Changes in Fluorescence Recovery After Photobleaching (FRAP) as an indicator of SOX9 transcription factor activity

Kannan Govindaraj, Jan Hendriks, Diane S. Lidke, Marcel Karperien, Janine N. Post (Corresponding Author)

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Abstract

Cells respond to their environment via an intricate cellular signaling network, directing cell fate. Changes in cell fate are characterized by changes in gene transcription, dictated by (master) transcription factor activity. SOX9 is the master transcription factor for chondrocyte development. Its impaired function is implicated in osteoarthritis and growth disorders, such as dwarfism. However, the factors regulating SOX9 transcriptional activity are not yet fully mapped. Current methods to study transcription factor activity are indirect and largely limited to quantification of SOX9 target gene and protein expression levels after several hours or days of stimulation, leading to poor temporal resolution. We used Fluorescence Recovery After Photobleaching (FRAP) to study the mobility of SOX9 and correlated the changes in mobility to changes in its transcriptional activity by cross-validating with chromatin immunoprecipitation and qPCR. We show that using FRAP, we can quantify the changes in SOX9 mobility on short time scales as an indication of transcriptional activity, which correlated to changes of SOX9 DNA-binding and long-term target gene expression.

Original languageEnglish
Pages (from-to)107-117
Number of pages11
JournalBiochimica et Biophysica Acta - Gene Regulatory Mechanisms
Volume1862
Issue number1
Early online date20 Nov 2018
DOIs
Publication statusPublished - Jan 2019

Fingerprint

SOX9 Transcription Factor
Fluorescence Recovery After Photobleaching
Photobleaching
Transcription Factors
Fluorescence
Recovery
Growth Disorders
Cell signaling
Gene Expression
Dwarfism
Chromatin Immunoprecipitation
Transcription
Chondrocytes
Gene expression
Osteoarthritis
Chromatin
Genes
DNA
Proteins

Keywords

  • FRAP
  • Osteoarthritis
  • SOX9
  • Transcription factor dynamics
  • Transcriptional activity
  • Cartilage

Cite this

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abstract = "Cells respond to their environment via an intricate cellular signaling network, directing cell fate. Changes in cell fate are characterized by changes in gene transcription, dictated by (master) transcription factor activity. SOX9 is the master transcription factor for chondrocyte development. Its impaired function is implicated in osteoarthritis and growth disorders, such as dwarfism. However, the factors regulating SOX9 transcriptional activity are not yet fully mapped. Current methods to study transcription factor activity are indirect and largely limited to quantification of SOX9 target gene and protein expression levels after several hours or days of stimulation, leading to poor temporal resolution. We used Fluorescence Recovery After Photobleaching (FRAP) to study the mobility of SOX9 and correlated the changes in mobility to changes in its transcriptional activity by cross-validating with chromatin immunoprecipitation and qPCR. We show that using FRAP, we can quantify the changes in SOX9 mobility on short time scales as an indication of transcriptional activity, which correlated to changes of SOX9 DNA-binding and long-term target gene expression.",
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AU - Hendriks, Jan

AU - Lidke, Diane S.

AU - Karperien, Marcel

AU - Post, Janine N.

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AB - Cells respond to their environment via an intricate cellular signaling network, directing cell fate. Changes in cell fate are characterized by changes in gene transcription, dictated by (master) transcription factor activity. SOX9 is the master transcription factor for chondrocyte development. Its impaired function is implicated in osteoarthritis and growth disorders, such as dwarfism. However, the factors regulating SOX9 transcriptional activity are not yet fully mapped. Current methods to study transcription factor activity are indirect and largely limited to quantification of SOX9 target gene and protein expression levels after several hours or days of stimulation, leading to poor temporal resolution. We used Fluorescence Recovery After Photobleaching (FRAP) to study the mobility of SOX9 and correlated the changes in mobility to changes in its transcriptional activity by cross-validating with chromatin immunoprecipitation and qPCR. We show that using FRAP, we can quantify the changes in SOX9 mobility on short time scales as an indication of transcriptional activity, which correlated to changes of SOX9 DNA-binding and long-term target gene expression.

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