Journal: bioRxiv

Article Title: Bone morphogenetic protein 4 reduces global H3K4me3 to inhibit proliferation and promote differentiation of human neural stem cells

doi: 10.1101/2020.01.22.915934

Figure Lengend Snippet: A. Gross anatomy of partial fetal SVZ architecture and localization of H3K4me3 positive cells. View of fetal brain in the area of the lateral ventricle (LV), showing the SVZ with H3K4me3 positive cells. Top panel: H&E staining (LV and SVZ are separated by a green dashed line). Bottom panel: immunostaining with H3K4me3 antibody (LV and SVZ are separated by a red dashed line). B. Immunofluorescence of human StemPro® NSC neurospheres positive for NESTIN, SOX2, CD133 and OCT4 and differentiated cells positive for MUSHASHI, GFAP, TUJ1 and O4. C. Real-time PCR showing hSETD1A and WDR82 expression in spheres and differentiated human StemPro® NSCs. D. Western blots using total protein extracts and histone from un- (UD) and -differentiated (Diff) cells. Error bars show the standard error of three independent experiments (** p<0.01).

Article Snippet: Human short interference RNA (siRNA) against human SETD1A (Gene ID 9739, Cat# SR306505), short hairpin RNA (shRNA) against WDR82 (Gene ID 80335, Cat#TG301034), and scrambled control shRNA cassette in pGFP-V-RS Vector (Cat#TR30013) were purchased from Origene (Rockville, MD, USA).

Techniques: Staining, Immunostaining, Immunofluorescence, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: bioRxiv

Article Title: Bone morphogenetic protein 4 reduces global H3K4me3 to inhibit proliferation and promote differentiation of human neural stem cells

doi: 10.1101/2020.01.22.915934

Figure Lengend Snippet: A. Western blots shows WDR82 and human SETD1A/B expression following 100ng/ml BMP4 treatment. B. Representative images and quantitative graphs showing sphere formation in human StemPro® NSCs following treatment with siRNA for hSETD1A (siSETD1A) or shRNA for WDR82 (shWDR82) vectors versus controls (control siRNA, siCtrl and scrambled shRNA, scrCtrl). C and D. Real-time PCR (C) and western blots (D) showing expression of hSETD1A, WDR82, OCT4, CCND1 and NESTIN, following treatments as in B. E. Real-time PCR using DNA from ChIP with rabbit IgG and H3K4me3 and detected with promoter primers for OCT4, CCND1 and NESTIN following treatment with siSETD1A, shWDR82 or siCtrl, scrCtrl, in StemPro® NSCs. Error bars show the standard error of three independent experiments. (* p<0.05, ** p<0.01)

Article Snippet: Human short interference RNA (siRNA) against human SETD1A (Gene ID 9739, Cat# SR306505), short hairpin RNA (shRNA) against WDR82 (Gene ID 80335, Cat#TG301034), and scrambled control shRNA cassette in pGFP-V-RS Vector (Cat#TR30013) were purchased from Origene (Rockville, MD, USA).

Techniques: Western Blot, Expressing, shRNA, Control, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Bone morphogenetic protein 4 reduces global H3K4me3 to inhibit proliferation and promote differentiation of human neural stem cells

doi: 10.1101/2020.01.22.915934

Figure Lengend Snippet: A. Representative images and quantitative graphs show sphere formation from HAs following transfection with WDR82 (pcDNA3-WDR82) or human SETD1A (pET28-hSETD1A-MHL) expression plasmids, in comparison to controls (pcDNA3 and pET28- MHL). B and C. Real-time PCR (B) and western blots (C) show expression of hSETD1A, WDR82, OCT4, CCND1 and NESTIN. D. ChIP with rabbit IgG and H3K4me3 combined with real-time PCR using promoter primers for OCT4, CCND1 and NESTIN following HA transfection. Error bars show the standard deviation of three independent experiments. (* p<0.05, ** p<0.01)

Article Snippet: Human short interference RNA (siRNA) against human SETD1A (Gene ID 9739, Cat# SR306505), short hairpin RNA (shRNA) against WDR82 (Gene ID 80335, Cat#TG301034), and scrambled control shRNA cassette in pGFP-V-RS Vector (Cat#TR30013) were purchased from Origene (Rockville, MD, USA).

Techniques: Transfection, Expressing, Comparison, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation

Journal: bioRxiv

Article Title: Bone morphogenetic protein 4 reduces global H3K4me3 to inhibit proliferation and promote differentiation of human neural stem cells

doi: 10.1101/2020.01.22.915934

Figure Lengend Snippet: Schematic diagram showing that BMP4 decreases levels of H3K4me3 mediated by hSETD1A-WDR82 at promoters of key regulators to promote differentiation and inhibit proliferation of human neural stem cells (NSCs).

Article Snippet: Human short interference RNA (siRNA) against human SETD1A (Gene ID 9739, Cat# SR306505), short hairpin RNA (shRNA) against WDR82 (Gene ID 80335, Cat#TG301034), and scrambled control shRNA cassette in pGFP-V-RS Vector (Cat#TR30013) were purchased from Origene (Rockville, MD, USA).

Techniques:

Journal: Science advances

Article Title: Cla4 phosphorylates histone methyltransferase Set1 to prevent its degradation by the APC/C Cdh1 complex.

doi: 10.1126/sciadv.adi7238

Figure Lengend Snippet: Fig. 1. Set1 is degraded by the APC/CCdh1 complex. (A) Set1 protein levels were cell cycle regu- lated. Yeast cells were arrested in G1 phase using α-factor for 3 hours and then released into fresh YPD medium. Cells were harvested at in- dicated time points and then sub- jected to immunoblot analysis (left panel) and flow cytometry analysis (right panel). (B) Set1 protein levels were reduced in G1 phase. Cells were arrested in G1, S, and G2-M using α- factor (10 μg/ml), 10 μM HU, and 8 μM nocodazole (Noco) for 3 hours, respectively. (C) Immunoblot analy- sis of Set1 in cells treated with 5 mM MG132 or 5 mM PMSF for 2 hours. Cells were treated with dimethyl sulfoxide (DMSO) and isopropanol as controls. (D) Diagram showing the D- box (226-RNSL-229) within WT Set1 and mutated D-box (226-KNSA-229) within Set1-KNSA mutant. (E) Im- munoblot analysis of Set1 in WT and Set1-KNSA mutant. (F) Immunoblot analysis of Set1 in WT, cdh1Δ, and Cdh1 overexpression (CDH1 OE) cells. Cells transfected with empty vector (EV) were used as controls. (G) Im- munoblot analysis of Set1 in WT and cdh1Δ mutant synchronized at G1, S, and G2-M phases by α-factor, HU, or Noco for 3 hours, respectively. (H) Analysis of Set1 ubiquitination in WT, cdh1Δ, and Set1-KNSA mutants. (I) In vitro ubiquitination assay showing that Cdh1 can mediate Set1 polyu- biquitination. (J and K) Set1 inter- acted with Cdh1 as determined by Co-IP and reciprocal IP. Cells ex- pressing untagged endogenous Cdh1 were used as a negative control. (L) In vitro Co-IP showing that Cdh1 preferentially bound WT Set1 but not Set1-KNSA mutant. The purified FLAG-Set1 and FLAG-Set1- KNSA were incubated with the purified recombinant His-Cdh1. Set1 was immunoprecipitated with anti- FLAG agarose. For (B), (C), and (E), data represent the mean ± SE of three biological independent exper- iments. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The antibody against Set1-S228p (1:500) was custom-made in ABclonal.

Techniques: Western Blot, Flow Cytometry, Mutagenesis, Over Expression, Transfection, Plasmid Preparation, Ubiquitin Proteomics, In Vitro, Co-Immunoprecipitation Assay, Negative Control, Purification, Incubation, Recombinant, Immunoprecipitation

Journal: Science advances

Article Title: Cla4 phosphorylates histone methyltransferase Set1 to prevent its degradation by the APC/C Cdh1 complex.

doi: 10.1126/sciadv.adi7238

Figure Lengend Snippet: Fig. 2. Cla4 phosphorylates Set1-S228 to stabilize Set1. (A) Set1 was phosphorylated at S228 inside cells. Left panel: Diagram showing the predicted Set1-S228 phosphorylation site within the D-box. Right panel: Immunoblot analysis of Set1-S228 phosphorylation in WT, Set1-S228A, and Set1-S228E mutants with anti–Set1- S228p antibody. (B) Immunoblot analysis of Set1 protein levels in WT, Set1-S228A, and Set1-S228E mutants. (C) Immunoblot analysis of Set1 stability in WT and Set1-S228A mutant. WT and Set1-S228A mutant were treated with cycloheximide (CHX) (0.1 μg/ml) for 0 to 40 min. (D) Liquid chromatography–mass spectrometry analysis of proteins copurified with untagged FLAG-Set1. The identified unique peptides and sequence coverage were listed. (E) Immunoblot analysis of Set1 in WT, cla4Δ, cka1Δ, skm1Δ, and sky1Δ mutants. (F and G) Set1 interacted with Cla4 as determined by Co-IP assay (F) and reciprocal IP (G). Cells expressing untagged endogenous Cla4 were used as a negative control in (F). (H) Analysis of the effect of Cla4 on Set1-S228 phosphorylation. (I) In vitro kinase assay showing the purified recombinant His- Cla4 phosphorylated Set1 peptide. (J) Set1 was phosphorylated at S228 by the purified recombinant His-Cla4 in vitro. (K and L) Immunoblot analysis of Cla4, Set1, and Set1-S228p in cells that were arrested by α-factor, HU, or Noco of 3 hours. For (B), (C), and (E), data represent the mean ± SE of three biological independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The antibody against Set1-S228p (1:500) was custom-made in ABclonal.

Techniques: Phospho-proteomics, Western Blot, Mutagenesis, Liquid Chromatography, Mass Spectrometry, Sequencing, Co-Immunoprecipitation Assay, Expressing, Negative Control, In Vitro, Kinase Assay, Purification, Recombinant

Journal: Science advances

Article Title: Cla4 phosphorylates histone methyltransferase Set1 to prevent its degradation by the APC/C Cdh1 complex.

doi: 10.1126/sciadv.adi7238

Figure Lengend Snippet: Fig. 3. Set1-S228 phosphorylation prevents Cdh1-mediated Set1 degradation. (A) Cdh1 preferentially interacted with Set1-S228A mutant as determined by Co-IP. FLAG-Set1 and FLAG-Set1-S228A were immunoprecipitated with anti-FLAG agarose and then incubated with purified recombinant His-Cdh1. (B) Isothermal titration calorimetry (ITC) assay showing that Cdh1 had a higher binding affinity toward Set1-S228 peptide than Set1-S228p peptide. (C) Cla4-catalyzed Set1-S228 phosphory- lation reduced the interaction between Set1 and Cdh1. FLAG-Set1 and FLAG-Set1-S228A were immobilized with anti-FLAG agarose. The purified recombinant His-Cla4 was added to phosphorylate Set1 for 2 hours. After His-Cla4 was washed away, the immobilized Set1 was incubated with the purified recombinant His-Cdh1 for 1 hour. (D) Immunoblot analysis of Set1 in WT, cla4Δ, cdh1Δ, and cla4Δ cdh1Δ mutants. (E) Immunoblot analysis of Set1 in WT, Set1-S228A, Set1-S228E, cdh1Δ, cdh1Δ Set1-S228A, and cdh1Δ Set1-S228E mutants. (F) Analysis of the ubiquitination of Set1 in WT, Set1-S228A, and Set1-S228E mutants. (G) In vitro ubiquitination assay showing that Cla4- catalyzed Set1-S228 phosphorylation inhibited Cdh1-mediated Set1 polyubiquitination. The immobilized FLAG-Set1 was first phosphorylated by His-Cla4. After His-Cla4 was washed away, phosphorylated Set1 was then incubated with Cdh1, Ube1, Ubc4, adenosine triphosphate (ATP), and free ubiquitin. The unphosphorylated Set1 was used as a control. For (D) and (E), data represent the mean ± SE of three biological independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The antibody against Set1-S228p (1:500) was custom-made in ABclonal.

Techniques: Phospho-proteomics, Mutagenesis, Co-Immunoprecipitation Assay, Immunoprecipitation, Incubation, Purification, Recombinant, Isothermal Titration Calorimetry, Binding Assay, Western Blot, Ubiquitin Proteomics, In Vitro, Control

Journal: Science advances

Article Title: Cla4 phosphorylates histone methyltransferase Set1 to prevent its degradation by the APC/C Cdh1 complex.

doi: 10.1126/sciadv.adi7238

Figure Lengend Snippet: Fig. 4. The PAF complex facilitates Cla4 to phosphorylate Set1-S228 and stabilize Set1. (A) Immunoblot analysis of Set1 in WT and PAF complex mutants. (B) Immunoblot analysis of Set1-S228 phosphorylation in WT, paf1Δ, and rtf1Δ mutants. (C) IP–mass spectrometry analysis of proteins copurified with Set1 and Cla4. (D) Cla4 interacted with PAF complex as determined by Co-IP assay. (E) Loss of Paf1 reduced Set1-Cla4 interaction. (F) In vitro Co-IP assay showing that PAF complex enhances Set1-Cla4 interaction. The purified FLAG-Set1 was incubated with His-Cla4 with or without purified PAF complex (Ctr9-TAP). (G) PAF complex facilitates Cla4 to phosphorylate Set1-S228. The purified His-Set1 was incubated with His-Cla4 with or without purified PAF complex (Ctr9-TAP). (H) Immunoblot analysis of Set1 ubiquitination in WT and paf1Δ mutant. (I) Immunoblot analysis of Set1 in WT, paf1Δ, cdh1Δ, and paf1Δ cdh1Δ mutants. (J) Immunoblot anal- ysis of Set1 in WT and cdh1Δ mutant when treated with 6AU for 0 to 45 min. (K) Box plots showing the normalized read intensity of Set1 occupancy at chromatin in cdh1Δ WT Set1, cdh1Δ Set1- S228A, and cdh1Δ Set1-S228E mutants. Center- lines denote medians, box limits denote 25th and 75th percentiles, and whiskers denote maximum and minimum values. Two-sided Wilcoxon test in R (package ggpval) was used for statistical analy- sis. (L) ChIP-seq tracks of Set1 occupancy at indi- cated genes in cdh1Δ WT Set1, cdh1Δ Set1-S228A, and cdh1Δ Set1-S228E mutants. (M) ChIP-qPCR analysis of Set1 occupancy at indicated genes in cdh1Δ WT Set1, cdh1Δ Set1-S228A, and cdh1Δ Set1-S228E mutants. (N) Nucleosomes facilitates Cla4 to phosphorylate Set1-S228. The purified His- Set1 was incubated with His-Cla4 and PAF complex with or without nucleosomes. (O) Diagram showing that Cla4-mediated Set1-S228 phosphorylation was facilitated by PAF complex and RNA Pol II. For (M), data represent the mean ± SE of three biological independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The antibody against Set1-S228p (1:500) was custom-made in ABclonal.

Techniques: Western Blot, Phospho-proteomics, Mass Spectrometry, Co-Immunoprecipitation Assay, In Vitro, Purification, Incubation, Ubiquitin Proteomics, Mutagenesis, ChIP-sequencing, ChIP-qPCR

Journal: Science advances

Article Title: Cla4 phosphorylates histone methyltransferase Set1 to prevent its degradation by the APC/C Cdh1 complex.

doi: 10.1126/sciadv.adi7238

Figure Lengend Snippet: Fig. 5. Cla4-catalyzed Set1-S228 phosphorylation promotes Set1-catalyzed H3K4me3. (A) Immunoblot analysis of H3K4me1, H3K4me2, and H3K4me3 in WT, cla4Δ, cdh1Δ, and cla4Δ cdh1Δ mutants. (B) Immunoblot analysis of H3K4me1, H3K4me2, and H3K4me3 in WT, Set1-S228A, and Set1-S228E mutants. (C) Immunoblot analysis of H3K4me3 in cdh1Δ WT Set1, cdh1Δ Set1-S228A, and cdh1Δ Set1-S228E mutants. (D) Immunoblot analysis of H3K4me1, H3K4me2, and H3K4me3 in WT, cla4Δ, Set1-S228E, and cla4Δ Set1-S228E mutants. (E) Immunoblot analysis of H3K4me1, H3K4me2, and H3K4me3 in WT, paf1Δ, cdh1Δ, and paf1Δ cdh1Δ mutants. (F) Average metagene profiles of H3K4me3 in WT, Set1-S228A, and Set1-S228E mutants. (G) Venn diagram of genes that overlap among the Set1-peak, Paf1-peak, and Cla4-peak gene sets as determined by ChIP-seq. The numbers of binding sites are represented. (H) Box plots showing the normalized read intensity of H3K4me3 in WT, Set1-S228A, and Set1- S228E mutants. Centerlines denote medians, box limits denote 25th and 75th percentiles, and whiskers denote maximum and minimum values. Two-sided Wilcoxon test in R (package ggpval) was used for statistical analysis. (I) ChIP-seq tracks of Set1, Cla4, and Paf1 occupancy in WT cells as well as H3K4me3/H3 enrichment in WT, Set1- S228A, and Set1-S228E mutants at indicated genes. For (A) to (C), data represent the mean ± SE of three biological independent experiments. For (D), data represent the mean ± SE of four biological independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The antibody against Set1-S228p (1:500) was custom-made in ABclonal.

Techniques: Phospho-proteomics, Western Blot, ChIP-sequencing, Binding Assay

Journal: Science advances

Article Title: Cla4 phosphorylates histone methyltransferase Set1 to prevent its degradation by the APC/C Cdh1 complex.

doi: 10.1126/sciadv.adi7238

Figure Lengend Snippet: Fig. 6. Cla4 and Cdh1 regulate gene transcription and chronological life span by affecting Set1 stability. (A) ChIP-seq tracks of Set1, Cla4, and Paf1 occupancy at eight core histone genes. (B) RT-qPCR analysis of core histone gene transcription in WT, Set1- S228A, and Set1-S228E mutants. HHT2 encodes histone H3; HHF2 encodes histone H4; HTA2 encodes histone H2A; HTB2 encodes histone H2B. (C) Immunoblot anal- ysis of core histones in WT, Set1-S228A, and Set1-S228E mutants. (D) Volcano plots for genes differentially ex- pressed in WT and Set1-S228A mutant. Differential ex- pression levels of aligned sequences were calculated using significant thresholds set at log2(FC) ≤ −0.75, log2(FC) ≥ 0.75, P < 0.05. Red color designates signifi- cantly down-regulated genes, and blue color desig- nates significantly up-regulated genes. (E) Heatmap showing the transcription of eight core histone genes in WT and Set1-S228A mutant. (F) GSEA analysis of genes involved in cell cycle, DNA replication, and mismatch repair in WT and Set1-S228A mutant. (G) Box plots showing the normalized read intensity of H3K4me3 at subtelomeric regions on 16 chromosomes in WT, Set1- S228A, and Set1-S228E mutants. The subtelomeric regions refer to 0 to 50 kb from the nearest left or right telomeres. (H) RT-qPCR analysis of the transcription of telomere-proximal genes in WT, Set1-S228A, and Set1- S228E mutants. (I) Heatmap showing the transcription of the longevity pathway genes in WT and Set1-S228A mutant. (J) Chronological life-span analysis of WT, Set1- S228A, and Set1-S228E mutants. (K) Chronological life- span analysis of WT, cla4Δ, Set1-S228E, and Set1-S228E cla4Δ mutants. For (B), (C), (H), (J), and (K), data repre- sent the mean ± SE of three biological independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The antibody against Set1-S228p (1:500) was custom-made in ABclonal.

Techniques: ChIP-sequencing, Quantitative RT-PCR, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Bone morphogenetic protein 4 reduces global H3K4me3 to inhibit proliferation and promote differentiation of human neural stem cells

doi: 10.1101/2020.01.22.915934

Figure Lengend Snippet: A. Western blots shows WDR82 and human SETD1A/B expression following 100ng/ml BMP4 treatment. B. Representative images and quantitative graphs showing sphere formation in human StemPro® NSCs following treatment with siRNA for hSETD1A (siSETD1A) or shRNA for WDR82 (shWDR82) vectors versus controls (control siRNA, siCtrl and scrambled shRNA, scrCtrl). C and D. Real-time PCR (C) and western blots (D) showing expression of hSETD1A, WDR82, OCT4, CCND1 and NESTIN, following treatments as in B. E. Real-time PCR using DNA from ChIP with rabbit IgG and H3K4me3 and detected with promoter primers for OCT4, CCND1 and NESTIN following treatment with siSETD1A, shWDR82 or siCtrl, scrCtrl, in StemPro® NSCs. Error bars show the standard error of three independent experiments. (* p<0.05, ** p<0.01)

Article Snippet: Human His-tagged SETD1A expression pET28-SETD1A-MHL plasmid and its control pET28-MHL were gifts from Cheryl Arrowsmith (Addgene plasmid # 32868 and #26096, respectively).

Techniques: Western Blot, Expressing, shRNA, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Bone morphogenetic protein 4 reduces global H3K4me3 to inhibit proliferation and promote differentiation of human neural stem cells

doi: 10.1101/2020.01.22.915934

Figure Lengend Snippet: A. Representative images and quantitative graphs show sphere formation from HAs following transfection with WDR82 (pcDNA3-WDR82) or human SETD1A (pET28-hSETD1A-MHL) expression plasmids, in comparison to controls (pcDNA3 and pET28- MHL). B and C. Real-time PCR (B) and western blots (C) show expression of hSETD1A, WDR82, OCT4, CCND1 and NESTIN. D. ChIP with rabbit IgG and H3K4me3 combined with real-time PCR using promoter primers for OCT4, CCND1 and NESTIN following HA transfection. Error bars show the standard deviation of three independent experiments. (* p<0.05, ** p<0.01)

Article Snippet: Human His-tagged SETD1A expression pET28-SETD1A-MHL plasmid and its control pET28-MHL were gifts from Cheryl Arrowsmith (Addgene plasmid # 32868 and #26096, respectively).

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation

Journal: Neuron

Article Title: Recapitulation and reversal of schizophrenia-related phenotypes in Setd1a -deficient mice

doi: 10.1016/j.neuron.2019.09.014

Figure Lengend Snippet: (A) Permutation tests (n=20,000, randomization method: circularRandomizeRegions) indicate there is significant overlaps between conserved and chromatin accessible Setd1a ChIP-Seq peaks (P = 0.0055, Z-score = 2.9) and established SCZ-GWAS loci identified. X-axis is the number of overlaps, Y-axis is the density of expected number of overlaps determined by permutation. EVperm, Expected average number of overlaps by permutation, EVobs, actual observed number of overlaps. Permutation P value threshold = 0.05.

Article Snippet: Setd1a CDS vector , Origene , Cat# MR215352.

Techniques: ChIP-sequencing

Journal: Neuron

Article Title: Recapitulation and reversal of schizophrenia-related phenotypes in Setd1a -deficient mice

doi: 10.1016/j.neuron.2019.09.014

Figure Lengend Snippet: (A) Representative ChIP-Seq locus (Kmt2a gene). Normalized reads per genomic coverage from PFC ChIP-Seq for Setd1a (black) and chromatin marks (H3K4me1, red; H3K4me2, orange; H3K4me3, blue; H3K27ac, green) are shown. Black boxes below the Setd1a track depict significant peaks that passed all quality checks (see Methods).

Article Snippet: Setd1a CDS vector , Origene , Cat# MR215352.

Techniques: ChIP-sequencing

Journal: Neuron

Article Title: Recapitulation and reversal of schizophrenia-related phenotypes in Setd1a -deficient mice

doi: 10.1016/j.neuron.2019.09.014

Figure Lengend Snippet: (A-B) Volcano plots of differentially expressed genes in whole-PFC RNA-Seq of Setd1a+/− mice are depicted in (A). 342 differentially expressed genes (FDR<5%) are shown in red, while non-significant changes are shown in grey (left panel). Of the 342 differentially expressed genes, 271 are Setd1a targets (orange and blue, right panel). We partitioned these genes in 4 categories based on the binding regions of Setd1a in the PFC. We defined ‘Setd1a bound promoters up-regulated genes’ and ‘Setd1a bound promoters down-regulated genes’ as genes in which promoters are bound by Setd1a. We defined ‘Setd1a bound enhancers up-regulated genes’ and ‘Setd1a bound enhancers down-regulated genes’ as genes in which enhancers, but not promoters, are bound by Setd1a. Genes with Setd1a binding at both promoters and enhancers, were partitioned into the “Setd1a-promoter bound” classes. A summary of the number of genes in each category is shown in (B).

Article Snippet: Setd1a CDS vector , Origene , Cat# MR215352.

Techniques: RNA Sequencing Assay, Binding Assay

Journal: Neuron

Article Title: Recapitulation and reversal of schizophrenia-related phenotypes in Setd1a -deficient mice

doi: 10.1016/j.neuron.2019.09.014

Figure Lengend Snippet: (A) Restoration of knockout-first allele after TAM treatment. For the rescue experiments, Setd1a+/− mice were crossed to R26FlpoER mice. FLP (Flpo) induced globally by TAM converts the “knockout-first” allele to a conditional allele, restoring gene activity.

Article Snippet: Setd1a CDS vector , Origene , Cat# MR215352.

Techniques: Knock-Out, Activity Assay

Journal: Neuron

Article Title: Recapitulation and reversal of schizophrenia-related phenotypes in Setd1a -deficient mice

doi: 10.1016/j.neuron.2019.09.014

Figure Lengend Snippet: (A) Neurod6 locus. ChIP-Seq of LSD1 (grey), Setd1a (black), Mef2 (dark blue), H3K27ac (green), H3K4me3 (light blue) is shown. Grey, black and dark blue boxes below Lsd1, Setd1a and Mef2 tracks respectively represent significant peaks that passed all quality checks (see methods).

Article Snippet: Setd1a CDS vector , Origene , Cat# MR215352.

Techniques: ChIP-sequencing

Journal: Neuron

Article Title: Recapitulation and reversal of schizophrenia-related phenotypes in Setd1a -deficient mice

doi: 10.1016/j.neuron.2019.09.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Setd1a CDS vector , Origene , Cat# MR215352.

Techniques: Virus, Recombinant, Luciferase, Plasmid Preparation, Software

Journal: Cell

Article Title: A non-catalytic function of SETD1A regulates Cyclin-K and the DNA damage response

doi: 10.1016/j.cell.2018.01.032

Figure Lengend Snippet: A. A volcano plot is used to depict RNA-seq data. Three independent samples on Setd1a+/+ and Setd1aΔ/Δ leukemia cells were analyzed. Significantly downregulated DNA repair genes classified by GO analysis (see also Figure S4A) are shown as red dots.

Article Snippet: Myc- and DDK-tagged human SETD1A ( {"type":"entrez-nucleotide","attrs":{"text":"NM_014712","term_id":"1519314153","term_text":"NM_014712"}} NM_014712 ) and SETD1B ( {"type":"entrez-nucleotide","attrs":{"text":"NM_015048","term_id":"210032579","term_text":"NM_015048"}} NM_015048 ) cDNA were obtained from Origene and DDK-tagged human CCNK ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001099402","term_id":"1653961938","term_text":"NM_001099402"}} NM_001099402 ) cDNA was obtained from GenScript.

Techniques: RNA Sequencing

Journal: Cell

Article Title: A non-catalytic function of SETD1A regulates Cyclin-K and the DNA damage response

doi: 10.1016/j.cell.2018.01.032

Figure Lengend Snippet: A. The human SETD1A deletion mutant constructs are shown as schematic illustrations.

Article Snippet: Myc- and DDK-tagged human SETD1A ( {"type":"entrez-nucleotide","attrs":{"text":"NM_014712","term_id":"1519314153","term_text":"NM_014712"}} NM_014712 ) and SETD1B ( {"type":"entrez-nucleotide","attrs":{"text":"NM_015048","term_id":"210032579","term_text":"NM_015048"}} NM_015048 ) cDNA were obtained from Origene and DDK-tagged human CCNK ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001099402","term_id":"1653961938","term_text":"NM_001099402"}} NM_001099402 ) cDNA was obtained from GenScript.

Techniques: Mutagenesis, Construct

Journal: Cell

Article Title: A non-catalytic function of SETD1A regulates Cyclin-K and the DNA damage response

doi: 10.1016/j.cell.2018.01.032

Figure Lengend Snippet: A. Relative expression level of Setd1a in MLL-AF9 leukemia cells was quantified by qRT-PCR at 4 days post-tamoxifen treatment. This experiment was repeated 3 times with 3 biological replicates in each experiment.

Article Snippet: Myc- and DDK-tagged human SETD1A ( {"type":"entrez-nucleotide","attrs":{"text":"NM_014712","term_id":"1519314153","term_text":"NM_014712"}} NM_014712 ) and SETD1B ( {"type":"entrez-nucleotide","attrs":{"text":"NM_015048","term_id":"210032579","term_text":"NM_015048"}} NM_015048 ) cDNA were obtained from Origene and DDK-tagged human CCNK ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001099402","term_id":"1653961938","term_text":"NM_001099402"}} NM_001099402 ) cDNA was obtained from GenScript.

Techniques: Expressing, Quantitative RT-PCR

Journal: Cell

Article Title: A non-catalytic function of SETD1A regulates Cyclin-K and the DNA damage response

doi: 10.1016/j.cell.2018.01.032

Figure Lengend Snippet: A. SETD1A-binding proteins were identified by Co-IP MS. Proteins were visualized by silver stain (left) and excised from the gel. 68 proteins were identified as SETD1A-binding proteins (right box outlined in grey) and 11 proteins were classified as DNA damage response-associated proteins in GO analysis (red circle). The ranks of signal intensity are shown in brackets.

Article Snippet: Myc- and DDK-tagged human SETD1A ( {"type":"entrez-nucleotide","attrs":{"text":"NM_014712","term_id":"1519314153","term_text":"NM_014712"}} NM_014712 ) and SETD1B ( {"type":"entrez-nucleotide","attrs":{"text":"NM_015048","term_id":"210032579","term_text":"NM_015048"}} NM_015048 ) cDNA were obtained from Origene and DDK-tagged human CCNK ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001099402","term_id":"1653961938","term_text":"NM_001099402"}} NM_001099402 ) cDNA was obtained from GenScript.

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Silver Staining

Journal: Cell

Article Title: A non-catalytic function of SETD1A regulates Cyclin-K and the DNA damage response

doi: 10.1016/j.cell.2018.01.032

Figure Lengend Snippet: A. Density of ChIP-seq reads for SETD1A (red) and RNAP2 (blue) of SETD1A positive TSS (n=18396) from Ren shRNA or Setd1a shRNA–expressing NIH3T3 cells are shown. Regions are ranked according to log2 fold change of SETD1A signal intensity between Ren and Setd1a shRNA.

Article Snippet: Myc- and DDK-tagged human SETD1A ( {"type":"entrez-nucleotide","attrs":{"text":"NM_014712","term_id":"1519314153","term_text":"NM_014712"}} NM_014712 ) and SETD1B ( {"type":"entrez-nucleotide","attrs":{"text":"NM_015048","term_id":"210032579","term_text":"NM_015048"}} NM_015048 ) cDNA were obtained from Origene and DDK-tagged human CCNK ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001099402","term_id":"1653961938","term_text":"NM_001099402"}} NM_001099402 ) cDNA was obtained from GenScript.

Techniques: ChIP-sequencing, shRNA, Expressing

Journal: Cell

Article Title: A non-catalytic function of SETD1A regulates Cyclin-K and the DNA damage response

doi: 10.1016/j.cell.2018.01.032

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Myc- and DDK-tagged human SETD1A ( {"type":"entrez-nucleotide","attrs":{"text":"NM_014712","term_id":"1519314153","term_text":"NM_014712"}} NM_014712 ) and SETD1B ( {"type":"entrez-nucleotide","attrs":{"text":"NM_015048","term_id":"210032579","term_text":"NM_015048"}} NM_015048 ) cDNA were obtained from Origene and DDK-tagged human CCNK ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001099402","term_id":"1653961938","term_text":"NM_001099402"}} NM_001099402 ) cDNA was obtained from GenScript.

Techniques: Mass Spectrometry, Knock-In, shRNA, Recombinant, Plasmid Preparation, Software