Elucidating protein

DNA complex by oligonucleotide DNA affinity purification

Teddy T C Yang, Chi Wing Chow

Research output: Chapter in Book/Report/Conference proceedingChapter

3 Citations (Scopus)

Abstract

Transcription factors recruit a wide variety of associated co-factors to regulate gene expression. These co-factors include protein kinases, phosphatases, deacetylases, methylases, and ubiquitin ligases, etc. To identify novel protein kinases associated with transcription factor NFAT, we took advantage of the increased ability of DNA binding and used an oligonucleotide affinity-binding approach. Coupling with in-gel kinase assays to detect phosphotransferase activity, we were able to identify p90 ribosomal S6 kinase (RSK) and p70 S6 kinase (S6K) that are present in the NFAT:DNA complex. We further demonstrated that RSK and S6K binds to and physically interacts with NFATc4. Similar oligonucleotide affinity-binding approach can be coupled with other enzymatic reactions, such as dephosphorylation, deacetylation, methylation, ubiquitination, etc. Mass spectrometry can also be carried out to systemically identify these transcription co-factors in the protein:DNA complex. Lastly, gene-specific enhancer elements can also be devised based on their respective sequence to identify distinctive protein:DNA complexes.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
Pages75-84
Number of pages10
Volume809
DOIs
StatePublished - 2012

Publication series

NameMethods in Molecular Biology
Volume809
ISSN (Print)10643745

Fingerprint

Oligonucleotides
Ribosomal Protein S6 Kinases
Transcription Factors
DNA
Protein Kinases
Proteins
90-kDa Ribosomal Protein S6 Kinases
Phosphotransferases
70-kDa Ribosomal Protein S6 Kinases
Phosphoprotein Phosphatases
Ubiquitination
Ligases
Ubiquitin
Methylation
Mass Spectrometry
Gels
Gene Expression
Genes

Keywords

  • Oligonucleotide DNA affinity purification
  • Protein kinases
  • Transcription activation complex
  • Transcription co-factors
  • Transcription factor NFAT

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Yang, T. T. C., & Chow, C. W. (2012). Elucidating protein: DNA complex by oligonucleotide DNA affinity purification. In Methods in Molecular Biology (Vol. 809, pp. 75-84). (Methods in Molecular Biology; Vol. 809). https://doi.org/10.1007/978-1-61779-376-9_5

Elucidating protein : DNA complex by oligonucleotide DNA affinity purification. / Yang, Teddy T C; Chow, Chi Wing.

Methods in Molecular Biology. Vol. 809 2012. p. 75-84 (Methods in Molecular Biology; Vol. 809).

Research output: Chapter in Book/Report/Conference proceedingChapter

Yang, TTC & Chow, CW 2012, Elucidating protein: DNA complex by oligonucleotide DNA affinity purification. in Methods in Molecular Biology. vol. 809, Methods in Molecular Biology, vol. 809, pp. 75-84. https://doi.org/10.1007/978-1-61779-376-9_5
Yang TTC, Chow CW. Elucidating protein: DNA complex by oligonucleotide DNA affinity purification. In Methods in Molecular Biology. Vol. 809. 2012. p. 75-84. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-61779-376-9_5
Yang, Teddy T C ; Chow, Chi Wing. / Elucidating protein : DNA complex by oligonucleotide DNA affinity purification. Methods in Molecular Biology. Vol. 809 2012. pp. 75-84 (Methods in Molecular Biology).
@inbook{975638aa69914d4281952f6bba36832c,
title = "Elucidating protein: DNA complex by oligonucleotide DNA affinity purification",
abstract = "Transcription factors recruit a wide variety of associated co-factors to regulate gene expression. These co-factors include protein kinases, phosphatases, deacetylases, methylases, and ubiquitin ligases, etc. To identify novel protein kinases associated with transcription factor NFAT, we took advantage of the increased ability of DNA binding and used an oligonucleotide affinity-binding approach. Coupling with in-gel kinase assays to detect phosphotransferase activity, we were able to identify p90 ribosomal S6 kinase (RSK) and p70 S6 kinase (S6K) that are present in the NFAT:DNA complex. We further demonstrated that RSK and S6K binds to and physically interacts with NFATc4. Similar oligonucleotide affinity-binding approach can be coupled with other enzymatic reactions, such as dephosphorylation, deacetylation, methylation, ubiquitination, etc. Mass spectrometry can also be carried out to systemically identify these transcription co-factors in the protein:DNA complex. Lastly, gene-specific enhancer elements can also be devised based on their respective sequence to identify distinctive protein:DNA complexes.",
keywords = "Oligonucleotide DNA affinity purification, Protein kinases, Transcription activation complex, Transcription co-factors, Transcription factor NFAT",
author = "Yang, {Teddy T C} and Chow, {Chi Wing}",
year = "2012",
doi = "10.1007/978-1-61779-376-9_5",
language = "English (US)",
isbn = "9781617793752",
volume = "809",
series = "Methods in Molecular Biology",
pages = "75--84",
booktitle = "Methods in Molecular Biology",

}

TY - CHAP

T1 - Elucidating protein

T2 - DNA complex by oligonucleotide DNA affinity purification

AU - Yang, Teddy T C

AU - Chow, Chi Wing

PY - 2012

Y1 - 2012

N2 - Transcription factors recruit a wide variety of associated co-factors to regulate gene expression. These co-factors include protein kinases, phosphatases, deacetylases, methylases, and ubiquitin ligases, etc. To identify novel protein kinases associated with transcription factor NFAT, we took advantage of the increased ability of DNA binding and used an oligonucleotide affinity-binding approach. Coupling with in-gel kinase assays to detect phosphotransferase activity, we were able to identify p90 ribosomal S6 kinase (RSK) and p70 S6 kinase (S6K) that are present in the NFAT:DNA complex. We further demonstrated that RSK and S6K binds to and physically interacts with NFATc4. Similar oligonucleotide affinity-binding approach can be coupled with other enzymatic reactions, such as dephosphorylation, deacetylation, methylation, ubiquitination, etc. Mass spectrometry can also be carried out to systemically identify these transcription co-factors in the protein:DNA complex. Lastly, gene-specific enhancer elements can also be devised based on their respective sequence to identify distinctive protein:DNA complexes.

AB - Transcription factors recruit a wide variety of associated co-factors to regulate gene expression. These co-factors include protein kinases, phosphatases, deacetylases, methylases, and ubiquitin ligases, etc. To identify novel protein kinases associated with transcription factor NFAT, we took advantage of the increased ability of DNA binding and used an oligonucleotide affinity-binding approach. Coupling with in-gel kinase assays to detect phosphotransferase activity, we were able to identify p90 ribosomal S6 kinase (RSK) and p70 S6 kinase (S6K) that are present in the NFAT:DNA complex. We further demonstrated that RSK and S6K binds to and physically interacts with NFATc4. Similar oligonucleotide affinity-binding approach can be coupled with other enzymatic reactions, such as dephosphorylation, deacetylation, methylation, ubiquitination, etc. Mass spectrometry can also be carried out to systemically identify these transcription co-factors in the protein:DNA complex. Lastly, gene-specific enhancer elements can also be devised based on their respective sequence to identify distinctive protein:DNA complexes.

KW - Oligonucleotide DNA affinity purification

KW - Protein kinases

KW - Transcription activation complex

KW - Transcription co-factors

KW - Transcription factor NFAT

UR - http://www.scopus.com/inward/record.url?scp=84555196027&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84555196027&partnerID=8YFLogxK

U2 - 10.1007/978-1-61779-376-9_5

DO - 10.1007/978-1-61779-376-9_5

M3 - Chapter

SN - 9781617793752

VL - 809

T3 - Methods in Molecular Biology

SP - 75

EP - 84

BT - Methods in Molecular Biology

ER -