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

核因子κB（NF-κB）介导的信号通路通常通过κB抑制蛋白（IκB）的作用维持沉默状态，IκB的经典功能是将NF-κB隔离于细胞质基质中。已有研究证实，IκB家族成员具备另一项染色质相关功能，可影响细胞干性与细胞分化，但NF-κB是否参与该功能尚未被排除。NFKI-1与IKB-1是秀丽隐杆线虫（Caenorhabditis elegans）中的IκB同源蛋白，该物种本身缺乏NF-κB核效应因子。nfki-1与ikb-1突变体存在发育缺陷，其表型模拟多梳基因以及脱甲基酶基因utx-1的突变，这提示秀丽隐杆线虫的IκB蛋白参与染色质调控，该结论得到多条证据的支持：(i) 我们在细胞核中检测到NFKI-1；(ii) NFKI-1与IKB-1可结合组蛋白与多梳蛋白；(iii) NFKI-1与IKB-1在体内可结合染色质；(iv) nfki-1与ikb-1的突变会改变染色质修饰标记。由此可见，古老的IκB抑制蛋白可能通过发挥核功能调控基因表达与个体发育。为研究nfki-1(cer1)与ikb-1(nr2027)突变体中的染色质改变，我们在L4期（同步化后于25℃培养36小时）对H3K27me3与H3K36me3两种染色质修饰标记进行染色质免疫共沉淀测序（ChIP-seq），所用线虫提取物与RNA测序（RNA-seq）实验的提取物一致。cer1等位基因是通过成簇规律间隔短回文重复序列相关系统（CRISPR）构建的，在nfki-1外显子2附近存在368 bp的缺失突变。nr2027是ikb-1的1911 bp缺失突变，其缺失序列包含锚蛋白重复结构域的编码区（Pujol等，2001）。N2（布里斯托品系）作为野生型对照。为检测nfki-1与ikb-1的直接染色质靶标，我们在L1期（同步化后先喂食5小时，随后于25℃饥饿3小时）对内源标记菌株CER425[ikb-1(syb267[ikb-1::mCherry])I;nfki-1(cer102[nfki-1::3xFLAG])]X进行ChIP-seq，每个菌株设置3次独立生物学重复。测序采用Illumina HiSeq 2000平台，进行50 bp单端测序。