Journal: bioRxiv

Article Title: Resistance of estrogen receptor function to BET bromodomain inhibition is mediated by transcriptional coactivator cooperativity

doi: 10.1101/2024.07.25.605008

Figure Lengend Snippet: a , Schematic of the knockin strategy for BRD4-dTAG. b , Agarose gel electrophoretic analysis of PCR products from MCF7 parental cells and MCF7 BRD4-dTAG knockin cells. c , Immunoblot for BRD4 expression in BRD4-dTAG cells treated with dTAG-7 or dTAG V -1 at indicated concentrations for indicated times. dBET6 was added for 2 hours to show changes in Pol II CTD Ser2-P. d , Immunoblot showing effects of different 2-hour treatments on Pol II and H2Bub1 in BRD4-dTAG cells. e , Percentage of MCF7 BRD4-dTAG cell survival after treatment with or without BET inhibitors and dTAG-7 (250 nM) for 6 days. Data shown as mean ± s.d. from biological triplicates. f , Immunoblot showing reduced ER expression in BRD4-dTAG cells upon overnight treatment with JQ1 and dTAG-7. g,h, qPCR of MYC, Cyclin D1 ( h ) and ESR1-Y537 g, mRNA levels in MCF7 BRD4-dTAG ESR1-Y537S cells upon overnight treatment with BET inhibitors and dTAG-7 (500 nM). Because ESR1-Y537S-IRES-GFP is transcribed as a single transcript, ESR1 -Y537S RNA level was monitored by GFP RNA. All cells were grown in normal full medium containing 10% FBS. Data shown as mean ± s.d. from independent triplicates. p values from two-tailed t-tests are depicted with asterisks.

Article Snippet: ERα (#D8H8, WB), H2Bub1 (#5546), MED26 (#13641), and Rpb1 NTD (#D8L4Y, ChIP) antibodies were from Cell Signaling.

Techniques: Knock-In, Agarose Gel Electrophoresis, Western Blot, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: B4 Raf-like MAPKKK RAF24 regulates Arabidopsis thaliana flowering time through HISTONE MONO-UBIQUITINATION 2

doi: 10.1101/2024.06.12.598286

Figure Lengend Snippet: (A) and (B) Loss of RAF24 increases H2Bub1 levels. Immunoblotting analysis of 10d-old WT and raf24-2 plants constitutively over-expressing TAP-HUB2. 50 μg of protein samples were loaded in each lane. Biological replicates (n=3) were examined and quantified with ImageJ software, with levels of H2Bub1 normalized to H2B levels. (C) and (D) Over-expression of TAP-HUB2 in raf24-2 also leads to an early flowering phenotype. Two independent lines of 29d-old WT and raf24-2 plants (denoted by the number) constitutively expressing TAP-HUB2 were grown and imaged (C), with flowering time determined by days to bolting (upper) and rosette number at bolting (bottom) (D). Letters indicate significant differences between genotypes (one-way ANOVA p-value < 0.05 ).

Article Snippet: After blocking 1 hour with 5% (w/v) non-fat milk, PVDF membranes were incubated overnight with anti-H2B (1:2500, abcam, ab1790), anti-H2Bub1 (1:1000, Cell Signaling, #5546), anti-GFP (1:2000, ChromoTek, PABG1), anti-HA(1:1000, Invitrogen, 26183), anti-H3 (1:2000, Invitrogen, PA5-16183) or anti-PAP (1:1000, SIGMA, P1291) followed by a 2-hour incubation with secondary antibodies, anti-rabbit (1:10000, Invitrogen, G-21234) or anti-mouse (1:5000, Invitrogen, G-21040).

Techniques: Western Blot, Expressing, Software, Over Expression

Journal: bioRxiv

Article Title: B4 Raf-like MAPKKK RAF24 regulates Arabidopsis thaliana flowering time through HISTONE MONO-UBIQUITINATION 2

doi: 10.1101/2024.06.12.598286

Figure Lengend Snippet: (A) and (B) Comparison of flowering time in raf4-2 plants constitutively over-expressing HA-HUB2 with no (S314S), phospho-ablative (S314A) and phospho-mimetic (S314D) mutations (A). Flowering time was determined by days to bolting and rosette number at bolting (B). Letters indicate significant differences between genotypes (one-way ANOVA p-value < 0.05). (C) Immunoblot analysis of raf24-2 plants constitutively over-expressing phospho-ablative (HA-HUB2 S314A ), phospho-mimetic (HA-HUB2 S314D ), which finds HA-HUB2 S314D maintains lower H2Bub1 levels. 50 μg of protein samples were loaded in each lane. Equal amounts of HA-HUB over-expression in the raf24-2 background was observed across the lines depicted here (Supplemental Figure 7).

Article Snippet: After blocking 1 hour with 5% (w/v) non-fat milk, PVDF membranes were incubated overnight with anti-H2B (1:2500, abcam, ab1790), anti-H2Bub1 (1:1000, Cell Signaling, #5546), anti-GFP (1:2000, ChromoTek, PABG1), anti-HA(1:1000, Invitrogen, 26183), anti-H3 (1:2000, Invitrogen, PA5-16183) or anti-PAP (1:1000, SIGMA, P1291) followed by a 2-hour incubation with secondary antibodies, anti-rabbit (1:10000, Invitrogen, G-21234) or anti-mouse (1:5000, Invitrogen, G-21040).

Techniques: Comparison, Expressing, Western Blot, Over Expression

Journal: bioRxiv

Article Title: B4 Raf-like MAPKKK RAF24 regulates Arabidopsis thaliana flowering time through HISTONE MONO-UBIQUITINATION 2

doi: 10.1101/2024.06.12.598286

Figure Lengend Snippet: (A) In WT, the presence of RAF24 phosphorylates SnRK2s to induce phosphorylation of HUB2, ensuring appropriately tuned HUB2-ligase activity. This in turn optimizes HUB2ub1 levels to maintain proper transcriptional activation of FLC expression and flowering time . Upon the loss of RAF24, SnRK2 fails to phosphorylate HUB2, causing an over-accumulation of H2Bub1 and reduced transcriptional activation of FLC expression. This results in earlier flowering (B) Both the phosphoproteomic and HUB2-TAP pulldowns find that RAF24 acts upstream of a wide variety of floral regulators, including BES1 and FBH3 as well as HUB2 to fine-tune flower time. Identification of both known HUB2 interactors (with straight line) and new, candidate HUB2 interactors (with a dash line) related to flowering time indicates that RAF24 possesses versatile functions / mechanisms in controlling flowering time.

Article Snippet: After blocking 1 hour with 5% (w/v) non-fat milk, PVDF membranes were incubated overnight with anti-H2B (1:2500, abcam, ab1790), anti-H2Bub1 (1:1000, Cell Signaling, #5546), anti-GFP (1:2000, ChromoTek, PABG1), anti-HA(1:1000, Invitrogen, 26183), anti-H3 (1:2000, Invitrogen, PA5-16183) or anti-PAP (1:1000, SIGMA, P1291) followed by a 2-hour incubation with secondary antibodies, anti-rabbit (1:10000, Invitrogen, G-21234) or anti-mouse (1:5000, Invitrogen, G-21040).

Techniques: Activity Assay, Activation Assay, Expressing

Journal: Nature Communications

Article Title: Two DOT1 enzymes cooperatively mediate efficient ubiquitin-independent histone H3 lysine 76 tri-methylation in kinetoplastids

doi: 10.1038/s41467-024-46637-6

Figure Lengend Snippet: a Human active-state DOT1L- Xenopus nucleosome cryo-EM structure (PDB: 6NQA). Key interactions are shown. 1: DNA binding, 2: H4-tail-DOT1L interactions, 3: acidic patch-DOT1L arginine anchor interactions, and 4: H2Bub1-DOT1L interactions. The positively-charged H2B K108 and DOT1L K270 residues would cause a repulsion. b Model of a DOT1A-nucleosome complex obtained by superimposing the DOT1A crystal structure (Molecule I, PDB: 8FJN) onto the human active-state DOT1L- Xenopus nucleosome cryo-EM structure (PDB: 6NQA). c Most protected histone residues by wild-type DOT1A from X-ray footprinting. d Sequence alignment of motif VI, two potential DOT1A/DOT1B arginine anchor residues, and two DOT1L arginine anchor residues. e Methyltransferase activity of wild-type Δ42-DOT1A and the Δ42-DOT1A R254A/R260A double mutant (RR/AA) toward the wild-type T. brucei nucleosome. Data are presented as mean values ± SD of n independent experiments ( n = 19 for WT Δ42-DOT1A, n = 3 for R254A/R260A Δ42-DOT1A, n = 8 for No Enzyme). Data were analyzed by one-way ANOVA followed by Tukey’s post-hoc test. Adjusted P value between WT Δ42-DOT1A vs. R254A/R260A Δ42-DOT1A is <0.001, WT Δ42-DOT1A vs. No Enzyme is <0.001, R254A/R260A Δ42-DOT1A vs. No Enzyme is >0.99. f Methyltransferase activity of human wild-type DOT1L (residues 1–420) and human DOT1L (residues 1–420) Lys270Asp mutant toward human un-ubiquitinated nucleosome. Data are presented as mean values ± SD of n independent experiments ( n = 3 for WT DOT1L (1–420), n = 2 for K270D DOT1L (1–420), n = 8 for No Enzyme). Data were analyzed by one-way ANOVA followed by Tukey’s post-hoc test. Adjusted P value between WT DOT1L (1–420) vs. K270D DOT1L (1–420) is 0.002, DOT1L (1–420) vs. No Enzyme is <0.001, K270D DOT1L (1-420) vs. no enzyme is 0.001. g Sequence alignment of histone H2B αC helix of human, Xenopus, yeast, and trypanosomes (Tryp). h Methyltransferase activity of wild-type Δ42-DOT1A and D247K Δ42-DOT1A mutant. Data are presented as mean values ± SD of n independent experiments ( n = 19 for WT Δ42-DOT1A, n = 5 for Δ42-DOT1A D247K, n = 8 for No Enzyme). Data was analyzed by one-way ANOVA followed by Tukey’s post-hoc test. Adjusted P value between WT Δ42-DOT1A vs. D247K Δ42-DOT1A is <0.001, WT Δ42-DOT1A vs. No Enzyme is <0.001, D247K Δ42-DOT1A vs. No Enzyme is >0.99. Source data are provided as a Source Data file.

Article Snippet: H2Bub1 was loaded on a SP cation exchange column (Cytiva), and H2Bub1-containing fractions were dialyzed against water, frozen, and lyophilized.

Techniques: Cryo-EM Sample Prep, Binding Assay, Footprinting, Sequencing, Activity Assay, Mutagenesis

Journal: Cell Communication and Signaling : CCS

Article Title: USP22 supports the aggressive behavior of basal-like breast cancer by stimulating cellular respiration

doi: 10.1186/s12964-023-01441-5

Figure Lengend Snippet: Mammary tissue-specific loss of Usp22 impairs the stem cell-like properties of the growing murine mammary gland: A Schematic representation of the two transgenes of the MMTV-cre; Usp22 fl/fl mouse model. B Whole mounts staining of mammary glands showing a significant decrease of mammary duct branching density in MMTV-cre; Usp22 fl/fl mice compared to the control group with representative brightfield pictures (left panel) and the respective quantification of branching density and number of ex vivo cultured mammospheres (right panel). C Mammosphere formation assay mammary epithelial cells from MMTV; Usp22 wt/wt and MMTV; Usp22 fl/fl mice. D Hematoxylin and eosin staining (left panel) and immunohistochemical detection of H2Bub1 (right panel) on mammary gland sections from MMTV-cre; Usp22 fl/fl mice and MMTV-cre mice. E-I Brightfield pictures (white scale bar: 100 µm) ( E ), western blot analysis of USP22 protein ( F ), growth kinetics ( G ), colony formation assay ( H ) and mammosphere formation assay ( I ) in siControl- and siUSP22-treated MCF10A and MCF12A cells. J Gene set enrichment analysis (GSEA) performed on the high-throughput RNA sequencing data of siControl- and siUSP22-treated MCF10A cells (accession number: E-MTAB-8247). Statistics: B (right panel): One-way Anova; C, G (based on the area under the curve = AUC); I: Student t-test. * p <0.05, *** p <0.005. All experiments were performed in at least three biological replicates

Article Snippet: Anti-H2Bub1 antibody (mouse, homemade at 1:50 dilution) or anti-USP22 (Sigma-Aldrich, HPA044980 at 1:100 dilution) was diluted on blocking solution and samples were treated overnight.

Techniques: Staining, Ex Vivo, Cell Culture, Tube Formation Assay, Immunohistochemistry, Western Blot, Colony Assay, High Throughput Screening Assay, RNA Sequencing Assay