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Dimer Formation - Fx Dimer - Biology

Dimer Formation - Fx Dimer - Biology



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Dimer Formation - Fx Dimer

Single-molecule analysis reveals agonist-specific dimer formation of µ-opioid receptors

G-protein-coupled receptors (GPCRs) are key signaling proteins that mostly function as monomers, but for several receptors constitutive dimer formation has been described and in some cases is essential for function. Using single-molecule microscopy combined with super-resolution techniques on intact cells, we describe here a dynamic monomer–dimer equilibrium of µ-opioid receptors (µORs), where dimer formation is driven by specific agonists. The agonist DAMGO, but not morphine, induces dimer formation in a process that correlates both temporally and in its agonist- and phosphorylation-dependence with β-arrestin2 binding to the receptors. This dimerization is independent from, but may precede, µOR internalization. These data suggest a new level of GPCR regulation that links dimer formation to specific agonists and their downstream signals.


INTRODUCTION

Classic cadherins are a family of adhesion transmembrane receptors that are responsible for the structural integrity and the specific architecture of all solid tissues in vertebrates. Malfunctions in the cadherin adhesion system are often regarded as a factor in tumor cell invasion and metastasis (Takeichi, 1995 Provost and Rimm, 1999 Patel et al., 2003 Gumbiner, 2005). It is widely accepted that cell–cell adhesion is produced by the homodimerization of cadherin molecules exposed on opposing cells. This interaction obviously determines many critical parameters of cell–cell adhesion including its strength, plasticity, and stability. Although extensive work has been done to characterize the molecular details of cadherin adhesion interactions, many basic aspects of this process remain unknown.

The principal question that is yet to be answered is the strength of the individual cadherin adhesion bonds. The uncertainty arises from two contradictory groups of observations (reviewed in Troyanovsky, 2005 Mege et al., 2006). On one hand, numerous biophysical experiments with recombinant cadherin fragments have shown that the lifetime of such a bond is limited to the millisecond range. On the other hand, remarkably stable cadherin homodimers with an undetectable dissociation rate were demonstrated in cultured cells by coimmunoprecipitation experiments. Consequently, there are two principally different models of cadherin adhesion. A low-affinity cadherin adhesion model suggests that the strength of a cell–cell adhesive contact is mediated by the clustering of cadherin receptors via cytoplasmic interactions (Yap et al., 1998 Kusumi et al., 1999). According to this model, the clustering of the short-lived adhesive bonds provides the essential stability for the entire junction. Little is known, however, about the molecular details of cadherin clustering. Moreover, some data clearly contradict the “clustering-stability” hypothesis. For example, in some experiments E-cadherin mutants entirely lacking the intracellular region (and thus disconnected from the hypothetical intracellular clustering machinery) provided an adhesive force sufficient to aggregate cells in the aggregation assay (Ozawa and Kemler, 1998). Such data circumstantially support an alternative, high-affinity model of cadherin adhesion. By this model, the cell–cell adhesion is based on the continuous formation of high-affinity cadherin adhesive dimers, which dissociate under a strict cellular control (Troyanovsky et al., 2006). The most obscure aspect of the latter model is the mechanism of high-affinity cadherin dimerization: why was this process never detected in vitro? It is also not clear why, if adhesion is based on stable dimers, does cadherin recruitment into junctions depend on intracellular cadherin–catenin interactions?

The prime objective for this work was to clarify these two questions, very critical for high-affinity model of cadherin adhesion. We first asked whether stable cadherin dimers identical to those detected in cells could be assembled in vitro. To answer this question, we studied cadherin dimerization on the surface of agarose beads using a site-specific cross-linking assay. In complete agreement with the published in vitro experiments, we showed that under physiological conditions cadherin formed unstable homodimers that immediately dissociated after the depletion of calcium ions. However, under destabilizing conditions (such as at pH 5, in the presence of cadmium ions or at high temperature) E-cadherin produced stable dimers. By all parameters these dimers were indistinguishable from those detected in living cells. These experiments clearly showed that stable dimers are formed in living cells as a result of a specific reaction. This reaction can be one of the important regulatory steps of cadherin-based adhesion.

We then studied a tailless cadherin mutant Ec1Δ(748-882)M. This mutant, which is unable to interact with any known intracellular cadherin partners, neither is recruited into intercellular junctions nor forms adhesive dimers (Chitaev and Troyanovsky, 1998). According to the low-affinity model of cadherin adhesion, such a phenotype is based on the disconnection of this mutant from the intracellular clustering machinery. However, we now found that the inactivation of clathrin endocytosis by several different small interfering RNAs (siRNAs) completely restored the recruitment of this mutant into cell–cell junctions. Furthermore, even a complete depolymerization of actin filaments by latrunculin A did not prevent the clustering of this mutant. These observations showed that the recruitment of cadherin into junctions can be based solely on extracellular interactions. Taken together, our study presents new, critical evidence supporting the high-affinity model of cadherin adhesion.


Abstract

In vivo molecular imaging with target-specific activatable “smart” probes, which yield fluorescence only at the intended target, enables sensitive and specific cancer detection. Dimerization and fluorescence quenching has been shown to occur in concentrated aqueous solutions of various fluorophores. Here, we hypothesized that fluorophore dimerization and quenching after conjugation to targeting proteins can occur at low concentration. This dimerization can be exploited as a mechanism for fluorescence activation. Rhodamine derivatives were conjugated to avidin and trastuzumab, which target d -galactose receptor and HER2/neu antigen, respectively. After conjugation, a large proportion of R6G and TAMRA formed H-type dimers, even at low concentrations, but could be fully dequenched upon dissociation of the dimers to monomers. To demonstrate the fluorescence activation effect during in vivo fluorescence endoscopic molecular imaging, a highly quenched probe, avidin-TAMRA, or a minimally quenched probe, avidin-Alexa488, was administered into mice with ovarian metastases to the peritoneum. The tumors were clearly visualized with avidin-TAMRA, with low background fluorescence in contrast, the background fluorescence was high for avidin-Alexa488. Thus, H-dimer formation as a mechanism of fluorescence quenching could be used to develop fluorescence activatable probes for in vivo molecular imaging.


Results

Chromosome Dimer Formation in V. cholerae

The growth of V. cholerae strains deficient in CDR was directly compared to the growth of their parental strain in competition experiments in rich media (Figure 2). These experiments revealed a defect of 5.8% and 3% per cell per generation for Δdif1 and Δdif2 cells, respectively, compared to their wild type counterparts. Since these growth defects were entirely suppressed in a recA background (Figure 2), they directly reflect the rates of dimer formation on chromosome I and II, fdimer Chr1 and fdimer Chr2 (See Material and Methods). The 8.6% growth defect of xerC Vc cells, which was also suppressed in a recA background, reflects the total rate of chromosome dimer formation in V. cholerae, fdimer Chr1+2 (Figure 2). Interestingly, fdimer Chr1+2 equals 1−(1−fdimer Chr1 )(1−fdimer Chr2 ), indicating that dimer formation on the two V. cholerae chromosomes is independent.


RESULTS

FOXA1 forms a highly cooperative homodimer on a compact DNA element

By scoring for the enrichment of co-occurring position weight matrices in cell-type-specific DNase hypersensitive (HS) regions in 78 human cell lines, we previously identified candidate TF dimer configurations that could contribute to their cell-type specific functions ( 28, 29). Two palindromic FOXA1 motifs constituted top hits of this analysis. First, a compact composite motif termed ‘diverging’ DIV (D0) motif was found where the AT-rich core of the forkhead motif ( TA TTT) overlap so that the central TA dinucleotide is shared by juxtaposed half-sites (AAA TA TTT). We also identified a less compact composite motif with forkhead motifs arranged in alternative directions which we called ‘converging’ or CON (C0) motif (Figure 1A). We were interested in both motifs for their strong enrichment in the breast cancer cell line MCF7 and prostate cancer cell line LNCaP.

To test whether FOXA1 homodimerises on these sequences, we established quantitative electrophoretic mobility shift assays (EMSAs) using the purified DNA binding domain (DBD) of FOXA1 (henceforth termed FOXA1). In the absence of DNA, FOXA1 does not dimerize but forms a monodisperse monomer as judged from calibrated size-exclusion chromatograms ( Supplementary Figure S1D ). We first performed EMSAs with 1 nM DNA probe encoding the FOXA1 monomer element and a concentration series of FOXA and measured a mean Kd_monomer of 2.4 ± 1.06 nM (n = 3, Figure 1B). Similar titrations in the presence of DIV DNA showed that dimeric FOXA1 bands begin to form at the lowest FOXA1 concentrations (0.61 nM) indicating a dimerization with strongly positive cooperativity (Figure 1C). An apparent binding affinity Kd_app for the binding of FOXA1 to DIV DNA was determined to be 4.5 ± 1.6 nM by regarding monomeric and dimeric states as an overall bound DNA fraction. To quantify the efficiency of the FOXA1 to homodimerize, we next estimated the cooperativity factor (ω) under equilibrium conditions at a DNA concentration of 100 nM. These measurements provide ratios of equilibrium binding constants (ω = Kd_monomer/Kd_dimer). Here, Kd_dimer represents the dissociation constant for the binding of a second FOXA1 molecule to a pre-formed FOXA1/DNA complex. Thus, cooperativity factors indicate how two TF molecules influence their mutual occupancies on a given DNA element with composite binding sites ( 34, 38). If ω > 1, TFs bind with positive cooperativity if ω<1, TFs bind with negative cooperativity or, in other words, compete and if ω = 1 binding is independent or non-cooperative ( 49). Using these assays, we compared the binding of FOXA1 to co-motifs enriched in DNase-HS regions (D0/DIV and C0/CON motifs) with control elements where the spacing between half-sites was altered (D2, C1 and C-1). FOXA1 binds DIV DNA with highly positive cooperativity (ω = 56.3 ± 11.9 Figure 1D– F, Supplementary Figure S1E , Supplementary Table S1 ). For the C0 DNA a profoundly lower cooperativity factor was measured (ω = 1.8 ± 1.3). If the half-site spacing is perturbed, the dimerization is impeded for both configurations of DNA elements. To further dissect the reliance of cooperative binding on half-site spacing, we inserted spacers from 1 to 10 bp between the half sites of the DIV (Figure 1E and F, Supplementary Figure S1F , Supplementary Table S1 ). This experiment revealed that cooperative dimerization of FOXA1 on DNA strictly depends on compact half-site spacing. Addition of spacers separating the FOXA1 core motifs decreased ω by at least an order of magnitude and in the case of the D3 configuration completely obliterated the formation of dimeric complexes. While the forkhead DBD is sufficient for cooperative dimerization, this binding mode is also retained in the context of recombinantly purified full-length mouse FoxA1 protein ( Supplementary Figure S1G ).

Structural modeling suggests alternative mechanisms for the dimer formation

To understand the basis for FOXA1 dimerization on DIV DNA, we constructed a series of FOXA1 DBD dimer models. First, we used a published FOXA3 DBD-DNA crystal structure (PDB ID 1VTN ( 22)) to produce FOXA1 homology models. The FOXA3 DBD spans about 25% of the full-length protein and differs by five amino acids from the FOXA1 DBD. Next, dimer models were generated by the superposition of two binary FOXA1/DNA models onto ideal B-DNA templates containing DIV sequences ( Supplementary Figure S1A-C ). To maintain the experimental DNA curvature, we concatenated experimental DNA fragments followed by removal of the B-DNA template. Finally, the complex models were energy minimized allowing for structural adjustments of both the protein and the DNA components leading to models with curved DNA. Notably, the DNA element of the 1VTN (CGTTG) model differs at several key positions from the forkhead consensus (TATTT) ( Supplementary Figure S1B ). Therefore, the curvature of the DNA of a binary complex with FOXA1 may not be correctly represented in presently available models. Further, profound structural changes of the DNA could accompany the assembly of dimeric complexes.

A conserved feature in forkhead DBD/DNA complexes is a bidentate hydrogen bound of Asn165 with Adenine A’4 (5′-T1A2T3T4T5-3′/3′-A’1T’2A’3A’4A’5-5′) ( Supplementary Figure S1A ). We first performed superpositions that maintain the interaction of Asn165 with A’4 in the final complex models. In these models we could not observe any intermolecular protein-protein interactions between juxtaposed FOXA1 molecules suggesting that the dimer formation could be facilitated allosterically through DNA (Figure 2A). However, as the DIV is AT-rich we surmised that in the context of a dimeric complex Asn165 could be induced to switch Adenines and bind to A’3 or A’5 instead. To test this possibility, we constructed nine alternative models with all possible combinations of Asn165 with Adenines 3–5 in either half site ( Supplementary Figure S1A, B ). Out of the nine models three are symmetric, that is, Asn165 binds the same Adenine in either of the two half-sites (Figure 2A– C). Out of the three symmetric models for FOXA1 homodimer, the D0-M4 model (where Asn165 binds A’5) shows and extensive hydrophobic protein-protein contact interface formed predominantly by Val229 and Ala232 (Figure 2B and Supplementary Figure S1C ). We also generated structure models for all DIV control motif configurations with spacer lengths 1–10 ( Supplementary Figure S2A, B ). In this set of models, severe clashes were observed for the D3 configuration ( Supplementary Figure S2B ) consistent with the absence of any dimer band in EMSAs (Figure 1E). Remaining configurations showed moderate structural clashes, which could possibly be relieved by conformational adjustments of the protein and changed shape of the DNA ( Supplementary Figure S2A ).

Structural model of dimeric FOXA1/DNA complexes. Structural models were constructed using FOXA1 homology models generated using the FOXA3/DNA crystal structures (PDB ID 1VTN) as template. The modeling strategy is outlined in Supplementary Figure S1A–C . The interaction of Asn165 with an Adenine is a critical mediator of the DNA recognition of forkhead DBDs. Based on the alignment with binary forkhead DBD/DNA structures Asn165 is expected to interact with A4’ in both 5′-T1A2T3T4T5-3′/3′-A’1T’2A’3A’4A’5-5′ DIV half sites (A, D0_M1). We surmised that in the context of a homodimeric complex Asn165 could switch Adenines leading to alternative models where Asn165 contacts A5’ (B, model D0_M4) or A3’ (C, model D0_M7). Left panels show overviews and right panels are zoomed in views highlighting amino acids V229 and A232 exposed to the neighboring molecule that could mediate the dimer formation. The DNA is shown as gray tube, protein helices, sheets or loops are in red, blue and yellow cartoons, respectively. The molecular surface is shown in transparent green. Selected amino acids are labeled and shown as ball-and-sticks. The DNA sequence used to construct the models is shown and nucleotides (or their reverse-complement) contacted by Asn165 are in red.

Structural model of dimeric FOXA1/DNA complexes. Structural models were constructed using FOXA1 homology models generated using the FOXA3/DNA crystal structures (PDB ID 1VTN) as template. The modeling strategy is outlined in Supplementary Figure S1A–C . The interaction of Asn165 with an Adenine is a critical mediator of the DNA recognition of forkhead DBDs. Based on the alignment with binary forkhead DBD/DNA structures Asn165 is expected to interact with A4’ in both 5′-T1A2T3T4T5-3′/3′-A’1T’2A’3A’4A’5-5′ DIV half sites (A, D0_M1). We surmised that in the context of a homodimeric complex Asn165 could switch Adenines leading to alternative models where Asn165 contacts A5’ (B, model D0_M4) or A3’ (C, model D0_M7). Left panels show overviews and right panels are zoomed in views highlighting amino acids V229 and A232 exposed to the neighboring molecule that could mediate the dimer formation. The DNA is shown as gray tube, protein helices, sheets or loops are in red, blue and yellow cartoons, respectively. The molecular surface is shown in transparent green. Selected amino acids are labeled and shown as ball-and-sticks. The DNA sequence used to construct the models is shown and nucleotides (or their reverse-complement) contacted by Asn165 are in red.

The D0-M4 model (Figure 2B) appears similar to the non-cooperative D2 model that was generated by separating the otherwise overlapping FOXA1 half-sites of the DIV ( Supplementary Figure S2A ). However, a superposition between the 2 models reveals that the beta-sheets of the FOXA1 monomers are closer to each other in the D2 model, potentially leading to a suboptimal interaction interface. Moreover, the DNA sequence differs between the two models, leading to alternative DNA shapes that may cause modifications of the predicted protein–protein interaction interface ( Supplementary Figure S2A ). Thus, the D0_M4 model cannot be invalidated due to the lack of positive cooperativity on the D2 element (Figure 1D– F). We propose that D0_M1, D0_M4 and D0_M7 all represent potentially valid initial models for the FOXA1 dimerization on the DIV motif based on the currently available data. Notably, only the D0_M1 model has the direct readout of the DNA sequence similar to that observed for the consensus sequence. Further validation of the models with experiments and molecular dynamics simulation is needed.

Notably, an Ala232Val mutation has been reported to drive prostate cancer ( 50). We decided to test whether mutations to residue Ala232 influence cooperative dimerization on the DNA. However, mutations associated with prostate cancer and 10 other substitutions only mildly influence the cooperativity on the DIV sequence (<1.5-fold change to ω, Supplementary Figure S2C ). As a more rigorous test we also constructed double mutants where both of the putative interface residues Val229 and Ala232 were concurrently mutated to acidic glutamates (Val229Glu/Ala232Glu and Val229Arg/Ala232Arg). EMSAs showed that the Val229Arg/Ala232Arg double mutation does not influence DNA binding ( Supplementary Figure S2D ). However, surprisingly, the Val229Glu/Ala232Glu double mutations lead to a marked increase in the cooperativity although the affinity for monomeric binding is reduced.

In the absence of experimental structures of a ternary FOXA1/DIV complex, the curvature of the bound DNA, the protein-DNA contact interface and the structural basis for dimerization remains hypothetical. The mechanism for the cooperative dimer formation could be due to two basic mechanisms. First, direct protein-protein interactions that would require major structural adjustments and including contact interface switching or the deformation of DNA. Second, DNA-mediated allosteric mechanism could facilitate cooperative DNA recognition by FOXA1. Such mechanisms have been described for a growing number of TF dimer pairs and appear to be common theme in TF biology ( 51–53).

FOXA1 strongly binds to DIV loci in human cancer cells

FOXA1 is a master regulator of endodermal cell and tissue types but also associated with tumorigenesis in several cancers including breast, prostate and liver cancer (( 9), reviewed in ( 8)). Accordingly, FOXA1 expression is highest in normal liver and intestine epithelium as well as breast, prostate, gastrointestinal and liver cancer cell lines amongst a panel of 562 samples analyzed by the FANTOM5 consortium ( 54) (Figure 3A). To study the relevance of the DIV to define the genomic binding profiles of FOXA1, we next defined four categories of binding locations in publicly available ChIP-seq datasets (Figure 3B). Category ‘D’ contains sites with matches to the DIV PWMs in the peak region (1.1e+5 instances in the human genome hg38, ‘D’). Category ‘C’ are control dimer sites that have matches to motifs where the dimer promoting configuration is disrupted by introduction of 1–10 spacers (D1-D10, 5.1e+5 loci in hg38, Supplementary Figure S3A ). Category ‘C’ controls for the preference for a specific configuration but maintains the number of half sites similar to DIV locations. Next, we defined locations where FOXA1 binds exclusively as monomer (‘M’). Lastly, remaining locations are designated ‘N’ for ‘no motif’ for the lack obvious matches to any of the three types of FOXA1 motifs (Figure 3B). FOXA1 ChIP-seq datasets from MCF7, T47D and HepG2 cells typically contain 1000–2000 matches to the DIV and show an enrichment for the DIV as compared to the dimer control with respect to the genome-wide DIV/control ratio ( Supplementary Figure S3B ). As a proxy for cooperative dimerization in a cellular context, we compared the ChIP-seq signals over the four classes of binding sites. FOXA1 loci with DIV signatures in T47D, MCF7 and HepG2 cells exhibit significantly stronger ChIP-seq read intensities compared to regions with alternative motif matches (Figure 3C, Supplementary S3C ). As an alternative analysis we ranked FOXA1 ChIP-seq peaks in MCF7, T47D and HepG2 cells by signal strength and divided them into deciles (Figure 3D). Next we quantified the fractional occurrence of the four binding categories per decile (Figure 3D). This analysis shows that FOXA1/DIV peaks are concentrated in the top deciles to a larger degree than the three other binding categories.

FOXA1 strongly binds to DIV sequences in the context of chromatin. (A) FOXA1 expression measured by the FANTOM 5 consortium in 562 cell and tissue types. Each dot represents a cell or tissue type and selected samples with highest FOXA1 expression are marked. (B) Schematic how ChIP-seq peaks were categorized based on the absence/presence of monomer, DIV or control dimer (DIV1-10) motifs. (C) Boxplot to compare ChIP-seq scores (ENCODE narrowPeak signal values) in the four FOXA1 ChIP-seq peak categories defined in (B) using data from T47D, HepG2 and MCF7 cells. P-values are calculated using pairwise comparisons with the unpaired Wilcoxon rank sum test (R function pairwise.wilcox.test) and adjusted using the Holm method (***P < 0.001). (D) ChIP-seq peaks were ranked by signal values and divided into deciles (top decile = 1, bottom decile = 10, shown as boxplots) and the fractional counts of the four binding categories per decile are shown as proportional barplots.

FOXA1 strongly binds to DIV sequences in the context of chromatin. (A) FOXA1 expression measured by the FANTOM 5 consortium in 562 cell and tissue types. Each dot represents a cell or tissue type and selected samples with highest FOXA1 expression are marked. (B) Schematic how ChIP-seq peaks were categorized based on the absence/presence of monomer, DIV or control dimer (DIV1-10) motifs. (C) Boxplot to compare ChIP-seq scores (ENCODE narrowPeak signal values) in the four FOXA1 ChIP-seq peak categories defined in (B) using data from T47D, HepG2 and MCF7 cells. P-values are calculated using pairwise comparisons with the unpaired Wilcoxon rank sum test (R function pairwise.wilcox.test) and adjusted using the Holm method (***P < 0.001). (D) ChIP-seq peaks were ranked by signal values and divided into deciles (top decile = 1, bottom decile = 10, shown as boxplots) and the fractional counts of the four binding categories per decile are shown as proportional barplots.

We also inspected ChIP-seq signals for alternative chromatin associated factors GATA3, the co-activator Histone acetyltransferase P300 (EP300), CTCF and c-Jun ( Supplementary Figure S3E ). We did not observe increased signals over DIV sequences as compared to other binding site categories indicating that the effect is specific for FOXA1. We conclude that FOXA1 associates with the DIV more strongly than with monomeric or alternative dimer sites in a chromatin context because of the highly cooperative homodimerization promoted by this binding site. We next performed gene ontology analysis using gene sets linked to FOXA1/DIV binding events in T47D or MCF7 cells with signatures of changed expression after chemical or genetic perturbation ( Supplementary Figure S3E ). Amongst the top most significantly enriched gene sets bound by FOXA1/DIV are genes up-regulated in xenografts resistant to endocrine therapy ( 55), differentially upregulated in luminal as compared to basal or mesenchymal breast cancer cell lines ( 56), down regulated in breast cancer cells depleted of ESR1 ( 57) and other gene sets associated with response to nuclear receptor signaling and oncogenic pathways. This suggests that genes associated with FOXA1/DIV locations are sensitive to perturbation of pathways relevant to cancer progression in particular in breast cancer models.

FOXA1 homodimerizes on DIV motifs to regulate enhancers near genes implicated in cancer progression

We next selected five FOXA1/DIV loci associated with genes responsive to perturbation studies that reproducibly showed strong ChIP-seq signals in MCF7 cells (Figure 4A, Supplementary Figure S3E ). These loci are located either in introns or within 50 kb upstream of the TSS of genes with a strong expression in MCF7 cells and other relevant cell and tissue types ( Supplementary Figure S4A and B ). Moreover, these genes were reported to play critical roles in cancer or essential cellular processes including ESR1 ( 58), PVT1/MYC ( 59), ATP9A ( 60), QSOX1 ( 61) and KAT6B ( 62) ( Supplementary Figure S4A ). PVT1 encodes for a long non-coding RNA that resides in the 8q24 locus shared with the oncogene MYC ( 63). This locus is strongly amplified across a panel of malignant cancers and a marker for poor prognosis ( 64, 65). We performed EMSAs using sequence derived from these five endogenous loci as well as two types of mutants (Figure 4B– D). First, we engineered these loci by introducing 3 bp spacers between the half sites reminiscent to the D3 element that is incompatible with dimeric binding (Figure 1E, ‘Monomer’, Supplementary Table S1 ). Moreover, we mutated both half-sites to generate DNA elements with completely destroyed FOXA1 consensus (‘No binding’, Supplementary Table S1 ). Results show a strongly positive cooperativity for FOXA1 homodimerization on all five DIV sequences with the highest ω value for the PVT1/MYC locus (Figure 4B– E). Introduction of the 3bp spacer destroys dimeric binding but leaves the affinity for monomeric binding unaffected (Figure 4C). Degeneration of both FOXA1 half-sites abolishes FOXA1 binding almost completely at the concentrations tested (Figure 4D). To test whether binding with purified components is associated with gene regulation in a context of cells and chromatin, we designed two reporter assays to validate the enhancer activity of these loci. First, we cloned ∼500 bp fragments encompassing the FOXA1-bound DIV sequences tested by EMSA into Tol2 vectors. The Tol2 system enables transposase-mediated integration into the genome of MCF7 cells that strongly express FOXA1 ( Supplementary Figure S4C ). Additionally, we designed a luciferase reporter assay. The latter was performed in T47D cells expressing FOXA1 endogenously and in the colon cancer cell line HCT116 where FOXA1 is not normally expressed ( 66) ( Supplementary Figure S4C ). The expression levels of the Tol2-GFP reporters driven by the various enhancer constructs were quantified in MCF7 cells using FACS and expression was scored taking the fraction of GFP positive cells as well as the median GFP signal into account ( Supplementary Figure S4D ). These expression levels show that sequences containing DIV motifs exhibit a strong enhancer activity (Figure 4F, ‘Dimer’). The expression of GFP reporters where the DIV was mutated so FOXA1 can only bind monomerically (‘Monomer’) or with two disrupted half-sites (‘No binding’) showed a significantly depleted reporter activity suggesting cooperative dimerization is required for full reporter activation (Figure 4F). Analogously, luciferase reporter activity was reduced in T47D cells for 4 out of 5 sequences when the homoderimic binding was disrupted (Figure 4G). Lastly, luciferase reporter activity was strongly elevated in HCT116 devoid of endogenous FOXA1 upon the exogenous provision of FOXA1 indicating that reporter activation is FOXA1-dependent (Figure 4H). Collectively, these results show that FOXA1 dimerizes cooperatively on endogenous DNA sequences and that the dimeric FOXA1/DIV configuration is required for the efficient activation of these enhancer sequences.

FOXA1/DIV binding regulates gene expression in cancer cells. (A) FOXA1 ChIP-seq peaks in MCF7 cells (accession numbers indicated) at five DIV loci near genes with potential roles in oncogenesis (see also Supplementary Figure S4A and B ). (B) EMSAs using Cy5 labeled DNA elements derived from the five endogenous DIV loci. (C) EMSAs where the five DIV DNA elements were mutated to monomer binding sites by adding a 3 base-pair spacer destroying dimeric binding but leaving monomeric binding intact (‘Monomer’). (D) EMSA where both half sites of the DIV elements were mutated abolishing binding (‘No binding’). (E) Homodimer cooperativity value ( ⁠|$omega$|⁠ ) shown as mean ± SD from n |$geq$| 3 measurements. (F) Total GFP fluorescence quantified by FACS analysis using Tol2 constructs containing DIV enhancers or the two types of mutated DIV sites (‘Monomer’ or ‘No Binding’) integrated into the genomes of MCF7 cells. FACS plots and photographs of cells are in Supplementary Figure S4D . (G) Dual Luciferase reporter assay using the T47D cell line endogenously expressing FOXA1. (H) Dual Luciferase assay in HCT116 cells co-transfected with FOXA1 expression plasmids (filled bars) or the pcDNA3 control (empty bars). Empty vector is the luciferase reporter without inserted DIV enhancer. Reporter signals in (F) and (G) were normalized to ‘Monomer’ values. Luciferase and Tol2 reporters were constructed as outlined in Supplementary Figure S4C . The mean ± SD of three biological replicates is shown in (F), (G) and (H). P-values were calculated using the unpaired two-tailed Student's t-test (***P< 0.001 **P< 0.01*P< 0.05).

FOXA1/DIV binding regulates gene expression in cancer cells. (A) FOXA1 ChIP-seq peaks in MCF7 cells (accession numbers indicated) at five DIV loci near genes with potential roles in oncogenesis (see also Supplementary Figure S4A and B ). (B) EMSAs using Cy5 labeled DNA elements derived from the five endogenous DIV loci. (C) EMSAs where the five DIV DNA elements were mutated to monomer binding sites by adding a 3 base-pair spacer destroying dimeric binding but leaving monomeric binding intact (‘Monomer’). (D) EMSA where both half sites of the DIV elements were mutated abolishing binding (‘No binding’). (E) Homodimer cooperativity value ( ⁠|$omega$|⁠ ) shown as mean ± SD from n |$geq$| 3 measurements. (F) Total GFP fluorescence quantified by FACS analysis using Tol2 constructs containing DIV enhancers or the two types of mutated DIV sites (‘Monomer’ or ‘No Binding’) integrated into the genomes of MCF7 cells. FACS plots and photographs of cells are in Supplementary Figure S4D . (G) Dual Luciferase reporter assay using the T47D cell line endogenously expressing FOXA1. (H) Dual Luciferase assay in HCT116 cells co-transfected with FOXA1 expression plasmids (filled bars) or the pcDNA3 control (empty bars). Empty vector is the luciferase reporter without inserted DIV enhancer. Reporter signals in (F) and (G) were normalized to ‘Monomer’ values. Luciferase and Tol2 reporters were constructed as outlined in Supplementary Figure S4C . The mean ± SD of three biological replicates is shown in (F), (G) and (H). P-values were calculated using the unpaired two-tailed Student's t-test (***P< 0.001 **P< 0.01*P< 0.05).

DIV sequences mediate chromatin opening at locations pre-bound by FOXA1 upon inhibition of the PI3K pathway

As FOXA1 is designated as a hallmark pioneer TF, we next asked whether the FOXA1/DIV configuration is involved in the regulation of chromatin accessibility. To address this question we became interested in a study that explored chromatin changes in the breast cancer cell line T47D in response to treatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor BYL719 (henceforth termed BYL) ( 67). The PI3K pathway is hyperactive in ∼70% of breast tumors and therefore an attractive target for anti-cancer therapies ( 68). However, PI3K kinase inhibition can lead to a potent compensatory response and cancer relapse, presumably facilitated by ERα-associated regulatory programs. To study the molecular mechanism for this process, Toska and colleagues compared the binding profile of FOXA1 and the chromatin accessibility measured by ATAC-seq in the absence and presence of BYL ( 67). Intriguingly, the authors reported a motif to become enriched in the FOXA1 binding landscape after BYL719 treatment representing a perfect match to the DIV. However, the authors refer to this motif as ‘Homeobox’ motif because of missing annotations of the DIV in common motif databases. We therefore decided to probe whether FOXA1/DIV configurations contribute to the compensatory chromatin remodeling in response to PI3K pathway inhibition.

Peaks of the DIV category (‘D’) exhibit stronger ChIP-seq signals than loci with monomeric sites (‘M’), sites with perturbed dimer configurations (‘C’) and sites without detectable FOXA1 consensus element (‘N’) under both DMSO and BYL conditions (Figure 5A and B). We grouped genomic locations according to the closed-open dynamics measured by ATAC-seq in the absence or presence of PI3K inhibition. Permanently open (PO) sites are open under both DMSO and BYL conditions close-to-open (CO) sites gain accessibility and open-to-close (OC) sites loose accessibility in response to BYL treatment (Figure 5C). We found that majority of loci belong to the OC category indicating lost accessibility upon BYL treatment. However, the FOXA1 ChIP-seq signal is predominantly associated with the PO or CO categories but barely detectable in the OC category (Figure 5C). This suggests that the absence of FOXA1 sensitizes genomic locations for closing whilst the presence of FOXA1 could have two roles. First, FOXA1 could function to maintain the open chromatin state of PO sites. Second, closed sites pre-bound by FOXA1 could become open in response to PI3K inhibition. We next inspected ATAC-seq signals over locations pre-bound by FOXA1 under DMSO conditions. We found that locations without matches to forkhead binding motifs (‘N’) are mostly pre-opened under DMSO conditions and show only a marginal increase in ATAC-seq signals after BYL treatment (Figure 5D and E, Supplementary Figure S5A ). However, strikingly, FOXA1/DIV locations pre-bound at DMSO conditions are mostly closed but show a strong increase in ATAC-seq signal following PI3K pathway inhibition (Figure 5D and E, Supplementary Figure S5A ). Consistently, a high proportion of FOXA1/DIV sites bound under DMSO conditions maps to locations of the CO category (Figure 5F). Whilst, FOXA1/C and FOXA1/M sites also show a preference for CO locations this association is more significant for FOXA1/DIV sites (Fisher's exact test P = 2.4e–05 (DIV versus control dimer)). We conclude that locations pre-bound by FOXA1 under DMSO conditions are subject to a closed-to-open transition upon PI3K inhibition and this effect is most profound for the subset of FOXA1/DIV location. The PVT1/MYC and KAT6B loci illustrate this effect with equally strong ChIP-seq signals under DMSO and BYL719 conditions but a strong increase of ATAC-seq signal after PI3K inhibition (Figure 5G). We next tested whether the DIV loci near the PVT1/MYC and KAT6B genes respond to PI3K inhibition in our luciferase reporter assay. Sequences with intact DIV elements show an elevated reporter activity in response to BYL treatment whilst sites with mutated DIV elements show no significant response (Figure 5H). However, plasmid-based reporter constructs are unlikely to exactly resemble the chromatin configurations of the endogenous loci. Therefore, we cannot be certain that the observed changes to reporter activity are due to the same chromatin closed-open dynamics apparent in the ATAC-seq data. Nevertheless, this analysis suggests that of blocking the PI3K pathway leads to chromatin remodeling at pre-bound FOXA1/DIV locations that could impact the transcriptional output.

FOXA1 bound DIV locations are associated with chromatin dynamics. (A, B) Boxplot of ChIP-seq scores (summit height of fragment pileup) from FOXA1 data in T47D cells comparing peak categories for the subsets containing DIV motifs (D), control motifs (C), monomer motifs (M) and no FOXA1 motif (N) (see Figure 2B). T47D cells were exposed to DMSO (A) or to the PI3K pathway inhibitor BYL719 (B) ( 67). (C). Heatmap of ATAC-seq reads as well as FOXA1 ChIP-seq reads under DMSO or BYL719 treatment conditions in three categories of accessibility patterns: PO (permanently open in DMSO and BYL), CO (closed in DMSO but open in BYL) and OC (open in DMSO but closed in BYL). (D) ATAC-seq read heatmaps under DMSO or BYL719 treatment conditions facetted by the four FOXA1 ChIP-seq peak categories defined in the DMSO condition. The lower panels are aggregate pileups of ATAC-seq signals. (E) Boxplot of the ratio between ATAC-seq read counts between BYL719 treated T47D cells and non-treated (DMSO) T47D cells in the four categories of FOXA1 binding sites. (F) Fractional barplots showing the relative proportions of FOXA1 peak categories and sites not bound by FOXA1 (‘Unbound’) associated with PO, OC or CO sites defined according to accessibility patterns measured by ATAC-seq (see panel C). (G) Genome browser plot examples of ChIP-seq and ATAC-seq signals at FOXA1/DIV sites near the PVT1/MYC and KAT6B loci. Black boxes mark locations with DIV motif while green box show alternative location showing a disappearance of ATAC-seq signals. (H) Bar plot of the Luciferase/Renilla signal measured in T47D cells treated with DMSO (red) or BYL719 (blue) using DIV element containing luciferase reporter from the PVT1/MYC and KAT6B loci and mutated ‘no binding’ controls. The mean ± SD of 3 biological replicates is shown. P-values in (H) were calculated using the unpaired two-tailed Student's t-test (***P< 0.001 **P< 0.01 *P< 0.05). P-values in (A), (B) and (E) are calculated using Wilcoxon rank sum test and adjusted using the Holm method ( ***P< 0.001).

FOXA1 bound DIV locations are associated with chromatin dynamics. (A, B) Boxplot of ChIP-seq scores (summit height of fragment pileup) from FOXA1 data in T47D cells comparing peak categories for the subsets containing DIV motifs (D), control motifs (C), monomer motifs (M) and no FOXA1 motif (N) (see Figure 2B). T47D cells were exposed to DMSO (A) or to the PI3K pathway inhibitor BYL719 (B) ( 67). (C). Heatmap of ATAC-seq reads as well as FOXA1 ChIP-seq reads under DMSO or BYL719 treatment conditions in three categories of accessibility patterns: PO (permanently open in DMSO and BYL), CO (closed in DMSO but open in BYL) and OC (open in DMSO but closed in BYL). (D) ATAC-seq read heatmaps under DMSO or BYL719 treatment conditions facetted by the four FOXA1 ChIP-seq peak categories defined in the DMSO condition. The lower panels are aggregate pileups of ATAC-seq signals. (E) Boxplot of the ratio between ATAC-seq read counts between BYL719 treated T47D cells and non-treated (DMSO) T47D cells in the four categories of FOXA1 binding sites. (F) Fractional barplots showing the relative proportions of FOXA1 peak categories and sites not bound by FOXA1 (‘Unbound’) associated with PO, OC or CO sites defined according to accessibility patterns measured by ATAC-seq (see panel C). (G) Genome browser plot examples of ChIP-seq and ATAC-seq signals at FOXA1/DIV sites near the PVT1/MYC and KAT6B loci. Black boxes mark locations with DIV motif while green box show alternative location showing a disappearance of ATAC-seq signals. (H) Bar plot of the Luciferase/Renilla signal measured in T47D cells treated with DMSO (red) or BYL719 (blue) using DIV element containing luciferase reporter from the PVT1/MYC and KAT6B loci and mutated ‘no binding’ controls. The mean ± SD of 3 biological replicates is shown. P-values in (H) were calculated using the unpaired two-tailed Student's t-test (***P< 0.001 **P< 0.01 *P< 0.05). P-values in (A), (B) and (E) are calculated using Wilcoxon rank sum test and adjusted using the Holm method ( ***P< 0.001).

Disease-associated SNPs within the DIV motif induce allele-specific dimer formation and expression activity

We surmised that cooperative FOXA1 homodimerisation on DIV sequences is necessary to execute a functional outcome on a subset of genomic loci. If the dimer motif is perturbed, a locus could loose the ability to effectively recruit FOXA1. Or, alternatively, monomeric FOXA1 could be unable to trigger a regulatory response despite effective recruitment. As a consequence, aberrant cellular responses could be evoked contributing to human diseases. To test this hypothesis we interrogated genome-wide association studies (GWAS) for variants affecting FOXA1 dimerization. We considered 25 218 disease-associated SNPs available from public resources (http://www.gwascentral.org, data released January 2016) and extracted further SNPs in linkage disequilibrium with them (proxy LD SNPs) using Haploreg (http://archive.broadinstitute.org/mammals/haploreg/haploreg.php) with parameter r 2 > 0.8 in the European population. This way, we obtained a set of 606 094 candidate SNPs. We then intersected SNP coordinates with genome-wide DIV coordinates affecting the central TA site (AAA TA TTT), which we assumed to be most critical for the cooperative homodimerization of FOXA1. This way, we obtained 23 candidate SNPs whose minor allele may perturb FOXA1 homodimerization and forkhead-dependent transcriptional networks. To determine whether FOXA1 exhibits allelic differences in dimer formation, we performed EMSAs on all 23 SNP candidates with either major or minor allele sequences. Using this strategy, we identified 15 SNPs that profoundly perturb FOXA1 dimerization (Figure 6A, Table 1, Supplementary Figure S6A ). We further inspected each of the 15 SNPs in the dataset provided by the genotype tissue expression (GTEx) consortium (https://gtexportal.org/) and found that six of the SNPs or their LD SNPs constitute GTEx expression quantitative trait locus (eQTLs, Figure 6A, Table 1). In some instances, e.g. rs2097744 that is associated with non-small cell lung cancer, the minor allele completely disrupted dimeric FOXA1 binding (Figure 6B). In other cases, the minor alleles decreased the cooperativity of FOXA1 dimerization ( Supplementary Table S1 ). We next focused on SNPs supported by genomic annotations in tissues relying on the activity of FOXA1 or related forkhead family proteins.

Disease-associated SNPs perturb dimerization and gene expression. (A) Flowchart to select SNPs for functional evaluation. (B) EMSAs for the rs2097744 locus where the minor allele completely disrupts dimerization. (C) ChIP-seq profiles of histone marks at the rs2941742 locus in various cell lines. (D) EMSA comparing dimer formation for major and minor alleles of rs2941742. (E) Cooperativity factor for EMSA in D (mean ± SD, n = 5). (F) Dual luciferase assay with exogenously supplied FOXA1 in HCT116 cells using both alleles of rs2941742 (mean ± SD, n = 5). (G) FOXA1 ChIP-seq profiles from several human cancer cell lines at the rs5414555835 locus (an alternative ID of the same locus is rs67668514). (H) EMSAs using the major and the minor allele of rs5414555835. I. Dual luciferase assay for the two alleles of the rs5414555835 locus in HCT116 cells. Filled bars are for exogenously provided full length FOXA1 and empty bars for the pcDNA3 vector controls (mean ± SD, n = 3 biological replicates). P-values were calculated using the unpaired two-tailed Student's t-test (**P< 0.01).

Disease-associated SNPs perturb dimerization and gene expression. (A) Flowchart to select SNPs for functional evaluation. (B) EMSAs for the rs2097744 locus where the minor allele completely disrupts dimerization. (C) ChIP-seq profiles of histone marks at the rs2941742 locus in various cell lines. (D) EMSA comparing dimer formation for major and minor alleles of rs2941742. (E) Cooperativity factor for EMSA in D (mean ± SD, n = 5). (F) Dual luciferase assay with exogenously supplied FOXA1 in HCT116 cells using both alleles of rs2941742 (mean ± SD, n = 5). (G) FOXA1 ChIP-seq profiles from several human cancer cell lines at the rs5414555835 locus (an alternative ID of the same locus is rs67668514). (H) EMSAs using the major and the minor allele of rs5414555835. I. Dual luciferase assay for the two alleles of the rs5414555835 locus in HCT116 cells. Filled bars are for exogenously provided full length FOXA1 and empty bars for the pcDNA3 vector controls (mean ± SD, n = 3 biological replicates). P-values were calculated using the unpaired two-tailed Student's t-test (**P< 0.01).

SNP ID . Location (hg38) . Gene . Disease association . Annotation . Luciferase . GTEx .
. . . . . Major . Minor . eQTL .
rs2858870 chr6:32604474 HLA-DRB1 Nodular sclerosis Hodgkin lymphoma Promoter histone marks, enhancer histone marks, and DNase marker of lymphoblastoid Cells, blood and mucle cells N
rs2097744 chr7:118382988 LSM8, ANKRD7 Response to platinum-based chemotherapy in non-small-cell lung cancer ++ ++ Y
rs767441 chr15:48613619 FBN1 Breast cancer Promoter histone marks and enhancer histone marks of adipocyte, muscle and lung cells N
rs2104047 chr14:68287700 RAD51B Primary biliary cirrhosis Promoter histone marks and enhancer histone marks of blood cells, muscle cells, thymus and hematopoietic stem cells ++ ++ N
rs2941742 chr6:151691853 ESR1 Bone mineral density (hip) Promoter histone marks and enhancer histone marks of osteoblast cells, muscle cells, blood cells and liver cells ++ + N
rs541455835 (rs67668514 *) chr17:46099939 KANSL1, MAPT Parkinson's disease Promoter histone marks and enhancer histone marks of brain cells, liver cells and blood cells ++ +++ Y
rs281038 chr5:156653467 SGCD Anthropometric traits Enhancer histone marks of foreskin melanocyte primary cells + Y (rs157350)
rs6990531 chr8:80483511 ZBTB10 Eating disorders Promoter histone marks and enhancer histone marks of brain, muscle and blood cells + ++ N
rs7697634 chr4:17965123 LCORL Height Promoter histone mark of liver and enhancer histone mark of pancreatic islets ++ + Y
rs7957274 chr12:21197462 SLCO1B1 blood metabolite measurement Promoter histone marks and enhancer histone marks of ESC and blood cells + ++ N
rs11716984 chr3:121643560 HCLS1 neuropsychological test Promoter histone marks and enhancer histone marks of ESC, iPSC and blood cells ++ Y
rs62288111 chr3:190946175 SNAR-I, GMNC Alzheimers disease Promoter histone mark of adult liver tissue and enhancer histone mark of hESC Derived CD56+ ectoderm cultured cells + ++ N
rs34466261 chr7:104835293 LHFPL3 Obesity Enhancer histone mark of HUES48 ESC cells + N
rs2149943 chr10:107811551 SORCS1 Prion diseases Enhancer histone mark of ES-UCSF4 cells and pancreatic islets ++ + N
rs7007731 chr8:76783496 ZFHX4 age at menarche Enhancer histone mark of Mesenchymal cells ++ Y (rs4735738)
SNP ID . Location (hg38) . Gene . Disease association . Annotation . Luciferase . GTEx .
. . . . . Major . Minor . eQTL .
rs2858870 chr6:32604474 HLA-DRB1 Nodular sclerosis Hodgkin lymphoma Promoter histone marks, enhancer histone marks, and DNase marker of lymphoblastoid Cells, blood and mucle cells N
rs2097744 chr7:118382988 LSM8, ANKRD7 Response to platinum-based chemotherapy in non-small-cell lung cancer ++ ++ Y
rs767441 chr15:48613619 FBN1 Breast cancer Promoter histone marks and enhancer histone marks of adipocyte, muscle and lung cells N
rs2104047 chr14:68287700 RAD51B Primary biliary cirrhosis Promoter histone marks and enhancer histone marks of blood cells, muscle cells, thymus and hematopoietic stem cells ++ ++ N
rs2941742 chr6:151691853 ESR1 Bone mineral density (hip) Promoter histone marks and enhancer histone marks of osteoblast cells, muscle cells, blood cells and liver cells ++ + N
rs541455835 (rs67668514 *) chr17:46099939 KANSL1, MAPT Parkinson's disease Promoter histone marks and enhancer histone marks of brain cells, liver cells and blood cells ++ +++ Y
rs281038 chr5:156653467 SGCD Anthropometric traits Enhancer histone marks of foreskin melanocyte primary cells + Y (rs157350)
rs6990531 chr8:80483511 ZBTB10 Eating disorders Promoter histone marks and enhancer histone marks of brain, muscle and blood cells + ++ N
rs7697634 chr4:17965123 LCORL Height Promoter histone mark of liver and enhancer histone mark of pancreatic islets ++ + Y
rs7957274 chr12:21197462 SLCO1B1 blood metabolite measurement Promoter histone marks and enhancer histone marks of ESC and blood cells + ++ N
rs11716984 chr3:121643560 HCLS1 neuropsychological test Promoter histone marks and enhancer histone marks of ESC, iPSC and blood cells ++ Y
rs62288111 chr3:190946175 SNAR-I, GMNC Alzheimers disease Promoter histone mark of adult liver tissue and enhancer histone mark of hESC Derived CD56+ ectoderm cultured cells + ++ N
rs34466261 chr7:104835293 LHFPL3 Obesity Enhancer histone mark of HUES48 ESC cells + N
rs2149943 chr10:107811551 SORCS1 Prion diseases Enhancer histone mark of ES-UCSF4 cells and pancreatic islets ++ + N
rs7007731 chr8:76783496 ZFHX4 age at menarche Enhancer histone mark of Mesenchymal cells ++ Y (rs4735738)

15 SNPs that show allelic differences in the homodimeric binding of FOXA1 to sites with DIV motifs. ‘+’ indicates luciferase expression level increased comparing to empty vector and ‘–’ that it decreased. denotes a previously used alternative

SNP ID . Location (hg38) . Gene . Disease association . Annotation . Luciferase . GTEx .
. . . . . Major . Minor . eQTL .
rs2858870 chr6:32604474 HLA-DRB1 Nodular sclerosis Hodgkin lymphoma Promoter histone marks, enhancer histone marks, and DNase marker of lymphoblastoid Cells, blood and mucle cells N
rs2097744 chr7:118382988 LSM8, ANKRD7 Response to platinum-based chemotherapy in non-small-cell lung cancer ++ ++ Y
rs767441 chr15:48613619 FBN1 Breast cancer Promoter histone marks and enhancer histone marks of adipocyte, muscle and lung cells N
rs2104047 chr14:68287700 RAD51B Primary biliary cirrhosis Promoter histone marks and enhancer histone marks of blood cells, muscle cells, thymus and hematopoietic stem cells ++ ++ N
rs2941742 chr6:151691853 ESR1 Bone mineral density (hip) Promoter histone marks and enhancer histone marks of osteoblast cells, muscle cells, blood cells and liver cells ++ + N
rs541455835 (rs67668514 *) chr17:46099939 KANSL1, MAPT Parkinson's disease Promoter histone marks and enhancer histone marks of brain cells, liver cells and blood cells ++ +++ Y
rs281038 chr5:156653467 SGCD Anthropometric traits Enhancer histone marks of foreskin melanocyte primary cells + Y (rs157350)
rs6990531 chr8:80483511 ZBTB10 Eating disorders Promoter histone marks and enhancer histone marks of brain, muscle and blood cells + ++ N
rs7697634 chr4:17965123 LCORL Height Promoter histone mark of liver and enhancer histone mark of pancreatic islets ++ + Y
rs7957274 chr12:21197462 SLCO1B1 blood metabolite measurement Promoter histone marks and enhancer histone marks of ESC and blood cells + ++ N
rs11716984 chr3:121643560 HCLS1 neuropsychological test Promoter histone marks and enhancer histone marks of ESC, iPSC and blood cells ++ Y
rs62288111 chr3:190946175 SNAR-I, GMNC Alzheimers disease Promoter histone mark of adult liver tissue and enhancer histone mark of hESC Derived CD56+ ectoderm cultured cells + ++ N
rs34466261 chr7:104835293 LHFPL3 Obesity Enhancer histone mark of HUES48 ESC cells + N
rs2149943 chr10:107811551 SORCS1 Prion diseases Enhancer histone mark of ES-UCSF4 cells and pancreatic islets ++ + N
rs7007731 chr8:76783496 ZFHX4 age at menarche Enhancer histone mark of Mesenchymal cells ++ Y (rs4735738)
SNP ID . Location (hg38) . Gene . Disease association . Annotation . Luciferase . GTEx .
. . . . . Major . Minor . eQTL .
rs2858870 chr6:32604474 HLA-DRB1 Nodular sclerosis Hodgkin lymphoma Promoter histone marks, enhancer histone marks, and DNase marker of lymphoblastoid Cells, blood and mucle cells N
rs2097744 chr7:118382988 LSM8, ANKRD7 Response to platinum-based chemotherapy in non-small-cell lung cancer ++ ++ Y
rs767441 chr15:48613619 FBN1 Breast cancer Promoter histone marks and enhancer histone marks of adipocyte, muscle and lung cells N
rs2104047 chr14:68287700 RAD51B Primary biliary cirrhosis Promoter histone marks and enhancer histone marks of blood cells, muscle cells, thymus and hematopoietic stem cells ++ ++ N
rs2941742 chr6:151691853 ESR1 Bone mineral density (hip) Promoter histone marks and enhancer histone marks of osteoblast cells, muscle cells, blood cells and liver cells ++ + N
rs541455835 (rs67668514 *) chr17:46099939 KANSL1, MAPT Parkinson's disease Promoter histone marks and enhancer histone marks of brain cells, liver cells and blood cells ++ +++ Y
rs281038 chr5:156653467 SGCD Anthropometric traits Enhancer histone marks of foreskin melanocyte primary cells + Y (rs157350)
rs6990531 chr8:80483511 ZBTB10 Eating disorders Promoter histone marks and enhancer histone marks of brain, muscle and blood cells + ++ N
rs7697634 chr4:17965123 LCORL Height Promoter histone mark of liver and enhancer histone mark of pancreatic islets ++ + Y
rs7957274 chr12:21197462 SLCO1B1 blood metabolite measurement Promoter histone marks and enhancer histone marks of ESC and blood cells + ++ N
rs11716984 chr3:121643560 HCLS1 neuropsychological test Promoter histone marks and enhancer histone marks of ESC, iPSC and blood cells ++ Y
rs62288111 chr3:190946175 SNAR-I, GMNC Alzheimers disease Promoter histone mark of adult liver tissue and enhancer histone mark of hESC Derived CD56+ ectoderm cultured cells + ++ N
rs34466261 chr7:104835293 LHFPL3 Obesity Enhancer histone mark of HUES48 ESC cells + N
rs2149943 chr10:107811551 SORCS1 Prion diseases Enhancer histone mark of ES-UCSF4 cells and pancreatic islets ++ + N
rs7007731 chr8:76783496 ZFHX4 age at menarche Enhancer histone mark of Mesenchymal cells ++ Y (rs4735738)

15 SNPs that show allelic differences in the homodimeric binding of FOXA1 to sites with DIV motifs. ‘+’ indicates luciferase expression level increased comparing to empty vector and ‘–’ that it decreased. denotes a previously used alternative

We became particularly interested in SNP rs2941742 located within an intron region of the ESR1 gene (Figure 6C). This SNP maps to a region with the enhancer mark H3K27ac and the promoter mark H3K4me3 in osteoblasts, as well as H3K27ac marks in muscle and bone marrow cells (Figure 6C). The SNP rs2941742 is in LD with rs2941740 (r 2 = 0.98 in the European population) linked to aberrant bone mineral density (BMD)—a trait used in the clinic to diagnose osteoporosis and the estimation of fracture risk ( 69). Interestingly, ESR1 is a gene relevant for bone metabolism as osteoporosis mainly affects post-menopausal women with depressed levels of its activating ligand estrogen ( 70). EMSAs showed that the minor allele reduced the homodimer cooperativity of FOXA1 to the rs2941742 locus 20-fold whilst monomeric binding is not affected (Figure 6D and E). Moreover, luciferase assays revealed reduced reporter activity for the minor as compared to the major allele that is dependent on exogenous FOXA1 addition (Figure 6F). It is therefore conceivable that the perturbation of FOXA1 dimers or of related forkhead TFs on the rs2941742 locus modifies ESR1 regulation and contributes to the etiology of osteoporosis.

rs5414555835 (also annotated with ID rs67668514) maps to a locus bound by FOXA1 in various cancer cell lines (Figure 6G) and displays active histone marks in liver and brain cells (Table 1). This SNP is in LD with rs17577094 at r 2 = 0.97 in the European population and maps to the chr17q21.31/MAPT locus ( 71) reported to be strongly associated with Parkinson's disease (PD) ( 72). MAPT encodes for the Tau protein whose de-regulation and aberrant folding is a major cause for the disease progression. Interestingly, rs541455835 is a GTEx eQTL associated with allele specific changes in MAPT expression in various tissues ( Supplementary Figure S6B ). Moreover, rs541455835 shows a strong difference in binding and reporter gene expression in an allele-specific and FOXA1-dependent manner (Figure 6H and I). Notably, FOXA1 and FOXA2 are critical for the function of adult dopaminergic neurons ( 73). For example, gene delivery of FOXA2 in a mouse model for PD protected midbrain dopaminergic neurons and alleviated motor deficits ( 74). Therefore, the modulation of FOXA1/2 dimerization on the MAPT locus could hamper the neuroprotective roles of FOXA1/2 and contribute to PD. Overall, we observed significant differences of reporter expression between major and minor allele sequences for 10 of the 15 tested SNPs ( Supplementary Figure S6C ), five of which are also eQTLs.


MATERIALS AND METHODS

Reagents and Antibodies

Human recombinant EGF was purchased from Boehringer-Mannheim Co. (Indianapolis, IN). Rabbit anti-EGFR antibody, mouse anti-Cbl monoclonal antibody (mAb), rabbit anti–hemagglutinin-tag antibody, rabbit anti-myc antibody, and rabbit anti–signal transducer and activator of transcription (STAT) 5 were all acquired from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Mouse anti-EGFR mAb (clone LA1) was purchased from Upstate Biotechnology Inc. (Charlottesville, VA), mouse anti-EGFR mAb (clone EGFR.1) from Neo Markers, horseradish peroxidase (HRP)-conjugated goat antimouse immunoglobulin G (IgG) from Zymed Laboratories Inc. (San Francisco, CA), HRP-conjugated goat anti-rabbit IgG from Chemicon International Inc. (Temecula, CA), mouse anti-phosphotyrosine mAb (6D12) from MBL Co (Nagoya, Japan), and mouse anti-Flag mAb (M2) from Sigma Chemical Co. (St. Louis, MO). Rabbit anti–mitogen-activated protein kinase (MAPK) and phospho-MAPK antibody were purchased from New England Biolabs Inc. (Beverly, MA).

Plasmid Construction

A plasmid encoding a glutathione S-transferase (GST) fusion protein containing the EGF-like domain of proHB-EGF, corresponding to amino acids 106–149 of human proHB-EGF, was constructed by insertion of the corresponding cDNA sequences of proHB-EGF into the EcoRI/BamHI sites of the pGEX-3X plasmid (Pharmacia). The inserted DNA fragment encoding proHB-EGF was prepared by polymerase chain reaction using plasmid pRTHG-1 (Mitamura et al., 1995) as a template. The resulting GST fusion protein, referred to as HB1, encompasses the entire EGF-like domain. Next, HB2, a GST fusion protein containing a mutated EGF-like domain of proHB-EGF, was produced: The coding sequence of proHB-EGF cDNA was mutated from 379 CGGAAA to CTTTCA and from 388 AAG to GAC. These substitutions resulted in amino acid alterations from 110 Arg- 111 Lys to Leu-Ser and 113 Lys to Asp. cDNA of the resulting mutant proHB-EGF, corresponding to amino acids 106–149 and containing the above substitutions, was inserted into theEcoRI/BamHI sites of the pGEX-3X plasmid. Truncated EGFR mutants were constructed: pRc/CMV-HA was constructed by the insertion of a DNA fragment encoding the HA-tag epitope into theXbaI site of pRc/CMV (Invitrogen, San Diego, CA). Deletion of EGFR was generated by polymerase chain reaction using pTJNEO-EGFR (Gotoh et al., 1992) as the template, and synthesized products were inserted between the HindIII andXbaI sites of pRc/CMV-HA. The sequence of each EGFR mutant was confirmed by sequence analysis.

Purification of GST Fusion Protein

The GST fusion proteins were purified with glutathione Sepharose 4B (Pharmacia, Piscataway, NJ) according to the manufacturer's instructions. GST-HB1 and GST-HB2, eluted from glutathione Sepharose, were dialyzed against HEPES-buffered saline (20 mM HEPES, 150 mM NaCl, pH 7.2) for use in the following experiments. Protein concentrations were determined by the Bradford method using BSA as a standard.

Cell Culture and Transfection

Ba/F3 cells were cultured in RPMI 1640 medium containing 10% fetal calf serum (FCS) and 5% WEHI-3 cell-conditioned medium as a source of interleukin 3 (IL-3). Stable transformants of Ba/F3 cells expressing EGFR or EGFR-EpoR were obtained by selection in medium containing G418 as previously described (Iwamoto et al., 1999). COS-7 cells were maintained in DMEM with 10% FCS. Chinese hamster ovary (CHO) cells were cultured in Ham's F12 medium with 10% FCS. Transfection was carried out by electroporation (Gene Pulser,Bio-Rad, Richmond, CA) according to the manufacturer's instructions.

Treatment with EGF Ligands

Before cross-linking and coimmunoprecipitation assays, cells indicated were incubated with 100 nM of EGF or the recombinant forms of HB-EGF for 3 min, washed with PBS, and then used for further analysis.

Chemical Cross-linking

Chemical cross-linking was carried out as described previously, with minor modifications (Iwamoto et al., 1994). Briefly, the cells were washed with PBS (137 mM NaCl, 0.67 mM KCl, 8 mM Na2HPO4, 1.4 mM KH2PO4) three times and incubated for 30 min at 4°C with 1 mM dithiobis-(sulfosuccinimdylpropionate) (DTSSP) (Pierce Chemical Co., Rockford, IL) in PBS, followed by washing three times with Tris-buffered saline (TBS) (20 mM Tris-HCl, 100 mM NaCl, pH 7.5) before use in the following studies.

Immunoprecipitation and Immunoblotting

Cells were lysed with 1% Triton X-100 in lysis buffer (0.15 M NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaVO4, 50 mM Tris pH 7.5) and then centrifuged for 20 min at 15,000 × g. The supernatants were cleared with Sepharose 4B for 1 h and then incubated with primary antibody for 2 h, followed by addition of Sepharose 4B-conjugated secondary antibody. The Sepharose beads were washed three times with lysis buffer and once with deionized water and then were boiled for 5 min in SDS-PAGE sample buffer with or without 50 mM dithiothreitol. Samples were run on SDS-PAGE and electrotransferred to an Immobilon membrane. The membrane was blocked with 3% skim milk in TBS (20 mM Tris, 0.1 M NaCl, pH 7.5) at 37°C for 1 h, then incubated with primary antibody in TBS containing 1% skim milk at room temperature for 1 h. Next, the membrane was washed four times with TTBS (TBS containing 0.05% Tween 20), incubated with HRP-conjugated secondary antibody, and finally analyzed with an ECL-Western blotting kit (Amersham International plc, Buckinghamshire, England).

DNA Synthesis Assay

Cells were seeded into 24-well plates at a density of 5 × 10 4 cells/well with or without each EGF ligand in fresh RPMI 1640 medium containing 10% serum, then cultured at 37°C for 24 h and incubated with [ 3 H]thymidine (37 kBq/ml) for 4 h. Cells were harvested, and radioactivity incorporated into DNA was determined as described previously (Iwamotoet al., 1999). The rate of DNA synthesis was expressed as a percentage of the average of the maximum value (40,000 cpm).

Preparation of Fab Fragment

Anti-EGFR mAb (EGFR.1), 1 mg/ml, was incubated with papain (0.1 mg/ml) in PBS at 37°C for 18 h, at which point iodoacetamide (30 mM) was added to stop the reaction. The mixture was dialyzed in PBS at 4°C for 12 h and then purified with goat antimouse IgG Fc antibody conjugated to Sepharose 4B. The size of the Fab fragment was checked by SDS-PAGE, and the Fab concentration was determined by measuring absorbance at 280 nm.


What are the benefits of hot-start technology?

  • Prevents extension of primers binding to template sequences with low homology (mispriming)
  • Prevents extension of primers binding to each other (primer-dimer formation) during reaction setup
  • Increases sensitivity and yield of the desired target fragments
  • Enables PCR setup on high-throughput or automated liquid-handling platforms as reactions are stable at room temperature without compromising specificity

Dimer

A dimer is a molecule composed of two subunits linked together. It is a special case of a polymer. Among the most common dimers are certain types of sugar sucrose, for example, is a dimer of a glucose molecule and a fructose molecule.

Primer dimer
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A Primer dimer (PD) is a potential by-product in PCR, a common biotechnological method. As its name implies, a PD consists of primer molecules that have attached (hybridized) to each other because of strings of complementary bases in the primers.

Dimercaprol
Dimercaprol is a synthetic compound produced for the treatment of lewisite (arsenic based chemical weapon) and poisoning by heavy metals. Dimercaprol itself is a toxic drug but small therapeutic dosage can be used.

ic /die-MARE-ick/ adj. In chemistry, composed of two parts.
dimethyl sulfate (DMS) /die-METH-əl/ A colorless oily liquid with an onionish aroma. Commonly used as a reagent for methylation of amines, phenols, and thiols.

Trans-dimer synthesis
A process which permits nucleotides to be inserted opposite a pyrimidine

. Because this process is not based upon complementary base pairing, the wrong base pairs may be inserted, resulting in a mutation.

captopropanol, an antidote for poisoning by arsenite which reacts with reduced lipoic acid.
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ization activates the tyrosine-kinase section of the receptors, each of which then adds phosphate from ATP to the tyrosine tail of the other polypeptide.
The fully activated receptor proteins activate a variety of specific relay proteins that bind to specific phosphorylated tyrosine molecules.

model.[35] This model assumed that a single PrPSc molecule binds to a single PrPC molecule and catalyzes its conversion into PrPSc.

s
Fortunately, most cells are able to repair damaged DNA. This can be achieved in two ways: repair enzymes called photolyase can break the covalent bond, using light as an energy-source for bond cleavage.

- A form of DNA damage that results from radiation. Adjacent thymines on the same strand of DNA form a bond that results in a bulky adduct that can impede DNA replication.

and, depending on the identity of its partner, will activate a different set of genes.

with another ethylene molecule, then the double carbon bonds in that ethylene molecule break and go find another ethylene molecule to interact with and form a trimer, or three unit molecule, and so on until you end up with a very long polymer.

Two of the monomer units form a coiled-coil

Nucleotide excision repair is particularly important in correcting thymine

, two thymine nucleotides adjacent to each other on one strand are covalently bonded to each other rather than their complementary bases.

Kinetic studies have indicated that EI acts as a

, to hydrolyze PEP into pyruvate and inorganic phosphate although not as efficiently as full length EI.

Leucine zipper proteins A family of DNA-binding proteins that require a

ize by virtue of an alpha helical region that contains leucine at every seventh position. Because 3.

ization domain and an N-terminal DNA binding
domain.
Zinc-finger - a conserved DNA-binding motif composed of protein domains folded .

contains an alpha and a beta tubulin). Microtubules play a role in cell division they are components of cilia and flagella and they also form centrioles.
The Cytoskeleton and Cell Movement - Image Diversity: microtubules tubulin .

Cysteine can react with itself to form an oxidized

Common structural motif in some

ization domain and N-terminal DNA-binding domain. (Figure 10-43)
leukemia
Cancer of white blood cells and their precursors.

The structural motif known as the leucine-zipper consists of a leucine repeat region, which forms an alpha helix with a hydrophobic region responsible for

of 34 kD subunits. Binds actin with Kd of around 25M.
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Protein that forms microtubules, 55-kd polypeptides of α-tubulin (alpha-) and β-tubulin (beta-) form a a

, which is the basic subunit of microtubules. A third type of tubulin γ-tubulin (gamma-) is located at the centrosome. Cytoskeleton 2 Microtubules
tumour necrosis factor
(tumor necrosis factor, TNF) .

Dominant-negative mutation: A (heterozygous) dominant mutation on one allele blocking the activity of wild-type protein still encoded by the normal allele (often by

ising with it) causing a loss-of-function phenotype. The phenotype is indistinguishable from that of homozygous dominant mutation.

excision repair Means by which cells are able to repair certain kinds of damage (

ized pyrimidines) in their DNA.
excitation Electron movement caused by light striking the chlorophyll molecule.

[L. carbo, charcoal + hydro, water]
A sugar (monosaccharide) or one of its

s (disaccharides) or polymers (polysaccharides).
carbon cycle .

AP-1 is actually a complex between c-fos protein and c-jun protein, or sometimes is just c-jun

s. The AP-1 site consensus sequence is (C/G)TGACT(C/A)A. Also known as the TPA-response element (TRE). [TPA is a phorbol ester, tetradecanoyl phorbol acetate, which is a chemical tumor promoter] .

Microtubules are small hollow cylinders (25 nm in diameter and from 200 nm-25 µm in length). These microtubules are composed of a globular protein tubulin. Assembly brings the two types of tubulin (alpha and beta) together as

s, which arrange themselves in rows.

by the repairing enzymes for example ultra violet radiation can cause a damage because it causes two neighboring thymines in the DNA sequence to get joined to each other instead of joining with the adenines across from them and then when the repair enzymes cut out those two joined together thymines or thymine


DISCUSSION

In this study, we demonstrate that FOXA1 can form a DNA-dependent homodimer in the presence of a palindromic DNA element with overlapping half-sites. Unlike some other TFs such as SOX9 that form dimers on flexibly spaced composite elements ( 39), FOXA1 dimerization relies on precise half-site spacing. Interestingly, the DIV motif was also detected, but not validated, using methyl-SELEX with full length FOXA1 ( 33) and high-throughput SELEX ( 32) for members of the FOXC subfamily. Further, we noticed that in a recently reported crystal structure of DNA bound FOXO1 an arrangement of crystallographically stacked DNA helices resembling the DIV configuration ( Supplementary Figure S7 ) ( 75). However, the authors did not test cooperative dimer formation on such an element. Lastly, ChIP-exonuclease sequencing studies indicated the presence of various forms of composite forkhead elements. First, two clustered forkhead sites (termed mesas) resembling a widely spaced CON element (Figure 1A) were reported ( 31). Second, a DIV signature was seen in glucocorticoid receptor (GR) ChIP-exo data showing signatures of cooperative binding and implying roles of the DIV for GR recruitment to chromatin ( 30). Collectively, the DIV motif could be a broadly used forkhead recognition sequence relevant for TFs beyond FOXA1. As a consequence, the dimer-modifying GWAS SNPs reported here could elicit their phenotypic consequence by perturbing regulatory programs of any forkhead protein. SNPs rs2941742 and rs541455835 are the most interesting candidates for a forkhead associated disease mechanism. They localize to distal enhancer of ESR1 and MAPT genes, which are related to osteoporosis or Parkinson's disease, respectively. The rs2941742[G] osteoporosis risk allele leads to a near 100-fold decrease in FOXA1 homodimer cooperativity, and causes depressed reporter gene expression. Whilst FOXA1, to our knowledge, has not been implicated in osteoporosis, the FOXO group plays an important role in bone metabolism by regulating the redox balance, protein synthesis and differentiation in the osteoblast lineage (reviewed in ( 76)). Similarly, rs541455835 risk alleles promote modulation in gene expression through perturbation of FOXA1 homodimerisation. Several studies found that the original GWAS SNPs linked to the dimer modifying SNPs (rs2941740 for rs2941742 rs17577094 for rs67668514) were associated with eQTLs ( 77, 78). Importantly, rs17577094 not only affects MAPT gene expression in the brain, but also in other tissues including the breast where the rs67668514 locus shows strong FOXA1 ChIP-seq signals. We also found that rs767441[C], which is one of the breast cancer risk-associated SNPs reported by Cowper-Sallari et al ( 26), destroyed dimeric binding without changing the affinity for monomeric binding. Genome editing studies provide the means to test whether perturbing forkhead DBD dimerization on disease-associated loci influences disease progression. Collectively, the identification of disease associated SNPs at regulatory genomic regions that reduce FOXA1 dimerization and perturb reporter gene expression corroborates our hypothesis that FOXA1 dimerization is critical for its regulatory function and contributes to disease progression.

While we showed that FOXA1/DIV configurations are linked to highly expressed genes, these binding events do not appear to act as simple transcriptional amplifier. Accordingly, the effects of minor alleles of the studied SNPs are diverse. Reporter assays showed that the presence of minor alleles could lead to elevated, reduced or unchanged expression levels relatively to major alleles (Figure 6E, F and I). Likewise, when the expression of eQTLs is compared across tissues, the minor allele can be associated with increased expression levels in one tissue but with reduced expression levels in another ( Supplementary Figure S6B ). This implicates that regulatory outcomes of FOXA1/DIV complexes are dependent on the overall sequence as well as the cellular context. For example, whether a FOXA1/DIV complex recruits co-activators or repressors could be influenced by peripheral sequences and change from one cell to another. The association of DIV sequences with open-close dynamics of chromatin suggests a role of FOXA1/DIV complex in chromatin remodeling. An important question is whether the regulatory consequence of FOXA1 dimers on DIV motifs is different than other configurations such as monomers, heterodimers or homodimers on CON sequences. Clearly, we identified enhancers that rely on FOXA1/DIV dimers for their activity. An intriguing possibility is that the DIV motif not only affects the strength and dynamics of binding but also has qualitative effects. A possible mechanism is that only when bound to DIV sequences FOXA1 would recruit a set of co-factors that in turn may trigger transactivation, chromatin looping or the setting of epigenetic marks in a cell type dependent manner. By contrast, alternative FOXA1 configurations induced by other DNA motifs could lead to disparate nuclear processes. Such a mechanism could explain the context specific activities of master TFs such as FOXA1 allowing them to regulate different sets of genes in a multitude of cells and at different development stages. Collectively, homodimeric FOXA1 critically contributes to its genomic binding landscape and its regulatory activity, likely by influencing chromatin dynamics and by modulating its interactome.


Watch the video: BC-331-10A 01-10V LED dimmer, push dimmer (August 2022).