Unknown,Transcriptomics,Genomics,Proteomics

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Psoralen binding across the Escherichia coli chromosome

ABSTRACT: We used trimethylpsoralen intercalation to map supercoiling across the E. coli chromosome. We treated E. coli cells with trimethylpsoralen and exposed them to UV light. Psoralen enters cells, intercalates between DNA base pairs, and crosslinks the two strands of DNA at a rate proportional to the local superhelical density. We then fragmented DNA and hybridized crosslinked and non-crosslinked DNA fragments separately to tiling microarrays. In total 14 samples; 6 for wild-type in stationary phase (3 replicates x 2 samples as there are crosslinked and non-crosslinked samples for each replicate); 4 for wild-type in mid-exponential phase (2 replicates x 2 samples); 4 for the delta hupAB mutant in stationary phase (2 replicates x 2 samples)

ORGANISM(S): Escherichia coli str. K-12 substr. MG1655

SUBMITTER: Avantika Lal

PROVIDER: E-GEOD-77687 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The airways of the human lung are lined by an epithelium made up of ciliated and secretory luminal cells and undifferentiated p63+ Krt5+ basal cells. The integrity of this epithelium and its ability to act as a selective barrier are critical for normal lung function. In other epithelia there is evidence that transcription factors of the evolutionarily conserved grainyheadlike (GRHL) family play key roles in co-ordinating the expression of numerous proteins required for epithelial morphogenesis, differentiation, remodeling and repair. However, little is known about their function in the adult lung. Here, we focus on the role of GRHL2 in primary human bronchial epithelial (HBE) cells, using either shRNA or a dominant negative protein (DN-GRHL2) to inhibit its function. We follow changes in epithelial phenotype, and in gene transcription using RNA-seq or microarray analysis, both in undifferentiated basal cells and in cells differentiating in air-liquid interface culture into a mucociliary epithelium with transepithelial electrical resistance. We identify several hundreds of genes that are directly or indirectly regulated by GRHL2. Using ChIP-seq to map sites of GRHL2 binding in the basal cells we identify 7,687 potential primary targets, and confirm that GRHL2 binding is strongly enriched near GRHL-regulated genes. Different subsets of the large cohort of potential GRHL2 targets appear to be active in basal and differentiated cells. Taken together, the results strongly support the hypothesis that GRHL2 plays a key role in regulating many physiological functions of human airway epithelium, including those involving cell adhesion, polarity and morphogenesis. Frozen primary human bronchial epithelial (HBE) cells were obtained from three donors and grown to confluence in standard culture conditions. Cells were crosslinked with formaldehyde and collected in aliquots of ~20M cells per donor. ChIP-seq for GRHL2 (Antibody: Sigma HPA004820) was then performed relative to an input control for each donor.

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Psoralen binding across the Escherichia coli chromosome

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