Journal: Biogerontology

Article Title: Histone lysine methyltransferases and their specific methylation marks show significant changes in mouse testes from young to older ages

doi: 10.1007/s10522-025-10187-1

Figure Lengend Snippet: Primary antibodies used in immunohistochemical staining

Article Snippet: SETDB1 , 1/500 , Bioss , bs-11670R.

Techniques: Immunohistochemical staining

Journal: Biogerontology

Article Title: Histone lysine methyltransferases and their specific methylation marks show significant changes in mouse testes from young to older ages

doi: 10.1007/s10522-025-10187-1

Figure Lengend Snippet: The cellular distribution and relative level of the SETDB1 protein in the postnatal mouse testes. a Representative micrographs of SETDB1 immunostaining in the young (n = 10), prepubertal (n = 8), pubertal (n = 8), postpubertal (n = 12), and aged (n = 17) groups. The micrographs were captured at 400 × original magnification. Scale bars are equal to 50 µm. b SETDB1 levels in the testes from 1- to 52-week-old ages and c postnatal testes from the young to the aged groups. d SETDB1 levels per seminiferous tubule in each age and e. each group. f SETDB1 levels in germinal epithelial cells of the postnatal testis groups. The data were analyzed using one-way ANOVA followed by Tukey’s post hoc test. P < 0.05 was considered statistically significant. We present values as mean ± standard deviation (SD). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Y young, PreP prepubertal, P pubertal; PostP postpubertal, A aged, SG spermatogonium, EP early prophase spermatocyte, P pachytene spermatocyte, RS round spermatid, ES elongating spermatid, EdS elongated spermatid, S Sertoli cell, L lumen, IT intertubular area

Article Snippet: SETDB1 , 1/500 , Bioss , bs-11670R.

Techniques: Immunostaining, Standard Deviation

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) Reporter architecture and characterization of its integration site. A reporter containing EGFP driven by the elongation factor 1α promoter (pEF), preceded by five Tet Operator sites (5xTetO), and a hygromycin resistance gene driven by the SV40 early promoter > 5kb upstream was site-specifically integrated into the genome of HEK293 cells. UCSC browser snapshot of ENCODE H3K4me3, H3K36me3, and H3K9me3 ChIP-seq tracks shows the euchromatic integration site. Two replicates of ChIP (colored) and input (gray) signals are overlaid. The H3K9me3 track on a KRAB-Znf gene cluster is shown as a reference for an H3K9me3-enriched region. (B) Schematic diagram of the inducible reporter system for studying heterochromatin. The rTetR-fused heterochromatin protein is stably co-expressed with the reporter and binds to TetO sites upon addition of doxycycline (Dox), and is released upon Dox removal. Analysis of transcriptional silencing efficiency and deposition and spreading of heterochromatin marks upon Dox supplementation and its removal after silencing establishment enables temporal evaluation of heterochromatin establishment, spreading, and maintenance. Red bars A–C denote amplicons used for qPCR analysis. (C) Artificial recruitment of HP1α leads to reporter silencing. GFP signal was measured by flow cytometry at different time points after rTetR-HP1α recruitment to the reporter. Dox was removed after 14 days of recruitment, and GFP signal measurement continued for an additional 15 days. The black line indicates the gating threshold to distinguish between GFP-positive (GFP+, yellow shading) and GFP-negative (GFP-, gray shading) cell populations. Dox-treated (blue) and control (gray) samples are shown. Right: Quantification of three biological replicates at indicated times. SD is plotted, but is too small to show for most points. (D) HP1α tethering induces H3K9me3 deposition over EGFP and the hygromycin resistance gene. H3K9me3 ChIP-qPCR was performed 3 days after HP1α recruitment. H3K9me3 ChIP-qPCR analysis was performed with and without Dox at three regions over the reporter as indicated in panel B. Dots correspond to independent biological replicates; bars indicate mean and SD. (E) SetDB1 is required for reporter silencing upon HP1α tethering. SetDB1 was knocked out in HEK293 cells that stably express both the reporter and rTetR-HP1α. Two single clones were isolated and validated by PCR (data not shown) and western blot (top). HP1α recruitment to the reporter was performed in SetDB1-WT cells and the two SetDB1-KO clones. GFP signals were measured by flow cytometry at day 5 (orange) and day 14 (green) after Dox treatment, or without Dox (gray). (F) SetDB1 knockdown decreases H3K9me3 deposition upon HP1α tethering. Control siRNA and siRNA against SetDB1 were transfected into cells stably expressing both the reporter and rTetR-HP1α. RNAi efficiency was determined 5 days post-siRNA transfection (left). HP1α was recruited to the reporter two days after siRNA transfection, and H3K9me3 ChIP-qPCR was performed 3 days later. Two regions are detected as described above. Dots correspond to independent biological replicates; bars indicate mean and SD.

Article Snippet: We cloned the BSD-P2A-mCherry-SetDB1 (WT or mutant) fragment into an AAV zinc finger donor vector (Addgene, 22075) containing a promoter-less splice-acceptor and homology arms against the AAVS1 locus by replacing the puromycin resistance gene of the vector backbone.

Techniques: ChIP-sequencing, Stable Transfection, Flow Cytometry, Control, Clone Assay, Isolation, Western Blot, Knockdown, Transfection, Expressing

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) Artificial recruitment of SetDB1 leads to efficient silencing of the reporter. Left: Schematic diagram of the inducible reporter system. Right: rTetR-SetDB1 was recruited to the reporter upon Dox supplementation in the medium, and GFP signal was measured by flow cytometry at different time points. After 14 days of Dox treatment, Dox was removed from the medium, and flow cytometry measurements continued for an additional 15 days. Data represent the mean of 3 biological replicates. SD are plotted but are too small to be visible. (B-C) SetDB1 tethering induces de novo H3K9me3 and HP1 deposition along the reporter sequence. H3K9me3 ChIP-qPCR was performed after 3 and 14 days of SetDB1 recruitment (B). HP1α ChIP-qPCR was performed after 14 days of SetDB1 recruitment (C). The locations of amplicons along the reporter are indicated in . Dots represent independent biological replicates; bars indicate the mean ± SD.

Article Snippet: We cloned the BSD-P2A-mCherry-SetDB1 (WT or mutant) fragment into an AAV zinc finger donor vector (Addgene, 22075) containing a promoter-less splice-acceptor and homology arms against the AAVS1 locus by replacing the puromycin resistance gene of the vector backbone.

Techniques: Flow Cytometry, Sequencing

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 physically interacts with HP1α. EGFP-SetDB1, purified from HEK293T cells using wash conditions of different stringency (150 mM–1 M NaCl), was used to pull down HP1α-Flag-His purified from E. coli . Proteins were detected using anti-GFP and anti-Flag antibodies. (B) The chromodomain (CD), but not the chromoshadow (CSD) domain, of HP1α shows a strong interaction with SetDB1. A diagram of HP1α protein structure is shown at the top. Full-length (FL) or truncated fragments of EGFP-HP1α were transiently co-expressed with Flag-SetDB1 in HEK293T cells. Co-immunoprecipitation was performed using GFP nanotrap beads. KAP1, a known interactor of HP1α CSD, was detected for comparison. (C) HP1α mutants that disrupt the HP1-H3K9me3 interaction also impair HP1-SetDB1 interaction. WT or mutant EGFP-HP1α was transiently co-expressed with Flag-SetDB1 in HEK293T cells, followed by co-immunoprecipitation using GFP nanotrap beads and western blot analysis. (D) H3K9me3 peptide competes with SetDB1 for HP1α binding. EGFP-SetDB1 was purified from HEK293T cells using GFP nanotrap beads. HP1α-Flag-His purified from E. coli and synthesized histone peptides (methylated or unmethylated) were incubated with the bead-coupled EGFP-SetDB1. HP1α was detected in input, IP, and flowthrough fractions by western blot.

Article Snippet: We cloned the BSD-P2A-mCherry-SetDB1 (WT or mutant) fragment into an AAV zinc finger donor vector (Addgene, 22075) containing a promoter-less splice-acceptor and homology arms against the AAVS1 locus by replacing the puromycin resistance gene of the vector backbone.

Techniques: Purification, Immunoprecipitation, Comparison, Mutagenesis, Western Blot, Binding Assay, Synthesized, Methylation, Incubation

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: WT or W174A mutant EGFP-HP1α and Flag-SetDB1 were transiently co-expressed in HEK293T cells and co-immunoprecipitated using GFP nanotrap beads. The indicated proteins were detected by western blot. Cells co-expressing EGFP and Flag-SetDB1 were used as a negative control.

Article Snippet: We cloned the BSD-P2A-mCherry-SetDB1 (WT or mutant) fragment into an AAV zinc finger donor vector (Addgene, 22075) containing a promoter-less splice-acceptor and homology arms against the AAVS1 locus by replacing the puromycin resistance gene of the vector backbone.

Techniques: Mutagenesis, Immunoprecipitation, Western Blot, Expressing, Negative Control

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: Biotin-conjugated H3K9me3 peptide was incubated with cell lysate containing EGFP-HP1α and its respective mutants. Pulldown was performed using streptavidin beads, and EGFP-HP1α signal was detected by western blot. EGFP and EGFP-SetDB1 were used as negative controls.

Article Snippet: We cloned the BSD-P2A-mCherry-SetDB1 (WT or mutant) fragment into an AAV zinc finger donor vector (Addgene, 22075) containing a promoter-less splice-acceptor and homology arms against the AAVS1 locus by replacing the puromycin resistance gene of the vector backbone.

Techniques: Incubation, Western Blot

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) Two different regions (aa 410-666 and aa 666-1291) are predicted to interact with HP1α. Full-length (FL) or truncated EGFP-SetDB1 and HP1α-Flag were co-expressed in HEK293T cells. Co-immunoprecipitation was performed using GFP nanotrap beads, and western blot was carried out to detect the immunoprecipitating proteins. Numbers indicate the expressed amino acid stretches. (B) AlphaFold2 predicts two motifs on SetDB1 that interact with the HP1α chromodomain. Full-length HP1α was submitted to AlphaFold2-Multimer along with two different SetDB1 regions (aa 410-666 and aa 666-1291). The PAE (predicted aligned error) files show five different interaction models generated by AlphaFold2-Multimer for each prediction. The two SetDB1 regions with the lowest PAE values are labeled as motif 1 and motif 2. (C) The predicted structures formed by HP1α-SetDB1 interactions show high similarity to the reported HP1-H3K9me2 interaction. The HP1 chromodomain is shown in blue, with relevant residues forming the binding pocket in green (left, reported HP1-H3K9me2 interaction ) or in green with relevant residues marked in pink (middle and right, predicted HP1α-SetDB1 interactions). Histone H3, SetDB1 motif 1, and motif 2 are shown in yellow (main chain) and red (side chains).

Article Snippet: We cloned the BSD-P2A-mCherry-SetDB1 (WT or mutant) fragment into an AAV zinc finger donor vector (Addgene, 22075) containing a promoter-less splice-acceptor and homology arms against the AAVS1 locus by replacing the puromycin resistance gene of the vector backbone.

Techniques: Immunoprecipitation, Western Blot, Generated, Labeling, Binding Assay

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) Domain architecture of SetDB1. Two HP1α-interacting motifs, predicted by AlphaFold2-Multimer, are highlighted, along with their corresponding sequences. (B) Sequence alignment of the histone H3 tail and the two identified SetDB1 motifs. H3K9 and the corresponding lysine residues in SetDB1 are shown in red. (C) HP1α interacts exclusively with methylated SetDB1 peptides in vitro . HP1α-Flag-His, purified from E. coli , was incubated with biotin-conjugated, lysine-trimethylated (Me3) or unmethylated (Un-Me) histone H3 or SetDB1 peptides. Pulldown was performed using streptavidin beads, and HP1α-Flag-His was detected by western blot. The negative control contained no peptide in the pulldown assay. (D) SetDB1 HMM mutants lose trimethylation and HP1 interaction. HP1α-Flag-His, purified from E. coli , was incubated with cell lysates from HEK293T cells expressing EGFP-SetDB1 (WT or mutants) and immunoprecipitated using GFP nanotrap beads. Cells expressing EGFP alone served as a negative control. HMm indicates a mutant with lysine-to-alanine mutations at positions 489, 490, and 1162. H3K9me3 antibody was used to detect trimethylated SetDB1.

Article Snippet: We cloned the BSD-P2A-mCherry-SetDB1 (WT or mutant) fragment into an AAV zinc finger donor vector (Addgene, 22075) containing a promoter-less splice-acceptor and homology arms against the AAVS1 locus by replacing the puromycin resistance gene of the vector backbone.

Techniques: Sequencing, Methylation, In Vitro, Purification, Incubation, Western Blot, Negative Control, Expressing, Immunoprecipitation, Mutagenesis

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: Sequence alignments of SetDB1 in different species ( Homo sapiens (Hs), Bos taurus (Bt), Mus musculus (Mm), Gallus gallus (Gg), Xenopus laevis (Xi), Danio rerio (Dr), Bombyx mori (Bm), Drosophila melanogaster (Dm) ) were performed using NCBI-COBALT. A schematic of positions colored by percentage identity is shown. Histograms reflect conservation and consensus. SetDB1 motif 1 and motif 2 are marked by red bars. The regions containing motif 1 and motif 2 and their flanking sequences are magnified (bottom). Images are based on alignments edited in Jalview .

Article Snippet: We cloned the BSD-P2A-mCherry-SetDB1 (WT or mutant) fragment into an AAV zinc finger donor vector (Addgene, 22075) containing a promoter-less splice-acceptor and homology arms against the AAVS1 locus by replacing the puromycin resistance gene of the vector backbone.

Techniques: Sequencing

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) EGFP-SetDB1 and mutants were expressed and immunoprecipitated from HEK293T cells. Endogenous HP1α and MPP8 were detected by western blot. (B) EGFP-SetDB1 was co-expressed with MPP8-Flag or HP1α-Flag in HEK293T cells and immunoprecipitated using GFP nanotrap beads. Overexpressed HP1α and MPP8 were detected by Western blot using an anti-Flag antibody.

Article Snippet: We cloned the BSD-P2A-mCherry-SetDB1 (WT or mutant) fragment into an AAV zinc finger donor vector (Addgene, 22075) containing a promoter-less splice-acceptor and homology arms against the AAVS1 locus by replacing the puromycin resistance gene of the vector backbone.

Techniques: Immunoprecipitation, Western Blot

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 K490 and K1162 can be tri-methylated in vivo . EGFP-SetDB1 was purified from HEK293T cells, digested with thermolysin, and analyzed by tandem mass spectrometry. Representative MS2 spectra of peptides with trimethylation at K490 (left, VAKKSTS, 424.7452 m/z) and K1162 (right, VAVKSTRGFA, 560.8273 m/z) are shown. The b and y ion peaks corresponding to the trimethylated peptides are labeled. The “-C3H9N” fragment indicates trimethylation. Carbamylation is an artificial modification occurring during thermolysin digestion. (B) SetDB1 methylates HMM motifs in vitro . EGFP-SUMO EU -SetDB1 was expressed in HEK293T cells, immunopurified using GFP nanotrap beads, and SetDB1 was eluted by SUMO protease SENP EUB cleavage (left). Quantification of the methyltransferase activity of purified SetDB1 on histone H3 tail or SetDB1 HMM-containing peptides was performed using a commercial kit. Methylation of H3K9 was quantified using an HRP-conjugated secondary antibody-color development system at OD450 (right). Dots represent independent biological replicates; bars indicate mean ± SD. (C) SetDB1 lacking methyltransferase activity cannot efficiently interact with HP1α. EGFP-SetDB1-WT or methyltransferase-dead mutants were transiently expressed in SetDB1-knockout HEK293T cells. Cell lysates were incubated with HP1α-Flag-His purified from E. coli , followed by co-immunoprecipitation using GFP nanotrap beads and western blot analysis. EGFP alone was used as a negative control.

Article Snippet: We cloned the BSD-P2A-mCherry-SetDB1 (WT or mutant) fragment into an AAV zinc finger donor vector (Addgene, 22075) containing a promoter-less splice-acceptor and homology arms against the AAVS1 locus by replacing the puromycin resistance gene of the vector backbone.

Techniques: Methylation, In Vivo, Purification, Mass Spectrometry, Labeling, Modification, In Vitro, Activity Assay, Knock-Out, Incubation, Immunoprecipitation, Western Blot, Negative Control

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: WT or mutant EGFP-SUMO EU -SetDB1 was expressed and purified from HEK293T cells. SetDB1 was eluted from the beads using SUMO protease SENP EUB (left). Quantification of the methyltransferase activity of purified SetDB1 on histone H3 peptide was performed using a commercial kit. The amount of methylated H3K9 was quantified using an HRP-conjugated secondary antibody and color development system under OD450 (right). Dots represent independent biological replicates; bars indicate the mean ± SD.

Article Snippet: We cloned the BSD-P2A-mCherry-SetDB1 (WT or mutant) fragment into an AAV zinc finger donor vector (Addgene, 22075) containing a promoter-less splice-acceptor and homology arms against the AAVS1 locus by replacing the puromycin resistance gene of the vector backbone.

Techniques: Mutagenesis, Purification, Activity Assay, Methylation

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: EGFP-SetDB1-WT or methyltransferase-dead mutants (H1224K or C1226A) were transiently expressed in HEK293T cells that endogenously express SetDB1. Cell lysates were incubated with HP1α-Flag-His purified from E. coli , followed by co-immunoprecipitation using GFP nanotrap beads and western blot analysis. EGFP alone was used as a negative control. Both EGFP-SetDB1 methylation and its interaction with HP1α were strongly reduced in the mutants.

Article Snippet: We cloned the BSD-P2A-mCherry-SetDB1 (WT or mutant) fragment into an AAV zinc finger donor vector (Addgene, 22075) containing a promoter-less splice-acceptor and homology arms against the AAVS1 locus by replacing the puromycin resistance gene of the vector backbone.

Techniques: Incubation, Purification, Immunoprecipitation, Western Blot, Negative Control, Methylation

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: We cloned the BSD-P2A-mCherry-SetDB1 (WT or mutant) fragment into an AAV zinc finger donor vector (Addgene, 22075) containing a promoter-less splice-acceptor and homology arms against the AAVS1 locus by replacing the puromycin resistance gene of the vector backbone.

Techniques: Stable Transfection, Western Blot, Immunofluorescence, Staining

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) Schematic diagram of cells engineered to analyze the effects of HMM mutation (HMm) on SetDB1 (top) and HP1α (bottom) induced heterochromatin. For SetDB1 tethering, WT or HMm rTetR-SetDB1 was stably expressed in cells harboring the reporter. For HP1 tethering, WT or HMm mCherry-SetDB1 was stably expressed in SetDB1-KO cells expressing the reporter and rTetR-HP1α. (B) Reporter silencing upon HP1α and SetDB1 tethering is unaffected by SetDB1-HMM mutation. GFP signal was measured by flow cytometry upon SetDB1 or HP1α recruitment to the reporter over different periods (4, 7, and 14 days). The histograms (left) show results from representative single clones. The black line indicates the gating threshold to distinguish between GFP-positive (GFP+, yellow shading) and GFP-negative (GFP-, gray shading) cell populations. Dox-treated (purple/blue/orange) and no-Dox control (gray) samples are shown. Bar graphs (right) show the quantitative results from different clones. Dots represent single clones; bars indicate mean ± SD. (C) Local deposition of H3K9me3 and HP1α is unaffected by SetDB1-HMM mutation, but their spreading to distal regions is disrupted. H3K9me3 ChIP-qPCR (left) and HP1α ChIP-qPCR (right) were performed after 7 days of SetDB1 (top) or HP1α (bottom) recruitment. Dots represent independent biological replicates; bars indicate mean ± SD. See for amplicon locations. (D) Defective SetDB1 histone mimic methylation impairs heterochromatin maintenance upon SetDB1 (purple) and HP1α (blue) recruitment. Dox was removed after 14 days of recruitment, and GFP signal was measured by flow cytometry at different time points after Dox removal. The histograms (left) show results from representative single clones. Bar graphs (right) show the quantitative results from different clones. Dots represent single clones; bars indicate mean ± SD. Numbers indicate mean values. Details and color scheme for histograms are the same as in panel B.

Article Snippet: We cloned the BSD-P2A-mCherry-SetDB1 (WT or mutant) fragment into an AAV zinc finger donor vector (Addgene, 22075) containing a promoter-less splice-acceptor and homology arms against the AAVS1 locus by replacing the puromycin resistance gene of the vector backbone.

Techniques: Mutagenesis, Stable Transfection, Expressing, Flow Cytometry, Clone Assay, Control, Amplification, Methylation

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: WT or HMm SetDB1 was recruited to the reporter, or HP1α was recruited to the reporter in SetDB1-KO cells rescued with either WT or HMm SetDB1. The expression of the hygromycin resistance gene located 5.5 kb upstream of the tethering site was detected by RT-qPCR. GAPDH was used as an internal reference, and the repression fold was calculated as the fold difference in hygromycin expression in the absence or presence of Dox. The amplicon used for qPCR analysis is shown as a red bar.

Article Snippet: We cloned the BSD-P2A-mCherry-SetDB1 (WT or mutant) fragment into an AAV zinc finger donor vector (Addgene, 22075) containing a promoter-less splice-acceptor and homology arms against the AAVS1 locus by replacing the puromycin resistance gene of the vector backbone.

Techniques: Expressing, Quantitative RT-PCR, Amplification

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) Genome-wide H3K9me3 distribution changes in SetDB1-HMM mutant. Circos plot showing H3K9me3 signal differences between cells stably expressing SetDB1-HMm or SetDB1-WT in a SetDB1-KO background. Outer gray tiles represent chromosomes. In the middle circle, black lines indicate 1Mb genomic windows where H3K9me3 ChIP/Input signal >1.5 in the WT rescue strain (defined as heterochromatic region, “het”). Green regions show no significant H3K9me3 enrichment (defined as euchromatin region, “eu”). The inner circle shows log2-transformed H3K9me3 signal changes (positive: orange, negative: blue) in SetDB1-HMm versus WT rescue strain. Data represent the average of two biological replicates. All subsequent ChIP-Seq and RNA-Seq data are from these two rescue strains. (B) The effect of SetDB1-HMm on H3K9 trimethylation varies across genomic regions. Scatterplot shows H3K9me3 ChIP/Input signals in 1Mb genomic windows (gw) from SetDB1-HMm versus WT rescue strain. Data represent the average of two biological replicates. Black dashed lines indicate a 1.5-fold difference. Gray dashed lines separate heterochromatic (H3K9me3 ChIP/Input >1.5) and euchromatic (H3K9me3 ChIP/Input <1.5) windows for both WT and HMm strains. Genomic windows from different chromosomes and with varying fold changes are depicted in different colors. (C) Euchromatin regions with low but detectable H3K9me3 signal gain H3K9me3 in SetDB1-HMM mutant. UCSC browser snapshot shows a representative euchromatin region with elevated H3K9me3. Tracks show overlaid ChIP (colored) and Input (gray) signals from two replicates. Numbers on the right show the normalized ChIP/Input signal for the manually selected genomic interval indicated by the black bar. (D) SetDB1-HMm leads to significant loss of H3K9me3 on ChrX. Left: Dot plot showing the distribution of H3K9me3 ChIP signal in 1Mb genomic windows from ChrX in both WT and SetDB1-HMm strains. Data represent the average of two biological replicates. The black line indicates the median. p<0.0001 is denoted as ****. Right: UCSC browser snapshot of a representative locus on ChrX. Tracks show overlaid ChIP (colored) and Input (gray) signals for two replicates. Numbers on the right show the normalized ChIP/Input signal for the manually selected genomic interval indicated by the black bar. (E) SetDB1-HMm leads to H3K9me3 loss over KRAB-Znf genes. Dot plot shows H3K9me3 signals on individual KRAB-Znf genes in SetDB1-WT and SetDB1-HMm strains. KRAB-Znf genes on Chr19 and on other chromosomes are plotted separately. Data represent the average of two biological replicates. The black line indicates the median. p<0.0001 is denoted as ****, p<0.05 is denoted as *. (F) H3K9me3 spreading on KRAB-Znf genes is impaired in SetDB1-HMM mutant. Left: Metaplot of H3K9me3 distributions over gene bodies of all KRAB-Znf genes in SetDB1-WT (blue) and HMm (orange) strains. Data is average of two biological replicates. Right: UCSC browser snapshot of a representative KRAB-Znf cluster on Chr19. Tracks show overlaid ChIP (colored) and Input (gray) signals for two replicates.

Article Snippet: We cloned the BSD-P2A-mCherry-SetDB1 (WT or mutant) fragment into an AAV zinc finger donor vector (Addgene, 22075) containing a promoter-less splice-acceptor and homology arms against the AAVS1 locus by replacing the puromycin resistance gene of the vector backbone.

Techniques: Genome Wide, Mutagenesis, Stable Transfection, Expressing, Transformation Assay, ChIP-sequencing, RNA Sequencing Assay

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: H3K9me3 ChIP-seq was performed in SetDB1-KO cells in which SetDB1 loss was rescued by stably expressing either HMm or WT SetDB1. Dot plots show the fold change in H3K9me3 in SetDB1-HMm rescue strain compared to WT rescue strain in 1Mb genomic windows (gw) along the chromosomes. Gray dashed lines indicate a 1.5-fold (1.5x) change. Windows with >1.5x increase in H3K9me3 signal are shown in yellow.

Article Snippet: We cloned the BSD-P2A-mCherry-SetDB1 (WT or mutant) fragment into an AAV zinc finger donor vector (Addgene, 22075) containing a promoter-less splice-acceptor and homology arms against the AAVS1 locus by replacing the puromycin resistance gene of the vector backbone.

Techniques: ChIP-sequencing, Stable Transfection, Expressing

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) Violin plots show the proportion of gene (left) and protein-coding gene (right) annotations in the sequence of 1Mb genomic windows. Windows are categorized based on whether the H3K9me3 signal increases by ≥1.5x (yellow) or <1.5x (grey) in SetDB1-HMm rescue strain compared to WT rescue strain. The solid line indicates the median, and the dashed lines represent quantiles. p-value < 0.0001 is indicated by ****. (B) Gene type distribution in regions showing ≥1.5x elevated H3K9me3 signal in SetDB1-HMM mutant (left) and genome-wide (right). (C) Most genes in regions with ≥1.5x elevated H3K9me3 signal in SetDB1-HMM mutant show no or low expression. A dot plot (left) shows log-transformed RPKM values (from RNA-Seq of the WT rescue strain) for genes located in 1Mb windows with ≥1.5x or <1.5x increase in H3K9me3 signal in SetDB1-HMm rescue strain compared to WT rescue strain. The right panel shows the proportions of genes with different expression levels in both groups. Numbers indicate the percentage of genes within each group.

Article Snippet: We cloned the BSD-P2A-mCherry-SetDB1 (WT or mutant) fragment into an AAV zinc finger donor vector (Addgene, 22075) containing a promoter-less splice-acceptor and homology arms against the AAVS1 locus by replacing the puromycin resistance gene of the vector backbone.

Techniques: Sequencing, Mutagenesis, Genome Wide, Expressing, Transformation Assay, RNA Sequencing Assay

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: H3K9me3 signal decreased significantly more on ChrX and Chr19 compared to other chromosomes in SetDB1-HMm rescue strain compared to WT rescue strain. Dot plots (bottom) show fold changes of H3K9me3 (HMm/WT) in 1Mb genomic windows. The percentage of windows with ≥1.5x, 1.3-1.5x, or <1.3x loss of H3K9me3 in the mutant was calculated for each group. p-value < 0.0001 is indicated by ****.

Article Snippet: We cloned the BSD-P2A-mCherry-SetDB1 (WT or mutant) fragment into an AAV zinc finger donor vector (Addgene, 22075) containing a promoter-less splice-acceptor and homology arms against the AAVS1 locus by replacing the puromycin resistance gene of the vector backbone.

Techniques: Mutagenesis

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 dot plot shows H3K9me3 enrichment in 1Mb genomic windows across different chromosomes in SetDB1-WT rescue strain. Numbers indicate the percentage of heterochromatic windows (H3K9me3 ChIP/Input >1.5) for each chromosome.

Article Snippet: We cloned the BSD-P2A-mCherry-SetDB1 (WT or mutant) fragment into an AAV zinc finger donor vector (Addgene, 22075) containing a promoter-less splice-acceptor and homology arms against the AAVS1 locus by replacing the puromycin resistance gene of the vector backbone.

Techniques:

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) H3K9me3 loss correlates with increased gene expression on ChrX. RNA-seq data were analyzed by DESeq2 using 3 replicates for WT and 2 replicates for SetDB1-HMM mutant. ChrX genes located in 1Mb heterochromatic windows with ≥1.5x loss of H3K9me3 in the mutant are shown in blue; all other genes are shown in grey. ChrX genes with significantly increased expression in the mutant (FDR < 0.05) are listed. (B) Heterochromatic KRAB-Znf genes that show ≥1.5x loss of H3K9me3 exhibit mildly increased gene expression in SetDB1-HMm rescue strain. KRAB-Znf genes with ≥1.5x loss of H3K9me3 in the mutant are shown in orange (FDR ≥ 0.05) or red (FDR < 0.05), while all other genes are shown in grey.

Article Snippet: We cloned the BSD-P2A-mCherry-SetDB1 (WT or mutant) fragment into an AAV zinc finger donor vector (Addgene, 22075) containing a promoter-less splice-acceptor and homology arms against the AAVS1 locus by replacing the puromycin resistance gene of the vector backbone.

Techniques: Expressing, RNA Sequencing Assay, Mutagenesis

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) KRAB-Znf genes on Chr19 reside in heterochromatin. Violin plots show the percentage of KRAB-Znf gene annotations within 1Mb windows on Chr19. Windows are categorized based on whether they are heterochromatic (H3K9me3 ChIP/Input ≥1.5 in WT rescue strain) or euchromatic (H3K9me3 ChIP/Input <1.5 in WT rescue strain). p-value < 0.0001 is indicated by ****. (B) Regions on Chr19 that lose H3K9me3 signal in SetDB1-HMm strain are enriched in KRAB-Znf genes. Violin plots show the percentage of KRAB-Znf gene annotations within 1Mb windows on Chr19. Windows are categorized based on whether H3K9me3 signal loss in SetDB1-HMm rescue strain was ≥1.3x or <1.3x. p-value < 0.01 is indicated by **.

Article Snippet: We cloned the BSD-P2A-mCherry-SetDB1 (WT or mutant) fragment into an AAV zinc finger donor vector (Addgene, 22075) containing a promoter-less splice-acceptor and homology arms against the AAVS1 locus by replacing the puromycin resistance gene of the vector backbone.

Techniques:

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: HP1 dimers were shown to stabilize condensed heterochromatin by binding H3K9me3 marks on adjacent nucleosomes. SetDB1 auto-methylates its HMMs creating sites for HP1 binding. In the indirect read-write model, SetDB1 is recruited to chromatin by HP1. In regions with lower density of the H3K9me3 mark, such as newly replicated chromatin (left) and the border region of heterochromatin (right), some HP1 dimers are not binding two histone tails, allowing one CD to bind and recruit SetDB1. SetDB1 methylates adjacent histone tails, maintaining heterochromatin integrity and enabling its spreading into adjacent regions. Accumulation of H3K9me3 displaces SetDB1 from chromatin as the H3K9me3 mark and SetDB1 compete for HP1 binding. Release of SetDB1 enables its redistribution to new, partially methylated regions.

Article Snippet: We cloned the BSD-P2A-mCherry-SetDB1 (WT or mutant) fragment into an AAV zinc finger donor vector (Addgene, 22075) containing a promoter-less splice-acceptor and homology arms against the AAVS1 locus by replacing the puromycin resistance gene of the vector backbone.

Techniques: Binding Assay, Methylation

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Modulation of SETDB1 activity by APQ ameliorates heterochromatin condensation, motor function, and neuropathology in a Huntington’s disease mouse model

doi: 10.1080/14756366.2021.1900160

Figure Lengend Snippet: Structures of recently reported SETDB1 inhibitors, nogalamycin and VH06.

Article Snippet: SETDB1 enzyme activity was analysed using commercially available SETBD1 chemiluminescent assay kit (BPS Bioscience, San Diego, CA, #51056) according to the manufacturer’s instructions.

Techniques:

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Modulation of SETDB1 activity by APQ ameliorates heterochromatin condensation, motor function, and neuropathology in a Huntington’s disease mouse model

doi: 10.1080/14756366.2021.1900160

Figure Lengend Snippet: Primary screening of our compounds via cell-based and dot blot assay. (a) A diagram representing a generation of Tet-inducible SETDB1 cell line. (b, c) SETDB1 induction by doxycycline (Doxy) increased H3K9me3 level. (d) SETDB1 inhibitor candidates were determined by the dot blot assay. Inhibitors were treated to cells (in that SETDB1 was already induced by Doxy for 24 h) for 12 h. Histone protein was isolated by acidic extraction method and H3K9me3 level.

Article Snippet: SETDB1 enzyme activity was analysed using commercially available SETBD1 chemiluminescent assay kit (BPS Bioscience, San Diego, CA, #51056) according to the manufacturer’s instructions.

Techniques: Dot Blot, Isolation

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Modulation of SETDB1 activity by APQ ameliorates heterochromatin condensation, motor function, and neuropathology in a Huntington’s disease mouse model

doi: 10.1080/14756366.2021.1900160

Figure Lengend Snippet: APQ ligand interaction diagram inside the SET domain. (a) SET domain represented in ribbon format. APQ and SAM showed by space-filling cpk presentation in magenta and blue colour, respectively. The hydrogen bond donor–acceptor surface area has shown around APQ. (b) APQ in magenta stick format interacts with Tyr1265, Ala855, Tyr832, Val1232, Asp851, and Asn1266. Binding site residues showed in white stick format. Pi-pi stacking, pi-sigma, pi-alkyl, and hydrophobic interactions are shown in pink, purple, light pink, and light green colour, respectively. (c) Schematic representation of SETDB1 domain organisation.

Article Snippet: SETDB1 enzyme activity was analysed using commercially available SETBD1 chemiluminescent assay kit (BPS Bioscience, San Diego, CA, #51056) according to the manufacturer’s instructions.

Techniques: Binding Assay

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Modulation of SETDB1 activity by APQ ameliorates heterochromatin condensation, motor function, and neuropathology in a Huntington’s disease mouse model

doi: 10.1080/14756366.2021.1900160

Figure Lengend Snippet: APQ reduces H3K9me3 level in HD striatal cells. (a) APQ decreases the level of H3K9me3 in HD ( Q111/Q111 ) striatal cells. (b, c) APQ reduces the immunoreactivity of H3K9me3 ( Q111/Q111 ) striatal cells. (d) FRET imaging analysis showing that APQ effectively modulates FRET/ECFP ratios of H3K9 methylation in Q111. APQ represses the transcriptional activity of Setdb1/Eset in a dose-dependent manner. The data represents the average of three separate experiments. Significantly different at * p < .05, ** p < .001.

Article Snippet: SETDB1 enzyme activity was analysed using commercially available SETBD1 chemiluminescent assay kit (BPS Bioscience, San Diego, CA, #51056) according to the manufacturer’s instructions.

Techniques: Imaging, Methylation, Activity Assay

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Modulation of SETDB1 activity by APQ ameliorates heterochromatin condensation, motor function, and neuropathology in a Huntington’s disease mouse model

doi: 10.1080/14756366.2021.1900160

Figure Lengend Snippet: SETDB1 inhibitor (APQ) reduces H3K9me3 immunoreactivity and level in the striatum of HD transgenic (YAC128) mice. (a) APQ administration significantly decreased medium H3K9me3 immunoreactivity in the striatum of YAC128 mice ( n = 5) compared to vehicle-treated YAC128 mice ( n = 5). (b) APQ administration significantly reduced H3K9me3 intensity in medium spiny neurons in the striatum of YAC128 mice. Significantly different from WT at ** p <.01. Significantly different from YAC128 mice at ## p <.01. (c) Acid histone extraction and Western blot analysis showed that APQ selectively decreased H3K9me3 level but not H3K9me1 and H3K9me2 levels in the striatum of YAC128 mice compared to vehicle-treated YAC128 mice. (d) Densitometry analysis showed that APQ significantly reduced H3K9me3 level in the striatum of YAC128 mice. H3K9me3 levels were normalised to histone H3 levels. Significantly different from WT at ** p <.01. Significantly different from YAC128 mice at # p <.05.

Article Snippet: SETDB1 enzyme activity was analysed using commercially available SETBD1 chemiluminescent assay kit (BPS Bioscience, San Diego, CA, #51056) according to the manufacturer’s instructions.

Techniques: Transgenic Assay, Western Blot

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Modulation of SETDB1 activity by APQ ameliorates heterochromatin condensation, motor function, and neuropathology in a Huntington’s disease mouse model

doi: 10.1080/14756366.2021.1900160

Figure Lengend Snippet: SETDB1 inhibitor (APQ) modulates medium spiny neuronal activity in the striatum of HD transgenic (YAC128) mice. (a, b) Confocal microscopy showed that APQ administration restored DARPP32 and calbindin immunoreactivity in the striatum of YAC128 mice. (c) Densitometry analysis showed that APQ significantly improved DARPP32 level in the striatum of YAC128 mice. Significantly different from WT at ** p <.01. Significantly different from YAC128 mice at ## p <.01. (d) Densitometry analysis showed that APQ significantly improved calbindin level in the striatum of YAC128 mice. (e) DAPI staining showed that APQ rescued the nuclei size in the striatum of YAC128 mice. (f) Densitometry analysis showed that APQ significantly improved the nuclei size in the striatum of YAC128 mice. Significantly different from WT at ** p <.01. Significantly different from YAC128 mice at ## p <.01.

Article Snippet: SETDB1 enzyme activity was analysed using commercially available SETBD1 chemiluminescent assay kit (BPS Bioscience, San Diego, CA, #51056) according to the manufacturer’s instructions.

Techniques: Activity Assay, Transgenic Assay, Confocal Microscopy, Staining

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Modulation of SETDB1 activity by APQ ameliorates heterochromatin condensation, motor function, and neuropathology in a Huntington’s disease mouse model

doi: 10.1080/14756366.2021.1900160

Figure Lengend Snippet: SETDB1 inhibitor (APQ) modulates H3K9me3-landscaped epigenomes in the striatum of HD transgenic (YAC128) mice. (a) A scheme illustrating H3K9me-ChIP sequencing analysis in APQ-administered YAC128 mice. (b) Volcano plots presented that H3K9me-landscaped epigenomes are altered in APQ-administered YAC128 mice. (c) Pie graphs exhibited that APQ affects the landscaping of H3K9me in the promoter regions of epigenomes in YAC128 mice. (d) Network analysis showed that APQ modulates remodelling of H3K9me-landscaped epigenomes (red nodules) that are associated with chromatin modification, transcription regulation, and nervous system development pathway in YAC128 mice. (e) Immunohistochemistry showed that APQ reduces SUV39H1 immunoreactivity (brown) in the striatal neurons of YAC128 mice. The nuclei (blue) were counterstained with haematoxylin. (f) Densitometry analysis showed that APQ significantly reduced SUV39H1 level in the striatal neurons of YAC128 mice. Significantly different from WT at ** p <.01. Significantly different from YAC128 mice at ## p <.01.

Article Snippet: SETDB1 enzyme activity was analysed using commercially available SETBD1 chemiluminescent assay kit (BPS Bioscience, San Diego, CA, #51056) according to the manufacturer’s instructions.

Techniques: Transgenic Assay, ChIP-sequencing, Modification, Immunohistochemistry