Journal: Genes & Development

Article Title: H3K9 trimethylation in active chromatin restricts the usage of functional CTCF sites in SINE B2 repeats

doi: 10.1101/gad.349282.121

Figure Lengend Snippet: Effect of SETDB1 depletion on H3K9me3 profiles in untreated and LPS-stimulated macrophages. ( A ) Snapshot showing H3K9me3 profiles in control and SETDB1-depleted untreated mouse bone marrow-derived macrophages in a 7-Mb genomic region. Compartments A (red) and B (blue) are indicated. Normalized H3K9me3 counts are reported. Data obtained with one of two distinct nontargeting (NT) or SETDB1 targeting shRNAs are shown. ( B ) H3K9me3 signal as a function of Hi-C compartment eigenvalue score. Shown are the H3K9me3 read counts per kilobase per compartment on the Y -axes compared with the compartment score on the X -axes in either compartment A (red) or compartment B (blue). Compartments were identified using Hi-C contact data at 40-kb resolution. ( C ) Mean read counts per kilobase in inactive ( left ) and active ( right ) compartments in control (NT sh; gray) and SETDB1-depleted (SETDB1 sh; orange) macrophages. Two-way ANOVA with Tukey multiple comparison test. (**) Adjusted P -value < 0.01. ( D ) H3K9me3 metaprofiles at genomic regions ( n = 7543) showing reduced H3K9me3 in SETDB1-depleted untreated macrophages. Shown is the metaplot of the replicate average of H3K9me3 signal per base pair in control (gray) and SETDB1-depleted (orange) conditions. ( E ) Cumulative megabases of genomic regions showing loss of H3K9me3 in SETDB1-depleted and untreated macrophages using different thresholds (adjusted P -value and fold change computed via DESeq2) for differential peak calling. ( F ) Effects of 2 h of LPS stimulation on the genomic profiles of H3K9me3 in untreated macrophages transduced with a nontargeting (NT) shRNA. ( G ) Effects of 2 h of LPS stimulation on the 7543 H3K9me3 blocks methylated by SETDB1. ( H ) H3K9me3 signal intensities at the 7543 H3K9me3 blocks methylated by SETDB1 in LPS-stimulated macrophages transduced with either a nontargeting (NT, X -axis) or a SETDB1 targeting ( Y -axis) shRNA.

Article Snippet: BMDM cells obtained from Rosa26-Cas9 knock-in mice ( ) were coinfected with two lentiGuide-Puro (Addgene plasmid 52963) plasmids, each one expressing one sgRNA of 15 pairs targeting the CTCF motif upstream of the Tmem154 locus (chromosome 3: 84,657,704–84,659,779) or two irrelevant target guides and the PLKO.1 lentiviral shRNA vectors targeting Setdb1 or PLKO.1 nontargeting. sgRNAs were designed using the CRISPR design tool CHOPCHOP ( https://chopchop.cbu.uib.no ) and cloned into the lentiGuide-Puro plasmid according to the manufacturer's instruction.

Techniques: Control, Derivative Assay, Hi-C, Comparison, Transduction, shRNA, Methylation

Journal: Genes & Development

Article Title: H3K9 trimethylation in active chromatin restricts the usage of functional CTCF sites in SINE B2 repeats

doi: 10.1101/gad.349282.121

Figure Lengend Snippet: Impact of SETDB1 depletion on the macrophage epigenome. ( A ) Genomic occupancy of PU.1 in control (NT sh; X -axis) and SETDB1-depleted (SETDB1 sh; Y -axis) untreated macrophages. PU.1 peaks were called using MACS2 with an FDR ≤10 −15 and retaining only peaks with overlap >50% in the two replicates ( n = 38,580). ( B ) Overlap between sites showing H3K9me3 signal loss in SETDB1-depleted macrophages and enhancers active in other cell types or tissues. ( C ) H3K27ac genomic distribution in control and SETDB1-depleted macrophages. ChIP-seq data sets were generated using cells transduced with two nontargeting (NT) or two SETDB1 targeting shRNAs. ( D ) Overlap of H3K27ac gain with genomic regions showing H3K9me3 reduction in SETDB1-depleted macrophages. ( E ) Overlap of genomic regions characterized by reduced H3K9me3 and gained H3K27ac in SETDB1-depleted macrophages with enhancers active in other lineages ( left ) or PU.1 peaks ( right ). ( F ) Genomic regions showing a gain in H3K27ac were divided based on the overlap with reduced H3K9me3 and analyzed for chromatin accessibility by ATAC-seq and PU.1 genomic occupancy. Shown is the metaplot of the replicate average of H3K27ac signal per base pair for the conditions reported. ( G ) Effects of LPS stimulation on H3K27ac at regions showing H3K27ac gain upon SETDB1 depletion. Shown are the box plots for the normalized reads per kilobase per individual replicates. Two-way ANOVA with Tukey multiple comparison test. (****) P < 0.0001. ( H ) Overrepresentation of transcription factor motifs at genomic sites showing increased H3K27ac in SETDB1-depleted macrophages, divided based on the overlap with H3K9me3 loss. Enrichment was calculated using all accessible sites as background sequences.

Article Snippet: BMDM cells obtained from Rosa26-Cas9 knock-in mice ( ) were coinfected with two lentiGuide-Puro (Addgene plasmid 52963) plasmids, each one expressing one sgRNA of 15 pairs targeting the CTCF motif upstream of the Tmem154 locus (chromosome 3: 84,657,704–84,659,779) or two irrelevant target guides and the PLKO.1 lentiviral shRNA vectors targeting Setdb1 or PLKO.1 nontargeting. sgRNAs were designed using the CRISPR design tool CHOPCHOP ( https://chopchop.cbu.uib.no ) and cloned into the lentiGuide-Puro plasmid according to the manufacturer's instruction.

Techniques: Control, ChIP-sequencing, Generated, Transduction, Comparison

Journal: Genes & Development

Article Title: H3K9 trimethylation in active chromatin restricts the usage of functional CTCF sites in SINE B2 repeats

doi: 10.1101/gad.349282.121

Figure Lengend Snippet: SETDB1 deposited H3K9me3 and control of CTCF recruitment to SINE B2 repeats. ( A ) Enrichment of different repeat families at genomic regions showing H3K9me3 loss in SETDB1-depleted macrophages. Reported is the Bonferroni adjusted P -value and the log 2 scaled ratio between expected and observed frequencies. ( B ) Motif overrepresentation analysis at repeats contained in genomic regions showing H3K9me3 reduction ( left ) or H3K9me3 loss and concurrent H3K27ac gain ( right ) upon SETDB1 depletion. Data are separately shown for LTR and SINE retrotransposons. Enrichment was computed using HOMER , providing distinct custom sets of background sequences: sequences of all LTRs and SINEs in the mm10 genome for the 7543 H3K9me3 set and sequences of LTRs and SINEs within the 7543 H3K9me3 sites for the 257 H3K9me3 sites with concurrent hyperacetylation. ( C ) CTCF genomic distribution in control and SETDB1-depleted macrophages. CTCF ChIP-seq was carried out using cells transduced with either two nontargeting (NT) shRNAs or two SETDB1 targeting shRNAs. ( D ) CTCF binding at genomic regions showing reduced H3K9me3 in SETDB1-depleted macrophages. ( E ) Quantitation of the effects shown in D reporting the normalized read counts per kilobase of CTCF in the various conditions. Two-way ANOVA with Tukey multiple comparison test. (****) P < 0.0001. ( F ) CTCF peaks called as up-regulated (red), unaffected (gray), or down-regulated (blue) upon SETDB1 depletion. ( G ) Association of constitutive CTCF peaks and peaks induced upon SETDB1 depletion with different families of repeat elements. Shown is the repeat density per base pair. ( H ) Distance between CTCF peaks and H3K27ac peaks induced upon SETDB1 depletion. ( Left ) Distance between gained H3K27ac peaks ( n = 579) and constitutive (gray) or induced (red) CTCF peaks. (****) P < 0.0001, Mann–Whitney test. ( Right ) Bootstrap resampling test assessing the mean distance between randomly selected CTCF peaks and gained H3K27ac ( P < 0.0001, Wilcoxon signed rank test). Mean distance between induced CTCF peaks and up-regulated H3K27ac sites is shown with a dashed green line. ( I ) Cumulative distribution of the H3K9me3 signal centered on the summits of the CTCF peaks induced upon SETDB1 depletion. ( J ) Heat maps displaying CTCF and H3K9me3 ChIP-seq signals for constitutive and induced CTCF peaks in both control (NT sh) and SETDB1-depleted (SETDB1 sh) cells. Enlarged view for the 1644 induced CTCF peaks is shown at the right of each heat map. ( K ) Inverse relationship between CTCF binding intensity (±400 bp surrounding CTCF peak summit) and H3K9me3. CTCF peaks were divided into quintiles based on increasing H3K9me3 signal. Two-way ANOVA with Tukey multiple comparison test. (****) P < 0.0001. ( L ) CpG DNA methylation at both CTCF-induced and not induced peaks. In case multiple CTCF motifs were associated with a single peak, only methylation data at the best motif were reported.

Article Snippet: BMDM cells obtained from Rosa26-Cas9 knock-in mice ( ) were coinfected with two lentiGuide-Puro (Addgene plasmid 52963) plasmids, each one expressing one sgRNA of 15 pairs targeting the CTCF motif upstream of the Tmem154 locus (chromosome 3: 84,657,704–84,659,779) or two irrelevant target guides and the PLKO.1 lentiviral shRNA vectors targeting Setdb1 or PLKO.1 nontargeting. sgRNAs were designed using the CRISPR design tool CHOPCHOP ( https://chopchop.cbu.uib.no ) and cloned into the lentiGuide-Puro plasmid according to the manufacturer's instruction.

Techniques: Control, ChIP-sequencing, Transduction, Binding Assay, Quantitation Assay, Comparison, MANN-WHITNEY, DNA Methylation Assay, Methylation

Journal: Genes & Development

Article Title: H3K9 trimethylation in active chromatin restricts the usage of functional CTCF sites in SINE B2 repeats

doi: 10.1101/gad.349282.121

Figure Lengend Snippet: Effect of SETDB1 depletion on chromatin organization in macrophages. ( A ) Insulation scores were measured at insulation sites in control (NT sh) and SETDB1-depleted (SETDB1 sh) macrophages, and differences were calculated. Insulation sites were identified genome-wide as point of local minima in the insulation score at 10-kb resolution. ( B ) CTCF peaks enhanced in SETDB1-depleted macrophages are associated with increased insulation capacity at pre-existing insulation sites. (**) P < 0.01, Mann–Whitney test. ( C ) Heat maps showing insulation scores in control macrophages (white/blue color palette) and the log 2 fold change (white/red color palette) between control and SETDB1-depleted macrophages (log 2 of the ratios between minimum–maximum normalized SETDB1 sh and NT sh bins) for genomic areas upstream of and downstream from the strand-oriented CTCF motifs. ( D ) Proportion of constitutive ( top ) and induced ( bottom ) CTCF peaks found at insulation sites. ( E ) Aggregate region analysis (ARA) showing Hi-C contact probabilities (i.e., average observed over expected contacts) in control (NT sh) and SETDB1-depleted (SETDB1 sh) macrophages. Plots are centered on either constitutive ( top ) or inducible ( bottom ) CTCF peaks matching insulation sites. Observed versus expected interaction ratios are shown together with the difference between control and SETDB1-depleted conditions. ( F ) Aggregate plots as in E centered on CTCF peaks elsewhere within TADs. ( G ) Frequency distribution ( top panel) of the differences in loop intensities normalized to local background ( bottom panel) in SETDB1-depleted versus control macrophages. ( H ) Aggregate plots ( left ) centered on down-regulated ( top ), invariant ( middle ), and up-regulated ( bottom ) loops in SETDB1-depleted versus control macrophages. ( I ) Box plots of the distribution of the fold changes of contact intensities per loop over the surrounding local background in control and SETDB1-depleted macrophages. Two-way ANOVA with Tukey multiple comparison test. (****) P < 0.0001, (**) P < 0.01, (*) P < 0.05. ( J ) Distance between induced CTCF peaks and induced loops. ( Left ) Distance between induced CTCF and invariant (gray), down-regulated (cyan), or induced (red) loop anchors. One-way ANOVA with Tukey multiple comparison test. (****) P < 0.0001. ( Right ) Bootstrap randomization test assessing the mean distance between randomly placed anchors and induced CTCF peaks ( P < 0.0001, Wilcoxon signed rank test). Mean distance between induced CTCF peaks and up-regulated loops is shown with a dashed green line.

Article Snippet: BMDM cells obtained from Rosa26-Cas9 knock-in mice ( ) were coinfected with two lentiGuide-Puro (Addgene plasmid 52963) plasmids, each one expressing one sgRNA of 15 pairs targeting the CTCF motif upstream of the Tmem154 locus (chromosome 3: 84,657,704–84,659,779) or two irrelevant target guides and the PLKO.1 lentiviral shRNA vectors targeting Setdb1 or PLKO.1 nontargeting. sgRNAs were designed using the CRISPR design tool CHOPCHOP ( https://chopchop.cbu.uib.no ) and cloned into the lentiGuide-Puro plasmid according to the manufacturer's instruction.

Techniques: Insulation, Control, Genome Wide, MANN-WHITNEY, Hi-C, Comparison

Journal: Genes & Development

Article Title: H3K9 trimethylation in active chromatin restricts the usage of functional CTCF sites in SINE B2 repeats

doi: 10.1101/gad.349282.121

Figure Lengend Snippet: Basal gene expression and its correlation with altered chromatin organization in SETDB1-depleted macrophages. ( A ) Heat map showing H3K27ac ChIP-seq signal in control macrophages (NT sh) at anchors of induced loops centered on the strongest H3K27ac peak. Each anchor pair was oriented in order to display the highest H3K27ac signal in the heat map at the right . The two heat maps were ranked based on the mean signal in anchors 1 and 2. ( B ) H3K27ac at induced loops is enhanced upon deletion of SETDB1. Wilcoxon matched-pairs signed rank test. (****) P < 0.0001. ( C ) Differentially expressed genes in untreated control and SETDB1-depleted macrophages. Data are from n = 2 independent biological replicates, each one done in duplicate with both shRNAs (total n = 4 samples per group). ( D ) Distance between up-regulated and down-regulated genes in SETDB1-depleted macrophages and induced CTCF peaks. (****) P < 0.0001, Mann–Whitney test. ( E ) Bootstrap randomization test assessing the mean distance between randomly placed anchors and up-regulated genes. The mean distance between induced loops and up-regulated genes is shown with a dashed green line. ( F ) Aggregate plots showing intensities of loops associated with genes up-regulated or down-regulated in SETDB1-depleted macrophages. ( Right ) Box plots showing the distribution of the fold changes of contact intensities per loop over the surrounding local background in control and SETDB1-depleted macrophages. Wilcoxon matched-pairs signed rank test. (****) P < 0.0001. ( G ) Genomic snapshot showing a genomic interval containing the Tmem154 gene. Data shown from top to bottom include H3K27ac, RNA-seq, contact density plot generated using Tmem154 as viewpoint (yellow highlight and red dashed square), loops identified via cLoop divided into up-regulated (red) and down-regulated (cyan), identified inducible CTCF peak upstream of Tmem154 (green), and normalized contact maps in control and SETDB1-depleted macrophages. ( H ) Zoomed-in view of the region surrounding Tmem154 showing H3K9me3 and CTCF peaks regulated by SETDB1 depletion. The M1-type and M2-type CTCF motifs contained in the SINE B2 elements are shown.

Article Snippet: BMDM cells obtained from Rosa26-Cas9 knock-in mice ( ) were coinfected with two lentiGuide-Puro (Addgene plasmid 52963) plasmids, each one expressing one sgRNA of 15 pairs targeting the CTCF motif upstream of the Tmem154 locus (chromosome 3: 84,657,704–84,659,779) or two irrelevant target guides and the PLKO.1 lentiviral shRNA vectors targeting Setdb1 or PLKO.1 nontargeting. sgRNAs were designed using the CRISPR design tool CHOPCHOP ( https://chopchop.cbu.uib.no ) and cloned into the lentiGuide-Puro plasmid according to the manufacturer's instruction.

Techniques: Gene Expression, ChIP-sequencing, Control, MANN-WHITNEY, RNA Sequencing, Generated

Journal: Genes & Development

Article Title: H3K9 trimethylation in active chromatin restricts the usage of functional CTCF sites in SINE B2 repeats

doi: 10.1101/gad.349282.121

Figure Lengend Snippet: Effects of SETDB1 depletion on LPS-regulated gene expression and its correlation with chromatin folding. ( A ) H3K27ac peaks significantly changed in LPS-stimulated macrophages (2 h). Up-regulated peaks are shown in red, and down-regulated peaks are shown in blue. Data are from n = 2 biological replicates. ( B , C ) Effects of SETDB1 depletion on LPS-regulated H3K27ac are shown as metaplots ( B ) or box plots of normalized and centered read counts per kilobase across all conditions ( C ). Two-way ANOVA with Tukey multiple comparison test. (****) P < 0.0001. ( D ) Distance between LPS-induced H3K27ac peaks and CTCF peaks induced upon SETDB1 depletion (expected distances were computed through 10,000 bootstrap resamplings of all CTCF peaks). One-way ANOVA with Tukey multiple comparison test. (****) P < 0.0001. ( E ) Gene expression changes induced by LPS stimulation (2 h) of mouse macrophages. ( F ) Effect of SETDB1 depletion on both LPS-induced (red) and down-regulated (blue) genes. ( G ) Quantification of the effects shown in F reporting the normalized and centered read counts per kilobase across all conditions. Two-way ANOVA with Tukey multiple comparison test. (****) P < 0.0001. ( H ) Representative snapshots of LPS-inducible and SETDB1-regulated genes. ( I ) Distance between LPS-regulated genes and CTCF peaks induced in SETDB1-depleted macrophages (expected distances were computed through bootstrap resampling of all CTCF peaks). One-way ANOVA with Tukey multiple comparison test. (****) P < 0.0001. ( J ) Genomic snapshot showing the LPS-inducible Egr2 gene. The heat maps display normalized contact densities in control ( top ) and SETDB1-depleted ( bottom ) macrophages. The contact density plot generated using Egr2 as the viewpoint is shown together with the position of induced (red) and constitutive (gray) CTCF peaks.

Article Snippet: BMDM cells obtained from Rosa26-Cas9 knock-in mice ( ) were coinfected with two lentiGuide-Puro (Addgene plasmid 52963) plasmids, each one expressing one sgRNA of 15 pairs targeting the CTCF motif upstream of the Tmem154 locus (chromosome 3: 84,657,704–84,659,779) or two irrelevant target guides and the PLKO.1 lentiviral shRNA vectors targeting Setdb1 or PLKO.1 nontargeting. sgRNAs were designed using the CRISPR design tool CHOPCHOP ( https://chopchop.cbu.uib.no ) and cloned into the lentiGuide-Puro plasmid according to the manufacturer's instruction.

Techniques: Gene Expression, Comparison, Control, Generated

Journal: Experimental and therapeutic medicine

Article Title: SETDB1 induces lenalidomide resistance in multiple myeloma cells via epithelial‑mesenchymal transition and PI3K/AKT pathway activation.

doi: 10.3892/etm.2023.11973

Figure Lengend Snippet: Figure 1. SETDB1 expression in MM is associated with poor survival and lenalidomide resistance. Kaplan‑Meier analysis of (A) progression‑free and (B) overall survival using GEO datasets (GSE136324). (C) Analysis of SETDB1 expression in lenalidomide‑resistant and ‑sensitive MM cell lines in a GEO cohort (GSE165557). SETDB1, SET domain bifurcated histone lysine methyltransferase 1; MM, multiple myeloma; GEO, Gene Expression Omnibus.

Article Snippet: Human SETDB1 (NM_001145415; Origene) was amplified and cloned into the lentiviral vector, pCDH‐CMV‐MCS‐EF1‐Pur (Asia‐Vector Biotechnology) and named SETDB1‐OE.

Techniques: Expressing, Gene Expression

Journal: Experimental and therapeutic medicine

Article Title: SETDB1 induces lenalidomide resistance in multiple myeloma cells via epithelial‑mesenchymal transition and PI3K/AKT pathway activation.

doi: 10.3892/etm.2023.11973

Figure Lengend Snippet: Figure 2. Construction of stable SETDB1‑OE and ‑knockdown multiple myeloma cell lines. (A) mRNA expression of SETDB1 in 293T cells was detected using reverse transcription‑quantitative PCR, SETDB1‑sh1 were selected for use in subsequent experiments. SETDB1 expression levels were normalized to GAPDH mRNA. ***P<0.001 vs. NC. #P<0.05, ###P<0.001 vs. shSETDB1‑NC. Western blot analysis following (B) OE and (C) knockdown of SETDB1. SETDB1, SET domain bifurcated histone lysine methyltrans‑ ferase 1; NC, negative control; OE, overexpression; sh, short hairpin RNA; Ctrl, control.

Article Snippet: Human SETDB1 (NM_001145415; Origene) was amplified and cloned into the lentiviral vector, pCDH‐CMV‐MCS‐EF1‐Pur (Asia‐Vector Biotechnology) and named SETDB1‐OE.

Techniques: Expressing, Western Blot, Knockdown, Negative Control, Over Expression, shRNA, Control

Journal: Experimental and therapeutic medicine

Article Title: SETDB1 induces lenalidomide resistance in multiple myeloma cells via epithelial‑mesenchymal transition and PI3K/AKT pathway activation.

doi: 10.3892/etm.2023.11973

Figure Lengend Snippet: Figure 3. SETDB1 expression affects apoptosis of U266 and RPMI‑8226 cells and is associated with lenalidomide resistance. (A) Apoptosis was measured using flow cytometry. (B) Statistical analysis of the apoptosis. **P<0.01, ***P<0.001; ###P<0.001. (C) Cell viability was measured using Cell Counting Kit‑8 assay following lenalidomide treatment. (D) IC50 values. IC50, half maximal inhibitory concentration; SETDB1, SET domain bifurcated histone lysine methyltrans‑ ferase 1; NC, negative control; OE, overexpression; sh, short hairpin RNA.

Article Snippet: Human SETDB1 (NM_001145415; Origene) was amplified and cloned into the lentiviral vector, pCDH‐CMV‐MCS‐EF1‐Pur (Asia‐Vector Biotechnology) and named SETDB1‐OE.

Techniques: Expressing, Flow Cytometry, CCK-8 Assay, Concentration Assay, Negative Control, Over Expression, shRNA

Journal: Experimental and therapeutic medicine

Article Title: SETDB1 induces lenalidomide resistance in multiple myeloma cells via epithelial‑mesenchymal transition and PI3K/AKT pathway activation.

doi: 10.3892/etm.2023.11973

Figure Lengend Snippet: Figure 4. SETDB1 expression is associated with PI3K/AKT signaling and epithelial‑mesenchymal transition activation in multiple myeloma cells. (A) Gene set enrichment analysis illustrating that SETDB1 expression is positively associated with the PI3K/AKT/mTOR signaling pathway. Western blot analysis in (B) U266 and (C) RPMI‑8226 cells. ***P<0.001 vs. SETDB1‑NC. ##P<0.01, ###P<0.001 vs. shSETDB1‑NC. SETDB1, SET domain bifurcated histone lysine methyltransferase 1; NC, negative control; OE, overexpression; sh, short hairpin RNA; N‑cad, N‑cadherin; FDR, false discovery rate; NES, normalized enrichment score; p, phosphorylated.

Article Snippet: Human SETDB1 (NM_001145415; Origene) was amplified and cloned into the lentiviral vector, pCDH‐CMV‐MCS‐EF1‐Pur (Asia‐Vector Biotechnology) and named SETDB1‐OE.

Techniques: Expressing, Activation Assay, Western Blot, Negative Control, Over Expression, shRNA

Journal: Experimental and therapeutic medicine

Article Title: SETDB1 induces lenalidomide resistance in multiple myeloma cells via epithelial‑mesenchymal transition and PI3K/AKT pathway activation.

doi: 10.3892/etm.2023.11973

Figure Lengend Snippet: Figure 5. PI3K/AKT signaling and epithelial‑mesenchymal transition contribute to SETDB1‑induced lenalidomide resistance in multiple myeloma. (A) Flow cytometry analysis of apoptosis of U266 and RPMI‑8226 cells after treatment with LY294002 and SETDB1 overexpression. (B) Statistical analysis of apop‑ tosis in each group. **P<0.01, ***P<0.001 vs. SETDB1‑NC; ###P<0.001 vs. LY294002 + SETDB1‑NC. (C) IC50 values of lenalidomide were determined by Cell Counting Kit‑8 assay. LY294002 significantly reduced the IC50 value of lenalidomide, whereas SETDB1 overexpression attenuated this effect. (D) Western blot analysis revealed that the expression of N‑cadherin and vimentin, as well as the phosphorylation of PI3K and AKT, were inhibited by LY294002; these effects were counteracted by SETDB1 overexpression. ***P<0.001 vs. SETDB1‑NC; #P<0.05, ##P<0.01 and ###P<0.001 vs. LY294002 + SETDB1‑NC. SETDB1, SET domain bifurcated histone lysine methyltransferase 1; NC, negative control; OE, overexpression; IC50, half maximal inhibitory concentration; N‑cad, N‑cadherin; p, phosphorylation.

Article Snippet: Human SETDB1 (NM_001145415; Origene) was amplified and cloned into the lentiviral vector, pCDH‐CMV‐MCS‐EF1‐Pur (Asia‐Vector Biotechnology) and named SETDB1‐OE.

Techniques: Flow Cytometry, Over Expression, CCK-8 Assay, Western Blot, Expressing, Phospho-proteomics, Negative Control, Concentration Assay

Journal: BMC biology

Article Title: SETDB1 targeting SESN2 regulates mitochondrial damage and oxidative stress in renal ischemia-reperfusion injury.

doi: 10.1186/s12915-024-02048-z

Figure Lengend Snippet: Fig. 8 HP1β interacted with SETDB1 to jointly regulate SESN2. A CoIP results of SETDB1 and different HP1. B WB detection of HP1α and HP1β protein levels. C ChIP detection of the enrichment of HP1α and HP1β on SESN2 promoter in vivo. D Re-ChIP detection of the enrichment of SETDB1 combined with HP1β on SESN2 promoter in vivo. E WB detection of HP1β and Sestrin2 protein levels. F Detection of ROS levels in HK-2 cells by flow cytometry. G Representative images of Mito Tracker Red CMXRos in HK-2 cells (left) and related quantitative analysis (right). Bar = 30 μm. Values are expressed as the mean ± SEM. N = 3. The ** represents differences between groups, and p < 0.01

Article Snippet: To inhibit SETDB1 activity in vivo, (R,R)-59 (MedChemExpress, HY-141539, USA) was injected intraperitoneally at a concentration of 25 mg/kg or 50 mg/kg and a volume of 100 μl for 3 consecutive days before surgery in mice.

Techniques: In Vivo, Flow Cytometry

Journal: BMC biology

Article Title: SETDB1 targeting SESN2 regulates mitochondrial damage and oxidative stress in renal ischemia-reperfusion injury.

doi: 10.1186/s12915-024-02048-z

Figure Lengend Snippet: Fig. 9 SETDB1 collaborates with heterochromatin HP1β, facilitating the labeling of H3K9me3 on the SESN2 promoter and impeding SESN2 expression. Loss of Sestrin2 induces mitochondrial damage and subsequent oxidative stress

Article Snippet: To inhibit SETDB1 activity in vivo, (R,R)-59 (MedChemExpress, HY-141539, USA) was injected intraperitoneally at a concentration of 25 mg/kg or 50 mg/kg and a volume of 100 μl for 3 consecutive days before surgery in mice.

Techniques: Labeling, Expressing

Journal: Nucleic Acids Research

Article Title: SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response

doi: 10.1093/nar/gks868

Figure Lengend Snippet: SPOC1 protein interacts with KAP-1 and enzymatically active H3K9 KMTs. ( A ) In GST-pulldown experiments with GST-SPOC1 wt, GST-SPOC1 (amino acids 1–100) or GST alone and chromatin extracts from U2OS cells, only the GST-SPOC1-full-length protein precipitated the KAP-1 protein, as detected on the immunoblot (lower panel). Amounts of GST proteins used were visualized by Amido black staining of the blots (upper panel). Asterisks mark the positions of full-length GST-proteins. ( B ) LUMIER-assay for mapping the KAP-1/SPOC1 interaction. The indicated combinations of SPOC1 and KAP-1, ProteinA (ProtA) and luciferase (Luc) fusion proteins were coexpressed. Luc activity coprecipitated with IgG-beads was measured (Z-scores). Significant KAP-1-interaction is only evident between both full-length proteins, or the C-terminal half of SPOC1 and the N-terminal 400 amino acids of KAP1, as detected by significant Luc activity in the corresponding pellets. Z -score ≥ 0.5 indicates an interaction. Mean values ± SD of three independent experiments each performed in triplicate are given. ( C ) Immunoprecipitation of SPOC1 from HeLa nuclear extracts with anti-SPOC1 (lane 2) or with control antibodies (lane 1) and analysis of pelleted material by immunoblotting shows specific coprecipitation of G9a and SETDB1 with SPOC1. ( D ) Immunoprecipitation (IP) of endogenous GLP or SUV39H1 from C2C12 cell extracts and immunoblotting with the indicated Abs (IB Abs) against GLP (PP-B0422-00), SUV39H1 (07-550), SETDB1 (ab12317) and SPOC1 (6F6). Unlike the control Ab, GLP and SUV39H1 Abs precipitated all associated KMTs as well as SPOC1. ( E ) Nuclear extracts of control C2C12 cells (lane 1) or cells expressing HA-FLAG-tagged SETDB1 wt (lane 2) or SETDB1 H1224K mutant (lane 3) were analyzed on immunoblots (IB Ab.) with antibodies against FLAG (F1804), G9A (D141-3), GLP (PP-B0422-00), SUV39H1 (07-550) and SPOC1 (6F6). After anti-FLAG-SETDB1 immunoprecipitation (IP) the immunoblot detected specific coprecipitation of SPOC1 by wt SETDB1 (lane 5) but much less by enzymatically inactive SETDB1 H1224K (lane 6). (F) In vitro analysis of histone H3 methlylation by proteins immunoprecipitated by control, SPOC1, G9A, or SETDB1 antibodies. Left panel: autoradiography and SimplyBlue staining of the assay products separated by SDS–PAGE. Right panel: immunoblot which demonstrates that SPOC1, G9A and SETDB1 were immunoprecipitated specifically from the HeLa nuclear extracts. ( G ) In total lysates from HeLa-S3 cell lines constitutively expressing HA-FLAG-SETDB1 (HA-SETDB1), lentiviral-mediated SPOC1wt expression elevates H3K9me3 levels compared to control cells (REV), as detected by immunblotting with Abs against the indicated proteins (left). The SPOC1 PHD mutant (SPOC1mut) unable to bind to H3K4me3 only marginally increased H3K9me3 levels compared to SPOC1 wt.

Article Snippet: Indirect immunofluroescence and immunoblotting were performed using antibodies to γH2AX (05-636; Millipore), 53BP1 (NB100-304; Novus), β-actin (A-5441; Sigma), ATM (1549-1, Epitomics), pATM-S1981 (2152-1; Epitomics), Chk2 (sc-56297; Santa Cruz), pChk2-T68 (2661; Cell signaling), CtIP (D4) (sc-271339; Sigma), FLAG (F1804; Sigma), G9A (D141-3; Clinisciences), G9A (A300-933A; Bethyl), GAPDH (sc-32233; Santa Cruz), GLP (PP-B0422-00; R&D Systems), GLP (Bethyl; A301-642A), H3 (1326-1; Epitomics), H3K9me3 (07-442; Upstate), HP1-α (05-689; Upstate), HP1-β (ab10811; Abcam), HP1-γ (ab10817; Abcam), Tif1-β/KAP-1 (MAB3662; Millipore), pKAP-1-S824 (A300-767A-1; Bethyl), SETDB1 (ab12317; Abcam), SETDB1 (A300-121A; Bethyl), SUV39H1 (05-615; Upstate), SUV39H1 (07-550; Upstate).

Techniques: Western Blot, Staining, Luciferase, Activity Assay, Immunoprecipitation, Control, Expressing, Mutagenesis, In Vitro, Autoradiography, SDS Page