An astrocyte‐specific enhancer of the aquaporin‐4 gene functions through a consensus sequence of POU transcription factors in concert with multiple upstream elements

Abstract

J. Neurochem. (2012) 120, 899–912. Abstract Aquaporin‐4, a predominant water channel in the brain, is specifically expressed in astrocyte endfeet and plays a central role in water homeostasis, neuronal activity, and cell migration in the brain. It has two dominant isoforms called M1 and M23, whose mRNA is driven by distinct promoters located upstream of exons 0 and 1 of the aquaporin‐4 gene, respectively. To identify cis‐acting elements responsible for the astrocyte‐specific transcription of M1 mRNA, the promoter activity of the 5′‐flanking region upstream of exon 0 in primary cultured mouse astrocytes was examined by luciferase assay, and sequences, where nuclear factors bind, were identified by electrophoretic mobility shift assay. An astrocyte‐specific activity enhancing transcription from the M1 promoter was observed within ∼2 kb from the transcriptional start sites of M1 mRNA. At least five elements clustered within the 286‐bp region were found to function as a novel astrocyte‐specific enhancer. Among the five elements, a consensus sequence of Pit‐1/Oct/Unc‐86 (POU) transcription factors was indispensable to the astrocyte‐specific enhancer since disruption of the POU motif completely abolished the enhancer activity in astrocytes. However, the POU motif alone had little activity, indicating the requirement for cooperation with other upstream elements to exert full enhancer activity.

The dual effects of the astrocyte‐specific enhancer of the AQP4 M1 promoter

“…We demonstrated in the previous report that an ~ 2‐kb fragment of the 5′ flanking region of exon 0 of mAQP4 (−5005/−3057) has a promoter activity specifically functioning in astrocytes, and that deleting the 1168‐bp fragment within this region (−4617/−3450) results in a 2.8‐fold increase in the promoter activity (Fig. , E0‐ Nhe I Δ Afl III‐ Ase I ). Thus, we next examined whether the combination of this promoter (designated as the M1 promoter in this report) and the M23 core promoter would cooperatively drive downstream luciferase cDNA in astrocytes.…”

“…*( P < 0.01), significantly different versus the E0‐ Nhe I Δ Afl III‐ Ase I fragment (Abe et al . ) by Dunnett's test. Abbreviations: SD , splice donor.…”

“…Recently, we identified a novel enhancer contributing to transcription of the AQP4 M1 mRNA in astrocytes (astrocyte-specific enhancer, ASE) located in a region approximately 1.5-kb upstream of the transcriptional start site [18]. In this enhancer, at least five cis-acting elements, including a consensus sequence for POU transcription factors, are involved [18]. Here, we investigated the relationship between the ASE and the M1 and M23 promoters, demonstrating that the ASE of the M1 promoter has not only an enhancer effect on the M1 promoter in astrocytes but also a silencer effect on both promoters in cells lacking AQP4.…”

The transmembrane transporter domain of glutamate transporters is a process tip localizer

“…Primary cultures of mouse astrocytes were prepared from C57BL/6JJ P0 mice as described previously40. On DIV 14–21, the astrocytes are seeded in 24-well plates containing uncoated glass coverslips at 1 × 10 5 cells/well.…”

Corrigendum

“…In Abe et al . (), doi: , labeling errors were identified in Figs and after publication. The correct Figs and are provided here.…”