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rabbit polyclonal anti-aff1  (Bethyl)


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    Bethyl rabbit polyclonal anti-aff1
    a – d Western blots showing knockdown (KD) efficiency of the shRNA’s targeting <t>AFF1</t> or AFF4. ( n = 3 biological replicates, AFF1 sh1 P = 0.0131, AFF1 sh2 P = 0.0227, AFF4 sh1 P < 0.0001, AFF4 sh2 P < 0.0001, two-tailed, unpaired t test, data are presented as mean values ± standard deviation). e – h Representative images and quantification of clonogenic assays comparing keratinocytes expressing shRNA’s targeting AFF1 or AFF4, versus non-targeting control shRNA ( n = 2–3 technical replicates). i , j Representative images and red:green fluorescence-quantification of epidermal tissue sections. Each piece of epidermal tissue was regenerated with 50% of GFP-labeled keratinocytes expressing non-targeting control shRNA and 50% of DsRed-labeled keratinocytes expressing non-targeting control shRNA (left), AFF1-targeting shRNA 1 (middle) or AFF4-targeting shRNA 1 (right). DsRed-labeled cells are represented in magenta. (scale bar = 100 µm, n = 15, **** P < 0.0001, two-tailed, unpaired t test, box plot represents first through third quartiles, minima, and maxima). k Comparison of genes differentially expressed in RNA-seq with AFF1 KD, AFF4 KD, and in both AFF1 and AFF4 KD conditions. l Top Gene Ontology (GO) terms of genes differentially expressed in both AFF1 and AFF4 KD RNA-seq data sets (two-tailed, Fisher’s exact test). m Top GO terms of genes differentially expressed in AFF1 but not AFF4 KD RNA-seq (two-tailed, Fisher’s exact test). n , o qRT-PCR comparing the expression of differentiation-activating TFs between non-targeting control and AFF1 or AFF4 KD ( n = 3 technical replicates, data are presented as mean values ± standard deviation). p Principal Component Analysis (PCA) of AFF1 KD, AFF4 KD, and KL 24-hour RNA-seq data sets. q – s qRT-PCR comparing mRNA levels of differentiation-activating TFs between DMSO control and KL2 treatment with non-targeting control or AFF1 knockdown using two independent shRNA’s. Control non-targeting KL is relative to non-targeting DMSO, AFF1 sh1 KL is relative to AFF1 sh1 DMSO, and AFF1 sh2 KL is relative to AFF1 sh2 DMSO ( n = 3 technical replicates, data are presented as mean values ± standard deviation). Source data are provided as a Source Data file.
    Rabbit Polyclonal Anti Aff1, supplied by Bethyl, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/anti+aff1/Rabbit+Albumin+Antibody/pmc09338292-358-16-19
    Average 90 stars, based on 1 article reviews
    rabbit polyclonal anti-aff1 - by Bioz Stars, 2026-10
    90/100 stars

    Images

    1) Product Images from "CDK9 activity switch associated with AFF1 and HEXIM1 controls differentiation initiation from epidermal progenitors"

    Article Title: CDK9 activity switch associated with AFF1 and HEXIM1 controls differentiation initiation from epidermal progenitors

    Journal: Nature Communications

    doi: 10.1038/s41467-022-32098-2

    a – d Western blots showing knockdown (KD) efficiency of the shRNA’s targeting AFF1 or AFF4. ( n = 3 biological replicates, AFF1 sh1 P = 0.0131, AFF1 sh2 P = 0.0227, AFF4 sh1 P < 0.0001, AFF4 sh2 P < 0.0001, two-tailed, unpaired t test, data are presented as mean values ± standard deviation). e – h Representative images and quantification of clonogenic assays comparing keratinocytes expressing shRNA’s targeting AFF1 or AFF4, versus non-targeting control shRNA ( n = 2–3 technical replicates). i , j Representative images and red:green fluorescence-quantification of epidermal tissue sections. Each piece of epidermal tissue was regenerated with 50% of GFP-labeled keratinocytes expressing non-targeting control shRNA and 50% of DsRed-labeled keratinocytes expressing non-targeting control shRNA (left), AFF1-targeting shRNA 1 (middle) or AFF4-targeting shRNA 1 (right). DsRed-labeled cells are represented in magenta. (scale bar = 100 µm, n = 15, **** P < 0.0001, two-tailed, unpaired t test, box plot represents first through third quartiles, minima, and maxima). k Comparison of genes differentially expressed in RNA-seq with AFF1 KD, AFF4 KD, and in both AFF1 and AFF4 KD conditions. l Top Gene Ontology (GO) terms of genes differentially expressed in both AFF1 and AFF4 KD RNA-seq data sets (two-tailed, Fisher’s exact test). m Top GO terms of genes differentially expressed in AFF1 but not AFF4 KD RNA-seq (two-tailed, Fisher’s exact test). n , o qRT-PCR comparing the expression of differentiation-activating TFs between non-targeting control and AFF1 or AFF4 KD ( n = 3 technical replicates, data are presented as mean values ± standard deviation). p Principal Component Analysis (PCA) of AFF1 KD, AFF4 KD, and KL 24-hour RNA-seq data sets. q – s qRT-PCR comparing mRNA levels of differentiation-activating TFs between DMSO control and KL2 treatment with non-targeting control or AFF1 knockdown using two independent shRNA’s. Control non-targeting KL is relative to non-targeting DMSO, AFF1 sh1 KL is relative to AFF1 sh1 DMSO, and AFF1 sh2 KL is relative to AFF1 sh2 DMSO ( n = 3 technical replicates, data are presented as mean values ± standard deviation). Source data are provided as a Source Data file.
    Figure Legend Snippet: a – d Western blots showing knockdown (KD) efficiency of the shRNA’s targeting AFF1 or AFF4. ( n = 3 biological replicates, AFF1 sh1 P = 0.0131, AFF1 sh2 P = 0.0227, AFF4 sh1 P < 0.0001, AFF4 sh2 P < 0.0001, two-tailed, unpaired t test, data are presented as mean values ± standard deviation). e – h Representative images and quantification of clonogenic assays comparing keratinocytes expressing shRNA’s targeting AFF1 or AFF4, versus non-targeting control shRNA ( n = 2–3 technical replicates). i , j Representative images and red:green fluorescence-quantification of epidermal tissue sections. Each piece of epidermal tissue was regenerated with 50% of GFP-labeled keratinocytes expressing non-targeting control shRNA and 50% of DsRed-labeled keratinocytes expressing non-targeting control shRNA (left), AFF1-targeting shRNA 1 (middle) or AFF4-targeting shRNA 1 (right). DsRed-labeled cells are represented in magenta. (scale bar = 100 µm, n = 15, **** P < 0.0001, two-tailed, unpaired t test, box plot represents first through third quartiles, minima, and maxima). k Comparison of genes differentially expressed in RNA-seq with AFF1 KD, AFF4 KD, and in both AFF1 and AFF4 KD conditions. l Top Gene Ontology (GO) terms of genes differentially expressed in both AFF1 and AFF4 KD RNA-seq data sets (two-tailed, Fisher’s exact test). m Top GO terms of genes differentially expressed in AFF1 but not AFF4 KD RNA-seq (two-tailed, Fisher’s exact test). n , o qRT-PCR comparing the expression of differentiation-activating TFs between non-targeting control and AFF1 or AFF4 KD ( n = 3 technical replicates, data are presented as mean values ± standard deviation). p Principal Component Analysis (PCA) of AFF1 KD, AFF4 KD, and KL 24-hour RNA-seq data sets. q – s qRT-PCR comparing mRNA levels of differentiation-activating TFs between DMSO control and KL2 treatment with non-targeting control or AFF1 knockdown using two independent shRNA’s. Control non-targeting KL is relative to non-targeting DMSO, AFF1 sh1 KL is relative to AFF1 sh1 DMSO, and AFF1 sh2 KL is relative to AFF1 sh2 DMSO ( n = 3 technical replicates, data are presented as mean values ± standard deviation). Source data are provided as a Source Data file.

    Techniques Used: Western Blot, Two Tailed Test, Standard Deviation, Expressing, shRNA, Fluorescence, Labeling, RNA Sequencing Assay, Quantitative RT-PCR

    a qRT-PCR showing mRNA levels of differentiation activators with 3-hour CDK9 inhibitor (CDK9i) treatment with flavopiridol (Flav) or NVP2 relative to DMSO control ( n = 3 technical replicates, data are presented as mean values ± standard deviation). b qRT-PCR showing the expression of differentiation-activating TFs in keratinocytes treated with KL alone or in combination with CDK9 inhibitors, as compared to DMSO control ( n = 3 technical replicates, data are presented as mean values ± standard deviation). c Western blot showing knockdown (KD) efficiency of HEXIM1 shRNA’s ( n = 3 biological replicates, quantified in Supplementary Fig. ). d Clonogenic assay of human keratinocytes expressing HEXIM1-shRNA’s or non-targeting control shRNA. e Quantification of colonies >1 mm 2 in HEXIM1 KD conditions relative to control ( n = 3/group, **** P < 0.0001, two-tailed, unpaired t test, data are presented as mean values ± standard deviation). f qRT-PCR comparing the mRNA levels of differentiation activators in HEXIM1 KD relative to control ( n = 3 technical replicates, data are presented as mean values ± standard deviation). g – i qRT-PCR showing the relative expression of differentiation-activating TFs between DMSO control and KL treatment where control non-targeting KL is relative to non-targeting DMSO, HEXIM1 sh1 KL is relative to HEXIM1 sh1 DMSO, and HEXIM1 sh2 KL is relative to HEXIM1 sh2 DMSO ( n = 3 technical replicates, data are presented as mean values ± standard deviation). j Venn diagram comparing genes differentially expressed in AFF1 KD versus HEXIM1 KD RNA-seq (Fisher’s exact test, two-Tail, P = 2 × 10 −220 ). k Heatmap showing shared genes significantly changed (fold change ≥2, P < 0.05, two-tailed, Wald test) in AFF1 KD RNA-seq or HEXIM1 KD RNA-seq. l Top Gene Ontology terms of the genes differentially expressed in AFF1 and HEXIM1 KD RNA-seq (two-tailed, Fisher’s exact test). m , n Co-immunoprecipitation of HA-AFF1 western blot showing interactions between AFF1 and HEXIM1, CDK9, and phosphorylated threonine 186 CDK9 (P-CDK) in progenitors (UD) and in differentiated (DF) keratinocytes ( n = 3 biological replicates). Source data are provided as a Source Data file.
    Figure Legend Snippet: a qRT-PCR showing mRNA levels of differentiation activators with 3-hour CDK9 inhibitor (CDK9i) treatment with flavopiridol (Flav) or NVP2 relative to DMSO control ( n = 3 technical replicates, data are presented as mean values ± standard deviation). b qRT-PCR showing the expression of differentiation-activating TFs in keratinocytes treated with KL alone or in combination with CDK9 inhibitors, as compared to DMSO control ( n = 3 technical replicates, data are presented as mean values ± standard deviation). c Western blot showing knockdown (KD) efficiency of HEXIM1 shRNA’s ( n = 3 biological replicates, quantified in Supplementary Fig. ). d Clonogenic assay of human keratinocytes expressing HEXIM1-shRNA’s or non-targeting control shRNA. e Quantification of colonies >1 mm 2 in HEXIM1 KD conditions relative to control ( n = 3/group, **** P < 0.0001, two-tailed, unpaired t test, data are presented as mean values ± standard deviation). f qRT-PCR comparing the mRNA levels of differentiation activators in HEXIM1 KD relative to control ( n = 3 technical replicates, data are presented as mean values ± standard deviation). g – i qRT-PCR showing the relative expression of differentiation-activating TFs between DMSO control and KL treatment where control non-targeting KL is relative to non-targeting DMSO, HEXIM1 sh1 KL is relative to HEXIM1 sh1 DMSO, and HEXIM1 sh2 KL is relative to HEXIM1 sh2 DMSO ( n = 3 technical replicates, data are presented as mean values ± standard deviation). j Venn diagram comparing genes differentially expressed in AFF1 KD versus HEXIM1 KD RNA-seq (Fisher’s exact test, two-Tail, P = 2 × 10 −220 ). k Heatmap showing shared genes significantly changed (fold change ≥2, P < 0.05, two-tailed, Wald test) in AFF1 KD RNA-seq or HEXIM1 KD RNA-seq. l Top Gene Ontology terms of the genes differentially expressed in AFF1 and HEXIM1 KD RNA-seq (two-tailed, Fisher’s exact test). m , n Co-immunoprecipitation of HA-AFF1 western blot showing interactions between AFF1 and HEXIM1, CDK9, and phosphorylated threonine 186 CDK9 (P-CDK) in progenitors (UD) and in differentiated (DF) keratinocytes ( n = 3 biological replicates). Source data are provided as a Source Data file.

    Techniques Used: Quantitative RT-PCR, Standard Deviation, Expressing, Western Blot, Clonogenic Assay, shRNA, Two Tailed Test, RNA Sequencing Assay, Immunoprecipitation

    a Venn diagram showing the significant overlap between AFF1 and HEXIM1 ChIP-seq peaks (Fisher’s exact test, two-tailed, P = 1×10 −268 ). b Violin plot showing the significant enrichment of total Pol II counts at ChIP-seq categories: AFF1 unique peaks, HEXIM1 unique peaks, and peaks with Pol II but no HEXIM1 or AFF1 relative to overlapping AFF1 and HEXIM1 peaks (**** P < 0.0001, two-tailed, unpaired t test). c Pie chart showing the peak distribution of AFF1 and HEXIM1 ChIP-seq data in promoters (transcription start site (TSS) ± 1 kb), gene bodies (TSS ± 1 kb through transcription end site), or intergenic regions. d Average profile plot showing the overlapping enrichment of AFF1 and HEXIM1 ChIP-seq signal near the promoters of their shared target genes. e Shared target genes among AFF1 ChIP-seq, HEXIM1 ChIP-seq, and the significantly upregulated genes in keratinocytes with 3-hr KL treatment. f Relative expression of the 92 AFF1-HEXIM1-KL(3 hr) shared target genes, ranked from low to high, in keratinocytes treated with KL versus DMSO for 3 hours. The top two upregulated genes ATF3 and DUSP1, as well as RND3, are highlighted in orange. g Heatmap showing differential expression of rapid-response and differentiation-activating genes with 1-hour and 3-hour nascent RNA-seq. h , i qRT-PCR showing the upregulation of ATF3, DUSP1, and RND3 with AFF1 or HEXIM1 knockdown ( n = 3 technical replicates, data are presented as mean values ± standard deviation). j – l Genome browser tracks showing the enrichment of AFF1, HEXIM1, CDK9, undifferentiated (UD) Pol II, and differentiated (DF) Pol II ChIP-seq signal at ATF3, DUSP1, and RND3. Source data are provided as a Source Data file.
    Figure Legend Snippet: a Venn diagram showing the significant overlap between AFF1 and HEXIM1 ChIP-seq peaks (Fisher’s exact test, two-tailed, P = 1×10 −268 ). b Violin plot showing the significant enrichment of total Pol II counts at ChIP-seq categories: AFF1 unique peaks, HEXIM1 unique peaks, and peaks with Pol II but no HEXIM1 or AFF1 relative to overlapping AFF1 and HEXIM1 peaks (**** P < 0.0001, two-tailed, unpaired t test). c Pie chart showing the peak distribution of AFF1 and HEXIM1 ChIP-seq data in promoters (transcription start site (TSS) ± 1 kb), gene bodies (TSS ± 1 kb through transcription end site), or intergenic regions. d Average profile plot showing the overlapping enrichment of AFF1 and HEXIM1 ChIP-seq signal near the promoters of their shared target genes. e Shared target genes among AFF1 ChIP-seq, HEXIM1 ChIP-seq, and the significantly upregulated genes in keratinocytes with 3-hr KL treatment. f Relative expression of the 92 AFF1-HEXIM1-KL(3 hr) shared target genes, ranked from low to high, in keratinocytes treated with KL versus DMSO for 3 hours. The top two upregulated genes ATF3 and DUSP1, as well as RND3, are highlighted in orange. g Heatmap showing differential expression of rapid-response and differentiation-activating genes with 1-hour and 3-hour nascent RNA-seq. h , i qRT-PCR showing the upregulation of ATF3, DUSP1, and RND3 with AFF1 or HEXIM1 knockdown ( n = 3 technical replicates, data are presented as mean values ± standard deviation). j – l Genome browser tracks showing the enrichment of AFF1, HEXIM1, CDK9, undifferentiated (UD) Pol II, and differentiated (DF) Pol II ChIP-seq signal at ATF3, DUSP1, and RND3. Source data are provided as a Source Data file.

    Techniques Used: ChIP-sequencing, Two Tailed Test, Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Standard Deviation

    a – c Keratinocytes expressing HA-CDK9 were treated with DMSO control, KL, or TPA. Immunoprecipitation using the HA antibody was performed from the lysate of these keratinocytes, followed by western blot probing for HA and HEXIM1. d – g Violin and ECDF plots showing HA-CDK9 traveling ratio (TR) with KL or TPA relative to DMSO control at 92, direct, rapid-response genes (**** P < 0.001 *** P < 0.01, d , e KL P = 0.0002, f , g P = 0.0041, two-tailed, unpaired t test). A higher travel ratio indicates high proportion of CDK9 binding in the gene body. h Genome browser tracks comparing HA-CDK9 ChIP-seq enrichment at ATF3 among the DMSO control, KL treatment, or TPA treatment. i Illustration of the working model. In the progenitor state, AFF1 and HEXIM1 cooperatively hold CDK9 in an inactive state. With SEC disruption (KL) or PKC signaling (TPA), CDK9 from SEC-AFF1 is rapidly released into an active form. This rapid switch allows the activation of SEC-direct-target genes such as ATF3, which can further promote the expression of differentiation-activating transcription factors (GRHL3, PRDM1, ZNF750, and OVOL1, etc) to advance the terminal differentiation process. Source data are provided as a Source Data file.
    Figure Legend Snippet: a – c Keratinocytes expressing HA-CDK9 were treated with DMSO control, KL, or TPA. Immunoprecipitation using the HA antibody was performed from the lysate of these keratinocytes, followed by western blot probing for HA and HEXIM1. d – g Violin and ECDF plots showing HA-CDK9 traveling ratio (TR) with KL or TPA relative to DMSO control at 92, direct, rapid-response genes (**** P < 0.001 *** P < 0.01, d , e KL P = 0.0002, f , g P = 0.0041, two-tailed, unpaired t test). A higher travel ratio indicates high proportion of CDK9 binding in the gene body. h Genome browser tracks comparing HA-CDK9 ChIP-seq enrichment at ATF3 among the DMSO control, KL treatment, or TPA treatment. i Illustration of the working model. In the progenitor state, AFF1 and HEXIM1 cooperatively hold CDK9 in an inactive state. With SEC disruption (KL) or PKC signaling (TPA), CDK9 from SEC-AFF1 is rapidly released into an active form. This rapid switch allows the activation of SEC-direct-target genes such as ATF3, which can further promote the expression of differentiation-activating transcription factors (GRHL3, PRDM1, ZNF750, and OVOL1, etc) to advance the terminal differentiation process. Source data are provided as a Source Data file.

    Techniques Used: Expressing, Immunoprecipitation, Western Blot, Two Tailed Test, Binding Assay, ChIP-sequencing, Activation Assay

    Related Articles

    Chromatin Immunoprecipitation:

    Article Title: AFF1 inhibits adipogenic differentiation via targeting TGM2 transcription
    Article Snippet: Chromatin immunoprecipitation (ChIP) assays were performed utilizing EZ‐ZymeTM Chromatin Prep Kit (#17‐375, Millipore) and EZ‐Magna ChIPTM HiSens Chromatin Immunoprecipitation Kit (#17‐10461, Millipore) according to the manufacturer's protocol. .. The antibodies used for ChIP assay were anti‐AFF1 (#A302‐344A, Bethyl, 4 μg/test) and control IgG (#CS200581, Millipore, 4 μg/test). ..

    Article Title: DOT1L-controlled cell-fate determination and transcription elongation are independent of H3K79 methylation
    Article Snippet: .. The following antibodies were used in this study: anti-DOT1L [generated in house ( 17 ), 1: 500 for Western blotting]; anti-H3K79me1 (generated in house, 1:1,000 for Western blotting, 1:100 for ChIP-seq); anti-H3K79me2 (Abcam 3594, 1 μg/mL for Western blotting); anti-H3K79me2 (generated in house, 1:100 for ChIP-seq); anti-H3K79me3 (Abcam 2621, 1 μg/mL for Western blotting, 1:100 for ChIP-seq); anti-GFAP (Abcam 7260, 1:1,000 for immunostaining); anti-βIII tubulin/TUJ1 (Millipore MAB1637, 1:50 for immunostaining); anti-AFF1 (Bethyl A302-344A, 1:1,000 for Western blotting); anti-AF9 (generated in house, 1:4,000 for Western blotting); anti-ENL (Cell Signaling Technology 14893, 1:1,000 for Western blotting); anti-RBBP5 (Bethyl A300-109A, 1:5,000 for Western blotting); anti-AF10 (Santa Cruz 53156, 1:100 for ChIP-seq); and anti-RNA Pol II (Cell Signaling Technology 14958, 1:100 for ChIP-seq). ..

    Control:

    Article Title: AFF1 inhibits adipogenic differentiation via targeting TGM2 transcription
    Article Snippet: Chromatin immunoprecipitation (ChIP) assays were performed utilizing EZ‐ZymeTM Chromatin Prep Kit (#17‐375, Millipore) and EZ‐Magna ChIPTM HiSens Chromatin Immunoprecipitation Kit (#17‐10461, Millipore) according to the manufacturer's protocol. .. The antibodies used for ChIP assay were anti‐AFF1 (#A302‐344A, Bethyl, 4 μg/test) and control IgG (#CS200581, Millipore, 4 μg/test). ..

    Generated:

    Article Title: DOT1L-controlled cell-fate determination and transcription elongation are independent of H3K79 methylation
    Article Snippet: .. The following antibodies were used in this study: anti-DOT1L [generated in house ( 17 ), 1: 500 for Western blotting]; anti-H3K79me1 (generated in house, 1:1,000 for Western blotting, 1:100 for ChIP-seq); anti-H3K79me2 (Abcam 3594, 1 μg/mL for Western blotting); anti-H3K79me2 (generated in house, 1:100 for ChIP-seq); anti-H3K79me3 (Abcam 2621, 1 μg/mL for Western blotting, 1:100 for ChIP-seq); anti-GFAP (Abcam 7260, 1:1,000 for immunostaining); anti-βIII tubulin/TUJ1 (Millipore MAB1637, 1:50 for immunostaining); anti-AFF1 (Bethyl A302-344A, 1:1,000 for Western blotting); anti-AF9 (generated in house, 1:4,000 for Western blotting); anti-ENL (Cell Signaling Technology 14893, 1:1,000 for Western blotting); anti-RBBP5 (Bethyl A300-109A, 1:5,000 for Western blotting); anti-AF10 (Santa Cruz 53156, 1:100 for ChIP-seq); and anti-RNA Pol II (Cell Signaling Technology 14958, 1:100 for ChIP-seq). ..

    Western Blot:

    Article Title: DOT1L-controlled cell-fate determination and transcription elongation are independent of H3K79 methylation
    Article Snippet: .. The following antibodies were used in this study: anti-DOT1L [generated in house ( 17 ), 1: 500 for Western blotting]; anti-H3K79me1 (generated in house, 1:1,000 for Western blotting, 1:100 for ChIP-seq); anti-H3K79me2 (Abcam 3594, 1 μg/mL for Western blotting); anti-H3K79me2 (generated in house, 1:100 for ChIP-seq); anti-H3K79me3 (Abcam 2621, 1 μg/mL for Western blotting, 1:100 for ChIP-seq); anti-GFAP (Abcam 7260, 1:1,000 for immunostaining); anti-βIII tubulin/TUJ1 (Millipore MAB1637, 1:50 for immunostaining); anti-AFF1 (Bethyl A302-344A, 1:1,000 for Western blotting); anti-AF9 (generated in house, 1:4,000 for Western blotting); anti-ENL (Cell Signaling Technology 14893, 1:1,000 for Western blotting); anti-RBBP5 (Bethyl A300-109A, 1:5,000 for Western blotting); anti-AF10 (Santa Cruz 53156, 1:100 for ChIP-seq); and anti-RNA Pol II (Cell Signaling Technology 14958, 1:100 for ChIP-seq). ..

    Immunostaining:

    Article Title: DOT1L-controlled cell-fate determination and transcription elongation are independent of H3K79 methylation
    Article Snippet: .. The following antibodies were used in this study: anti-DOT1L [generated in house ( 17 ), 1: 500 for Western blotting]; anti-H3K79me1 (generated in house, 1:1,000 for Western blotting, 1:100 for ChIP-seq); anti-H3K79me2 (Abcam 3594, 1 μg/mL for Western blotting); anti-H3K79me2 (generated in house, 1:100 for ChIP-seq); anti-H3K79me3 (Abcam 2621, 1 μg/mL for Western blotting, 1:100 for ChIP-seq); anti-GFAP (Abcam 7260, 1:1,000 for immunostaining); anti-βIII tubulin/TUJ1 (Millipore MAB1637, 1:50 for immunostaining); anti-AFF1 (Bethyl A302-344A, 1:1,000 for Western blotting); anti-AF9 (generated in house, 1:4,000 for Western blotting); anti-ENL (Cell Signaling Technology 14893, 1:1,000 for Western blotting); anti-RBBP5 (Bethyl A300-109A, 1:5,000 for Western blotting); anti-AF10 (Santa Cruz 53156, 1:100 for ChIP-seq); and anti-RNA Pol II (Cell Signaling Technology 14958, 1:100 for ChIP-seq). ..

    other:

    Article Title: ELL-associated factors EAF1/2 negatively regulate HIV-1 transcription through inhibition of Super Elongation Complex formation.
    Article Snippet: This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record.. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article.. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.



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    Image Search Results


    a – d Western blots showing knockdown (KD) efficiency of the shRNA’s targeting AFF1 or AFF4. ( n = 3 biological replicates, AFF1 sh1 P = 0.0131, AFF1 sh2 P = 0.0227, AFF4 sh1 P < 0.0001, AFF4 sh2 P < 0.0001, two-tailed, unpaired t test, data are presented as mean values ± standard deviation). e – h Representative images and quantification of clonogenic assays comparing keratinocytes expressing shRNA’s targeting AFF1 or AFF4, versus non-targeting control shRNA ( n = 2–3 technical replicates). i , j Representative images and red:green fluorescence-quantification of epidermal tissue sections. Each piece of epidermal tissue was regenerated with 50% of GFP-labeled keratinocytes expressing non-targeting control shRNA and 50% of DsRed-labeled keratinocytes expressing non-targeting control shRNA (left), AFF1-targeting shRNA 1 (middle) or AFF4-targeting shRNA 1 (right). DsRed-labeled cells are represented in magenta. (scale bar = 100 µm, n = 15, **** P < 0.0001, two-tailed, unpaired t test, box plot represents first through third quartiles, minima, and maxima). k Comparison of genes differentially expressed in RNA-seq with AFF1 KD, AFF4 KD, and in both AFF1 and AFF4 KD conditions. l Top Gene Ontology (GO) terms of genes differentially expressed in both AFF1 and AFF4 KD RNA-seq data sets (two-tailed, Fisher’s exact test). m Top GO terms of genes differentially expressed in AFF1 but not AFF4 KD RNA-seq (two-tailed, Fisher’s exact test). n , o qRT-PCR comparing the expression of differentiation-activating TFs between non-targeting control and AFF1 or AFF4 KD ( n = 3 technical replicates, data are presented as mean values ± standard deviation). p Principal Component Analysis (PCA) of AFF1 KD, AFF4 KD, and KL 24-hour RNA-seq data sets. q – s qRT-PCR comparing mRNA levels of differentiation-activating TFs between DMSO control and KL2 treatment with non-targeting control or AFF1 knockdown using two independent shRNA’s. Control non-targeting KL is relative to non-targeting DMSO, AFF1 sh1 KL is relative to AFF1 sh1 DMSO, and AFF1 sh2 KL is relative to AFF1 sh2 DMSO ( n = 3 technical replicates, data are presented as mean values ± standard deviation). Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: CDK9 activity switch associated with AFF1 and HEXIM1 controls differentiation initiation from epidermal progenitors

    doi: 10.1038/s41467-022-32098-2

    Figure Lengend Snippet: a – d Western blots showing knockdown (KD) efficiency of the shRNA’s targeting AFF1 or AFF4. ( n = 3 biological replicates, AFF1 sh1 P = 0.0131, AFF1 sh2 P = 0.0227, AFF4 sh1 P < 0.0001, AFF4 sh2 P < 0.0001, two-tailed, unpaired t test, data are presented as mean values ± standard deviation). e – h Representative images and quantification of clonogenic assays comparing keratinocytes expressing shRNA’s targeting AFF1 or AFF4, versus non-targeting control shRNA ( n = 2–3 technical replicates). i , j Representative images and red:green fluorescence-quantification of epidermal tissue sections. Each piece of epidermal tissue was regenerated with 50% of GFP-labeled keratinocytes expressing non-targeting control shRNA and 50% of DsRed-labeled keratinocytes expressing non-targeting control shRNA (left), AFF1-targeting shRNA 1 (middle) or AFF4-targeting shRNA 1 (right). DsRed-labeled cells are represented in magenta. (scale bar = 100 µm, n = 15, **** P < 0.0001, two-tailed, unpaired t test, box plot represents first through third quartiles, minima, and maxima). k Comparison of genes differentially expressed in RNA-seq with AFF1 KD, AFF4 KD, and in both AFF1 and AFF4 KD conditions. l Top Gene Ontology (GO) terms of genes differentially expressed in both AFF1 and AFF4 KD RNA-seq data sets (two-tailed, Fisher’s exact test). m Top GO terms of genes differentially expressed in AFF1 but not AFF4 KD RNA-seq (two-tailed, Fisher’s exact test). n , o qRT-PCR comparing the expression of differentiation-activating TFs between non-targeting control and AFF1 or AFF4 KD ( n = 3 technical replicates, data are presented as mean values ± standard deviation). p Principal Component Analysis (PCA) of AFF1 KD, AFF4 KD, and KL 24-hour RNA-seq data sets. q – s qRT-PCR comparing mRNA levels of differentiation-activating TFs between DMSO control and KL2 treatment with non-targeting control or AFF1 knockdown using two independent shRNA’s. Control non-targeting KL is relative to non-targeting DMSO, AFF1 sh1 KL is relative to AFF1 sh1 DMSO, and AFF1 sh2 KL is relative to AFF1 sh2 DMSO ( n = 3 technical replicates, data are presented as mean values ± standard deviation). Source data are provided as a Source Data file.

    Article Snippet: Antibodies used for western blotting included mouse monoclonal anti-Lamin A/C (E1) (Santa Cruz Biotechnology, sc-376248, 1:1000), rabbit polyclonal anti-AFF1 (Bethyl, A-3020344A-T, 1:500), rabbit polyclonal anti-AFF4 (Abclonal, A4644, 1:1000), rabbit polyclonal anti-HEXIM1 (Bethyl, A303-112A-T, 1:1000), rabbit monoclonal anti-ATF-3 (E9J4N) (Cell Signaling, 18665, 1:1000), rabbit monoclonal anti-DUSP1 (E8L7D) (Cell Signaling, 48625, 1:1000), mouse monoclonal anti-RhoE (RND3) (Cell Signaling, 3664, 1:300), Rabbit anti-PhosphoThr186 CDK9 (Cell Signaling, 2549, 1:1000), Rabbit anti-CDK9 (C12F7) (Cell Signaling, 2316, 1:1000), Rabbit anti-HA (C29F4) (Cell Signaling, 3724, 1:1000), Rabbit anti-DNMT1 (D63A6) (Cell Signaling, 5032, 1:1000), and Rabbit anti-MYC (D84C12) (Cell Signaling, 5605, 1:1000).

    Techniques: Western Blot, Two Tailed Test, Standard Deviation, Expressing, shRNA, Fluorescence, Labeling, RNA Sequencing Assay, Quantitative RT-PCR

    a qRT-PCR showing mRNA levels of differentiation activators with 3-hour CDK9 inhibitor (CDK9i) treatment with flavopiridol (Flav) or NVP2 relative to DMSO control ( n = 3 technical replicates, data are presented as mean values ± standard deviation). b qRT-PCR showing the expression of differentiation-activating TFs in keratinocytes treated with KL alone or in combination with CDK9 inhibitors, as compared to DMSO control ( n = 3 technical replicates, data are presented as mean values ± standard deviation). c Western blot showing knockdown (KD) efficiency of HEXIM1 shRNA’s ( n = 3 biological replicates, quantified in Supplementary Fig. ). d Clonogenic assay of human keratinocytes expressing HEXIM1-shRNA’s or non-targeting control shRNA. e Quantification of colonies >1 mm 2 in HEXIM1 KD conditions relative to control ( n = 3/group, **** P < 0.0001, two-tailed, unpaired t test, data are presented as mean values ± standard deviation). f qRT-PCR comparing the mRNA levels of differentiation activators in HEXIM1 KD relative to control ( n = 3 technical replicates, data are presented as mean values ± standard deviation). g – i qRT-PCR showing the relative expression of differentiation-activating TFs between DMSO control and KL treatment where control non-targeting KL is relative to non-targeting DMSO, HEXIM1 sh1 KL is relative to HEXIM1 sh1 DMSO, and HEXIM1 sh2 KL is relative to HEXIM1 sh2 DMSO ( n = 3 technical replicates, data are presented as mean values ± standard deviation). j Venn diagram comparing genes differentially expressed in AFF1 KD versus HEXIM1 KD RNA-seq (Fisher’s exact test, two-Tail, P = 2 × 10 −220 ). k Heatmap showing shared genes significantly changed (fold change ≥2, P < 0.05, two-tailed, Wald test) in AFF1 KD RNA-seq or HEXIM1 KD RNA-seq. l Top Gene Ontology terms of the genes differentially expressed in AFF1 and HEXIM1 KD RNA-seq (two-tailed, Fisher’s exact test). m , n Co-immunoprecipitation of HA-AFF1 western blot showing interactions between AFF1 and HEXIM1, CDK9, and phosphorylated threonine 186 CDK9 (P-CDK) in progenitors (UD) and in differentiated (DF) keratinocytes ( n = 3 biological replicates). Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: CDK9 activity switch associated with AFF1 and HEXIM1 controls differentiation initiation from epidermal progenitors

    doi: 10.1038/s41467-022-32098-2

    Figure Lengend Snippet: a qRT-PCR showing mRNA levels of differentiation activators with 3-hour CDK9 inhibitor (CDK9i) treatment with flavopiridol (Flav) or NVP2 relative to DMSO control ( n = 3 technical replicates, data are presented as mean values ± standard deviation). b qRT-PCR showing the expression of differentiation-activating TFs in keratinocytes treated with KL alone or in combination with CDK9 inhibitors, as compared to DMSO control ( n = 3 technical replicates, data are presented as mean values ± standard deviation). c Western blot showing knockdown (KD) efficiency of HEXIM1 shRNA’s ( n = 3 biological replicates, quantified in Supplementary Fig. ). d Clonogenic assay of human keratinocytes expressing HEXIM1-shRNA’s or non-targeting control shRNA. e Quantification of colonies >1 mm 2 in HEXIM1 KD conditions relative to control ( n = 3/group, **** P < 0.0001, two-tailed, unpaired t test, data are presented as mean values ± standard deviation). f qRT-PCR comparing the mRNA levels of differentiation activators in HEXIM1 KD relative to control ( n = 3 technical replicates, data are presented as mean values ± standard deviation). g – i qRT-PCR showing the relative expression of differentiation-activating TFs between DMSO control and KL treatment where control non-targeting KL is relative to non-targeting DMSO, HEXIM1 sh1 KL is relative to HEXIM1 sh1 DMSO, and HEXIM1 sh2 KL is relative to HEXIM1 sh2 DMSO ( n = 3 technical replicates, data are presented as mean values ± standard deviation). j Venn diagram comparing genes differentially expressed in AFF1 KD versus HEXIM1 KD RNA-seq (Fisher’s exact test, two-Tail, P = 2 × 10 −220 ). k Heatmap showing shared genes significantly changed (fold change ≥2, P < 0.05, two-tailed, Wald test) in AFF1 KD RNA-seq or HEXIM1 KD RNA-seq. l Top Gene Ontology terms of the genes differentially expressed in AFF1 and HEXIM1 KD RNA-seq (two-tailed, Fisher’s exact test). m , n Co-immunoprecipitation of HA-AFF1 western blot showing interactions between AFF1 and HEXIM1, CDK9, and phosphorylated threonine 186 CDK9 (P-CDK) in progenitors (UD) and in differentiated (DF) keratinocytes ( n = 3 biological replicates). Source data are provided as a Source Data file.

    Article Snippet: Antibodies used for western blotting included mouse monoclonal anti-Lamin A/C (E1) (Santa Cruz Biotechnology, sc-376248, 1:1000), rabbit polyclonal anti-AFF1 (Bethyl, A-3020344A-T, 1:500), rabbit polyclonal anti-AFF4 (Abclonal, A4644, 1:1000), rabbit polyclonal anti-HEXIM1 (Bethyl, A303-112A-T, 1:1000), rabbit monoclonal anti-ATF-3 (E9J4N) (Cell Signaling, 18665, 1:1000), rabbit monoclonal anti-DUSP1 (E8L7D) (Cell Signaling, 48625, 1:1000), mouse monoclonal anti-RhoE (RND3) (Cell Signaling, 3664, 1:300), Rabbit anti-PhosphoThr186 CDK9 (Cell Signaling, 2549, 1:1000), Rabbit anti-CDK9 (C12F7) (Cell Signaling, 2316, 1:1000), Rabbit anti-HA (C29F4) (Cell Signaling, 3724, 1:1000), Rabbit anti-DNMT1 (D63A6) (Cell Signaling, 5032, 1:1000), and Rabbit anti-MYC (D84C12) (Cell Signaling, 5605, 1:1000).

    Techniques: Quantitative RT-PCR, Standard Deviation, Expressing, Western Blot, Clonogenic Assay, shRNA, Two Tailed Test, RNA Sequencing Assay, Immunoprecipitation

    a Venn diagram showing the significant overlap between AFF1 and HEXIM1 ChIP-seq peaks (Fisher’s exact test, two-tailed, P = 1×10 −268 ). b Violin plot showing the significant enrichment of total Pol II counts at ChIP-seq categories: AFF1 unique peaks, HEXIM1 unique peaks, and peaks with Pol II but no HEXIM1 or AFF1 relative to overlapping AFF1 and HEXIM1 peaks (**** P < 0.0001, two-tailed, unpaired t test). c Pie chart showing the peak distribution of AFF1 and HEXIM1 ChIP-seq data in promoters (transcription start site (TSS) ± 1 kb), gene bodies (TSS ± 1 kb through transcription end site), or intergenic regions. d Average profile plot showing the overlapping enrichment of AFF1 and HEXIM1 ChIP-seq signal near the promoters of their shared target genes. e Shared target genes among AFF1 ChIP-seq, HEXIM1 ChIP-seq, and the significantly upregulated genes in keratinocytes with 3-hr KL treatment. f Relative expression of the 92 AFF1-HEXIM1-KL(3 hr) shared target genes, ranked from low to high, in keratinocytes treated with KL versus DMSO for 3 hours. The top two upregulated genes ATF3 and DUSP1, as well as RND3, are highlighted in orange. g Heatmap showing differential expression of rapid-response and differentiation-activating genes with 1-hour and 3-hour nascent RNA-seq. h , i qRT-PCR showing the upregulation of ATF3, DUSP1, and RND3 with AFF1 or HEXIM1 knockdown ( n = 3 technical replicates, data are presented as mean values ± standard deviation). j – l Genome browser tracks showing the enrichment of AFF1, HEXIM1, CDK9, undifferentiated (UD) Pol II, and differentiated (DF) Pol II ChIP-seq signal at ATF3, DUSP1, and RND3. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: CDK9 activity switch associated with AFF1 and HEXIM1 controls differentiation initiation from epidermal progenitors

    doi: 10.1038/s41467-022-32098-2

    Figure Lengend Snippet: a Venn diagram showing the significant overlap between AFF1 and HEXIM1 ChIP-seq peaks (Fisher’s exact test, two-tailed, P = 1×10 −268 ). b Violin plot showing the significant enrichment of total Pol II counts at ChIP-seq categories: AFF1 unique peaks, HEXIM1 unique peaks, and peaks with Pol II but no HEXIM1 or AFF1 relative to overlapping AFF1 and HEXIM1 peaks (**** P < 0.0001, two-tailed, unpaired t test). c Pie chart showing the peak distribution of AFF1 and HEXIM1 ChIP-seq data in promoters (transcription start site (TSS) ± 1 kb), gene bodies (TSS ± 1 kb through transcription end site), or intergenic regions. d Average profile plot showing the overlapping enrichment of AFF1 and HEXIM1 ChIP-seq signal near the promoters of their shared target genes. e Shared target genes among AFF1 ChIP-seq, HEXIM1 ChIP-seq, and the significantly upregulated genes in keratinocytes with 3-hr KL treatment. f Relative expression of the 92 AFF1-HEXIM1-KL(3 hr) shared target genes, ranked from low to high, in keratinocytes treated with KL versus DMSO for 3 hours. The top two upregulated genes ATF3 and DUSP1, as well as RND3, are highlighted in orange. g Heatmap showing differential expression of rapid-response and differentiation-activating genes with 1-hour and 3-hour nascent RNA-seq. h , i qRT-PCR showing the upregulation of ATF3, DUSP1, and RND3 with AFF1 or HEXIM1 knockdown ( n = 3 technical replicates, data are presented as mean values ± standard deviation). j – l Genome browser tracks showing the enrichment of AFF1, HEXIM1, CDK9, undifferentiated (UD) Pol II, and differentiated (DF) Pol II ChIP-seq signal at ATF3, DUSP1, and RND3. Source data are provided as a Source Data file.

    Article Snippet: Antibodies used for western blotting included mouse monoclonal anti-Lamin A/C (E1) (Santa Cruz Biotechnology, sc-376248, 1:1000), rabbit polyclonal anti-AFF1 (Bethyl, A-3020344A-T, 1:500), rabbit polyclonal anti-AFF4 (Abclonal, A4644, 1:1000), rabbit polyclonal anti-HEXIM1 (Bethyl, A303-112A-T, 1:1000), rabbit monoclonal anti-ATF-3 (E9J4N) (Cell Signaling, 18665, 1:1000), rabbit monoclonal anti-DUSP1 (E8L7D) (Cell Signaling, 48625, 1:1000), mouse monoclonal anti-RhoE (RND3) (Cell Signaling, 3664, 1:300), Rabbit anti-PhosphoThr186 CDK9 (Cell Signaling, 2549, 1:1000), Rabbit anti-CDK9 (C12F7) (Cell Signaling, 2316, 1:1000), Rabbit anti-HA (C29F4) (Cell Signaling, 3724, 1:1000), Rabbit anti-DNMT1 (D63A6) (Cell Signaling, 5032, 1:1000), and Rabbit anti-MYC (D84C12) (Cell Signaling, 5605, 1:1000).

    Techniques: ChIP-sequencing, Two Tailed Test, Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Standard Deviation

    a – c Keratinocytes expressing HA-CDK9 were treated with DMSO control, KL, or TPA. Immunoprecipitation using the HA antibody was performed from the lysate of these keratinocytes, followed by western blot probing for HA and HEXIM1. d – g Violin and ECDF plots showing HA-CDK9 traveling ratio (TR) with KL or TPA relative to DMSO control at 92, direct, rapid-response genes (**** P < 0.001 *** P < 0.01, d , e KL P = 0.0002, f , g P = 0.0041, two-tailed, unpaired t test). A higher travel ratio indicates high proportion of CDK9 binding in the gene body. h Genome browser tracks comparing HA-CDK9 ChIP-seq enrichment at ATF3 among the DMSO control, KL treatment, or TPA treatment. i Illustration of the working model. In the progenitor state, AFF1 and HEXIM1 cooperatively hold CDK9 in an inactive state. With SEC disruption (KL) or PKC signaling (TPA), CDK9 from SEC-AFF1 is rapidly released into an active form. This rapid switch allows the activation of SEC-direct-target genes such as ATF3, which can further promote the expression of differentiation-activating transcription factors (GRHL3, PRDM1, ZNF750, and OVOL1, etc) to advance the terminal differentiation process. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: CDK9 activity switch associated with AFF1 and HEXIM1 controls differentiation initiation from epidermal progenitors

    doi: 10.1038/s41467-022-32098-2

    Figure Lengend Snippet: a – c Keratinocytes expressing HA-CDK9 were treated with DMSO control, KL, or TPA. Immunoprecipitation using the HA antibody was performed from the lysate of these keratinocytes, followed by western blot probing for HA and HEXIM1. d – g Violin and ECDF plots showing HA-CDK9 traveling ratio (TR) with KL or TPA relative to DMSO control at 92, direct, rapid-response genes (**** P < 0.001 *** P < 0.01, d , e KL P = 0.0002, f , g P = 0.0041, two-tailed, unpaired t test). A higher travel ratio indicates high proportion of CDK9 binding in the gene body. h Genome browser tracks comparing HA-CDK9 ChIP-seq enrichment at ATF3 among the DMSO control, KL treatment, or TPA treatment. i Illustration of the working model. In the progenitor state, AFF1 and HEXIM1 cooperatively hold CDK9 in an inactive state. With SEC disruption (KL) or PKC signaling (TPA), CDK9 from SEC-AFF1 is rapidly released into an active form. This rapid switch allows the activation of SEC-direct-target genes such as ATF3, which can further promote the expression of differentiation-activating transcription factors (GRHL3, PRDM1, ZNF750, and OVOL1, etc) to advance the terminal differentiation process. Source data are provided as a Source Data file.

    Article Snippet: Antibodies used for western blotting included mouse monoclonal anti-Lamin A/C (E1) (Santa Cruz Biotechnology, sc-376248, 1:1000), rabbit polyclonal anti-AFF1 (Bethyl, A-3020344A-T, 1:500), rabbit polyclonal anti-AFF4 (Abclonal, A4644, 1:1000), rabbit polyclonal anti-HEXIM1 (Bethyl, A303-112A-T, 1:1000), rabbit monoclonal anti-ATF-3 (E9J4N) (Cell Signaling, 18665, 1:1000), rabbit monoclonal anti-DUSP1 (E8L7D) (Cell Signaling, 48625, 1:1000), mouse monoclonal anti-RhoE (RND3) (Cell Signaling, 3664, 1:300), Rabbit anti-PhosphoThr186 CDK9 (Cell Signaling, 2549, 1:1000), Rabbit anti-CDK9 (C12F7) (Cell Signaling, 2316, 1:1000), Rabbit anti-HA (C29F4) (Cell Signaling, 3724, 1:1000), Rabbit anti-DNMT1 (D63A6) (Cell Signaling, 5032, 1:1000), and Rabbit anti-MYC (D84C12) (Cell Signaling, 5605, 1:1000).

    Techniques: Expressing, Immunoprecipitation, Western Blot, Two Tailed Test, Binding Assay, ChIP-sequencing, Activation Assay

    Journal: iScience

    Article Title: A natural product targets BRD4 to inhibit phase separation and gene transcription

    doi: 10.1016/j.isci.2021.103719

    Figure Lengend Snippet:

    Article Snippet: Antibodies used in this study are as follows: AFF1 (Bethyl Laboratories, Cat# A302-344A), AFF4 (Abcam, Cat# ab57077), ELL2 (Bethyl Laboratories, Cat# A302-505A), MePCE (Bethyl Laboratories, Cat# A304-184A), CYCT1 (Santa Cruz Biotechnology, Cat# sc-10750), normal rabbit IgG (Santa Cruz Biotechnology, Cat# sc-2027); Anti-BRD4, -LARP7, -HEXIM1 and -CDK9 were generated in our own laboratory and have been described previously( ; ).

    Techniques: Recombinant, Virus, SYBR Green Assay, Plasmid Preparation, Mutagenesis, Expressing, Software

    The AFF4 form of the super elongation complex is preferentially required for rapid transcriptional induction upon heat shock. ( A ) Design of auxin degron tagging of the super elongation complex (SEC) scaffolding proteins, AFF1 and AFF4. AFF1-AID and AFF4-AID single degron cells were generated independently using the double selection strategy shown for the BET degron cells. ( B ) Western blotting with the indicated antibodies shows the degradation of nuclear AFF1 or AFF4 in the corresponding degron cells within 2 and 24 h of auxin treatment. ( C ) Genome browser overlay of Pol II occupancy at the representative heat shock-induced genes before (dark color) or after 1h heat shock (light colors) in AFF1 or AFF4 degron cells ±auxin for 2 h. ( D ) ECDF plots of the log 2 fold change of PRR for the 227 HS-induced genes after 1 h heat shock. AFF4 depletion by auxin treatment leads to reduced heat shock induction as indicated by the leftward shift in the PRR, while AFF1 depletion by auxin treatment does not affect heat shock induction. ( E ) Metagene analysis of the log 2 fold change (FC) of Pol II occupancy of 42°C over 37°C for the 227 HS-induced genes ±auxin for 2 h in AFF1-AID and AFF4-AID cells or ±6-h KL-1 (20 µM) treatment in AFF4-AID cells.

    Journal: Genes & Development

    Article Title: Acute perturbation strategies in interrogating RNA polymerase II elongation factor function in gene expression

    doi: 10.1101/gad.346106.120

    Figure Lengend Snippet: The AFF4 form of the super elongation complex is preferentially required for rapid transcriptional induction upon heat shock. ( A ) Design of auxin degron tagging of the super elongation complex (SEC) scaffolding proteins, AFF1 and AFF4. AFF1-AID and AFF4-AID single degron cells were generated independently using the double selection strategy shown for the BET degron cells. ( B ) Western blotting with the indicated antibodies shows the degradation of nuclear AFF1 or AFF4 in the corresponding degron cells within 2 and 24 h of auxin treatment. ( C ) Genome browser overlay of Pol II occupancy at the representative heat shock-induced genes before (dark color) or after 1h heat shock (light colors) in AFF1 or AFF4 degron cells ±auxin for 2 h. ( D ) ECDF plots of the log 2 fold change of PRR for the 227 HS-induced genes after 1 h heat shock. AFF4 depletion by auxin treatment leads to reduced heat shock induction as indicated by the leftward shift in the PRR, while AFF1 depletion by auxin treatment does not affect heat shock induction. ( E ) Metagene analysis of the log 2 fold change (FC) of Pol II occupancy of 42°C over 37°C for the 227 HS-induced genes ±auxin for 2 h in AFF1-AID and AFF4-AID cells or ±6-h KL-1 (20 µM) treatment in AFF4-AID cells.

    Article Snippet: BRD3 (#A302-368A) and AFF1 (#A302-345A) antibodies were obtained from Bethyl Laboratories.

    Techniques: Scaffolding, Generated, Selection, Western Blot

    The SEC is essential for rapid transcriptional induction by heat shock. ( A ) Design of AFF1-AFF4 double degron DLD-1 cells. Neomycin was used for selection of AFF1-AID homozygous knock-in clones, which were subsequently used for a homozygous knock-in of the mAID tag for AFF4 using hygromycin selection. ( B ) Western blot analysis demonstrating the degradation of both AFF1 and AFF4 after auxin treatment for 2 h, compared with the parental DLD-1 cells. ( C ) Metagene analysis showing the log 2 FC of Pol II occupancy of 42°C over 37°C for the 227 HS-induced genes before and after simultaneous depletion of AFF1 and AFF4. Decreased induction of Pol II occupancy in the gene bodies during heat shock is observed. ( D ) Genome browser overlay of Pol II occupancy at representative heat shock genes before (dark color) and after (light color) heat shock for 1 h, ±auxin depletion of AFF1 and AFF4 for 2 h, or KL-1 treatment (20 µM) for 6 h. ( E ) Heat map showing the log 2 fold change of Pol II coverage around the TSS for the 227 HS-induced genes for auxin treatment of the AFF1–AFF4 double degron cells and KL-1 treatment of AFF4 degron cells. ( F ) Venn diagram showing the overlap of the 227 HS-induced genes whose induction is impaired by auxin depletion of AFF1 and AFF4 or by KL-1 treatment or both treatments. ( G ) Western blotting with the indicated antibodies showing attenuated HSP70 induction after 6-h heat shock after dual depletion of AFF1 and AFF4. Cells were treated with H2O or auxin for 2 h at 37°C before 42°C heat shock for 6 h. ( H ) Quantification of HSP70 protein levels ±auxin for three independent replicates as in G . Western signal intensity of HSP70 was first converted to fold change of actin within groups and then normalized to the condition of H2O treatment without heat shock. Circled dots represent the median. Replicate 2 is shown in G .

    Journal: Genes & Development

    Article Title: Acute perturbation strategies in interrogating RNA polymerase II elongation factor function in gene expression

    doi: 10.1101/gad.346106.120

    Figure Lengend Snippet: The SEC is essential for rapid transcriptional induction by heat shock. ( A ) Design of AFF1-AFF4 double degron DLD-1 cells. Neomycin was used for selection of AFF1-AID homozygous knock-in clones, which were subsequently used for a homozygous knock-in of the mAID tag for AFF4 using hygromycin selection. ( B ) Western blot analysis demonstrating the degradation of both AFF1 and AFF4 after auxin treatment for 2 h, compared with the parental DLD-1 cells. ( C ) Metagene analysis showing the log 2 FC of Pol II occupancy of 42°C over 37°C for the 227 HS-induced genes before and after simultaneous depletion of AFF1 and AFF4. Decreased induction of Pol II occupancy in the gene bodies during heat shock is observed. ( D ) Genome browser overlay of Pol II occupancy at representative heat shock genes before (dark color) and after (light color) heat shock for 1 h, ±auxin depletion of AFF1 and AFF4 for 2 h, or KL-1 treatment (20 µM) for 6 h. ( E ) Heat map showing the log 2 fold change of Pol II coverage around the TSS for the 227 HS-induced genes for auxin treatment of the AFF1–AFF4 double degron cells and KL-1 treatment of AFF4 degron cells. ( F ) Venn diagram showing the overlap of the 227 HS-induced genes whose induction is impaired by auxin depletion of AFF1 and AFF4 or by KL-1 treatment or both treatments. ( G ) Western blotting with the indicated antibodies showing attenuated HSP70 induction after 6-h heat shock after dual depletion of AFF1 and AFF4. Cells were treated with H2O or auxin for 2 h at 37°C before 42°C heat shock for 6 h. ( H ) Quantification of HSP70 protein levels ±auxin for three independent replicates as in G . Western signal intensity of HSP70 was first converted to fold change of actin within groups and then normalized to the condition of H2O treatment without heat shock. Circled dots represent the median. Replicate 2 is shown in G .

    Article Snippet: BRD3 (#A302-368A) and AFF1 (#A302-345A) antibodies were obtained from Bethyl Laboratories.

    Techniques: Selection, Knock-In, Clone Assay, Western Blot

    AFF1 occupies the NTS downstream region and inhibits NTS expression. A , KM plot showing the overall survival of lung cancer patients stratified by AFF1 expression level. B , Gene ontology (GO) analysis of the top ten genes that were most upregulated after AFF1 knockdown. C , ChIP-seq genome browser track (upper, blue ) showing the occupancy of AFF1 at the NTS downstream region. RNA-seq genome browser track (lower, purple ) showing increased NTS expression upon AFF1 knockdown.

    Journal: The Journal of Biological Chemistry

    Article Title: Suppression of the NTS-CPS1 regulatory axis by AFF1 in lung adenocarcinoma cells

    doi: 10.1016/j.jbc.2021.100319

    Figure Lengend Snippet: AFF1 occupies the NTS downstream region and inhibits NTS expression. A , KM plot showing the overall survival of lung cancer patients stratified by AFF1 expression level. B , Gene ontology (GO) analysis of the top ten genes that were most upregulated after AFF1 knockdown. C , ChIP-seq genome browser track (upper, blue ) showing the occupancy of AFF1 at the NTS downstream region. RNA-seq genome browser track (lower, purple ) showing increased NTS expression upon AFF1 knockdown.

    Article Snippet: Antibody against AFF1 (A302-344A) was purchased from Bethyl.

    Techniques: Expressing, Knockdown, ChIP-sequencing, RNA Sequencing

    The AFF1-bound NTS downstream region acts as a NTS enhancer. A , cartoon model illustrating generation of A549 cell lines without the AFF1-bound NTS downstream region. B , RT-qPCR showing the expression level of NTS after removal of the AFF1-bound NTS downstream region in A549 cells. Significant differences are marked with an asterisk ( t -test, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). Error bars represent standard deviations; n = 3. C , Western blot showing HA tagged VP64 or KRAB can be detected in A549 cells after lentiviral transduction of the CRISPRa and CRISPRi vectors. D , qPCR analysis after HA ChIP showing that specific sgRNAs were able to guide HA tagged VP64 or KRAB to the AFF1-bound NTS downstream region. The HEMO gene serves as a negative control for ChIP-qPCR. Error bars represent standard deviations; n = 3. E , RT-qPCR showing the expression level of NTS after CRISPR interference and activation of the AFF1-bound NTS downstream region. Significant differences are marked with an asterisk ( t -test, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). Error bars represent standard deviations; n = 3. (F) 4C-seq showing the physical association between the AFF1-bound NTS downstream region and the NTS promoter region.

    Journal: The Journal of Biological Chemistry

    Article Title: Suppression of the NTS-CPS1 regulatory axis by AFF1 in lung adenocarcinoma cells

    doi: 10.1016/j.jbc.2021.100319

    Figure Lengend Snippet: The AFF1-bound NTS downstream region acts as a NTS enhancer. A , cartoon model illustrating generation of A549 cell lines without the AFF1-bound NTS downstream region. B , RT-qPCR showing the expression level of NTS after removal of the AFF1-bound NTS downstream region in A549 cells. Significant differences are marked with an asterisk ( t -test, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). Error bars represent standard deviations; n = 3. C , Western blot showing HA tagged VP64 or KRAB can be detected in A549 cells after lentiviral transduction of the CRISPRa and CRISPRi vectors. D , qPCR analysis after HA ChIP showing that specific sgRNAs were able to guide HA tagged VP64 or KRAB to the AFF1-bound NTS downstream region. The HEMO gene serves as a negative control for ChIP-qPCR. Error bars represent standard deviations; n = 3. E , RT-qPCR showing the expression level of NTS after CRISPR interference and activation of the AFF1-bound NTS downstream region. Significant differences are marked with an asterisk ( t -test, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). Error bars represent standard deviations; n = 3. (F) 4C-seq showing the physical association between the AFF1-bound NTS downstream region and the NTS promoter region.

    Article Snippet: Antibody against AFF1 (A302-344A) was purchased from Bethyl.

    Techniques: Quantitative RT-PCR, Expressing, Western Blot, Transduction, Negative Control, ChIP-qPCR, CRISPR, Activation Assay

    NTS is required for the proper activation of CPS1, FGG, GPX2 in A549 cells. A , ChIP-seq genome browser track (upper, blue ) showing that AFF1 was not detected at the CPS1 , FGG , and GPX2 loci. RNA-seq genome browser track (lower, purple ) showing increased CPS1 , FGG , and GPX2 expression upon AFF1 knockdown. B , Western blot showing that the protein level of CPS1 was increased upon AFF1 knockdown. Tubulin was used as a loading control. C , RT-qPCR showing that the RNA levels of CPS1, FGG, and GPX2 were reduced in the NTS-en deleted A549 cells. Significant differences are marked with an asterisk ( t -test, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). Error bars represent standard deviations; n = 3. D , Western blot showing that the protein level of CPS1 was reduced in the NTS-en deleted A549 cells. Tubulin was used as a loading control. E , RT-qPCR showing the expression level of CPS1, FGG, and GPX2 after AFF1 and NTS double knockdown in A549 cells. Significant differences are marked with an asterisk ( t -test, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). Error bars represent standard deviations; n = 3. F , Cartoon model illustrating that AFF1 suppresses NTS expression through binding to NTS - en (upper) and that AFF1 inhibits the expression of CPS1, FGG, GPX2 via NTS (lower).

    Journal: The Journal of Biological Chemistry

    Article Title: Suppression of the NTS-CPS1 regulatory axis by AFF1 in lung adenocarcinoma cells

    doi: 10.1016/j.jbc.2021.100319

    Figure Lengend Snippet: NTS is required for the proper activation of CPS1, FGG, GPX2 in A549 cells. A , ChIP-seq genome browser track (upper, blue ) showing that AFF1 was not detected at the CPS1 , FGG , and GPX2 loci. RNA-seq genome browser track (lower, purple ) showing increased CPS1 , FGG , and GPX2 expression upon AFF1 knockdown. B , Western blot showing that the protein level of CPS1 was increased upon AFF1 knockdown. Tubulin was used as a loading control. C , RT-qPCR showing that the RNA levels of CPS1, FGG, and GPX2 were reduced in the NTS-en deleted A549 cells. Significant differences are marked with an asterisk ( t -test, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). Error bars represent standard deviations; n = 3. D , Western blot showing that the protein level of CPS1 was reduced in the NTS-en deleted A549 cells. Tubulin was used as a loading control. E , RT-qPCR showing the expression level of CPS1, FGG, and GPX2 after AFF1 and NTS double knockdown in A549 cells. Significant differences are marked with an asterisk ( t -test, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). Error bars represent standard deviations; n = 3. F , Cartoon model illustrating that AFF1 suppresses NTS expression through binding to NTS - en (upper) and that AFF1 inhibits the expression of CPS1, FGG, GPX2 via NTS (lower).

    Article Snippet: Antibody against AFF1 (A302-344A) was purchased from Bethyl.

    Techniques: Activation Assay, ChIP-sequencing, RNA Sequencing, Expressing, Knockdown, Western Blot, Control, Quantitative RT-PCR, Binding Assay

    NTS and IL6 oppositely regulates the expression of CPS1, FGG, and GPX2. A , Heatmap showing the expression of several cytokines after AFF1 knockdown in A549 cells. B , RT-qPCR showing the expression level of IL6 in the NTS-en deleted A549 cells. Significant differences are marked with an asterisk ( t -test, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). Error bars represent standard deviations; n = 3. C , RT-qPCR showing the upregulation of IL6 after NTS knockdown in A549 cells. Significant differences are marked with an asterisk ( t -test, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). Error bars represent standard deviations; n = 3. D–F, Western blot showing that the protein level of IL6 was reduced upon AFF1 knockdown ( D ), but increased after NTS depletion ( E and F ). Tubulin was used as a loading control. G , RT-qPCR showing the upregulation of CPS1, FGG, and GPX2 after IL6 knockdown in A549 cells. Significant differences are marked with an asterisk ( t -test, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). Error bars represent standard deviations; n = 3. H , Western blot showing that the protein level of CPS1 was increased upon IL6 knockdown. Tubulin was used as a loading control.

    Journal: The Journal of Biological Chemistry

    Article Title: Suppression of the NTS-CPS1 regulatory axis by AFF1 in lung adenocarcinoma cells

    doi: 10.1016/j.jbc.2021.100319

    Figure Lengend Snippet: NTS and IL6 oppositely regulates the expression of CPS1, FGG, and GPX2. A , Heatmap showing the expression of several cytokines after AFF1 knockdown in A549 cells. B , RT-qPCR showing the expression level of IL6 in the NTS-en deleted A549 cells. Significant differences are marked with an asterisk ( t -test, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). Error bars represent standard deviations; n = 3. C , RT-qPCR showing the upregulation of IL6 after NTS knockdown in A549 cells. Significant differences are marked with an asterisk ( t -test, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). Error bars represent standard deviations; n = 3. D–F, Western blot showing that the protein level of IL6 was reduced upon AFF1 knockdown ( D ), but increased after NTS depletion ( E and F ). Tubulin was used as a loading control. G , RT-qPCR showing the upregulation of CPS1, FGG, and GPX2 after IL6 knockdown in A549 cells. Significant differences are marked with an asterisk ( t -test, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). Error bars represent standard deviations; n = 3. H , Western blot showing that the protein level of CPS1 was increased upon IL6 knockdown. Tubulin was used as a loading control.

    Article Snippet: Antibody against AFF1 (A302-344A) was purchased from Bethyl.

    Techniques: Expressing, Knockdown, Quantitative RT-PCR, Western Blot, Control