Paired-Homeodomain Transcription Factor PAX4 Acts as a Transcriptional Repressor in Early Pancreatic Development

Abstract

The paired-homeodomain transcription factor PAX4 is expressed in the developing pancreas and along with PAX6 is required for normal development of the endocrine cells. In the absence of PAX4, the numbers of insulin-producing beta cells and somatostatin-producing delta cells are drastically reduced, while the numbers of glucagon-producing alpha cells are increased. To gain insight into PAX4 function, we cloned a full-length Pax4 cDNA from a beta-cell cDNA library and identified a bipartite consensus DNA binding sequence consisting of a homeodomain binding site separated from a paired domain binding site by 15 nucleotides. The paired half of this consensus sequence has similarities to the PAX6 paired domain consensus binding site, and the two proteins bind to common sequences in several islet genes, although with different relative affinities. When expressed in an alpha-cell line, PAX4 represses transcription through the glucagon or insulin promoters or through an isolated PAX4 binding site. This repression is not simply due to competition with the PAX6 transcriptional activator for the same binding site, since PAX4 fused to the unrelated yeast GAL4 DNA binding domain also represses transcription through the GAL4 binding site in the alpha-cell line and to a lesser degree in beta-cell lines and NIH 3T3 cells. Repressor activity maps to more than one domain within the molecule, although the homeodomain and carboxyl terminus give the strongest repression. PAX4 transcriptional regulation apparently plays a role only early in islet development, since Pax4 mRNA as determined by reverse transcriptase PCR peaks at embryonic day 13.5 in the fetal mouse pancreas and is undetectable in adult islets. In summary, PAX4 can function as a transcriptional repressor and is expressed early in pancreatic development, which may allow it to suppress alpha-cell differentiation and permit beta-cell differentiation.

p38 MAPK Is Involved in Activin A- and Hepatocyte Growth Factor-mediated Expression of Pro-endocrine Gene Neurogenin 3 in AR42J-B13 Cells

“…Reporter Gene Constructs and Expression Plasmids-To generate reporter plasmids, fragments of the 5Ј region of NEUROG3 obtained by restriction digestion or PCR using the pFOX Luc1 NEUROG3-5.7kb plasmid (13) as a template were ligated upstream of the luciferase gene in pFOX Luc1 plasmids or upstream of the thymidine kinase (TK) minimal promoter in pFOX Luc1TK plasmids (14). Mutagenesis of the reporter gene constructs was performed using a Quick Change sitedirected mutagenesis kit according to the manufacturer's instructions (Stratagene, La Jolla, CA).…”

Autoregulation and Maturity Onset Diabetes of the Young Transcription Factors Control the Human PAX4 Promoter

“…Construction of pBAT12.mPAX4, the DNA plasmid expressing mouse PAX4 under the control of the human cytomegalovirus (CMV) immediate-early gene promoter, has been described previously (16). Mutations in the paired domain (S43P and R44P) and the homeodomain (N220P and R222P) in the mouse PAX4 cDNA were produced in the pBAT12.mPAX4 plasmid and confirmed by sequencing.…”

“…One of these islet cell-type determination factors is the paired homeodomain transcription factor PAX4. PAX4 functions as a potent transcriptional repressor and is expressed only transiently in the fetal pancreas, peaking during the period of beta and delta cell differentiation (16). Mice with a targeted disruption of the pax4 gene have a marked decrease in beta and delta cells with a commensurate increase in alpha cells (17), suggesting that PAX4 functions by repressing the alpha cell differentiation program and thereby deviates cells from a default alpha cell fate to an alternate beta or delta cell fate.…”

Transcription factor regulation of pancreatic organogenesis, differentiation and maturation

“…54 Pax4 is expressed at e9.5 in the dorsal and ventral buds but is limited to the first and second wave b-cells; however, it is down-regulated shortly after birth and undetectable in adult islets. [184][185][186] Additionally, lineage tracing revealed that Pax4 C cells represent specified endocrine progenitors that may commit to endocrine cell fate. 187,188 Pax4 is expressed downstream of Ngn3 and its expression is lost in Ngn3 ¡/¡ mice but not vice versa.…”

“…186 Also, Pax4 is a repressor of both ghrelin expression and Pax6-mediated glucagon expression; hence there is elevated expression of these 2 cell types in Pax4 mutants. 185,188,189 A loss in Pax4 expression inhibits Pdx1, Hb9 and insulin mRNA in b-cell precursors. 190 Ectopic expression of Pax4 and Arx prompts the formation of b-and a-cell lineage respectively.…”