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

NF-?B-mediated signaling is maintained silent by the action of I?B proteins, whose canonical role is to sequester NF-?B in the cytoplasm. An alternative chromatin role for I?B members have been shown to affect stemness and cell differentiation but the involvement of NF-?B in this function has not been excluded. NFKI-1 and IKB-1 are I?B homologs in Caenorhabditis elegans, which lacks NF-?B 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 I?B inhibitors may exert nuclear functions regulating of gene expression and development. Overall design: To study chromatin alterations in nfki-1(cer1) and ikb-1(nr2027) mutants, we performed ChIP-seq for H3K27me3 and H3K36me3 chromatin marks at L4 stage (36 hours at 25ºC after synchronization, from the same worm extracts used for RNA-seq). cer1 allele is a CRISPR-generated 368 bp deletion near nfki-1 exon 2. 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 as a wildtype control. To detect direct chromatin targets of nfki-1 and ikb-1, we performed ChIP-seq at L1 (5 hours fed 3 hours at 25ºC starved after synchronization) on the endogenous tagged strain CER425[ikb-1(syb267[ikb-1::mCherry])I;nfki-1(cer102[nfki-1::3xFLAG)])X], using 3 independent biological replicates per strain. 50 bp single-end sequencing was done on an Illumina HiSeq 2000.