Specific GATA-binding elements in the GnRH promoter are required for gene expression pulse activity: Role of GATA-4 and GATA-5 in this intermittent process

Gilles M. Leclerc, Sudeep K. Bose, Fredric R. Boockfor

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Recent evidence reveals that several GATA factors act as versatile transcriptional modulators in neuroendocrine gene expression. The rat GnRH promoter is expressed in an episodic fashion that requires a portion of the promoter termed the neuron-specific enhancer (NSE) for activity. In this study, we examined whether certain GATA regulatory elements in the NSE are necessary for this intermittent activity. When injected into individual living GT1-7 cells, luciferase reporter constructs containing mutations of either GATA-A- or GATA-B-binding sites resulted in a marked reduction in gene expression pulse frequency, while mutations of both sites virtually abolished pulses. In subsequent studies, RT-PCR and western blot analysis revealed for the first time that GATA-5 and GATA-6 were expressed in GT1-7 cells, but electrophoretic mobility shift assays demonstrated further that GATA-5 bound to one of these GATA sites: GATA-A. Chromatin immunoprecipitation analysis revealed that all three factors, GATA-4, GATA-5, and GATA-6, were associated with the GnRH promoter in vivo. Interestingly though, immunoneutralization of GATA-5 or GATA-4 (reported to bind GATA-B) abolished gene expression pulses, but injection of GATA-6 antibody did not, indicating that of these factors just GATA-5 and GATA-4 are critical for intermittent activity. Finally, gel shift competition experiments revealed an interaction between proteins binding at the GATA-A site and those associating with an adjacent OCT1 site, previously shown to be necessary for pulse formation. These findings indicate that episodic GnRH gene expression pulses are mediated by GATA-5 and GATA-4, likely acting through the GATA-binding sites in the GnRH NSE region. Moreover, our observations that factors associated with GATA sites may also interact with OCT1 sites and that both are critical for pulse activity raise the intriguing possibility that GnRH pulse elaboration is a highly complex process that may require the coordinated interaction of several NSE-binding elements of the GnRH promoter.

Original languageEnglish
Pages (from-to)1-16
Number of pages16
JournalNeuroendocrinology
Volume88
Issue number1
DOIs
StatePublished - Jul 1 2008
Externally publishedYes

Fingerprint

Gonadotropin-Releasing Hormone
GATA Transcription Factors
Gene Expression
Neurons
Binding Sites
Chromatin Immunoprecipitation
Electrophoretic Mobility Shift Assay
Mutation Rate
Luciferases
Protein Binding
Western Blotting
Gels
Polymerase Chain Reaction
Mutation
Injections
Antibodies

Keywords

  • Cellular pulsatility
  • Fluorescent EMSA
  • GATA-5
  • GATA-6
  • GT1-7 cells, microinjection
  • Luciferase reporter gene
  • Single-cell imaging

ASJC Scopus subject areas

  • Endocrinology
  • Neuroscience(all)

Cite this

Specific GATA-binding elements in the GnRH promoter are required for gene expression pulse activity : Role of GATA-4 and GATA-5 in this intermittent process. / Leclerc, Gilles M.; Bose, Sudeep K.; Boockfor, Fredric R.

In: Neuroendocrinology, Vol. 88, No. 1, 01.07.2008, p. 1-16.

Research output: Contribution to journalArticle

@article{79fc002f3bce410a95ca7f23a4085f0b,
title = "Specific GATA-binding elements in the GnRH promoter are required for gene expression pulse activity: Role of GATA-4 and GATA-5 in this intermittent process",
abstract = "Recent evidence reveals that several GATA factors act as versatile transcriptional modulators in neuroendocrine gene expression. The rat GnRH promoter is expressed in an episodic fashion that requires a portion of the promoter termed the neuron-specific enhancer (NSE) for activity. In this study, we examined whether certain GATA regulatory elements in the NSE are necessary for this intermittent activity. When injected into individual living GT1-7 cells, luciferase reporter constructs containing mutations of either GATA-A- or GATA-B-binding sites resulted in a marked reduction in gene expression pulse frequency, while mutations of both sites virtually abolished pulses. In subsequent studies, RT-PCR and western blot analysis revealed for the first time that GATA-5 and GATA-6 were expressed in GT1-7 cells, but electrophoretic mobility shift assays demonstrated further that GATA-5 bound to one of these GATA sites: GATA-A. Chromatin immunoprecipitation analysis revealed that all three factors, GATA-4, GATA-5, and GATA-6, were associated with the GnRH promoter in vivo. Interestingly though, immunoneutralization of GATA-5 or GATA-4 (reported to bind GATA-B) abolished gene expression pulses, but injection of GATA-6 antibody did not, indicating that of these factors just GATA-5 and GATA-4 are critical for intermittent activity. Finally, gel shift competition experiments revealed an interaction between proteins binding at the GATA-A site and those associating with an adjacent OCT1 site, previously shown to be necessary for pulse formation. These findings indicate that episodic GnRH gene expression pulses are mediated by GATA-5 and GATA-4, likely acting through the GATA-binding sites in the GnRH NSE region. Moreover, our observations that factors associated with GATA sites may also interact with OCT1 sites and that both are critical for pulse activity raise the intriguing possibility that GnRH pulse elaboration is a highly complex process that may require the coordinated interaction of several NSE-binding elements of the GnRH promoter.",
keywords = "Cellular pulsatility, Fluorescent EMSA, GATA-5, GATA-6, GT1-7 cells, microinjection, Luciferase reporter gene, Single-cell imaging",
author = "Leclerc, {Gilles M.} and Bose, {Sudeep K.} and Boockfor, {Fredric R.}",
year = "2008",
month = "7",
day = "1",
doi = "10.1159/000115952",
language = "English",
volume = "88",
pages = "1--16",
journal = "Neuroendocrinology",
issn = "0028-3835",
publisher = "S. Karger AG",
number = "1",

}

TY - JOUR

T1 - Specific GATA-binding elements in the GnRH promoter are required for gene expression pulse activity

T2 - Role of GATA-4 and GATA-5 in this intermittent process

AU - Leclerc, Gilles M.

AU - Bose, Sudeep K.

AU - Boockfor, Fredric R.

PY - 2008/7/1

Y1 - 2008/7/1

N2 - Recent evidence reveals that several GATA factors act as versatile transcriptional modulators in neuroendocrine gene expression. The rat GnRH promoter is expressed in an episodic fashion that requires a portion of the promoter termed the neuron-specific enhancer (NSE) for activity. In this study, we examined whether certain GATA regulatory elements in the NSE are necessary for this intermittent activity. When injected into individual living GT1-7 cells, luciferase reporter constructs containing mutations of either GATA-A- or GATA-B-binding sites resulted in a marked reduction in gene expression pulse frequency, while mutations of both sites virtually abolished pulses. In subsequent studies, RT-PCR and western blot analysis revealed for the first time that GATA-5 and GATA-6 were expressed in GT1-7 cells, but electrophoretic mobility shift assays demonstrated further that GATA-5 bound to one of these GATA sites: GATA-A. Chromatin immunoprecipitation analysis revealed that all three factors, GATA-4, GATA-5, and GATA-6, were associated with the GnRH promoter in vivo. Interestingly though, immunoneutralization of GATA-5 or GATA-4 (reported to bind GATA-B) abolished gene expression pulses, but injection of GATA-6 antibody did not, indicating that of these factors just GATA-5 and GATA-4 are critical for intermittent activity. Finally, gel shift competition experiments revealed an interaction between proteins binding at the GATA-A site and those associating with an adjacent OCT1 site, previously shown to be necessary for pulse formation. These findings indicate that episodic GnRH gene expression pulses are mediated by GATA-5 and GATA-4, likely acting through the GATA-binding sites in the GnRH NSE region. Moreover, our observations that factors associated with GATA sites may also interact with OCT1 sites and that both are critical for pulse activity raise the intriguing possibility that GnRH pulse elaboration is a highly complex process that may require the coordinated interaction of several NSE-binding elements of the GnRH promoter.

AB - Recent evidence reveals that several GATA factors act as versatile transcriptional modulators in neuroendocrine gene expression. The rat GnRH promoter is expressed in an episodic fashion that requires a portion of the promoter termed the neuron-specific enhancer (NSE) for activity. In this study, we examined whether certain GATA regulatory elements in the NSE are necessary for this intermittent activity. When injected into individual living GT1-7 cells, luciferase reporter constructs containing mutations of either GATA-A- or GATA-B-binding sites resulted in a marked reduction in gene expression pulse frequency, while mutations of both sites virtually abolished pulses. In subsequent studies, RT-PCR and western blot analysis revealed for the first time that GATA-5 and GATA-6 were expressed in GT1-7 cells, but electrophoretic mobility shift assays demonstrated further that GATA-5 bound to one of these GATA sites: GATA-A. Chromatin immunoprecipitation analysis revealed that all three factors, GATA-4, GATA-5, and GATA-6, were associated with the GnRH promoter in vivo. Interestingly though, immunoneutralization of GATA-5 or GATA-4 (reported to bind GATA-B) abolished gene expression pulses, but injection of GATA-6 antibody did not, indicating that of these factors just GATA-5 and GATA-4 are critical for intermittent activity. Finally, gel shift competition experiments revealed an interaction between proteins binding at the GATA-A site and those associating with an adjacent OCT1 site, previously shown to be necessary for pulse formation. These findings indicate that episodic GnRH gene expression pulses are mediated by GATA-5 and GATA-4, likely acting through the GATA-binding sites in the GnRH NSE region. Moreover, our observations that factors associated with GATA sites may also interact with OCT1 sites and that both are critical for pulse activity raise the intriguing possibility that GnRH pulse elaboration is a highly complex process that may require the coordinated interaction of several NSE-binding elements of the GnRH promoter.

KW - Cellular pulsatility

KW - Fluorescent EMSA

KW - GATA-5

KW - GATA-6

KW - GT1-7 cells, microinjection

KW - Luciferase reporter gene

KW - Single-cell imaging

UR - http://www.scopus.com/inward/record.url?scp=47549106165&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=47549106165&partnerID=8YFLogxK

U2 - 10.1159/000115952

DO - 10.1159/000115952

M3 - Article

C2 - 18259093

AN - SCOPUS:47549106165

VL - 88

SP - 1

EP - 16

JO - Neuroendocrinology

JF - Neuroendocrinology

SN - 0028-3835

IS - 1

ER -