Journal: Nucleic Acids Research

Article Title: GSE1 links the HDAC1/CoREST co-repressor complex to DNA damage

doi: 10.1093/nar/gkad911

Figure Lengend Snippet: Identification of specific GSE1/CoREST complex with multi-enzymatic eraser activities. (A) Histone western blot detecting levels of ubiquitination of H2B at K120 upon treatment with etoposide for 6, 12 and 24 h. C-terminal histone H3 was used as a loading control. (B) Volcano plot displaying the interactome of GSE1 in HAP1 cells determined by AP-MS. Significant interactors (≥1.5-fold change [FC] over wildtype cells [dashed line], adjusted P -value ≤ 0.05) in GSE1 V5-tagged cells over HAP1 WT cells are shown with red dots. Gene names of known subunits of the HDAC1/CoREST complex are highlighted, GSE1 is highlighted with bold red text. (C) STRING DB-based protein interaction network of the GSE1 interactome. High-confidence interactions (confidence score ≥ 0.7) were selected. Strength of the associations is represented by the thickness of the edges. (D) Western blot depicting immunoprecipitated endogenous USP22 in HAP1 WT, HDAC1 KO and GSE1 KO cells. Antibodies used for detection are depicted on the right. (E) Potential mechanism of DNA damage regulation by GSE1. Proper initiation and orchestration of downstream signalling during DNA damage is highly dependent on the induction of Serine 139 phosphorylation of histone variant H2AX. To sustain proper signalling resulting in efficient DNA repair, a chromatin environment suitable for the recruitment of effector molecules of DNA damage signalling pathway is one of the essential requirements. Here we show a novel role of a neglected protein GSE1, a subunit of a specific CoREST/HDAC1 complex, in DDR. We propose that during DNA damage, GSE1/CoREST/HDAC1 complex can bind USP22 that can in return deubiquitinate histone H2B at lysine 120. This eraser event is necessary for sufficient γH2AX signalling as reported previously ( , ). In the absence of GSE1, USP22 cannot bind to GSE1/CoREST/HDAC1 complex and H2B lysine 120 remains ubiquitinated. This inadequate removal of the ubiquitin mark might interfere with γH2AX formation leading to reduced γH2AX signal and downstream signalling.

Article Snippet: Antibodies: GSE1 (KIAA0182) (Proteintech, 24947–1-AP, 1:1000), HDAC1 SAT 208 (Seiser lab, polyclonal rabbit, 1: 10 000), HDAC2 clone 3F3 (Seiser/Ogris lab, monoclonal mouse, 1:1000), CoREST (Millipore, 07-455, 1:1000), β-actin (Abcam, ab8226, 1:1000), cleaved caspase-3 (Cell Signalling, 9661, 1:1000), γH2AX (Millipore, JBW301, 1:1000), vinculin (Cell Signalling, 13901, 1:1000), LSD1 (Cell Signalling, 2139, 1:1000), USP22 (Novus Biologicals, NBP1-49644, 1:1000).

Techniques: Western Blot, Immunoprecipitation, Variant Assay

Journal: The EMBO Journal

Article Title: The SAGA complex regulates early steps in transcription via its deubiquitylase module subunit USP22

doi: 10.15252/embj.2019102509

Figure Lengend Snippet: A, B ChIP‐qPCR for USP22 at the CHOP and ERP70 loci before and after Thaps treatment, with and without shRNA‐mediated depletion of USP22. Three independent experiments are represented as mean ± SEM, with significance measured by Student’s t ‐test. *** P < 0.005. X‐axis labels on CHOP indicate coordinates relative to the TSS. C ChIP‐qPCR for USP22 and ATXN7L3 at bound ( GRP78 , SEL1L ) and unbound ( FNDC3B ) genes. Three independent experiments are represented as mean ± SEM. Desert serves as a negative control. D Venn diagram overlap of SAGA peaks prior to filtering for ER stress responsiveness as measured by increases in SAGA subunit ChIP‐seq signal following Thaps treatment.

Article Snippet: Lysate concentration was determined using the bicinchoninic acid (BCA) assay and analyzed by SDS–PAGE using antibodies against USP22 (Novus Biologicals #: NBP1‐49644), PARP (Cell Signaling #: 9532), caspase 3 (Cell Signaling #: 9662), actin (Santa Cruz #: sc‐8432), GAPDH (Cell Signaling #: 2118), CHOP (Cell Signaling #: 2895), GRP78 (Cell Signaling #: 3177), Ub‐H2B (Millipore #: 05‐1312), histone H2B (Santa Cruz #: sc‐10108), PERK (Cell Signaling # 5683), TBP (Abcam #: ab818), TFIIF (Abcam #: ab28179), Pol II (Santa Cruz #: sc‐9001), MED24 (Bethyl Laboratories #: A301‐472A), MED16 (Abcam #: ab130996), ORC2 (BD Pharmingen #: 559266) and β‐tubulin (Sigma‐Aldrich #: T4026), ubiquitin (Santa Cruz #: sc‐9133), ATF4 (Cell Signaling #: 11815), ATF6 (Abcam #: ab11909), Xbp1s (Cell Signaling #: 12782), GCN5 (Santa Cruz #: sc‐20698), ATXN7L3 (Bethyl Laboratories #: A302‐800A), MED16 (Abcam #: ab130996), and pan‐14‐3‐3 (Santa Cruz #: sc‐1657).

Techniques: shRNA, Negative Control, ChIP-sequencing

Journal: The EMBO Journal

Article Title: The SAGA complex regulates early steps in transcription via its deubiquitylase module subunit USP22

doi: 10.15252/embj.2019102509

Figure Lengend Snippet: A Cells were treated for 2 h with 100 nM Thapsigargin in the presence or absence of shRNAs targeting USP22. Whole‐cell lysate was subjected to immunoblotting with the indicated antibodies. B HCT116 cells were treated with 100 nM Thapsigargin for the indicated times in the presence or absence of shRNA targeting USP22. RNA was isolated and subjected to qRT–PCR against the indicated transcripts. Three independent experiments are represented as mean ± SEM. C, D Cells were treated as in (A) and subjected to ChIP‐qPCR for ATF4 and XBP1s at the indicated gene enhancers and promoters. Three independent experiments are represented as mean ± SEM. Source data are available online for this figure.

Article Snippet: Lysate concentration was determined using the bicinchoninic acid (BCA) assay and analyzed by SDS–PAGE using antibodies against USP22 (Novus Biologicals #: NBP1‐49644), PARP (Cell Signaling #: 9532), caspase 3 (Cell Signaling #: 9662), actin (Santa Cruz #: sc‐8432), GAPDH (Cell Signaling #: 2118), CHOP (Cell Signaling #: 2895), GRP78 (Cell Signaling #: 3177), Ub‐H2B (Millipore #: 05‐1312), histone H2B (Santa Cruz #: sc‐10108), PERK (Cell Signaling # 5683), TBP (Abcam #: ab818), TFIIF (Abcam #: ab28179), Pol II (Santa Cruz #: sc‐9001), MED24 (Bethyl Laboratories #: A301‐472A), MED16 (Abcam #: ab130996), ORC2 (BD Pharmingen #: 559266) and β‐tubulin (Sigma‐Aldrich #: T4026), ubiquitin (Santa Cruz #: sc‐9133), ATF4 (Cell Signaling #: 11815), ATF6 (Abcam #: ab11909), Xbp1s (Cell Signaling #: 12782), GCN5 (Santa Cruz #: sc‐20698), ATXN7L3 (Bethyl Laboratories #: A302‐800A), MED16 (Abcam #: ab130996), and pan‐14‐3‐3 (Santa Cruz #: sc‐1657).

Techniques: Western Blot, shRNA, Isolation, Quantitative RT-PCR

Journal: The EMBO Journal

Article Title: The SAGA complex regulates early steps in transcription via its deubiquitylase module subunit USP22

doi: 10.15252/embj.2019102509

Figure Lengend Snippet: A Flowchart depicting ChIP‐seq subsetting. B Metagene profiles for Pol II (top) and Ub‐H2B (bottom) ChIP‐seq for all genes not directly bound by USP22 and insensitive to USP22 knockdown, termed “Insensitive” genes. C, D ChIP‐qPCR for ATXN7L3 and GCN5 at the indicated loci, before and after ER stress, in the presence and absence of shRNA targeting USP22, represented from three independent experiments as mean ± SEM.

Article Snippet: Lysate concentration was determined using the bicinchoninic acid (BCA) assay and analyzed by SDS–PAGE using antibodies against USP22 (Novus Biologicals #: NBP1‐49644), PARP (Cell Signaling #: 9532), caspase 3 (Cell Signaling #: 9662), actin (Santa Cruz #: sc‐8432), GAPDH (Cell Signaling #: 2118), CHOP (Cell Signaling #: 2895), GRP78 (Cell Signaling #: 3177), Ub‐H2B (Millipore #: 05‐1312), histone H2B (Santa Cruz #: sc‐10108), PERK (Cell Signaling # 5683), TBP (Abcam #: ab818), TFIIF (Abcam #: ab28179), Pol II (Santa Cruz #: sc‐9001), MED24 (Bethyl Laboratories #: A301‐472A), MED16 (Abcam #: ab130996), ORC2 (BD Pharmingen #: 559266) and β‐tubulin (Sigma‐Aldrich #: T4026), ubiquitin (Santa Cruz #: sc‐9133), ATF4 (Cell Signaling #: 11815), ATF6 (Abcam #: ab11909), Xbp1s (Cell Signaling #: 12782), GCN5 (Santa Cruz #: sc‐20698), ATXN7L3 (Bethyl Laboratories #: A302‐800A), MED16 (Abcam #: ab130996), and pan‐14‐3‐3 (Santa Cruz #: sc‐1657).

Techniques: ChIP-sequencing, shRNA

Journal: The EMBO Journal

Article Title: The SAGA complex regulates early steps in transcription via its deubiquitylase module subunit USP22

doi: 10.15252/embj.2019102509

Figure Lengend Snippet: High‐throughput proteomic analysis of Pol II following depletion of USP22, with significance measured by Student’s t ‐test. Specific ubiquitylated lysines detected are highlighted in red text. HCT116 cells were treated for 2 h with 100 nM Thapsigargin in the presence or absence of shRNA targeting USP22. Cells were harvested and fractionated, and fractions were subjected to immunoblotting with the indicated antibodies. Loading controls ORC2 and β‐tubulin are duplicate images from Fig . In vitro deubiquitylation UbiTest of endogenously ubiquitylated Pol II. HCT116 cells were treated with MG132 in the presence or absence of shRNA targeting USP22. Lysates were generated using buffer containing protease cocktail inhibitor, pan‐DUB inhibitor PR619, and the JAMM protease inhibitor o ‐phenanthroline. Ubiquitylated proteins were purified on ubiquitin‐binding resin, and eluates were either undigested (lanes 1 and 2) or digested with USP2 to strip polyubiquitin (lanes 3 and 4) and reduce target proteins to unit length. Digestion reactions were subjected to immunoblotting with the indicated antibodies. shRNA control USP22 and loading controls actin and Ub are duplicate images from Fig . In vitro deubiquitylation of endogenously ubiquitylated proteins. HCT116 cells were treated as in (C). Ubiquitylated proteins were purified on ubiquitin‐binding resin, and eluates were either undigested (lane 3) or digested with non‐specific DUB (“USP2”, lane 4) or human DUB module (“USP22”, lane 5) to reduce target protein ubiquitylation. Digestion reactions were subjected to immunoblotting with the indicated antibodies. Source data are available online for this figure.

Article Snippet: Lysate concentration was determined using the bicinchoninic acid (BCA) assay and analyzed by SDS–PAGE using antibodies against USP22 (Novus Biologicals #: NBP1‐49644), PARP (Cell Signaling #: 9532), caspase 3 (Cell Signaling #: 9662), actin (Santa Cruz #: sc‐8432), GAPDH (Cell Signaling #: 2118), CHOP (Cell Signaling #: 2895), GRP78 (Cell Signaling #: 3177), Ub‐H2B (Millipore #: 05‐1312), histone H2B (Santa Cruz #: sc‐10108), PERK (Cell Signaling # 5683), TBP (Abcam #: ab818), TFIIF (Abcam #: ab28179), Pol II (Santa Cruz #: sc‐9001), MED24 (Bethyl Laboratories #: A301‐472A), MED16 (Abcam #: ab130996), ORC2 (BD Pharmingen #: 559266) and β‐tubulin (Sigma‐Aldrich #: T4026), ubiquitin (Santa Cruz #: sc‐9133), ATF4 (Cell Signaling #: 11815), ATF6 (Abcam #: ab11909), Xbp1s (Cell Signaling #: 12782), GCN5 (Santa Cruz #: sc‐20698), ATXN7L3 (Bethyl Laboratories #: A302‐800A), MED16 (Abcam #: ab130996), and pan‐14‐3‐3 (Santa Cruz #: sc‐1657).

Techniques: High Throughput Screening Assay, shRNA, Western Blot, In Vitro, Generated, Protease Inhibitor, Purification, Binding Assay, Stripping Membranes

Journal: The EMBO Journal

Article Title: The SAGA complex regulates early steps in transcription via its deubiquitylase module subunit USP22

doi: 10.15252/embj.2019102509

Figure Lengend Snippet: HCT116 human colorectal adenocarcinoma cells were treated for 2 h with 100 nM Thapsigargin to induce ER stress. Cells were cross‐linked, harvested, and subjected to high‐throughput ChIP‐sequencing for SAGA members USP22, GCN5, and ATXN7L3. A Genome browser images of ChIP‐seq for SAGA proteins before and after induction of ER stress at ER stress response genes CHOP, ERP70, and GRP78. ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952). B Heat map of USP22, GCN5, and ATXN7L3 association at the TSS for 29 genes whose promoters undergo significant recruitment of USP22 following induction of ER stress. C Mean ChIP‐seq signal for USP22, GCN5, and ATXN7L3 surrounding gene promoters from (B). Nonparametric Mann–Whitney analysis of 7 bins (gray shade) surrounding maximal peak in Thaps‐treated conditions is reported. D–F Linear regression analysis of SAGA member recruitment to target gene promoters following induction of ER stress.

Article Snippet: Lysate concentration was determined using the bicinchoninic acid (BCA) assay and analyzed by SDS–PAGE using antibodies against USP22 (Novus Biologicals #: NBP1‐49644), PARP (Cell Signaling #: 9532), caspase 3 (Cell Signaling #: 9662), actin (Santa Cruz #: sc‐8432), GAPDH (Cell Signaling #: 2118), CHOP (Cell Signaling #: 2895), GRP78 (Cell Signaling #: 3177), Ub‐H2B (Millipore #: 05‐1312), histone H2B (Santa Cruz #: sc‐10108), PERK (Cell Signaling # 5683), TBP (Abcam #: ab818), TFIIF (Abcam #: ab28179), Pol II (Santa Cruz #: sc‐9001), MED24 (Bethyl Laboratories #: A301‐472A), MED16 (Abcam #: ab130996), ORC2 (BD Pharmingen #: 559266) and β‐tubulin (Sigma‐Aldrich #: T4026), ubiquitin (Santa Cruz #: sc‐9133), ATF4 (Cell Signaling #: 11815), ATF6 (Abcam #: ab11909), Xbp1s (Cell Signaling #: 12782), GCN5 (Santa Cruz #: sc‐20698), ATXN7L3 (Bethyl Laboratories #: A302‐800A), MED16 (Abcam #: ab130996), and pan‐14‐3‐3 (Santa Cruz #: sc‐1657).

Techniques: High Throughput Screening Assay, ChIP-sequencing, Binding Assay, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: The SAGA complex regulates early steps in transcription via its deubiquitylase module subunit USP22

doi: 10.15252/embj.2019102509

Figure Lengend Snippet: HCT116 cells were treated with 100 nM Thapsigargin for the indicated times in the presence or absence of shRNA targeting USP22. Cells were harvested, and whole‐cell lysate was subjected to immunoblotting with the indicated antibodies. Cells were treated as in (A), but over an extended series of time points. RNA was isolated and subjected to qRT–PCR against the indicated transcripts. Three independent experiments are represented as mean ± SEM. A Tetracycline (Tet)‐inducible, shRNA‐resistant USP22 transgene was stably introduced into HCT116 cells. Cells were treated as in (A), with and without expression of the transgene via Tet treatment. Whole‐cell lysates were subjected to immunoblotting with the indicated antibodies. RNA was isolated from conditions described in (C) subjected to qRT–PCR analysis against the CHOP mRNA. Three independent experiments are represented as mean ± SEM, with significance measured by Student’s t ‐test. *** P < 0.005. Source data are available online for this figure.

Article Snippet: Lysate concentration was determined using the bicinchoninic acid (BCA) assay and analyzed by SDS–PAGE using antibodies against USP22 (Novus Biologicals #: NBP1‐49644), PARP (Cell Signaling #: 9532), caspase 3 (Cell Signaling #: 9662), actin (Santa Cruz #: sc‐8432), GAPDH (Cell Signaling #: 2118), CHOP (Cell Signaling #: 2895), GRP78 (Cell Signaling #: 3177), Ub‐H2B (Millipore #: 05‐1312), histone H2B (Santa Cruz #: sc‐10108), PERK (Cell Signaling # 5683), TBP (Abcam #: ab818), TFIIF (Abcam #: ab28179), Pol II (Santa Cruz #: sc‐9001), MED24 (Bethyl Laboratories #: A301‐472A), MED16 (Abcam #: ab130996), ORC2 (BD Pharmingen #: 559266) and β‐tubulin (Sigma‐Aldrich #: T4026), ubiquitin (Santa Cruz #: sc‐9133), ATF4 (Cell Signaling #: 11815), ATF6 (Abcam #: ab11909), Xbp1s (Cell Signaling #: 12782), GCN5 (Santa Cruz #: sc‐20698), ATXN7L3 (Bethyl Laboratories #: A302‐800A), MED16 (Abcam #: ab130996), and pan‐14‐3‐3 (Santa Cruz #: sc‐1657).

Techniques: shRNA, Western Blot, Isolation, Quantitative RT-PCR, Stable Transfection, Expressing

Journal: The EMBO Journal

Article Title: The SAGA complex regulates early steps in transcription via its deubiquitylase module subunit USP22

doi: 10.15252/embj.2019102509

Figure Lengend Snippet: HCT116 cells were treated for 2 h with 100 nM Thapsigargin in the presence or absence of shRNA targeting USP22. Cells were cross‐linked, harvested, and subjected to high‐throughput ChIP‐sequencing for RNA Pol II and Ub‐H2B. Genome browser images of ChIP‐seq for Pol II and Ub‐H2B before and after induction of ER stress, with and without USP22 knockdown, at ER stress response genes CHOP, ERP70, and GRP78. ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952). Metagene profiles for Pol II (upper panel) and Ub‐H2B (middle panel) ChIP‐seq for USP22‐dependent, USP22‐bound genes. Nonparametric Mann–Whitney analyses of 7 bins (gray shade) surrounding TSS (TSS) and the midpoint of the profiles (Body) in Thaps‐treated conditions are reported. mRNA analyses of select bound gene transcripts are presented (lower panel). Three independent experiments are represented as mean ± SEM, with significance measured by Student’s t ‐test. * P < 0.05, ** P < 0.01, and *** P < 0.005. Metagene profiles for Pol II (upper panel) and Ub‐H2B (middle panel) ChIP‐seq for USP22‐dependent, USP22‐unbound genes. Nonparametric Mann–Whitney analyses of 7 bins (gray shade) surrounding TSS (TSS) and the midpoint of the profiles (Body) in Thaps‐treated conditions are reported. mRNA analysis of select unbound gene transcripts are presented (lower panel). Three independent experiments are represented as mean ± SEM, with significance measured by Student’s t ‐test. ** P < 0.01 and *** P < 0.005.

Article Snippet: Lysate concentration was determined using the bicinchoninic acid (BCA) assay and analyzed by SDS–PAGE using antibodies against USP22 (Novus Biologicals #: NBP1‐49644), PARP (Cell Signaling #: 9532), caspase 3 (Cell Signaling #: 9662), actin (Santa Cruz #: sc‐8432), GAPDH (Cell Signaling #: 2118), CHOP (Cell Signaling #: 2895), GRP78 (Cell Signaling #: 3177), Ub‐H2B (Millipore #: 05‐1312), histone H2B (Santa Cruz #: sc‐10108), PERK (Cell Signaling # 5683), TBP (Abcam #: ab818), TFIIF (Abcam #: ab28179), Pol II (Santa Cruz #: sc‐9001), MED24 (Bethyl Laboratories #: A301‐472A), MED16 (Abcam #: ab130996), ORC2 (BD Pharmingen #: 559266) and β‐tubulin (Sigma‐Aldrich #: T4026), ubiquitin (Santa Cruz #: sc‐9133), ATF4 (Cell Signaling #: 11815), ATF6 (Abcam #: ab11909), Xbp1s (Cell Signaling #: 12782), GCN5 (Santa Cruz #: sc‐20698), ATXN7L3 (Bethyl Laboratories #: A302‐800A), MED16 (Abcam #: ab130996), and pan‐14‐3‐3 (Santa Cruz #: sc‐1657).

Techniques: shRNA, High Throughput Screening Assay, ChIP-sequencing, Binding Assay, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: The SAGA complex regulates early steps in transcription via its deubiquitylase module subunit USP22

doi: 10.15252/embj.2019102509

Figure Lengend Snippet: A Schematic of the CHOP locus. Black boxes indicate amplicons for subsequent ChIP‐qPCR analysis. B HCT116 cells were treated for 2 h with 100 nM Thapsigargin in the presence or absence of shRNA targeting USP22. Cells were cross‐linked, harvested, and subjected to ChIP‐qPCR for total RNA Pol II, pSer5 Pol II, and pSer2 Pol II at the loci across CHOP (indicated in (A)). Three independent experiments are represented as mean ± SEM. C ChIP‐qPCR for total RNA Pol II, pSer5 Pol II, and pSer2 Pol II at ERP70 , GRP78 , and a gene desert ( Prom = gene promoter and Coding = downstream coding region—see Appendix Table for primer sequences). Three independent experiments are represented as mean ± SEM. with significance measured by Student’s t ‐test. * P < 0.05, ** P < 0.01, and *** P < 0.005. D–G Cells were treated as in (B) and subjected to ChIP‐qPCR for TBP, TFIIB, TFIIF, and TAF7 at the indicated gene promoters. Three independent experiments are represented as mean ± SEM, with significance measured by Student’s t ‐test. * P < 0.05 and *** P < 0.005. H An aliquot of cells from (D‐G) were harvested for whole‐cell lysate and subjected to immunoblotting with the indicated antibodies. Source data are available online for this figure.

Article Snippet: Lysate concentration was determined using the bicinchoninic acid (BCA) assay and analyzed by SDS–PAGE using antibodies against USP22 (Novus Biologicals #: NBP1‐49644), PARP (Cell Signaling #: 9532), caspase 3 (Cell Signaling #: 9662), actin (Santa Cruz #: sc‐8432), GAPDH (Cell Signaling #: 2118), CHOP (Cell Signaling #: 2895), GRP78 (Cell Signaling #: 3177), Ub‐H2B (Millipore #: 05‐1312), histone H2B (Santa Cruz #: sc‐10108), PERK (Cell Signaling # 5683), TBP (Abcam #: ab818), TFIIF (Abcam #: ab28179), Pol II (Santa Cruz #: sc‐9001), MED24 (Bethyl Laboratories #: A301‐472A), MED16 (Abcam #: ab130996), ORC2 (BD Pharmingen #: 559266) and β‐tubulin (Sigma‐Aldrich #: T4026), ubiquitin (Santa Cruz #: sc‐9133), ATF4 (Cell Signaling #: 11815), ATF6 (Abcam #: ab11909), Xbp1s (Cell Signaling #: 12782), GCN5 (Santa Cruz #: sc‐20698), ATXN7L3 (Bethyl Laboratories #: A302‐800A), MED16 (Abcam #: ab130996), and pan‐14‐3‐3 (Santa Cruz #: sc‐1657).

Techniques: shRNA, Western Blot

Journal: The EMBO Journal

Article Title: The SAGA complex regulates early steps in transcription via its deubiquitylase module subunit USP22

doi: 10.15252/embj.2019102509

Figure Lengend Snippet: High‐throughput proteomic analysis of Mediator tail subunits MED16 and MED24 following depletion of USP22, with significance measured by Student’s t ‐test. Specific ubiquitylated lysines detected are highlighted in red text. HCT116 cells were treated for 2 h with 100 nM Thapsigargin in the presence or absence of shRNA targeting USP22. Cells were harvested and fractionated, and fractions were subjected to immunoblotting with the indicated antibodies. In vitro deubiquitylation UbiTest of endogenously ubiquitylated MED24. HCT116 cells were treated with MG132 in the presence or absence of shRNA targeting USP22. Lysates were generated using buffer containing protease cocktail inhibitor, pan‐DUB inhibitor PR619, and the JAMM protease inhibitor o ‐phenanthroline. Ubiquitylated proteins were purified on ubiquitin‐binding resin, and eluates were either undigested (lanes 1 and 2) or digested with USP2 to strip polyubiquitin (lanes 3 and 4) and reduce target proteins to unit length. Digestion reactions were subjected to immunoblotting with the indicated antibodies. Source data are available online for this figure.

Article Snippet: Lysate concentration was determined using the bicinchoninic acid (BCA) assay and analyzed by SDS–PAGE using antibodies against USP22 (Novus Biologicals #: NBP1‐49644), PARP (Cell Signaling #: 9532), caspase 3 (Cell Signaling #: 9662), actin (Santa Cruz #: sc‐8432), GAPDH (Cell Signaling #: 2118), CHOP (Cell Signaling #: 2895), GRP78 (Cell Signaling #: 3177), Ub‐H2B (Millipore #: 05‐1312), histone H2B (Santa Cruz #: sc‐10108), PERK (Cell Signaling # 5683), TBP (Abcam #: ab818), TFIIF (Abcam #: ab28179), Pol II (Santa Cruz #: sc‐9001), MED24 (Bethyl Laboratories #: A301‐472A), MED16 (Abcam #: ab130996), ORC2 (BD Pharmingen #: 559266) and β‐tubulin (Sigma‐Aldrich #: T4026), ubiquitin (Santa Cruz #: sc‐9133), ATF4 (Cell Signaling #: 11815), ATF6 (Abcam #: ab11909), Xbp1s (Cell Signaling #: 12782), GCN5 (Santa Cruz #: sc‐20698), ATXN7L3 (Bethyl Laboratories #: A302‐800A), MED16 (Abcam #: ab130996), and pan‐14‐3‐3 (Santa Cruz #: sc‐1657).

Techniques: High Throughput Screening Assay, shRNA, Western Blot, In Vitro, Generated, Protease Inhibitor, Purification, Binding Assay, Stripping Membranes

Journal: The EMBO Journal

Article Title: The SAGA complex regulates early steps in transcription via its deubiquitylase module subunit USP22

doi: 10.15252/embj.2019102509

Figure Lengend Snippet: HCT116 cells were treated for 2 h with 100 nM Thapsigargin in the presence or absence of shRNA targeting USP22. Cells were cross‐linked, harvested, and subjected to high‐throughput ChIP‐sequencing for Mediator subunits MED1 (middle) and MED16 (tail). A Genome browser image of ChIP‐seq for middle and tail Mediator subunits and H3K4me3 HiChIP tracks before and after induction of ER stress at ER stress response gene BHLHE40 . H3K4me1 and H3K27ac tracks are from the ENCODE Consortium (GEO accessions GSM945858 and GSM945853). ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952). B H3K4me3 HiChIP signal (log2) at promoter–enhancer loops before and after induction of ER stress or USP22 depletion at indicated bound group genes. C, D Mean ChIP‐seq signal MED1 and MED16 surrounding gene promoters from gene groups defined in Fig . E, F Linear regression analysis of Mediator and USP22 recruitment to USP22‐bound target gene promoters following induction of ER stress.

Article Snippet: Lysate concentration was determined using the bicinchoninic acid (BCA) assay and analyzed by SDS–PAGE using antibodies against USP22 (Novus Biologicals #: NBP1‐49644), PARP (Cell Signaling #: 9532), caspase 3 (Cell Signaling #: 9662), actin (Santa Cruz #: sc‐8432), GAPDH (Cell Signaling #: 2118), CHOP (Cell Signaling #: 2895), GRP78 (Cell Signaling #: 3177), Ub‐H2B (Millipore #: 05‐1312), histone H2B (Santa Cruz #: sc‐10108), PERK (Cell Signaling # 5683), TBP (Abcam #: ab818), TFIIF (Abcam #: ab28179), Pol II (Santa Cruz #: sc‐9001), MED24 (Bethyl Laboratories #: A301‐472A), MED16 (Abcam #: ab130996), ORC2 (BD Pharmingen #: 559266) and β‐tubulin (Sigma‐Aldrich #: T4026), ubiquitin (Santa Cruz #: sc‐9133), ATF4 (Cell Signaling #: 11815), ATF6 (Abcam #: ab11909), Xbp1s (Cell Signaling #: 12782), GCN5 (Santa Cruz #: sc‐20698), ATXN7L3 (Bethyl Laboratories #: A302‐800A), MED16 (Abcam #: ab130996), and pan‐14‐3‐3 (Santa Cruz #: sc‐1657).

Techniques: shRNA, High Throughput Screening Assay, ChIP-sequencing, HiChIP, Binding Assay