Journal: Bioscience Reports

Article Title: A regulatory circuitry comprising TP53, miR-29 family, and SETDB1 in non-small cell lung cancer

doi: 10.1042/BSR20180678

Figure Lengend Snippet: ( A ) Dual luciferase assay of 293T cells co-transfected with the luciferase constructs containing the SETDB1 3′-UTR_WT or SETDB1 3′-UTR_Mut as well as miR-29s mimics or Scr NC. ( B ) SETDB1 protein levels was assessed in PC14, A549, and BEAS-2B cells. ( C ) SETDB1 mRNA levels and ( D ) SETDB1 protein levels after transfection of miR-29s mimics or Scr NC into A549 and PC14 cells. ( E ) SETDB1 mRNA levels and ( F ) SETDB1 protein levels after transfection of miR-29s inhibitors or Scr NC into BEAS-2B cells. * P <0.05; ** P <0.01; *** P <0.001

Article Snippet: The siRNAs specifically targetting SETDB1 (siSETDB1) and TP53 (siTP53) were obtained commercially (GenePharma, Shanghai, China).

Techniques: Luciferase, Transfection, Construct

Journal: Bioscience Reports

Article Title: A regulatory circuitry comprising TP53, miR-29 family, and SETDB1 in non-small cell lung cancer

doi: 10.1042/BSR20180678

Figure Lengend Snippet: miR-29s mRNA expression after transfection of pEGFP-TP53 vector or pEGFP-N1 control vector into ( A ) A549 cells and ( B ) PC14 cells. ( C ) SETDB1 protein level and ( D ) SETDB1 mRNA expression after transfection of pEGFP-TP53 vector or pEGFP-N1 control vector into A549 and PC14 cells. ( E ) miR-29s mRNA expression, ( F ) SETDB1 mRNA expression and ( G ) SETDB1 protein level after transfection of siTP53 or Scr NC into BEAS-2B cells. ** P <0.01; *** P <0.001

Article Snippet: The siRNAs specifically targetting SETDB1 (siSETDB1) and TP53 (siTP53) were obtained commercially (GenePharma, Shanghai, China).

Techniques: Expressing, Transfection, Plasmid Preparation, Control

Journal: Bioscience Reports

Article Title: A regulatory circuitry comprising TP53, miR-29 family, and SETDB1 in non-small cell lung cancer

doi: 10.1042/BSR20180678

Figure Lengend Snippet: ( A ) TP53 mRNA levels and ( B ) TP53 protein levels ( C , D ) miR-29s levels after transfection of siSETDB1 scrambled oligonucleotides as the negative control into A549 and PC14 cells. ( E ) TP53 mRNA levels and ( F ) TP53 protein levels and ( G ) miR-29s levels after transfection of pcDNA3.1-SETDB1 vector or pcDNA3.1 control vector into BEAS-2B cells. ** P <0.01; *** P <0.001

Article Snippet: The siRNAs specifically targetting SETDB1 (siSETDB1) and TP53 (siTP53) were obtained commercially (GenePharma, Shanghai, China).

Techniques: Transfection, Negative Control, Plasmid Preparation, Control

Journal: Bioscience Reports

Article Title: A regulatory circuitry comprising TP53, miR-29 family, and SETDB1 in non-small cell lung cancer

doi: 10.1042/BSR20180678

Figure Lengend Snippet: ( A ) SETDB1 mRNA levels and ( B ) TP53 mRNA levels in primary NSCLC samples and their paired adjacent normal samples. ( C ) SETDB1 and TP53 mRNA levels were inversely correlated in primary NSCLC samples. ( D – F ) Pearson correlation analysis between miR-29s and SETDB1 mRNA levels, as well as between miR-29s and TP53 mRNA levels in primary NSCLC samples. *** P <0.001

Article Snippet: The siRNAs specifically targetting SETDB1 (siSETDB1) and TP53 (siTP53) were obtained commercially (GenePharma, Shanghai, China).

Techniques:

Journal: Bioscience Reports

Article Title: A regulatory circuitry comprising TP53, miR-29 family, and SETDB1 in non-small cell lung cancer

doi: 10.1042/BSR20180678

Figure Lengend Snippet: ( A ) TP53 and Suv39h1 mRNA levels after transfection of miR-29s mimics or Scr NC into A549 and PC14 cells. ( B ) TP53 and Suv39h1 mRNA levels after transfection of miR-29s inhibitors or Scr NC into BEAS-2B cells. Histone H3K9 methylation status in ( C ) NSCLC cells (A549 and PC14) with overexpressed miR-29s and ( D ) BEAS-2B cells with downexpressed miR-29s were examined by Western blot using antibodies specific for H3K9me2 and H3K9me3 (di- and trimethylation of H3K9, respectively); expression levels of histone H3 protein were used as an internal control. ( E ) Schematic illustration of the proposed TP53/ miR-29s /SETDB1 regulatory circuitry in NSCLC. ** P <0.01; *** P <0.001

Article Snippet: The siRNAs specifically targetting SETDB1 (siSETDB1) and TP53 (siTP53) were obtained commercially (GenePharma, Shanghai, China).

Techniques: Transfection, Methylation, Western Blot, Expressing, Control

Journal: Nucleic Acids Research

Article Title: Cockayne syndrome group B deficiency reduces H3K9me3 chromatin remodeler SETDB1 and exacerbates cellular aging

doi: 10.1093/nar/gkz568

Figure Lengend Snippet: SETDB1 expression decreases with chronological aging. Immunofluorescence (IF) analysis of normal human fibroblast from healthy female (ages: 20, 40 and 60) and male (age: 20, 40 and 60) donors. IF was performed with (A) anti-CSB (red) and (B) anti-SETDB1 (green). Cell nuclei were stained with DAPI. Representative images are maximum intensity z-projection of all the slices from z-stacks. Quantification of mean fluorescence intensity of z-stacks of cells from different sex and aged groups, or different aged groups to measure staining intensity. For each cell line from an individual, a total of 19 and 13 cells were analyzed for CSB and SETDB1, respectively. Error bars represents SEM. (C) Brain lysates of young, middle and old mice were probed for the indicated proteins on western blot. Blot density was measured and normalized to GAPDH (H3K9me3 was normalized to H3). Scale bar, 30 μm. Error bars represent SD. NS, not significant; *, <0.05; **, <0.01.

Article Snippet: Dox-inducible GFP-tagged lentiviral SUV39H1 or SETDB1 constructs were designed and ordered from VectorBuilder Inc. (Santa Clara, CA, USA).

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence, Western Blot

Journal: Nucleic Acids Research

Article Title: Cockayne syndrome group B deficiency reduces H3K9me3 chromatin remodeler SETDB1 and exacerbates cellular aging

doi: 10.1093/nar/gkz568

Figure Lengend Snippet: H3K9me3 recovery by SETDB1 reduces PAR production by oxidative stress. Tet-inducible SETDB1 (A) or SUV39H1 (B) expressing CSB-deficient (CSB−) cells were treated with 10 or 50 ng/ml of doxycycline for 72 h. Whole cell lysates were probed for the indicated proteins on western blot. Sample loading quantities were normalized with either total H3 or GAPDH. Tet-inducible SETDB1 (C) or SUV39H1 (E) cells were treated with DMSO or 50 ng doxycycline for 72 h and treated with 2.5 mM H2O2 for 30 min before ADPr-ChAP sample preparation. Chromatin samples prepared for ADPr-ChAP were probed for nuclear PAR analysis. (D and F) ADPr-ChAP were performed from chromatin preparations from C and E and analyzed for real-time qPCR on indicated genomic locus. Error bars represent SD. *, <0.05.

Article Snippet: Dox-inducible GFP-tagged lentiviral SUV39H1 or SETDB1 constructs were designed and ordered from VectorBuilder Inc. (Santa Clara, CA, USA).

Techniques: Expressing, Western Blot, Sample Prep

Journal: Nucleic Acids Research

Article Title: Cockayne syndrome group B deficiency reduces H3K9me3 chromatin remodeler SETDB1 and exacerbates cellular aging

doi: 10.1093/nar/gkz568

Figure Lengend Snippet: SETDB1-induction restores mitochondrial homeostasis in CS fibroblasts. (A) Doxycycline-induced HMTase expression was probed on western blot (CSB+; CSB-corrected cells, SUV; SUV39H1-inducible CSB-deficient cells, SET; SETDB1-inducible CSB-deficient cells). (B) H3K9me3-ChIP and ADPr-ChAP sequencing reads of SETDB1-inducible CSB-deficient (CSB- rtTA SETDB1) cells were aligned on TSS. (C and D) Merged dataset profiles of aligned sequencing reads from (B). Seahorse extracellular flux analysis representative trace (E) and quantification of basal respiration (F), proton leak (G), maximal respiration (H), complex IV function (I), spare capacity (J), ATP-linked respiration (K) and non-mitochondrial respiration (L) in CSB-corrected (CSB+) and SUV39H1/SETDB1-inducible CSB-deficient cells. (n = 5; data are represented as mean ± SEM) *P< 0.05, **P< 0.01, ***P< 0.001 One-way ANOVA.

Article Snippet: Dox-inducible GFP-tagged lentiviral SUV39H1 or SETDB1 constructs were designed and ordered from VectorBuilder Inc. (Santa Clara, CA, USA).

Techniques: Expressing, Western Blot, Sequencing

Journal: Nucleic Acids Research

Article Title: Cockayne syndrome group B deficiency reduces H3K9me3 chromatin remodeler SETDB1 and exacerbates cellular aging

doi: 10.1093/nar/gkz568

Figure Lengend Snippet: Schematic diagram of CSB-SETDB1 axis. In CS, as well as chronological aging, decreased CSB causes decreased SETDB1 expression, which could lead to reduced H3K9me3 on TSS regions, triggering accumulation of PAR and mitochondrial abnormalities.

Article Snippet: Dox-inducible GFP-tagged lentiviral SUV39H1 or SETDB1 constructs were designed and ordered from VectorBuilder Inc. (Santa Clara, CA, USA).

Techniques: Expressing

Journal: BMC Cancer

Article Title: Histone methyltransferase SETDB1 promotes cells proliferation and migration by interacting withTiam1 in hepatocellular carcinoma

doi: 10.1186/s12885-018-4464-9

Figure Lengend Snippet: Tiam1 could interacted with SETDB1directly by GST- pull down and Cross-linking assays. A Schematic representation of the domain structure of Tiam1. PCER; C685;C751 and C1199 were four truncations constructed containing different domains. B Interaction sites was verified by GST-pull down and westernblot assays. Proteins pulled down by agarose beads with GST tag further were detected by westernblot, (a) purified Tiam1-PCER protein labelled with GST was co-incubated with purified SETDB1 and detected by westernblot after elution, as shown in figure.a, Tiam1-PCER could be detected but not STEDB1, indicating the Tiam1-PCER fragment has no binding sties with SETDB1 (b,c,d) purified Tiam1-C685 protein was obtained as indicated, as shown in above figures, Tiam1-C685, C751 and C1199 could be detected as well as STEDB1, indicating the these fragments have binding sties with SETDB1. C Cross-linking assay confirmed Tiam1could interacted with SETDB1

Article Snippet: The purified protein SETDB1 was acquired with TNT® Quick Coupled Transcription/Translation Systems (Promega,USA).

Techniques: Construct, Purification, Incubation, Binding Assay

Journal: BMC Cancer

Article Title: Histone methyltransferase SETDB1 promotes cells proliferation and migration by interacting withTiam1 in hepatocellular carcinoma

doi: 10.1186/s12885-018-4464-9

Figure Lengend Snippet: SETDB1 promoted cell proliferation both in vivo and vitro in HCC. a The expression of SETDB1 was detected by westernblot after transfecting with overexpression and knockdown virus. b Overexpression of SETDB1 could promote cellsproliferation ability was detected by CCK8 and Plate clony formation assays. ** P <0.01. c knockdown of SETDB1 could inhibit cellsproliferation ability was detected by CCK8 and Plate clony formation assays. ** P <0.01. d SETDB1 could promote HCC cell proliferation in vitro by xenografts experiments. ** P <0.01

Article Snippet: The purified protein SETDB1 was acquired with TNT® Quick Coupled Transcription/Translation Systems (Promega,USA).

Techniques: In Vivo, Expressing, Over Expression, In Vitro

Journal: BMC Cancer

Article Title: Histone methyltransferase SETDB1 promotes cells proliferation and migration by interacting withTiam1 in hepatocellular carcinoma

doi: 10.1186/s12885-018-4464-9

Figure Lengend Snippet: SETDB1 promoted cell metastasis both in vivo and vitro in HCC. a and b Transwell and Boyden assays indicate SETDB1 could enhance cell migration and invasion. The invasive cells were stained and counted under microscope at 24–30 h after reseeding. Original magnification, × 400. ** P <0.01, as compared LV-SET with WT and LV-con groups, or LV-SET with WT and LV-shcon groups. c SETDB1 significantly promoted HCC tumorigenicity in vivo as demonstrated by an orthotopic tumor implantation experiment in nude mice. d SETDB1 increased lung metastasis in an orthotopic tumor implantation model in nude mice. Hematoxylin and eosin staining confirming the formation of HCC tumor foci in the lungs. e SETDB1 modulates EMT-related genes expression

Article Snippet: The purified protein SETDB1 was acquired with TNT® Quick Coupled Transcription/Translation Systems (Promega,USA).

Techniques: In Vivo, Migration, Staining, Microscopy, Tumor Implantation, Expressing

Journal: BMC Cancer

Article Title: Histone methyltransferase SETDB1 promotes cells proliferation and migration by interacting withTiam1 in hepatocellular carcinoma

doi: 10.1186/s12885-018-4464-9

Figure Lengend Snippet: SETDB1 expression is associated with Tiam1expression in HCC cancer. a The expression of SETDB1 and Tiam1 in HCC biopsy samples and control normal samples detected by qRT-PCR. b SETDB1correlates positively with Tiam1 in HCC tissue samples. c The expression of SETDB1 in HCC tissues and adjacent normal tissue were detected by IHC

Article Snippet: The purified protein SETDB1 was acquired with TNT® Quick Coupled Transcription/Translation Systems (Promega,USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: BMC Cancer

Article Title: Histone methyltransferase SETDB1 promotes cells proliferation and migration by interacting withTiam1 in hepatocellular carcinoma

doi: 10.1186/s12885-018-4464-9

Figure Lengend Snippet: Tiam1 is a functional target of SETDB1. a The expression of Tiam1 was identified by westernblot analysis. b Knockdown of Tiam1 could offset the effect of SETDB1 on cell migration ability by transwell assay. c Knockdown of Tiam1 could decrease the number of colonies caused by SETDB1

Article Snippet: The purified protein SETDB1 was acquired with TNT® Quick Coupled Transcription/Translation Systems (Promega,USA).

Techniques: Functional Assay, Expressing, Migration, Transwell Assay

Journal: Oncotarget

Article Title: Whole exome and targeted deep sequencing identify genome-wide allelic loss and frequent SETDB1 mutations in malignant pleural mesotheliomas

doi: 10.18632/oncotarget.7032

Figure Lengend Snippet: Eleven high-confidence ^a non-synonymous variants identified by whole exome ^b and targeted deep sequencing ^c

Article Snippet: We designed another targeted panel of SETDB1 covering all 21 coding exons spanning 6.05 kb (99.76% coverage) (CureSeq Inc, Brisbane, CA).

Techniques: Sequencing, Mutagenesis

Journal: Oncotarget

Article Title: Whole exome and targeted deep sequencing identify genome-wide allelic loss and frequent SETDB1 mutations in malignant pleural mesotheliomas

doi: 10.18632/oncotarget.7032

Figure Lengend Snippet: SETDB1 mutations identified in 78 MPMs from 69 patients and previously reported

Article Snippet: We designed another targeted panel of SETDB1 covering all 21 coding exons spanning 6.05 kb (99.76% coverage) (CureSeq Inc, Brisbane, CA).

Techniques: Mutagenesis

Journal: Oncotarget

Article Title: Whole exome and targeted deep sequencing identify genome-wide allelic loss and frequent SETDB1 mutations in malignant pleural mesotheliomas

doi: 10.18632/oncotarget.7032

Figure Lengend Snippet: Two types of deletion mutations from MPM patients, shown by an Integrative Genomics Viewer (IGV). A. 17 bp deletion (677_693del17) and B. In-frame deletion (3747_3749del) of SETDB1 were identified from different MPM patients.

Article Snippet: We designed another targeted panel of SETDB1 covering all 21 coding exons spanning 6.05 kb (99.76% coverage) (CureSeq Inc, Brisbane, CA).

Techniques:

Journal: Oncotarget

Article Title: Whole exome and targeted deep sequencing identify genome-wide allelic loss and frequent SETDB1 mutations in malignant pleural mesotheliomas

doi: 10.18632/oncotarget.7032

Figure Lengend Snippet: Nine SETDB1 somatic mutations identified in this study and reported previously were mapped in domains of SETDB1 . Four of five truncating mutations were located at the N-terminal (5′) regions before Tudor (Tud) domains. All three missense and one deletion mutation were found in the SET domain. Tud: Tudor; MBD: methyl-CpG-binding domain; pS: pre-SET; PS: post-SET [ – ].

Article Snippet: We designed another targeted panel of SETDB1 covering all 21 coding exons spanning 6.05 kb (99.76% coverage) (CureSeq Inc, Brisbane, CA).

Techniques: Mutagenesis, 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) 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: SetDB1 KO single clones were validated by genomic PCR, sequencing (Plasmidsaurus, Premium PCR) and Western blot.

Techniques: ChIP-sequencing, Stable Transfection, Flow Cytometry, Control, ChIP-qPCR, 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: SetDB1 KO single clones were validated by genomic PCR, sequencing (Plasmidsaurus, Premium PCR) and Western blot.

Techniques: Flow Cytometry, Sequencing, ChIP-qPCR

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: SetDB1 KO single clones were validated by genomic PCR, sequencing (Plasmidsaurus, Premium PCR) and Western blot.

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: SetDB1 KO single clones were validated by genomic PCR, sequencing (Plasmidsaurus, Premium PCR) and Western blot.

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: SetDB1 KO single clones were validated by genomic PCR, sequencing (Plasmidsaurus, Premium PCR) and Western blot.

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: SetDB1 KO single clones were validated by genomic PCR, sequencing (Plasmidsaurus, Premium PCR) and Western blot.

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: SetDB1 KO single clones were validated by genomic PCR, sequencing (Plasmidsaurus, Premium PCR) and Western blot.

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: SetDB1 KO single clones were validated by genomic PCR, sequencing (Plasmidsaurus, Premium PCR) and Western blot.

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: SetDB1 KO single clones were validated by genomic PCR, sequencing (Plasmidsaurus, Premium PCR) and Western blot.

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: SetDB1 KO single clones were validated by genomic PCR, sequencing (Plasmidsaurus, Premium PCR) and Western blot.

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: SetDB1 KO single clones were validated by genomic PCR, sequencing (Plasmidsaurus, Premium PCR) and Western blot.

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: SetDB1 KO single clones were validated by genomic PCR, sequencing (Plasmidsaurus, Premium PCR) and Western blot.

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: SetDB1 KO single clones were validated by genomic PCR, sequencing (Plasmidsaurus, Premium PCR) and Western blot.

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: SetDB1 KO single clones were validated by genomic PCR, sequencing (Plasmidsaurus, Premium PCR) and Western blot.

Techniques: Mutagenesis, Stable Transfection, Expressing, Flow Cytometry, Clone Assay, Control, ChIP-qPCR, 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: SetDB1 KO single clones were validated by genomic PCR, sequencing (Plasmidsaurus, Premium PCR) and Western blot.

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: SetDB1 KO single clones were validated by genomic PCR, sequencing (Plasmidsaurus, Premium PCR) and Western blot.

Techniques: Genome Wide, Mutagenesis, Stable Transfection, Expressing, Transformation Assay, ChIP-sequencing, RNA 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: 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: SetDB1 KO single clones were validated by genomic PCR, sequencing (Plasmidsaurus, Premium PCR) and Western blot.

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: SetDB1 KO single clones were validated by genomic PCR, sequencing (Plasmidsaurus, Premium PCR) and Western blot.

Techniques: Sequencing, Mutagenesis, Genome Wide, Expressing, Transformation Assay, RNA 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: 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: SetDB1 KO single clones were validated by genomic PCR, sequencing (Plasmidsaurus, Premium PCR) and Western blot.

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: SetDB1 KO single clones were validated by genomic PCR, sequencing (Plasmidsaurus, Premium PCR) and Western blot.

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: SetDB1 KO single clones were validated by genomic PCR, sequencing (Plasmidsaurus, Premium PCR) and Western blot.

Techniques: Gene Expression, RNA Sequencing, Mutagenesis, 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) 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: SetDB1 KO single clones were validated by genomic PCR, sequencing (Plasmidsaurus, Premium PCR) and Western blot.

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: SetDB1 KO single clones were validated by genomic PCR, sequencing (Plasmidsaurus, Premium PCR) and Western blot.

Techniques: Binding Assay, Methylation