Crystallization and X-ray diffraction analysis of the HMG domain of the chondrogenesis master regulator Sox9 in complex with a ChIP-Seq-identified DNA element

Saravanan Vivekanandan, Balasubramanian Moovarkumudalvan, Julien Lescar, Prasanna Kolatkar

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Sox9 is a fundamental sex-determining gene and the master regulator of chondrogenesis, and is involved in the development of various vital organs such as testes, kidney, heart and brain, and in skeletal development. Similar to other known Sox transcription factors, Sox9 recognizes and binds DNA with the consensus sequence C(T/A)TTG(T/A)(T/A) through the highly conserved HMG domain. Nonetheless, the molecular basis of the functional specificity of Sox9 in key developmental processes is still unclear. As an initial step towards a mechanistic understanding of Sox9 transcriptional regulation, the current work describes the details of the purification of the mouse Sox9 HMG domain (mSox9HMG), its crystallization in complex with a ChIP-Seq-identified FOXP2 promoter DNA element and the X-ray diffraction data analysis of this complex. The mSox9HMG-FOXP2 promoter DNA complex was crystallized by the hanging-drop vapour-diffusion method using 20% PEG 3350 in 200mM sodium/potassium phosphate with 100mM bis-tris propane at pH 8.5. The crystals diffracted to 2.7Å resolution and the complex crystallized in the tetragonal space group P41212, with unit-cell parameters a = b = 99.49, c = 45.89Å. Crystal-packing parameters revealed that asymmetric unit contained one mSox9HMG-FOXP2 promoter DNA complex with an estimated solvent content of 64%.

Original languageEnglish
Pages (from-to)1437-1441
Number of pages5
JournalActa Crystallographica Section F:Structural Biology Communications
Volume71
DOIs
Publication statusPublished - 2015

Fingerprint

Chondrogenesis
regulators
Crystallization
X-Ray Diffraction
X ray diffraction analysis
deoxyribonucleic acid
crystallization
mice
DNA
diffraction
x rays
testes
potassium phosphates
Crystals
Consensus Sequence
kidneys
Regulator Genes
purification
propane
organs

Keywords

  • chondrogenesis
  • FOXP2 promoter
  • HMG domain
  • sex-determining gene
  • Sox9
  • transcription factor

ASJC Scopus subject areas

  • Biophysics
  • Genetics
  • Structural Biology
  • Condensed Matter Physics
  • Biochemistry

Cite this

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title = "Crystallization and X-ray diffraction analysis of the HMG domain of the chondrogenesis master regulator Sox9 in complex with a ChIP-Seq-identified DNA element",
abstract = "Sox9 is a fundamental sex-determining gene and the master regulator of chondrogenesis, and is involved in the development of various vital organs such as testes, kidney, heart and brain, and in skeletal development. Similar to other known Sox transcription factors, Sox9 recognizes and binds DNA with the consensus sequence C(T/A)TTG(T/A)(T/A) through the highly conserved HMG domain. Nonetheless, the molecular basis of the functional specificity of Sox9 in key developmental processes is still unclear. As an initial step towards a mechanistic understanding of Sox9 transcriptional regulation, the current work describes the details of the purification of the mouse Sox9 HMG domain (mSox9HMG), its crystallization in complex with a ChIP-Seq-identified FOXP2 promoter DNA element and the X-ray diffraction data analysis of this complex. The mSox9HMG-FOXP2 promoter DNA complex was crystallized by the hanging-drop vapour-diffusion method using 20{\%} PEG 3350 in 200mM sodium/potassium phosphate with 100mM bis-tris propane at pH 8.5. The crystals diffracted to 2.7{\AA} resolution and the complex crystallized in the tetragonal space group P41212, with unit-cell parameters a = b = 99.49, c = 45.89{\AA}. Crystal-packing parameters revealed that asymmetric unit contained one mSox9HMG-FOXP2 promoter DNA complex with an estimated solvent content of 64{\%}.",
keywords = "chondrogenesis, FOXP2 promoter, HMG domain, sex-determining gene, Sox9, transcription factor",
author = "Saravanan Vivekanandan and Balasubramanian Moovarkumudalvan and Julien Lescar and Prasanna Kolatkar",
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T1 - Crystallization and X-ray diffraction analysis of the HMG domain of the chondrogenesis master regulator Sox9 in complex with a ChIP-Seq-identified DNA element

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AU - Moovarkumudalvan, Balasubramanian

AU - Lescar, Julien

AU - Kolatkar, Prasanna

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N2 - Sox9 is a fundamental sex-determining gene and the master regulator of chondrogenesis, and is involved in the development of various vital organs such as testes, kidney, heart and brain, and in skeletal development. Similar to other known Sox transcription factors, Sox9 recognizes and binds DNA with the consensus sequence C(T/A)TTG(T/A)(T/A) through the highly conserved HMG domain. Nonetheless, the molecular basis of the functional specificity of Sox9 in key developmental processes is still unclear. As an initial step towards a mechanistic understanding of Sox9 transcriptional regulation, the current work describes the details of the purification of the mouse Sox9 HMG domain (mSox9HMG), its crystallization in complex with a ChIP-Seq-identified FOXP2 promoter DNA element and the X-ray diffraction data analysis of this complex. The mSox9HMG-FOXP2 promoter DNA complex was crystallized by the hanging-drop vapour-diffusion method using 20% PEG 3350 in 200mM sodium/potassium phosphate with 100mM bis-tris propane at pH 8.5. The crystals diffracted to 2.7Å resolution and the complex crystallized in the tetragonal space group P41212, with unit-cell parameters a = b = 99.49, c = 45.89Å. Crystal-packing parameters revealed that asymmetric unit contained one mSox9HMG-FOXP2 promoter DNA complex with an estimated solvent content of 64%.

AB - Sox9 is a fundamental sex-determining gene and the master regulator of chondrogenesis, and is involved in the development of various vital organs such as testes, kidney, heart and brain, and in skeletal development. Similar to other known Sox transcription factors, Sox9 recognizes and binds DNA with the consensus sequence C(T/A)TTG(T/A)(T/A) through the highly conserved HMG domain. Nonetheless, the molecular basis of the functional specificity of Sox9 in key developmental processes is still unclear. As an initial step towards a mechanistic understanding of Sox9 transcriptional regulation, the current work describes the details of the purification of the mouse Sox9 HMG domain (mSox9HMG), its crystallization in complex with a ChIP-Seq-identified FOXP2 promoter DNA element and the X-ray diffraction data analysis of this complex. The mSox9HMG-FOXP2 promoter DNA complex was crystallized by the hanging-drop vapour-diffusion method using 20% PEG 3350 in 200mM sodium/potassium phosphate with 100mM bis-tris propane at pH 8.5. The crystals diffracted to 2.7Å resolution and the complex crystallized in the tetragonal space group P41212, with unit-cell parameters a = b = 99.49, c = 45.89Å. Crystal-packing parameters revealed that asymmetric unit contained one mSox9HMG-FOXP2 promoter DNA complex with an estimated solvent content of 64%.

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