Project description:Transcriptional profiling of Mycobacterium smegmatis comparing the effects of wild type levels of CarD expression with CarD depletion. Transcriptional profile of the mgm1701 control strain was compared with that of mgm1703, a strain where CarD transcription is regulated by a Tet-On system. Both strains were grown in the absence of anhydrotetracline for 13 hours in nutrient rich broth to allow for depletion of CarD transcription in mgm1703.
Project description:Human fibroblast of different age were investigated for their gene expression profiles in quiescence. Cultures were established from skin samples derived from young and old donors, obtained from the NIA Aging Cell Repository (Coriell Institute for Medical Research, Camden, NJ). All cell lines originated from 2 mm punch biopsies taken from the medial aspect of the upper arm. The donors were members of the Baltimore Longitudinal Study of Aging (BLSA) where they were characterized as "healthy" indviduals. The cell lines investigated had normal karyotypes.
Project description:Transcriptional profiling of Mycobacterium smegmatis comparing the effects of wild type levels of CarD expression with CarD depletion. Transcriptional profile of the mgm1701 control strain was compared with that of mgm1703, a strain where CarD transcription is regulated by a Tet-On system. Both strains were grown in the absence of anhydrotetracline for 13 hours in nutrient rich broth to allow for depletion of CarD transcription in mgm1703. Two condition experiment, control (mgm1701) vs CarD depletion (mgm1703). 3 biological replicates of each strain.
Project description:Genome-wide transcriptome analysis of expression changes in EBV transformed cell lines from the Coriell Cell Repository in Parkinson and Control subjects.
Project description:CarD is an essential mycobacterial protein that we had previously shown to bind the RNA polymerase (RNAP) and affect the transcriptional profile of M. smegmatis and Mycobacterium tuberculosis. For this reason, we suspected that CarD was directly regulating transcriptional complexes but we did not know at what stage of CarD was functioning and at which genes CarD interacted with the RNAP. To determine in which stage of the transcription cycle (initiation, elongation, or termination) CarD acts, we used Chromatin Immunoprecipitation sequencing (ChIP-seq) to survey the distribution of CarD throughout the M. smegmatis chromosome. Specific antibodies targeting core RNAPb, RNAPσ, or a hemagglutinin (HA) epitope fused to CarD (CarD-HA) were used to co-immunoprecipitate associated DNA. CarD-HA was immunoprecipitated from the M. smegmatis Mc2155 ΔcarD attB::tetcarD-HA strain and unfused HA was immunoprecipitated from the Mc2155 attB::pmsg431 strain with monoclonal antibodies specific for HA (Sigma). RNAP β and σ were immunoprecipitated from M. smegmatis ΔcarD attB::tetcarD-HA with monoclonal antibodies specific for these subunits (Neoclone, Madison, WI; 8RB13 for β, 2G10 for σ). Co-precipitated DNA was sequenced using a SOLiD sequencer (Life Technologies), which provided sufficient reads for 100-fold coverage of the genome. The number of sequence reads per base pair was normalized to the total number of reads and expressed as a log2 value. The reads per base pair from the HA-alone sample served as the background and was subtracted from the other datasets. We found that CarD was never present on the genome in the absence of RNAP. However, whereas RNAP core enzyme was found throughout transcribed regions of the genome, CarD was primarily associated with promoter regions and highly correlated with RNAPσ. The colocalization of σA and CarD led us to propose that in vivo, CarD associates with RNAP initiation complexes at most promoters and is therefore a global regulator of transcription initiation.