Project description:Turnover and exchange of nucleosomal histones and their variants, a process long believed to be static in post-replicative cells, remains largely unexplored in brain. Here, we describe a novel mechanistic role for HIRA (histone cell cycle regulator) and proteasomal degradation associated histone dynamics in the regulation of activity-dependent transcription, synaptic connectivity and behavior. We uncover a dramatic developmental profile of nucleosome occupancy across the lifespan of both rodents and humans, with the histone variant H3.3 accumulating to near saturating levels throughout the neuronal genome by mid-adolescence. Despite such accumulation, H3.3 containing nucleosomes remain highly dynamic–in a modification independent manner–to control neuronal- and glial- specific gene expression patterns throughout life. Manipulating H3.3 dynamics in both embryonic and adult neurons confirmed its essential role in neuronal plasticity and cognition. Our findings establish histone turnover as a critical, and previously undocumented, regulator of cell-type specific transcription and plasticity in mammalian brain. All ChIP-seq samples were generated to test the impact of neuronal activity/adult physiological plasticity on histone turnover in the central nervous system. This was tested in cultured neurons and astrocytes, FACS purified neurons or FACS purified Glia.
Project description:Treatment of stationary growth phase Staphylococcus aureus SA113 with 100-fold of the MIC of the lipopeptide antibiotic daptomycin leaves alive a small fraction of drug tolerant albeit genetically susceptible bacteria. This study shows that cells of this subpopulation exhibit active metabolism even hours after the onset of the drug challenge. Isotopologue profiling using fully 13C-labeled glucose revealed de novo biosynthesis of the amino acids Ala, Asp, Glu, Ser, Gly and His. The isotopologue composition in Asp and Glu suggested an increased activity of the TCA cycle under daptomycin treatment compared to unaffected stationary growth phase cells. Microarray analysis showed differential expression of specific genes 10 minutes and 3 hours after addition of the drug. Besides factors involved in drug response, a number of metabolic genes appear to shape the signature of daptomycin-tolerant S. aureus cells. These observations will be useful towards the development of new strategies against persisters and related forms of bacterial cells with downshifted physiology. Altogether 12 samples were analysed. Bacteria were treated with Daptomycin and samples were taken 10 minutes (3 replicates) and 3 hours later (3 replicates). For each time-point untreated cultures were sampled as controls (3 replicates for each time-point).
Project description:Two Acinetobacter baumannii strains with low susceptibility to fosmidomycin and two reference with high susceptibility to fosmidomycin were DNA-sequenced to investigate the genomic determinants of fosmidomycin resistance.
Project description:Although desert dust is known to cause increased respiratory morbidity and mortality, the underlying biological pathways remain unclear. We used RNA-seq on an advanced human alveolar in vitro model to find yet unidentified genes dysregulated by Saharan dust exposure. For comparison, DQ12 quartz dust was used as a well-established pulmonary toxicant. Co-cultures of A549 cells and phorbol 12-myristate-13-acetate (PMA)-differentiated THP-1 cells were cultivated at the air-liquid interface (ALI) for one day before exposure. For exposure, a Vitrocell Cloud 12α system was used. In the exposure chamber, SD or DQ12 suspensions were nebulized onto ALI co-cultures. In parallel, in the control chamber, the vehicle was nebulized onto ALI co-cultures. After exposure for 24 h, RNA was isolated and used for RNA-seq.
Project description:The nigrostriatal circuitry in the substantia nigra (SN) exerts control over the motor system and the loss of dopaminergic neurons (DAns) in the SN is the primary cause of Parkinson’s disease (PD). We have performed RNA-seq, ChIP-seq, and ATAC-seq analysis of the ventral midbrains of three widely used mouse strains, C57BL/6J, A/J and DBA/2J strains and found 1000-1200 genes to be differentially expressed between each of the strains. Such extensive transcriptome changes could be due to altered activity of upstream transcription factors (TFs) or due to cumulative regulatory variation across genes.
Project description:Candida albicans is an opportunistic pathogenic fungus that is able to assume several morphologies, including yeast and hyphal growth forms. The hyphal morphology can be induced by various environmental stimuli and is accompanied by expression of a large set of hyphal-specific genes (HSGs). Cell cycle and morphogenetic programs are interconnected: notably, inhibition of cell cycle progression often causes a switch to filamentous growth. Here we identify and characterize CaNrm1, a C. albicans homolog of the S. cerevisiae Whi5 and Nrm1 transcription inhibitors that, analogous to mammalian Rb, regulate the cell cycle transcription program in the G1 phase and at the G1/S transition. CaNRM1 is able to complement the phenotypes of both whi5 and nrm1 mutants in S. cerevisiae. Deletion of CaNRM1 causes a reduction in cell size and results in increased resistance to hydroxyurea (HU), an inhibitor of DNA replication; analysis of the expression of ribonucleotide reductase, the target of HU, suggests that its transcriptional induction in response to HU is mainly dependent upon CaNrm1. Genetic epistasis analysis suggests that CaNrm1 interacts with the SBF and MBF transcription factors in S. cerevisiae and with the MBF functional homolog in C. albicans. At the transcription level, deletion of CaNRM1 causes an induction of many G1 and G1/S-specific genes. Induction of the HSGs is dampened under certain conditions in the Canrm1-/- mutant, suggesting that the cell cycle transcription program influences the morphogenetic transcription program of C. albicans. One aspect of the characterization of the C. albicans Whi5/Nrm1 gene was to examine how this molecule influences global gene expression. To that end, we isolated total RNA from log-phase nrm1-/nrm1- (as well as parent/background strain) cells in two independent replicate experiments. These samples were processed, labeled, and subsequently used for hybridization of custom C. albicans Affymetrix arrays.
Project description:Constitutive overexpression of the Mdr1 efflux pump is an important mechanism of acquired drug resistance in the yeast Candida albicans. The zinc cluster transcription factor Mrr1 is a central regulator of MDR1 expression, but other transcription factors have also been implicated in MDR1 regulation. To better understand how MDR1-mediated drug resistance is achieved in this important fungal pathogen, we studied the interdependence of Mrr1 and two other MDR1 regulators, Upc2 and Cap1, in the control of MDR1 expression. A mutated, constitutively active Mrr1 could upregulate MDR1 and confer drug resistance in the absence of Upc2 or Cap1. On the other hand, Upc2 containing a gain-of-function mutation only slightly activated the MDR1 promoter, and this activation depended on the presence of a functional MRR1 gene. In contrast, a C-terminally truncated, activated form of Cap1 could upregulate MDR1 in a partially Mrr1-independent fashion. The induction of MDR1 expression by toxic chemicals occurred independently of Upc2, but required the presence of Mrr1 and also partially depended on Cap1. Transcriptional profiling and in vivo DNA binding studies showed that a constitutively active Mrr1 binds to and upregulates most of its direct target genes in the presence or absence of Cap1. Therefore, Mrr1 and Cap1 cooperate in the environmental induction of MDR1 expression in wild-type C. albicans, but gain-of-function mutations in either of the two transcription factors can independently mediate efflux pump overexpression and drug resistance. We endeavored to determine how the function of a gain-of-function allele of MRR1 (shown to confer high-level azole resistance) is affected when the CAP1 gene is disrupted.
Project description:Illumina HiSeq technology was used to generate mRNA profiles from Tulasnella calospora mycorrhizal protocorms compared to free-living mycelium . Protocorms and control mycelium were harvested after 30 days and used for RNA extraction. Reads of 2X100bp were generated and aligned to Tulasnella calospora transcripts (http://genome.jgi-psf.org/Tulca1) using CLC Genomics Workbench 6. mRNA profiles from Tulasnella calospora mycorrhizal protocorms and free-living mycelium were generated by paired-end (2x100bp) Illumina HiSeq2000 sequencing. Three biological replicates were sequenced for mycorrhizal and mycelium samples.
Project description:Here, we report the comparison of transcriptomes of Anabaena sp. PCC7120 and a FurC-overexpressing derivative strain grown under standard conditions (BG11) and after 48 hours of nitrogen step-down (BG110). Anabaena sp PCC7120 is a cyanobacterium that differentiates specialized nitrogen-fixing cells called heterocysts. Our data suggests that FurC directly controls the regulation of heterocyst differentiation and nitrogen fixation in this cyanobacterium. In addition, we found that FurC is also clearly involved in the regulation of several genes belonging to different functional categories, such as iron metabolism, photosynthesis and regulatory functions.