cJun modulates Gγ-globin gene expression via an upstream cAMP response element

Sirisha Kodeboyina, Parimaladevi Balamurugan, Li Liu, Betty Sue Pace

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

The upstream Gγ-globin gene cAMP response element (G-CRE) was previously shown to play a role in drug-mediated fetal hemoglobin induction. This effect is achieved via p38 mitogen activated protein kinase (MAPK)-dependent CREB1 and ATF-2 phosphorylation and G-CRE transactivation. Since this motif is also a predicted consensus binding site for cJun we extended our analysis to determine the ability of cJun to transactivate γ-globin through the G-CRE. Using chromatin immunoprecipitation assays we showed comparable in vivo cJun and CREB1 binding to the G-CRE region. Protein-protein interactions were confirmed between cJun/ATF-2 and CREB1/ATF-2 but not between CREB1 and cJun. However, we observed cJun and CREB1 binding to the G-CRE in vitro by electrophoretic mobility shift assay. Promoter pull-down assay followed by sequential western blot analysis confirmed co-localization of cJun, CREB1, and ATF-2 on the G-CRE. To show functional relevance, enforced expression studies with pLen-cJun and a Gγ-promoter (- 1500 to + 36) luciferase reporter were completed; we observed a concentration-dependent increase in luciferase activity with pLen-cJun similar to that produced by CREB1 enforced expression. Moreover, the G/A mutation at -1225 in the G-CRE abolished cJun transactivation. Finally, enforced cJun expression in K562 cells and normal primary erythroid progenitors enhanced endogenous γ-globin gene expression. We conclude that these data indicate that cJun activates the Gγ-globin promoter via the G-CRE in a manner comparable with CREB1 and propose a model for γ-globin activation based on DNA-protein interactions in the G-CRE.

Original languageEnglish (US)
Pages (from-to)7-15
Number of pages9
JournalBlood Cells, Molecules, and Diseases
Volume44
Issue number1
DOIs
StatePublished - Jan 1 2010
Externally publishedYes

Fingerprint

Globins
Response Elements
Gene Expression
Genes
Luciferases
Transcriptional Activation
Fetal Hemoglobin
Proteins
K562 Cells
Chromatin Immunoprecipitation
Electrophoretic Mobility Shift Assay
p38 Mitogen-Activated Protein Kinases
Western Blotting
Binding Sites
Phosphorylation
Mutation
DNA

Keywords

  • ATF-2
  • CREB1
  • cAMP response element
  • cJun
  • γ-globin

ASJC Scopus subject areas

  • Molecular Medicine
  • Hematology
  • Molecular Biology
  • Cell Biology

Cite this

cJun modulates Gγ-globin gene expression via an upstream cAMP response element. / Kodeboyina, Sirisha; Balamurugan, Parimaladevi; Liu, Li; Pace, Betty Sue.

In: Blood Cells, Molecules, and Diseases, Vol. 44, No. 1, 01.01.2010, p. 7-15.

Research output: Contribution to journalArticle

Kodeboyina, Sirisha ; Balamurugan, Parimaladevi ; Liu, Li ; Pace, Betty Sue. / cJun modulates Gγ-globin gene expression via an upstream cAMP response element. In: Blood Cells, Molecules, and Diseases. 2010 ; Vol. 44, No. 1. pp. 7-15.
@article{5b4e05ab08e5458bb72b89792a3f3c8a,
title = "cJun modulates Gγ-globin gene expression via an upstream cAMP response element",
abstract = "The upstream Gγ-globin gene cAMP response element (G-CRE) was previously shown to play a role in drug-mediated fetal hemoglobin induction. This effect is achieved via p38 mitogen activated protein kinase (MAPK)-dependent CREB1 and ATF-2 phosphorylation and G-CRE transactivation. Since this motif is also a predicted consensus binding site for cJun we extended our analysis to determine the ability of cJun to transactivate γ-globin through the G-CRE. Using chromatin immunoprecipitation assays we showed comparable in vivo cJun and CREB1 binding to the G-CRE region. Protein-protein interactions were confirmed between cJun/ATF-2 and CREB1/ATF-2 but not between CREB1 and cJun. However, we observed cJun and CREB1 binding to the G-CRE in vitro by electrophoretic mobility shift assay. Promoter pull-down assay followed by sequential western blot analysis confirmed co-localization of cJun, CREB1, and ATF-2 on the G-CRE. To show functional relevance, enforced expression studies with pLen-cJun and a Gγ-promoter (- 1500 to + 36) luciferase reporter were completed; we observed a concentration-dependent increase in luciferase activity with pLen-cJun similar to that produced by CREB1 enforced expression. Moreover, the G/A mutation at -1225 in the G-CRE abolished cJun transactivation. Finally, enforced cJun expression in K562 cells and normal primary erythroid progenitors enhanced endogenous γ-globin gene expression. We conclude that these data indicate that cJun activates the Gγ-globin promoter via the G-CRE in a manner comparable with CREB1 and propose a model for γ-globin activation based on DNA-protein interactions in the G-CRE.",
keywords = "ATF-2, CREB1, cAMP response element, cJun, γ-globin",
author = "Sirisha Kodeboyina and Parimaladevi Balamurugan and Li Liu and Pace, {Betty Sue}",
year = "2010",
month = "1",
day = "1",
doi = "10.1016/j.bcmd.2009.10.002",
language = "English (US)",
volume = "44",
pages = "7--15",
journal = "Blood Cells, Molecules, and Diseases",
issn = "1079-9796",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - cJun modulates Gγ-globin gene expression via an upstream cAMP response element

AU - Kodeboyina, Sirisha

AU - Balamurugan, Parimaladevi

AU - Liu, Li

AU - Pace, Betty Sue

PY - 2010/1/1

Y1 - 2010/1/1

N2 - The upstream Gγ-globin gene cAMP response element (G-CRE) was previously shown to play a role in drug-mediated fetal hemoglobin induction. This effect is achieved via p38 mitogen activated protein kinase (MAPK)-dependent CREB1 and ATF-2 phosphorylation and G-CRE transactivation. Since this motif is also a predicted consensus binding site for cJun we extended our analysis to determine the ability of cJun to transactivate γ-globin through the G-CRE. Using chromatin immunoprecipitation assays we showed comparable in vivo cJun and CREB1 binding to the G-CRE region. Protein-protein interactions were confirmed between cJun/ATF-2 and CREB1/ATF-2 but not between CREB1 and cJun. However, we observed cJun and CREB1 binding to the G-CRE in vitro by electrophoretic mobility shift assay. Promoter pull-down assay followed by sequential western blot analysis confirmed co-localization of cJun, CREB1, and ATF-2 on the G-CRE. To show functional relevance, enforced expression studies with pLen-cJun and a Gγ-promoter (- 1500 to + 36) luciferase reporter were completed; we observed a concentration-dependent increase in luciferase activity with pLen-cJun similar to that produced by CREB1 enforced expression. Moreover, the G/A mutation at -1225 in the G-CRE abolished cJun transactivation. Finally, enforced cJun expression in K562 cells and normal primary erythroid progenitors enhanced endogenous γ-globin gene expression. We conclude that these data indicate that cJun activates the Gγ-globin promoter via the G-CRE in a manner comparable with CREB1 and propose a model for γ-globin activation based on DNA-protein interactions in the G-CRE.

AB - The upstream Gγ-globin gene cAMP response element (G-CRE) was previously shown to play a role in drug-mediated fetal hemoglobin induction. This effect is achieved via p38 mitogen activated protein kinase (MAPK)-dependent CREB1 and ATF-2 phosphorylation and G-CRE transactivation. Since this motif is also a predicted consensus binding site for cJun we extended our analysis to determine the ability of cJun to transactivate γ-globin through the G-CRE. Using chromatin immunoprecipitation assays we showed comparable in vivo cJun and CREB1 binding to the G-CRE region. Protein-protein interactions were confirmed between cJun/ATF-2 and CREB1/ATF-2 but not between CREB1 and cJun. However, we observed cJun and CREB1 binding to the G-CRE in vitro by electrophoretic mobility shift assay. Promoter pull-down assay followed by sequential western blot analysis confirmed co-localization of cJun, CREB1, and ATF-2 on the G-CRE. To show functional relevance, enforced expression studies with pLen-cJun and a Gγ-promoter (- 1500 to + 36) luciferase reporter were completed; we observed a concentration-dependent increase in luciferase activity with pLen-cJun similar to that produced by CREB1 enforced expression. Moreover, the G/A mutation at -1225 in the G-CRE abolished cJun transactivation. Finally, enforced cJun expression in K562 cells and normal primary erythroid progenitors enhanced endogenous γ-globin gene expression. We conclude that these data indicate that cJun activates the Gγ-globin promoter via the G-CRE in a manner comparable with CREB1 and propose a model for γ-globin activation based on DNA-protein interactions in the G-CRE.

KW - ATF-2

KW - CREB1

KW - cAMP response element

KW - cJun

KW - γ-globin

UR - http://www.scopus.com/inward/record.url?scp=73049085606&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=73049085606&partnerID=8YFLogxK

U2 - 10.1016/j.bcmd.2009.10.002

DO - 10.1016/j.bcmd.2009.10.002

M3 - Article

C2 - 19861239

AN - SCOPUS:73049085606

VL - 44

SP - 7

EP - 15

JO - Blood Cells, Molecules, and Diseases

JF - Blood Cells, Molecules, and Diseases

SN - 1079-9796

IS - 1

ER -