Functional characterization of mel-28 / elys

Resumen

The nuclear envelope (NE) is a hallmark of eukaryotic cells. The NE encloses the nuclear genome and separates it from the cytoplasm allowing to the cell a high level of organization and regulation of transcription and translation. The NE is formed by four major components: the inner nuclear membrane (INM), the outer nuclear membrane (ONM), the nuclear lamina (NL) and the nuclear pore complexes (NPCs; Hetzer, 2010). Transport of macromolecules between the nucleus and the cytoplasm is regulated by NPCs, which are constituted of 30 different nucleoporins (Nups). Nups can be classified into three general classes. The first class is formed by approximately 15 Nups rich in phenylalanine-glycine (FG) repeats and that constitute peripheral and transport channel components of the NPC. The second class is composed by the transmembrane Nups NDC1, POM121, GP210 and POM33/TMEM33 (Chadrin et al., 2010). And, the third class denotes the structural scaffold of the NPC, including the Nup MEL-28/ELYS and the complexes NUP107-160 and NUP205-188-93 (Rabut, Doye, & Ellenberg, 2004). The association of Nups with chromatin is one of the initial steps in nuclear reorganization, suggesting that they could have a role in the chromatin organization through mitosis and, consequently, in the propagation of epigenetic memory. This Thesis is focused on a conserved Nup known as MEL-28 in Caenorhabditis elegans and ELYS in vertebrates. MEL-28/ELYS plays a critical role in post-mitotic NPC reassembly through recruitment of the NUP107-160 complex and is required for correct segregation of mitotic chromosomes (Fernandez & Piano, 2006; Franz et al., 2007; Galy, Askjaer, Franz, López-Iglesias, & Mattaj, 2006a). MEL-28 has a dynamic behavior: it localizes to NPCs and chromatin during interphase and shuttles to spindle microtubules and kinetochores during cell division. Several studies done in C. elegans and HeLa cells have reported that mutations or knockdown of MEL-28/ELYS produces defects in NE morphology and in NPC assembly as well as defects in chromatin segregation, mitotic spindle assembly and nucleocytoplasmic transport (Fernandez & Piano, 2006; Galy, Askjaer, Franz, López-Iglesias, et al., 2006a; Beth A Rasala, Orjalo, Shen, Briggs, & Forbes, 2006). However, it is unknown how MEL-28 localization and activity is regulated. The general objective of my Ph.D. project is the analysis of chromatin organization through identification of chromatin domains bound by MEL-28. For this purpose, we evaluate the function of MEL-28/ELYS according to its location, either when located in NPC or in the nucleoplasm using C. elegans as model organism. The results of this thesis are present in chapters IV and V; besides, in chapter III (Materials and Methods), we describe the DamID-seq workflow from sample preparation to bioinformatics analysis, which was published in the book “The Nuclear Envelope: Methods and Protocols”, chapter name: “DamID Analysis of Nuclear Organization in Caenorhabditis elegans”, (Gómez-Saldivar, Meister, & Askjaer, 2016). DamID is based on the in vivo expression of a chromatin-associated protein of interest fused to the Escherichia coli DNA adenine methyltransferase, which produces unique identification tags at binding site in the genome (Van Steensel & Henikoff, 2000). This marking is simple, highly specific and can be mapped by sensitive enzymatic and next generation sequencing (NGS) approaches. In chapter IV: “Identification of Conserved MEL-28/ELYS Domains with Essential Roles in Nuclear Assembly and Chromosome Segregation”, (Gómez-Saldivar, Fernandez, et al., 2016), we present a systematic, functional and structural analysis of MEL-28 in C. elegans early development and human ELYS in cultured cells. In collaboration with Dr. Anita Fernandez from Fairfield University (USA), Dr. Yasushi Hiraoka and Dr. Tokuko Haraguchi from Graduate School of Frontier Biosciences, Osaka University (Japan), we have generated and examined the expression and localization of full-length and almost 50 different MEL-28/ELYS truncations that lack different domains and fused these to GFP to track their localization by confocal microscopy. We have identified functional domains responsible for NPC and kinetochore localization, chromatin binding, mitotic spindle matrix association and chromosome segregation. Surprisingly, we found that perturbations to MEL-28’s conserved AT-hook and loop2 domains do not affect MEL-28 localization although they disrupt MEL-28 function and delay cell cycle progression. Specifically, deletion of the AT-hook domain from MEL- 28 activates the ATR DNA damage checkpoint. Our analyses also uncover a novel meiotic role of MEL-28, intervening in the proper development of anaphase I and II. Together, these results show that MEL-28 has conserved structural domains that are essential for its fundamental roles in NPC assembly and chromosome segregation during meiosis and mitosis. In chapter V: “Characterization of genome-nucleoporin interaction in Caenorhabditis elegans” (manuscript in preparation), we propose the DamID technique as an attractive method to globally characterize chromatin organization in C. elegans. In this part of our research, we have used DamID-array and DamID-sequencing to identify the chromatin regions with which MEL-28 associates. Interestingly, MEL-28 is enriched at transcribed genes and correlates positively with active histone marks (H3K4me3 and H3K36me3), suggesting that it may be involved in regulation of gene expression. In addition, we have performed DamID with two MEL-28 fragments that are unable to bind to NPCs, which were characterized in chapter IV. In general, the binding profile of MEL-28 fragments is very similar to the full-length protein, suggesting that MEL-28-chromatin interaction is placed mostly in the nucleoplasm and not in the nuclear periphery. Besides, we compared the MEL-28 chromatin profile with the profile of another Nup, NPP-22/NDC1, which is permanently anchored to the nuclear pore complex. Surprisingly, despite its location this protein interacts with the genome in a very similar percentage than the mobile Nup MEL-28; although we saw a wide reduction of interaction with the sex chromosome. Likewise, we found that the chromatin association profile of NPP-22 was more similar to the profile of the nuclear lamina protein LMN-1 (González-Aguilera et al., 2014) than to MEL-28’s profile, suggesting that individual Nups interact with specific chromatin domains. Interestingly, GO-term analysis reveals that MEL-28-associated genes are related to embryonic, larval and reproductive development, as well as with mitotic processes. This suggests that MEL-28 has postembryonic functions that have not yet been studied. On the other hand, genes placed in the DNA interaction domains of NPP-22 and LMN-1, are involved with processes more typical from the NE, such as signaling pathways, sensorial perception, metabolism, and innate immune response. Finally, in the appendix I, we attach the article: “Differential spatial and structural organization of the X chromosome underlies dosage compensation in C. elegans”, (Sharma et al., 2014), a research work done in collaboration with Dr. Peter Meister from University of Bern (Switzerland). There, we performed MEL-28 DamID in males and hermaphrodites, and this study revealed new information about the mechanism of dosage compensation. Dosage compensation is a genetic regulatory mechanism, which operates to equalize expression of genes that are present on the X chromosome, and thus ensure that these genes are expressed equally in the two sexes. In C. elegans, dosage compensation is achieved by two-fold down-regulation of gene expression from both X chromosomes in hermaphrodites. We found that in males, the single X chromosome interacts with NPC proteins, while in hermaphrodites; the dosage compensation complex impairs this interaction altering X chromosome localization, and probably its transcription.