The homeobox gene GAX activates p21WAF1/CIP1 expression in vascular endothelial cells through direct interaction with upstream AT-rich sequences

Yun Chen, Alejandro D. Leal, Sejal Patel, David H. Gorski

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

Tumors secrete pro-angiogenic factors to induce the ingrowth of blood vessels from the surrounding stroma, the end targets of which are vascular endothelial cells (ECs). The homeobox gene GAX inhibits angiogenesis and induces p21WAF1/CIP1 expression in vascular ECs. To elucidate the mechanism through which GAX activates p21WAF1/CIP1 expression, we constructed GAX cDNAs with deletions of the N-terminal domain, the homeodomain, or the C-terminal domain and then assessed these constructs for their ability to activate p21WAF1/CIP1. There was an absolute requirement for the homeodomain, whereas deleting the C-terminal domain decreased but did not abolish transactivation of the p21WAF1/CIP1 promoter by GAX. Deleting the N-terminal domain did abolish transactivation. Next, we performed chromatin immunoprecipitation and found, ∼15 kb upstream of the p21 WAF1/CIP1 ATG codon, an ATTA-containing GAX-binding site (designated A6) with a sequence similar to that of other homeodomain-binding sites. GAX was able to bind to A6 in a homeodomain-dependent manner and thereby activate the expression of a reporter gene coupled to this sequence, and this activation was abolished by mutating specific residues in this sequence. On the basis of the sequence of A6, we were then able to locate other ATTA-containing sequences that also bound GAX and activated transcription in reporter constructs. Finally, we found that the ability of these GAX deletions to induce G0/G 1 arrest correlates with their ability to transactivate the p21 WAF1/CIP1 promoter. We conclude that GAX activates p21 WAF1/CIP1 through multiple upstream AT-rich sequences. Given the multiple biological activities of GAX in regulating EC function, identification of a putative GAX-binding site will allow the study of how GAX activates or represses other downstream targets to inhibit angiogenesis.

Original languageEnglish (US)
Pages (from-to)507-517
Number of pages11
JournalJournal of Biological Chemistry
Volume282
Issue number1
DOIs
StatePublished - Jan 5 2007
Externally publishedYes

Fingerprint

AT Rich Sequence
Homeobox Genes
Endothelial cells
Endothelial Cells
Genes
Binding Sites
Transcriptional Activation
Angiogenesis Inducing Agents
Chromatin Immunoprecipitation
Blood vessels
Transcription
Bioactivity
Reporter Genes
Codon
Chromatin
Blood Vessels
Tumors
Complementary DNA
Chemical activation
Neoplasms

ASJC Scopus subject areas

  • Biochemistry

Cite this

The homeobox gene GAX activates p21WAF1/CIP1 expression in vascular endothelial cells through direct interaction with upstream AT-rich sequences. / Chen, Yun; Leal, Alejandro D.; Patel, Sejal; Gorski, David H.

In: Journal of Biological Chemistry, Vol. 282, No. 1, 05.01.2007, p. 507-517.

Research output: Contribution to journalArticle

@article{5b300bcb4448492eaa9f0b295f8a92d4,
title = "The homeobox gene GAX activates p21WAF1/CIP1 expression in vascular endothelial cells through direct interaction with upstream AT-rich sequences",
abstract = "Tumors secrete pro-angiogenic factors to induce the ingrowth of blood vessels from the surrounding stroma, the end targets of which are vascular endothelial cells (ECs). The homeobox gene GAX inhibits angiogenesis and induces p21WAF1/CIP1 expression in vascular ECs. To elucidate the mechanism through which GAX activates p21WAF1/CIP1 expression, we constructed GAX cDNAs with deletions of the N-terminal domain, the homeodomain, or the C-terminal domain and then assessed these constructs for their ability to activate p21WAF1/CIP1. There was an absolute requirement for the homeodomain, whereas deleting the C-terminal domain decreased but did not abolish transactivation of the p21WAF1/CIP1 promoter by GAX. Deleting the N-terminal domain did abolish transactivation. Next, we performed chromatin immunoprecipitation and found, ∼15 kb upstream of the p21 WAF1/CIP1 ATG codon, an ATTA-containing GAX-binding site (designated A6) with a sequence similar to that of other homeodomain-binding sites. GAX was able to bind to A6 in a homeodomain-dependent manner and thereby activate the expression of a reporter gene coupled to this sequence, and this activation was abolished by mutating specific residues in this sequence. On the basis of the sequence of A6, we were then able to locate other ATTA-containing sequences that also bound GAX and activated transcription in reporter constructs. Finally, we found that the ability of these GAX deletions to induce G0/G 1 arrest correlates with their ability to transactivate the p21 WAF1/CIP1 promoter. We conclude that GAX activates p21 WAF1/CIP1 through multiple upstream AT-rich sequences. Given the multiple biological activities of GAX in regulating EC function, identification of a putative GAX-binding site will allow the study of how GAX activates or represses other downstream targets to inhibit angiogenesis.",
author = "Yun Chen and Leal, {Alejandro D.} and Sejal Patel and Gorski, {David H.}",
year = "2007",
month = "1",
day = "5",
doi = "10.1074/jbc.M606604200",
language = "English (US)",
volume = "282",
pages = "507--517",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "1",

}

TY - JOUR

T1 - The homeobox gene GAX activates p21WAF1/CIP1 expression in vascular endothelial cells through direct interaction with upstream AT-rich sequences

AU - Chen, Yun

AU - Leal, Alejandro D.

AU - Patel, Sejal

AU - Gorski, David H.

PY - 2007/1/5

Y1 - 2007/1/5

N2 - Tumors secrete pro-angiogenic factors to induce the ingrowth of blood vessels from the surrounding stroma, the end targets of which are vascular endothelial cells (ECs). The homeobox gene GAX inhibits angiogenesis and induces p21WAF1/CIP1 expression in vascular ECs. To elucidate the mechanism through which GAX activates p21WAF1/CIP1 expression, we constructed GAX cDNAs with deletions of the N-terminal domain, the homeodomain, or the C-terminal domain and then assessed these constructs for their ability to activate p21WAF1/CIP1. There was an absolute requirement for the homeodomain, whereas deleting the C-terminal domain decreased but did not abolish transactivation of the p21WAF1/CIP1 promoter by GAX. Deleting the N-terminal domain did abolish transactivation. Next, we performed chromatin immunoprecipitation and found, ∼15 kb upstream of the p21 WAF1/CIP1 ATG codon, an ATTA-containing GAX-binding site (designated A6) with a sequence similar to that of other homeodomain-binding sites. GAX was able to bind to A6 in a homeodomain-dependent manner and thereby activate the expression of a reporter gene coupled to this sequence, and this activation was abolished by mutating specific residues in this sequence. On the basis of the sequence of A6, we were then able to locate other ATTA-containing sequences that also bound GAX and activated transcription in reporter constructs. Finally, we found that the ability of these GAX deletions to induce G0/G 1 arrest correlates with their ability to transactivate the p21 WAF1/CIP1 promoter. We conclude that GAX activates p21 WAF1/CIP1 through multiple upstream AT-rich sequences. Given the multiple biological activities of GAX in regulating EC function, identification of a putative GAX-binding site will allow the study of how GAX activates or represses other downstream targets to inhibit angiogenesis.

AB - Tumors secrete pro-angiogenic factors to induce the ingrowth of blood vessels from the surrounding stroma, the end targets of which are vascular endothelial cells (ECs). The homeobox gene GAX inhibits angiogenesis and induces p21WAF1/CIP1 expression in vascular ECs. To elucidate the mechanism through which GAX activates p21WAF1/CIP1 expression, we constructed GAX cDNAs with deletions of the N-terminal domain, the homeodomain, or the C-terminal domain and then assessed these constructs for their ability to activate p21WAF1/CIP1. There was an absolute requirement for the homeodomain, whereas deleting the C-terminal domain decreased but did not abolish transactivation of the p21WAF1/CIP1 promoter by GAX. Deleting the N-terminal domain did abolish transactivation. Next, we performed chromatin immunoprecipitation and found, ∼15 kb upstream of the p21 WAF1/CIP1 ATG codon, an ATTA-containing GAX-binding site (designated A6) with a sequence similar to that of other homeodomain-binding sites. GAX was able to bind to A6 in a homeodomain-dependent manner and thereby activate the expression of a reporter gene coupled to this sequence, and this activation was abolished by mutating specific residues in this sequence. On the basis of the sequence of A6, we were then able to locate other ATTA-containing sequences that also bound GAX and activated transcription in reporter constructs. Finally, we found that the ability of these GAX deletions to induce G0/G 1 arrest correlates with their ability to transactivate the p21 WAF1/CIP1 promoter. We conclude that GAX activates p21 WAF1/CIP1 through multiple upstream AT-rich sequences. Given the multiple biological activities of GAX in regulating EC function, identification of a putative GAX-binding site will allow the study of how GAX activates or represses other downstream targets to inhibit angiogenesis.

UR - http://www.scopus.com/inward/record.url?scp=33846967463&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33846967463&partnerID=8YFLogxK

U2 - 10.1074/jbc.M606604200

DO - 10.1074/jbc.M606604200

M3 - Article

VL - 282

SP - 507

EP - 517

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 1

ER -