anti med1  (Santa Cruz Biotechnology)

Journal: Nature Chemical Biology

Article Title: MED1 IDR deacetylation controls stress responsive genes through RNA Pol II recruitment

doi: 10.1038/s41589-025-02035-7

Figure Lengend Snippet: a , Workflow depicting the depletion of MED1 by CRISPR/Cas9. Cas9 (FLAG-tagged) was stably expressed in cells and lentiviral infection was used to introduce plasmids expressing gRNAs targeting MED1 exons into pools of cells to deplete MED1. b , Immunoblotting confirming depletion of MED1 by targeting 3 independent regions (designated KO-1, 2, and 3) in exons of the MED1 gene (with a gRNA targeting the mouse Rosa26 locus as a non-targeting control) in MCF7 and T47D cells, as well as comparable ERα levels in control and KO cells. Immunoblots are representative of three independent experiments. c , Cell growth attenuated by MED1 depletion in either MCF7 or T47D cells, measured by CellTiter Blue assay. N = 3 (biological replicates) for each cell type. Data points report mean ± SEM. Two-sided unpaired t-test for each of KO cell type versus control for D6 data. d , Attenuated colony formation in MCF7 cells with MED1 KO compared with control, observed by staining with crystal violet for cells growing for 16 days after initial seeding.

Article Snippet: To identify residues of MED1 with acetylation, the endogenous MED1 was immunoprecipitated from whole-cell lysate of HEK293T by anti-MED1 antibody (Bethyl/Fortis, A300-793, 1:800) followed by conjugation of protein A Sepharose CL-4B beads (Amersham, 17-0963-03).

Techniques: CRISPR, Stable Transfection, Infection, Introduce, Expressing, Western Blot, Control, CtB Assay, Staining

Journal: Nature Chemical Biology

Article Title: MED1 IDR deacetylation controls stress responsive genes through RNA Pol II recruitment

doi: 10.1038/s41589-025-02035-7

Figure Lengend Snippet: a – c , Poly(A) RNA-seq for control and MED1 KO MCF7 cells. Two biological replicate samples were detected for control or each of KO cell types. a , A volcano plot showing the adjusted P value (two-sided) and fold-change of each detected gene (combined from KO-1, -2, and -3) where the cut-off to define DEGs (FC > 2, P adj < 0.05) is indicated. b , A clustered heatmap of Z-scores of genes with differential expression with P adj < 0.05 from MED1 KO (-1, -2 and -3) versus control cells. Colors in scale represent relative expression levels: blue (Z ≤ −2), white (Z = 0), and red (Z ≥ +2), with a linear gradient for intermediate values. c , Gene ontology analyses with adjusted P values with KEGG gene lists for up-regulated and down-regulated DEGs, respectively. d , Venn diagram showing overlap of genomic peaks of MED1, MED17 and Pol II. e – g , Analyses for peaks classified by occupancy of MED1, MED17 and Pol II. e , Pie diagrams showing constitution of each type of these peaks for types of regulatory elements. See Methods for the definition of regulatory elements. f , Genomic annotation of MED1 + MED17 + Pol II + peaks that cannot be categorized into pre-defined types of regulatory elements. g , Pie diagrams showing constitution of each type of these peaks for association with up-, down-regulated genes or no-change-genes of MED1 KO versus control cells. h , Density of MED1 occupancy at active promoters associated with plotted genes with stratification of genes based on their fold-change values detected from RNA-seq of MED1 KO versus control cells (designated STRONG UP, MILD UP, NO-CHANGE, MILD DOWN, STRONG DOWN). Boxes span the 25 th -75 th percentiles (center line indicates median), whiskers extend to 1.5 X IQR, and points beyond represent outliers. Parentheses indicate sample sizes.

Article Snippet: To identify residues of MED1 with acetylation, the endogenous MED1 was immunoprecipitated from whole-cell lysate of HEK293T by anti-MED1 antibody (Bethyl/Fortis, A300-793, 1:800) followed by conjugation of protein A Sepharose CL-4B beads (Amersham, 17-0963-03).

Techniques: RNA Sequencing, Control, Quantitative Proteomics, Expressing

Journal: Nature Chemical Biology

Article Title: MED1 IDR deacetylation controls stress responsive genes through RNA Pol II recruitment

doi: 10.1038/s41589-025-02035-7

Figure Lengend Snippet: a , b , Detection of acetylation of FLAG-tagged Mediator subunits transiently expressed in HEK293T cells treated with Trichostatin A (TSA, 0.5 μM for 24 h) and nicotinamide (NAM, 5 mM for 3/6 h), referred to as T + N3 or T + N6 in the panel. Immunoblotting images ( a ) and densitometry quantification with AceK immunoblot signals normalized by the FLAG signals ( b ) are shown. c , Silver staining (left) and immunoblotting (right) confirming the yield and integrity of Mediator (presence of MED1 and MED17) purified from HeLa-S cells and the absence of p300 and SIRT1. d , Workflow for mass spectrometry identification of acetylation sites from immunoprecipitated endogenous MED1 from HEK293T cells with overexpression of p300 with or without A485. e , Immunoblotting confirming the overexpression of p300 and acetylation of MED1 in IP-Mass Spectrometry samples. Densitometry analysis was performed on immunoblotting images, and the normalized acetyl-lysine levels (relative to IP target protein, MED1) are shown. f , Analysis of MED1 protein sequence by VL-XT algorithm showing a disordered region (963-1320 aa) with high PONDR score. g , Multiple sequence alignments indicating that the 6 acetylation sites (arrows) in the MED1 IDR are conserved among species. h , Immunostaining with anti-FLAG antibodies combined with DAPI staining for MCF7 cells with ectopic expression of FLAG-tagged WT or 6KR MED1. Scale bar is 20 μm. For panels a (MED1 test), c , e , h , the results are representative of three independent experiments.

Article Snippet: To identify residues of MED1 with acetylation, the endogenous MED1 was immunoprecipitated from whole-cell lysate of HEK293T by anti-MED1 antibody (Bethyl/Fortis, A300-793, 1:800) followed by conjugation of protein A Sepharose CL-4B beads (Amersham, 17-0963-03).

Techniques: Western Blot, Silver Staining, Purification, Mass Spectrometry, Immunoprecipitation, Over Expression, Sequencing, Immunostaining, Staining, Expressing

Journal: Nature Chemical Biology

Article Title: MED1 IDR deacetylation controls stress responsive genes through RNA Pol II recruitment

doi: 10.1038/s41589-025-02035-7

Figure Lengend Snippet: a , Immunoblotting showing enhanced lysine acetylation (referred to as AceK) of FLAG–MED1 in HEK293T cells following overexpression of WT but not D1399Y mutant p300 (human and mouse, with 98% sequence identity to human CBP). IB, immunonlot. b , Immunoblotting showing enhanced acetylation of FLAG–MED1 in HEK293T cells following overexpression of p300 and its suppression by the p300 inhibitor A485 but not C646 or SGC-CBP30. Cells were treated with these inhibitors at the concentrations indicated for 24 h. c , Immunoblotting showing enhanced acetylation of FLAG–MED1 following overexpression of p300 or CBP but not GCN5, PCAF, TIP60 and MOF in HEK293T cells. d , Immunoblotting showing enhanced acetylation of endogenous MED1 in HEK293T cells following overexpression of p300 or CBP. e , Immunoblotting showing enhanced acetylation of endogenous MED1 from HEK293T cells with overexpression of p300 at variable concentrations. f , Immunoblotting showing enhanced acetylation of MED1 (within the purified Mediator complex) by p300 and acetyl-CoA. Effects of variable p300 concentrations (top) and variable incubation times (bottom) were tested. Autoacetylation of p300 was also observed. g , Signal of digested peptides with acetylation plotted as a function of amino acid sequence. Signals from control samples and from p300 overexpression samples with or without A485 were compared and the two acetylation clusters are indicated. h , Analysis of MED1 protein sequence by MobiDB showing a disordered region (amino acids 947–1573). i , Immunoblotting showing enhanced acetylation of WT but not 6KR FLAG–MED1 in HEK293T cells following overexpression of p300. Effects of variable p300 concentrations were tested. For d , e , IgG was used as an IP control. For a – e , i , densitometry analysis was performed on immunoblotting images and the normalized protein levels (relative to the IP target protein, either FLAG-tagged or endogenous MED1) are shown. For a – f , i , immunoblots are representative of three independent experiments.

Article Snippet: To identify residues of MED1 with acetylation, the endogenous MED1 was immunoprecipitated from whole-cell lysate of HEK293T by anti-MED1 antibody (Bethyl/Fortis, A300-793, 1:800) followed by conjugation of protein A Sepharose CL-4B beads (Amersham, 17-0963-03).

Techniques: Western Blot, Over Expression, Mutagenesis, Sequencing, Purification, Incubation, Control

Journal: Nature Chemical Biology

Article Title: MED1 IDR deacetylation controls stress responsive genes through RNA Pol II recruitment

doi: 10.1038/s41589-025-02035-7

Figure Lengend Snippet: a , Immunoblotting showing enhanced acetylation of FLAG-MED1 in HEK293T cells treated with Ex527. b , Immunoblotting showing enhanced acetylation of endogenous MED1 in MCF7 cells treated with Sirtinol. c , Immunoblotting showing reduced acetylation of FLAG-MED1 in MCF7 cells treated with CoCl 2 (24 h). HIF-1α was detected to demonstrate the hypoxic response. d , Immunoblotting showing reduced acetylation of FLAG-MED1 in MCF7 cells under heat shock (42 °C, 2 h), while treatment of Ex527 (50 μM) prevented this effect. e , Immunoblotting showing elevated SIRT1 detected in association with endogenous AFF4 in T47D cells treated with DFOM (200 μM, 6 h). f , Immunoblotting showing elevated SIRT1 detected for association with endogenous CDK9 in MCF7 cells treated with DFOM (200 μM, 18 h), while equivalent association of Cyclin T1 with CDK9 with or without DFOM treatment was detected. g , Immunoblotting showing elevated SIRT1 detected in association with endogenous AFF4 in MCF7 cells challenged by heat shock (42゜C, 2 h), while equivalent association of CDK9 with AFF4 with or without heat shock was detected. h , Chart showing SEC components, SIRT1 and other AFF4-binding proteins detected by IP-Mass Spectrometry in each sample with area values and visualized with a color scale. The bait protein, AFF4, does not show a significant difference in levels among samples. i , IGV snapshots showing elevated occupancy of AFF4 and SIRT1 in promoter-proximal regions of representative genes in DFOM-treated (200 μM, 6 h) MCF7 cells detected by CUT&RUN-seq. For panels b and e – g , IgG was used as an IP control. For panels a – g , densitometry analysis was performed on immunoblotting images, and the normalized protein levels (relative to the IP target protein, FLAG tag of MED1 or endogenous MED1, AFF4 or CDK9) are shown, and these immunoblots are representative of three independent experiments.

Article Snippet: To identify residues of MED1 with acetylation, the endogenous MED1 was immunoprecipitated from whole-cell lysate of HEK293T by anti-MED1 antibody (Bethyl/Fortis, A300-793, 1:800) followed by conjugation of protein A Sepharose CL-4B beads (Amersham, 17-0963-03).

Techniques: Western Blot, Binding Assay, Mass Spectrometry, Control, FLAG-tag

Journal: Nature Chemical Biology

Article Title: MED1 IDR deacetylation controls stress responsive genes through RNA Pol II recruitment

doi: 10.1038/s41589-025-02035-7

Figure Lengend Snippet: a , Immunoblotting showing enhanced acetylation of endogenous MED1 in MCF7 cells treated with Ex527, where the effect was attenuated by cotreatment with A485 (6 μM, 24 h). b , Immunoblotting showing enhanced acetylation of WT but not 6KR FLAG–MED1 in MCF7 cells treated with Ex527 (100 μM, 24 h). A control sample (ctrl IP) was included, in which the lysate of normal MCF7 cells was immunoprecipitated with anti-FLAG. c , Immunoblotting showing enhanced acetylation of WT but not 6KR MED1 in T47D cells where endogenous MED1 was depleted by CRISPR–Cas9 (referred to as WT/KO and 6KR/KO) treated with Ex527 (25 and 100 μM for 4 h). Unmodified T47D cells with IgG used as the IP control. d , Immunoblotting showing reduced acetylation of WT FLAG–MED1 in MCF7 cells treated with various concentrations of DFOM (24 h). HIF1α was detected to demonstrate the induction of hypoxic response. e , Immunoblotting showing reduced acetylation of WT FLAG–MED1 in MCF7 cells treated with DFOM (100 μM, 24 h), while cotreatment of Ex527 (50 μM) prevented this effect. f , Immunoblotting showing reduced acetylation of WT FLAG–MED1 in MCF7 cells under 42 °C heat shock for various durations. g , Immunoblotting showing reduced acetylation of WT FLAG–MED1 in MCF7 cells treated with various concentrations of H 2 O 2 (4 h). h , Immunoblotting showing reduced acetylation of endogenous MED1 in MCF7 cells treated with H 2 O 2 or DFOM (either with 200 μM, 24 h), while cotreatment of Ex527 (50 μM, 24 h) prevented this effect. i , Immunoblotting showing elevated SIRT1 detected for association with endogenous AFF4 in MCF7 cells treated with DFOM (16 h), while equivalent association of cyclin T1 with AFF4 with or without DFOM treatment was detected. j , AlphaFold 3 prediction of structure of the SEC associated with SIRT1 (green). The overall structure (top) is shown in cartoon form and the close-up views of the SIRT1–SEC interface (bottom left and bottom right) are shown in cartoon form and surface representation. The extended fraction of SIRT1 inserting into the pocket formed by AFF4 (dark blue) and CDK9 (light blue) is indicated with a dashed circle. For a , h , IgG was used as an IP control. For a – i , densitometry analysis was performed on immunoblotting images and the normalized protein levels (relative to the IP target protein, FLAG tag of MED1 or endogenous MED1 or AFF4) are shown; these immunoblots are representative of three independent experiments.

Article Snippet: To identify residues of MED1 with acetylation, the endogenous MED1 was immunoprecipitated from whole-cell lysate of HEK293T by anti-MED1 antibody (Bethyl/Fortis, A300-793, 1:800) followed by conjugation of protein A Sepharose CL-4B beads (Amersham, 17-0963-03).

Techniques: Western Blot, Control, Immunoprecipitation, CRISPR, FLAG-tag

Journal: Nature Chemical Biology

Article Title: MED1 IDR deacetylation controls stress responsive genes through RNA Pol II recruitment

doi: 10.1038/s41589-025-02035-7

Figure Lengend Snippet: a , Workflow depicting the methods employed to replace endogenous MED1 with ectopic MED1 in MCF7 or T47D cells. Lentiviruses expressing gRNAs targeting MED1 were transfected into pools of cells expressing ectopic HA-tagged MED1 (WT or 6KR/Q mutant) with CRISPR-mutated target sequences. b , c , Immunoblotting confirming efficient depletion of MED1, expression of HA-tagged WT and 6KR/Q MED1 (after CRISPR KO) at levels comparable to that of endogenous MED1 in MCF7 ( b ) and T47D ( c ) cells. d – f , Poly(A) RNA-seq for 6KR/KO and WT/KO MCF7 cells in steady state. Three biological replicates were detected for each cell type. d , Volcano plot showing Fold-change versus Adjusted P value (two-sided) for each detected gene, with the cut-off for defining DEGs (FC > 2, P adj < 0.05) indicated. e , GSEA comparing the 6KR/KO versus WT/KO gene rank list with KEGG (left) and GOBP (right) gene lists. Adjusted P values are shown in addition to NES reported by bars. f , GSEA revealing that the up-regulated (upper) and down-regulated (lower) genes in MED1 KO versus control cells (strong or mild) are enriched, respectively, in the sets of down-regulated and up-regulated genes of 6KR/KO versus WT/KO cells. NES values are shown. g , Detection by qRT-PCR showing stronger induction of representative hypoxia-induced genes in 6KR/KO versus WT/KO T47D cells treated with 200 μM DFOM. For each of WT/KO or 6KR/KO cells, expression of the gene at each condition was normalized by its expression at control condition in this cell type. N = 3 (biological replicates) for each group. Bars report mean ± SEM. If < 0.05, the P values of two-sided unpaired t-test comparing cell types under specific conditions are shown. For panels b and c , immunoblots are representative of three independent experiments.

Article Snippet: To identify residues of MED1 with acetylation, the endogenous MED1 was immunoprecipitated from whole-cell lysate of HEK293T by anti-MED1 antibody (Bethyl/Fortis, A300-793, 1:800) followed by conjugation of protein A Sepharose CL-4B beads (Amersham, 17-0963-03).

Techniques: Expressing, Transfection, Mutagenesis, CRISPR, Western Blot, RNA Sequencing, Control, Quantitative RT-PCR

Journal: Nature Chemical Biology

Article Title: MED1 IDR deacetylation controls stress responsive genes through RNA Pol II recruitment

doi: 10.1038/s41589-025-02035-7

Figure Lengend Snippet: a , GSEA showing that the upregulated (left) and downregulated (right) genes in 6KR/KO versus WT/KO cells were enriched in the set of downregulated and upregulated genes of MED1 -KO (versus control) cells, respectively. The cutoff for defining DEGs was fold change (FC) > 2 and adjusted P < 0.05. NES (normalized enrichment score) values are shown. b , c , Detection by RT–qPCR showing stronger induction of representative stress-induced genes in 6KR/KO versus WT/KO MCF7 cells. b , Genes induced by DFOM. c , Genes induced 42 °C heat shock for 60 or 120 min. For each of WT/KO or 6KR/KO cells, expression of the gene at each condition was normalized by its expression at control condition in this cell type. For b , c , n = 4 (biological replicates) for each group. Bars report the mean ± s.e.m. If <0.05, the P values of a two-sided unpaired t -test comparing cell types are shown. d – i , Poly(A) RNA-seq for MCF7 cells under H 2 O 2 stress, comparing 6KR/KO versus WT/KO or 6KQ/KO versus WT/KO. Three biological replicates were detected for each cell type in each condition. d , Volcano plots showing the log 2 FC versus Wald statistics (two-sided) after H 2 O 2 treatment for each detected gene in tests of WT/KO, 6KR/KO and 6KQ/KO cells, with the cutoff for defining DEGs (FC > 2, P < 0.05) indicated. e , A clustered heat map of z scores of genes showing differential expression by H 2 O 2 treatment (adjusted P < 0.05) in any of the WT/KO, 6KR/KO or 6KQ/KO cells. A division of six groups for these genes is shown. Colors in the scale represent relative expression levels: blue ( z ≤ −3), white ( z = 0), and red (Z ≥ 3), with a linear gradient for intermediate values. f , A box plot showing the FC by H 2 O 2 treatment for group B genes ( n = 1,601) in WT/KO, 6KR/KO and 6KQ/KO cells. g , FC values (H 2 O 2 treatment versus control condition in each cell type) of representative stress-protective genes in group B. If <0.05, the P values from a two-sided unpaired t -test are shown. h , A box plot showing FC by H 2 O 2 treatment for group F genes ( n = 8481) in WT/KO, 6KR/KO and 6KQ/KO cells. i , GO analyses with adjusted P values for GOBP (left) and KEGG (right) gene lists for group F genes. For f , h , boxes span the 25th–75th percentiles, the center line indicates the median, whiskers extend to 1.5× the interquartile range (IQR) and points beyond represent outliers. P values of a two-sided unpaired t -test comparing cell types are shown.

Article Snippet: To identify residues of MED1 with acetylation, the endogenous MED1 was immunoprecipitated from whole-cell lysate of HEK293T by anti-MED1 antibody (Bethyl/Fortis, A300-793, 1:800) followed by conjugation of protein A Sepharose CL-4B beads (Amersham, 17-0963-03).

Techniques: Control, Quantitative RT-PCR, Expressing, RNA Sequencing, Quantitative Proteomics

Journal: Nature Chemical Biology

Article Title: MED1 IDR deacetylation controls stress responsive genes through RNA Pol II recruitment

doi: 10.1038/s41589-025-02035-7

Figure Lengend Snippet: a – c , Density metaplots for predefined MED1 peaks (further classified according to their co-occupancy with MED17 and Pol II) ( a ), ER + active enhancers ( b ) and active promoters ( c ), showing the occupancy of MED1 (by detecting MED1 or its HA tag), MED17, Pol II (RPB1 S5P) and ERα (only in b ), with a comparison between 6KR/KO and WT/KO MCF7 cells. d , Heat maps of predefined active promoters showing occupancy of (ectopic HA-tagged) MED1, MED17 and Pol II (RPB1 S5P) with comparisons between 6KR/KO and WT/KO MCF7 cells. e – g , Metagene density plot ( e ) and heat map ( f ) for genes associated with predefined active promoters showing elevated occupancy of transcriptionally engaged Pol II (RPB1 S5P) across genic regions in 6KR/KO versus WT/KO MCF7 cells. The pausing index for each gene associated with these promoters was calculated accordingly and is shown with a box plot ( g ). The boxes span the 25th–75th percentiles, the center line indicates the median, whiskers extend to 1.5× the IQR and points beyond represent outliers. The P value of a two-sided unpaired t -test comparing cell types is shown ( n = 11,395 for each box). h , i , Metagene density plot ( h ) and heat map ( i ) for genes associated with predefined active promoters showing comparable occupancy of elongating Pol II (by detecting phosphorylation of RPB1 CTD S2 across genic regions in 6KR/KO versus WT/KO MCF7 cells. j , IGV snapshots of regulatory regions of two representative genes, TFF1 and GREB1 , with occupancy of HA (MED1), MED17 and Pol II (RPB1 S5P) shown for WT/KO and 6KR/KO cells. Epigenetic marks to identify enhancers and promoters are also shown.

Article Snippet: To identify residues of MED1 with acetylation, the endogenous MED1 was immunoprecipitated from whole-cell lysate of HEK293T by anti-MED1 antibody (Bethyl/Fortis, A300-793, 1:800) followed by conjugation of protein A Sepharose CL-4B beads (Amersham, 17-0963-03).

Techniques: Comparison, Phospho-proteomics

Journal: Nature Chemical Biology

Article Title: MED1 IDR deacetylation controls stress responsive genes through RNA Pol II recruitment

doi: 10.1038/s41589-025-02035-7

Figure Lengend Snippet: a , IGV snapshots of representative stress-induced genes with occupancy of HA (MED1) and Pol II (RPB1 S5P) shown for WT/KO and 6KR/KO cells. b , Comparable levels of representative Mediator subunits detected for association with immunoprecipitated endogenous MED17 in WT/KO, 6KR/KO and 6KQ/KO MCF7 cells. IgG was used as the IP control. c , Workflow depicting sequential ChIP-seq by anti-FLAG antibodies and pan-anti-acetyl-lysine antibodies to detect acetylation levels of FLAG-tagged MED1 (and its associated proteins) in genomic locations. d , Metaplots of occupancy of FLAG and acetylation for pre-defined MED1 peaks (from ChIP-seq in untreated MCF7) in High Ac, Mid Ac and Low Ac groups. e , Range-based heatmap of the occupancy of FLAG-MED1, acetyl-lysine (2 nd ChIP), MED17, Pol II (RPB1) and p300 at pre-defined MED1 peaks (from ChIP-seq in untreated MCF7) that are stratified into 3 tiers based on relative acetyl-lysine density (lower 25%, middle 50% and upper 75% percentile, referred to as Low Ac, Mid Ac, High Ac). f , Metaplots of occupancy of MED17, Pol II (RPB1 S5P) and p300 for pre-defined MED1 peaks (from ChIP-seq in untreated MCF7) with comparison among High Ac, Mid Ac and Low Ac groups. g , h , Pie diagram showing the constitution of High/Mid/Low Ac groups of MED1 peaks for occupancy of MED17 and Pol II ( g ), and types of regulatory elements ( h ).

Article Snippet: To identify residues of MED1 with acetylation, the endogenous MED1 was immunoprecipitated from whole-cell lysate of HEK293T by anti-MED1 antibody (Bethyl/Fortis, A300-793, 1:800) followed by conjugation of protein A Sepharose CL-4B beads (Amersham, 17-0963-03).

Techniques: Immunoprecipitation, Control, ChIP-sequencing, Comparison

Journal: Nature Chemical Biology

Article Title: MED1 IDR deacetylation controls stress responsive genes through RNA Pol II recruitment

doi: 10.1038/s41589-025-02035-7

Figure Lengend Snippet: a , Colloidal blue staining for HIS-tagged EGFP-fused MED1 IDRs (6KR or 6KQ, 963-1320 aa) purified from E.coli . b , Reduced saturation concentration for 6KQ versus 6KR MED1 IDR. N = 4 (biological replicates). P value of two-sided unpaired t-test is shown. c , Colloidal blue staining for WT MED1 IDR fragment (fused to EGFP) and CBP (GST-tagged) purified from E.coli (left), and immunoblotting confirming the enforced acetylation of MED1 IDR (right). d , Droplets formed by WT MED1 IDR (fused with EGFP), with enhanced in-droplet partitioning of the acetylated versus control MED1 IDR. Representative images are shown (left) where the scale bars are 10 μm. The partition coefficient (inside versus outside droplets) is plotted, comparing control and the acetylated MED1 IDR (right). Bars report mean ± SEM. P value of two-sided unpaired t-test is shown. The parentheses show the numbers of droplets analyzed (across 3 biological replicates). e , Reduced saturation concentration for CBP-acetylated versus control WT MED1 IDR. N = 4 (biological replicates). P value of two-sided unpaired t-test is shown. f , g , Faster recovery of droplets formed by CBP-treated versus control WT MED1 IDR revealed by FRAP. Images for representative droplets ( f ) and recovery curves ( g ) are shown. Scale bar for images is 10 μm. Data points report mean ± SEM. Control and CBP-acetylated MED1 IDRs were tested for 6 and 7 droplets (across 3 biological replicates), respectively. P values (two-sided unpaired t-test) for each data point are shown. h , RNA Pol II CTD heptad sequences used in the Pol II co-condensation experiments (left) and colloidal blue staining of the mCherry-fused Pol II CTD heptads (right). i , j , Immunoblotting detection of Mediator subunits and Pol II (RPB1) remaining on beads after washing the purified Mediator complex from HeLa-S cells by buffers with increasing concentrations of salt (100/150/200/300 mM KCl) ( i ) or buffers with 1,6- or 2,5-Hexanediol (Hexn) at 2% or 10% concentration ( j ). For panels a , c , h – j , gel staining and immunoblots are representative of three independent experiments.

Article Snippet: To identify residues of MED1 with acetylation, the endogenous MED1 was immunoprecipitated from whole-cell lysate of HEK293T by anti-MED1 antibody (Bethyl/Fortis, A300-793, 1:800) followed by conjugation of protein A Sepharose CL-4B beads (Amersham, 17-0963-03).

Techniques: Staining, Purification, Concentration Assay, Western Blot, Control

Journal: Nature Chemical Biology

Article Title: MED1 IDR deacetylation controls stress responsive genes through RNA Pol II recruitment

doi: 10.1038/s41589-025-02035-7

Figure Lengend Snippet: a , Droplet formation of EGFP-fused MED1 IDR showing enhanced partitioning of 6KQ versus 6KR MED1 IDR. Left, representative images are shown. Scale bar, 10 μm. Right, the partition coefficient of MED1 IDR (inside versus outside droplets) is plotted. Bars report the mean ± s.e.m. P values (two-sided unpaired t -test) and the numbers of droplets analyzed (across three biological replicates) are indicated. b , Differed turbidity (absorbance at 350 nm) for 6KQ versus 6KR MED1 IDRs. PEG and protein concentrations are indicated ( n = 7 for each condition; across three biological replicates). Bars indicate the mean ± s.e.m. If <0.05, the P values of a two-sided unpaired t -test are shown. c , d , Faster recovery of 6KQ relative to 6KR MED1 IDR droplets observed by FRAP. Images for representative droplets ( c ) are shown. Scale bar, 10 μm. In recovery curves ( d ), data points report the mean ± s.e.m. 6KR and 6KQ IDRs were tested for 10 and 12 droplets (across three biological replicates), respectively. P values (two-sided unpaired t -test) for each time point are shown. e , f , Increased incorporation of a mCherry-fused Pol II CTD heptad fragment into droplets formed by EGFP-fused 6KR ( e ) or nonacetylated WT ( f ) MED1 IDR, compared to the droplets formed by 6KQ ( e ) or acetylated WT ( f ) MED1 IDR. Left, representative images are shown. Scale bar, 10 μm. Right, the partition coefficient of the mCherry–Pol II fragment (inside versus outside MED1 IDR droplets) is plotted. P values of a two-sided unpaired t -test are shown. The parentheses show the numbers of MED1 IDR droplets analyzed across three biological replicates. g , Increased RNA Pol II enrichment was observed in condensates formed by the 6KR MED1 IDR compared to 6KQ in cells. Left, schematic of LacO array in cells where LacI fused with MED1 IDR forms condensates. Middle, immunofluorescence staining of Pol II in LacO array reporter cells with ectopic expression of GFP–LacI (control or fused with 6KR/Q MED1 IDR). Representative images are shown. Scale bar, 10 μm. Right, the partition coefficient of Pol II (intensity inside versus outside LacI puncta) is plotted for control and 6KR/Q MED1 IDR-fused GFP–LacI. Numbers of cells analyzed in each group (across three biological replicates) are shown in parentheses. P values of a two-sided unpaired t -test are shown. h , i , Metaplots ( h ) and heat maps ( i ) showing Pol II occupancy at high-Ac or low-Ac MED1 peaks (taking top and bottom 10%, respectively, from sequential ChIP-seq experiment) with or without 1,6-hexanediol (Hexn) treatment in MCF7 cells. j , ChIP–qPCR detection of Pol II occupancy at TSS of TFF1 gene showing increasing in 6KR/KO versus WT/KO MCF7 cells and reduction in both cells with 1,6-hexanediol treatment ( n = 6 biological replicates for Pol II and n = 2 biological replicates for nonspecific IgG control in each group). Bars report the mean ± s.e.m. P values of a two-sided unpaired t -test are shown.

Article Snippet: To identify residues of MED1 with acetylation, the endogenous MED1 was immunoprecipitated from whole-cell lysate of HEK293T by anti-MED1 antibody (Bethyl/Fortis, A300-793, 1:800) followed by conjugation of protein A Sepharose CL-4B beads (Amersham, 17-0963-03).

Techniques: Immunofluorescence, Staining, Expressing, Control, ChIP-sequencing, ChIP-qPCR

Journal: Nature Chemical Biology

Article Title: MED1 IDR deacetylation controls stress responsive genes through RNA Pol II recruitment

doi: 10.1038/s41589-025-02035-7

Figure Lengend Snippet: a , Measurement of tumor volume when mice bearing tumors of WT/KO or 6KR/KO MCF7 cells were sacrificed 133 or 95 days after implantation, respectively. Data are mean ± SEM. Number of tumors measured in each group is shown in parentheses. b , Immunoblotting showing comparable expression of HA-tagged (WT or 6KR) MED1 in WT/KO and 6KR/KO MCF7 tumors. Size-matched WT/KO and 6KR/KO MCF7 tumors were collected from NSG mice. Images with densitometry results (normalized by β-actin) are shown and plotted (right). c , Immunoblotting showing increased expression of NRF2 and NDRG1 in 6KR/KO versus WT/KO MCF7 tumors. Images with densitometry results (normalized by β-actin) are shown. d , e , Poly(A) RNA-seq for 6KR/KO and WT/KO MCF7 tumors developed in NSG mice. Three biological replicates were detected for each type of tumor. d , GSEA comparing the gene rank list with KEGG (left) and GOBP (right) pathway gene lists. Adjusted P values are noticed in addition to NES reported by bars. e , Fold change values of select stress-activated genes detected by RNA-seq. For b , c , each group has 3 tumors as biological replicates, the tests used the same samples, and the immunoblots are representative of three technical replicates. For a , b , the n.s. (no significance) indicates a p value > 0.05 (two-sided unpaired t-test).

Article Snippet: To identify residues of MED1 with acetylation, the endogenous MED1 was immunoprecipitated from whole-cell lysate of HEK293T by anti-MED1 antibody (Bethyl/Fortis, A300-793, 1:800) followed by conjugation of protein A Sepharose CL-4B beads (Amersham, 17-0963-03).

Techniques: Western Blot, Expressing, RNA Sequencing

Journal: Nature Chemical Biology

Article Title: MED1 IDR deacetylation controls stress responsive genes through RNA Pol II recruitment

doi: 10.1038/s41589-025-02035-7

Figure Lengend Snippet: A model showing deacetylation of MED1 in promoter-proximal regions by SEC-associated SIRT1 under stress conditions and enhanced incorporation of Pol II through IDR-mediated interaction between MED1 IDR and Pol II CTD (shown in upper inset images). Thus, through increased RNA Pol II association, deacetylation of MED1 amplifies induction of cyto-protective genes and mitigates suppression of growth-supportive genes in stress conditions (shown in lower inset images). In parallel with the extensively-studied action of SEC/P-TEFb on Pol II pausing-release, we propose a SEC-SIRT1-MED1 axis that enables the sequential entry and action of Pol II to support survival and growth of stressed cells. This image was created with BioRender.

Article Snippet: To identify residues of MED1 with acetylation, the endogenous MED1 was immunoprecipitated from whole-cell lysate of HEK293T by anti-MED1 antibody (Bethyl/Fortis, A300-793, 1:800) followed by conjugation of protein A Sepharose CL-4B beads (Amersham, 17-0963-03).

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