The C. elegans gene lin-29 is required for the terminal differentiation of the lateral hypodermal seam cells during the larval-to-adult molt. lin-29 protein accumulates in the nuclei of these cells, consistent with its predicted role as a zinc finger transcription factor. The earliest detectable LIN-29 accumulation in seam cell nuclei is during the last larval stage (L4), following the final seam cell division, which occurs during the L3-to-L4 molt. LIN-29 accumulates in all hypodermal nuclei during the L4 stage. The time of LIN-29 appearance in the hypodermis is controlled by the heterochronic gene pathway: LIN-29 accumulates in the hypodermis abnormally early, during the third larval stage, in loss-of-function lin-14, lin-28 and lin-42 mutants, and fails to accumulate in the hypodermis of lin-4 mutants. LIN-29 also accumulates stage-specifically in the nuclei of a variety of non-hypodermal cells during development. Its accumulation is dependent upon the upstream heterochronic genes in some, but not all, of these non-hypodermal cells (Bettinger, 1996).
C. elegans vulval development culminates during exit from the L4-to-adult molt with the formation of an opening through the adult hypodermis and cuticle that is used for egg laying and mating. Vulva formation requires the heterochronic gene lin-29, which triggers hypodermal cell terminal differentiation during the final molt. lin-29 mutants are unable to lay eggs or mate because no vulval opening forms; instead, a protrusion forms at the site of the vulva. It has been demonstrated, through analysis of genetic mosaics, that lin-29 is absolutely required in a small subset of lateral hypodermal seam cells, adjacent to the vulva, for wild-type vulva formation and egg laying. However, lin-29 function is not strictly limited to the lateral hypodermis: (1) LIN-29 accumulates in many non-hypodermal cells with known roles in vulva formation or egg laying; (2) animals homozygous for one lin-29 allele, ga94, have the vulval defect and cannot lay eggs, despite having a terminally differentiated adult lateral hypodermis; (3) vulval morphogenesis and egg laying requires lin-29 activity within the EMS lineage, a lineage that does not generate hypodermal cells (Bettinger, 1997).
The C. elegans gene lin-29 encodes a zinc-finger transcription factor that is required for hypodermal cell terminal differentiation and proper vulva morphogenesis. lin-29 is also required in males for productive mating. lin-29 males can perform the early mating behaviors including response to hermaphrodite contact and vulva location, but they do not perform the subsequent steps of vulva attachment via spicule insertion and sperm transfer. Consistent with this observation, lin-29 mutant spicules are on average 43% shorter than wild-type spicules while other male mating structures appear unaltered. In lin-29 mutants, spicule development goes awry after the generation of spicule cells, when spicule morphogenesis occurs in wild-type males. LIN-29 accumulates in many cells of the wild-type male tail, including those that form the spicules. It has been demonstrated, through analysis of genetic mosaics, that the formation of wild-type-length spicules requires lin-29(+) in the AB.p lineage, the lineage that gives rise to the spicules and other male copulatory structures. Mosaic analysis also reveals a role for lin-29(+) in the P1 lineage, which mainly produces sex muscles, cells of the somatic gonad, and body wall muscles (Euling, 1999).
The development of a connection between the uterus and the vulva in the nematode C. elegans requires specification of a uterine cell called the 'utse', and its attachment to the vulva and the epidermal seam cells. The uterine pi cells generate the utse and uv1 cells, which also connect the uterus to the vulva. The uterine anchor cell (AC) induces the vulva through LIN-3/epidermal growth factor (EGF) signaling, and the pi cells through LIN-12/Notch signaling. A gene required for seam cell maturation is also required for specification of the utse and for vulval differentiation, and thus helps to coordinate development of the vulval-uterine-seam cell connection. The egl-29 gene is necessary for induction of uterine pi cells; it is allelic to lin-29, which encodes a zinc finger transcription factor that is necessary for the terminal differentiation of epidermal seam cells. In the uterus, lin-29 functions upstream of lin-12 in the induction of pi cells and is necessary to maintain expression in the AC of lag-2, which encodes a ligand for LIN-12. It is concluded that the lin-29 gene controls gene expression in the epidermal seam cells, uterus and vulva, and may help to coordinate the terminal development of these three tissues by regulating the timing of late gene expression during organogenesis (Newman, 2000).
Null mutations in the C. elegans heterochronic gene lin-41 cause precocious expression of adult fates at larval stages. Increased lin-41 activity causes the opposite phenotype, reiteration of larval fates. let-7 mutations cause similar reiterated heterochronic phenotypes that are suppressed by lin-41 mutations, showing that lin-41 is negatively regulated by let-7. lin-41 negatively regulates the timing of LIN-29 adult specification transcription factor expression. lin-41 encodes an RBCC protein, and two elements in the lin-41 3'UTR are complementary to the 21 nucleotide let-7 regulatory RNA. A lin-41::GFP fusion gene is downregulated in the tissues affected by lin-41 at the time that the let-7 regulatory RNA is upregulated. It is suggested that late larval activation of let-7 RNA expression downregulates LIN-41 to relieve inhibition of lin-29 (Slack, 2000).
p130(cas) (Cas) is a docking protein that contains an SH3 domain and multiple tyrosine residues. p130(cas) is located at focal adhesions, is tyrosine phosphorylated in response to integrin stimulation, and is thought to transmit signals, via c-Crk and other proteins, for the remodeling of actin stress fibers and cell movement. In a search for the ligands of the SH3 domain of p130(cas) by far-Western screening, a novel protein named CIZ (for Cas-interacting zinc finger protein) was cloned. CIZ consists of the following: a putative leucine zipper; a serine/threonine-rich region; a proline-rich sequence; five, six, or eight Krüppel-type C(2)H(2) zinc fingers, and the glutamine-alanine repeat. CIZ binds Cas in cells and is located in the nucleus and at focal adhesions. CIZ has been identified as a nucleocytoplasmic shuttling protein, by using the transient interspecies heterokaryon formation assay. In order to search for the targets of CIZ in nucleus, the DNA binding consensus of CIZ has been identified as (G/C)AAAAA(A) by cyclic amplification and selection of targets analysis. The consensus-like sequences are found in several promoters of matrix metalloproteinases (MMPs), which are the enzymes used to degrade the extracellular matrix proteins. CIZ binds to a consensus-like sequence in the MMP-1 (collagenase) promoter. Overexpression of CIZ upregulates the transcriptions from MMP-1, MMP-3 (stromelysin), and MMP-7 (matrilysin) promoters, and this transactivation is enhanced in the presence of Cas. Furthermore, the stable overexpression of CIZ promotes the production of MMP-7 in culture medium. In summary, CIZ, a novel zinc finger protein, binds Cas, is a nucleocytoplasmic shuttling protein, and regulates the expression of MMPs (Nakamoto, 2000).
In Caenorhabditis elegans, a well-defined pathway of heterochronic genes ensures the proper timing of stage-specific developmental events. During the final larval stage, an upregulation of the let-7 microRNA indirectly activates the terminal differentiation factor and central regulator of the larval-to-adult transition, LIN-29, via the downregulation of the let-7 target genes lin-41 and hbl-1. This study identifies a new heterochronic gene, mab-10, and shows that mab-10 encodes a NAB (NGFI-A-binding protein) transcriptional co-factor. MAB-10 acts with LIN-29 to control the expression of genes required to regulate a subset of differentiation events during the larval-to-adult transition, and the NAB-interaction domain of LIN-29 is conserved in Kruppel-family EGR (early growth response) proteins. A similar interaction between Drosophila NAB and the two Drosophila LIN-29 homologs RN and SQZ was reported recently. In mammals, EGR proteins control the differentiation of multiple cell lineages, and EGR-1 acts with NAB proteins to initiate menarche by regulating the transcription of the luteinizing hormone β subunit. Genome-wide association studies of humans and various studies of mouse recently have implicated the mammalian homologs of the C. elegans heterochronic gene lin-28 in regulating cellular differentiation and the timing of menarche. This work suggests that human homologs of multiple C. elegans heterochronic genes might act in an evolutionarily conserved pathway to promote cellular differentiation and the onset of puberty (Harris, 2011).
This study identified mab-10 as a new heterochronic gene that is required for specific aspects of the larval-to-adult transition, specifically molting cycle exit and seam cell exit from the cell cycle. mab-10 encodes the only C. elegans NAB transcriptional co-factor. NAB proteins are thought to physically interact with Kruppel family EGR transcription factors to regulate their activity (Harris, 2011).
Previous work demonstrated that MAB-10 (then known only as the C. elegans NAB protein R166.1) could interact with mammalian EGR proteins in a yeast two-hybrid assay; no corresponding C. elegans EGR protein was identified. This study has demonstrate that MAB-10 interacts with the terminal differentiation factor LIN-29 through an evolutionarily conserved NAB binding domain (R1 domain) and that MAB-10 is required for a subset of LIN-29-dependent activities. This work identifies LIN-29 as a C. elegans EGR-like protein and demonstrates that the C. elegans heterochronic pathway controls the timing of NAB/EGR-mediated differentiation (Harris, 2011).
Several experiments using mammalian tissue culture suggest that NAB proteins negatively regulate EGR activity by binding EGR proteins at specific target genes and preventing EGR-mediated transcription. However, loss of either EGR2 function or NAB function in mice and humans results in hypomyelination, suggesting that EGR and NAB proteins need not act antagonistically in vivo (Harris, 2011).
In C. elegans, MAB-10 and LIN-29 both act to promote terminal differentiation and the onset of adulthood. Furthermore, mab-10 promotes the formation of precocious adult alae in a lin-41 mutant background, suggesting that MAB-10 does not specifically act to control genes required for exit from the molting cycle and seam cell exit from the cell cycle, but more likely acts as a general enhancer of LIN-29 activity (Harris, 2011).
EGR and NAB proteins have been shown to operate in a negative-feedback loop wherein an EGR protein promotes the expression of its NAB co-factor, which then inhibits EGR activity. mab-10 transcription does not depend on LIN-29, despite a dramatic increase of mab-10 transcription during the L4 stage. Thus, mab-10 is not a transcriptional target of LIN-29 (Harris, 2011).
Whereas mab-10 is not a transcriptional target of LIN-29, MAB-10::GFP localization to seam cell nuclei during the L4 stage required LIN-29, indicating that LIN-29 might promote MAB-10 seam cell nuclear localization via a post-transcriptional mechanism or via direct physical interaction (Harris, 2011).
This work demonstrates that MAB-10 and LIN-29 do not operate in a negative-feedback loop. It is proposed that other components of the heterochronic pathway directly regulate mab-10 transcription to temporally regulate MAB-10/LIN-29 activity and that LIN-29 or some factor downstream of LIN-29 controls MAB-10/LIN-29 activity by promoting the accumulation of MAB-10 in seam cell nuclei (Harris, 2011).
By showing that MAB-10 acts with LIN-29 through an evolutionarily conserved EGR R1 domain, LIN-29 and the Drosophila LIN-29 homologs RN and SQZ are identified as EGR-like molecules. It is proposed that NAB proteins and EGR proteins act together in temporal developmental programs to control terminal differentiation. In Drosophila, the LIN-29 homolog SQZ acts with Drosophila NAB to control neuroblast differentiation. In C. elegans, LIN-29 and MAB-10 act together to control the differentiation of a hypodermal stem cell lineage during the transition from larva to adult by regulating the expression of the nuclear hormone receptors nhr-23 and nhr-25 and the cell cycle regulator cki-1. Recently, a study of C. elegans demonstrated that nhr-25 is itself a heterochronic gene and possibly functions with lin-29 to promote aspects of the larval-to-adult transition, including seam cell exit from the cell cycle. Though the mechanism by which nhr-25 regulates seam cell exit from the cell cycle is not known, it is speculated that LIN-29 and NHR-25 might act together to promote cki-1 expression (Harris, 2011).
EGR proteins were originally identified as immediate-early genes and generally have been regarded as differentiation factors. Like mab-10 and lin-29 mutants, Nab and Egr mutant mice are defective in the terminal differentiation of several cell lineages. For example, in Schwann cells, EGR2 promotes the expression of P27, the homolog of C. elegans CKI-1, and acts with NAB proteins to promote terminal differentiation. Mammalian homologs of other C. elegans heterochronic genes also control differentiation. Similar to the role of LIN-28 in C. elegans, mammalian LIN28 and LIN28B promote stem cell identity and prevent differentiation by repressing the let-7 microRNA gene. As in C. elegans, increasing levels of let-7 drive differentiation, and the mouse homolog of LIN-41, LIN41, has been shown to be a let-7 target acting in stem cell niches to prevent premature differentiation (Harris, 2011).
Mammalian LIN-28 controls the timing of the onset of puberty in mice and possibly humans. Mice lacking EGR1 function, like lin-29 mutants of C. elegans, fail to undergo puberty. EGR1 and NAB proteins act with SF1, the homolog of C. elegans NHR-25, in the gonadotrope lineage of the pituitary gland to regulate the expression of luteinizing hormone and the onset of puberty. The molecular mechanism by which mammalian LIN-28 regulates the onset of puberty is not known. This work raises the possibility that homologs of C. elegans heterochronic genes might act in an evolutionarily conserved pathway that controls the terminal differentiation of cell lineages and the onset of adulthood by regulating the activity of NAB and EGR proteins (Harris, 2011).
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