Journal: bioRxiv

Article Title: Intrinsic immunity against HAdV is achieved by a novel epigenetic silencing complex

doi: 10.1101/2025.02.10.637372

Figure Lengend Snippet: H1299 shCRL, shMPP8, sh PPHLN1, and shTASOR were infected with HAdV-wt at a MOI of 50 and harvested 48 h pi. (A) Viral particles were isolated from HAdV-infected cells by three cycles of freeze and thaw. HEK293 cells were reinfected with a serial dilution of the viral particles and virus yield was determined using quantitative E2A immunofluorescence staining. Bar charts represents mean values of three independent biological replicates. H1299 shCRL cells were set to 100% and the remaining samples were normalized. Statistically significant differences were determined using an one-way ANOVA and Dunnet’s T3 test with the GraphPad Prism10 software. ns = not significant; * p ≤ 0.05. (B) Whole cell lysates were prepared from some of the cells, from which virus particles were isolated. Proteins were separated by SDS PAGE and subjected to immunoblotting using mAb AC-15 (β-actin), rabbit pAb MPP8, rabbit pAb PPHLN1, rabbit pAb TASOR, and mouse mAb E2A. β-actin serves as a loading control. Molecular weights in kDa are indicated in the left, proteins are indicated on the right. (C,D) Total mRNA was isolated using TRIzol reagent, reverse transcribed and subjected to qPCR using oligonucleotides specific for viral transcripts (C) E1A and (D) Hexon. The data was normalized on the respective GAPDH, actin and RPL30 mRNA levels. Bar charts represents mean values of three independent biological replicates measured in technical triplicates. Statistically significant differences were determined using an unpaired student’s t test with the GraphPad Prism10 software. ns = not significant; ** p ≤ 0.01. (E) Whole cell lysates were prepared, proteins separated by SDS PAGE and subjected to immunoblotting using mAb AC- 15 (β-actin), rabbit pAb MPP8, mouse mAb E1A, mouse mAb E1B-55K, mouse mAb E2A, mouse mAb E4orf6, rat mAb E4orf3, and rabbit pAb Capsid. β-actin serves as a loading control. Molecular weights in kDa are indicated in the left, proteins are indicated on the right. (F) Densitometric analysis of detected band was performed using ImageJ (version 1.54m) to quantify protein levels. Relative protein levels were normalized to the respective housekeeping β-actin steady state levels. Bar charts represent mean values and standard deviations are based on two independent experiments. Statistically significant differences were determined using an unpaired student’s t test with the GraphPad Prism10 software. ns = not significant; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: Primary antibodies used for immunoblotting are mouse mAb AC-15 (anti-β-actin; Sigma-Aldrich), mouse mAb MPP8 (sc-398598; Santa Cruz Biotechnology), rabbit pAb MPP8 (HPA040035; Sigma-Aldrich), rabbit pAb PPHLN1 (HPA0038902; Sigma-Aldrich), rabbit pAb TASOR (FAM208A; NBP1-90673; Novus Biologicals), rabbit pAb NP220 (A301-547A; Biomol) mouse mAb Ubiquitin (P4D1; Cell Signaling), rabbit pAb PML (ab72137, abcam), mouse mAb FLAG(M2) (F1804, Sigma Aldrich), mouse mAb E1A (M73; sc-25, Santa Cruz Biotechnology; [ ]), mouse mAb E1B-55K (4E8, [ ]), mouse mAb E2A (B6-8, [ ]), rat mAb E4orf3 (6A11, [ ]), mouse mAb E4orf6 (RSA3, [ ]), and rabbit pAb Capsid (L133, [ ]).

Techniques: Infection, Isolation, Serial Dilution, Virus, Immunofluorescence, Staining, Software, SDS Page, Western Blot, Control, Reverse Transcription

Journal: bioRxiv

Article Title: Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin

doi: 10.1101/2025.01.21.634156

Figure Lengend Snippet: (A) SetDB1-HMm stabilizes the SetDB1 cofactor ATF7IP. WT or HMm mCherry-SetDB1 was stably expressed in SetDB1-KO HEK293 cells. Proteins were detected from total cell lysates by Western blot using antibodies against SetDB1, ATF7IP, and Tubulin. (B) SetDB1-HMm is present in the nucleus. WT or HMm mCherry-SetDB1 was stably expressed in SetDB1-KO HEK293 cells. Immunofluorescence was performed using an antibody against SetDB1. Nuclei were stained with DAPI.

Article Snippet: Anti-H3K9me3 (Abcam, ab8898), anti-SetDB1 (Proteintech, 11231-1-AP), anti-FLAG (Sigma, F1804), rabbit polyclonal anti-GFP (Chen et al., 2016), anti-MPP8 (Proteintech, 16796-1-AP), anti-HP1α (Cell Signaling, 2616), anti-KAP1 (Invitrogen, MA1-2023), anti-ATF7IP (Proteintech, 14699-1-AP), anti-Tubulin (Sigma, T5168), IRDye® anti-rabbit and anti-mouse secondary antibodies (LICORbio, 926-32210, 926-32211, 926-68071, 926-68070), HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (Cell Signaling, 7074 and 7076), Alexa Fluor 647-conjugated anti-rabbit secondary antibody (Invitrogen, A21245).

Techniques: Stable Transfection, Western Blot, Immunofluorescence, Staining

Journal: Molecular cell

Article Title: Co-transcriptional genome surveillance by HUSH is coupled to termination machinery

doi: 10.1016/j.molcel.2023.04.014

Figure Lengend Snippet: (A) Log of label-free quantification (LFQ) intensity for HA immunoprecipitation (IP) followed by mass-spectrometry in TASOR-AGH (y axis) and untagged control mESCs (x axis). Colored dots highlight associations of proteins with the HUSH complex (blue) or with RNA polymerase II (red). (B) Same as (A), for MPP8-AGH IP-MS. (C) Western blot showing coIP of MPP8 and WDR82 with TASOR (HA IP) in the TASOR-AGH cell line. Samples were incubated with RNaseA at 2 ng/μL during the IP. Inputs (in), non-bound flowthrough (NB), and HA peptide eluted (E) fractions at shown. (D) Enrichment of MPP8 irCLIP-seq for various genomic features. y axis was calculated by normalizing the number of RT stops to both length and level of transcription, as measured by PRO-seq. Exons were annotated by custom transcriptome models derived from RNA-seq of WT mESCs. (E) Aggregate plot showing MPP8 CLIP-seq normalized read count (y axis) relative to the distance from HUSH or MPP8-only ChIP peaks (x axis). HUSH peaks were divided into H3K9me3-positive (blue), H3K9me3-negative (purple), or MPP8-only regions based on the classification in . (F) Heatmaps showing ChIP-seq coverage for RNA polymerase II using different antibodies that recognize various C-terminal domain (CTD) modifications, centered and sorted as in . The x axis represents distance from MPP8 ChIP peak in kb.

Article Snippet: Antibodies used in this paper include: MPP8 (Proteintech, 16796–1), GFP (Thermo Fisher Scientific, A-11122), H3K9me3 (Abcam, ab8898), WDR82 (Cell Signaling, #99715), and H3K4me3 (Abcam, ab8580).

Techniques: Quantitative Proteomics, Immunoprecipitation, Mass Spectrometry, Control, Protein-Protein interactions, Western Blot, Incubation, Derivative Assay, RNA Sequencing, ChIP-sequencing

Journal: Molecular cell

Article Title: Co-transcriptional genome surveillance by HUSH is coupled to termination machinery

doi: 10.1016/j.molcel.2023.04.014

Figure Lengend Snippet: (A) Heatmap showing odds ratio values for overlap between H3K4me3, H3K9me3, MPP8, TASOR, and WDR82 ChIP-seq peaks. Peaks were called using MACS2 with broad peak settings. Statistics were calculated by BEDTools fisher. The number of peaks for respective proteins is indicated at the bottom. (B) Heatmaps of WDR82 ChIP-seq coverage in WT mESCs, centered and sorted as in . The x axis represents distance from MPP8 ChIP peak in kb. (C) Western blot of WT and TASOR-AGH mESC total cell lysates. Control and WDR82 KO clones from each cell line are shown. Membranes were probed with antibodies against WDR82, L1-ORF1p, and HSP90 as a loading control. (D) Heatmaps showing TASOR (left) or MPP8 (middle) differential ChIP-seq coverage in WDR82 KO – WT backgrounds compared with reanalyzed ChIP-seq coverage for TRIM28 (right). Signal is centered and sorted as in . The x axis represents distance from MPP8 ChIP peak in kb. (E) Aggregate plot showing TASOR ChIP-seq coverage (y axis) relative to the distance from HUSH ChIP peak (x axis) in WT (red) and WDR82 KO (blue) mESCs. (F) Top: browser tracks of TASOR, MPP8 and WDR82 ChIP-seq in WT and WDR82 KO mESCs. Bottom: browser tracks of PRO-seq in WT (orange) and WDR82 KO (blue) mESCs for the positive (+) and negative (−) DNA strands. The arrow marks the observed shifts in ChIP-seq and PRO-seq signals in WDR82 KO samples. (G) Aggregate plot showing TASOR ChIP-seq coverage individually scaled to span (0,1) interval in each sample (y axis) to visualize changes in plot shape, relative to the distance from HUSH ChIP peak (x axis) in WT (red) and WDR82 KO (blue) mESCs. (H) Heatmaps showing subtraction of PRO-seq coverage (WDR82 KO – WT), for sense (S, left) and antisense (AS, right) strands. Heatmaps were centered and sorted as in . The x axis represents distance from MPP8 ChIP peak in kb.

Article Snippet: Antibodies used in this paper include: MPP8 (Proteintech, 16796–1), GFP (Thermo Fisher Scientific, A-11122), H3K9me3 (Abcam, ab8898), WDR82 (Cell Signaling, #99715), and H3K4me3 (Abcam, ab8580).

Techniques: ChIP-sequencing, Western Blot, Control, Clone Assay

Journal: Molecular cell

Article Title: Co-transcriptional genome surveillance by HUSH is coupled to termination machinery

doi: 10.1016/j.molcel.2023.04.014

Figure Lengend Snippet: (A) Western blot in CPSF3-AGH mESCs, either with or without auxin treatment for 6 or 12 hours. Membranes were probed with antibodies against CPSF3 and β-actin. (B) Average plot of CPSF3 ChIP-seq coverage at H3K9me3-negative HUSH peaks in CPSF3-AGH cells with or without auxin represented by blue or red lines, respectively. The x axis indicates distance from HUSH ChIP peak in kb. (C) Average plot of MPP8 ChIP-seq coverage at H3K9me3-negative HUSH peaks in CPSF3-AGH cells with or without auxin represented by blue or red lines, respectively. The x axis indicates distance from HUSH ChIP peak in kb. (D) Fraction of HUSH peaks that overlap GENCODE-annotated transcription termination sites (TTS) for H3K9me3-positive and H3K9me3-negative regions. (E) Aggregate plots of TASOR, MPP8, WDR82, RNAPII (8WG16), and NELF ChIP-seq coverage centered on unique TSS, midpoints, or TTS (based on polyA) for H3K9me3-negative HUSH sites. Plotted are the median (solid line) and 95% CI of 1,000× bootstrap. The ordinate represents genomic distance relative to the feature in kb and the abscissa represents coverage (reads per billion per base per region).

Article Snippet: Antibodies used in this paper include: MPP8 (Proteintech, 16796–1), GFP (Thermo Fisher Scientific, A-11122), H3K9me3 (Abcam, ab8898), WDR82 (Cell Signaling, #99715), and H3K4me3 (Abcam, ab8580).

Techniques: Western Blot, ChIP-sequencing

Journal: Molecular cell

Article Title: Co-transcriptional genome surveillance by HUSH is coupled to termination machinery

doi: 10.1016/j.molcel.2023.04.014

Figure Lengend Snippet: (A) Schematic model for the mechanism of HUSH binding to endogenous, non-TE gene targets absent H3K9me3 (top-left), and silencing of young, transcriptionally-active TEs (top-right). HUSH associates with termination factors WDR82 and CPSF and tracks with RNAPII to survey transcribed portion of the genome. Loss of WDR82 results in transcriptional readthrough by RNAPII and prevents stable interaction of HUSH with chromatin at both H3K9me3-positive and -negative HUSH targets. We hypothesize that recognition of a cryptic TTS in an L1 or at the Sox2–1kb allele may provide a trigger H3K9me3. (B) Summary of the different regions and relevant features that are associated with H3K9me3 and/or HUSH ChIP-seq signals.

Article Snippet: Antibodies used in this paper include: MPP8 (Proteintech, 16796–1), GFP (Thermo Fisher Scientific, A-11122), H3K9me3 (Abcam, ab8898), WDR82 (Cell Signaling, #99715), and H3K4me3 (Abcam, ab8580).

Techniques: Binding Assay, ChIP-sequencing

Journal: Molecular cell

Article Title: Co-transcriptional genome surveillance by HUSH is coupled to termination machinery

doi: 10.1016/j.molcel.2023.04.014

Figure Lengend Snippet:

Article Snippet: Antibodies used in this paper include: MPP8 (Proteintech, 16796–1), GFP (Thermo Fisher Scientific, A-11122), H3K9me3 (Abcam, ab8898), WDR82 (Cell Signaling, #99715), and H3K4me3 (Abcam, ab8580).

Techniques: Virus, Recombinant, Protease Inhibitor, Multiplex Assay, Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation, CRISPR, Software, Flow Cytometry