Ancestral function of Inhibitors-of-kappaB regulates Caenorhabditis elegans development (RNA-Seq)

NF-κB-mediated signaling is maintained silent by the action of IκB proteins, whose canonical role is to sequester NF-κB in the cytoplasm. An alternative chromatin role for IκB members have been shown to affect stemness and cell differentiation but the involvement of NF-κB in this function has not been excluded. NFKI-1 and IKB-1 are IκB homologs in Caenorhabditis elegans, which lacks NF-κB nuclear effectors. nfki-1 and ikb-1 mutants present developmental defects that phenocopy mutations in Polycomb genes and demethylases as utx-1. suggesting a role for C. elegans IKB proteins in chromatin regulation, which is supported by various lines of evidence:(i) we detected NFKI-1 in the nucleus; (ii) NFKI-1 and IKB-1 bind to histones and Polycomb proteins, (iii) NFKI-1 and IKB-1 bind to chromatin in vivo, and (iv) mutations in nfki-1 and ikb-1 alter chromatin marks . Thus, ancestral IκB inhibitors may exert nuclear functions regulating of gene expression and development. To study the transcriptional alterations in nfki-1 and ikb-1 mutants during animal development, we performed RNA-seq at two different timepoints: L1 (5 hours fed and 3 hours starved at 25ºC after synchronization, 1 replicate per strain) and L4 (36 hours at 25ºC after synchronization, 2 replicates per strain) developmental stages. We used different mutant strains for nfki-1 and ikb-1 to avoid mutant-specific alterations. cer1 allele is a CRISPR-generated 368 bp deletion near nfki-1 exon 2. cer2 is a 434 bp deletion and 50 bp insertion at exon 2. Both modifications generate premature stop codons. cer9 allele is a 462 bp deletion that includes the ATG start codon of ikb-1. nr2027 is a 1911 bp deletion of ikb-1 that includes the ankyrin-repeat domains encoding sequence (Pujol et al., 2001). N2(Bristol) was used for all analyses as wildtype (WT) control. 50 bp single-end sequencing was done on an Illumina HiSeq 2000.