Journal: bioRxiv

Article Title: Systematic Dissection of Sequence Features Affecting the Binding Specificity of a Pioneer Factor Reveals Binding Synergy Between FOXA1 and AP-1

doi: 10.1101/2023.11.08.566246

Figure Lengend Snippet: a) Workflows for ChIP-ISO (1), EMSA-seq (2), and neural network analysis (3). b) Reproducibility of FOXA1 ChIP-ISO signal across two biological replicates. Black / gray dots represent sequences containing WT / mutated FOXA1 motifs. r: Pearson correlation. c) Histogram of the FOXA1 ChIP-ISO signals with the entire ISO library, fit by two Gaussian peaks (green and yellow: low and high peaks, respectively; red: superposition of the two). d) Comparison of FOXA1 ChIP-ISO signals with low-throughput ChIP-qPCR signals from three biological replicates over three individual ISO library sequences.

Article Snippet: A pooled equimolar mixture of the ChIP-ISO synthetic oligonucleotide library was PCR-amplified and purified via agarose gel purification (Thermo Scientific K0691).

Techniques: Comparison

Journal: bioRxiv

Article Title: Systematic Dissection of Sequence Features Affecting the Binding Specificity of a Pioneer Factor Reveals Binding Synergy Between FOXA1 and AP-1

doi: 10.1101/2023.11.08.566246

Figure Lengend Snippet: a) Diagram of ChIP-ISO library oligonucleotide design. The variable region of each sequence spans 193 bp, as indicated in blue. The BsaI / BbsI is the restriction enzyme recognition sites engineered in the 18 bp flanking primer sequences. b) Table summarizing the three subsets of our ChIP-ISO oligonucleotide library. c) Plasmid construction strategy. We start off with the native CCND1 enhancer sequence, cloned into the pAAVS1-Nst-MCS plasmid background. We then delete a 193 bp region containing all three FOXA1 binding sites, as well as a BbsI cutting site, and replace it with two BsaI cutting sites. Finally, we ligate the 193 bp ChIP-ISO oligonucleotides (blue) into this plasmid using the BsaI sites. Grey arrows represent primers used for ChIP-ISO amplicon sequencing. d) Strategy for integration of the plasmid library into the AAVS1 site using CRISPR/Cas9. Double-strand break is generated at the AAVS1 site by pSpCas9 (1.1). The region containing library and background sequences, as well as the selection marker (Neo), is integrated into the AAVS1 site (inside the first intron of PPP1R12C gene) by homology-directed repair. If integrated successfully, the promoterless Neo gene will be transcribed together with the endogenous PPP1R12C gene, and RNA splicing will happen between exon 1 and the splicing acceptor (SA) site. A 2A peptide is engineered before the Neo gene to make sure the translated Neo protein is folded independently. HA-L, left homologous arm; HA-R, right homologous arm. WT, a primer pair amplifying unintegrated AAVS1 site; KI, a primer pair amplifying AAVS1 site with successful integration. e) A549 colonies after integration with no Cas9 (left, control), eSpCas9, and wt Cas9. The colonies are selected with G418 for 12 days, and stained with methylene blue. f) PCR test on mixed colonies after integration. The “KI” primer pair (see d) amplifies across the integration junction, and therefore the band is only visible when the plasmid sequence is integrated into the right locus. g) Same as in panel f except that the PCR is carried out in 18 single colonies. 15 out of the 18 colonies show the right PCR band. h) Strategy of amplicon sequencing. After first round of PCR using the primer pair in panel c, we perform BbsI digestion, which cuts the native CCND1 enhancers, but not the synthetic ones. The second round PCR (with sequencing adaptors) can therefore selectively amplify the ISO library for sequencing.

Article Snippet: A pooled equimolar mixture of the ChIP-ISO synthetic oligonucleotide library was PCR-amplified and purified via agarose gel purification (Thermo Scientific K0691).

Techniques: Sequencing, Plasmid Preparation, Clone Assay, Binding Assay, Amplification, CRISPR, Generated, Selection, Marker, Staining

Journal: bioRxiv

Article Title: Systematic Dissection of Sequence Features Affecting the Binding Specificity of a Pioneer Factor Reveals Binding Synergy Between FOXA1 and AP-1

doi: 10.1101/2023.11.08.566246

Figure Lengend Snippet: a) Map of the 193bp portion of the CCND1e explored in this study (chr11:69,654,913-69,655,105). Three FOXA1 motifs (orientations depicted by arrow directions) and motifs of potential co-binding TFs are labeled. b) ChIP-ISO signals over CCND1e variants containing scrambled sequences in a 10bp moving window (step size: 3bp). Bar: averaged ChIP-ISO signal; Dot: data from individual replica; X: missing data. Gray box highlights an area where the scrambles lead to particularly low FOXA1 ChIP-ISO signals, indicating that these sequences are critical for FOXA1 binding. c) The effect on FOXA1 binding by manipulating individual FOXA1 motifs (left) or mutating co-factor motifs (right). Each FOXA1 motif is either mutated (mut), orientation-reversed (rev), or converted into the strongest consensus (con). Both tables list the fold change and statistical significance of FOXA1 ChIP-ISO signal caused by these sequence variations. Two-tailed paired t-test. d) Example FOXA1 ChIP-ISO signals over CCND1e variants containing 0, 1, 2, or 3 original (ori) FOXA1 motifs or 1 consensus (con) FOXA1 motif. X: missing data. e) Box-and-whisker plots showing FOXA1 ChIP-ISO signals over otherwise identical CCND1e sequences containing WT or mutated AP-1, CEBPB, or SP1 motifs. Paired sequences are connected by a line. ****: p < 0.0001, ***: p < 0.001, **: p < 0.01, and ns: non-significant based on two-tailed paired t-test (same below, unless specified). f) Pearson correlation coefficient between FOXA1 ChIP-ISO signals and CCND1 sequence variables, calculated using the total set or the subset containing an AP-1 motif. These numbers reflect the level of impact of each variable on FOXA1 binding. g) FOXA1 ChIP-ISO signal as a function of the linear distance between the 3rd FOXA1 motif and the AP-1 motif. h) Relation between the FOXA1 ChIP-ISO signal and the total FOXA1 motif score over CCND1 variants ± AP-1 / CEBPB motifs. i) Effect of FOXA1 on AP-1 binding. Bar plots show FOXA1 and FOSL2 ChIP-qPCR for three biological replicates over integrated WT CCND1e or a variant with all three FOXA1 motifs mutated (Knock-in). ChIP-qPCR over a positive / negative control locus (P and N) and the native CCND1e are also shown. Error bars represent standard error (same below, unless specified).

Article Snippet: A pooled equimolar mixture of the ChIP-ISO synthetic oligonucleotide library was PCR-amplified and purified via agarose gel purification (Thermo Scientific K0691).

Techniques: Binding Assay, Labeling, Sequencing, Two Tailed Test, Whisker Assay, Variant Assay, Knock-In, Negative Control

Journal: bioRxiv

Article Title: Systematic Dissection of Sequence Features Affecting the Binding Specificity of a Pioneer Factor Reveals Binding Synergy Between FOXA1 and AP-1

doi: 10.1101/2023.11.08.566246

Figure Lengend Snippet: a) Genomic tracks of TF and histone modification ChIP-seq and ATAC-seq signals at the CCND1 enhancer in WT A549 cells. b) Conservation of DNA sequence within the ∼200 bp CCND1 enhancer. Blue: conserved nucleotide, brown: non-conserved. TF motifs are outlined and color coded. Vertical black lines marks the exact 193 bp sequences use in our ISO library. c) Manipulation of the FOXA1 motifs in the CCND1 enhancer. Each motif is mutated, orientation reversed, or converted into a perfect consensus. d) Low-throughput FOXA1 and FOSL2 ChIP-qPCR for three biological replicates showing the impact of the AP-1 motif on their binding. P: positive control, Native: native CCND1 enhancer, Knock-in = AAVS1-integrated CCND1 enhancer (wt or AP-1 mut), N: negative control. *: p < 0.05, **: p < 0.01.

Article Snippet: A pooled equimolar mixture of the ChIP-ISO synthetic oligonucleotide library was PCR-amplified and purified via agarose gel purification (Thermo Scientific K0691).

Techniques: Modification, ChIP-sequencing, Sequencing, Binding Assay, Positive Control, Knock-In, Negative Control

Journal: bioRxiv

Article Title: Systematic Dissection of Sequence Features Affecting the Binding Specificity of a Pioneer Factor Reveals Binding Synergy Between FOXA1 and AP-1

doi: 10.1101/2023.11.08.566246

Figure Lengend Snippet: a) Bioinformatic analysis of FOXA1/TF co-binding. Top: Schematics of co-binding events. Bottom: for each TF, the dot plot shows the percentage of the overlapped FOXA1 ChIP-seq peaks (x axis) and the enrichment of its motif within FOXA1 peaks (y axis). TFs chosen for further analysis are labeled. AP-1 subunits are indicated in blue. b) Heatmaps of ChIP-seq / ATAC-seq and the corresponding intensity profiles in WT A549 cells over FOXA1 binding regions separated based on FOXA1 motif scores. Sequences in the top section contain strong consensus motifs (score >16), and the ones at the bottom contain weak motifs (score < 12). c) The effect on FOXA1 binding by mutating co-factor motifs. The ISO set used in panel c-e is derived from genomic sequences that show overlapped FOXA1 and TF ChIP-seq peaks in A549 and contain both motifs in proximity (<30bp). The table lists the fold change and statistical significance of FOXA1 ChIP-ISO signal upon mutations of co-factor motifs in these sequences. Two-tailed paired t-test. d & e) Box-and-whisker plots showing the changes of FOXA1 ChIP-ISO signal upon mutations of AP-1 (d) or CEBPB motif (e). f) DeepLIFT-Shap feature attribution scores highlighting features used by a sequence-trained CNN to predict FOXA1 binding in A549 cells at the CCND1e. g) Top 5 motifs detected by TF-MoDISco in the genome-wide DeepLIFT-Shap positive feature attribution scores at sites predicted by the CNN to be bound by FOXA1. The TF family of matching motifs is annotated for each motif, as is the number of seqlets used by TF-MoDISco to construct each motif.

Article Snippet: A pooled equimolar mixture of the ChIP-ISO synthetic oligonucleotide library was PCR-amplified and purified via agarose gel purification (Thermo Scientific K0691).

Techniques: Binding Assay, ChIP-sequencing, Labeling, Derivative Assay, Genomic Sequencing, Two Tailed Test, Whisker Assay, Sequencing, Genome Wide, Construct

Journal: bioRxiv

Article Title: Systematic Dissection of Sequence Features Affecting the Binding Specificity of a Pioneer Factor Reveals Binding Synergy Between FOXA1 and AP-1

doi: 10.1101/2023.11.08.566246

Figure Lengend Snippet: a) The same as lower panel, except the x axis represents the percentage of a TF ChIP-seq peaks overlapped with FOXA1 peaks ( is the percentage of the overlapped FOXA1 peaks). b) Two examples of native sequence with co-binding events included in our ISO library. Top: a sequence with both FOSL2 and FOXA1 ChIP-seq peaks and their motifs in proximity. Dotted lines represent the boundaries of the ∼200 bp library sequence. Blue bar: FOSL2 motif, orange bar: FOXA1 motif. Bottom: Same as top, but for a sequence with CEBPB / FOXA1 overlap. c) Histogram showing distributions of scores given by a sequence-trained CNN to sequences that were tested by ChIP-ISO, where the sequences are grouped according to ChIP-ISO signal. The CNN is trained to predict FOXA1 ChIP-seq data in A549 cells. d) Heatmap showing DeepLIFT-Shap feature attribution scores from the sequence-trained CNN at eight CCND1 enhancer variants. e) Top two motifs detected by TF-MoDISco in the genome-wide DeepLIFT-Shap negative feature attribution scores. The TF family of matching motifs is annotated for each motif, as is the number of seqlets used by TF-MoDISco to construct each motif.

Article Snippet: A pooled equimolar mixture of the ChIP-ISO synthetic oligonucleotide library was PCR-amplified and purified via agarose gel purification (Thermo Scientific K0691).

Techniques: ChIP-sequencing, Sequencing, Binding Assay, Genome Wide, Construct

Journal: bioRxiv

Article Title: Systematic Dissection of Sequence Features Affecting the Binding Specificity of a Pioneer Factor Reveals Binding Synergy Between FOXA1 and AP-1

doi: 10.1101/2023.11.08.566246

Figure Lengend Snippet: a) A representative EMSA-seq gel conducted on the ISO library with increasing levels of purified recombinant mouse FOXA1. The lower asterisk represents unbound oligonucleotides, and the upper asterisk indicates FOXA1-bound oligonucleotides. L: 100bp DNA ladder. b) FOXA1 bound fraction as a function of FOXA1 concentration measured by EMSA-seq. Data for a subset of the library, CCND1e variants, is plotted here. Green, yellow, orange, and red curves correspond to CCND1 variants with three, two, one, and zero FOXA1 motifs. The dotted line marks the fraction bound at 15 nM FOXA1, which is used to represent “b g strength in” in c-e. c) Pearson correlation coefficient between FOXA1 binding strength in vitro and CCND1 sequence variables. d) Correlation between the binding strength in vitro and total (summed) FOXA1 motif score for each sequence. e) Correlation between the binding strength in vivo (measured by ChIP-ISO) and that in vitro (EMSA-seq). f) Representative EMSA gels with FOXA1 titration ± AP-1 (left) or AP-1 titration ± FOXA1 (right) using a CCND1 variant with the first two FOXA1 motifs mutated (FOXA112_mut). Different populations are labeled on the right side of the gel (FOXA1 = orange, AP-1 = blue). g) Quantification of the EMSA gel in panel f. Error bar represents standard error for three replicates. h & i) Same as f & g but using different DNA templates and with two replicates performed on each template. Top: CCND1 variant with all three FOXA1 motifs mutated (FOXA1all_mut). Bottom: CCND1 variant with the first two FOXA1 motif and AP-1 motif mutated (FOXA112_mut, AP-1mut).

Article Snippet: A pooled equimolar mixture of the ChIP-ISO synthetic oligonucleotide library was PCR-amplified and purified via agarose gel purification (Thermo Scientific K0691).

Techniques: Purification, Recombinant, Concentration Assay, Binding Assay, In Vitro, Sequencing, In Vivo, Titration, Variant Assay, Labeling

Journal: bioRxiv

Article Title: Systematic Dissection of Sequence Features Affecting the Binding Specificity of a Pioneer Factor Reveals Binding Synergy Between FOXA1 and AP-1

doi: 10.1101/2023.11.08.566246

Figure Lengend Snippet: a) Genomic tracks of TF and histone modification ChIP-seq and ATAC-seq signals at the AAVS1 safe-harbor locus in WT A549 cells. Arrow indicates locus where library sequences were inserted. The insertion site is near active histone marks, but not the repressive ones. b) ChIP-seq signals of repressive histone marks near the native sequences included in our ISO library in . c) Heatmap of FOXA1, H3K9me3, H3K27me3, FOSL2, and Jun ChIP-seq signals for a set of ChIP-ISO library sequences derived from H3K27me3-marked genomic loci (top) and a set of control sequences with comparable FOXA1 binding level derived from euchromatic loci (bottom). d) Violin plot of H3K27me3 ChIP-seq signals for sequences in panel c at their native genomic loci (left) and ChIP-ISO signals of the same sequences at AAVS1 (right). Dark green: H3K27me3 set, and light green: control set from panel c. ****: p < 0.0001 and ns: non-significant. e) Same as panel d, but for FOXA1.

Article Snippet: A pooled equimolar mixture of the ChIP-ISO synthetic oligonucleotide library was PCR-amplified and purified via agarose gel purification (Thermo Scientific K0691).

Techniques: Modification, ChIP-sequencing, Derivative Assay, Binding Assay

Journal: bioRxiv

Article Title: Systematic Dissection of Sequence Features Affecting the Binding Specificity of a Pioneer Factor Reveals Binding Synergy Between FOXA1 and AP-1

doi: 10.1101/2023.11.08.566246

Figure Lengend Snippet: a) ChIP-ISO test cases where strong / weak FOXA1 motifs show low / high FOXA1 binding (set 1 and 2, respectively). Left: FOXA1 motif scores. Right: heatmap of FOXA1 ChIP-seq signals in WT A549. b) Heatmap of FOXA1 ChIP-ISO signals (left) and EMSA-seq signals (fraction bound at 15 nM FOXA1, right) for sequences in panel a. c) Heatmap of FOSL2, JUN, and CEBPB ChIP-seq signals in WT A549 (left) and number of FOXA1, FOSL2, and CEBPB motifs for sequences in panel a. d) Heatmap of FOXA1, H3K9me3, H3K27me3, FOSL2, and Jun ChIP-seq signals in WT A549 for a set of ChIP-ISO library sequences derived from H3K9me3-marked regions (top) and a control set with comparable FOXA1 binding derived from euchromatic loci (bottom). e) Violin plot of H3K9me3 ChIP-seq signals for sequences in panel d at their native genomic loci (left) and ChIP-ISO signals of the same sequences at AAVS1 (right). f) Same as panel e, but for FOXA1. g) Correlation between FOXA1 ChIP-ISO and ChIP-seq signals for all sequences derived from the native genome in our library. Orange: sequences from euchromatin, blue: H3K9me3-marked heterochromatin, green: H3K27me3-marked heterochromatin. h) Precision recall curves showing performance of neural networks trained on FOXA1 ChIP-seq data in A549 cells. Each plot shows performance of a CNN trained using only sequence (blue lines); Bichrom trained using sequence and H3K9me3 (orange lines); Bichrom trained using sequence and ATAC-seq (green lines); and Bichrom trained using sequence and a selection of five histone marks (red lines). The left plot shows the performance of the neural networks across all held-out test sites, while the right plot shows performance at FOXA1 motif instances that overlap H3K9me3 or H3K27me3 peaks. i) Schematic of CRISPRi method where KRAB-dCas9 is induced by doxycycline to ectopically write H3K9me3 to the endogenous CCND1e in A549 cells. This system is used to measure the effect of H3K9me3 deposition on FOXA1 binding. j) H3K9me3 (left) and FOXA1 (right) ChIP-qPCR signals on three biological replicates at a positive control region (P), CCND1e (C1 and C2), and a negative control region (N) with (orange) or without (beige) KRAB-dCas9 induction. ****: p < 0.0001 and ns: non-significant based on two-tailed unpaired t-test.

Article Snippet: A pooled equimolar mixture of the ChIP-ISO synthetic oligonucleotide library was PCR-amplified and purified via agarose gel purification (Thermo Scientific K0691).

Techniques: Binding Assay, ChIP-sequencing, Derivative Assay, Sequencing, Selection, Positive Control, Negative Control, Two Tailed Test