Dynamic binding of RBPJ is determined by Notch signaling status

Abstract

Notch signaling plays crucial roles in mediating cell fate choices in all metazoans largely by specifying the transcriptional output of one cell in response to a neighboring cell. The DNA-binding protein RBPJ is the principle effector of this pathway in mammals and, together with the transcription factor moiety of Notch (NICD), regulates the expression of target genes. The prevalent view presumes that RBPJ statically occupies consensus binding sites while exchanging repressors for activators in response to NICD. We present the first specific RBPJ chromatin immunoprecipitation and high-throughput sequencing study in mammalian cells. To dissect the mode of transcriptional regulation by RBPJ and identify its direct targets, whole-genome binding profiles were generated for RBPJ; its coactivator, p300; NICD; and the histone H3 modifications H3 Lys 4 trimethylation (H3K4me3), H3 Lys 4 monomethylation (H3K4me1), and histone H3 Lys 27 acetylation (H3K27ac) in myogenic cells under active or inhibitory Notch signaling conditions. Our results demonstrate dynamic binding of RBPJ in response to Notch activation at essentially all sites co-occupied by NICD. Additionally, we identify a distinct set of sites where RBPJ recruits neither NICD nor p300 and binds DNA statically, irrespective of Notch activity. These findings significantly modify our views on how RBPJ and Notch signaling mediate their activities and consequently impact on cell fate decisions.

Notch1 maintains dormancy of olfactory horizontal basal cells, a reserve neural stem cell

“…FIMO (find individual motif occurances) scanning using the generally acknowledged consensus binding motif for RBPJ (41,42) revealed not only scattered binding sites far upstream, but also two distinct clusters, one located 250 bp upstream from the TSS and a smaller one directly at the TSS, supporting direct RBPJ regulation of the ΔN-p63 locus. In addition, analysis using a longer consensus binding motif that enriches for sites of coincident RBPJ/NICD binding (42) predicted two such sites in the more upstream area, consistent with published reports suggesting that in general, NICD/RBPJ binding occurs further away from the TSS, whereas NICD-independent RBPJ binding is more enriched closer to the TSS (Fig.…”

SpDamID: Marking DNA Bound by Protein Complexes Identifies Notch-Dimer Responsive Enhancers

“…oPossum analysis showed that 1622 out of 2403 Notch dependent open chromatin identified by FAIRE in mK4 cells have a Runx1 motif within 100bp of a RBP motif (Z-score of 176.331). Similarly, "dynamic" RBP ChIPed sites identified in C2C12 cells (Castel et al, 2013) displayed greater labeling by mature complexes than by the N1/RBP immature complex ( Figure S6F). The strength of this approach is demonstrated by the ability of SpDamID to distinguish monomeric from dimeric binding, and detect DNA-bridged DAM complementation when noninteracting factors (Notch and Runx1) bind simultaneously to the same enhancer, a nontrivial tasks with antibody-based methodologies.…”

“…It has been observed that some sites in the genome are occupied only when Notch signaling is active ("dynamic sites"; (Bray and Bernard, 2010;Castel et al, 2013;Housden et al, 2013;Krejci et al, 2009;Wang et al, 2014;Wang et al, 2011). These dynamic sites show increased recoverability by RBP ChIP when NICD is present, leading to the proposal that the NICD/RBP complex acts as a pioneer factor to modify chromatin at these sites (Castel et al, 2013).…”

“…These dynamic sites show increased recoverability by RBP ChIP when NICD is present, leading to the proposal that the NICD/RBP complex acts as a pioneer factor to modify chromatin at these sites (Castel et al, 2013). These reports led us investigate chromatin opening in response to Notch binding via FAIRE.…”

“…SpDamID-seq reads were mapped to the genomic locations of the FAIRE peaks (either total FAIRE sites or Notch-dependent FAIRE sites that open in response to Notch) or the RBP ChIP peaks that were induced by Notch signaling in C2C12 cells (Castel et al, 2013) using the bam coverage tool in the deeptools package of Galaxy. Heat maps and graphs of the SpDamID read coverage of these sites were generated using the computeMatrix and heatmapper tools of the deeptools package.…”