SILAC-based quantitative proteomics approach to identify transcription factors interacting with a novel cis-regulatory element

Ngo Tat Trung, Rudolf Engelke, Gerhard Mittler

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

The motif TMTCGCGANR (M being C or A, R being A or G, and N any nucleotide) called M8 was discovered as a putative cis-regulatory element present in 368 human gene promoters. Of these, 236 (64%) are conserved within promoter sequences of four related organisms: human, mouse, rat and dog. However, transcription factors (TFs) interacting with the M8 motif has not yet been described. We previously reported the use of quantitative proteomics coupled to one-step DNA affinity purification as a means of screening for TFs associated with given functional DNA elements. The procedure is performed in-vitro employing SILAC-labeled nuclear extracts and making use of a well-characterized cis-regulatory motifs. Building on that, in this study we have combined our method with statistical analysis to filter out false positive hits from the one-step DNA affinity pull-down experiments. This resulted in the identification of zinc finger BED domain-containing protein 1 (ZBED1), alpha globin transcription factor CP2 (TFCP2), upstream binding protein 1 (UBP) and transcription factor CP2 like 1(TFCP2L1), as specific M8 interacting factors. We validated our screen demonstrating the in vivo binding of alpha globin transcription factor TFCP2 to selected genes harboring M8-containing promoters using ChIP (chromatin immuno-precipitation) assays. This not only implicates a functional role of the above proteins in regulating M8 motif containing genes, but also suggests the potential use of our approach to decipher protein-DNA interactions occurring in living cells.

Original languageEnglish
Pages (from-to)82-87
Number of pages6
JournalJournal of Proteomics and Bioinformatics
Volume7
Issue number3
DOIs
Publication statusPublished - 1 Jan 2014

Fingerprint

Transcription factors
Proteomics
Transcription Factors
DNA
alpha-Globins
Genes
Proteins
Zinc Fingers
Nucleotides
Chromatin
Purification
Zinc
Rats
Assays
Statistical methods
Carrier Proteins
Screening
Cells
Dogs
Experiments

Keywords

  • M8 motif
  • Quanitative proteomics
  • SILAC

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Computer Science Applications
  • Cell Biology

Cite this

SILAC-based quantitative proteomics approach to identify transcription factors interacting with a novel cis-regulatory element. / Trung, Ngo Tat; Engelke, Rudolf; Mittler, Gerhard.

In: Journal of Proteomics and Bioinformatics, Vol. 7, No. 3, 01.01.2014, p. 82-87.

Research output: Contribution to journalArticle

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