Project description:Genome-wide occupancy analysis of TBX5, NKX2-5 and GATA4 in differentiating WT, Nkx2-5KO (NKO), Tbx5KO (TKO) and Nkx2-5;Tbx5KO (DKO) cells at the cardiac precursor (CP) and cardiomyocyte (CM) differentiation stages. Analysis of TBX5, NKX2-5 and GATA4 occupancy a and gene expression in WT, Tbx5KO, Nkx2-5KO and DoubleKO precursor (CP) and mature (CM) in vitro differentiated cardiomyocytes. We performed ChIP-exo experiments for NKX2-5, TBX5 and GATA4 in differentiating WT, Nkx2-5KO (NKO), Tbx5KO (TKO) and Nkx2-5;Tbx5KO (DKO) cells at the CP and CM stages. ChIP-exo was performed according to methods published previously (Boyer et al., 2005; Serandour et al., 2013; Wamstad et al., 2012) using specific antisera to TBX5 (sc-17866 XS, lot no. B1213), NKX2-5 (sc-8697 XS, lot no. B1213), and GATA4 (sc-1237 XS, lot no. B1213) on chromatin isolated from 10 mill. cells. Re-ChIP-exo was performed from 40 mill cells, combining methods published previously (Serandour et al., 2013; Shankaranarayanan et al., 2011). ChIP-exo Analysis: Barcoded libraries were single-end 50bp sequenced on an Illumina HiSeq 2500 instrument. Reads were trimmed using the fastq-mcf program (Aronesty, 2011), and then aligned to the mm9 mouse genome assembly using bowtie2 (ChIP-seq, ChIP-exo) (Langmead and Salzberg, 2012). Samtools was then used to retain reads that had a mapq score of 30 or greater (Li et al., 2009), thereby ensuring that each mapped uniquely to the genome assembly. The 5' -most position of reads that mapped to the reference strand and the 3Ã¢??-most position of reads that mapped to the non-reference strand were identified for each read as the actual edges of each exonuclease-treated fragment. The genome was divided into 20bp genomic bins and a normalized tag density was calculated for each genomic bin 'i' as follows: tag density(i)=([#tags within 75bp of i] X [total# genomic bins])/(total #of tags) To identify broad regions of binding, bins with tag densities of greater than 100 were merged to generate a peak list for each sample. Within 1kb of each region, strand-specific single-base-resolution tag densities were calculated for each dataset by dividing each region into 1bp bins, then counting the number of tags within 5bp of each bin. For each region of binding, the footprint for each bound region was defined as the span from the peak position of '+' strand binding to the peak position of '-' strand binding as seen from the high-resolution tag densities. For subsequent analyses, any overlapping footprint for replica/factor/stage/genotype was merged to generate an average footprint list. Only average footprints present in at least two replicas of the same factor, stage and genotype were considered (Final Average Footprints).

Project description:Background: Cardiac transcription factors are master regulators during heart development. Recently, some were shown to transdifferentiate noncardiac mesoderm cells and cardiac fibroblasts into cardiomyocytes. However, the individual roles of each transcription factors in activating cardiac gene program have not been elucidated. We examined cardiac-specific and genome-wide gene expression in fibroblasts induced with cardiac transcription factors Nkx2.5 (N), Tbx5 (T), Gata4 (G), Myocardin (M) alone or different combinations. Methodology/Principal Findings: We applied different combinations of human Nkx2.5 (N), Tbx5 (T), Gata4 (G) and Myocardin (M) lentiviruses into 10T1/2 fibroblasts. Immunostaining and quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that N, T, G or M alone did not induce expression of cardiac marker genes M-NM-1-myosin heavy chain (M-NM-1MHC) and cardiac troponin T (cTnT). Only T+M and T+G+M combinations induced M-NM-1MHC and cTnT expression. Microarray-based gene ontology analysis revealed that T alone inhibited most genes involved in cardiac-related processes and activated genes involved in Wnt receptor signaling pathway and in aberrant processes. M alone inhibited genes involved in Wnt receptor signaling pathway and activated genes involved in cardiac-related processes and in aberrant processes. G alone inhibited genes involved in ectoderm development. T+G+M combination was the most effective activator of genes associated with cardiac-related processes including muscle cell differentiation, sarcomere, striated muscle contraction, regulation of heart contraction, and glucose metabolism and fatty acid oxidation (two significant forms of cardiomyocyte energy metabolism). And unlike T, M, G alone or T+M, T+G+M did not activate genes associated with aberrant processes. Conclusions: Tbx5, Gata4 and Myocardin play different roles in activating cardiac gene program and in avoiding aberrant gene program activation. The combination of T+G+M activated cardiac gene program and avoided aberrant gene program activation. Two weeks after doxycline induction, total RNA was isolated from 10T1/2-tTA cells infected with different combinations of Tbx5, Gata4, and Myocardin lentiviruses. Biological triplicated.

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Mus musculus

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