Journal: PLoS Genetics

Article Title: Trim33 Binds and Silences a Class of Young Endogenous Retroviruses in the Mouse Testis; a Novel Component of the Arms Race between Retrotransposons and the Host Genome

doi: 10.1371/journal.pgen.1005693

Figure Lengend Snippet: ( A ) Representative GFP profiles for MommeD44 wildtype (black) and heterozygous (red) mice are shown by the numbers of erythrocytes expressing GFP along a log scale of fluorescence. A GFP gate shows cells positive for GFP expression. ( B ) Genetic mapping of phenotypically heterozygous and wildtype F2 (C57/FVB) mice indicates the boundaries for the MommeD44 mutation. FVB/C57 genotype SNPs are shaded yellow and C57/C57 genotype SNPs are shaded blue. Numbers of mice representing each SNP profile are indicated. ( C ) The MommeD44 mutation is located in the Bromodomain of Trim33 and changes a leucine to a stop codon. ( D ) Mice heterozygous for the MommeD44 mutation had significantly reduced levels of Trim33 mRNA, normalized to the housekeeping gene Hprt , in the testis. * p value ≤ 0.05, error bars indicate SEM, n ≥ 3 mice per genotype. ( E ) Embryos from heterozygous intercrosses show that MommeD44 homozygous embryos die during early development. Astericies indicate abnormal embryos, represented by E10.5 MD44/MD44 image. ( F ) The weight of MommeD44 heterozygous mice ( MD44 / + ) at weaning. The weight of each mouse was normalized to the average weight of wildtype mice from each litter, error bars indicate SEM,* p value ≤ 0.05.

Article Snippet: To identify the binding sites of Trim33, across the genome, the same anti-Trim33 antibody (Bethyl Laboratories A301-060A) was used to perform ChIP in adult testis followed by high throughput sequencing of Trim33-bound DNA.

Techniques: Expressing, Fluorescence, Mutagenesis

Journal: PLoS Genetics

Article Title: Trim33 Binds and Silences a Class of Young Endogenous Retroviruses in the Mouse Testis; a Novel Component of the Arms Race between Retrotransposons and the Host Genome

doi: 10.1371/journal.pgen.1005693

Figure Lengend Snippet: ( A ) ChIP-seq was performed for Trim33 in adult testis. From the 9,109 detected peaks, the percentage that overlap with promoters (2Kb from a TSS), intragenic sequence and intergenic sequence are shown. ( B ) Trim33 occupancy over all RefSeq gene bodies is shown as read tag density along the transcription unit, including 2Kb up and downstream of the transcriptional start and stop site. ( C ) Read density for Trim33 and ENCODE histone marks were calculated across a 6Kb region centred on the 30,000 RefSeq TSSs. Datasets were clustered by read density. ( D ) The GREAT tool was used to test if regions associated with Trim33 peaks were significantly associated with expression of genes in a specific tissue/stage.

Article Snippet: To identify the binding sites of Trim33, across the genome, the same anti-Trim33 antibody (Bethyl Laboratories A301-060A) was used to perform ChIP in adult testis followed by high throughput sequencing of Trim33-bound DNA.

Techniques: ChIP-sequencing, Sequencing, Expressing

Journal: PLoS Genetics

Article Title: Trim33 Binds and Silences a Class of Young Endogenous Retroviruses in the Mouse Testis; a Novel Component of the Arms Race between Retrotransposons and the Host Genome

doi: 10.1371/journal.pgen.1005693

Figure Lengend Snippet: ( A ) Trim33 enrichment at retrotransposon classes that show at least a 1.25 fold change over input. RLTR10B and, to a lesser extent RLTR10B2, are enriched in Trim33 binding. ( B ) Trim33 binds to subsets of RLTR10B (166/478) and RLTR10B2 (100/850) repeats in the genome. ( C ) Read density for Trim33 is shown across the RLTR10B and RLTR10B2 element unit (shaded) ± 2Kb. All reads are shown in red and separately all reads with a Bowtie2 read quality of > 20 are shown in black. ( D ) The MEME-chip tool was used to identify enriched motifs in the sequences that overlapped Trim33 binding peaks across the genome in testis. The most significant of these matched a Myb transcription factor consensus sequence within RLTR10B elements. ( E ) Heat plot of ChIP-seq read density, clustered by similarity, for Trim33 and input for all RLTR10B and RLTR10B2 elements, against publically available ChIP-seq data for the A-Myb (plus input), GEO accession GSE44690, in testis and the testis Encode datasets for H3K27ac and H3K27me3. Each element shown is generated from 1Kb either side of the centre of RLTR10Bs and RLTR10B2s in the genome. ( F ) Significant Trim33 ChIP-seq peaks across the genome were compared to significant A-Myb ChIP-seq peaks across the genome, the overlap is shown.

Article Snippet: To identify the binding sites of Trim33, across the genome, the same anti-Trim33 antibody (Bethyl Laboratories A301-060A) was used to perform ChIP in adult testis followed by high throughput sequencing of Trim33-bound DNA.

Techniques: Binding Assay, Sequencing, ChIP-sequencing, Generated

Journal: PLoS Genetics

Article Title: Trim33 Binds and Silences a Class of Young Endogenous Retroviruses in the Mouse Testis; a Novel Component of the Arms Race between Retrotransposons and the Host Genome

doi: 10.1371/journal.pgen.1005693

Figure Lengend Snippet: ( A ) HEK293T cells were transfected with HA-tagged ubiquitin and FLAG-tagged A-MYB in the presence or in the absence of GFP-tagged TRIM33. To detect ubiquitinated A-MYB, immunoprecipitation was performed on the lysates using anti-FLAG affinity beads and a Western blot was performed using anti-HA antibodies (top panel). To detect the immunoprecipitated A-MYB, the Western blot was probed with anti-FLAG antibodies (middle panel). Expression of tagged TRIM33 and the loading control HSP70 is shown in the bottom panel. ( B ) HEK293T cells were transfected with HA-tagged ubiquitin and FLAG-tagged A-MYB and either a control or TRIM33 shRNA expression vector. A-MYB was immunoprecipitated and Western blotting was performed as in ( A ) to detect ubiquitinated A-MYB (top panel). Lysates were subjected to Western blotting with anti-TRIM33 antibody to show knock down and anti-γ-Tubulin antibody as a loading control (bottom panels). ( C ) The protein stability of A-MYB in the presence and in the absence of TRIM33 was monitored using the protein translation inhibitor cycloheximide (CHX). Cells were transfected as in ( A ) and CHX was added at 10 μg/ml and samples were analyzed by Western blots at various time points, as shown. HSP70 was used as the loading control. ( D ) FLAG-tagged A-MYB and GFP-tagged TRIM33 were co-expressed in HEK293T cells. Lysates were subjected to immunoprecipitation with anti-FLAG beads or, as a control, anti-IgG beads, and co-immunoprecipitation of TRIM33 with A-MYB was detected by Western blotting with anti-GFP and anti-FLAG antibodies, respectively (top panels). Expression of tagged Trim33 in lysate is shown by Western blotting with anti-GFP antibody and as a loading control anti-HSP70 is also shown (bottom panels).

Article Snippet: To identify the binding sites of Trim33, across the genome, the same anti-Trim33 antibody (Bethyl Laboratories A301-060A) was used to perform ChIP in adult testis followed by high throughput sequencing of Trim33-bound DNA.

Techniques: Transfection, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Expressing, Control, shRNA, Plasmid Preparation, Knockdown

Journal: PLoS Genetics

Article Title: Trim33 Binds and Silences a Class of Young Endogenous Retroviruses in the Mouse Testis; a Novel Component of the Arms Race between Retrotransposons and the Host Genome

doi: 10.1371/journal.pgen.1005693

Figure Lengend Snippet: ( A ) The Mean Intensity plot of RNA-seq data shows the log2 fold change of transcripts between wildtype (n = 3) and heterozygotes (n = 4), along a log2 scale of expression intensity (i.e. wildtype levels multiplied by heterozygous levels). Black triangles represent the 39 genes predicted to be significantly differentially expressed between the two sample groups. Trim33 is indicated as significantly lower in heterozygotes. ( B ) Trim33 binding at Nmnat3 . Visual inspection of the RNA-seq reads suggested that this gene was expressed from an upstream RLTR10B. ( C ) Reads from each individual repeat were mapped to a Repbase consensus sequence. Those repeat classes at which the RPKM was < 10 were excluded. The ratio of RPKM in wildtypes versus that in heterozygotes is shown on the Y-axis. Trim33 ChIP enrichment at each repeat class is shown on the X-axis. Only one class, RLTR10B (indicated), was bound by Trim33 (seven fold enriched) more than threefold above Input values and was at least 1.3 fold upregulated in heterozygous mutant mouse testis.

Article Snippet: To identify the binding sites of Trim33, across the genome, the same anti-Trim33 antibody (Bethyl Laboratories A301-060A) was used to perform ChIP in adult testis followed by high throughput sequencing of Trim33-bound DNA.

Techniques: RNA Sequencing, Expressing, Binding Assay, Sequencing, Mutagenesis

Journal: PLoS Genetics

Article Title: Trim33 Binds and Silences a Class of Young Endogenous Retroviruses in the Mouse Testis; a Novel Component of the Arms Race between Retrotransposons and the Host Genome

doi: 10.1371/journal.pgen.1005693

Figure Lengend Snippet: An RLTR10B element that acts as an alternative promoter of a nearby gene is shown. A-Myb recruits RNA Pol II and drives transcription. Trim33, acting as an E3 ubiquitin ligase, ubiquitinates and destabilises A-Myb, resulting in decreased expression of the locus.

Article Snippet: To identify the binding sites of Trim33, across the genome, the same anti-Trim33 antibody (Bethyl Laboratories A301-060A) was used to perform ChIP in adult testis followed by high throughput sequencing of Trim33-bound DNA.

Techniques: Ubiquitin Proteomics, Expressing

Journal: Nature communications

Article Title: Trim33 masks a non-transcriptional function of E2f4 in replication fork progression.

doi: 10.1038/s41467-023-40847-0

Figure Lengend Snippet: Fig. 3 | Deletion of Trim33 promotes E2f4 interactions with chromatin and with the Recql helicase. a Genome browser tracks of E2f4 ChIP-seq in p19/Nras Trim33- WT and Trim33-KO cells; n = 2. b Quantification of ChIP-seq tag coverage at E2f4 peaks in p19/Nras Trim33-WT and Trim33-KO cells. c Relationship between changes (log2FC) in E2f4 binding (normalized E2f4 ChIP-seq tags) and mRNA expression (log2FC) of the encoded gene for p19/Nras Trim33-KO vs Trim33-WT cells. Each dot is a bin of 5 genes sorted by the change in mRNA levels. Trendlines are derived from linear regression analysis. d LC-MS/MS analysis of E2f4 interactome in p19/Nras Trim33-WT and Trim33-KO cells. Known interaction partners are highlighted in blue. e PLA analysis of Recql-E2f4 association in p19/Nras Trim33-WT and Trim33- KO cells stably expressing HA-tagged E2f4 or vector Ctrl; from left, n = 49,49,50,50

Article Snippet: For mass spectrometry analysis of E2f4 interactions, three independent immunoprecipitation reactions with E2f4 antibodies (Proteintech 10923-1-AP) and Isotype Control rabbit IgG (Cell Signaling 3900) from the TNT-150 lysates of Trim33-WT and Trim33-KO p19/Nras cells were performed.

Techniques: ChIP-sequencing, Binding Assay, Expressing, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Stable Transfection, Plasmid Preparation