Journal: bioRxiv

Article Title: CRISPR tiling deletion screens reveal functional enhancers of neuropsychiatric risk genes and allelic compensation effects (ACE) on transcription

doi: 10.1101/2024.10.08.616922

Figure Lengend Snippet: a, Flow cytometry plots showing the expression of EGFP and mCherry reporters in APP-EGFP/mCherry, SIN3A-EGFP/mCherry, FMR1-mCherry, and MECP2-EGFP reporter cell lines. The expression of reporters was checked in both iPSCs and excitatory neurons. Gray lines are signals from negative control cells, WTC11 i 3 N. b, RNA-seq data shows the expression of APP , FMR1, MECP2, and SIN3A in iPSCs and 2-week excitatory neurons. The genes were ranked on RPKM. c, The expression of cell type marker genes in iPSCs and excitatory neurons.

Article Snippet: To construct the SIN3A promoter reporter plasmids, we modified one lentivirus EGFP reporter plasmid (Addgene, #137725) by replacing the scaffold-attached region (SAR) , with human anti-repressor element 40 , and used the modified plasmid as a backbone for SIN3A promoter reporter plasmid cloning.

Techniques: Flow Cytometry, Expressing, Negative Control, RNA Sequencing Assay, Marker

Journal: bioRxiv

Article Title: CRISPR tiling deletion screens reveal functional enhancers of neuropsychiatric risk genes and allelic compensation effects (ACE) on transcription

doi: 10.1101/2024.10.08.616922

Figure Lengend Snippet: a, The workflow of identifying enhancers of APP , FMR1, MECP2 , and SIN3A in iPSC-induced excitatory neurons using CRISPR tilling deletion screening. b, The P value distribution of enriched pgRNAs (log2FC>0) in each screen. The positive control pgRNAs targeting EGFP and mCherry and some of the test pgRNAs are significantly enriched in each screen. The negative control pgRNAs are not significantly enriched. c, The distribution of identified enhancers of APP , FMR1, MECP2 , and SIN3A , relative to TSS of each target gene. d, Upset plot showing the overlap between identified enhancers and each chromatin feature. The numbers in each row and column indicate the total number of enhancers in each category. e, The percentage of enhancers interacting and not interacting with target promoters based on H3K4me3 PLAC-seq data.

Article Snippet: To construct the SIN3A promoter reporter plasmids, we modified one lentivirus EGFP reporter plasmid (Addgene, #137725) by replacing the scaffold-attached region (SAR) , with human anti-repressor element 40 , and used the modified plasmid as a backbone for SIN3A promoter reporter plasmid cloning.

Techniques: CRISPR, Positive Control, Negative Control

Journal: bioRxiv

Article Title: CRISPR tiling deletion screens reveal functional enhancers of neuropsychiatric risk genes and allelic compensation effects (ACE) on transcription

doi: 10.1101/2024.10.08.616922

Figure Lengend Snippet: a, Genome browser screenshot showing gene body enhancer of FMR1 and sgRNAs targeting FMR1 promoter and enhancer. b, Flow cytometry plots showing the significant downregulation of FMR1-mCherry expression after deleting FMR1 promoter and FMR1-E1 enhancer in both iPSCs and excitatory neurons. Positive controls (black line) are the FMR1-mCherry reporter cells. c, Genome browser screenshot showing identified enhancers of MECP2 and sgRNAs targeting MECP2 promoter and enhancers. d, Single clones of MECP2 promoter or enhancers deletion showing significant downregulation of MECP2-EGFP in both iPSCs and excitatory neurons. Positive controls (black line) are the MECP2-EGFP cells. C1 and C2 indicate two independent clones. e, RT-qPCR results showing the significant downregulation of MECP2 expression in each clone ( P < 0.05 for all the clones, two-tailed two-sample t -test; n = 2). Data are mean ± SEM. f, Flow cytometry plots showing the significant downregulation of APP-EGFP or APP-mCherry in APP promoter and APP-E3 deletion cells. Positive controls (black line) are the APP-EGFP/mCherry reporter cells. g, Flow cytometry plots showing the downregulation of SIN3A-EGFP or SIN3A-mCherry in SIN3A promoter and SIN3A-E4 deletion cells. Red dashed lines indicate the position of SIN3A-EGFP/mCherry double positive cells. h, The genome browser screenshot showing the CTCF ChIP-seq signal in SIN3A-E4 enhancer region in WTC11 iPSCs. The CTCF motif was obtained from JASPAR. Two sgRNAs were designed to target the CTCF motif. PAM sequences were in red. i, Flow cytometry plots showing the downregulation of SIN3A-EGFP or SIN3A-mCherry in sgRNA1 and sgRNA2 infected cells. j, The editing outcomes of sgRNA 1 in the cells of SIN3A-EGFP-/SIN3A-mCherry+. k, The enrichment of disease-associated CNVs in distal cCREs identified in diverse cell types in the human body. Heatmap shows the data from diseases with at least 10 CNVs and P value less than 1×10 -5 in at least one cell type.

Article Snippet: To construct the SIN3A promoter reporter plasmids, we modified one lentivirus EGFP reporter plasmid (Addgene, #137725) by replacing the scaffold-attached region (SAR) , with human anti-repressor element 40 , and used the modified plasmid as a backbone for SIN3A promoter reporter plasmid cloning.

Techniques: Flow Cytometry, Expressing, Clone Assay, Quantitative RT-PCR, Two Tailed Test, ChIP-sequencing, Infection

Journal: bioRxiv

Article Title: CRISPR tiling deletion screens reveal functional enhancers of neuropsychiatric risk genes and allelic compensation effects (ACE) on transcription

doi: 10.1101/2024.10.08.616922

Figure Lengend Snippet: a,b, Flow cytometry plots showing the percentage of cells with reduced expression of SIN3A-EGFP or SIN3A-mCherry in each condition. The negative control is the WTC11 i 3 N cells. The positive control is the SIN3A-EGFP/mCherry reporter cells. c,d, Bar graphs showing the significance of the relative enrichment of cells with reduced expression of SIN3A-EGFP or SIN3A-mCherry compared to positive control cells. P values were determined using the two-sided Fisher’s exact test. * P < 0.0001.

Article Snippet: To construct the SIN3A promoter reporter plasmids, we modified one lentivirus EGFP reporter plasmid (Addgene, #137725) by replacing the scaffold-attached region (SAR) , with human anti-repressor element 40 , and used the modified plasmid as a backbone for SIN3A promoter reporter plasmid cloning.

Techniques: Flow Cytometry, Expressing, Negative Control, Positive Control

Journal: bioRxiv

Article Title: CRISPR tiling deletion screens reveal functional enhancers of neuropsychiatric risk genes and allelic compensation effects (ACE) on transcription

doi: 10.1101/2024.10.08.616922

Figure Lengend Snippet: a , CRISPResso2 analysis of the targeted sequencing data shows the genome editing outcomes at the CTCF motif in the cells with reduced expression of SIN3A-EGFP or SIN3A-mCherry.

Article Snippet: To construct the SIN3A promoter reporter plasmids, we modified one lentivirus EGFP reporter plasmid (Addgene, #137725) by replacing the scaffold-attached region (SAR) , with human anti-repressor element 40 , and used the modified plasmid as a backbone for SIN3A promoter reporter plasmid cloning.

Techniques: Sequencing, Expressing

Journal: bioRxiv

Article Title: CRISPR tiling deletion screens reveal functional enhancers of neuropsychiatric risk genes and allelic compensation effects (ACE) on transcription

doi: 10.1101/2024.10.08.616922

Figure Lengend Snippet: a , The percentage of copy number variants (CNVs) with experimental evidence-based functional consequences. Numbers are displayed in the format of CNVs with functional consequences / total CNVs in each category. b , The classification of copy number variants (size ≥ 50bp) in ClinVar. c , The overlap between CNVs and coding regions, promoter regions, and distal cCREs. Numbers are displayed in the format of overlapping CNVs / total CNVs in each category. P values determined by two-sided Fisher’s exact test. * P < 1×10 -15 . d , The overlap between SIN3A enhancers, SIN3A gene, and genetic variants including heterozygous deletions from Witteveen-Kolk syndrome patients and two copy number loss variants in ClinVar. e , The overlap between MECP2 enhancer and copy number variants in MECP2 locus. In total, 155 clinical deletion/copy number loss variants overlapping with MECP2 coding regions were interpreted as pathogenic variants and associated with Rett syndrome. RCV000142850 is a 4.3kb copy number loss variant located in the 3’UTR of MECP2 , and it was interpreted as a pathogenic variant.

Article Snippet: To construct the SIN3A promoter reporter plasmids, we modified one lentivirus EGFP reporter plasmid (Addgene, #137725) by replacing the scaffold-attached region (SAR) , with human anti-repressor element 40 , and used the modified plasmid as a backbone for SIN3A promoter reporter plasmid cloning.

Techniques: Functional Assay, Variant Assay

Journal: bioRxiv

Article Title: CRISPR tiling deletion screens reveal functional enhancers of neuropsychiatric risk genes and allelic compensation effects (ACE) on transcription

doi: 10.1101/2024.10.08.616922

Figure Lengend Snippet: a, Genome browser screenshot showing enhancers of SIN3A and sgRNAs targeting SIN3A promoter and enhancers. b, Flow cytometry plots showing the significant downregulation of SIN3A-EGFP and SIN3A-mCherry expression after deleting SIN3A enhancers. Positive controls (black lines) are SIN3A-EGFP/mCherry reporter cells. c, The model of the allelic expression pattern of SIN3A and the associated genotype. d, Sanger sequencing shows the SNP in SIN3A intron. e, Allelic gene expression analysis using the SNP located in SIN3A intron shows dominant expression from one allele in G-M+ (SIN3A-EGFP-/SIN3A-mCherry+) and G+M-(SIN3A-EGFP+/SIN3A-mCherry-) clones in both iPSCs and 2-week excitatory neurons. C1 and C2 indicate two independent clones, and each clone has three biological replicates. Dark blue color indicates the C allele, and orange color indicates the T allele. f, RT-qPCR results showing the total SIN3A expression in each clone relative to GAPDH . Each clone has three biological replicates. P values were determined using the two-tailed two-sample t -test.

Article Snippet: To construct the SIN3A promoter reporter plasmids, we modified one lentivirus EGFP reporter plasmid (Addgene, #137725) by replacing the scaffold-attached region (SAR) , with human anti-repressor element 40 , and used the modified plasmid as a backbone for SIN3A promoter reporter plasmid cloning.

Techniques: Flow Cytometry, Expressing, Sequencing, Clone Assay, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: CRISPR tiling deletion screens reveal functional enhancers of neuropsychiatric risk genes and allelic compensation effects (ACE) on transcription

doi: 10.1101/2024.10.08.616922

Figure Lengend Snippet: a, Sanger sequencing data showing the genotype of each allele of SIN3A enhancer and SIN3A . P1 and P2 alleles are identified using the phased variants in WTC11 genome. Both SIN3A enhancer region and SIN3A region are amplified using genomic DNA from indicated cells, and the phased variants in amplified regions are confirmed using Sanger sequencing.

Article Snippet: To construct the SIN3A promoter reporter plasmids, we modified one lentivirus EGFP reporter plasmid (Addgene, #137725) by replacing the scaffold-attached region (SAR) , with human anti-repressor element 40 , and used the modified plasmid as a backbone for SIN3A promoter reporter plasmid cloning.

Techniques: Sequencing, Amplification

Journal: bioRxiv

Article Title: CRISPR tiling deletion screens reveal functional enhancers of neuropsychiatric risk genes and allelic compensation effects (ACE) on transcription

doi: 10.1101/2024.10.08.616922

Figure Lengend Snippet: a, Flow cytometry plots showing the expression of SIN3A-EGFP and SIN3A-mCherry in control cells (SIN3A-EGFP/mCherry reporter cells) and cells infected with pgRNAs targeting SIN3A promoter and SIN3A-E4 enhancer. The dates refer to the days following the lentivirus infection. b, Dot plots showing the expression trend of SIN3A-EGFP and SIN3A-mCherry signals in the cells with reduced expression level of SIN3A-EGFP or SIN3A-mCherry in panel a. Trendlines are based on logarithmic model. c, Allelic promoter and enhancer deletion-induced downregulation of SIN3A . Dots indicate the levels of SIN3A-EGFP or SIN3A-mCherry in cells with allelic promoter or enhancer deletions. The black dashed line indicates allelic expression levels from wild-type cells. d, The ACE rate of SIN3A enhancer E4 deletion. The average downregulation and transcriptional compensation resulting from enhancer deletion on the EGFP and mCherry alleles were used to calculate the slope between each pair of adjacent time points. e, Flow cytometry plots showing the SIN3A-EGFP and SIN3A-mCherry signals from each clone in iPSCs and neurons. Positive control is SIN3A-EGFP/mCherry reporter cells. C1 and C2 indicate two independent clones of each genotype. f, Flow cytometry plots showing the SIN3A-EGFP and SIN3A-mCherry signals in the cells with and without ectopic SIN3A expression. SIN3A-EGFP/mCherry reporter cells were used as control.

Article Snippet: To construct the SIN3A promoter reporter plasmids, we modified one lentivirus EGFP reporter plasmid (Addgene, #137725) by replacing the scaffold-attached region (SAR) , with human anti-repressor element 40 , and used the modified plasmid as a backbone for SIN3A promoter reporter plasmid cloning.

Techniques: Flow Cytometry, Expressing, Control, Infection, Positive Control, Clone Assay

Journal: bioRxiv

Article Title: CRISPR tiling deletion screens reveal functional enhancers of neuropsychiatric risk genes and allelic compensation effects (ACE) on transcription

doi: 10.1101/2024.10.08.616922

Figure Lengend Snippet: a, The SIN3A promoter P1 controlled SIN3A-P2A-BFP expression cassette. b, RT-qPCR results show the expression levels of SIN3A in control condition and overexpression conditions. Data are mean ± SD from three technical replicates. c, WashU Epigenome Browser snapshot showing SIN3A transcripts from refGene, SIN3A promoter deletion region in validation experiments, two promoter regions used for SIN3A promoter reporter assay, ATAC-seq signal in WTC11 iPSCs, and SIN3A ChIP-seq signals in H1 cells. d , The expression of SIN3A transcripts from long read RNA-seq data in WTC11 cells. Data are mean ± SEM from three biological replicates.

Article Snippet: To construct the SIN3A promoter reporter plasmids, we modified one lentivirus EGFP reporter plasmid (Addgene, #137725) by replacing the scaffold-attached region (SAR) , with human anti-repressor element 40 , and used the modified plasmid as a backbone for SIN3A promoter reporter plasmid cloning.

Techniques: Expressing, Quantitative RT-PCR, Control, Over Expression, Reporter Assay, ChIP-sequencing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: CRISPR tiling deletion screens reveal functional enhancers of neuropsychiatric risk genes and allelic compensation effects (ACE) on transcription

doi: 10.1101/2024.10.08.616922

Figure Lengend Snippet: a, Flow cytometry plots showing the EGFP expression from SIN3A promoter reporters. b, shRNA-mediated downregulation of SIN3A . c,d, SIN3A promoter reporters show significantly higher EGFP intensity in cells with SIN3A shRNA, compared to cells with control shRNA. P values in panels b-d were determined using the two-tailed two-sample t -test. e, The working model of allelic enhancer deletion-induced ACE. SIN3A is evenly expressed from two alleles in wild-type cells. Allelic enhancer deletion causes downregulation of SIN3A from the enhancer deletion allele (sky blue dashed line), which triggers ACE from the intact allele (sky blue solid line). Allelic partial promoter deletion causes partial downregulation of SIN3A (orange dashed line) without ACE (orange solid line).

Article Snippet: To construct the SIN3A promoter reporter plasmids, we modified one lentivirus EGFP reporter plasmid (Addgene, #137725) by replacing the scaffold-attached region (SAR) , with human anti-repressor element 40 , and used the modified plasmid as a backbone for SIN3A promoter reporter plasmid cloning.

Techniques: Flow Cytometry, Expressing, shRNA, Control, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: TET1 modulates H4K16 acetylation by controlling auto-acetylation of hMOF to affect gene regulation and DNA repair function

doi: 10.1093/nar/gkw919

Figure Lengend Snippet: Integrative genomic analysis of published ChIP-seq data sets in mESCs show highly similar binding patterns between Tet1 and H4K16ac. ( A ) Pearson correlation between Tet1 and other 102 DBPs (Supplementary Table S1) in the 2000 bp TSS flanking regions. DeepTools was employed to calculate the correlation of ChIP-seq data. Color bar represents correlation coefficient. ( B ) Further correlation between Tet1, other 13 DBPs (Kdm2a, Dpy30, Mof, Sin3a, Lsd1, Oct4, p300, Dax1, Dmap1, Max, Nanog, Myc and Tip60) and 6 histone modifications (H3K4me3, H3K9me3, H3K9ac, H3K27ac, H3K36me3 and H4K16ac) in 2000 bp TSS flanking regions. ( C ) Correlation of Tet1, Sin3a, Mof, H4K16ac, H3K27ac, H3K9ac and H3K4m3. ( D ) The bindings of Tet1, Sin3a, Mof and H4K16ac are commonly enriched in the TSS regions. ( E ) Seven distinct distribution among Tet1/Sin3a/Mof complex, PRC2 complex and Sin3a/Hdac1/Hdac2 complex were generated by ChromHMM. ( F ) The enriched pathways in cluster M6, including DNA repair pathways marked as red color.

Article Snippet: TET1 cDNA was purchased from Origene (RC218608), cDNA for hMOF (NM_032188) , SIN3A (NM_001145357), and Histone H4 (NM_003541) were generated from cDNA library and confirmed by DNA sequencing, and then subcloned into pcDNA3.0 vector, followed by sequencing validation.

Techniques: ChIP-sequencing, Binding Assay, Generated

Journal: Nucleic Acids Research

Article Title: TET1 modulates H4K16 acetylation by controlling auto-acetylation of hMOF to affect gene regulation and DNA repair function

doi: 10.1093/nar/gkw919

Figure Lengend Snippet: TET1 forms a chromatin complex with hMOF, and SIN3A. ( A ) Nuclear extracts from HEK293T cells were added to a 12–30% sucrose gradient, and fractions were assayed by immunoblotting. The fraction numbers and 660 kDa molecular mass standard are given across the top. The larger fraction numbers indicate the fraction with smaller molecular weight. ( B ) TET1 and SIN3A were shown interacting with hMOF using nuclear protein immunoprecipitations (IPs) in TET1 overexpressing HEK293T cells. ( C ) Schematic representation of TET1 fragments, including Flag-F1, Flag-F2, and Flag-F3 (CXXC: binding CpG islands; CD: Cysteine-rich domain; DSBH: double stranded β-helix). ( D ) HEK293T cells were transiently transfected with Flag-TET1-F1, Flag-TET1-F2 and Flag-TET1-F3, respectively. Nuclear protein IPs were performed using a Flag-tag antibody, followed by western blot analysis using indicated antibodies. ( E ) GST and GST-F3 were expressed in BL21 cells and purified following pGEX-GST-vector's manual. His-SIN3A and His-hMOF1 were also expressed in BL21 and purified and. Pull down assays were performed using a GST-tag antibody.

Article Snippet: TET1 cDNA was purchased from Origene (RC218608), cDNA for hMOF (NM_032188) , SIN3A (NM_001145357), and Histone H4 (NM_003541) were generated from cDNA library and confirmed by DNA sequencing, and then subcloned into pcDNA3.0 vector, followed by sequencing validation.

Techniques: Western Blot, Molecular Weight, Binding Assay, Transfection, FLAG-tag, Purification, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: TET1 modulates H4K16 acetylation by controlling auto-acetylation of hMOF to affect gene regulation and DNA repair function

doi: 10.1093/nar/gkw919

Figure Lengend Snippet: TET1 facilitates HRR and NHEJ via modulating auto-acetylation of hMOF. ( A ) Frequency of HRR after TET1, TET2 or TET3 depletion (** P < 0.01). ( B ) Frequency of NHEJ after TaET1, TET2 or TET3 depletion (** P < 0.01). ( C ) Frequency of HRR in TET1-knockdown cells, with or without overexpressing hMOF or hMOF (K274R) respectively (** P < 0.01). ( D ) Frequency of NHEJ in TET1-knockdown cells, with or without overexpressing hMOF or hMOF (K274R), respectively (** P < 0.01). ( E, F ) TET1 stable knockdown cells were transfected with shRNA. Transfectants were treated with x-ray or bleomycin with increasing concentrations as indicated for 72h. Relative cell proliferation was determined by MTT assay. P value was calculated by unpaired Student's t test. ( G ) The left graph depictes that TET1 forms a chromatin complex with SIN3A and hMOF. SIN3A may function as a scaffold on chromatin. The right graph depicted the process that TET1 depletion promotes auto-acetylation of hMOF to dissociate from chromatin and subsequently induces hypo-acetylation of H4K16, ultimately leading to down-regulation of DNA repair genes and defect of DNA repair.

Article Snippet: TET1 cDNA was purchased from Origene (RC218608), cDNA for hMOF (NM_032188) , SIN3A (NM_001145357), and Histone H4 (NM_003541) were generated from cDNA library and confirmed by DNA sequencing, and then subcloned into pcDNA3.0 vector, followed by sequencing validation.

Techniques: Transfection, shRNA, MTT Assay