Project description:Plakoglobin (PG; γ-Catenin, JUP) is a protein with controversial function. First described as a component of intercellular junctions, it has remained unresolved whether this near-ubiquitously expressed protein acts as a genuine transcriptional regulator in adult tissue like its closest relative β-catenin. Here we have mapped the global gene targets of PG by ChIP-chip in differentiating skin keratinocytes. Applying a peak algorithm, over 5’000 high-confidence PG target promoters were identified and 2’000 for β-catenin with an overlap of 38%. Bioinformatics analyses most significantly associated PG target genes with the Wnt signaling pathway as well as relevant pathways for keratinocyte differentiation. Using a combination of wild-type, PG, β-catenin and PG/β-catenin double null keratinocytes, PG was functionally validated as a LEF/TCF-dependent transcriptional regulator. These data challenge the current understanding of Wnt signaling, one of the most important pathways in tissue homeostasis, by identifying PG as a potent LEF/TCF-dependent transcriptional regulator which functionally overlaps with β-catenin.
Project description:ChIP-seq is used to map transcription factor occupancy and generate epigenetic profiles genome-wide. The requirement of nano-scale ChIP DNA for generation of sequencing libraries has impeded ChIP-seq on in vivo tissues of low cell numbers. We describe a robust, simple and scalable methodology for ChIP-seq of low-abundant cell populations, reliably amplifying 50 pg of ChIP DNA, corresponding to ~30,000 input cells for transcription factor ChIP (CEBPA) and 3,000 cells for histone mark ChIP (H3K27me3). This represents a significant advance compared to existing technologies, which involve complex and time-consuming steps of pre-amplification, making them susceptible to experimental biases. ChIP-seq of histone modifications H3K27me3 (2 biological replicates (I+II) , 2 ng input), H3K4me3 (2 biological replicates (II+III), 2 ng input), transcription factor CEBPA (2 biological replicates (I+II), 300 pg input), 4 diluted CEBPA libraries (pool of ChIP from 3 biol. replicates (I+II+III) 3x 100 pg input, 1x 50 pg). Additonal ChIP-seq using 10,000 cells, 1 biological replicate of each H3K4me3 and CEBPA.
Project description:Despite much investigation, mechanisms conferring stage specific responsiveness of the corpus luteum (CL) to prostaglandin F2 (PG) are unknown. The objective of this study was to identify PG- induced changes in transcriptome of bovine CL specific to d 11 ( PG responsive) but not d 4 (PG refractory) CL associated with luteolysis. CL were collected from heifers at 0, 4 and 24 h following PG injection on d 4 and 11 of the estrous cycle (n = 5 animals/treatment) and isolated RNA labeled and hybridized to Affymetrix GeneChip Bovine Genome Arrays. At 4 and 24 h post PG respectively, 221 (d 4) and 661 (d 11) and 248 (d 4) and 1419 (d 11) regulated genes were identified.
Project description:The autonomic nervous system is derived from the neural crest and supplies motor innervation to the smooth muscle of visceral organs, including the lower urinary tract (bladder and urethra, LUT). In rodents, autonomic innervation of the LUT is supplied by the major pelvic ganglia (PG) that lie near the neck of the bladder and proximal urethra. Compared to other autonomic ganglia, the PG are unique in that they harbor both sympathetic and parasympathetic neurons. The coordinated activity of PG neurons is critical for normal functioning of the LUT – however, surprisingly little is known about how PG neuronal diversity is established or what molecular factors control PG development. In this study we conducted transcriptome profiling of Sox10-H2BVenus+ sacral neural crest (NC) progenitors to discover candidate genes involved in PG neurogenesis.
Project description:Endogenous peptidoglycan (PG) hydrolysing enzymes, the autolysins, are needed to relax the rigid PG sacculus to allow bacterial cell growth and separation. PG of pathogens and commensal bacteria may also be degraded by hydrolases of animal origin (lysozymes), which act as antimicrobials. The genetic mechanisms regulating PG resistance to hydrolytic degradation was dissected in the Gram-positive bacterium Lactococcus lactis. We found that the ability of L. lactis to counteract PG hydrolysis depends on the degree of acetylation. Overexpression of PG O-acetylase, encoded by oatA, led to bacterial growth arrest, indicating potential lethality of oatA and a need for its tight regulation. A novel regulatory factor SpxB (previously denoted as YneH), exerts a positive effect on oatA expression. Our results indicate that SpxB binding to RNA polymerase constitutes a previously missing link in the multi-step response to cell envelope stress, provoked by PG hydrolysis with lysozyme. We suggest that the two component system CesSR responds to this stress by inducing SpxB, thus favoring its interactions with RNA polymerase. Induction of PG O-acetylation by this cascade renders it resistant to hydrolysis. Keywords: mutant, lysozym
Project description:Chloroplasts and chromoplasts of tomato fruits contain lipid droplets known as plastoglobules (PG). Recent studies have shown that PG act as a metabolic sink to store triacylglycerol (TAG), carotenoids, vitamin E, and K as well as other lipophile compounds. In addition, it has been shown that proteins associated with PG contribute to prenyl lipid metabolic pathways and homeostasis. Proteome studies in Arabidopsis and bell pepper, have demonstrated that PG contains a specific proteome containing around 30 proteins. So far, the tomato PG proteome particularly that of red fruit (chromoplast) has not yet been established. We have investigated the chromoplast PG proteome and metabolome and identified 17 new candidate proteins of chromoplast PG with known PG proteins. Interestingly, most of the carotenoid biosynthetic pathway enzymes such as PSY‐1, PDS, ZDS, CRTISO, and β‐LYC are enriched in chromoplast PG compared with chloroplast PG. In this study, we propose a model of chromoplast PG acting as a carotenoid biosynthesis platform.