FoxA2, Nkx2.2, and PDX-1 regulate islet beta-cell-specific mafA expression through conserved sequences located between base pairs -8118 and -7750 upstream from the transcription start site.
The MafA transcription factor is both critical to islet beta-cell function and has a unique pancreatic cell-type-specific expression pattern. To localize the potential transcriptional regulatory region(s) involved in directing expression to the beta cell, areas of identity within the 5' flanking region of the mouse, human, and rat mafA genes were found between nucleotides -9389 and -9194, -8426 and -8293, -8118 and -7750, -6622 and -6441, -6217 and -6031, and -250 and +56 relative to the transcription start site. The identity between species was greater than 75%, with the highest found between bp -8118 and -7750 ( approximately 94%, termed region 3). Region 3 was the only upstream mammalian conserved region found in chicken mafA (88% identity). In addition, region 3 uniquely displayed beta-cell-specific activity in cell-line-based reporter assays. Important regulators of beta-cell formation and function, PDX-1, FoxA2, and Nkx2.2, were shown to specifically bind to region 3 in vivo using the chromatin immunoprecipitation assay. Mutational and functional analyses demonstrated that FoxA2 (bp -7943 to -7910), Nkx2.2 (bp -7771 to -7746), and PDX-1 (bp -8087 to -8063) mediated region 3 activation. Consistent with a role in transcription, small interfering RNA-mediated knockdown of PDX-1 led to decreased mafA mRNA production in INS-1-derived beta-cell lines (832/13 and 832/3), while MafA expression was undetected in the pancreatic epithelium of Nkx2.2 null animals. These results suggest that beta-cell-type-specific mafA transcription is principally controlled by region 3-acting transcription factors that are essential in the formation of functional beta cells.
Raum
Jeffrey C
JC
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medical Center,723 Light Hall, Nashville, TN 37232, USA.
Gerrish
Kevin
K
Artner
Isabella
I
Henderson
Eva
E
Guo
Min
M
Sussel
Lori
L
Schisler
Jonathan C
JC
Newgard
Christopher B
CB
Stein
Roland
R
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002645:Chickens; D005786:Gene Expression Regulation; D051542:Hepatocyte Nuclear Factor 3-beta / Q000235:genetics / Q000378:metabolism*; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000502:physiology*; D051473:Maf Transcription Factors, Large / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D016415:Sequence Alignment; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D029961:Zebrafish Proteins
C496503:Foxa2 protein, mouse; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; C485159:MAFA protein, human; D051473:Maf Transcription Factors, Large; C000715631:NKX2-2 protein, human; C000715627:Nkx2-2 protein, mouse; C000715628:Nkx2-2 protein, rat; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D015534:Trans-Activators; D014157:Transcription Factors; D029961:Zebrafish Proteins; C000715630:nkx2.2b protein, zebrafish; C084010:pancreatic and duodenal homeobox 1 protein; D051542:Hepatocyte Nuclear Factor 3-beta