Mist1 is a basic helix-loop-helix (bHLH) transcription factor that is highly expressed in the adult pancreas. The mouse Mist1 gene has been sequenced and its genomic structure determined. Fluorescence in situ hybridization mapping located the Mist1 gene to the telomere of mouse chromosome 5 at position 5G2-5G3, an area that is syntenic to human chromosome 13q and which contains several additional pancreatic regulatory genes including IPF1 and CDX (Pin, 1999).
Basic helix-loop-helix (bHLH) proteins often belong to a family of transcription factors that bind to the DNA target sequence -CANNTG- (E-box) that is present in the promoter or enhancer regions of numerous developmentally regulated genes. A novel bHLH factor, termed Mist1, was identified by virtue of its ability to interact with E-box regulatory elements in a yeast 'one-hybrid' screening procedure. Northern analysis revealed that Mist1 transcripts are expressed in several adult tissues, including stomach, liver, lung, and spleen but no expression is detected in the heart, brain, kidney, or testis. During mouse embryogenesis, Mist1 mRNA is first observed at E10.5 in the primitive gut and in the developing lung bud. Expression persists through E16.5 and remains restricted primarily to the epithelial lining. Mist1 is also detected in skeletal muscle tissues beginning at E12.5, persisting throughout all embryonic stages examined although in older embryos and in the adult expression becomes severely reduced. At later developmental times, Mist1 transcripts also are found in the pancreas, submandibular gland, and adult spleen. As predicted, the Mist1 protein is nuclear and binds efficiently to E-box sites as a homodimer. Mist1 also is capable of binding to E-box elements when complexed as a heterodimer with the widely expressed E-proteins, E12 and E47. Surprisingly, although Mist1 binds to E-boxes in vivo, the Mist1 protein lacks a functional transcription activation domain. These observations suggest that Mist1 may function as a unique regulator of gene expression in several different embryonic and postnatal cell lineages (Lemercier, 1997).
A good model system to examine aspects of positive and negative transcriptional regulation is the muscle-specific regulatory factor, MyoD, which is a basic helix-loop-helix (bHLH) transcription factor. Although MyoD has the ability to induce skeletal muscle terminal differentiation in a variety of non-muscle cell types, MyoD activity itself is highly regulated through protein-protein interactions involving several different co-factors. A novel bHLH protein, Mist1, influences MyoD function. Mist1 accumulates in myogenic stem cells (myoblasts) and then decreases as myoblasts differentiate into myotubes. Mist1 functions as a negative regulator of MyoD activity, preventing muscle differentiation and the concomitant expression of muscle-specific genes. Mist1-induced inhibition occurs through a combination of mechanisms, including the formation of inactive MyoD-Mist1 heterodimers and occupancy of specific E-box target sites by Mist1 homodimers. Mist1 lacks a classic transcription activation domain and instead possesses an N-terminal repressor region capable of inhibiting heterologous activators. Thus, Mist1 may represent a new class of repressor molecules that play a role in controlling the transcriptional activity of MyoD, ensuring that expanding myoblast populations remain undifferentiated during early embryonic muscle formation (Lemercier, 1998).
Transcription factors of the basic Helix-Loop-Helix (bHLH) protein family play key roles in several developmental processes. Mist1 belongs to this group of proteins and shares several properties with the other family members. For example, Mist1 is capable of dimerization with the ubiquitously expressed E2A bHLH proteins and exhibits a strong DNA-binding activity to the core E-box sequence. Using in-situ hybridization and Northern blot hybridization, Mist1 mRNA has been detected in a variety of embryonic and adult rodent tissues. To understand the molecular mechanisms involved in the expression of the gene, the rat Mist1 gene has been cloned and 2.5 kb of its 5' flanking region have been analyzed. The Mist1 gene spans over 5 kilobases and is composed of two exons separated by a unique intron. The entire coding region is localized in the second exon. Sequence analysis of the promoter region indicates an absence of TATA-box or CAAT-box sequence, but several consensus Sp1-binding sites are present near the transcription start site. Deletion analysis of the promoter region identified a 272 bp proximal fragment to be sufficient to drive expression of a reporter gene in NIH3T3 fibroblasts. Subsequent deletion of potential Sp1 sites results in a marked decrease in promoter activity. Electrophoretic mobility shift assays reveal that Sp1 binds to two different regions in the proximal promoter, a typical Sp1 site located at (-38; -33) and a G/C-rich region between (-67; -62). These data suggest that the basal expression of this TATA-less gene might be driven by general transcription factors, such as Sp1 (Lemercier, 2000).
Mist1 is a basic helix-loop-helix transcription factor that represses E-box-mediated transcription. Previous studies have suggested that the Mist1 gene is expressed in a wide range of tissues, although a complete characterization of Mist1 protein accumulation in the adult organism has not been described. In an effort to identify specific cell types that contain the Mist1 protein, antibodies specific for Mist1 were generated and used in Western blot and immunohistochemical assays. These studies show that the Mist1 protein is present in many different tissues but that it is restricted to cell types that are exclusively secretory in nature. Pancreatic acinar cells, serous or seromucous cells of the salivary glands, chief cells of the stomach, and secretory cells of the prostate and seminal vesicle show high levels of Mist1 protein, whereas nonserous exocrine cells, including the mucus-producing cells of the salivary glands, remain Mist1 negative. These results identify Mist1 as the first transcription factor that exhibits this unique serous-specific expression pattern and suggest that Mist1 may have a key role in establishing and maintaining a pathway responsible for the exocytosis of serous secretions (Pin, 2000).
The pancreas is a complex organ that consists of separate endocrine and exocrine cell compartments. Although great strides have been made in identifying regulatory factors responsible for endocrine pancreas formation, the molecular regulatory circuits that control exocrine pancreas properties are just beginning to be elucidated. In an effort to identify genes involved in exocrine pancreas function, Mist1, a basic helix-loop-helix transcription factor expressed in pancreatic acinar cells, has been identified. Mist1-null [Mist1(KO)] mice exhibit extensive disorganization of exocrine tissue and intracellular enzyme activation. The exocrine disorganization is accompanied by increases in p8, RegI/PSP, and PAP1/RegIII gene expression, mimicking the molecular changes observed in pancreatic injury. By 12 m, Mist1(KO) mice develop lesions that contain cells coexpressing acinar and duct cell markers. Analysis of the factors involved in cholecystokinin (CCK) signaling reveal inappropriate levels of the CCK receptor A and the inositol-1,4,5-trisphosphate receptor 3, suggesting that a functional defect exists in the regulated exocytosis pathway of Mist1(KO) mice. Based on these observations, it is proposed that Mist1(KO) mice represent a new genetic model for chronic pancreas injury and that the Mist1 protein serves as a key regulator of acinar cell function, stability, and identity (Pin, 2001).
Gap junctions are intercellular channels that provide direct passage of small molecules between adjacent cells. In pancreatic acini, the connexin26 (Cx26) and connexin32 (Cx32) proteins form functional channels that coordinate the secretion of digestive enzymes. Although the function of Cx26/Cx32 gap junctions are well characterized, the regulatory circuits that control the spatial and temporal expression patterns of these connexin genes are not known. In an effort to identify the molecular pathways that regulate connexin gene expression, Cx26 and Cx32 gene activities were examined in mice lacking the basic helix-loop-helix transcription factor Mist1 (Mist1KO). Mist1, Cx26 and Cx32 are co-expressed in most exocrine cell types, and acinar cells from Mist1KO mice exhibit a highly disorganized cellular architecture and an altered pattern of expression for several genes involved in regulated exocytosis. Analysis of Mist1KO mice reveals a dramatic decrease in both connexin proteins, albeit through different molecular mechanisms. Cx32 gene transcription is greatly reduced in all Mist1KO exocrine cells, while Cx26 gene expression remains unaffected. However, in the absence of Cx32 protein, Cx26 does not participate in gap junction formation, leading to a complete lack of intercellular communication among Mist1KO acinar cells. Additional studies testing Mist1 gene constructs in pancreatic exocrine cells confirm that Mist1 transcriptionally regulates expression of the Cx32 gene. It is concluded that Mist1 functions as a positive regulator of Cx32 gene expression and, in its absence, acinar cell gap junctions and intercellular communication pathways become disrupted (Rukstalis, 2003).
The pancreas consists of three main cell lineages (endocrine, exocrine, and duct) that develop from common primitive foregut precursors. The transcriptional network responsible for endocrine cell development has been studied extensively, but much less is known about the transcription factors that maintain the exocrine and duct cell lineages. One transcription factor that may be important to exocrine cell function is Mist1, a basic helix-loop-helix (bHLH) factor that is expressed in acinar cells. In order to perform a molecular characterization of this protein, coimmunoprecipitation and bimolecular fluorescence complementation assays, coupled with electrophoretic mobility shift assay studies, were used to show that Mist1 exists in vivo as a homodimer complex. Analysis of transgenic mice expressing a dominant-negative Mist1 transgene [Mist1(mutant basic)] [Mist1(MB)]) revealed the cell autonomous effect of inhibiting endogenous Mist1. Mist1(MB) cells become disorganized, exhibit a severe depletion of intercellular gap junctions, and express high levels of the glycoprotein clusterin, which has been shown to demarcate immature acinar cells. Inhibition of Mist1 transcriptional activity also leads to activation of duct-specific genes, such as cytokeratin 19 and cytokeratin 20, suggesting that alterations in the bHLH network produce a direct acinar-to-ductal phenotypic switch in mature cells. It is proposed that Mist1 is a key transcriptional regulator of exocrine pancreatic cells and that in the absence of functional Mist1, acinar cells do not maintain their normal identity (Zhu, 2004).
Ca2+ signaling and exocytosis are highly polarized functions of pancreatic acinar cells. The role of cellular architecture in these activities and the capacity of animals to tolerate aberrant acinar cell function are not known. A key regulator of acinar cell polarity is Mist1, a basic helix-loop-helix transcription factor. Ca2+ signaling and amylase release were examined in pancreatic acini of wild type and Mist1 null mice to gain insight into the importance of cellular architecture for Ca2+ signaling and regulated exocytosis. Mist1-/- acinar cells exhibit dramatically altered Ca2+ signaling with up-regulation of the cholecystokinin receptor but minimal effect upon expression of the M3 receptor. However, stimulation of inositol 1,4,5-trisphosphate production by cholecystokinin and carbachol is inefficient in Mist1-/- cells. Although agonist stimulation of Mist1-/- cells evokes a Ca2+ signal, often the Ca2+ increase is not in the form of typical Ca2+ oscillations but rather in the form of a peak/plateau-type response. Mist1-/- cells also display distorted apical-to-basal Ca2+ waves. The aberrant Ca2+ signaling is associated with mislocalization and reduced Ca2+ uptake by the mitochondria of stimulated Mist1-/- cells. Deletion of Mist1 also leads to mislocalization of the Golgi apparatus and markedly reduced digestive enzyme content. The combination of aberrant Ca2+ signaling and reduced digestive enzyme content results in poor secretion of digestive enzymes. Yet, food consumption and growth of Mist1-/- mice are normal for at least 32 weeks. These findings reveal that Mist1 is critical to normal organelle localization in exocrine cells and highlight the critical importance of maintaining cellular architecture and polarized localization of cellular organelles in generating a propagating apical-to-basal Ca2+ wave. The studies also reveal the spare capacity of the exocrine pancreas that allows normal growth and development in the face of compromised exocrine pancreatic function (Luo, 2005).
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