Unknown,Transcriptomics,Genomics,Proteomics

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Proteomic and Genomic Analysis of transcription factor TFII-I reveals insight into the response to cellular stress

ABSTRACT: We expressed biotin-tagged transcription factor TFII-I (GTF2I) in K562 cells and subjected crosslinked chromatin to streptavidin mediated pull down. We subjected the isolated DNA to Illumina sequencing. We identified 18920 TFII-I binding peaks. Nearest gene analysis revealed 5886 genes associated with a TFII-I binding peak in proliferating K562 cells. We generated two K562 single cell clones stably expressing bio-tagged TFII-I, and K562 cells expressing only the BirA protein biotin ligase from E.coli. These three clones were subjected to streptavidin mediated pull down and Illumina sequencing.

ORGANISM(S): Homo sapiens

SUBMITTER: Giorgio Papadopoulos

PROVIDER: E-GEOD-51065 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Genomic and proteomic analysis of transcription factor TFII-I reveals insight into the response to cellular stress.

Fan Alex Xiucheng AX Papadopoulos Giorgio L GL Hossain Mir A MA Lin I-Ju IJ Hu Jianhong J Tang Tommy Ming TM Kilberg Michael S MS Renne Rolf R Strouboulis John J Bungert Jörg J

Nucleic acids research 20140529 12

The ubiquitously expressed transcription factor TFII-I exerts both positive and negative effects on transcription. Using biotinylation tagging technology and high-throughput sequencing, we determined sites of chromatin interactions for TFII-I in the human erythroleukemia cell line K562. This analysis revealed that TFII-I binds upstream of the transcription start site of expressed genes, both upstream and downstream of the transcription start site of repressed genes, and downstream of RNA polymer ...[more]

PMID: 24875474

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Project description:The interaction between transcription factors and genomic DNA forms the basis for spatio-temporal control of gene expression. Therefore, these interactions and their impact on disease and cellular fate are extensively studied on a global level, mainly using techniques based on next-generation sequencing. These techniques, however, do not allow an unbiased study of proteins or entire protein complexes that bind to a certain DNA sequence. In recent years, DNA pull-downs followed by quantitative mass spectrometry were introduced to close this gap. Established protocols, however, are based on metabolic labeling techniques or require enormous amounts of cellular material, thus restricting the method to cell lines grown in culture. Furthermore, they require substantial amount of expertise, thus keeping this technique restricted to a limited number of laboratories. Here, we introduce a high-throughput compatible, LC-MS/MS based DNA pull-down that combines on-bead digestion with direct dimethyl labeling or label-free protein quantification. We demonstrate, that our method can efficiently identify transcription factors binding to their known consensus DNA motifs when using nuclear extracts from model cell lines. Subsequently, we apply the method to study DNA-protein interactions in primary foreskin fibroblasts and peripheral blood mononuclear cells (PBMCs) freshly isolated from human donors. We show that the same DNA sequence binds different sets of proteins in an established model cell line as opposed to PBMCs. This stresses the importance of selecting relevant cell extracts for any interaction in question. In-depth nuclear proteomes with absolute quantification of close to 7,000 proteins in K562 cells and PBMCs clearly link these differential interactions to differences in protein abundance. In conclusion, our approach, applicable to primary material and capable of profiling DNA-protein interactions in high-throughput, will likely prove itself as a useful screening platform and will provide invaluable functional data, for example through integration with large scale GWAS.

Dataset Information

Proteomic and Genomic Analysis of transcription factor TFII-I reveals insight into the response to cellular stress

Publications

Genomic and proteomic analysis of transcription factor TFII-I reveals insight into the response to cellular stress.

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