Transcriptional regulation of the murine Connexin40 promoter by cardiac factors Nkx2-5, GATA4 and Tbx5.
OBJECTIVE
Connexin40 (Cx40) is a gap junction protein expressed specifically in developing and mature atrial myocytes and cells of the conduction system. In this report, we identify cis-acting elements within the mouse Cx40 promoter and unravel part of the complex pathways involved in the cardiac expression of this gene.


METHODS
To identify the factors involved in the cardiac expression of Cx40, we used transient transfections in mammalian cells coupled with electrophoretic mobility shift assays (EMSA) and RT-PCR.


RESULTS
Within the promoter region, we identified the minimal elements required for transcriptional activity within 150 base pairs (bp) upstream of the transcriptional start site. Several putative regulatory sites for transcription factors were predicted within this region by computer analysis, and we demonstrated that the nuclear factors Sp1, Nkx2-5, GATA4 and Tbx5 could interact specifically with elements present in the minimal promoter region of the Cx40. Furthermore, co-transfection experiments showed the ability of Nkx2-5 and GATA4 to transactivate the minimal Cx40 promoter while Tbx5 repressed Nkx2-5/GATA4-mediated activation. Mutagenesis of the Nkx2-5 core site in the Cx40 promoter led to significantly decreased activity in rat smooth muscle cell line A7r5. Consistent with this, mouse embryos lacking Nkx2-5 showed a marked decrease in Cx40 expression.


CONCLUSIONS
In this work, we cloned the promoter region of the Cx40 and demonstrated that the core promoter was modulated by cardiac transcriptional factors Nkx2-5, Tbx5 and GATA4 acting together with ubiquitous Sp1.
Linhares
Vania L F
VL
Laboratório de Cardiologia Celular e Molecular-Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro 20941-900, Brazil.
Almeida
Norma A S
NA
Menezes
Diego C
DC
Elliott
David A
DA
Lai
Donna
D
Beyer
Eric C
EC
Campos de Carvalho
Antonio C
AC
Costa
Mauro W
MW
D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002478:Cells, Cultured; D017630:Connexins / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D050991:GATA4 Transcription Factor; D005786:Gene Expression Regulation; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D007136:Immunoglobulins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009206:Myocardium / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020825:T-Box Domain Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D000097003:Gap Junction alpha-5 Protein
D017630:Connexins; D004268:DNA-Binding Proteins; D050991:GATA4 Transcription Factor; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; D007136:Immunoglobulins; D012333:RNA, Messenger; D020825:T-Box Domain Proteins; D014157:Transcription Factors; D000097003:Gap Junction alpha-5 Protein; C484469:Nkx2-5 protein, mouse; C090698:SP1 antigen; C103172:T-box transcription factor 5