Mec-3 Gene Research Articles

MBL-1 and EEL-1 affect the splicing and protein levels of MEC-3 to control dendrite complexity.

Transcription factors (TFs) play critical roles in specifying many aspects of neuronal cell fate including dendritic morphology. How TFs are accurately regulated during neuronal morphogenesis is not fully understood. Here, we show that LIM homeodomain protein MEC-3, the key TF for C. elegans PVD dendrite morphogenesis, is regulated by both alternative splicing and an E3 ubiquitin ligase. The mec-3 gene generates several transcripts by alternative splicing. We find that mbl-1, the orthologue of the muscular dystrophy disease gene muscleblind-like (MBNL), is required for PVD dendrite arbor formation. Our data suggest mbl-1 regulates the alternative splicing of mec-3 to produce its long isoform. Deleting the long isoform of mec-3(deExon2) causes reduction of dendrite complexity. Through a genetic modifier screen, we find that mutation in the E3 ubiquitin ligase EEL-1 suppresses mbl-1 phenotype. eel-1 mutants also suppress mec-3(deExon2) mutant but not the mec-3 null phenotype. Loss of EEL-1 alone leads to excessive dendrite branches. Together, these results indicate that MEC-3 is fine-tuned by alternative splicing and the ubiquitin system to produce the optimal level of dendrite branches.

Imaging corneal pathology in a transgenic mouse model using nonlinear microscopy

A transgenic mouse model with a Clim [co-factor of LIM (a combination of first letters of Lin-11 (C. elegans), ISL1 (rat), and Mec-3 (C. elegans) gene names) domain proteins] gene partially blocked in the epithelial compartment of its tissues is used to establish the sensitivity of intrinsic reflectance nonlinear optical microscopy (NLOM) to stromal and cellular perturbations in the cornea. Our results indicate dysplasia in the squamous epithelium, irregular collagen arrays in the stroma, and a compromised posterior endothelium in the corneas of these mice. As suggested by biochemical data, the collagen alterations are likely due to collagen III synthesis and deposition during healing and remodeling of transgenic mice corneal stromas. All of the topographic features seen in NLOM images of normal and aberrant corneas are confirmed by coregistration with histological sections. In this work, we also use ratiometric redox fluorometry based on two-photon excited cellular fluorescence from reduced nicotinamide adenine dinucleotide (NAD)(P)H and oxidized flavin adenine dinucleotide (FAD) to study mitochondrial energy metabolism. Employing this method, we detect higher metabolic activity in the endothelial layer of cornea compared to an epithelial layer located further away from the metabolites. The combination of two-photon excited fluorescence (TPF) with second harmonic generation (SHG) signals allows imaging to aid in understanding the relationship between alternation of specific genes and structural changes in cells and extracellular matrix.

Expression and regulation of an FMRFamide-related neuropeptide gene family in Caenorhabditis elegans.

FMRFamide (Phe-Met-Arg-Phe-NH2) and related peptides (FaRPs) have been found throughout the animal kingdom, where they are involved in many behaviors. We previously identified 22 genes comprising the flp gene family that encodes FaRPs in Caenorhabditis elegans; in this paper we report the identification of another flp gene, flp-23. As a first step toward determining their functional roles in C. elegans, we examined the cell-specific expression pattern of the flp gene family. Of the 19 flp genes examined, each gene is expressed in a distinct set of cells; these cells include interneurons, motor neurons, and sensory neurons that are involved in multiple behaviors, as well as supporting cells, muscle cells, and epidermal cells. Several flp genes show sex-specific expression patterns. Furthermore, we find that expression of two flp genes changes in response to the developmental state of the animal. Many neurons express multiple flp genes. To investigate how flp genes are regulated in different neuronal subtypes, we examined flp expression in a small, well-defined subset of neurons, the mechanosensory neurons. Mutations in the unc-86 and mec-3 genes, which are necessary for the production and differentiation of the mechanosensory neurons, result in the complete loss of flp-4, flp-8, and flp-20 expression in mechanosensory neurons. Collectively, these data indicate that members of the flp gene family are likely to influence multiple behaviors and that their regulation can be dependent on the developmental state of the organism.

Checkpoint proteins influence telomeric silencing and length maintenance in budding yeast.

A complex network of surveillance mechanisms, called checkpoints, interrupts cell cycle progression when damage to the genome is detected or when cells fail to complete DNA replication, thus ensuring genetic integrity. In budding yeast, components of the DNA damage checkpoint regulatory network include the RAD9, RAD17, RAD24, MEC3, DDC1, RAD53, and MEC1 genes that are proposed to be involved in different aspects of DNA metabolism. We provide evidence that some DNA damage checkpoint components play a role in maintaining telomere integrity. In fact, rad53 mutants specifically enhance repression of telomere-proximal transcription via the Sir-mediated pathway, suggesting that Rad53 might be required for proper chromatin structure at telomeres. Moreover, Rad53, Mec1, Ddc1, and Rad17 are necessary for telomere length maintenance, since mutations in all of these genes cause a decrease in telomere size. The telomeric shortening in rad53 and mec1 mutants is further enhanced in the absence of SIR genes, suggesting that Rad53/Mec1 and Sir proteins contribute to chromosome end protection by different pathways. The finding that telomere shortening, but not increased telomeric repression of gene expression in rad53 mutants, can be suppressed by increasing dNTP synthetic capacity in these strains suggests that transcriptional silencing and telomere integrity involve separable functions of Rad53.

The Saccharomyces cerevisiae DNA damage checkpoint is required for efficient repair of double strand breaks by non-homologous end joining

In this work we report that the Saccharomyces cerevisiae RAD9, RAD24, RAD17, MEC1, MEC3 and RAD53 checkpoint genes are required for efficient non-homologous end joining (NHEJ). RAD9 and RAD24 function additionally in this process. Defective NHEJ in rad9Δ–rad24Δ, but not yku80Δ cells, is only partially rescued by imposing G1 or G2/M delays. Thus, checkpoint functions other than transient cell cycle delays may be required for normal levels of NHEJ. Epistasis analysis also indicated that YKU80 and RAD9/RAD24 function in the same pathway for repair of lesions caused by MMS and γ-irradiation. Unlike NHEJ, the checkpoint pathway is not required for efficient site-specific integration of plasmid DNA into the yeast genome, which is RAD52-dependent, but RAD51-independent.

Regulation of touch receptor differentiation by the Caenorhabditis elegans mec-3 and unc-86 genes.

The nematode Caenorhabditis elegans possesses six morphologically similar neurons that are responsible for sensing gentle touch to the body. Previous genetic studies identified genes that are necessary for the production and differentiation of these touch cells. In particular, unc-86 encodes a POU-type homeodomain protein needed for the production of the touch cells, while mec-3 encodes a LIM-type homeodomain protein needed for the differentiation of the touch cells. Molecular studies showed that MEC-3 and UNC-86 bind cooperatively to sites in the mec-3 promoter and can synergistically activate transcription from it in vitro. Here we show that UNC-86::MEC-3 hetero-oligomer-binding sites are also found in the promoters of two presumed targets of mec-3, the mec-4 and mec-7 genes, that are necessary for the function of the touch cells. These sites, which are well-conserved in the related nematode C. briggsae, are required for promoter activity. When one of the binding sites is cloned into a heterologous promoter, expression is found in the touch cells and two to four other cells that express mec-3 and unc-86. These data support a model in which touch-cell differentiation is specified, in part, by the UNC-86::MEC-3 hetero-oligomer and not by MEC-3 alone. Ectopic expression of mec-3, driven by a heat-shock promoter, also supports this hypothesis: the acquisition of touch-cell characteristics by several additional cells under these conditions required unc-86. Since the touch-cell lineages express UNC-86 before MEC-3, MEC-3 appears to modify the activity of UNC-86, leading to touch-cell-specific gene expression. Because both UNC-86 and MEC-3 have activation domains, the formation of the hetero-oligomer may create a strong activator. In the modification of UNC-86 function by MEC-3 in the touch cells, these studies provide an example of how the sequential activation of transcription factors can determine cell fate within particular cell lineages.

The novel DNA damage checkpoint protein ddc1p is phosphorylated periodically during the cell cycle and in response to DNA damage in budding yeast.

The DDC1 gene was identified, together with MEC3 and other checkpoint genes, during a screening for mutations causing synthetic lethality when combined with a conditional allele altering DNA primase. Deletion of DDC1 causes sensitivity to UV radiation, methyl methanesulfonate (MMS) and hydroxyurea (HU). ddc1Delta mutants are defective in delaying G1-S and G2-M transition and in slowing down the rate of DNA synthesis when DNA is damaged during G1, G2 or S phase, respectively. Therefore, DDC1 is involved in all the known DNA damage checkpoints. Conversely, Ddc1p is not required for delaying entry into mitosis when DNA synthesis is inhibited. ddc1 and mec3 mutants belong to the same epistasis group, and DDC1 overexpression can partially suppress MMS and HU sensitivity of mec3Delta strains, as well as their checkpoint defects. Moreover, Ddc1p is phosphorylated periodically during a normal cell cycle and becomes hyperphosphorylated in response to DNA damage. Both phosphorylation events are at least partially dependent on a functional MEC3 gene.

Immunohistochemical localization of cysteine-rich intestinal protein in rat small intestine.

Cysteine-rich intestinal protein (CRIP) is a LIM (cysteine-rich motif of leu-11, isl-1, and mec-3 genes) domain protein with a double zinc finger motif. The protein is abundantly expressed in the intestine, peritoneal macrophages, and peripheral blood mononuclear cells. The function of CRIP is not known. The purpose of this study was to determine the cellular distribution of CRIP in rat intestine, as an initial step toward eventual determination of a function. Immunohistochemical and immunogold labeling electron microscopy using a purified polyclonal rabbit antibody to a synthetic peptide representing a zinc finger domain of rat CRIP were carried out on sections of rat duodenum. Western blotting was used to detect signal specificity of the antibodies. These immunohistochemical and electron microscopy studies showed particularly high abundance of CRIP in the cytoplasmic granules of Paneth cells of the intestine. Some evidence of CRIP expression was also found in cells of the villus tip, but abundance was less than that found in the Paneth cells. The localization of CRIP in Paneth cells and its presence in mononuclear cells suggests that CRIP may be involved in host defense mechanisms and/or tissue differentiation/remodeling processes common to these cell types.

Yeast pip3/mec3 Mutants Fail To Delay Entry into S Phase and To Slow DNA Replication in Response to DNA Damage, and They Define a Functional Link between Mec3 and DNA Primase

The catalytic DNA primase subunit of the DNA polymerase alpha-primase complex is encoded by the essential PRI1 gene in Saccharomyces cerevisiae. To identify factors that functionally interact with yeast DNA primase in living cells, we developed a genetic screen for mutants that are lethal at the permissive temperature in a cold-sensitive pril-2 genetic background. Twenty-four recessive mutations belonging to seven complementation groups were identified. Some mutants showed additional phenotypes, such as increased sensitivity to UV irradiation, methyl methanesulfonate, and hydroxyurea, that were suggestive of defects in DNA repair and/or checkpoint mechanisms. We have cloned and characterized the gene of one complementation group, PIP3, whose product is necessary both for delaying entry into S phase or mitosis when cells are UV irradiated in G1 or G2 phase and for lowering the rate of ongoing DNA synthesis in the presence of methyl methanesulfonate. PIP3 turned out to be the MEC3 gene, previously identified as a component of the G2 DNA damage checkpoint. The finding that Mec3 is also required for the G1- and S-phase DNA damage checkpoints, together with the analysis of genetic interactions between a mec3 null allele and several conditional DNA replication mutations at the permissive temperature, suggests that Mec3 could be part of a mechanism coupling DNA replication with repair of DNA damage, and DNA primase might be involved in this process.

Promoter sequences for the establishment of mec-3 expression in the nematode Caenorhabditis elegans

In certain stereotyped lineages of Caenorhabditis elegans, the mec-3 gene is transcribed in neurons that are anterior daughters of cells containing the UNC-86 protein. UNC-86 binds to the mec-3 promoter and is necessary for transcription activation, but this protein is present in many cells that do not transcribe mec-3, including the posterior sister and parent cells of the mec-3-expressing neurons. To understand how the mec-3 promoter directs transcription in only a subset of cells that contain UNC-86, we have compared sequences within the promoter that are bound by UNC-86 in vitro with sequences that are necessary for early transcription of mec-3 in vivo. We find that upstream of the mec-3 start codon are two blocks of sequence that are each sufficient to generate the cellular pattern of mec-3 transcription. The proximal sequence includes three previously identified short regions that have been conserved in nematode evolution and each contains one high-affinity UNC-86 binding site. The recognition consensus sequence for UNC-86 is CATnnn T/A AAT, which is identical to the recognition sequence for the UNC-86-related mammalian transcription factor Brn-3. Adjacent to the UNC-86 recognition site is an additional sequence that is important for establishment of mec-3 expression and is presumably recognized by an unidentified transcription factor.

Activation of the mec-3 promoter in two classes of stereotyped lineages in Caenorhabditis elegans

The mec-3 gene of Caenorhabditis elegans encodes a homeodomain protein and is expressed in one of two cells upon asymmetric cell division. As a result of asymmetric mec-3 expression, the two sister cells express different fates, so mec-3 is a likely target for the machinery that mediates asymmetric cell division. The unc-86 gene encodes a homeodomain protein of the POU family, which activates mec-3 by binding to its promoter. The ten mec-3-expressing cells are a subset of the anterior daughters of UNC-86-containing cells. Posterior daughters of UNC-86-containing cells do not express mec-3, even though the UNC-86 protein is distributed into both daughter cells. Lineages that express the unc-86 and mec-3 genes can be grouped into two types: in Type I lineages, UNC-86 protein is first made in the immediate parent of the terminal mec-3-expressing cell, while in Type II lineages, UNC-86 is first made in the grandparent of the terminal mec-3-expressing cell. The purpose of experiments presented here is to understand the relationship between the mec-3 expression patterns in each type of lineage, and to determine the fundamental activity pattern of the mec-3 promoter. We find that in the Type I V5.pa lineage, mec-3-lacZ is first synthesized in the terminal PVDR neuron, one cell division after unc-86 is expressed. mec-3 expression in PVDR can occur by transcriptional regulation alone; segregation of the mec-3 RNA or protein is not required to explain the asymmetric expression of mec-3. In the Type II Q lineage, the mec-3 promoter activity can be detected in the immediate anterior daughter of the first unc-86-expressing cell, but when this cell divides, mec-3 is expressed in only one of its daughters at later times. It seems likely that, in the short-lived immediate anterior daughter cell in Type II lineages, mec-3 product does not accumulate to levels that can influence subsequent events. Our results suggest that the mec-3 promoter is activated in all anterior daughters of unc-86-expressing cells.

Genetic regulation of mec-3 gene expression implicated in the specification of the mechanosensory neuron cell types in Caenorhabditis elegans.

The mec-3 gene, a member of the LIM-homeodomain transcription factors, is required for touch receptor, FLP and PVD neurons to differentiate in the nematode Caenorhabditis elegans. Stably integrated transgenic strains with mec-3-lacZ fusion were generated by irradiating UV light to an unstable transgenic strain with the extrachromosomal DNA. Expression patterns of the mec-3-lacZ fusion were examined in mutant backgrounds (lin-4, lin-14, egl-44, egl-46 and sem-4 genes) which alter touch receptor-specific gene expression. In the lin-4 mutant background, ectopic mec-3-lacZ positive AVM/PVM-like cells were observed in 9% of the animals. By contrast, in the lin-14 mutant background, mec-3-lacZ staining in AVM/PVM cells was lost in 86% of the animals. In the egl-44 and egl-46 mutant backgrounds, expression pattern was the same as wild-type animals. In the sem-4 mutant background, more than half of the animals (54-69%) had ectopic staining cells in the tail in addition to the wild-type staining pattern. The modes of action of these genetically interacting genes in the differentiation of mechanosensory neurons are proposed.

Cell polarity and the mechanism of asymmetric cell division.

During development, one mechanism for generating different cell types is asymmetric cell division, by which a cell divides and contributes different factors to each of its daughter cells. Asymmetric cell division occurs throughout the eukaryotic kingdom, from yeast to humans. Many asymmetric cell divisions occur in a defined orientation. This implies a cellular mechanism for sensing direction, which must ultimately lead to differences in gene expression between two daughter cells. In this review, we describe two classes of molecules: regulatory factors that are differentially expressed upon asymmetric cell division, and components of a signal transduction pathway that may define cell polarity. The lin-11 and mec-3 genes of C. elegans, the Isl-1 gene of mammals and the HO gene of yeast, encode regulatory factors that determine cell type of one daughter after asymmetric cell division. The CDC24 and CDC42 genes of yeast affect both bud positioning and orientation of mating projections, and thus may define a general cellular polarity. We speculate that molecules such as Cdc24 and Cdc42 may regulate expression of genes such as lin-11, mec-3, Isl-1 and HO upon asymmetric cell division.

1108 Expression of the mec-3 gene and the mechanosensory neuron fate in the nematode C. elegans

1108 Expression of the mec-3 gene and the mechanosensory neuron fate in the nematode C. elegans

Regulation of the mec-3 gene by the C.elegans homeoproteins UNC-86 and MEC-3.

The mec-3 gene encodes a homeodomain protein with LIM repeats that is required for the specification of touch cell fate in Caenorhabditis elegans. Previous experiments suggested that mec-3 expression requires the product of the unc-86 gene, a POU-type homeoprotein, and mec-3 itself. We have analyzed the control of mec-3 expression by identifying potential cis regulatory elements in the mec-3 gene (by conservation in a related nematode and by DNase I footprinting using unc-86 and mec-3 proteins) and testing their importance by transforming C.elegans with mec-3lacZ fusions in which these sites have been mutagenized in vitro. Both unc-86 and mec-3 proteins bind specifically to the promoter of the mec-3 gene, suggesting that both proteins may be directly involved in the regulation of the mec-3 gene. In addition, the footprint pattern with mec-3 protein is altered in the presence of unc-86 protein. In vivo transformation experiments reveal that some of the binding regions of the two proteins are needed for general positive control and maintenance of mec-3 expression while others have no detectable, unique function. Interestingly, the unc-86 gene appears to be required not only to initiate mec-3 expression but also to maintain it.

Regulation of anterior cell-specific mec-3 expression during asymmetric cell division in C. elegans.

The homeobox-containing mec-3 gene of C. elegans is expressed in 10 mechanosensory neurons and is necessary for these cells to acquire their fate. All the mec-3-expressing cells are anterior daughters from an asymmetric cell division. In this paper, we examine the expression of a mec-3--lacZ fusion in the presence of mutations that may disrupt asymmetric cell division or anterior-posterior positional information, as well as mutations that may specifically alter mec-3 expression. A mutation in lin-17 causes production of additional mec-3-expressing cells and can have its effect on the cell division that produces a mec-3-expressing cell. In a lin-5 mutant, in which postembryonic blast cells do not complete cell division and become polyploid, blast cells that would give rise to mec-3-expressing daughters instead express mec-3 themselves. In a lin-12 glp-1 double mutant, which is disrupted for many cell interactions in which two cells compete for the same fate, mec-3 expression is unaffected. These results are consistent with a model for asymmetric cell division in which the mec-3-expressing cell and its sister are different immediately upon cell division, rather than acquiring differences through later interaction with each other or their surroundings. lin-17 mutant animals also show defects in the position of the PVM cell and the PLM axons. Animals mutant in unc-73 and unc-40, known to have axon outgrowth defects, also show errors in PVM position and a low frequency of additional mec-3-expressing cells, as well as occasional secondary vulval protrusions, a common phenotype of lin-17 animals. Many other mutations have either no effect on mec-3 expression or an effect that can be largely predicted from previously known phenotypes: these include mab-5, mig-1, unc-53, egl-5, lin-32, and egl-27. unc-11 shows an unexpected and specific defect in mec-3 expression in the PVD neurons, but not in the other mec-3-expressing cells. Two mutations that suppress the egg-laying defect of unc-86 have no effect on the mec-3 expression defect in an unc-86 mutant.

The LIM domain-containing homeo box gene Xlim-1 is expressed specifically in the organizer region of Xenopus gastrula embryos.

A novel cysteine-rich motif, named LIM, has been identified in the homeo box genes lin-11, Isl-1, and mec-3; the mec-3 and lin-11 genes determine cell lineages in Caenorhabditis elegans. We isolated LIM class homeo box genes from Xenopus laevis that are closely related to lin-11 and mec-3 in the LIM and homeo domains. This paper deals with one of these genes, Xlim-1. Xlim-1 mRNA is found at low abundance in the unfertilized egg, has a major expression phase at the gastrula stage, decreases, and rises again during the tadpole stage. In adult tissues the brain shows the highest abundance, by far, of Xlim-1 mRNA. The maternal and late expression phases of the Xlim-1 gene suggest that it has multiple functions at different stages of the Xenopus life cycle. In the gastrula embryo, Xlim-1 mRNA is localized in the dorsal lip and the dorsal mesoderm, that is, in the region of Spemann's organizer. Explant experiments showed that Xlim-1 mRNA is induced by the mesoderm-inducer activin A and by retinoic acid, which is not a mesoderm inducer but affects patterning during Xenopus embryogenesis; application of activin A and retinoic acid together results in synergistic induction. The structure, inducibility, and localized expression in the organizer of the Xlim-1 gene suggest that it has a role in establishing body pattern during gastrulation.

The mec-3 gene contains cis-acting elements mediating positive and negative regulation in cells produced by asymmetric cell division in Caenorhabditis elegans.

The homeo box-containing genes mec-3 and unc-86 are necessary to specify the fate of a defined set of mechanoreceptors in Caenorhabditis elegans. Previous experiments have shown that mec-3 expression can be divided into two phases: initial synthesis mediated in part by unc-86, and continued synthesis that requires mec-3 itself. We now identify sequences that have been conserved during Caenorhabditis evolution and are necessary for establishment, maintenance, and repression of mec-3 expression. Upstream of the start codon for the mec-3-coding sequence are four segments (regions I-IV) of 71, 29, 28, and 24 bp, which are almost identical between C. elegans and Caenorhabditis vulgarensis. Region I is the only conserved sequence that effects establishment of mec-3 synthesis. Maintenance of mec-3 expression is mediated primarily by region II. Repression appears to be controlled by several segments: Mutation of region III, region IV, and parts of region I in a mec-3-lacZ fusion results in beta-galactosidase expression in some non-mec-3-expressing sisters of mec-3-positive cells. These results indicate that the mec-3 5' region contains target sequences that mediate a genetic switch between alternative fates expressed by sister cells in a stereotyped cell lineage.

Insulin gene enhancer binding protein Isl-1 is a member of a novel class of proteins containing both a homeo-and a Cys–His domain

The activity of the rat insulin I gene enhancer is mainly dependent on two cis-acting protein-binding domains. Here we report the isolation of a complementary DNA encoding a protein, Isl-1, that binds to one of these domains. Isl-1 contains a homeodomain with greatest similarity to those of the Caenorhabditis elegans proteins encoded by mec-3 and lin-11. In addition, Isl-1, like the lin-11 and mec-3 gene products, contains a novel Cys-His domain which is reminiscent of known metal-binding regions. Together these proteins define a novel class of proteins containing both a homeo- and a Cys His-domain. Isl-1 is preferentially expressed in cells of pancreatic endocrine origin. If the structural homologies between Isl-1 and the C. elegans gene products reflect functional similarities, a role for Isl-1 in the development of pancreatic endocrine cells could be envisaged.

The mec-3 gene of Caenorhabditis elegans requires its own product for maintained expression and is expressed in three neuronal cell types.

The homeo-box-containing gene mec-3 of the nematode Caenorhabditis elegans, is expressed in several sensory neurons, as assayed by expression of a mec-3-lacZ fusion. These cells are the touch receptors, which mediate the response to gentle touch, and the FLP and PVD neurons. PVD mediates a response to harsh mechanical stimuli, and FLP has an ultrastructure suggestive of a mechanoreceptor, but its function is unknown. mec-3 is necessary for the differentiation of the touch receptors, because in mec-3 mutants, the touch receptors do not function and have none of their distinguishing features. mec-3 is also needed for PVD function: The PVD neurons no longer mediate a response to harsh mechanical stimuli in the mutants. The expression of the mec-3-lacZ fusion, and presumably mec-3 itself, is altered by mutations in several genes originally identified by their effects on touch cell development. unc-86, another homeo-box-containing gene, is necessary for all mec-3-lacZ expression, but also affects several other lineages and cells in which mec-3 is not expressed. mec-3 activity appears to be required for maintained expression of the mec-3-lacZ fusion in all cells in which it is expressed. In a mec-17 mutant, mec-3-lacZ expression is not maintained in the touch receptors, but is not affected in the FLP and PVD neurons. These findings suggest that combinatorial mechanisms of gene regulation control both the expression of mec-3 itself and its action in promoting the terminal differentiation of various cell types.