Journal: bioRxiv

Article Title: Interface-guided phenotyping of coding variants in the transcription factor RUNX1 with SEUSS

doi: 10.1101/2023.08.03.551876

Figure Lengend Snippet: An interface-guided Perturb-seq assay for coding variant phenotyping of RUNX1. a. 3D crystal structure of transcription factor CBF, consisting of RUNX1 Runt domain (purple) and CBFB (blue), interacting with DNA (yellow and pink strands) (PDB: 1h9d). b. Amino acid residue map of the RUNX1 Runt domain. Columns represent amino acid residues, while rows represent interaction partners of RUNX1. At each row, interface residues involved in interaction to the partner are highlighted black. Rows are hierarchically clustered. On top: 3D location annotations of each residue (core, intermediate, and surface), followed by VEST and FoldX scores of most damaging mutations targeting the residue. The darker the color, the more damaging (VEST) or destabilizing (FoldX) the mutation is. c. Schematic of lentiviral ORF vector containing the RUNX1 variant (WT, mutated, or GFP) and a 12 base pair barcode sequence unique to each variant for identification during single cell transcriptome sequencing. d. Experimental and computational overview: ORF variant library design, transduction, single cell RNA-sequencing of all 117 library elements, bulk RNA and ATAC-sequencing of 12 selected library elements, and computational analysis.

Article Snippet: The gene overexpression vector was generated from a modified lentiviral vector (Addgene #120426).

Techniques: Variant Assay, Mutagenesis, Plasmid Preparation, Sequencing, Transduction, RNA Sequencing 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 ) 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: Strain, strain background ( H. sapiens ) , HNF4A , K562 , Cat# CCL-243 (ATCC); RRID: CVCL_0004 , Infected with pINDUCER21 lentiviral vector ( ) (Addgene# 46948 ) carrying HNF4A ORF (Addgene# 120450 ) .

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: Strain, strain background ( H. sapiens ) , HNF4A , K562 , Cat# CCL-243 (ATCC); RRID: CVCL_0004 , Infected with pINDUCER21 lentiviral vector ( ) (Addgene# 46948 ) carrying HNF4A ORF (Addgene# 120450 ) .

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: Strain, strain background ( H. sapiens ) , HNF4A , K562 , Cat# CCL-243 (ATCC); RRID: CVCL_0004 , Infected with pINDUCER21 lentiviral vector ( ) (Addgene# 46948 ) carrying HNF4A ORF (Addgene# 120450 ) .

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: Strain, strain background ( H. sapiens ) , HNF4A , K562 , Cat# CCL-243 (ATCC); RRID: CVCL_0004 , Infected with pINDUCER21 lentiviral vector ( ) (Addgene# 46948 ) carrying HNF4A ORF (Addgene# 120450 ) .

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: Strain, strain background ( H. sapiens ) , HNF4A , K562 , Cat# CCL-243 (ATCC); RRID: CVCL_0004 , Infected with pINDUCER21 lentiviral vector ( ) (Addgene# 46948 ) carrying HNF4A ORF (Addgene# 120450 ) .

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: Strain, strain background ( H. sapiens ) , HNF4A , K562 , Cat# CCL-243 (ATCC); RRID: CVCL_0004 , Infected with pINDUCER21 lentiviral vector ( ) (Addgene# 46948 ) carrying HNF4A ORF (Addgene# 120450 ) .

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: Strain, strain background ( H. sapiens ) , HNF4A , K562 , Cat# CCL-243 (ATCC); RRID: CVCL_0004 , Infected with pINDUCER21 lentiviral vector ( ) (Addgene# 46948 ) carrying HNF4A ORF (Addgene# 120450 ) .

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: Strain, strain background ( H. sapiens ) , HNF4A , K562 , Cat# CCL-243 (ATCC); RRID: CVCL_0004 , Infected with pINDUCER21 lentiviral vector ( ) (Addgene# 46948 ) carrying HNF4A ORF (Addgene# 120450 ) .

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: Strain, strain background ( H. sapiens ) , HNF4A , K562 , Cat# CCL-243 (ATCC); RRID: CVCL_0004 , Infected with pINDUCER21 lentiviral vector ( ) (Addgene# 46948 ) carrying HNF4A ORF (Addgene# 120450 ) .

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: Strain, strain background ( H. sapiens ) , HNF4A , K562 , Cat# CCL-243 (ATCC); RRID: CVCL_0004 , Infected with pINDUCER21 lentiviral vector ( ) (Addgene# 46948 ) carrying HNF4A ORF (Addgene# 120450 ) .

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: Strain, strain background ( H. sapiens ) , HNF4A , K562 , Cat# CCL-243 (ATCC); RRID: CVCL_0004 , Infected with pINDUCER21 lentiviral vector ( ) (Addgene# 46948 ) carrying HNF4A ORF (Addgene# 120450 ) .

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: Strain, strain background ( H. sapiens ) , HNF4A , K562 , Cat# CCL-243 (ATCC); RRID: CVCL_0004 , Infected with pINDUCER21 lentiviral vector ( ) (Addgene# 46948 ) carrying HNF4A ORF (Addgene# 120450 ) .

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: Strain, strain background ( H. sapiens ) , HNF4A , K562 , Cat# CCL-243 (ATCC); RRID: CVCL_0004 , Infected with pINDUCER21 lentiviral vector ( ) (Addgene# 46948 ) carrying HNF4A ORF (Addgene# 120450 ) .

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

Journal: Cell Reports Medicine

Article Title: Castrate-resistant prostate cancer response to taxane is determined by an HNF1-dependent apoptosis resistance circuit

doi: 10.1016/j.xcrm.2024.101868

Figure Lengend Snippet:

Article Snippet: Lentivirus was made by transfecting Lenti-X 293T cells (Clontech 632180) with EF1a_HNF1A_P2A_Hygro_Barcode (Addgene plasmid # 120448) or empty vector EF1a_mCherry_P2A_ (Addgene plasmid # 135003) plus psPAX2 and VSV-G packaging plasmids using Lipofectamine 2000 (Invitrogen 11668019).

Techniques: Recombinant, Electron Microscopy, Plasmid Preparation, Software

Journal: Cell reports

Article Title: High-Throughput Screens of PAM-Flexible Cas9 Variants for Gene Knockout and Transcriptional Modulation

doi: 10.1016/j.celrep.2020.02.010

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pCC_03 - lenti-hU6-sgRNA{F+E)-EFS-xGas9-NLS-2A-Puro , This study , Addgene, plasmid #139088.

Techniques: Virus, Recombinant, DNA Extraction, DNA Purification, Staining, Plasmid Preparation, Software