|dc.description.abstract||The nuclear envelope (NE) is a hallmark of eukaryotic cells. It is composed by four major components: the inner and outer nuclear membranes (INM and ONM respectively), the nuclear lamina (NL) and the nuclear pore complexes (NPCs; Hetzer, 2010). Apart from the regulation of macromolecules transport between the nucleus and the cytoplasm, it is becoming increasingly clear that the NE plays a crucial role in nuclear organization and gene expression. Specific chromatin domains are anchored to the NE, which generally is associated with formation of heterochromatin and transcriptional silencing. Reflecting the importance of NE functions, alterations in its structure are associated with a broad range of human diseases, often referred to as laminopathies, and include cardiomyopathy, muscular dystrophy, neurological disorders, lipodystrophy and premature ageing. An example is Emery-Dreifuss muscular dystrophy (EDMD), which was initially linked to mutations in the EMD gene encoding the inner nuclear membrane protein emerin, but can also be caused by mutations in the nuclear lamina protein LMNA, SUN1, SUN2, SYNE1, SYNE2 and TMEM43.
Since most NE proteins are ubiquitously expressed, a main challenge is to understand how laminopathy mutations result in tissue-specific phenotypes. It is hypothesized that tissue-specific alteration in nuclear organization might be responsible for particular symptoms of laminopathies.
This thesis is focused on the INM protein EMR-1, the Caenorhabditis elegans ortholog of emerin. Recently, we found that EMR-1 is enriched in contractile tissues and is required for correct neuromuscular junction activity as shown by hypersensitivity to the cholinesterase inhibitor aldicarb (Gonzalez-Aguilera et al., 2014). DamID experiments demonstrated that emerin is enriched at genes involved in muscle and neuronal function and deletion of emerin causes local changes in nuclear architecture. Moreover, transcriptome analyses revealed that emerin is associated with gene repression, particularly of genes implicated in muscle and nervous system function (Gonzalez-Aguilera et al., 2014). Based on these results, we suggested that emerin might regulate nuclear organization in a tissue-specific manner. Analyses of tissue-specific interaction profiles between chromatin and NE proteins (EMR-1 and LMN-1) are relevant to understand the role of EMR-1 in the NMJ activity, but it has not been explored so far in intact organisms due to technical limitations. To characterize the role of emerin in nuclear organization during development in a tissue-specific way we developed a FLP-Frt recombination-based DamID system in the nematode Caenorhabditis elegans, which enables the identification of global NE-chromatin contacts in specific ectodermal, mesodermal and endodermal tissues in the physiological context of an intact animal.
On a different topic, it has been reported that NE morphology is prone to suffer from changes during normal aging and in progeria patients. Again, we proposed that alterations in nuclear organization through development and aging may have an impact on the aging-associated disorders. To characterize the changes in chromatin organization during aging, the already optimized FLP-Frt system has been used to perform temporal DamID in young vs. old animals.
Finally, Susan Gasser¿s laboratory showed that the chromodomain protein CEC-4 bound to the INM is required for peripheral anchoring of heterochromatin in embryonic cells via H3K9me. However, during differentiation, a second heterochromatin-sequestering pathway is induced. An RNA interference screen in the cec-4 background identified the chromodomain protein MRG-1 as a broadly expressed factor responsible of heterochromatin positioning during differentiation of intestinal cells via H3K36me. Since MRG-1 is exclusively bound to euchromatin, these results suggest that MRG-1 acts indirectly. Again, analyses of nuclear organization in WT, single and double mutants are relevant to understand the role of CEC-4 and MRG-1 in heterochromatin positioning during differentiation.
Thus, the main goal of this thesis is to explore the association between EMR-1 and chromatin during development in a tissue-specific manner as well as during aging, using the novel FLP-Frt based toolkit developed in this study.||es_ES