Diverse eukaryotic organisms share developmental transcription factors with homologous DNA-binding domains. The developmental regulator AbaA, a member of the ATTS/TEA (AbaA, TEF-1, TEC1, Scalloped/TEF-1, TEC1, AbaA) class of transcription factors of the filamentous fungus Aspergillus nidulans, induces pseudohyphal development in the yeast Saccharomyces cerevisiae. The S. cerevisiae homolog of AbaA, TEC1p, is required for this morphological transition. Evidence is provided that TEC1p functions in co-operation with STE12p to induce pseudohyphal development (Gavrias, 1996).

In yeast, an overlapping set of mitogen-activated protein kinase (MAPK) signaling components controls mating, haploid invasion, and pseudohyphal development. Paradoxically, a single downstream transcription factor, Ste12, is necessary for the execution of these distinct programs. Developmental specificity was found to require a transcription factor of the TEA/ATTS family, Tec1, which cooperates with Ste12 during filamentous and invasive growth. Purified derivatives of Ste12 and Tec1 bind cooperatively to enhancer elements called filamentation and invasion response elements (FREs), which program transcription that is specifically responsive to the MAPK signaling components required for filamentous growth. An FRE in the TEC1 promoter functions in a positive feedback loop required for pseudohyphal development (Madhani, 1997).

The transcription factor Tec1 is involved in pseudohyphal differentiation and agar-invasive growth of Saccharomyces cerevisiae cells. The sole element in the TEC1 promoter that has thus far been shown to control Tec1 function is the filament response element. The TEC1 promoter also contains several pheromone response element sequences that are likely to be functional: TEC1 transcription is induced by mating factor; it is cell cycle regulated and dependent on the Ste4, Ste18 and Ste5, all components of the mating factor signal transduction pathway. Using alleles of the transcription factor Ste12 that are defective in DNA binding, transcriptional induction or cooperativity with other transcription factors, little correlation is found between TEC1 transcript levels and agar-invasive growth (Oehlen, 1998).

Diploid yeast develop pseudohyphae in response to nitrogen starvation, while haploid yeast produce invasive filaments that penetrate the agar in rich medium. This study has identified the gene FLO11, which encodes a cell wall protein that is critically required for both invasion and pseudohyphae formation in response to nitrogen starvation. FLO11 encodes a cell surface flocculin with a structure similar to the class of yeast serine/threonine-rich GPI-anchored cell wall proteins. Cells of the Saccharomyces cerevisiae strain Sigma1278b with deletions of FLO11 do not form pseudohyphae as diploids nor invade agar as haploids. In rich media, FLO11 is regulated by mating type; it is expressed in haploid cells but not in diploids. However, upon transfer to nitrogen starvation media, FLO11 transcripts accumulate in diploid cells, but not in haploids. Overexpression of FLO11 in diploid cells, which are otherwise not invasive, enables them to invade agar. Thus, the mating type repression of FLO11 in diploids grown in rich media suffices to explain the inability of these cells to invade. The promoter of FLO11 contains a consensus binding sequence for Ste12p and Tec1p, proteins known to cooperatively activate transcription of Ty1 elements and the TEC1 gene during development of pseudohyphae. Yeast with a deletion of STE12 does not express FLO11 transcripts, indicating that STE12 is required for FLO11 expression. These ste12-deletion strains also do not invade agar. However, the ability to invade can be restored by overexpressing FLO11. Activation of FLO11 may thus be the primary means by which Ste12p and Tec1p cause invasive growth (Lo, 1998).

Specialized gene expression programs are induced by signaling pathways that act on transcription factors. Whether these transcription factors can function in multiple developmental programs through a global switch in promoter selection is not known. Genome-wide location analysis has been used to show that the yeast Ste12 transcription factor, which regulates mating and filamentous growth, is bound to distinct program-specific target genes dependent on the developmental condition. This condition-dependent distribution of Ste12 requires concurrent binding of the transcription factor Tec1 during filamentation and is differentially regulated by the MAP kinases Fus3 and Kss1. Program-specific distribution across the genome may be a general mechanism by which transcription factors regulate distinct gene expression programs in response to signaling (Zeitlinger, 2003).

In response to pheromone from a mating partner, Ste12 induces the expression of mating gene, whereas under certain starvation conditions, Ste12 induces genes involved in filamentous growth, which facilitates foraging for nutrients. Ste12 can activate transcription alone or in partnership with other transcription factors. Some mating genes are induced in response to pheromone by Ste12 alone, whereas other mating genes are regulated by Ste12 together with Mcm1. For its role in filamentous growth, Ste12 requires the function of Tec1, a well-conserved transcription factor of the ATTS/TEA (AbaA, TEF-1, TEC1, Scalloped/TEF-1, TEC1, AbaA) family. Ste12 binds cooperatively with Tec1 at filamentation-responsive elements (FREs) in vitro. These studies suggest that differential gene expression by Ste12 might be achieved through selective partnership with other transcription factors. It is not known, however, whether the two MAPK pathways that induce mating and filamentation differentially regulate Ste12 by activating distinct complexes already bound to DNA or by regulating the partnership and DNA binding behaviors of these complexes (Zeitlinger, 2003 and references therein).

Activation of Ste12 during mating and filamentation involves two different MAPK signal transduction pathways that share components. Since they share the MAPK kinase and the MAPK kinase, two MAPKs, Fus3 and Kss1, may be activated by the mating and filamentation pathways. Genetic studies indicate that Fus3 and Kss1 act in a partially redundant fashion during mating. Deletion of both MAPKs, but not either one alone, abolishes Ste12-dependent induction of mating genes in response to pheromone. The two MAPKs, however, have different effects on Ste12-dependent induction of filamentous growth. The kinase activity of Kss1 increases filamentation, whereas the kinase activity of Fus3 appears to suppress filamentation. The mechanism by which Fus3 and Kss1 might differentially regulate Ste12 is not clear. Genetic assays indicate that both MAPKs activate Ste12 through regulation of two inhibitors, Dig1 and Dig2. Ste12 can form a complex with these two proteins and is released from the complex upon activation by pheromone. Both MAPKs phosphorylate Dig1 and Dig2, as well as Ste12 (Zeitlinger, 2003 and references therein).

A genome-wide binding assay that combines chromatin immunoprecipitation (ChIP) with DNA microarray technology was used to study the binding behavior of Ste12 during mating and filamentation. Ste12 binds to distinct promoters in vivo under different conditions and the different sets of genes specify distinct developmental programs. The global switch in target gene specificity of Ste12 depends on the transcription factor Tec1 during filamentation and is differentially regulated by the two MAPKs. Both MAPKs have the ability to induce mating genes in response to pheromone, but Fus3 has an additional activity that inhibits Ste12 binding to filamentation genes under the same condition. Thus, regulation of Ste12 binding, and not selective activation of transcription factor complexes already bound to DNA, is the mechanism by which the two MAPKs regulate distinct gene expression programs and direct cells toward specific developmental fates (Zeitlinger, 2003 and references therein).

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).