RAB26 and RAB3D Are Direct Transcriptional Targets of MIST1 That Regulate Exocrine Granule Maturation

Abstract

Little is known about how differentiating cells reorganize their cellular structure to perform specialized physiological functions. MIST1, an evolutionarily conserved transcription factor, is required for the formation of large, specialized secretory vesicles in gastric zymogenic (chief) cells (ZCs) as they differentiate from their mucous neck cell progenitors. Here, we show that MIST1 binds to highly conserved CATATG E-boxes to directly activate transcription of 6 genes, including those encoding the small GTPases RAB26 and RAB3D. We next show that RAB26 and RAB3D expression is significantly downregulated in Mist1 ؊/؊ ZCs, suggesting that MIST1 establishes large secretory granules by inducing RAB transcription. To test this hypothesis, we transfected human gastric cancer cell lines stably expressing MIST1 with red fluorescent protein (RFP)-tagged pepsinogen C, a key secretory product of ZCs. Those cells upregulate expression of RAB26 and RAB3D to form large secretory granules, whereas control, non-MIST1-expressing cells do not. Moreover, granule formation in MIST1-expressing cells requires RAB activity because treatment with a RAB prenylation inhibitor and transfection of dominant negative RAB26 abrogate granule formation. Together, our data establish the molecular process by which a transcription factor can directly induce fundamental cellular architecture changes by increasing transcription of specific cellular effectors that act to organize a unique subcellular compartment.

MIST1 Links Secretion and Stress as both Target and Regulator of the Unfolded Protein Response

“…Peak calling on each data set was cross-referenced to identify only conserved binding regions using both MIST1 antibodies (see Materials and Methods). Consistent with previous reports on MIST1 DNA binding (18,51), CAGCTG (GC) and CATATG (TA) E-box sites were the most enriched motifs within all MIST1 peaks, with an expected enhanced distribution centered over the transcription start site (TSS), mirroring the genome-wide MIST1 binding distribution. As predicted, several previously confirmed MIST1 targets were well represented in this analysis, including members of the RAB family of cytoskeletal adapters (18) and the proapoptotic gene Htra2 (12).…”

“…MIST1's widespread transcriptional regulatory role throughout the secretory network establishes it as a key component in ensuring the proper function of these pathways. This broad role also explains, for the first time, the complex defective exocytosis phenotype associated with Mist1 KO mice (12,18,19,21,67). The transcriptional role of MIST1 during ER stress is primarily to augment the secretory pathway rather than to initiate new expression of target genes, consistent with previous classifications of MIST1 as a "scaling factor," a unique class of transcriptional regulators that serve to enhance the effects of other transcription complexes (67).…”

“…Secretory cells utilize transcription factor networks to alter their cytoskeletal arrangement, polarity, membrane protein composition, and organelle makeup to support the proper generation, storage, and release of protein products (8). Recently, the basic helix-loop-helix (bHLH) transcription factor MIST1 (encoded by Bhlha15) has emerged as a potent regulator of a wide variety of secretory cell functions and responses to stress (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Secretory cells lacking MIST1 exhibit structural and functional defects, including altered cytoskeletal organization, a change in cell polarity, and improper cell-to-cell communication (12,17,(19)(20)(21).…”

Rab3D regulates amylase levels, not agonist-induced amylase release, in AR42J cells

“…This issue remains to be resolved. However, another more recent study has demonstrated that the transcription factor MIST1 induces the formation of large granules in gastric chief cells by inducing expression of Rab3D and Rab26 [35]. Similarly, overexpression of mutant Rab3D interfered with the formation of Weibel-Palade bodies in platelets [36].…”

The Transcription Factor MIST1 Is a Novel Human Gastric Chief Cell Marker Whose Expression Is Lost in Metaplasia, Dysplasia, and Carcinoma

“…[31][32][33] This transition involves expression of the bHLH transcription factor MIST1, and genetic ablation in mice has demonstrated MIST1 as critically important for the structural features of the mature chief cell, including basal nuclear polarity and formation of large digestive enzyme containing granules. 28,34 In animal models, chronic Helicobacter colonization induces loss of parietal cells (ie, oxyntic atrophy) and concomitant metaplasia of the basally located chief cells. 11,31,[35][36][37] Specifically, chief cells regain proliferative potential and start reexpressing progenitor markers such as TFF2, MUC6, and the epitope for the lectin Griffonia Simplificolia, GS-II.…”