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Coordination of Developmental Timing and Maturation by the F-box Protein DRE-1/FBXO11

Horn, Moritz (2014) Coordination of Developmental Timing and Maturation by the F-box Protein DRE-1/FBXO11. PhD thesis, Max Planck Institute for Biology of Ageing.

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    Spatiotemporal control of cellular events is key to developmental progression in all organisms. Accordingly the abundance of regulatory proteins must be tightly controlled in a highly time- and tissue-specific manner. Pioneering work in the small roundworm Caenorhabditis elegans (C. elegans) has uncovered a network of genes, termed the heterochronic loci, which govern temporal coordination of developmental processes. Importantly this heterochronic network is remarkably conserved across species. There are currently over 30 identified heterochronic loci, which encode various transcriptional, translational and post-translational regulators including the first discovered microRNAs lin-4 and let-7. In 2007 our laboratory discovered a novel heterochronic gene, dre-1, which specifies late larval development in the C. elegans epidermis and gonad. Interestingly dre-1 encodes a highly conserved F-box protein; F-box proteins typically function as substrate recognition components of SCF E3-ubiquitin ligase complexes, thus conferring substrate specificity to the ubiquitin proteasome system (UPS). The initial analysis of DRE-1 and its mammalian homolog FBXO11 revealed their interaction with SCF complex components, suggesting conservation also on the functional level. Mutation of C. elegans dre-1 leads to a diverse array of developmental alterations including precocious terminal differentiation of epidermal blast cells, molting defects and alterations in gonadal outgrowth. Likewise, haploinsuffiency of Fbxo11 has been linked to craniofacial malformations such as cleft palates, as well as otitis media, an inflammation of the middle ear, in both mice and men. Furthermore, FBXO11 was recently shown to function as a tumor suppressor in lymphoid malignancies by controlling turnover of the oncoprotein BCL6. However, the DRE-1/FBXO11 substrate(s) coordinating developmental progression still remain to be elucidated. Thus the central questions of my thesis are: what are the substrates of DRE-1/FBXO11 in the heterochronic circuit, and how do they illuminate developmental timing events? In an effort to identify novel substrates of the SCFDRE-1/FBXO11 E3-ubiquitin ligase complex involved in developmental timing, I pursued an RNAi-based suppressor approach in C. elegans. I reasoned that client substrates should aberrantly accumulate in dre-1 mutants and cause developmental alterations across tissues. If so, then knockdown of such substrates would ameliorate related phenotypes. Here, I identified the highly conserved zinc-finger transcriptional repressor BLMP-1 as a new substrate of the SCFDRE-1 complex. Several lines of evidence reveal that BLMP-1 is a substrate of the SCFDRE-1 complex in developmental timing circuits. First, blmp-1 loss of function strongly suppressed dre-1 heterochronic phenotypes in epidermis and gonad and blmp-1 mutation itself exhibited developmental timing defects opposite to dre-1. blmp-1 also opposed dre-1 for other life history traits such as entry into the dauer diapause and adult longevity. Second, BLMP-1 protein was strikingly elevated upon depletion of dre-1 and other SCF complex components, as well as by inhibition of the proteasome. Moreover, BLMP-1 was regulated in a time- and tissue-specific manner by dre-1, consistent with corresponding phenotypes. Third, DRE-1 and BLMP-1 physically interacted in vivo in C. elegans and in vitro in a cell-based system. Importantly the role of DRE-1 in regulating BLMP-1 stability is evolutionary conserved, as direct protein interaction and degradation function was also observed for the human counterparts FBXO11 and BLIMP-1. In addition to blmp-1, I also investigated genetic interactions of dre-1 and cdt-2 in C. elegans, since CDT2 was recently identified as a FBXO11 substrate in mammalian systems. I found cdt-2 to genetically interact with dre-1 for specific processes in the epidermis. In particular gaps in an adult-specific cuticular structure occurring in dre-1 mutants could arise from elevated CDT-2 levels and a subsequent failure of cell cycle exit of epidermal blast cells, which synthesize these structures. My findings are in line with mammalian studies that established CDT2, which itself is part of an E3-ubiqitin ligase complex, as a critical cell cycle regulator by targeting substrates such as the CDK inhibitor p21 for proteasomal degradation. Altogether I conclude that dre-1 mutants’ diverse developmental alterations arise from aberrant accumulation of distinct target proteins. In particular, my work shows that spatiotemporal control of BLMP-1 protein by DRE-1 coordinates developmental timing and other life history traits in C. elegans. DRE-1, on the one hand, restrains BLMP-1 abundance in the epidermis to prevent precocious terminal differentiation and, on the other hand, triggers BLMP-1 degradation in distal tip cells to initiate dorso-ventral migratory events. The notion that DRE-1/BLMP-1 work together for multiple processes suggests these proteins comprise a two-protein module that may mediate related metazoan maturation processes. Taken together, my work fundamentally contributes to the understanding of developmental timing coordination by DRE-1/FBXO11 in C. elegans and might help to shed light on related processes in higher organisms.

    Item Type: Thesis (PhD thesis)
    Horn, MoritzMoritz.Horn@web.de
    URN: urn:nbn:de:hbz:38-56933
    Subjects: Life sciences
    Uncontrolled Keywords:
    developmental timingUNSPECIFIED
    Caenorhabditis elegansUNSPECIFIED
    ubiquitin ligaseUNSPECIFIED
    Faculty: Mathematisch-Naturwissenschaftliche Fakultät
    Divisions: Mathematisch-Naturwissenschaftliche Fakultät
    Language: English
    Date: 23 July 2014
    Date Type: Publication
    Date of oral exam: 30 June 2014
    Full Text Status: Public
    Date Deposited: 13 Aug 2014 13:57:39
    NameAcademic Title
    Antebi, AdamProf. Dr.
    Hoppe, ThorstenProf. Dr.
    Schwarz, GünterProf. Dr.
    URI: http://kups.ub.uni-koeln.de/id/eprint/5693

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