Targeting RNA polymerase I to treat MYC-driven cancer.

The MYC oncoprotein and transcription factor is dysregulated in a majority of human cancers and is considered a major driver of the malignant phenotype. As such, developing drugs for effective inhibition of MYC in a manner selective to malignancies is a 'holy grail' of transcription factor-based cancer therapy. Recent advances in elucidating MYC biology in both normal cells and pathological settings were anticipated to bring inhibition of tumorigenic MYC function closer to the clinic. However, while the extensive array of cellular pathways that MYC impacts present numerous fulcrum points on which to leverage MYC's therapeutic potential, identifying the critical target(s) for MYC-specific cancer therapy has been difficult to achieve. Somewhat unexpectedly, MYC's fundamental role in regulating the 'housekeeping' process of ribosome biogenesis, one of the most ubiquitously required and conserved cell functions, may provide the Achilles' heel for therapeutically targeting MYC-driven tumors.

Drosophila CtBP: a Hairy-interacting protein required for embryonic segmentation and hairy-mediated transcriptional repression.

hairy is a Drosophila pair-rule segmentation gene that functions genetically as a repressor. To isolate protein components of Hairy-mediated repression, we used a yeast interaction screen and identified a Hairy-interacting protein, the Drosophila homolog of the human C-terminal-binding protein (CtBP). Human CtBP is a cellular phosphoprotein that interacts with the C-terminus of the adenovirus E1a oncoprotein and functions as a tumor suppressor. dCtBP also interacts with E1a in a directed yeast two-hybrid assay. We show that dCtBP interacts specifically and directly with a small, previously uncharacterized C-terminal region of Hairy. dCtBP activity appears to be specific to Hairy of the Hairy/Enhancer of split [E(spl)]/Dpn basic helix-loop-helix protein class. We identified a P-element insertion within the dCtBP transcription unit that fails to complement alleles of a known locus, l(3)87De. We demonstrate that dCtBP is essential for proper embryonic segmentation by analyzing embryos lacking maternal dCtBP activity. While Hairy is probably not the only segmentation gene interacting with dCtBP, we show dose-sensitive genetic interactions between dCtBP and hairy mutations.

A network of interacting transcriptional regulators involved in Drosophila neural fate specification revealed by the yeast two-hybrid system.

Neural fate specification in Drosophila is promoted by the products of the proneural genes, such as those of the achaete-scute complex, and antagonized by the products of the Enhancer of split [E(spl)] complex, hairy, and extramacrochaetae. As all these proteins bear a helix-loop-helix (HLH) dimerization domain, we investigated their potential pairwise interactions using the yeast two-hybrid system. The fidelity of the system was established by its ability to closely reproduce the already documented interactions among Da, Ac, Sc, and Extramacrochaetae. We show that the seven E(spl) basic HLH proteins can form homo- and heterodimers inter-se with distinct preferences. We further show that a subset of E(spl) proteins can heterodimerize with Da, another subset can heterodimerize with proneural proteins, and yet another with both, indicating specialization within the E(spl) family. Hairy displays no interactions with any of the HLH proteins tested. It does interact with the non-HLH protein Groucho, which itself interacts with all E(spl) basic HLH proteins, but with none of the proneural proteins or Da. We investigated the structural requirements for some of these interactions by site-specific and deletion mutagenesis.

Tryptophan analog resistance mutations in Chlamydomonas reinhardtii.

Forty single gene mutations in Chlamydomonas reinhardtii were isolated based on resistance to the compound 5'-methyl anthranilic acid (5-MAA). In other organisms, 5-MAA is converted to 5'-methyltryptophan (5-MT) and 5-MT is a potent inhibitor of anthranilate synthase, which catalyzes the first committed step in tryptophan biosynthesis. The mutant strains fall into two phenotypic classes based on the rate of cell division in the absence of 5-MAA. Strains with class I mutations divide more slowly than wild-type cells. These 17 mutations map to seven loci, which are designated MAA1 to MAA7. Strains with class II mutations have generation times indistinguishable from wild-type cells, and 7 of these 23 mutations map to loci defined by class I mutations. The remainder of the class II mutations map to 9 other loci, which are designated MAA8-MAA16. The maa5-1 mutant strain excretes high levels of anthranilate and phenylalanine into the medium. In this strain, four enzymatic activities in the tryptophan biosynthetic pathway are increased at least twofold. These include the combined activities of anthranilate phosphoribosyl transferase, phosphoribosyl anthranilate isomerase, indoleglycerol phosphate synthetase and anthranilate synthase. The slow growth phenotypes of strains with class I mutations are not rescued by the addition of tryptophan, but the slow growth phenotype of the maa6-1 mutant strain is partially rescued by the addition of indole. The maa6-1 mutant strain excretes a fluorescent compound into the medium, and cell extracts have no combined anthranilate phosphoribosyl transferase, phosphoribosyl anthranilate isomerase and indoleglycerol phosphate synthetase activity. The MAA6 locus is likely to encode a tryptophan biosynthetic enzyme. None of the other class I mutations affected these enzyme activities. Based on the phenotypes of double mutant strains, epistatic relationships among the class I mutations have been determined.