## 2 Results
### Inc-_Nr6a1_ Protein Interactome  
To explore the molecular mechanisms underlying the biological activity of _Inc-Nr6a1_, we searched for proteins that were associated with _Inc-Nr6a1_ using the iDRIP in vivo technique (identification of direct RNA interacting proteins) [18] (Figure 7A). After UV crosslinking of NMuMG cells, Inc-_Nr6a1_-protein complexes were purified using _Inc-Nr6a1_ antisense biotinylated oligonucleotides. Different pools of biotinylated antisense probes were tested against the Inc-_Nr6a1_ isoforms. We achieved a pool of oligonucleotides that specifically retrieved over 30% of the Inc-_Nr6a1-1_ complexes, without enrichment of Inc-_Nr6a1-2_ complexes or housekeeping _Hprt_ mRNA. A _LacZ_ oligonucleotide pool that targeted the _lacZ_ mRNA did not retrieve _Hprt_ nor Inc-_Nr6a1-1_ RNAs (Figure 7B). It should be mentioned that we have been struggling to achieve a pool of biotinylated oligonucleotides that specifically retrieves the Inc-_Nr6a1-2_ isoform, but all attempts to solve this problem have so far been unsuccessful. After washing the complexes under denaturing conditions and treating them with DNAse I, the eluted proteins were analyzed by mass spectrometry. A total of 42 proteins were found to be enriched (\(\geq\)2-fold) over background (Figure 7C and Table S5). These proteins belonged to different functional groups: protein-folding chaperones (HSP90 AA1, HSPA5, HSPA1B, HSPA8, 14-3-ss/g/e/z/d), lamin binding (HNRNPK, PKP1), cell-cell junction organization (DSP, PKP1, ACTN4, JUP), translational elongation (EEF1A1, EEF2, EEF1G), glycolytic process (ALDOA, ENO1, PKM, GAPDH) and intermediate filament-based process (DSP, PLEC, PKP1, EVPL). Interactions of the most enriched proteins were confirmed by RNA immunoprecipitation (RIP) assay using specific and non-specific IgG antibodies (Figure 7D). Prelimin A/C was the most enriched protein by Inc-_Nr6a1-1_ probes. The subcellular location of Inc-_Nr6a1_ isoforms using _Hprt_, _U3_ and _Xist_ RNAs as indicators for cytosolic and soluble and insoluble nuclear reactions, respectively, resulted in nuclear enrichment located mainly in the chromatin fraction (Figure 7E). Since a significant proportion of Inc-_Nr6a1-1_ was located at the nuclear insoluble fraction and one of the major components of this fraction is chromatin, we studied whether Inc-_Nr6a1-1_ isoform could be interacting with DNA and modulating gene expression. The genome-wide binding profile of Inc-_Nr6a1-1_ was mapped using the ChIP technique (chromatin isolation via RNA immunoprecipitation) [19]. This technique allows the discovery of RNA-associated DNA sequences and mapping of genomic binding sites of chromatin-associated lncRNAs with high sensitivity and low background. Target lncRNA from glutaraldehyde electrophysiological cells was affinity captured using antisense-biotinylated oligonucleotides, and the IncRNA-associated DNA chromatin used to create a sequencing library. Under the conditions used for ChIP, more than 30% of the Inc-_Nr6a1-1_ RNA was retrieved from the control cells. Results of ChIP-seq analysis, using _Inc-Nr6a1_-depleted cells as the negative control, showed that there were no specific DNA binding sites for the Inc-_Nr6a1-1_ isoform. Thus, we concluded that nuclear Inc-_Nr6a1-1_ molecules bind Lamin A/C in regions not associated with the genome. Lamins associate with chromatin through large domainstermed lamina-associated domains or LADs, which contain mostly inactive genes and are located at the nuclear periphery [20]. Since Inc-_Nr6a1-1_ isoform interacted with Lamin A/C, but not with chromatin, we wondered if the depletion of _Lnc-Nr6a1_ could affect the specific Lamin A/C-associated genomic domains (LADs). Genomic regions that interact with Lamin A/C in the control and _Lnc-Nr6a1_-depleted cells were mapped by chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq).