Human Hand1 basic helix-loop-helix (bHLH) protein: extra-embryonic expression pattern, interaction partners and identification of its transcriptional repressor domains.
The basic helix-loop-helix (bHLH) transcription factor, Hand1, plays an important role in the development of the murine extra-embryonic trophoblast cell lineage. In the present study, we have analysed the expression of Hand1 in human extra-embryonic cell types and determined its binding specificity and transcriptional activity upon interaction with different class A bHLH factors. Northern blotting and in situ hybridization showed that Hand1 mRNA is specifically expressed in amnion cells at different stages of gestation. Accordingly, we demonstrate that the protein is exclusively produced in the amniotic epithelium in vivo and in purified amnion cells in vitro using a novel polyclonal Hand1 antiserum. Reverse transcriptase-PCR and immunohistochemical staining of blastocysts revealed the production of Hand1 mRNA and polypeptide in the trophectodermal cell layer. In the presence of E12/E47, Hand1 stimulated the transcription of luciferase reporters harbouring degenerate E-boxes, suggesting that E-proteins are potential dimerization partners in trophoblastic tumour and amnion cells. In contrast, Hand1 diminished E12/E47-dependent transcription of reporters containing perfect E-boxes by inhibiting the interaction of Hand1/E-protein heterodimers with the palindromic cognate sequence. Furthermore, we show that Hand1 down-regulated GAL-E12-dependent reporter expression, indicating that the protein can also act directly as a transcriptional repressor. Mutational analyses of GAL-Hand1 suggested that two protein regions located within its N-terminal portion mainly confer the repressing activity. In conclusion, human Hand1 may play an important role in the differentiation of the amniotic membrane and the pre-implanting trophoblast. Furthermore, the data suggest that Hand1 can act as a repressor by two independent mechanisms; sequestration of class A bHLH factors from E-boxes and inhibition of their transcriptional activity.
Knöfler
Martin
M
Department of Obstetrics and Gynecology, University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. martin.knoefler@akh-wien.ac.at
Meinhardt
Gudrun
G
Bauer
Sandra
S
Loregger
Thomas
T
Vasicek
Richard
R
Bloor
Debra J
DJ
Kimber
Susan J
SJ
Husslein
Peter
P
D051792:Basic Helix-Loop-Helix Proteins; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry*; D019281:Dimerization; D015536:Down-Regulation; D004622:Embryo, Mammalian / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008156:Luciferases / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014018:Tissue Distribution; D014157:Transcription Factors / Q000737:chemistry*; D014158:Transcription, Genetic; D014162:Transfection
D051792:Basic Helix-Loop-Helix Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D008156:Luciferases; D012333:RNA, Messenger; D014157:Transcription Factors