The START domain potentiates HD-ZIPIII transcriptional activity.

The CLASS III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIPIII) transcription factors (TFs) were repeatedly deployed over 725 million years of evolution to regulate central developmental innovations. The START domain of this pivotal class of developmental regulators was recognized over 20 years ago, but its putative ligands and functional contributions remain unknown. Here, we demonstrate that the START domain promotes HD-ZIPIII TF homodimerization and increases transcriptional potency. Effects on transcriptional output can be ported onto heterologous TFs, consistent with principles of evolution via domain capture. We also show the START domain binds several species of phospholipids, and that mutations in conserved residues perturbing ligand binding and/or its downstream conformational readout abolish HD-ZIPIII DNA-binding competence. Our data present a model in which the START domain potentiates transcriptional activity and uses ligand-induced conformational change to render HD-ZIPIII dimers competent to bind DNA. These findings resolve a long-standing mystery in plant development and highlight the flexible and diverse regulatory potential coded within this widely distributed evolutionary module.

Identifying Cancer-Relevant Mutations in the DLC START Domain Using Evolutionary and Structure-Function Analyses.

Rho GTPase signaling promotes proliferation, invasion, and metastasis in a broad spectrum of cancers. Rho GTPase activity is regulated by the deleted in liver cancer (DLC) family of bona fide tumor suppressors which directly inactivate Rho GTPases by stimulating GTP hydrolysis. In addition to a RhoGAP domain, DLC proteins contain a StAR-related lipid transfer (START) domain. START domains in other organisms bind hydrophobic small molecules and can regulate interacting partners or co-occurring domains through a variety of mechanisms. In the case of DLC proteins, their START domain appears to contribute to tumor suppressive activity. However, the nature of this START-directed mechanism, as well as the identities of relevant functional residues, remain virtually unknown. Using the Catalogue of Somatic Mutations in Cancer (COSMIC) dataset and evolutionary and structure-function analyses, we identify several conserved residues likely to be required for START-directed regulation of DLC-1 and DLC-2 tumor-suppressive capabilities. This pan-cancer analysis shows that conserved residues of both START domains are highly overrepresented in cancer cells from a wide range tissues. Interestingly, in DLC-1 and DLC-2, three of these residues form multiple interactions at the tertiary structural level. Furthermore, mutation of any of these residues is predicted to disrupt interactions and thus destabilize the START domain. As such, these mutations would not have emerged from traditional hotspot scans of COSMIC. We propose that evolutionary and structure-function analyses are an underutilized strategy which could be used to unmask cancer-relevant mutations within COSMIC. Our data also suggest DLC-1 and DLC-2 as high-priority candidates for development of novel therapeutics that target their START domain.

EMBRYONIC DEVELOPMENT AND A QUANTITATIVE MODEL OF PROGRAMMED DNA ELIMINATION IN MESOCYCLOPS EDAX (S. A. FORBES, 1891) (COPEPODA: CYCLOPOIDA).

The highly programmed fragmentation of chromosomes and elimination of large amounts of nuclear DNA from the presomatic cell lineages (i.e., chromatin diminution), occurs in the embryos of the freshwater zooplankton Mesocyclops edax (S. A. Forbes, 1891) (Crustacea: Copepoda). The somatic genome is reorganized and reduced to a size five times smaller even though the germline genome remains intact. We present the first comprehensive, quantitative model of DNA content throughout embryogenesis in a copepod that possesses embryonic DNA elimination. We used densitometric image analysis to measure the DNA content of polar bodies, germline and somatic nuclei, and excised DNA "droplets." We report: 1) variable DNA contents of polar bodies, some of which do not contain the amount corresponding to the haploid germline genome size; 2) presence of pronuclei in newly laid embryo sacs; 3) gonomeric chromosomes in the second to fourth cleavage divisions and in the primordial germ cell and primordial endoderm cell during the fifth cleavage division; 4) timing of early embryonic cell stages, elimination of DNA, and divisions of the primordial germ cell and primordial endoderm cell at 22°C; and 5) persistence of a portion of the excised DNA "droplets" throughout embryogenesis. DNA elimination is a trait that spans multiple embryonic stages and a knowledge of the timing and variability of the associated cytological events with DNA elimination will promote the study of the molecular mechanisms involved in this trait. We propose the "genome yolk hypothesis" as a functional explanation for the persistence of the eliminated DNA that might serve as a resource during postdiminution cleavage divisions.

Mutation Spectra of the MRN (MRE11, RAD50, NBS1/NBN) Break Sensor in Cancer Cells.

The MRN complex (MRE11, RAD50, NBS1/NBN) is a DNA double strand break sensor in eukaryotes. The complex directly participates in, or coordinates, several activities at the break such as DNA resection, activation of the DNA damage checkpoint, chromatin remodeling and recruitment of the repair machinery. Mutations in components of the MRN complex have been described in cancer cells for several decades. Using the Catalogue of Somatic Mutations in Cancer (COSMIC) database, we characterized all the reported MRN mutations. This analysis revealed several hotspot frameshift mutations in all three genes that introduce premature stop codons and truncate large regions of the C-termini. We also found through evolutionary analyses that COSMIC mutations are enriched in conserved residues of NBS1/NBN and RAD50 but not in MRE11. Given that all three genes are important to carcinogenesis, we propose these differential enrichment patterns may reflect a more severe pleiotropic role for MRE11.

Whole genome DNA methylation sequencing of the chicken retina, cornea and brain.

Whole genome bisulfite sequencing (WGBS) analysis of DNA methylation uses massively parallel next generation sequencing technology to characterize global epigenetic patterns and fluctuations throughout a range of tissue samples. Development of the vertebrate retina is thought to involve extensive epigenetic reprogramming during embryogenesis. The chicken embryo (Gallus gallus) is a classic model system for studying developmental biology and retinogenesis, however, there are currently no publicly available data sets describing the developing chicken retinal methylome. Here we used Illumina WGBS analysis to characterize genome-wide patterns of DNA methylation in the developing chicken retina as well as cornea and brain in an effort to further our understanding of retina-specific epigenetic regulation. These data will be valuable to the vision research community for correlating global changes in DNA methylation to differential gene expression between ocular and neural tissues during critical developmental time points of retinogenesis in the chicken retina.