Journal: bioRxiv

Article Title: WILD-seq: Clonal deconvolution of transcriptomic signatures of sensitivity and resistance to cancer therapeutics in vivo

doi: 10.1101/2021.12.09.471927

Figure Lengend Snippet: a. Lentiviral construct design. A PGK promoter drives expression of a transcript encoding zsGreen harbouring a WILD-seq barcode sequence in the 3’UTR. A spacer sequence and polyadenylation signal ensure that that the barcode is detectable as part of a standard oligo dT single cell RNA library preparation and sequencing pipeline. The barcode cassette comprises 2 distinct 12 nucleotide barcode sequences separated by a constant 20 nucleotide linker region. The library of barcode sequences was designed with Hamming distance 5 to allow for sequencing error correction. b. Schematic of WILD-seq method. Tumour cells are infected with the WILD-seq lentiviral library and an appropriate size population of zsGreen positive cells isolated, each of which will express a single unique WILD-seq barcode. This WILD-seq barcoded, heterogenous cell pool is then subjected to an intervention of interest (such as in vivo treatment of the implanted pool with a therapeutic agent) and subsequently analysed by single cell RNA sequencing using the 10X Genomics platform. An additional PCR amplification step is included that specifically enriches for the barcode sequence to increase the number of cells to which a WILD-seq barcode can be conclusively assigned. c. scRNA-seq of in vitro 4T1 WILD-seq cell pool. UMAP plot of in vitro cultured 4T1 WILD-seq cells. Cells for which a WILD-seq clonal barcode is identified are shown as dark grey or coloured spots. Cells which belong to five selected clonal lineages are highlighted. d. scRNA-seq of 4T1 WILD-seq tumours. UMAP plots of vehicle-treated 4T1 WILD-seq tumours generated by injecting the 4T1 WILD-seq pool into the mammary fatpad of BALB/c mice. Four independent experiments were performed each involving injection into 3 separate host animals. Six animals from experiments A and B received vehicle 1 (10% DMSO, 0.9%β- cyclodextrin) and six animals from experiments C and D received vehicle 2 (12.5%ethanol, 12.5% Kolliphor). e. Clonal representation. Proportion of tumour cells assigned to each clonal lineage based on the WILD-seq barcode (n = 1 for in vitro cultured cells, n = 6 for tumours from NSG mice, n = 12 for vehicle-treated tumours from BALB/c mice). Selected clones from the most abundant lineages are plotted. Data represents mean ± SEM. f. Principal component analysis of clonal transcriptomes. Pseudo-bulk analysis was performed by summing counts for all tumour cells expressing the same WILD-seq clonal barcode within an independent experiment. For in vivo tumour samples each point represents the combined cells from 3 animals. Principal component analysis of normalized pseudo-bulk count data showed separation of samples by origin with PC1 and PC2 and separation by clonality with PC3. g. Transcriptomic programs associated with principal components. The top/bottom 50 gene loadings of PC1, PC2 and PC3 were analysed using Enrichr – . h. Clonal transcriptomic signatures from vehicle-treated BALB/c tumours. An AUCell score enrichment was calculated for each clone and for each experiment by comparing cells of a specific clonal lineage of interest to all assigned tumour cells within the same experiment. All gene sets which showed consistent and statistically significant enrichment in one of the six most abundant clones across experiments are illustrated.

Article Snippet: The pHSW8 lentiviral backbone was constructed using a four-way Gibson Assembly (NEB) by inserting a reverse expression cassette, consisting of a PGK promoter, the zsGreen ORF, a cloning site for high-diversity barcode libraries and a synthetic polyA signal, into an empty pCCL-c-MNDU3-X backbone (#81071 Addgene).

Techniques: Construct, Expressing, Sequencing, RNA Library Preparation, Infection, Isolation, In Vivo, RNA Sequencing, Amplification, In Vitro, Cell Culture, Generated, Injection, Clone Assay

Journal: bioRxiv

Article Title: WILD-seq: Clonal deconvolution of transcriptomic signatures of sensitivity and resistance to cancer therapeutics in vivo

doi: 10.1101/2021.12.09.471927

Figure Lengend Snippet: 4T1 WILD-seq tumours were treated with the BET bromodomain inhibitor JQ1 or vehicle from 7 days post-implantation until endpoint (n = 4 mice per condition). Data represents mean ± SEM. b. scRNA-seq of JQ1-treated 4T1 WILD-seq tumours. UMAP plots of vehicle- or JQ1-treated 4T1 WILD-seq tumours. Combined cells from 2 independent experiments, each with 3 mice per condition are shown. Cells for which a WILD-seq clonal barcode is identified are shown as dark grey or coloured spots. Cells which belong to four selected clonal lineages are highlighted. c. JQ1-treatment results in a reduction in Cd8+ tumour-associated T-cells. Cells belonging to the T- cell compartment were computationally extracted from the single cell data and reclustered. Upper panels show combined UMAP plots from experiments A and B with Cd8a expression per cell illustrated enabling identification of the Cd8+ T cell cluster. Lower panels show neighborhood graphs of the results from differential abundance testing using Milo . Coloured nodes represent neighbourhoods with significantly different cell numbers between conditions (FDR < 0.05) and the layout of nodes is determined by the position of the neighborhood index cell in the UMAP panel above. Experiments A and B were analysed separately due to differences in cell numbers. d. Differential gene expression between JQ1- and vehicle-treated tumour cells. Single cell heatmap of expression for genes which are significantly and consistently down-regulated across clonal lineages (combined fisher p-value < 0.05 and mean logFC < −0.2 for both experiments).1600 cells are represented (400 per experiment/condition), grouped according to their clonal lineage. e. Differential gene set expression between JQ1 and vehicle-treated tumour cells. Median AUCell score per experiment/condition for selected gene sets. The 5 clonal lineages with the highest representation across experiments are shown. f. Clonal representation. Proportion of tumour cells assigned to each clonal lineage in experiment A based on the WILD-seq barcode (n = 3 tumours per condition). Clones which make up at least 2% of the assigned tumour cells under at least one condition are plotted. The most sensitive clone 473 is highlighted in blue and the most resistant clones 93, 439, 264 are highlighted in red. Data represents mean ± SEM. g. Clonal response to JQ1-treatment. Log 2 fold change in clonal proportions upon JQ1 treatment across experiments A and B. Fold change was calculated by comparing each JQ1- treated sample with the mean of the 3 corresponding vehicle-treated samples from the same experiment. p-value calculated by one-sample t-test vs a theoretical mean of 0. Data represents mean ± SEM. h. and i. Correlation of JQ1-response with baseline clonal transcriptomic signatures. Clonal gene set enrichment scores for vehicle-treated tumours were calculated by comparing cells of a specific clonal lineage of interest to all assigned tumour cells within the same experiment. Correlation between these scores and JQ1-treatment response (mean log 2 fold change clonal proportion JQ1 vs vehicle) was then calculated for each gene set. Selected gene sets with the highest positive or negative correlation values (Pearson correlation test) are shown. A positive correlation indicates a higher expression in resistant clones, whereas a negative correlation indicates a higher expression in sensitive clones. Resistant clonal lineages identified by barcodes 93, 264 and 439 were combined for the purpose of this analysis to have enough cells for analysis within the vehicle-treated samples.

Article Snippet: The pHSW8 lentiviral backbone was constructed using a four-way Gibson Assembly (NEB) by inserting a reverse expression cassette, consisting of a PGK promoter, the zsGreen ORF, a cloning site for high-diversity barcode libraries and a synthetic polyA signal, into an empty pCCL-c-MNDU3-X backbone (#81071 Addgene).

Techniques: Expressing, Gene Expression, Clone Assay

Journal: bioRxiv

Article Title: WILD-seq: Clonal deconvolution of transcriptomic signatures of sensitivity and resistance to cancer therapeutics in vivo

doi: 10.1101/2021.12.09.471927

Figure Lengend Snippet: a. Tumour growth curves with docetaxel treatment. 4T1 WILD-seq tumours were treated with docetaxel or vehicle (12.5% ethanol, 12.5% Kolliphor) from 7 days post-implantation for 2 weeks (n = 5 mice per condition). Dosing regimen was 12.5 mg/Kg docetaxel three times per week. Data represents mean ± SEM. b. scRNA-seq of docetaxel-treated 4T1 WILD- seq tumours. UMAP plots of vehicle- or docetaxel-treated 4T1 WILD-seq tumours. Combined cells from 2 independent experiments, each with 3 mice per condition are shown. Cells for which a WILD- seq clonal barcode is identified are shown as dark grey or coloured spots. Cells which belong to three selected clonal lineages are highlighted. c. Clonal representation. Proportion of tumour cells assigned to each clonal lineage in experiment C based on the WILD-seq barcode (n = 3 tumours per condition). Clones which make up at least 2% of the assigned tumour cells under at least one condition are plotted. The most sensitive clone 238 is highlighted in blue and the most resistant clone 679 is highlighted in red. Data represents mean ± SEM. d. Clonal response to docetaxel-treatment. Log 2 fold change in clonal proportions upon docetaxel treatment across experiments C and D. Fold change was calculated by comparing each docetaxel-treated sample with the mean of the 3 corresponding vehicle-treated samples from the same experiment. p-values calculated by one-sample t-test vs a theoretical mean of 0. Data represents mean ± SEM. e. and f. Correlation of docetaxel-response with baseline clonal transcriptomic signatures. Clonal gene set enrichment scores for vehicle-treated tumours were calculated by comparing cells of a specific clonal lineage of interest to all assigned tumour cells within the same experiment. Correlation between these scores and docetaxel-treatment response (mean log 2 fold change clonal proportion docetaxel vs vehicle) was then calculated for each gene set. Selected gene sets with the highest positive or negative correlation values (Pearson correlation test) are shown. A positive correlation indicates a higher expression in resistant clones, whereas a negative correlation indicates a higher expression in sensitive clones.

Article Snippet: The pHSW8 lentiviral backbone was constructed using a four-way Gibson Assembly (NEB) by inserting a reverse expression cassette, consisting of a PGK promoter, the zsGreen ORF, a cloning site for high-diversity barcode libraries and a synthetic polyA signal, into an empty pCCL-c-MNDU3-X backbone (#81071 Addgene).

Techniques: Clone Assay, Expressing

Journal: bioRxiv

Article Title: WILD-seq: Clonal deconvolution of transcriptomic signatures of sensitivity and resistance to cancer therapeutics in vivo

doi: 10.1101/2021.12.09.471927

Figure Lengend Snippet: a. D2A1 WILD-seq tumour growth curves with docetaxel treatment. D2A1 WILD-seq tumours were treated with docetaxel or vehicle from 7 days post-implantation for 2 weeks (n = 5 vehicle-treated mice, n = 4 docetaxel-treated mice). Data represents mean ± SEM. b. scRNA-seq of docetaxel-treated D2A1 WILD-seq tumours. UMAP plots of vehicle-treated D2A1 WILD-seq D2A1 tumours and reclustered barcoded-tumour cells from vehicle- and docetaxel-treated tumours. Combined cells from 3 mice per condition are shown. Cells for which a WILD-seq clonal barcode is identified are shown as dark grey or coloured spots. Cells which belong to five selected clonal lineages are highlighted. c. Comparison of EMT status of major 4T1 and D2A1 WILD-seq clones. Violin plot of AUCell scores from vehicle-treated tumour cells generated using the HOLLERN_EMT_BREAST_TUMOR_DN gene set, a set of genes that have low expression in murine mammary tumours of mesenchymal histology. 4T1 WILD-seq clones exhibit varying levels of expression of this geneset whereas D2A1 WILD-seq clones have consistently low levels of expression of these genes. d. Clonal representation. Proportion of tumour cells assigned to each clonal lineage based on the WILD-seq barcode (n = 3 tumours per condition). Clones which make up at least 2% of the assigned tumour cells under at least one condition are plotted. The most sensitive clones to docetaxel treatment 118, 2874 and 1072 are highlighted in blue and the most resistant clones 1240, 1197 and 751 are highlighted in red. Data represents mean ± SEM. e. Clonal transcriptomic signatures from vehicle-treated tumours. Heatmap of median AUCell scores per sample for each of the five most abundant clones. All gene sets which showed consistent and statistically significant enrichment (combined fisher p-value < 0.01 & mean log 2 enrichment > 0.1) in at least one of these clones are illustrated. f. Selected gene sets whose expression is associated with sensitivity to docetaxel. Median AUCell scores per sample for each of the five most abundant clones is plotted. g. Transcriptomic signatures associated with resistance to docetaxel. For vehicle-treated tumours, resistant clonal lineages identified by barcodes 1197, 751 and 1240 were combined to have enough cells for analysis. Gene sets with significantly enriched expression in these resistant clones in vehicle-treated tumours were determined (adjusted p-value < 0.01 & log 2 enrichment > 0.1). A heatmap of median AUCell scores per clone, per condition of these resistance-associated gene sets is plotted. h. Selected gene sets whose expression is enriched or depleted in resistant clones. Median AUCell scores per clone, per sample are plotted for samples with at least 20 cells per clone. Due to changes in clonal abundance with treatment some clones can only be assessed under vehicle- or docetaxel- treated conditions.

Article Snippet: The pHSW8 lentiviral backbone was constructed using a four-way Gibson Assembly (NEB) by inserting a reverse expression cassette, consisting of a PGK promoter, the zsGreen ORF, a cloning site for high-diversity barcode libraries and a synthetic polyA signal, into an empty pCCL-c-MNDU3-X backbone (#81071 Addgene).

Techniques: Comparison, Clone Assay, Generated, Expressing

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) Schematic of experimental design to infect K562 cells with FOXA1- or HNF4A-lentivirus and then perform functional assays on dox-induced cells. In CUT&Tag, a protein A-protein G fusion (pA/G) increases the binding spectrum for Fc-binding and allows Tn5 recruitment to antibody-labeled transcription factor (TF) binding sites. In ATAC-seq, Tn5 homes to any accessible site. And in RNA-seq, polyA RNA is captured and sequenced. ( B ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by FOXA1-HNF4A compared to the number actually activated. Both liver- (p<10 –38 ) and intestinal enrichment (p<10 –13 ) are significant. There are 242 total liver-enriched genes and 122 total intestine-enriched genes. ( C ) Genome browser view of a representative liver-specific locus ( ALB ) in FOXA1-HNF4A clonal line that shows uninduced and induced accessibility, FOXA1 binding, and HNF4A binding. ( D ) Heatmap showing uninduced and induced accessibility at all FOXA1-HNF4A co-bound sites within 50 kb of each FOXA1-HNF4A-activated liver-specific gene (n = 53). ( E ) Meta plot showing average signal across each site from ( D ).

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Functional Assay, Binding Assay, Labeling, RNA Sequencing

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: qPCR measurements made from RNA extracted from either the FOXA1 clonal line ( A–D ) or the HNF4A clonal line ( E–H ) that was treated with either increasing doxycycline concentrations or longer time periods. Expression is displayed as log10 fold induction over either 0 µg/ml doxycycline control (for concentration titration) or time 0 (for time titration). Each sample primer was normalized to the HPRT housekeeping gene. Doxycycline concentration titration measurements were made at 0, 0.01, 0.05, 0.1, 0.5, 2, and 5 µg/ml. Doxycycline treatment time measurements were made at 0, 6, 12, 24, 48, 72, and 96 hr.

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Expressing, Control, Concentration Assay, Titration

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) The number of genome-wide FOXA1 or HNF4A transcription factor binding sites (TFBS) in the induced (+dox) cells that overlap with an ATAC-seq peak in the uninduced (-dox) cells (‘accessible binding site’) or that do not overlap with an ATAC-seq peak in the uninduced (-dox) cells (‘inaccessible binding site’). ( B ) The number of inaccessible binding sites from ( A ) that overlap with an ATAC-seq peak in the induced (+dox) cells (‘opened’) or that do not overlap with an ATAC-seq peak (‘remained closed’). ( C ) The number of FOXA1 or HNF4A binding sites within 50 kb of each FOXA1-HNF4A co-activated gene characterized as either a ‘HepG2 binding site,’ where the TFBS overlaps a TFBS of FOXA1 or HNF4A in HepG2 liver cells, or as a ‘Novel K562 binding site,’ where the TFBS does not overlap with a HepG2 binding site.

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Genome Wide, Binding Assay

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by FOXA1 compared to the number actually activated. Liver enrichment (p<10 –4 ) is significant. There are 242 total liver-enriched genes. ( B ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by HNF4A compared to the number actually activated. Liver- (p<10 –8 ) and intestine enrichment (p<10 –15 ) are significant. There are 242 total liver-enriched genes and 122 total intestine-enriched genes. ( C ) 242 liver genes characterized as activated by Foxa1, HNF4A, both, or neither. ( D ) 122 intestine genes characterized as activated by FOXA1, HNF4A, both, or neither.

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques:

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) Genome browser view of a representative liver-specific locus ( ARG1 ) in FOXA1 clonal line showing uninduced and induced accessibility and FOXA1 binding. ( B ) Genome browser view of a representative liver-specific locus ( APOC3 ) in HNF4A clonal line showing uninduced and induced accessibility and HNF4A binding. ( C ) Heatmap of uninduced and induced accessibility at all FOXA1 binding sites within 50 kb of each FOXA1-activated liver-specific genes (n = 59). ( D ) Heatmap of uninduced and induced accessibility at all HNF4A binding sites within 50 kb of each HNF4A-activated liver-specific genes (n = 76). ( E ) Meta plot showing average signal across each site from ( C ). ( F ) Meta plot showing average signal across each site from ( D ). ( G ) Human FOXA1 and HNF4A sequence logo from JASPAR. ( H ) FOXA1 or HNF4A motif count within 500 bp centered upon FOXA1 or HNF4A binding sites within 50 kb of each FOXA1- or HNF4A-activated liver-specific genes, respectively. Motifs were called with FIMO using 1e-3 a p-value threshold. For each boxplot, the center line represents the median, the box represents the first to third quartiles, and the whiskers represent any points within 1.5× the interquartile range.

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Binding Assay, Sequencing

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) The number of genome-wide FOXA1 or HNF4A transcription factor binding sites (TFBS) in the induced (+dox) cells that overlap with an ATAC-seq peak in the uninduced (-dox) cells (‘aAccessible binding site’) or that do not overlap with an ATAC-seq peak in the uninduced (-dox) cells (‘inaccessible binding site’). ( B ) The number of inaccessible binding sites from ( A ) that overlap with an ATAC-seq peak in the induced (+dox) cells (‘opened’) or that do not overlap with an ATAC-seq peak (‘remained closed’). ( C ) The number of FOXA1 or HNF4A binding sites within 50 kb of each FOXA1- or HNF4A-activated gene characterized as either a ‘HepG2 binding site,’ where the TFBS overlaps a TFBS of FOXA1 or HNF4A in HepG2 liver cells, or as a ‘Novel K562 binding site,’ where the TFBS does not overlap with a HepG2 binding site.

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Genome Wide, Binding Assay

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) FIMO scans at p-value threshold 1e-3 for four most common proposed K562 pioneer factors (PFs) in either FoxA1 inaccessible binding sites (red), Hnf4a inaccessible binding sites (blue), or random equally lengthed binding sites (gray).

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Binding Assay

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) 1000 random 200 bp fragments were generated using BEDTools and then scanned for FOXA1 and HNF4A motifs with FIMO using 1e-3 a p-value threshold. Total motif count was divided by the number of non-N-containing random sequences (924) to identify motifs per random 200 bp fragment.

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Generated

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by FOXA1 at a lower doxycycline concentration (0.05 µg/ml) compared to the number actually activated. There are 242 total liver-enriched genes. ( B ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by HNF4A at a lower doxycycline concentration (0.05 µg/ml) compared to the number actually activated. Liver- (p<10 –5 ) and intestine enrichment (p<10 –14 ) are significant. There are 242 total liver-enriched genes and 122 total intestine-enriched genes. ( C, D ) Genome-wide FOXA1 ( C ) or HNF4A ( D ) binding sites classified as either events that occurred at sites that were accessible or inaccessible in the uninduced (-dox) state at 0.5 and 0.05 µg/ml doxycycline induction.

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Concentration Assay, Genome Wide, Binding Assay

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) Venn diagram of all liver genes categorized as either activated by FOXA1, HNF4A, FOXA1-HNF4A, some combination, or by none of the three cocktails. ( B ) Genome browser view of a representative liver-specific locus ( AMDHD1 ) showing examples of a co-bound site that is ‘FOXA1 pioneered’ (FP), ‘HNF4A pioneered’ (HP), and ‘cooperatively bound’ (CB). The first two tracks are FOXA1 and HNF4A binding in the FOXA1-HNF4A co-expression clone, and the last two tracks are FOXA1 and HNF4A binding in their individual expression clones. ( C ) List of the 31 liver genes that are only activated by FOXA1-HNF4A co-expression. The columns indicate how many co-bound FP, HP, or CB peaks exist within 100 kb of the gene. ( D ) Venn diagram of all genome-wide co-bound peaks categorized as either bound by FOXA1 individually (FP), HNF4A individually (HP), by both, or by neither (CB). ( E ) Overlap of FP, HP, and CB sites from ( D ) with ChromHMM annotations showing the fraction of each co-binding site type in each chromatin region.

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Binding Assay, Expressing, Clone Assay, Genome Wide

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) Venn diagram of all FOXA1-HNF4A-induced differentially accessible peaks categorized by whether the peak was also induced in the FOXA1 clone, HNF4A clone, neither, or both.

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques:

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) FOXA1 or HNF4A motif count at all genomic occurrences of the respective transcription factor’s (TF’s) accessible or inaccessible binding sites. ( B ) FOXA1 or HNF4A motif count in genome-wide inaccessible binding sites versus length-matched random inaccessible DNA sequences. ( C ) Receiver operating characteristic (ROC) curves for predictive power of using sequence motif content to predict accessible (left panels) or inaccessible (right panels) binding sites from random sequence. ( D ) Total FOXA1 and HNF4A motif count at all genomic occurrences of inaccessible co-binding versus length-matched random inaccessible DNA sequences. ( E ) FOXA1 or HNF4A motif count in respective FOXA1 or HNF4A pioneered sites versus in cooperative binding sites (where neither TF bound individually). ( F ) ROC curves for predictive power of using sequence motif content to predict accessible or inaccessible co-binding events from random sequence (top panels) or to predict FOXA1 or HNF4A pioneered events from cooperative binding events. All FIMO scans used 1e-3 as p-value threshold and were conducted on 500 bp of sequence centered upon the binding site.

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Binding Assay, Genome Wide, Sequencing

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet:

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Infection, Plasmid Preparation, cDNA Synthesis, SYBR Green Assay, Software

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) Schematic of experimental design to infect K562 cells with FOXA1- or HNF4A-lentivirus and then perform functional assays on dox-induced cells. In CUT&Tag, a protein A-protein G fusion (pA/G) increases the binding spectrum for Fc-binding and allows Tn5 recruitment to antibody-labeled transcription factor (TF) binding sites. In ATAC-seq, Tn5 homes to any accessible site. And in RNA-seq, polyA RNA is captured and sequenced. ( B ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by FOXA1-HNF4A compared to the number actually activated. Both liver- (p<10 –38 ) and intestinal enrichment (p<10 –13 ) are significant. There are 242 total liver-enriched genes and 122 total intestine-enriched genes. ( C ) Genome browser view of a representative liver-specific locus ( ALB ) in FOXA1-HNF4A clonal line that shows uninduced and induced accessibility, FOXA1 binding, and HNF4A binding. ( D ) Heatmap showing uninduced and induced accessibility at all FOXA1-HNF4A co-bound sites within 50 kb of each FOXA1-HNF4A-activated liver-specific gene (n = 53). ( E ) Meta plot showing average signal across each site from ( D ).

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Functional Assay, Binding Assay, Labeling, RNA Sequencing

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: qPCR measurements made from RNA extracted from either the FOXA1 clonal line ( A–D ) or the HNF4A clonal line ( E–H ) that was treated with either increasing doxycycline concentrations or longer time periods. Expression is displayed as log10 fold induction over either 0 µg/ml doxycycline control (for concentration titration) or time 0 (for time titration). Each sample primer was normalized to the HPRT housekeeping gene. Doxycycline concentration titration measurements were made at 0, 0.01, 0.05, 0.1, 0.5, 2, and 5 µg/ml. Doxycycline treatment time measurements were made at 0, 6, 12, 24, 48, 72, and 96 hr.

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Expressing, Control, Concentration Assay, Titration

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) The number of genome-wide FOXA1 or HNF4A transcription factor binding sites (TFBS) in the induced (+dox) cells that overlap with an ATAC-seq peak in the uninduced (-dox) cells (‘accessible binding site’) or that do not overlap with an ATAC-seq peak in the uninduced (-dox) cells (‘inaccessible binding site’). ( B ) The number of inaccessible binding sites from ( A ) that overlap with an ATAC-seq peak in the induced (+dox) cells (‘opened’) or that do not overlap with an ATAC-seq peak (‘remained closed’). ( C ) The number of FOXA1 or HNF4A binding sites within 50 kb of each FOXA1-HNF4A co-activated gene characterized as either a ‘HepG2 binding site,’ where the TFBS overlaps a TFBS of FOXA1 or HNF4A in HepG2 liver cells, or as a ‘Novel K562 binding site,’ where the TFBS does not overlap with a HepG2 binding site.

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Genome Wide, Binding Assay

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by FOXA1 compared to the number actually activated. Liver enrichment (p<10 –4 ) is significant. There are 242 total liver-enriched genes. ( B ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by HNF4A compared to the number actually activated. Liver- (p<10 –8 ) and intestine enrichment (p<10 –15 ) are significant. There are 242 total liver-enriched genes and 122 total intestine-enriched genes. ( C ) 242 liver genes characterized as activated by Foxa1, HNF4A, both, or neither. ( D ) 122 intestine genes characterized as activated by FOXA1, HNF4A, both, or neither.

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques:

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) Genome browser view of a representative liver-specific locus ( ARG1 ) in FOXA1 clonal line showing uninduced and induced accessibility and FOXA1 binding. ( B ) Genome browser view of a representative liver-specific locus ( APOC3 ) in HNF4A clonal line showing uninduced and induced accessibility and HNF4A binding. ( C ) Heatmap of uninduced and induced accessibility at all FOXA1 binding sites within 50 kb of each FOXA1-activated liver-specific genes (n = 59). ( D ) Heatmap of uninduced and induced accessibility at all HNF4A binding sites within 50 kb of each HNF4A-activated liver-specific genes (n = 76). ( E ) Meta plot showing average signal across each site from ( C ). ( F ) Meta plot showing average signal across each site from ( D ). ( G ) Human FOXA1 and HNF4A sequence logo from JASPAR. ( H ) FOXA1 or HNF4A motif count within 500 bp centered upon FOXA1 or HNF4A binding sites within 50 kb of each FOXA1- or HNF4A-activated liver-specific genes, respectively. Motifs were called with FIMO using 1e-3 a p-value threshold. For each boxplot, the center line represents the median, the box represents the first to third quartiles, and the whiskers represent any points within 1.5× the interquartile range.

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Binding Assay, Sequencing

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) The number of genome-wide FOXA1 or HNF4A transcription factor binding sites (TFBS) in the induced (+dox) cells that overlap with an ATAC-seq peak in the uninduced (-dox) cells (‘aAccessible binding site’) or that do not overlap with an ATAC-seq peak in the uninduced (-dox) cells (‘inaccessible binding site’). ( B ) The number of inaccessible binding sites from ( A ) that overlap with an ATAC-seq peak in the induced (+dox) cells (‘opened’) or that do not overlap with an ATAC-seq peak (‘remained closed’). ( C ) The number of FOXA1 or HNF4A binding sites within 50 kb of each FOXA1- or HNF4A-activated gene characterized as either a ‘HepG2 binding site,’ where the TFBS overlaps a TFBS of FOXA1 or HNF4A in HepG2 liver cells, or as a ‘Novel K562 binding site,’ where the TFBS does not overlap with a HepG2 binding site.

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Genome Wide, Binding Assay

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) FIMO scans at p-value threshold 1e-3 for four most common proposed K562 pioneer factors (PFs) in either FoxA1 inaccessible binding sites (red), Hnf4a inaccessible binding sites (blue), or random equally lengthed binding sites (gray).

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Binding Assay

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) 1000 random 200 bp fragments were generated using BEDTools and then scanned for FOXA1 and HNF4A motifs with FIMO using 1e-3 a p-value threshold. Total motif count was divided by the number of non-N-containing random sequences (924) to identify motifs per random 200 bp fragment.

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Generated

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by FOXA1 at a lower doxycycline concentration (0.05 µg/ml) compared to the number actually activated. There are 242 total liver-enriched genes. ( B ) The number of tissue-specific genes predicted from the hypergeometric distribution to be activated by HNF4A at a lower doxycycline concentration (0.05 µg/ml) compared to the number actually activated. Liver- (p<10 –5 ) and intestine enrichment (p<10 –14 ) are significant. There are 242 total liver-enriched genes and 122 total intestine-enriched genes. ( C, D ) Genome-wide FOXA1 ( C ) or HNF4A ( D ) binding sites classified as either events that occurred at sites that were accessible or inaccessible in the uninduced (-dox) state at 0.5 and 0.05 µg/ml doxycycline induction.

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Concentration Assay, Genome Wide, Binding Assay

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) Venn diagram of all liver genes categorized as either activated by FOXA1, HNF4A, FOXA1-HNF4A, some combination, or by none of the three cocktails. ( B ) Genome browser view of a representative liver-specific locus ( AMDHD1 ) showing examples of a co-bound site that is ‘FOXA1 pioneered’ (FP), ‘HNF4A pioneered’ (HP), and ‘cooperatively bound’ (CB). The first two tracks are FOXA1 and HNF4A binding in the FOXA1-HNF4A co-expression clone, and the last two tracks are FOXA1 and HNF4A binding in their individual expression clones. ( C ) List of the 31 liver genes that are only activated by FOXA1-HNF4A co-expression. The columns indicate how many co-bound FP, HP, or CB peaks exist within 100 kb of the gene. ( D ) Venn diagram of all genome-wide co-bound peaks categorized as either bound by FOXA1 individually (FP), HNF4A individually (HP), by both, or by neither (CB). ( E ) Overlap of FP, HP, and CB sites from ( D ) with ChromHMM annotations showing the fraction of each co-binding site type in each chromatin region.

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Binding Assay, Expressing, Clone Assay, Genome Wide

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) Venn diagram of all FOXA1-HNF4A-induced differentially accessible peaks categorized by whether the peak was also induced in the FOXA1 clone, HNF4A clone, neither, or both.

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques:

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet: ( A ) FOXA1 or HNF4A motif count at all genomic occurrences of the respective transcription factor’s (TF’s) accessible or inaccessible binding sites. ( B ) FOXA1 or HNF4A motif count in genome-wide inaccessible binding sites versus length-matched random inaccessible DNA sequences. ( C ) Receiver operating characteristic (ROC) curves for predictive power of using sequence motif content to predict accessible (left panels) or inaccessible (right panels) binding sites from random sequence. ( D ) Total FOXA1 and HNF4A motif count at all genomic occurrences of inaccessible co-binding versus length-matched random inaccessible DNA sequences. ( E ) FOXA1 or HNF4A motif count in respective FOXA1 or HNF4A pioneered sites versus in cooperative binding sites (where neither TF bound individually). ( F ) ROC curves for predictive power of using sequence motif content to predict accessible or inaccessible co-binding events from random sequence (top panels) or to predict FOXA1 or HNF4A pioneered events from cooperative binding events. All FIMO scans used 1e-3 as p-value threshold and were conducted on 500 bp of sequence centered upon the binding site.

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Binding Assay, Genome Wide, Sequencing

Journal: eLife

Article Title: A test of the pioneer factor hypothesis using ectopic liver gene activation

doi: 10.7554/eLife.73358

Figure Lengend Snippet:

Article Snippet: We used PCR to add V5 epitope tags to the 3′ end of FOXA1 (Addgene #120438, Watertown, MA) and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly (NEB #E2621L, Ipswich, MA) to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948).

Techniques: Infection, Plasmid Preparation, cDNA Synthesis, SYBR Green Assay, Software