Six touch receptor neurons with distinctive morphological features sense gentle touch in Caenorhabditis elegans. Previous studies have identified three genes (lin-32, unc-86 and mec-3) that regulate touch cell development. However, since other cell types also require these genes, it is suspected that other genes help restrict the expression of touch cell characteristics to the six neurons seen in the wild type. To identify such genes, mutants defective in genes required for the development of other C. elegans cells were examined for changes in the pattern of touch cell-specific features. Mutations in seven genes either reduce (lin-14) or increase (lin-4, egl-44, egl-46, sem-4, ced-3 and ced-4) the number of touch receptor-like cells. The combinatorial action of these genes, all of which are required for the production of many cell types, restrict the number of cells expressing touch receptor characteristics in wild-type animals by acting as positive and negative regulators and by removing cells by programmed cell death (Mitani, 1993).
In wild-type Caenorhabditis elegans, six cells develop as receptors for gentle touch. In egl-44 (Drosophila homolog: Scalloped) and egl-46 (Drosophila homologs: Nerfin I and II) mutants, two other neurons, the FLP cells, express touch receptor-like features. The FLP cells normally express lin-14 as well as unc-86 (Drosophila homolog ACJ6/IPOU and mec-3 that code for a LIM homeodomain transcription factor). These cells do not express touch cell characteristics because of the action of two genes, egl-44 and egl-46. Mutation of either egl gene results in a transformation of the FLP cells into cells that resemble the touch cells. Instead of differentiating as FLP neurons, the cells in the mutants express the mec-4 and mec-7 touch function genes and have processes that lie adjacent to the normal touch cell processes and that also have the large-diameter microtubules and extracellular matrix characteristic of the touch cells. egl-44 and egl-46 also affect the differentiation of other neurons, including the HSN neurons, two cells needed for egg laying (Wu, 2001).
The egl-44 gene encodes a putative transcription regulatory protein of 471 amino acids similar to transcription enhancer factor (TEF) proteins. TEF-1-like proteins, which have been found from yeast to humans, are involved in a variety of developmental processes. For example, mutations in the Drosophila TEF gene scalloped affect the development of sensory bristles and central neurons needed for taste, and human TEF-5 is expressed in the placenta and activates the chorionic somatomammotropin gene. The most conserved region among family members is the 70-amino-acid TEA/ATTS DNA-binding domain at the N terminus. EGL-44, the Drosophila TEF Scalloped (Sd), and the human TEF-5 protein (the human TEF most similar to EGL-44) are 82% identical in the TEA/ATTS DNA-binding domain. The egl-44 mutations are in this domain. The C-terminal half of EGL-44, Sd, and TEF-5 are also 47% identical, and this region in TEF-1 contains Pro-rich, STY-rich, and other sequences that are needed together for transcriptional activation. Although EGL-44 does not contain sequences that match the activation domains in other TEF proteins, its C-terminal half is rich in Pro, Ser, Thr, and Tyr (Wu, 2001).
egl-46 encodes a predicted protein of 286 amino acids. The most notable features of EGL-46 are three closely spaced zinc finger motifs in the C-terminal region of EGL-46. The first two zinc finger motifs are separated by 9 amino acids and may form a pair, and the third motif is 19 amino acids C-terminal to the first two. The second finger motif of EGL-46 conforms to the TFIIIA (C2H2) consensus, whereas the other two fingers differ slightly. In the first and third fingers, the last His is replaced by Cys; in the first finger, the spacing between the His and the last Cys residue differs from the consensus. Nonetheless, the overall spacing and the conservation of other residues indicate that these are variants of the TFIIIA type. These three zinc fingers may mediate DNA binding by EGL-46. Consistent with a role in the nucleus, EGL-46 contains a potential nuclear localization signal (amino acids 110-126). Also consistent with a role as a transcription factor, EGL-46 contains a glutamine-rich region (amino acids 61-75), which may act as a transcriptional activation domain. EGL-46 and several proteins with which it shares similarity appear to form a new family of zinc finger proteins. These proteins include the human and mouse IA-1 proteins, the mouse MLT-1 protein, a human protein tentatively named R-355C3p, and two proteins from Drosophila melanogaster, Nerfin-1 and Nerfin-2 (Stivers, 2000). No other closely similar sequences are found in the C. elegans genome. All seven proteins have three zinc finger regions; the mammalian proteins have two additional zinc finger sequences C-terminal to these three. The first two zinc fingers show considerable similarity and equal spacing in all seven proteins. The second zinc finger of the pair is 90% identical for all the proteins and has a conserved potential PKC phosphorylation site. The high degree of similarity in this zinc finger pair region indicates that the region is functionally important. The egl-46(n1127) mutation produces a Cys to Phe substitution in the first zinc finger of this pair. All seven proteins share an additional region of similarity N-terminal to the zinc fingers (26 amino acids in EGL-46) that contains a potential nuclear localization signal. N-terminal to this region, all seven proteins have regions that are proline-rich (although EGL-46 is less so than the others). The mammalian proteins also contain a short transcriptional repression domain, but this sequence is not conserved in EGL-46 or the Drosophila proteins (Wu, 2001).
Two features of egl-46 indicate that its product may be regulated posttranscriptionally: (1) the N-terminal region of the protein contains a 25-amino-acid putative PEST sequence that could target the protein for rapid degradation; (2) the egl-46 mRNA 3' UTR contains sequences that may target the mRNA for degradation. The 3' UTR contains three AUUUA motifs, which have been associated with RNA instability. More recent work, however, has found that a single AUUUA motif is not sufficient to cause mRNA instability and that a UUAUUUA(U/A)(U/A) sequence may be required. egl-46 has the core of this sequence, UAUUUAU, which when tested in three copies promoted mRNA degradation. Several other members of this gene family share these features. The Nerfin-1 (but not Nerfin-2), hIA-1 (but not mIA-1), MLT 1, and R-355C3p proteins have predicted PEST sequences. Nerfin-1, hIA-1, and mlt 1 mRNAs contain AUUUA repeats in their 3' UTRs. Little is known about the EGL-46-related proteins, although they are often found in neuronal tissues, and at least Nerfin-1 and IA-1 are found in dividing neural precursors. Of the Drosophila proteins, Nerfin-1 is distributed widely throughout the nervous system and is observed in many neuronal precursors; Nerfin-2 is found in only a few neurons (Stivers, 2000). The human IA-1 protein is found in many neuroendocrine tumor cell lines and virtually all small cell lung cancer cell lines. The mouse protein MLT 1 has been identified in brain and kidney (Wu, 2001).
Both egl-44 and egl-46 are expressed in FLP and HSN neurons (and other cells); expression of egl-46 is dependent on egl-44 in the FLP cells but not in the HSN cells. Wild-type touch cells express egl-46 but not egl-44. Moreover, ectopic expression of egl-44 in the touch cells prevents touch cell differentiation in an egl-46-dependent manner. The sequences of these genes and their nuclear location as seen with GFP fusions indicate that they repress transcription of touch cell characteristics in the FLP cells (Wu, 2001).
egl-44 appears to repress the expression of touch cell features in the FLP cells in two ways: (1) egl-44 is required for the wild-type levels of egl-46 expression in these neurons; (2) egl-44 must be expressed with egl-46 to repress touch cell differentiation. This conclusion is supported by the ectopic expression of these genes in the touch cells and by the finding that these cells normally express egl-46. (The expression of egl-46 in the touch cells may account for the minor touch cell process and morphological defects in egl-46 mutants. EGL-44 may interact directly with EGL-46, because TEF proteins in other organisms act as transcription cofactors (Wu, 2001).
These considerations extend the model of combinatorial control of touch cell development. In the six touch cells, unc-86 promotes mec-3 expression, and the UNC-86/MEC-3 heterodimer activates the expression of touch genes. In the FLP neurons, egl-44 promotes egl-46 expression, presumably with some other factor(s), and EGL-44 and EGL-46 together, presumably also with some other factor(s), inhibit touch gene expression to secure the normal differentiation of FLP neurons. Because mec-3 and unc-86 are expressed in FLP cells normally, it is possible that EGL-44 and EGL-46 repress touch cell fate by directly antagonizing activation by MEC-3 or/and UNC-86 (Wu, 2001).
Although both egl-44 and egl-46 are expressed in the HSN neurons, and expression of the wild-type genes in the HSN cells complements egl-44 and egl-46 mutations, the timing of their expression is unexpected given the phenotypes of the mutant cells. egl-44 and egl-46 mutations affect HSN differentiation in three ways: (1) the cells migrate further than wild-type cells; (2) their axons are misdirected, and (3) they have reduced production of the neurotransmitter serotonin. Of these three processes, only cell migration occurs in the embryos; the others arise as the animals become adults. In contrast, the HSN cells express egl-44 in the embryo and express egl-46 in the embryo and transiently and weakly in L2 larvae. The embryonic expression could underlie a role for these genes in the regulation of HSN migration. The expression pattern of these genes is less easily reconciled with the late larval outgrowth defects and adult serotonin defect (both of which are incompletely penetrant). One explanation is that these genes act early to establish the ability of the cells to generate serotonin or grow appropriately. If so, the genes could act indirectly within the HSN to influence these later traits. It has been suggested, for example, that interactions of the HSN cells with their muscle targets result in the lowered levels of serotonin in the mutant HSN cells. An early defect in the HSN cells could lead to these abnormal interactions. Alternatively, because the genes are expressed in many other cells, their influence on axonal outgrowth and/or serotonin production could be caused by the loss of gene activity in other cells; for example, some of the HSN phenotypes are not the result of the cell-autonomous action of the genes. The AVM and PVM touch cells also have a low penetrant outgrowth defect in egl-44 and egl-46 animals, but the cells do not detectably express egl-44. Perhaps the loss of egl-44 expression in the hypodermis underlies the touch cell and HSN outgrowth defects (Wu, 2001).
Caenorhabditis elegans polycystins LOV-1 and PKD-2 are expressed in the male-specific HOB neuron, and are necessary for sensation of the hermaphrodite vulva during mating. Male vulva location behavior and expression of lov-1 and pkd-2 in the ciliated sensory neuron HOB require the activities of transcription factor EGL-46 and to some extent also EGL-44. This EGL-46- regulated program is specific to HOB and is distinct from a general ciliogenic pathway functioning in all ciliated neurons. The ciliogenic pathway regulator DAF-19 affects downstream components of the HOB-specific program indirectly and is independent of EGL-46 activity. The sensory function of HOB requires the combined action of these two distinct regulatory pathways (Yu, 2003).
Because of its simple nervous system with invariant cell lineage and position, C. elegans provides an excellent model to study how diverse neuronal subtypes are specified. The anatomy and interconnectivity of all 118 hermaphrodite neuron types are known, as are the molecular details of many neuronal subtypes. The C. elegans male has 79 additional neurons, falling into 37 classes. Most of those male-specific neurons are located in the tail region and contribute to specific motor output during mating behavior (Yu, 2003).
During mating, the C. elegans male scans for the vulva by touching the hermaphrodite with the ventral side of his tail and backing along her body. If the vulva is not found, he turns at the hermaphrodite head or tail and scans the other side. The male hook sensillum is a copulatory structure that is located just anterior to the cloaca and mediates vulval location behavior. Intact wild-type males usually stop at their first or second vulval encounter. When the hook sensillum is ablated, operated males circle the hermaphrodite multiple times and fail to stop at the vulva. This defect is referred to as the Lov (location of the vulva defective) phenotype. The hook sensillum consists of five cells, including a structural cell and two ciliated sensory neurons HOA and HOB. The two hook neurons have large nuclei and send dendrites into the hook structure; however, their anatomy can be distinguished by cell morphology and synaptic contacts. Ablation of either HOA or HOB results in a Lov phenotype, indicating that HOA and HOB have non-redundant functions (Yu, 2003).
The C. elegans homologues of human autosomal dominant polycystic kidney disease genes PKD1 (lov-1) and PKD2 (pkd-2) are expressed in the HOB hook neuron. Human PKD genes, which encode divergent members of the TRP family of cation channels, possibly act in signal transduction important for renal epithelial differentiation, because mutations in PKD1 and PKD2 are associated with pathogenic renal cyst formation. In C. elegans, lov-1 and pkd-2 mutations disrupt vulva location behavior, consistent with a defect in HOB sensory function. Although LOV-1 and PKD-2 are localized in sensory cilia and cell bodies, the ultrastructure of cilia and dendrites appears normal in lov-1 and pkd-2 mutants (Yu, 2003).
Another class of genes required for vulva location affects the formation of ciliated endings in sensory neurons. This class includes che-3, daf-10, osm-5 and osm-6. che-3, osm-5 and osm-6 are required for most or all sensory cilia, while daf-10 functions only in a subset of ciliated sensory neurons. The hermaphrodite expression of osm-5, a homolog of the mouse autosomal recessive polycystic kidney disease (ARPKD) gene, and osm-6 has been shown to be regulated by a RFX transcription factor DAF-19, which plays a critical role in general sensory cilium differentiation (Yu, 2003).
An allele of egl-46, a putative zinc-finger transcription factor, was isolated in a screen for loci required for fate specification of C. elegans hook neuron HOB. egl-46 has been characterized as a gene, which, when mutated, affects the development of two mechanosensory neurons (FLP cells), as well as having defects in the hermaphrodite HSN egg-laying motoneurons (Desai, 1988; Desai and Horvitz, 1989). EGL-46 and the transcription enhancer factor (TEF) homolog EGL-44 (Drosophila homolog Scalloped) are expressed in the HOB hook neuron and are required for expression of genes encoding polycystins LOV-1 and PKD-2, homeodomain protein CEH-26, and neuropeptide-like protein NLP-8. egl-44 and egl-46 mutants are defective in vulva location behavior during mating, suggesting compromised normal HOB function. This HOB-specific pathway is distinct from the DAF-19-regulated general cilia formation pathway in sensory neurons. daf-19 acts independently of egl-44 and egl-46 to affect expression of downstream genes in the HOB-specific program, indicating that general and cell-specific regulatory factors work in concert to establish cell-specific features crucial for HOB neuronal function in sensory behavior (Yu, 2003).
To fulfill its sensory function, the HOB neuron must build specific structures and express appropriate molecules to receive and transduce signals. In a proposed model, the general cilium formation pathway governed by daf-19 programs HOB to have sensory cilia, and egl-46, partly with egl-44, regulates expression of genes in HOB involved in signal transduction cascades. These two pathways are distinct. Formation of the cilium structures is not necessary for HOB-specific gene expression, and regulators in the cell-specific pathway, egl-44 and egl-46, showed no obvious effect on the HOB expression of the cilium structure genes osm-5 and osm-6. However, these two pathways do interact: not only are they both necessary for HOB function, but the ciliogenic pathway regulator daf-19 has an effect on downstream components of the HOB-specific program without affecting egl-44 or egl-46 expression (Yu, 2003).
Previous studies have suggested that daf-19 is only required for genes functioning in common aspects of cilium formation. This study provides the first evidence that daf-19 is required for the expression of some cell-type-specific factors. It is proposed that daf-19 acts through some unknown factor(s) [which could be an X-box containing gene(s)] to modify HOB-specific gene expression. Stronger daf-19::gfp expression is observed in HOB than in HOA, but whether it is associated with additional daf-19 regulation of HOB-specific gene expression is not known. This daf-19 regulation is not limited to the HOB neuron as daf-19 also affects pkd-2 expression in the ray neurons and CEM neurons, indicating some general features are common in this subtype of ciliated sensory neurons. Coupled regulation of general neuronal features and cell-specific identities by multiple transcriptional factors has been found in several different organisms, such as specification of the C. elegans AIY interneuron, C. elegans olfactory neurons and vertebrate motoneurons, and thus might be a general aspect of the logic of neuronal cell type specification (Yu, 2003).
Both male hook neurons, HOA and HOB, play a role in vulva location behavior. They both detect the presence of a hermaphrodite vulva, and then produce a distinctive output. This output causes the male to stop at the vulva and to proceed to the next step of mating. One possible explanation for the functional non-redundancy of HOA and HOB is that they possess different sensory specificity, and hence respond to different cues from the vulva. Another possibility is HOA and HOB might receive the same cues at different times. egl-44 is broadly expressed in many cells of the male tail, but its expression is almost undetectable in HOA. None of the other genes, including egl-46 and its downstream targets in the HOB-specific program described in this study, is expressed in HOA. The unequal expression of those genes in the two hook neurons provides molecular evidence supporting distinct roles for HOA and HOB in mating (Yu, 2003).
egl-46 mutations result in an extra cell division in the terminal differentiation of the C. elegans Q neuroblast lineage (Desai, 1989). Loss of either egl-44 or egl-46 function does not cause a cell division defect or a failure in establishment of primary ciliated neural fate during HOB specification. This was determined by anatomical examination and by expression of the cilium structure genes, osm-5 and osm-6. In the non-sex-specific FLP cells, it has been shown that egl-44 and egl-46 act as transcriptional repressors (Wu, 2001). They promote the correct subtype of mechanosensory neurons by suppressing expression of genes dedicated to another subtype. Possible positive roles in gene transcription are implicated for egl-44 and egl-46 in the HSN neurons, but no target has been identified (Desai, 1989; Wu, 2001). These data suggest a positive effect of egl-44 and egl-46 on the expression of downstream HOB-specific genes. However, it has not been ruled out that EGL-44 and EGL-46 activate gene expression in HOB by repression of a repressor of HOB-specific genes (Yu, 2003).
It is proposed that the sensory abilities of the HOB neuron are established by individual cell-specific components regulated by egl-44 and egl-46. One of these components, ceh-26, is the C. elegans ortholog of Drosophila prospero (pros) gene. pros is involved in the initiation of differentiation in specific neurons following asymmetric cell division. However, expression of ceh-26 in HOB is not coupled with cell division. Instead, it is expressed at a much later stage, after basic features of cell fate have been established. Similar to HOB, ray B neurons express both egl-44 and egl-46, but unlike HOB, these neurons do not express ceh-26::gfp. Therefore, it is thought that co-expression of egl-44 and egl-46 is not sufficient to activate ceh-26::gfp in HOB and additional co-factors are also required. The other downstream components, lov-1, pkd-2 and nlp-8, encode proteins that are probably involved in HOB sensory input and output. LOV-1 and PKD-2 accumulate in the sensory cilia and have been proposed to act in a complex; a working model is that LOV-1 is a sensory receptor and PKD-2 is a channel protein. Neuropeptide-like protein NLP-8 might act as a neurotransmitter or neuromodulator released by HOB to mediate the response to the stimuli from the hermaphrodite vulva (Yu, 2003).
Potential mechanosensory and chemosensory interactions between the male and the hermaphrodite during mating is implied by the vulva location behavior itself, as well as by the requirement of functional ciliated sensory endings in the two hook neurons. Whether HOB is a mechanical sensor or a chemical sensor or both, as is the case for the polymodal ASH neuron, is not known. Because egl-44 and egl-46 distinguish between mechanosensory neuron subtypes during FLP fate specification, it is possible that these two genes regulate downstream targets that confer mechanosensory ability to the HOB neuron. If so, as members of TRP protein gene family, lov-1 and pkd-2 might be such targets. Known examples of TRP proteins that play a role in mechanotransduction include a C. elegans TRP protein OSM-9 and the Drosophila TRP-like NOMPC protein. Both of these TRP proteins are expressed in mechanosensory neurons and are involved in mechanosensory response (Yu, 2003).
Human PKD1 and PKD2 were identified as two loci responsible for the autosomal dominant polycystic kidney disease (ADPKD), a genetic disorder that causes renal failure at various ages of adulthood. Relatively little is known about the regulation of these PKD genes and possible alterations during the disease process. Expression of C. elegans PKD gene homologs, lov-1 and pkd-2, is affected by transcription factors egl-44 and egl-46. The mammalian TEF proteins, homologous to egl-44, have been implicated in multiple developmental processes. Specific expression in kidney was reported for multiple members of TEF proteins. C. elegans EGL-46 belongs to a novel zinc-finger protein subfamily. Identified close mammalian homologs of egl-46 includes insulinoma associated (IA) proteins, implicated in islet differentiation of the pancreas, and murine MLT 1 protein, silenced in the liver tumors, but their possible roles in the kidney have not been investigated. Progressive cyst formation in ADPKD is not restricted to kidney: involvement of the liver and the pancreas occurs, indicating that those organs suffer similar pathogenesis during progression of the disease. The demonstrated gene regulation network in HOB might reveal important insights into the regulation of human polycystin gene expression (Yu, 2003).
The dependence of ciliogenesis for the function of PKD-2 may be even more relevant to renal development in mammals. In C. elegans, the ARPKD homolog osm-5 is a direct target of the RFX factor DAF-19, making the requirement of DAF-19 activity for pkd-2 expression particularly interesting with regard to the link between ADPKD and ARPKD. Mammalian polycystins and the cilia of the kidney cells might participate in a common signaling pathway crucial for renal differentiation and function. This hypothesis implies that RFX factor(s) might play a role in the renal development (Yu, 2003).
A subtraction library was constructed from human insulinoma (beta cell tumor) and glucagonoma (alpha cell tumor) cDNA phagemid libraries. Differential screening of 153 clones with end-labeled mRNAs from insulinoma, glucagonoma, and HeLa cells resulted in the isolation of a novel cDNA clone designated IA-1. This cDNA clone has a 2838-base pair sequence consisting of an open reading frame of 1530 nucleotides, which translates into a protein of 510 amino acids with a pI value of 9.1 and a molecular mass of 52,923 daltons. At the 3'-untranslated region there are seven ATTTA sequences between two polyadenylation signals (AATAAA). The IA-1 protein can be divided into two domains based upon the features of its amino acid sequence. The NH2-terminal domain of the deduced protein sequence (amino acids 1-250) has four classical pro-hormone dibasic conversion sites and an amidation signal sequence, Pro-Gly-Lys-Arg. The COOH-terminal domain (amino acids 251-510) contains five putative 'zinc-finger' DNA-binding motifs of the form X3-Cys-X2-4-Cys-X12-His-X3-4-His-X4 which has been described as a consensus sequence for members of the Cys2-His2 DNA-binding protein class. Northern blot analysis revealed IA-1 mRNA in five of five human insulinoma and three of three murine insulinoma cell lines. Expression of this gene was undetectable in normal tissues. Additional tissue studies have revealed that the message is expressed in several tumor cell lines of neuroendocrine origin including pheochromocytoma, medullary thyroid carcinoma, insulinoma, pituitary tumor, and small cell lung carcinoma. The restricted tissue distribution and unique sequence motifs suggest that this novel cDNA clone may encode a protein associated with the transformation of neuroendocrine cells (Goto, 1992).
IA-1 is a novel cDNA originally isolated from a human insulinoma subtraction library (ISL-153). It encodes a protein containing both a zinc finger DNA-binding domain and a putative prohormone domain. IA-1 transcripts have been found thus far only in tumors of neuroendocrine origin. Clinical studies have shown that IA-1 is a sensitive marker for neuroendocrine differentiation of human lung tumors. The entire IA-1 gene and its 5'-upstream region have been cloned and sequenced from a human liver genomic library. In situ hybridization localized the IA-1 gene to the short arm of human chromosome 20. Sequence analysis and restriction enzyme mapping showed that the IA-1 gene is uninterrupted and appears to be intronless. Evidence that IA-1 is an intronless gene that can translate into protein was obtained from in vitro translation studies that showed that both IA-1 cDNA and IA-1 genomic DNA yielded identical protein products of approximately 61,000 daltons. Examination of the 5' upstream region (2090 base pairs) revealed several tissue-specific regulatory elements, including glucokinase upstream promoter elements and a Pit-1 factor binding site. The presence of several different upstream regulatory elements may account for IA-1 gene expression in different neuroendocrine tumors (Lan, 1994).
IA-1 is a novel zinc finger transcription factor with a restricted tissue distribution in the embryonic nervous system and tumors of neuroendocrine origin. The 1.7-kilobase 5'-upstream DNA sequence of the human IA-1 gene directs transgene expression predominantly in the developing nervous system including forebrain, midbrain, hindbrain, spinal cord, retina, olfactory bulb, and cerebellum; this pattern recapitulates the expression patterns of neuroendocrine tissues and childhood brain tumors. The IA-1 promoter deletion reporter gene constructs revealed that the sequence between -426 and -65 bp containing three putative E-boxes (~361 bp) upstream of the transcription start site is sufficient to confer tissue-specific transcriptional activity. Further mutation analysis revealed that the proximal E-box (E3) closest to the start site is critical to confer transcriptional activity. Electrophoretic mobility shift assay and transient transfection studies demonstrated that the NeuroD1 and E47 heterodimer are the key transcription factors that regulate the proximal E-box of the IA-1 promoter. Therefore, it is concluded that the IA-1 gene is developmentally expressed in the nervous system and the NeuroD1/E47 transcription factors up-regulate IA-1 gene expression through the proximal E-box element of the IA-1 promoter (Breslin, 2001).
A novel cDNA, insulinoma-associated antigen-1 (IA-1), containing five zinc-finger DNA-binding motifs, was isolated from a human insulinoma subtraction library. IA-1 expression is restricted to fetal but not adult pancreatic and brain tissues as well as tumors of neuroendocrine origin. Using various GAL4 DNA binding domain (DBD)/IA-1 fusion protein constructs, it was demonstrated that IA-1 functions as a transcriptional repressor and that the region between amino acids 168 and 263 contains the majority of the repressor activity. Using a selected and amplified random oligonucleotide binding assay and bacterially expressed GST-IA-1DBD fusion protein (257-510 a.a.), the consensus IA-1 binding sequence, TG/TC/TC/TT/AGGGGG/TCG/A, was identified. Further experiments showed that zinc-fingers 2 and 3 of IA-1 are sufficient to demonstrate transcriptional activity using an IA-1 consensus site containing a reporter construct. A database search with the consensus IA-1 binding sequence revealed target sites in a number of pancreas- and brain-specific genes consistent with its restricted expression pattern. The most significant matches were for the 5'-flanking regions of IA-1 and NeuroD/beta2 genes. Co-transfection of cells with either the full-length IA-1 or hEgr-1AD/IA-1DBD construct and IA-1 or NeuroD/beta2 promoter/CAT construct modulated CAT activity. These findings suggest that the IA-1 protein may be auto-regulated and play a role in pancreas and neuronal development, specifically in the regulation of the NeuroD/beta2 gene (Breslin, 2002).
The isolation and characterization has been described of the mouse homolog of the human zinc-finger transcription factor INSM1 (IA-1), and an interacting protein was identified. A 2.9-kb cDNA with an open reading frame of 1563 nucleotides, corresponding to a translated protein of 521 amino acids, was isolated from a mouse beta TC-1 cDNA library. Mouse INSM1 was found to be 86% identical to human INSM1 and both proteins contain proline-rich regions and multiple zinc-finger DNA-binding motifs. Sequencing of mouse Insm1 genomic DNA revealed that it is an intronless gene. Chromosomal mapping localized Insm1 to chromosome 2. Northern blot analysis showed that mouse Insm1 expression begins at 10.5 days in the embryo, decreases after 13.5 days, and is barely detected at 18.5 days. In mouse brain, Insm1 is strongly expressed for 2 weeks after birth but shows little or no expression thereafter. Transfection of cells with GFP-tagged INSM1 revealed that INSM1 is expressed exclusively in the nucleus. Proteins that interacted with INSM1 were identified by the yeast two-hybrid system and the binding of one of them, Cbl-associated protein (CAP), to INSM1 was confirmed by in vitro pull-down experiments, nuclear colocalization, and co-immunoprecipitation assays. Further studies showed that both INSM1 and CAP proteins were present in the nucleus of insulinoma cells and that endogenous INSM1 protein was co-precipitated with antibody to CAP. These findings raise the possibility that during embryo development CAP may enter the nucleus through its own nuclear localization signal or by binding to INSM1 (Xie, 2002).
The restriction of IA-1 gene expression in human fetal pancreata of different gestational stages was analyzed along with whether the expression of IA-1 gene is associated with rat AR42J cell differentiation into insulin-positive cells. To examine whether the IA-1 gene is associated with pancreatic endocrine cell differentiation, a rat pancreatic amphicrine cell line, AR42J, was used to investigate whether the expression of the IA-1 gene coincides with AR42J cells converting into either endocrine or exocrine lineage. A set of islet transcription factors was also analyzed that regulate key differentiation steps involved in activating the genes that confer the specialized functions of terminally differentiated pancreatic islet cells. When the AR42J cells were converted into insulin-positive cells induced by GLP-1, insulinoma conditioned-medium, or both, a significant elevated expression of mRNA for IA-1 and islet-specific transcription factors such as Pdx-1, NeuroD/beta2, and Nkx6.1 was observed. In contrast, dramatically decreased expression of mRNA for IA-1 and islet-specific transcription factors was displayed when AR42J cells were converted into the acinar-like phenotype by dexamethasone. It is concluded that the IA-1 gene is developmentally regulated in fetal pancreatic cells, and its expression pattern is consistent with parallel changes in islet-specific transcription factors during the endocrine differentiation of AR42J cells (Zhu, 2002).
Zhai, W., Jeong, H., Cui, L., Krainc, D. and Tjian, R. (2005). In vitro analysis of huntingtin-mediated transcriptional repression reveals multiple transcription factor targets. Cell 123(7): 1241-53. 16377565
Transcriptional dysregulation has emerged as a potentially important pathogenic mechanism in Huntington's disease, a neurodegenerative disorder associated with polyglutamine expansion in the huntingtin (htt) protein. This study reports the development of a biochemically defined in vitro transcription assay that is responsive to mutant htt. Both gene-specific activator protein Sp1 and selective components of the core transcription apparatus, including TFIID and TFIIF, are direct targets inhibited by mutant htt in a polyglutamine-dependent manner. The RAP30 subunit of TFIIF specifically interacts with mutant htt both in vitro and in vivo to interfere with formation of the RAP30-RAP74 native complex. Importantly, overexpression of RAP30 in cultured primary striatal cells protects neurons from mutant htt-induced cellular toxicity and alleviates the transcriptional inhibition of the dopamine D2 receptor gene by mutant htt. These results suggest a mutant htt-directed repression mechanism involving multiple specific components of the basal transcription apparatus (Zhai, 2005).
This study developed an in vitro transcription assay to dissect the potential molecular mechanisms employed by mutant htt to repress transcription of specific promoters (e.g., Sp1-dependent). Taking advantage of this well-defined in vitro transcription system, it was demonstrate that specific components (TFIID and TFIIF) of the transcriptional machinery are directly targeted by mutant htt. Importantly, these in vitro results correlate very well with the in vivo effects of mutant htt, such as the previously reported disruption of Sp1 and TAF4 interaction by mutant htt at the D2 promoter (versus NR1 promoter) in primary neurons. Bearing this principle in mind, it may be possible, in the future, to take advantage of this in vitro system to identify other potential direct targets and mechanisms of transcriptional dysregulation associated with other transcription pathways in HD. Secondly, this study demonstrates that soluble rather than aggregated forms of mutant htt may directly dysregulate transcription by interfering with specific components of the transcriptional preinitiation complex. The data suggest that transcriptional dysfunction may occur as a result of interference by the soluble forms of mutant htt early in disease before any aggregation is seen. In addition, this work suggests that mutant htt may act as a special class of transcriptional repressor or corepressor. This is a potentially important point because it suggests that one of the primary and direct effects of mutant htt on transcription is via specific repressor mechanisms, whereas other documented effects of htt such as activation of transcription may be compensatory or secondary. Finally, this work demonstrates that transcriptional repression by mutant htt is polyQ length dependent. This strongly confirms the observed toxic gain of function for mutant htt. Progressive expansion of polyQ in mutant htt appears to lead to more severe repression while little or no repression is seen with wt htt both in vitro and in vivo. The strong correlation between polyQ length and the efficiency of repression observed in vitro fits well with the documented timing and severity of HD onset. This striking finding further suggests that direct disruption of transcription integrity via aberrant interactions between mutant htt, Sp1, TFIID, and TFIIF are specific and may be significant for orchestrating the pathogenesis of HD (Zhai, 2005).
In this work, a variety of different htt N-terminal fragment constructs were used to take advantage of the various systems established by other HD researchers. Although truncated htt proteins might behave somewhat different from the intact protein, it is nevertheless believed that these in vitro and in vivo studies should be quite informative. Indeed, in vitro studies were inspired by previous findings showing that various truncated versions of mutant htt bearing different lengths of polyQ expansions are produced by proteolytic cleavage in vivo, resulting in fragments that can readily enter the nucleus. Thus, these in vitro studies largely attempt to recapitulate the situation that is thought to occur in vivo (Zhai, 2005).
The most striking finding from the in vitro studies was the identification of TFIIF as a novel direct target in mutant htt-mediated transcriptional repression. Although there have been reports linking TFIIF to the function of transcription activators and repressors, this study provides the first direct connection between TFIIF and transcriptional repression induced by a polyQ expansion protein. RAP30, a subunit of TFIIF, appears to consist of three functional domains. The N-terminal domain of RAP30 is thought to bind RAP74, the central region binds RNA Pol II, and the C-terminal domain binds DNA. In this study, it was found that mutant htt has a strong affinity for RAP30. Because RAP30 lacks a Q-rich domain, its interaction with mutant htt is likely mediated through an alternative interface. Crystal structure of the N-terminal fragments of RAP30 and RAP74 have been shown to adopt a triple-barrel structure with multiple β sheets. Since mutant htt favors the formation of an intramolecular β sheet structure, it is possible that the RAP30 mutant htt interaction involves contact between β sheet structures. Such a structure-based interference mechanism is consistent with the finding that expansion of glutamines in mutant htt enhanced its affinity for RAP30. Thus, mutant htt may target not only polyQ-containing proteins, but also non-polyQ proteins with specific β sheet structures. It should be noted that addition of Congo red, a β sheet-reactive reagent, to the in vitro system did not prevent mutant htt-mediated transcriptional repression, possibly due to its inability to prevent mutant htt from forming protofibrils in vitro (Zhai, 2005).
An important aspect revealed by this study is that mutant htt has a higher affinity for RAP30 than wt htt and may compete with RAP74 for interaction with RAP30. Because an intact TFIIF complex is required for efficient initiation and elongation of transcription at least for some promoters, it is hypothesized that TFIIF dissociation will contribute to transcriptional dysregulation by mutant htt. It is conceivable that mutant htt, which has a higher affinity for RAP30, when it accumulates in both the cytoplasm and nucleus could cause less TFIIF to be formed in the cytoplasm and more TFIIF to be disrupted in the nucleus. Such a scenario will likely result in a general decrease of transcription in HD cells, as has been observed. In several DNA microarray studies, the level of RNA Pol II large subunit has been shown to increase in mutant HD brain. Since the role of TFIIF in transcription is dependent on its interaction with RNA Pol II, it is speculate that elevated levels of RNA Pol II subunits in HD cells may arise as a compensatory mechanism triggered by decreased levels of TFIIF. However, in vitro, adding excess RNA Pol II did not rescue the htt-mediated repression (Zhai, 2005).
By contrast, the findings showed that overexpression of RAP30 is able to abrogate transcriptional repression and rescue the cellular toxicity induced by mutant htt in primary striatal neurons. There are two potential explanations. One possibility is for RAP30 to interact with mutant htt and compete it away from other htt-interacting partners. Another possibility is for RAP30 to drive the formation of more TFIIF complexes, thereby potentiating transcription of important genes involved in neuronal survival. An intriguing observation that was made is that overexpression of RAP74 alone can induce significant cellular toxicity in striatal neurons. This suggests that the chronic release of free RAP74 from TFIIF may contribute to the progressive nature of HD pathogenesis. Thus, the data favor the mechanism in which RAP30 can protect the striatal neurons by promoting TFIIF complex formation. To better understand how much the TFIIF-mediated mechanism contributes to the selective neuronal death during HD pathogenesis, it will be important to identify those genes whose transcription in striatal neurons is particularly sensitive to both mutant htt and RAP74 in future investigations (Zhai, 2005).
Taking the in vitro and in vivo observations together with previous studies, the following model is proposed for how mutant htt represses Sp1-dependent gene expression in neurons. In normal cells, Sp1 is recruited to GC-box-containing promoters through its DNA binding domain. Once bound to DNA, Sp1 utilizes its multiple glutamine-rich activation domains to target components of the basal transcription machinery, one of which is TAF4, a subunit of TFIID. In a multistep recruiting process involving TFIIA, TFIID, TFIIB, TFIIE, TFIIF, TFIIH, RNA Pol II, and CRSP, the preinitiation complex is then formed on activated promoters to potentiate transcription. In HD cells, soluble nuclear mutant htt fragment is free to bind Sp1 through direct protein interactions, thus sequestering this key transcriptional activator from binding to its cognate GC boxes. Furthermore, mutant htt can also prevent Sp1-mediated recruitment of TFIID through its interaction with TAF4. In the case where there is already an Sp1-TFIID complex formed at the promoter, mutant htt could subsequently disrupt the stepwise PIC assembly by targeting TFIIF, an essential transcription factor important for initiation, promoter escape, and elongation at certain promoters. It is anticipated that for different potential target genes, mutant htt will have differential effects because these multiple transcription factor targets may be differentially required for critical functions and rate-limiting transactions at specific gene promoters. In summary, this simple model describes one potential mechanism by which mutant htt can selectively target an activator (Sp1) and multiple components of the core machinery (TFIID and TFIIF) to interfere with various stages of the transcription process. It is anticipated that this model will undergo further refinements as more gene regulatory targets for mutant htt are identified and their molecular consequences determined (Zhai, 2005).
Neurogenin 3 (Ngn3) is key for endocrine cell specification in the embryonic pancreas and induction of a neuroendocrine cell differentiation program by misexpression in adult pancreatic duct cells. The gene encoding IA1, a zinc-finger transcription factor, as a direct target of Ngn3 and it forms a novel branch in the Ngn3-dependent endocrinogenic transcription factor network. During embryonic development of the pancreas, IA1 and Ngn3 exhibit nearly identical spatio-temporal expression patterns. However, embryos lacking Ngn3 fail to express IA1 in the pancreas. Upon ectopic expression in adult pancreatic duct cells Ngn3 binds to chromatin in the IA1 promoter region and activates transcription. Consistent with this direct effect, IA1 expression is normal in embryos mutant for NeuroD1, Arx, Pax4 and Pax6, regulators operating downstream of Ngn3. IA1 is an effector of Ngn3 function as inhibition of IA1 expression in embryonic pancreas decreases the formation of insulin- and glucagon-positive cells by 40%, while its ectopic expression amplifies neuroendocrine cell differentiation by Ngn3 in adult duct cells. IA1 is therefore a novel Ngn3-regulated factor required for normal differentiation of pancreatic endocrine cells (Mellitzer, 2006).
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