Rfx2 Stabilizes Foxj1 Binding at Chromatin Loops to Enable Multiciliated Cell Gene Expression

Abstract

Cooperative transcription factor binding at cis-regulatory sites in the genome drives robust eukaryotic gene expression, and many such sites must be coordinated to produce coherent transcriptional programs. The transcriptional program leading to motile cilia formation requires members of the DNA-binding forkhead (Fox) and Rfx transcription factor families and these factors co-localize to cilia gene promoters, but it is not clear how many cilia genes are regulated by these two factors, whether these factors act directly or indirectly, or how these factors act with specificity in the context of a 3-dimensional genome. Here, we use genome-wide approaches to show that cilia genes reside at the boundaries of topological domains and that these areas have low enhancer density. We show that the transcription factors Foxj1 and Rfx2 binding occurs in the promoters of more cilia genes than other known cilia transcription factors and that while Rfx2 binds directly to promoters and enhancers equally, Foxj1 prefers direct binding to enhancers and is stabilized at promoters by Rfx2. Finally, we show that Rfx2 and Foxj1 lie at the anchor endpoints of chromatin loops, suggesting that target genes are activated when Foxj1 bound at distal sites is recruited via a loop created by Rfx2 binding at both sites. We speculate that the primary function of Rfx2 is to stabilize distal enhancers with proximal promoters by operating as a scaffolding factor, bringing key regulatory domains bound by Foxj1 into close physical proximity and enabling coordinated cilia gene expression.

The Xenopus animal cap transcriptome: building a mucociliary epithelium

“…For each pairwise comparison, the method generates two lists of TFs predicted to control the transcriptome of the earlier (neg_rank list) or the later stage (pos_rank list), respectively ( Supplementary Table S11 ). From the group of confirmed epidermal TFs, this method predicts regulatory roles for E2F4/5 and KLF17 from the gastrula timepoint on, followed at the neurula stage by tp63, gata2, grhl1/3 and members of the Fox (foxj1/1.2, foxi1, foxa1, foxn4) and Rfx (rfx1, -2, -3) protein families, compatible with other reports ( 41 , 43 , 60 ).…”

“…However, the manual dissection of embryos inadvertently poses the risk of contaminating the tissue of interest with unwanted cells. Several studies in X. laevis have observed this problem, both with ACs and ectodermal explants ( 41 , 42 ). To obtain a quantitative estimate of such a contamination, we have measured by qRT/PCR the transcript levels of seven structural muscle genes, both in embryos and in the very same AC samples used for the RNA-Seq analysis.…”

“…We also included a core set of over 800 genes from X. laevis ACs, which have been linked to MCC differentiation by gain and loss of function approaches ( 41 ). This gene set is derived from experimentally maximized differences in mRNA abundance targetting MCC differentiation in ACs, while our analysis detects natural fluctuations of mRNA abundance for all epidermal cell types.…”

“…All these early factors are absent from the predicted TF network controlling the tailbud epidermis (Figure 9B ; Supplementary Table S9 , pos_rank list). Here, the algorithm selects several TFs, which have not been recognized before as epidermal regulators in Xenopus ( 41–43 ). Two of them are connected to epidermal differentiation only by expression (ELF1, KLF3; this paper and ( 28 )), but also the broadly expressed proto-oncogene c-fos belongs to this group.…”

Characterization of the human RFX transcription factor family by regulatory and target gene analysis

“…The presence of X-box motifs was shown to contribute to the activeness of both promoters and enhancers, whereby distal enhancers that harbor X-box motifs exhibited greater promoter activity than enhancers that lack them [ 76 ]. This phenomenon would fit a model where (as found in Xenopus leavis ) Rfx2 and Foxj1 coordinately regulate ciliary gene expression, with Rfx2 stabilizing Foxj1 binding at chromatin loops [ 77 ].…”