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. 2013 Jul 1;18(3):315–327. doi: 10.2478/s11658-013-0091-2

Characterization of the 5′-flanking region of the mouse asparagine-linked glycosylation 12 homolog gene

Kentaro Oh-Hashi 191,✉, Tomomi Tejima 191, Yoko Hirata 191,291, Kazutoshi Kiuchi 191,291
PMCID: PMC6275931  PMID: 23818223

Abstract

Recently, we characterized multiple roles of the endoplasmic reticulum stress responsive element (ERSE) in the promotion of a unique headto-head gene pair: mammalian asparagine-linked glycosylation 12 homolog (ALG12) and cysteine-rich with EGF-like domains 2 (CRELD2). This bidirectional promoter, which consists of fewer than 400 base pairs, separates the two genes. It has been demonstrated that the ALG12 promoter shows less transcriptional activity through ERSE, but its basic regulatory mechanism has not been characterized. In this study, we focused on well-conserved binding elements for the transcription factors for ATF6, NF-Y and YY1 and the Sp1 and Ets families in the 5’-flanking region of the mouse ALG12 gene. We characterized their dominant roles in regulating ALG12 promoter activities using several deletion and mutation luciferase reporter constructs. The ALG12 gene is expressed in three distinct cell lines: Neuro2a, C6 glioma and HeLa cells. The reporter activity in each cell line decreased similarly with serial deletions of the mouse ALG12 promoter. Mutations in the ERSE and adjacent NF-Y-binding element slightly affected reporter activity. Each of the mutations in the GC-rich sequence and YY1-binding element reduced ALG12 promoter activity, and the combination of these mutations additively decreased reporter activity. Each mutation in the tandem-arranged Ets-family consensus sequences partially attenuated ALG12 promoter activity, and mutations of all three Ets-binding elements decreased promoter activity by approximately 40%. Mutation of the three conserved regulatory elements (GC-rich, YY1 and Ets) in the ALG12 promoter decreased reporter activity by more than 90%. Our results suggest that the promoter activity of the mouse ALG12 gene is regulated in a similar manner in the three cell lines tested in this study. The well-conserved consensus sequences in the promoter of this gene synergistically contribute to maintaining basal gene expression.

Key words: ALG12, CRELD2, ERSE, Ets family, NF-Y, Sp1 family, YY1

Full Text

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Abbreviations used

ALG12

asparagine-linked glycosylation 12 homolog

ATF6

activating transcription factor 6

CDG

congenital disorders of glycosylation

CRELD2

cysteine-rich with EGF-like domains 2

ER

endoplasmic reticulum

ERSE

ER stress response element

Ets

v-ets erythroblastosis virus E26 oncogene homolog

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

HDAC

histone deacetylase

NF-Y

nuclear transcription factor Y

Rb

retinoblastoma

YY1

Yin Yang 1

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