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• W3135788595 abstract "Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The canonical Wnt pathway transcriptional co-activator β-catenin regulates self-renewal and differentiation of mammalian nephron progenitor cells (NPCs). We modulated β-catenin levels in NPC cultures using the GSK3 inhibitor CHIR99021 (CHIR) to examine opposing developmental actions of β-catenin. Low CHIR-mediated maintenance and expansion of NPCs are independent of direct engagement of TCF/LEF/β-catenin transcriptional complexes at low CHIR-dependent cell-cycle targets. In contrast, in high CHIR, TCF7/LEF1/β-catenin complexes replaced TCF7L1/TCF7L2 binding on enhancers of differentiation-promoting target genes. Chromosome confirmation studies showed pre-established promoter–enhancer connections to these target genes in NPCs. High CHIR-associated de novo looping was observed in positive transcriptional feedback regulation to the canonical Wnt pathway. Thus, β-catenin’s direct transcriptional role is restricted to the induction of NPCs, where rising β-catenin levels switch inhibitory TCF7L1/TCF7L2 complexes to activating LEF1/TCF7 complexes at primed gene targets poised for rapid initiation of a nephrogenic program. Introduction The now classical model of canonical Wnt signaling invokes two transcriptional states (Wiese et al., 2018; Steinhart and Angers, 2018). In the absence of Wnt ligand, HMG box family Tcf transcription factors bind enhancers of Wnt target genes recruiting co-repressors (Tle, Ctbp, and others) to silence target gene expression. In the cytoplasm, the transcriptional co-activator β-catenin is phosphorylated by an axin/GSK3β-dependent β-catenin destruction complex, resulting in ubiquitin-mediated proteasomal degradation. Upon Wnt ligand binding to Fzd receptor/Lrp co-receptors on the cell surface, the β-catenin destruction complex is sequestered to the activated receptor protein complex through axin interactions, removing β-catenin from GSK3β-directed, phosphorylation-mediated degradation (Schaefer and Peifer, 2019). As a result of increasing β-catenin levels, β-catenin is free to associate with TCF/LEF DNA binding partners, activating Wnt target gene transcription (Mosimann et al., 2009). While evidence suggests that all four mammalian Tcf family members are able to functionally interact with both Tle family co-repressors and β-catenin (Brantjes et al., 2001), a variety of studies in a range of biological systems indicate that Tcf7l1 predominantly acts as a repressor, Tcf7l2 as a context-dependent activator or repressor, and Tcf7 and Lef1 as transcriptional activators, of Wnt target gene expression (Lien and Fuchs, 2014). The adult (metanephric) mammalian kidney arises from distinct cell populations within the intermediate mesoderm (McMahon, 2016). All nephrons – the repeating functional unit of the kidney – arise from a small pool of a few hundred nephron progenitor cells (NPCs) established at the onset of kidney development (Short et al., 2014; Kobayashi et al., 2008). The subsequent balance in the maintenance, expansion, and commitment of NPCs is critical to ensuring a full complement of nephrons, approximately 14,000 in the mouse and 1 million in the human kidney (Bertram et al., 2011). A reduced nephron endowment has been associated with abnormal kidney function and disease susceptibility (Luyckx and Brenner, 2010; McMahon, 2016; Bertram et al., 2011). The maintenance and expansion of NPCs are supported by Fgf, Bmp, and Wnt signals produced by NPCs or adjacent mesenchymal interstitial progenitor and ureteric epithelial cell types (McMahon, 2016). Within this nephrogenic niche, ureteric epithelium-derived Wnt9b is thought to act on NPCs in a β-catenin-dependent transcriptional process to regulate NPC target gene expression and expansion of the nephron progenitor pool (Karner et al., 2011). The removal of Wnt9b from the ureteric epithelium and NPC-specific production of β-catenin also results in the failure of NPC differentiation (Carroll et al., 2005), whereas chemical inhibition of GSK3β (Davies and Garrod, 1995; Kuure et al., 2007), or genetic activation within NPCs of a β-catenin form insensitive to GSK phosphorylation-mediated proteasomal degradation, leads to Wnt9b-independent ectopic induction of differentiation-promoting gene targets (Park et al., 2007). Genetic and chemical modification of Wnt pathway components suggest different levels of β-catenin distinguish maintenance and commitment of NPCs (Ramalingam et al., 2018). Genomic analysis of β-catenin engagement at TCF/LEF recognition motifs within enhancers linked to genes driving NPC differentiation (Park et al., 2012), and subsequent transgenic studies demonstrating TCF/LEF-dependent activity of cis regulatory elements, provides strong evidence for a canonical Wnt/β-catenin/Tcf regulatory axis (Mosimann et al., 2009). Thus, canonical Wnt signaling directs opposing NPC programs: maintenance and expansion of uncommitted NPCs and their commitment to nephron formation. In this study, we employed an in vitro model to investigate the genomic regulatory mechanisms underlying the diverse action of canonical Wnt signaling in NPC programs. In this system, maintenance and expansion of NPCs, or their commitment to a nephrogenic program, are controlled by varying levels of CHIR99021 (CHIR) (Cohen and Goedert, 2004) supplemented to a chemically defined nephron progenitor expansion medium (NPEM) (Brown et al., 2015). CHIR binding to Gsk3β inhibits Gsk3β-mediated phosphorylation and proteasomal degradation of β-catenin (Yost et al., 1996; Aberle et al., 1997). Analysis of chromatin interactions and TCF/LEF factor engagement at DNA targets supports a model where β-catenin levels act as a key regulatory switch to modify TCF/LEF complex engagement at DNA targets and commitment of NPCs to a nephron-forming program. Results Elevated CHIR levels mediate a rapid inductive response in mouse NPCs A low level of CHIR (1.25 µM) is an essential component in NPEM medium supporting the expansion of NPCs while maintaining the nephron-forming competence (Brown et al., 2015). Within 3 days of elevating CHIR levels (3 µM), aggregate NPC cultures show a robust signature of nephron differentiation (Brown et al., 2015; Ramalingam et al., 2018). To develop this system further for detailed molecular characterization of CHIR/β-catenin-directed transcriptional events, we collected NPCs from E16.5 embryonic kidneys by magnetic-activated cell sorting (MACS; Brown et al., 2015). NPCs were cultured in NPEM supplemented with a maintenance level of CHIR (1.25 µM – low CHIR throughout) to promote self-renewal of NPCs. CHIR levels were then titrated to determine an effective concentration for a rapid activation of early target genes of NPC commitment, mirroring in vivo responses. As expected, low CHIR conditions maintained Six2, a key determinant of the NPC state, but did not induce expression of Jag1 (Figure 1A–C, Figure 1—figure supplement 1B), a Notch pathway ligand activity at early stage of mouse and human NPC commitment (Georgas et al., 2009; Lindström et al., 2018b). A significant increase of cellular and nuclear β-catenin (Figure 1A, B and Figure 3—figure supplement 1) was observed in 5 µM CHIR (‘high CHIR’ throughout), along with a strong inductive response: Six2 protein level persisted, but there was a robust induction of Jag1 (Figure 1A–C), mirroring early inductive events in the pre-tubular aggregate and renal vesicle in vivo (Lindström et al., 2018b; Mugford et al., 2009; Georgas et al., 2009; Xu et al., 2014a). We adopted this induction condition throughout the study. Figure 1 with 1 supplement see all Download asset Open asset Nephron progenitor expansion medium (NPEM) supplemented with differential levels of CHIR99021 (CHIR) models nephron progenitor cell maintenance or differentiation in a plate. (A) Immunofluorescence (IF) staining showing expression level of Six2, non-phospho (NP) β-catenin and Jag1 in nephron progenitor cell (NPC) cultured in NPEM supplemented with various CHIR dosages. (B) Relative intensity of IF signals from individual cells in experiment associated with A. (C) Heatmap/hierarchical cluster of expression levels of NPC signature, self-renewal, and differentiation marker genes. (D) Top five enriched Gene Ontology (GO) terms of indicated differentially expressed gene lists, analyzed by DAVID. Link to high-definition figure: https://www.dropbox.com/s/76chyrs07m0toen/Fig%201.pdf?dl=0. Next, we sought to systematically characterize gene expression profiles of NPCs in low and high CHIR conditions by mRNA-seq. Additionally, to explore the effect of low CHIR on NPCs, we generated data removing CHIR from the culture (‘No CHIR’ throughout) (Figure 1—figure supplement 1A). We also examined freshly isolated NPCs prior to culture. Low CHIR maintains expression of transcriptional regulators required for NPC specification and/or maintenance, including Pax2, Wt1, Hoxa/d11, and Sall1 (Figure 1C and Supplementary file 1; McMahon, 2016). In contrast, high CHIR led to a downregulation of regulators and markers of self-renewing NPCs, including Six2 (Self et al., 2006), Cited1 (Mugford et al., 2009), Osr1 (Xu et al., 2014a), and Eya1 (Xu et al., 2014b), and a concomitant increase in expression of genes associated with induction of nephrogenesis, such as Wnt4, Jag1, Lhx1, and Fgf8 (Figure 1C; Park et al., 2007). Trends in gene expression from mRNA-seq were confirmed by RT-qPCR analysis (Figure 1—figure supplement 1B). To examine biological processes at play in different NPC culture conditions, we performed Gene Ontology (GO) enrichment analysis of differentially expressed genes (differential expression analysis described in 'Materials and methods') with DAVID (Huang et al., 2009). Comparing input NPCs freshly isolated from the kidney cortex with NPC-free cortical preparations (NFC), the strong enrichment for NPC-relevant GO terms (Figure 1D, top panel) was consistent with a strong enrichment of Six2+ NPCs (more than 90% of isolated cells were Six2+). When NPCs cultured in high versus low CHIR conditions were compared, a strong enrichment was observed in terms associated with Wnt signaling pathway, as expected, on CHIR-mediated NPC induction (Figure 1D, middle and bottom panels). Although primary NPCs, and their counterpart in low CHIR, showed similar expression patterns for NPC self-renewal and differentiation markers, transcriptome-wide comparison of all samples clustered primary NPCs into a distinct group from NPCs cultured in either low or high CHIR (Figure 1—figure supplement 1C). This is explained by a pronounced metabolic shift in culture where there is a strong enrichment in GO analysis in metabolic processes such as sterol biosynthesis (Figure 1—figure supplement 1D). In addition, freshly isolated NPCs showed a low-level inductive signature reflecting a co-contribution of small numbers of early induced NPCs (expression of Fgf8, Wnt4, Lhx1, Heyl, Bmp4, Mafb, and Podxl, Figure 1C and Supplementary file 1). Within 24 hr, low CHIR culture stabilized an undifferentiated NPCs signature with the downregulation of induction markers (Figure 1C, Figure 1—figure supplement 1B, and Supplementary file 1). Importantly, these data show that NPEM culture establishes a more rigorous model for distinguishing uninduced versus induced NPC responses than is possible with the intrinsic heterogeneity within primary isolates of NPC populations. CHIR-mediated induction modifies the epigenomic profile of NPCs To investigate the chromatin landscape regulating NPCs, we integrated chromatin accessibility through ATAC-seq analysis (Buenrostro et al., 2013) with chromatin immunoprecipitation studies examining active (H3K27ac ChIP-seq) and repressive (H3K27me3 ChIP-seq) chromatin features, RNA Pol II recruitment (RNA Pol II Ser5P ChIP-Seq), and RNA-seq expression profiling (Figure 1—figure supplement 1A). Initially, we evaluated enhancers previously validated in transgenic studies (Park et al., 2012) associated with Six2 expression in uncommitted NPCs (Six2 distal enhancer; Six2DE) and Wnt4 activation on NPC differentiation (Wnt4 distal enhancer; Wnt4DE). In low CHIR conditions, the Six2DE shows an open and active configuration: an ATAC-seq peak, flanked by H3K27ac peaks with Pol II engagement at the enhancer and within the gene body. In high CHIR, ATAC-seq, H3K27ac, and Pol II ChIP-Seq signals were reduced, correlating with the downregulation of gene expression (Figure 2A). As predicted, the Wnt4DE displayed an opposite trend in the shift from low to high CHIR: the ATAC and H3K27ac ChIP signals increased together with increased Pol II engagement in the gene body. Surprisingly, a marked enhancer-specific Pol II ChIP-seq signature was visible in low CHIR NPC maintenance conditions and reduced on initiation of active transcription in high CHIR (Figure 2B). Figure 2 with 1 supplement see all Download asset Open asset High dosage of CHIR99021 (CHIR) triggered change of nephron progenitor cell (NPC) epigenome. (A, B) Genome browser view of RNA-Seq, ATAC-Seq, as well as Six2, H3K27ac, H3K27me3, and Ser5P ChIP-Seq data near Six2 (A) and Wnt4 (B) in low CHIR (left) and high CHIR (right) conditions. Black arrow indicates Six2DE and Wnt4DE, respectively. (C) Display of differentially accessible regions (DARs) generated by the indicated comparisons. (Left) Heatmaps showing log2 normalized read counts of top 500 most significant DARs; (middle) top five most significant Gene Ontology (GO) terms associated with the DARs; (right) top five most enriched motifs discovered de novo in the DARs; * indicates less well-conserved motifs for the factor. NFC: NPC-free cortex. Having validated the data sets at target loci, we examined the data sets more systematically for broad features of epigenetic regulation. We focused on the differentially accessible regions (DARs) enriched in uncultured NPCs relative to NFC as they reflect a general NPC-specific signature. NPC-specific DARs were significantly enriched in transcription factor binding sites for Six, Pax, and Hox factors consistent with the critical roles of Six1/2, Pax2, and Hox11 paralogues in NPC programs (Figure 2C; Naiman et al., 2017; Self et al., 2006; Wellik et al., 2002). Functionally, GO-GREAT analysis (McLean et al., 2010) predicted that these regions were enriched near genes linked to kidney development (Figure 2C). Hierarchical clustering of ATAC-Seq data was used to examine the relationship between CHIR dosage and the open chromatin landscape identifying DARs in NPCs cultured in low and high CHIR conditions (Figure 2—figure supplement 1A). Most of the DARs are distal to the transcriptional start site (TSS) of genes, indicative of enhancer elements (Figure 2—figure supplement 1B). The top two motifs identified in high CHIR-specific DARs were the Jun (AP-1) and TCF/LEF motifs (Figure 2C), supporting a β-catenin-driven increase in accessibility through engagement with TCF/LEF factors. Phosphorylated Jun has been detected in both NPCs and differentiates renal vesicles (Muthukrishnan et al., 2015). Further, Jun binds Tcf7l2 to cooperatively activate gene expression in Wnt-dependent intestinal tumors (Nateri et al., 2005). Elevated expression of Jun, Junb, and Jund in high CHIR (Supplementary file 1) suggests a potential interplay of Jun family members with β-catenin/Tcf-driven NPC differentiation. Statistical assessment (Supplementary file 2) determined that the Wnt4DE was significantly more accessible in high CHIR versus low CHIR conditions (peak ID 16567, adjusted p-value=0.02). The Six2DE showed a trend of greater accessibility in low CHIR versus high CHIR condition (peak ID 10164, adjusted p-value=0.13). These observations are consistent with differences in ATAC-Seq data comparing FACS-purified E16 and P2 Six2GFP+ cells, where P2 NPCs are thought to have enhanced differentiation capability (Hilliard et al., 2019). Differential expression and DNA binding of TCF/LEF family members in the regulation of NPC programs TCF/LEF factors directly bind to DNA and mediate the transcriptional response elicited by Wnt/β-catenin. Studies in other developmental systems have generally documented repressive roles for Tcf7l1 and Tcf7l2, and activating roles for Tcf7 and Lef1 (Lien and Fuchs, 2014). To examine the role of TCF/LEF factors directly in NPC maintenance and differentiation, we characterized expression of each of the four members (Tcf7l1, Tcf7l2, Tcf7, and Lef1). Of the four genes, Tcf7l1, Tcf7l2, and Tcf7 transcripts were expressed at low (2–10 TPM; Tcf7l2 and Tcf7) or moderate (50 TPM; Tcf7l1) levels in low CHIR NPC maintenance conditions. CHIR-mediated induction of NPCs resulted in a significant downregulation of Tcf7l1 expression, while expression of both Tcf7 and Lef1 was markedly upregulated (Figure 3A and Figure 1—figure supplement 1A,B'). The same general trend was observed examining the level of each protein in the nucleus of NPCs through quantitative immunofluorescence (Figure 3B, C) and western blot (Figure 3—figure supplement 1) analyses. Interestingly, there is a significant decrease in Tcf7l2 protein but not transcript level in high CHIR versus low CHIR condition, suggesting transcriptionally independent regulation of Tcf7l2 activity. To compare in vitro findings with NPCs in vivo, single-cell RNA-seq (scRNA-Seq) transcriptional profiles were examined in cells isolated from E16.5 kidney cortex. In agreement with in vitro data, Tcf7l1 transcripts were enriched in self-renewing NPCs while Lef1 levels were elevated in differentiated NPCs, though Tcf7 and Tcf7l2 expression levels were relatively low, with little variation between non- and early induced NPC states (Figure 3—figure supplement 2C–F). Figure 3 with 2 supplements see all Download asset Open asset Differential expression of TCF/LEF family transcription factors in nephron progenitor cell (NPC) in response to distinct level of CHIR99021 (CHIR). (A) Bar plots showing RNA-Seq measured expression levels of TCF/LEF family factors in NPC cultured in nephron progenitor expansion medium (NPEM) culture supplemented with various concentrations of CHIR. (B) Immunofluorescence (IF) staining of TCF/LEF family factors in NPEM cultured with conditions indicated. (C) Relative intensity of IF signals from individual cells in experiment associated with B. Link to high-definition figure: https://www.dropbox.com/s/pvhu1ffhoujt39p/Fig%203.pdf?dl=0. NFC: NPC-free cortex. To directly address TCF/LEF target interactions and β-catenin-mediated regulation of NPCs, we generated Lef1, Tcf7, Tcf7l1, Tcf7l2, and β-catenin ChIP-Seq data sets from freshly isolated, uncultured NPCs, and NPCs cultured in low and high CHIR (Figure 1—figure supplement 1A). Further, given the key role for Six2 in NPC maintenance and evidence supporting Six2 engagement in Tcf7l2 and β-catenin containing complexes (Park et al., 2012), we collected Six2 ChIP-seq data sets in the same conditions. Motif discovery of TCF/LEF/β-catenin ChIP-seq binding sites shows highest DNA motif enrichment as the TCF/LEF binding element, indicating a high specificity of the data sets and supporting direct TCF/LEF/β-catenin target interactions (Figure 4—figure supplement 1C). In addition, a Hox motif is highly enriched in TCF/LEF binding sites in both low CHIR and high CHIR conditions consistent with Hox11 paralog regulation of the NPC state (Wellik et al., 2002; Park et al., 2012). A Runx motif is enriched in TCF/LEF binding sites in high CHIR. Runx1 expression is upregulated in the same condition (Supplementary file 1), but the significance of possible Runx-dependent regulation remains to be determined. Consistent with the different levels of each protein in low and high CHIR conditions, Tcf7l1 engagement at DNA targets was reduced on NPC induction, while Tcf7, Lef1, and β-catenin showed a marked increase in DNA bound sites (Figure 4—figure supplement 1A). In both low and high CHIR conditions, β-catenin association overlapped extensively with the binding of cognate TCF/LEF factors specifically enriched in each condition (Figure 4—figure supplement 1B). In either CHIR condition, motif recovery suggests direct engagement through TCF/LEF binding sites (Figure 4—figure supplement 1C). This is consistent with previous findings in murine intestinal studies that localization of β-catenin is primarily dependent on TCF/LEF factors (Schuijers et al., 2014). Notably, hierarchical clustering indicates that the general feature of TCF/LEF factor binding in low CHIR and high CHIR was distinct and determined by the condition as different TCF/LEF factors targeted common genomic regions that differ between low CHIR and high CHIR conditions (Figure 4—figure supplement 1D). This observation was most evident examining Tcf7l2. Tcf7l2 mRNA and protein levels did not vary greatly between low and high CHIR conditions but Tcf7l2 DNA interactions differed significantly (Figure 4—figure supplement 1D). Our previous studies identified (Park et al., 2012) and functionally validated (O'Brien et al., 2018) a Wnt4 distal enhancer (Wnt4DE, Figure 2B) driving Wnt4 expression in response to NPC induction. This enhancer was shown to interact with Six2, Hoxd11, Osr1, and Wt1, critical determinants of the NPC state (O'Brien et al., 2018). Our data demonstrates that Tcf7l1 and Tcf7l2 bind to the Wnt4DE in low CHIR condition but were replaced by Tcf7 and Lef1 in high CHIR conditions (Figure 4A). This switch in TCF/LEF factor binding correlated with activation of Wnt4 expression, consistent with a potential repressive role for Tcf7l1/Tcf7l2 interactions and activating role for Tcf7/Lef1 engagement (Figure 4A). Interestingly, β-catenin was engaged at the Wnt4DE in low CHIR condition where Wnt4 was transcriptionally silent, though β-catenin binding at this enhancer was increased on high CHIR induction of NPCs (Figure 4A). In summary, though elevating β-catenin levels through high CHIR stabilization of GSK3β lead to some increase in binding of β-catenin at the Wnt4 enhancer, a marked switch in the binding signature of TCF/LEF factors is a more striking correlation with subsequent activation of Wnt4 transcription. Figure 4 with 3 supplements see all Download asset Open asset Increased CHIR99021 (CHIR) dosage induces a switch of TCF/LEF factors binding to the genome. (A, B) Genome browser view of Wnt4 enhancer locus showing ChIP-Seq signal of TCF/LEF factors in low and high CHIR conditions. (C) Histograms showing binding intensity of TCF/LEF factors and chromatin markers on the three sets of TCF/LEF binding sites assigned by overlap of low CHIR Tcf7l1 and high CHIR Lef1 binding sites. (D) Result from de novo motif discovery of the three sets of TCF/LEF binding sites described in A. (E) Top Gene Ontology (GO) terms associated with the corresponding sets of TCF/LEF binding sites shown in B. (F) Percentage of TCF/LEF target genes belonging to different sets in differential expressed genes specific to low CHIR or high CHIR conditions as described in Figure 1. (G) Venn diagram showing overlap of set 2 and 3 target genes assigned by GREAT (McLean et al., 2010). (H) Histograms showing quantification of reads from the indicated data sets in ±2 kb of β-catenin binding sites in high CHIR condition. As Tcf7l1 and Lef1 binding best distinguished distinct NPCs’ states, we performed a more extensive analysis of these factors. Comparing DNA regions bound by Tcf7l1 in low CHIR and those bound by Lef1 in high CHIR, we observed a significant overlap (p-value = 1e-569), though over half were unique to a data set (Figure 4C). We assigned the Tcf7l1/low CHIR-specific sites (see Supplementary file 3) as set 1 (also ‘lost,’ as no longer bound by TCF/LEF in high CHIR condition), overlapping sites as set 2 (also ‘switch,’ as TCF/LEF factors interchange at these sites between low and high CHIR conditions), and Lef1/high CHIR-specific sites as set 3 (also ‘de novo,’ as TCF/LEF binding sites arose on elevated CHIR levels). Interestingly, examining motif recovery across sets 1–3 indicated that only sets 2 and 3 showed a strong prediction for direct TCF/LEF binding (Figure 4D). Thus, Tcf7l1 likely associates with a large number of DNA regions without direct DNA binding at TCF/LEF response elements. The majority (73%) of Tcf7l1 sites bound in low CHIR with a predicted TCF/LEF motif overlapped with those bound by Lef1 in high CHIR (Figure 4—figure supplement 2A). In contrast, only 29% of Tcfl71 binding sites without a TCF/LEF motif prediction overlapped with Lef1 bound regions (Figure 4—figure supplement 2A). Thus, switching from a Tcf7l1 to a Lef1-centered DNA interaction at TCF/LEF response elements was a general feature of NPC induction response. Comparing with set 3 (de novo), set 2 (switch) sites displayed stronger binding of Tcf7l2, Tcf7, Lef1, and β-catenin, as well as higher level of markers of activated chromatin (ATAC-Seq and H3K27ac ChIP-Seq) in high CHIR condition (Figure 4C). Thus, the data suggests that TCF/LEF sites occupied by Tcf7l1 in low CHIR are poised for stronger binding (and therefore activation) by TCF/LEF activators in high CHIR condition. Interestingly, GO GREAT analysis of all three sets predicted expected kidney terms such as ‘metanephric nephron morphogenesis’ or ‘renal vesicle morphogenesis’ (Figure 4E), consistent with biologically relevant interactions, arguing against artifactual responses to culture or CHIR treatment. More than half of the potential target genes (assigned bioinformatically by GREAT) of set 2 ‘switch’ sites overlap with those of set 3 ‘de novo’ sites (60%; Figure 4G, p=5.9e-627), although the latter are much broader. For this overlapping group, pre-engagement by Tcf7l1 may increase the likelihood of additional engagement in neighboring regions of DNA by Lef1 and potentially reinforce transcriptional input into a common gene target. Examining chromatin features and RNA Pol II engagement, we observed stronger enrichment of active enhancer markers (H3K27ac and RNA Pol II) in low CHIR on set 1 sites predicting indirect Tcf7l1 engagement than set 2 sites, where Tcf7l1 was predicted to bind DNA directly through TCF/LEF motifs (Figure 4—figure supplement 2E). Thus, direct binding of TCF7L1/TCF7L2 on set 2 sites was associated with putative enhancers in a less active chromatin state in low CHIR condition. Consistent with this view, a direct analysis of the expression of target gene set of set 2 sites showed the highest expression in high CHIR conditions (Figure 4F), suggesting them tending to function in high CHIR. To examine whether elevated β-catenin correlates with chromatin activation, we examined change of active chromatin markers (ATAC-Seq and H3K27ac ChIP-Seq) and RNA Pol II loading on β-catenin bound sites. A dramatic elevation of all these signals in high CHIR condition (Figure 4H) is consistent with β-catenin engagement enhancing an open chromatin, active enhancer signature. Together, the data support the conclusion that high CHIR increased β-catenin association, correlating with switching of TCF/LEF factors at existing active chromatin, de novo opening of new chromatin sites, and an active transcription program initiating nephron formation. β-Catenin uses both pre-established and de novo enhancer–promoter loops to drive NPC differentiation program To examine genome organization and chromatin interactions at a higher level in self-renewing and induced NPC, Hi-C analysis (Rao et al., 2014) was performed to characterize global chromatin–chromatin interactions. Further, as CTCF is known to mediate long-range chromatin interactions common across cell types, we integrated a published CTCF ChIP-seq data set generated from NPCs isolated directly from the developing kidney (O'Brien et al., 2018). Analysis of two Hi-C data sets, using HiCCUPS within Juicer Tools (Durand et al., 2016), replicated 19,494 low CHIR and 20,729 high CHIR chromatin–chromatin interaction loops (Figure 5—figure supplement 1A). Almost half of all loops (40% low CHIR; 44% high CHIR), including those anchored on TSSs (35% low CHIR; 49% high CHIR), were unique to each culture condition (Figure 5B and Supplementary file 4). Given a focus on chromatin loops responding to β-catenin-mediated induction of NPCs, we examined loops anchored on β-catenin peaks identified in high CHIR conditions. Of the 5530 β-catenin-associated sites in high CHIR condition, 28% (1573) were located in loop anchors; 41% (647) of these connected to a TSS. Of the 647 peaks looped to TSSs, 57% (371) were connected by ‘conserved’ loops, shared between low CHIR and high CHIR conditions, the remainder appeared de novo under high CHIR conditions (Figure 5A). Figure 5 with 1 supplement see all Download asset Open asset β-Catenin activates gene expression through both stable and de novo enhancer–promoter loops. (A) β-Catenin binding sites that overlap with an anchor of chromatin loop in high CHIR99021 (CHIR), the proportion that connects to a transcriptional start site (TSS) (gray in the pie chart) and segregation between two types of loops defined in B. (B) Overlap of chromatin loops between low CHIR and high CHIR conditions. Examples (C, E) and schematics (D, F) of β-catenin utilizing low/high CHIR-shared enhancer–promoter loops to activate Wnt4 (C, D) or high CHIR-specific loops to activate Lef1 (E, F). Black arrow at the bottom indicates the β-catenin binding sites involved in the loops. To understand the biological consequences of these regulatory events, we identified promoters connected to β-catenin-bound enhancers (Supplementary file 5). Among those connected by conserved loops present in both CHIR" @default.
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• W3135788595 title "Decision letter: A β-catenin-driven switch in TCF/LEF transcription factor binding to DNA target sites promotes commitment of mammalian nephron progenitor cells" @default.
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