The biology of the DIMM/MIST1 family of TFs – organizers of cell specialization
MIST1 (aka BHLHA15) is a bHLH TF expressed in diverse secretory lineages that, in all cases studied so far, are long-lived, high capacity secretors of proteins. For example, its expression has been characterized in pancreatic and salivary gland acinar cells and in gastric chief (zymogenic) cells secreting digestive enzymes, in lacrimal glands secreting tear proteins, in epithelial cells of lactating mammary glands secreting milk proteins, in intestinal Paneth cells secreting anti-microbial agents, and in plasma cells secreting antibodies 3–8. MIST1-expressing cells are thus not related by lineage, only by functionality. MIST1 expression commences only during terminal differentiation of a cell and is sustained thereafter throughout its lifespan 8–10. With few exceptions 8, cells that normally would express MIST1 still form in its absence, but they are structurally and functionally abnormal.
In pancreatic and gastric digestive enzyme secreting cells, where MIST1 physiology has been most explored, MIST1 is required for basal polarization of nuclei, and for formation/maintenance of large, apical digestive-enzyme containing granules 3, 5, 6, 9 (Fig. 2, top). Thus, Mist1−/− gastric chief cells and pancreatic acinar cells show a dramatic reduction in apical cytoplasm with apically oriented nuclei and small secretory granules and dramatically reduced secretion of digestive enzymes in response to secretagogues 11. Under standard laboratory conditions, MIST1 deficient mice are viable with normal lifespan and reproductive capacity; however, as mice age, the long-lived acinar cells of the pancreas begin to degenerate in the absence of MIST1, undergoing acinar to ductal metaplasia by 9–10 months 5. Similarly, Mist1−/− acinar cells are also more sensitive to acute, chemically induced pancreatitis 12. Thus, the inability of specialized secretory cells to scale up regulated secretion in the absence of MIST1 makes them less able to adapt over the lifetime of the animal to their extracellular environment.
Figure 2. Genetic analyses of Mist1/DIMM functions reveal their scaling factor properties. Top: A schematic of results observed in a MIST1 loss of function model 3. During normal zymogenic chief cell development, the chief cell begins to express MIST1 as it arises from its mucous granule-containing neck cell precursor. MIST1 is required for the expansion of apical cytoplasm and formation/maintenance of large, zymogenic vesicles. In its absence, cell apices are stunted, vesicles are smaller, and nuclei are nearer the cell apex. Bottom: A schematic of results observed in a DIMM gain of function model 17. Drosophila photoreceptors project axons that terminate within the brain, and store the fast neurotransmitter histamine within small clear synaptic vesicles (SSV). SSVs are released from T-bar type active zones (AZ) and retrieved by endocytosis at glia invaginations – capitate projections (CP). Following misexpression of DIMM at moderate (top) or high (bottom) levels, the properties of the photoreceptor are transformed. Low-level DIMM misexpression results in accumulation of small dense-core vesicles (small DCV) in addition to the SSV. Hi-level DIMM misexpression results in loss of SSVs, AZs and CPs, and the heavy accumulation of large DCVs. With co-misexpression of a neuropeptide precursor transgene, the large DCVs process and store mature neuropeptide.
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The DIMM TF is a basic helix-loop-helix protein of the Atonal class normally restricted to diverse peptidergic neurosecretory neurons and endocrine cells in Drosophila13. DIMM is associated with secretion of most of the ∼35 families of insect neuropeptides. In the larval central nervous system, approximately 3% of neurons are DIMM-positive but they are not related by lineage, position, axonal projection, or specific peptide expression 14, 15. With loss of dimm function, neurosecretory neurons survive, express antecedent developmental markers of cell fate, but fail to display their normal accumulation of large amounts of secretory peptides or biosynthetic enzymes (the cells are “dimmed”) 15. DIMM expression commences only after cells undergo terminal divisions and for most DIMM cells, it is sustained thereafter throughout the cell's lifespan 14, 15. Cells that normally would express DIMM still form in its absence, but they are structurally and functionally abnormal. Hence, the DIMM loss of function phenotype argues for its role in cell structure and function and not in the specification of cell fate or in the control of cell survival.
DIMM over-expression in photoreceptors, a cell lineage that does not normally express DIMM, is sufficient to establish a neuroendocrine-type secretory apparatus 16. Ectopic DIMM over-expression leads to accumulation of large (∼60 nm) dense core vesicles (LDCVs), similar to those seen in normal DIMM expressing cells, and does so at the expense of small clear vesicles and pre-synaptic secretory machinery 17 (Fig. 2, bottom). The scaling aspect of DIMM actions is evident in this suppressive activity: small clear synaptic vesicles are either retained or completely lost depending on the level of DIMM misexpression. Additionally, DIMM misexpression causes loss of other components of fast neurotransmission within photoreceptors such as active zones, as well as a loss of its principal differentiated feature the rhabdomere, where rhodopsin normally is accumulated in high density. Interestingly, rhodopsin expression remains unchanged: only its normal trafficking to the rhabdomeric membranes is affected. Thus DIMM misexpression has major consequences on the details of subcellular domain organization, and not so much on the essential features of cell fate determination.
With combined co-misexpression of a pro-neuropeptide transgene, photoreceptors accumulate the ectopic neuropeptide within the large dense core vesicles and biochemically process the pro-form of the neuropeptide to its active form. Hence, the DIMM TF ramps up two cardinal features of the classic regulated secretory pathway – (i) packaging within a dedicated subcellular organelle (the LDCV) and (ii) processing by dedicated biosynthetic enzymes resident within the trans-Golgi and in secretory granules. Thus, DIMM is normally dedicated to scaling up regulated secretion (and scaling down other types of secretion) within that subset of peptidergic cells in Drosophila that have a strong “professional” dedication to the secretion of bioactive peptides.
The essential properties of DIMM and MIST1 suggest a new category of transcription factor activity that underlies subcellular scaling
DIMM and MIST1 are sequence orthologues, with high similarity throughout the bHLH region of the molecules. The fundamental similarities in molecular genetic studies of DIMM and MIST1 suggest that they regulate transcription of genes that perform a shared, evolutionarily-conserved purpose. But what kind of TFs are they? Unlike other TFs that are part of developmental cascades, MIST1/DIMM expression occurs predominantly in mature cells and persists well after development of those cells. Furthermore, MIST1/DIMM do not seem to affect cell specification (the qualitative features of cell development), because loss of mature, lineage-specific markers is not a phenotype seen following loss of MIST1/DIMM function, and there is no evidence of paralogous TFs in vertebrates or flies. Thus, the lack of cell specification phenotype is not likely to be due to redundancy. Rather, MIST1/DIMM loss of function causes cellular defects that are quantitative: they are essentially limited to a reduced capacity for regulated secretion of polypeptides; MIST1/DIMM gain of function is sufficient to re-direct cell resources to promote that subcellular process.
Here, we argue that MIST1/DIMM are prototypes for a new class of TFs that we term scaling factor TFs. These have critical distinguishing characteristics that we outline below.
They control entire subcellular domains
First and foremost, their target genes will dictate expansion of a specific subcellular domain or resource (Fig. 3). For MIST1/DIMM, this subcellular resource is the secretory vesicle trafficking apparatus, the extended domain that permits regulated peptide secretion. Note, however, that certain direct targets of SFs may not at first glance appear to fit the definition of a subcellular domain component, thus belying the original SF definition. However, such apparent contradictions may in truth reflect a lack of understanding for how such proteins actually operate.
Figure 3. Scaling factors function by scaling specific subcellular domains. In the illustration shown at center, an immature cell devotes resources equally to each of four subcellular domains – domains involved in protein synthesis (endoplasmic reticulum), energetics (mitochondria), protein degradation (lysosomes) and regulated secretion (secretory granules). We propose the concept of scaling factors (SFs), whose expression is induced during terminal cell fate specification. SFs regulate the distribution of a cell's resources as indicated by the four examples SF1-SF4. This figure highlights several features of SFs that are discussed in the text: namely, (i) that SFs are not required to generate particular subcellular domains, only to scale them appropriately; (ii) that each is devoted to a certain subcellular domain; and (iii) that SFs scale up single subcellular domains while repressing others.
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For example, we know that regulated secretion is profoundly decreased by loss of MIST1/DIMM, but MIST1/DIMM's effects on the secretory subcellular domain will not be exerted only by modulating expression of secretory vesicle-associated genes. Known MIST1 targets include Connexin32 (GJB1) 18 and SPCA2 (Atp2c2) 19, which mediate either cell-cell or intracellular transport of the calcium signal that regulates release of secretory vesicles; and p21 (CDKN1A) 20 which inhibits mitosis to allow development of an elaborate secretory vesicle apparatus.
Furthermore in the case of DIMM, its identified direct targets include genes that encode bona fide secretory granule proteins like PHM and cyt-b561-1, but also proteins like CAT-4, a putative arginine transporter 21. Such a transporter protein has never previously been implicated in secretory pathway regulation, yet down-regulation of CAT-4 RNA severely abrogates DIMM's ability to support a functional secretory pathway 21. Thus a general point is that the study of SFs like DIMM and MIST1 provides a basis to create testable hypotheses, and thereby potentially implicate critical components of subcellular domains that were not or could not be otherwise identified.
They control quantitative features
DIMM/MIST1 scale up the secretory apparatus, but they do not appear to be required for its establishment. Consistent with this scaling function MIST1 and DIMM are not exclusive regulators of their respective gene targets; indeed many of their target genes are still expressed at low levels even in their absence. Therefore the second critical characteristic of SFs is that they amplify expression of their target genes, rather than gate them – OFF to ON – in binary fashion 6, 15, 22, 23.
They persist for the life of the cells
MIST1/DIMM expression continues throughout cell lifespan. It follows that, because SFs are used by cells to maintain preferential, high levels of specific subcellular organelles, they must express the TFs themselves at persistent, high levels to maintain, in turn, high levels of all the constantly turning over protein components. SFs not only initiate a program of subcellular differentiation, but also contribute to the maintenance of cell specialization throughout a cell's lifespan (Fig. 4).
Figure 4. Scaling factors are persistently expressed and mediate cellular adaptive responses. SF levels fluctuate throughout a cell's lifespan, with higher levels generating greater specialization – in this example, more secretory granules. As long as SF levels remain within a certain range (in this example, 4–7), the cell functions normally; however, SF expression may fall outside the normal range, leading, in the depicted example, to a lack of balance among the cell's different subcellular domains and ultimately to cellular pathophysiology.
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