Whole Genome Re-Sequencing of Three Domesticated Chicken Breeds.

Chicken is one of the most popular domesticated species worldwide, as it can serve an important role in agricultural as well as biomedical research fields. Because it inhabits almost every continent and presents diverse morphology and traits, the need of genetic markers for distinguishing each breed for various purposes has increased. The whole genome sequencing of three different breeds (White Leghorn, Korean domestic, and Araucana) that show similar coloring patterns, with the exception of the White Leghorn breed, have confirmed previously reported genomic alterations and identified many novel variants. Additionally, the Whole Genome Re-Sequencing (WGRS) approach identified an approximately 4 kb insert within SLCO1B3 responsible for blue egg shell color. Targeted investigation of pigment-related genes corroborated previously reported non-synonymous mutations, and provided deeper insight into chicken coloring, where not a single but a combination of non-synonymous mutations in the MC1R gene is likely to be responsible for altered feather coloring.

Combinatorial regulation of the dev operon by MrpC2 and FruA during Myxococcus xanthus development.

Proper expression of the dev operon is important for normal development of Myxococcus xanthus. When starved, these bacteria coordinate their gliding movements to build mounds that become fruiting bodies as some cells differentiate into spores. Mutations in the devTRS genes impair sporulation. Expression of the operon occurs within nascent fruiting bodies and depends in part on C signaling. Here, we report that expression of the dev operon, like that of several other C-signal-dependent genes, is subject to combinatorial control by the transcription factors MrpC2 and FruA. A DNA fragment upstream of the dev promoter was bound by a protein in an extract containing MrpC2, protecting the region spanning positions -77 to -54. Mutations in this region impaired binding of purified MrpC2 and abolished developmental expression of reporter fusions. The association of MrpC2 and/or its longer form, MrpC, with the dev promoter region depended on FruA in vivo, based on chromatin immunoprecipitation analysis, and purified FruA appeared to bind cooperatively with MrpC2 to DNA just upstream of the dev promoter in vitro. We conclude that cooperative binding of the two proteins to this promoter-proximal site is crucial for dev expression. 5' deletion analysis implied a second upstream positive regulatory site, which corresponded to a site of weak cooperative binding of MrpC2 and FruA and boosted dev expression 24 h into development. This site is unique among the C-signal-dependent genes studied so far. Deletion of this site in the M. xanthus chromosome did not impair sporulation under laboratory conditions.

Transcription factor MrpC binds to promoter regions of hundreds of developmentally-regulated genes in Myxococcus xanthus.

BACKGROUND: Myxococcus xanthus is a bacterium that undergoes multicellular development when starved. Cells move to aggregation centers and form fruiting bodies in which cells differentiate into dormant spores. MrpC appears to directly activate transcription of fruA, which also codes for a transcription factor. Both MrpC and FruA are crucial for aggregation and sporulation. The two proteins bind cooperatively in promoter regions of some developmental genes. RESULTS: Chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) and bioinformatic analysis of cells that had formed nascent fruiting bodies revealed 1608 putative MrpC binding sites. These sites included several known to bind MrpC and they were preferentially distributed in likely promoter regions, especially those of genes up-regulated during development. The up-regulated genes include 22 coding for protein kinases. Some of these are known to be directly involved in fruiting body formation and several negatively regulate MrpC accumulation. Our results also implicate MrpC as a direct activator or repressor of genes coding for several transcription factors known to be important for development, for a major spore protein and several proteins important for spore formation, for proteins involved in extracellular A- and C-signaling, and intracellular ppGpp-signaling during development, and for proteins that control the fate of other proteins or play a role in motility. We found that the putative MrpC binding sites revealed by ChIP-seq are enriched for DNA sequences that strongly resemble a consensus sequence for MrpC binding proposed previously. MrpC2, an N-terminally truncated form of MrpC, bound to DNA sequences matching the consensus in all 11 cases tested. Using longer DNA segments containing 15 of the putative MrpC binding sites from our ChIP-seq analysis as probes in electrophoretic mobility shift assays, evidence for one or more MrpC2 binding site was observed in all cases and evidence for cooperative binding of MrpC2 and FruA was seen in 13 cases. CONCLUSIONS: We conclude that MrpC and MrpC2 bind to promoter regions of hundreds of developmentally-regulated genes in M. xanthus, in many cases cooperatively with FruA. This binding very likely up-regulates protein kinases, and up- or down-regulates other proteins that profoundly influence the developmental process.

Combinatorial regulation by MrpC2 and FruA involves three sites in the fmgE promoter region during Myxococcus xanthus development.

Starvation causes cells in a dense population of Myxococcus xanthus to change their gliding movements and construct mounds. Short-range C-signaling between rod-shaped cells within mounds induces gene expression that promotes differentiation into spherical spores. Several C-signal-dependent genes have been shown to be regulated by cooperative binding of two transcription factors to the promoter region. These FruA- and MrpC2-regulated genes (designated fmg) each exhibit a different arrangement of binding sites. Here, we describe fmgE, which appears to be regulated by three sites for cooperative binding of FruA and MrpC2. Chromatin immunoprecipitation analysis showed that association of MrpC2 and/or its longer form, MrpC with the fmgE promoter region, depends on FruA, consistent with cooperative binding of the two proteins in vivo. Electrophoretic mobility shift assays with purified His(10)-MrpC2 and FruA-His(6) indicated cooperative binding in vitro to three sites in the fmgE promoter region. The effects of mutations on binding in vitro and on expression of fmgE-lacZ fusions correlated site 1 (at about position -100 relative to the transcriptional start site) with negative regulation and site 2 (just upstream of the promoter) and site 3 (at about position +100) with positive regulation. Site 3 was bound by His(10)-MrpC2 alone, or the combination of His(10)-MrpC2 and FruA-His(6), with the highest affinity, followed by site 1 and then site 2, supporting a model in which site 3 recruits MrpC2 and FruA to the fmgE promoter region, site 1 competes with site 2 for transcription factor binding, and site 2 occupancy is required to activate the promoter but only occurs when C-signaling produces a high concentration of active FruA.

Combinatorial regulation of fmgD by MrpC2 and FruA during Myxococcus xanthus development.

Upon starvation, a dense population of rod-shaped Myxococcus xanthus bacteria coordinate their movements to construct mounds in which some of the cells differentiate to spherical spores. During this process of fruiting body formation, short-range C-signaling between cells regulates their movements and the expression of genes important for sporulation. C-signaling activates FruA, a transcription factor that binds cooperatively with another transcription factor, MrpC2, upstream of the fmgA and fmgBC promoters, activating transcription. We have found that a third C-signal-dependent gene, herein named fmgD, is subject to combinatorial control by FruA and MrpC2. The two proteins appear to bind cooperatively upstream of the fmgD promoter and activate transcription. FruA binds proximal to the fmgD promoter, as in the fmgBC promoter region, whereas MrpC2 binds proximal to the fmgA promoter. A novel feature of the fmgD promoter region is the presence of a second MrpC2 binding site partially overlapping the promoter and therefore likely to mediate repression. The downstream MrpC2 site appears to overlap the FruA site, so the two transcription factors may compete for binding, which in both cases appears to be cooperative with MrpC2 at the upstream site. We propose that binding of MrpC2 to the downstream site represses fmgD transcription until C-signaling causes the concentration of active FruA to increase sufficiently to outcompete the downstream MrpC2 for cooperative binding with the upstream MrpC2. This would explain why fmgD transcription begins later during development and is more dependent on C-signaling than transcription of fmgA and fmgBC.

Identification of genomic mutations associated with clinical outcomes of induction chemotherapy in patients with head and neck squamous cell carcinoma.

PURPOSE: We performed deep sequencing of target genes in head and neck squamous cell carcinoma (HNSCC) tumors to identify somatic mutations that are associated with induction chemotherapy (IC) response. METHODS: Patients who were diagnosed with HNSCC were retrospectively identified. Patients who were treated with IC were divided into two groups: good responders and poor responders by tumor response and progression-free survival. Targeted gene sequencing for 2404 somatic mutations of 44 genes was performed on HNSCC tissues. Mutations with total coverage of <500 were excluded, and the cutoff for altered allele frequency was >10 %. RESULTS: Of the 71 patients, 45 were treated upfront with IC. Mean total coverage was 1941 per locus, and 42.2 % of tumors had TP53 mutations. Thirty-three mutations in TP53, NOTCH3, FGFR2, FGFR3, ATM, EGFR, MET, PTEN, FBXW7, SYNE1, and SUFU were frequently altered in poor responders. Among the patients who were treated with IC, those with unfavorable genomic profiles had significantly poorer overall survival than those without unfavorable genomic profiles (hazard ratio 6.45, 95 % confidence interval 2.07-20.10, P < 0.001). CONCLUSIONS: Comprehensive analysis of mutation frequencies identified unfavorable genomic profiles, and the patients without unfavorable genomic profiles can obtain clinical benefits from IC in patients with HNSCC.

Efficient generation of transgenic cattle using the DNA transposon and their analysis by next-generation sequencing.

Here, we efficiently generated transgenic cattle using two transposon systems (Sleeping Beauty and Piggybac) and their genomes were analyzed by next-generation sequencing (NGS). Blastocysts derived from microinjection of DNA transposons were selected and transferred into recipient cows. Nine transgenic cattle have been generated and grown-up to date without any health issues except two. Some of them expressed strong fluorescence and the transgene in the oocytes from a superovulating one were detected by PCR and sequencing. To investigate genomic variants by the transgene transposition, whole genomic DNA were analyzed by NGS. We found that preferred transposable integration (TA or TTAA) was identified in their genome. Even though multi-copies (i.e. fifteen) were confirmed, there was no significant difference in genome instabilities. In conclusion, we demonstrated that transgenic cattle using the DNA transposon system could be efficiently generated, and all those animals could be a valuable resource for agriculture and veterinary science.

Whole genome comparison of donor and cloned dogs.

Cloning is a process that produces genetically identical organisms. However, the genomic degree of genetic resemblance in clones needs to be determined. In this report, the genomes of a cloned dog and its donor were compared. Compared with a human monozygotic twin, the genome of the cloned dog showed little difference from the genome of the nuclear donor dog in terms of single nucleotide variations, chromosomal instability, and telomere lengths. These findings suggest that cloning by somatic cell nuclear transfer produced an almost identical genome. The whole genome sequence data of donor and cloned dogs can provide a resource for further investigations on epigenetic contributions in phenotypic differences.