Characterization of Nfatc1 regulation identifies an enhancer required for gene expression that is specific to pro-valve endocardial cells in the developing heart

Bin Zhou, Bingruo Wu, Kevin L. Tompkins, Kathleen L. Boyer, Justin C. Grindley, H. Scott Baldwin

Research output: Contribution to journalArticle

58 Citations (Scopus)

Abstract

Nfatc1 is an endocardial transcription factor required for development of cardiac valves. Herein, we describe identification and characterization of a tissue-specific enhancer in the first intron of murine Nfatc1 that activates a heterogenic promoter and directs gene expression in a subpopulation of endocardial cells of the developing heart: the pro-valve endocardial cells. This enhancer activity begins on embryonic day (E) 8.5 in endocardial cells at the ventricular end of the atrioventricular canal, intensifies and extends from E9.5 to E11.5 in endocardium along the atrioventricular canal and outflow tract. By E12.5, the enhancer activity is accentuated in endocardial cells of forming valves. Sequential deletion analysis identified that a 250 bp DNA fragment at the 3′ end of the intron 1 is required for endocardial-specific activity. This region contains two short conserved sequences hosting a cluster of binding sites for transcription factors, including Nfat and Hox proteins. Electrophoresis mobility shift and chromatin immunoprecipitation assays demonstrated binding of Nfatc1 to the Nfat sites, and inactivation of Nfatc1 downregulated the enhancer activity in pro-valve endocardial cells. By contrast, mutation of the Hox site abolished its specificity, allowing gene expression in non pro-valve endocardium and extracardiac vasculature. Thus, autoregulation of Nfatc1 is required for maintaining high Nfatc1 expression in pro-valve endocardial cells, while suppression through the Hox site prevents its expression outside pro-valve endocardial cells during valve development. Our data demonstrate the first autonomous cell-specific enhancer for pro-valve endocardial cells and delineate a unique transcriptional mechanism that regulates endocardial Nfatc1 expression within developing cardiac valves.

Original languageEnglish (US)
Pages (from-to)1137-1146
Number of pages10
JournalDevelopment
Volume132
Issue number5
DOIs
StatePublished - Mar 2005
Externally publishedYes

Fingerprint

Gene Expression
Heart Valves
Endocardium
Introns
Transcription Factors
Conserved Sequence
Chromatin Immunoprecipitation
Electrophoresis
Homeostasis
Down-Regulation
Binding Sites
Mutation
DNA
Proteins

Keywords

  • Endocardium
  • Enhancer
  • Heart
  • Mouse
  • Nfatc1
  • Transcription

ASJC Scopus subject areas

  • Anatomy
  • Cell Biology

Cite this

Characterization of Nfatc1 regulation identifies an enhancer required for gene expression that is specific to pro-valve endocardial cells in the developing heart. / Zhou, Bin; Wu, Bingruo; Tompkins, Kevin L.; Boyer, Kathleen L.; Grindley, Justin C.; Baldwin, H. Scott.

In: Development, Vol. 132, No. 5, 03.2005, p. 1137-1146.

Research output: Contribution to journalArticle

Zhou, Bin ; Wu, Bingruo ; Tompkins, Kevin L. ; Boyer, Kathleen L. ; Grindley, Justin C. ; Baldwin, H. Scott. / Characterization of Nfatc1 regulation identifies an enhancer required for gene expression that is specific to pro-valve endocardial cells in the developing heart. In: Development. 2005 ; Vol. 132, No. 5. pp. 1137-1146.
@article{b871313035c648d7ba3c0fe70fe55a46,
title = "Characterization of Nfatc1 regulation identifies an enhancer required for gene expression that is specific to pro-valve endocardial cells in the developing heart",
abstract = "Nfatc1 is an endocardial transcription factor required for development of cardiac valves. Herein, we describe identification and characterization of a tissue-specific enhancer in the first intron of murine Nfatc1 that activates a heterogenic promoter and directs gene expression in a subpopulation of endocardial cells of the developing heart: the pro-valve endocardial cells. This enhancer activity begins on embryonic day (E) 8.5 in endocardial cells at the ventricular end of the atrioventricular canal, intensifies and extends from E9.5 to E11.5 in endocardium along the atrioventricular canal and outflow tract. By E12.5, the enhancer activity is accentuated in endocardial cells of forming valves. Sequential deletion analysis identified that a 250 bp DNA fragment at the 3′ end of the intron 1 is required for endocardial-specific activity. This region contains two short conserved sequences hosting a cluster of binding sites for transcription factors, including Nfat and Hox proteins. Electrophoresis mobility shift and chromatin immunoprecipitation assays demonstrated binding of Nfatc1 to the Nfat sites, and inactivation of Nfatc1 downregulated the enhancer activity in pro-valve endocardial cells. By contrast, mutation of the Hox site abolished its specificity, allowing gene expression in non pro-valve endocardium and extracardiac vasculature. Thus, autoregulation of Nfatc1 is required for maintaining high Nfatc1 expression in pro-valve endocardial cells, while suppression through the Hox site prevents its expression outside pro-valve endocardial cells during valve development. Our data demonstrate the first autonomous cell-specific enhancer for pro-valve endocardial cells and delineate a unique transcriptional mechanism that regulates endocardial Nfatc1 expression within developing cardiac valves.",
keywords = "Endocardium, Enhancer, Heart, Mouse, Nfatc1, Transcription",
author = "Bin Zhou and Bingruo Wu and Tompkins, {Kevin L.} and Boyer, {Kathleen L.} and Grindley, {Justin C.} and Baldwin, {H. Scott}",
year = "2005",
month = "3",
doi = "10.1242/dev.01640",
language = "English (US)",
volume = "132",
pages = "1137--1146",
journal = "Development (Cambridge)",
issn = "0950-1991",
publisher = "Company of Biologists Ltd",
number = "5",

}

TY - JOUR

T1 - Characterization of Nfatc1 regulation identifies an enhancer required for gene expression that is specific to pro-valve endocardial cells in the developing heart

AU - Zhou, Bin

AU - Wu, Bingruo

AU - Tompkins, Kevin L.

AU - Boyer, Kathleen L.

AU - Grindley, Justin C.

AU - Baldwin, H. Scott

PY - 2005/3

Y1 - 2005/3

N2 - Nfatc1 is an endocardial transcription factor required for development of cardiac valves. Herein, we describe identification and characterization of a tissue-specific enhancer in the first intron of murine Nfatc1 that activates a heterogenic promoter and directs gene expression in a subpopulation of endocardial cells of the developing heart: the pro-valve endocardial cells. This enhancer activity begins on embryonic day (E) 8.5 in endocardial cells at the ventricular end of the atrioventricular canal, intensifies and extends from E9.5 to E11.5 in endocardium along the atrioventricular canal and outflow tract. By E12.5, the enhancer activity is accentuated in endocardial cells of forming valves. Sequential deletion analysis identified that a 250 bp DNA fragment at the 3′ end of the intron 1 is required for endocardial-specific activity. This region contains two short conserved sequences hosting a cluster of binding sites for transcription factors, including Nfat and Hox proteins. Electrophoresis mobility shift and chromatin immunoprecipitation assays demonstrated binding of Nfatc1 to the Nfat sites, and inactivation of Nfatc1 downregulated the enhancer activity in pro-valve endocardial cells. By contrast, mutation of the Hox site abolished its specificity, allowing gene expression in non pro-valve endocardium and extracardiac vasculature. Thus, autoregulation of Nfatc1 is required for maintaining high Nfatc1 expression in pro-valve endocardial cells, while suppression through the Hox site prevents its expression outside pro-valve endocardial cells during valve development. Our data demonstrate the first autonomous cell-specific enhancer for pro-valve endocardial cells and delineate a unique transcriptional mechanism that regulates endocardial Nfatc1 expression within developing cardiac valves.

AB - Nfatc1 is an endocardial transcription factor required for development of cardiac valves. Herein, we describe identification and characterization of a tissue-specific enhancer in the first intron of murine Nfatc1 that activates a heterogenic promoter and directs gene expression in a subpopulation of endocardial cells of the developing heart: the pro-valve endocardial cells. This enhancer activity begins on embryonic day (E) 8.5 in endocardial cells at the ventricular end of the atrioventricular canal, intensifies and extends from E9.5 to E11.5 in endocardium along the atrioventricular canal and outflow tract. By E12.5, the enhancer activity is accentuated in endocardial cells of forming valves. Sequential deletion analysis identified that a 250 bp DNA fragment at the 3′ end of the intron 1 is required for endocardial-specific activity. This region contains two short conserved sequences hosting a cluster of binding sites for transcription factors, including Nfat and Hox proteins. Electrophoresis mobility shift and chromatin immunoprecipitation assays demonstrated binding of Nfatc1 to the Nfat sites, and inactivation of Nfatc1 downregulated the enhancer activity in pro-valve endocardial cells. By contrast, mutation of the Hox site abolished its specificity, allowing gene expression in non pro-valve endocardium and extracardiac vasculature. Thus, autoregulation of Nfatc1 is required for maintaining high Nfatc1 expression in pro-valve endocardial cells, while suppression through the Hox site prevents its expression outside pro-valve endocardial cells during valve development. Our data demonstrate the first autonomous cell-specific enhancer for pro-valve endocardial cells and delineate a unique transcriptional mechanism that regulates endocardial Nfatc1 expression within developing cardiac valves.

KW - Endocardium

KW - Enhancer

KW - Heart

KW - Mouse

KW - Nfatc1

KW - Transcription

UR - http://www.scopus.com/inward/record.url?scp=16844380032&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=16844380032&partnerID=8YFLogxK

U2 - 10.1242/dev.01640

DO - 10.1242/dev.01640

M3 - Article

VL - 132

SP - 1137

EP - 1146

JO - Development (Cambridge)

JF - Development (Cambridge)

SN - 0950-1991

IS - 5

ER -