to as \"pickle roots\". In an attempt to examine the breadth of PKL-dependent repression of embryo-specific differentiation pathways, we determined the extent to which a variety of embryo-specific compounds accumulate in pickle roots. We found that pickle roots accumulate triacylglycerol with a fatty acid composition that is similar to that found in seeds. The major seed storage proteins are also present in pickle roots. In addition to these two well-characterized seed storage compounds, we observed that pickle roots accumulate phytate, a form of stored phosphate that is preferentially accumulated in seeds. Seeds of members of the Brassicaceae also accumulate a variety of unique secondary metabolites, including sinapate esters and glucosinolates. Surprisingly, the levels of secondary metabolites in pickle roots were not suggestive of an embryonic differentiation state, but did reveal that a mutation in PKL results in substantial changes in root secondary metabolism. Taken together, these data suggest that PKL is responsible for regulating some but not all aspects of the embryonic program as it relates to the accumulation of embryo-specific metabolites.

AFLP markers in linkage disequilibrium with vH13, an avirulence gene in the Hessian fly (Mayetiola destructor) that conditions avirulence to resistance gene H13 in wheat (Triticum spp.), were discovered by bulked segregant analysis. Five AFLPs were converted into codominant site-specific markers that genetically mapped within 13 cM of this gene. Flanking markers used as probes positioned vH13 near the telomere of the short arm of Hessian fly chromosome X2. These results suggest that the X-linked avirulence genes vH6, vH9, and vH13 are present on Hessian fly chromosome X2 rather than on chromosome X1 as reported previously. Genetic complementation demonstrated that recessive alleles of vH13 were responsible for the H13-virulence observed in populations derived from four different states in the U.S.A.: Georgia, Maryland, Virginia, and Washington. Results support the hypothesis that a gene-for-gene interaction exists between wheat and Hessian fly.

The Hessian fly has an intimate genetic relationship with wheat, and the two species have co-evolved. The plant has developed genes that allow it to resist infestation by the fly. The fly has developed mutations in avirulence genes that allow it to overcome this resistance. This interaction has not been characterized at the molecular level, and much is to be learned about the interaction if durable resistance to the Hessian fly is to be developed into wheat cultivars. We are in the process of identifying and cloning the genes from the fly that are responsible for inducing a resistance response in the plant. A direct, map-based approach is being pursued because little biochemical information is available about the fly-wheat interaction. A genetic map of the Hessian fly was constructed to aid in predicting the potential success of a positional cloning approach, and to aid in the acquisition of molecular markers for Hessian fly avirulence loci. The Hessian fly has a relatively small genome (98 megabases, 545 cM) and a relatively high rate of recombination. About 180 kb represents one centimorgan of recombination. Thus, map-based cloning in the Hessian fly is feasible. Molecular markers were identified that are within walking distance of the vH13 locus—the locus that confers avirulence to the H13 wheat resistance gene. Positional cloning of this avirulence locus is underway.

A seed marks the transition between two developmental states; a plant is an embryo during seed formation, whereas it is a seedling after emergence from the seed. Two factors have been identified in Arabidopsis that play a role in establishment of repression of the embryonic state: PKL (PICKLE), which codes for a putative CHD3 chromatin remodeling factor, and gibberellin (GA), a plant growth regulator. Previous observations have also suggested that PKL mediates some aspects of GA responsiveness in the adult plant. To investigate possible mechanisms by which PKL and GA might act to repress the embryonic state, we further characterized the ability of PKL and GA to repress embryonic traits and reexamined the role of PKL in mediating GA-dependent responses. We found that PKL acts throughout the seedling to repress expression of embryonic traits. Although the ability of pkl seedlings to express embryonic traits is strongly induced by inhibiting GA biosynthesis, it is only marginally responsive to abscisic acid and SPY (SPINDLY), factors that have previously been demonstrated to inhibit GA-dependent responses during germination. We also observed that pkl plants exhibit the phenotypic hallmarks of a mutation in a positive regulator of a GA response pathway including reduced GA responsiveness and increased synthesis of bioactive GAs. These observations indicate that PKL may mediate a subset of GA-dependent responses during shoot development.

AFLP markers in linkage disequilibrium with vH13, an avirulence gene in the Hessian fly (Mayetiola destructor) that conditions avirulence to resistance gene H13 in wheat (Triticum spp.), were discovered by bulked segregant analysis. Five AFLPs were converted into codominant site-specific markers that genetically mapped within 13 cM of this gene. Flanking markers used as probes positioned vH13 near the telomere of the short arm of Hessian fly chromosome X2. These results suggest that the X-linked avirulence genes vH6, vH9, and vH13 are present on Hessian fly chromosome X2 rather than on chromosome X1 as reported previously. Genetic complementation demonstrated that recessive alleles of vH13 were responsible for the H13-virulence observed in populations derived from four different states in the U.S.A.: Georgia, Maryland, Virginia, and Washington. Results support the hypothesis that a gene-for-gene interaction exists between wheat and Hessian fly.