Binding of activating transcription factor 6 to the A5/Core of the rat insulin II gene promoter does not mediate its transcriptional repression

Abstract

Pancreatic β-cells have a well-developed endoplasmic reticulum (ER) due to their highly specialized secretory function to produce insulin in response to glucose and nutrients. It has been previously reported that overexpression of activating transcription factor 6 (ATF6) reduces insulin gene expression in part via upregulation of small heterodimer partner. In this study, we investigated whether ATF6 directly binds to the insulin gene promoter, and whether its direct binding represses insulin gene promoter activity. A bioinformatics analysis identified a putative ATF6 binding site in the A5/Core region of the rat insulin II gene promoter. Direct binding of ATF6 was confirmed using several approaches. Electrophoretic mobility shift assays in nuclear extracts from MCF7 cells, isolated rat islets and insulin-secreting HIT-T15 cells showed ATF6 binding to the native A5/Core of the rat insulin II gene promoter. Antibody-mediated supershift analyses revealed the presence of both ATF6 isoforms, ATF6α and ATF6β, in the complex. Chromatin immunoprecipitation assays confirmed the binding of ATF6α and ATF6β to a region encompassing the A5/Core of the rat insulin II gene promoter in isolated rat islets. Overexpression of the active (cleaved) fragment of ATF6α, but not ATF6β, inhibited the activity of an insulin promoter-reporter by 50%. However, the inhibitory effect of ATF6α was insensitive to mutational inactivation or deletion of the A5/Core. Therefore, although ATF6 binds directly to the A5/Core of the rat insulin II gene promoter, this direct binding does not appear to contribute to its repressive activity.

Pancreatic β-cells depend on basal expression of active ATF6α-p50 for cell survival even under nonstress conditions

“…3F). This effect has previously been shown (33) and is consistent with reports that active ATF6␣ overexpression downregulates the insulin gene in pancreatic ␤-cells (2,33). Overall, these data show that active ATF6␣-p50 expression in ␤-cells maintains GRP78 chaperone levels in basal, nonstressed cells and also maximizes induction of other ER stress response genes under ER stress conditions.…”

“…For each plasmid used, 1 g DNA was diluted in 50 l OptiMEM (GIBCO). Full-length FLAGtagged ATF6␣ and ATF6␣-p50 (aa1-377) were generated from the vector p3XFLAG-CMV-7.1-ATF6␣ (obtained from Dr. R. Prywes, Columbia University, New York) as previously described (2,42). The FLAG-tagged ATF6␤-p60 (aa1-392) was obtained from Dr. C. Glembotski (San Diego State University, San Diego, CA) (37).…”

“…In pancreatic ␤-cells an additional effect of maintaining some active ATF6␣ expression is that it can contribute to keeping insulin gene expression turned off under basal (low glucose) conditions. ATF6␣ has been shown to repress insulin gene transcription (2,33), which is something that is desired in pancreatic ␤-cells at basal glucose levels.…”

Paradoxical roles of ATF6α and ATF6β in modulating disease severity caused by mutations in collagen X

“…While both ATF6α and -β undergo proteolytic cleavage and become activated following accumulation of unfolded/misfolded protein within the ER (ER stress), surprisingly only ATF6α has been implicated in the pathology of many ER stress-associated diseases and has been the main focus of most studies [ [32] , [33] , [34] , [35] , [36] , [37] , [38] , [39] , [40] , [41] ]. Comparatively few studies have examined the function of ATF6β [ 11 , 18 , 20 , [23] , [24] , [25] , [26] , 36 , 42 ] and several of these have concluded that ATF6β either does not have any physiological significance or has a minor functional significance that is redundant with that of ATF6α [ 36 , 42 ].…”

ER stress‐related ATF6 upregulates CIP2A and contributes to poor prognosis of colon cancer

“…ATF6 is known to regulate ER stress response genes by recognizing a specific DNA sequence called the ER stress response element (ERSE, CCAAT‐N 9 ‐CCACG) (Haze et al ., ; Yoshida et al ., ) and the unfolded protein response element [UPRE, (G)(G)TGACGTG(G/A)], where the nucleotides in parentheses are less strongly maintained (Wang et al ., ). A recent study used bioinformatics analysis to reveal that ATF6 could also bind to the UPRE‐like sequence ‘TATGTGG’ within the insulin II promoter and regulate its gene expression (Amyot et al ., ). In the present study, we found a novel sequence ‘TTATGTGG’ within the CIP2A promoter responsible for ATF6 binding (Fig.…”