- Exploring the changing landscape of cell- to-cell variation after CTCF knockdown via single cell RNA-seq. - Based on single cell flow cytometry and single cell RNA-FISH analyses, our previous study showed that deletion of CTCF binding site led to a significantly increase of cellular variation of its target gene. - However, the effect of CTCF on genome-wide landscape of cell-to-cell variation remains unclear.. - Results: We knocked down CTCF in EL4 cells using shRNA, and conducted single cell RNA-seq on both wild type (WT) cells and CTCF-Knockdown (CTCF-KD) cells using Fluidigm C1 system. - Principal component analysis of single cell RNA-seq data showed that WT and CTCF-KD cells concentrated in two different clusters on PC1, indicating that gene expression profiles of WT and CTCF-KD cells were systematically different. - Interestingly, GO terms including regulation of transcription, DNA binding, zinc finger and transcription factor binding were significantly enriched in CTCF-KD-specific highly variable genes, implying tissue-specific genes such as transcription factors were highly sensitive to CTCF level. - The dysregulation of transcription factors potentially explains why knockdown of CTCF leads to systematic change of gene expression. - In contrast, housekeeping genes such as rRNA processing, DNA repair and tRNA processing were significantly enriched in WT-specific highly variable genes, potentially due to a higher cellular variation of cell activity in WT cells compared to CTCF-KD cells. - We further found that cellular variation-increased genes were significantly enriched in down-regulated genes, indicating CTCF knockdown simultaneously reduced the expression levels and increased the expression noise of its regulated genes.. - Conclusions: To our knowledge, this is the first attempt to explore genome-wide landscape of cellular variation after CTCF knockdown. - Our study not only advances our understanding of CTCF function in maintaining gene expression and reducing expression noise, but also provides a framework for examining gene function.. - Keywords: Single cell RNA-seq, Cell-to-cell variation, CTCF, Change of cellular variation, CTCF knockdown. - CTCF activates and silences gene expression by preventing the spread of heterochromatin and blocking of unrelated enhancer-promoter interactions [1, 16]. - In addition, chromatin loops mediated by CTCF and cohesin can tether distal enhancers to gene promoters and regulate its target gene expression . - For instance, inversion of a CTCF-binding site reconfigured the topology of chromatin loops and activated gene expression by creating a new chro- matin loop [21, 22].. - Our recent study demonstrated that CTCF played an important role in stabilizing enhancer-promoter interaction and redu- cing the gene expression noises in mammalian cells [18]. - In particular, we found that CTCF-KD or dele- tion of CTCF binding sites led to increased variation of cellular expression of GATA3, CD28, CD90 and CD5 [18]. - However, the genome-wide change of cell- to-cell variation after CTCF-KD remains unknown. - In this study, we conducted single cell RNA-seq on both WT and CTCF-KD cells to investigate the changing landscape of cell-to-cell variation at a genome-wide scale. - Interestingly, GO terms including regulation of transcription, DNA binding and Zinc finger were signifi- cantly enriched in CTCF-KD specific highly variable genes. - We also found that cellular variation-increased genes were significantly enriched in down-regulated genes, indicating knockdown of CTCF simultaneously reduced. - Efficient CTCF knockdown and single cell RNA-seq We knocked down CTCF in EL4 cells by short hairpin RNA (shRNA). - In order to investigate the changing landscape of cell-to- cell variation after CTCF knockdown, we successfully con- ducted single cell RNA-seq for shLuc#1, shLuc #2, shCTCF #1 and shCTCF#2 using 4 integrated fluidics cir- cuits (IFCs) (Fig. - We noticed the gene expression levels of pooled shLuc single cells were highly correlated with that of bulk data from our previous study [18] (r 2 = 0.86. - The gene expression of pooled single cells from shLuc #1 was also highly corre- lated with that of shLuc #2 (r 2 = 0.87. - In addition, the gene expression of pooled. - 1 Knockdown of CTCF and schema of single cell sequencing. - Western blot analysis of CTCF in luciferase control (shLuc) and CTCF-KD cells (shCTCF#1 and shCTCF#2). - The expression level of CTCF was normalized to GAPDH. - Schema of single cell RNA sequencing using Fluidigm C1 system. - single cell repeats and bulk cell repeats in CTCF-KD cells were highly correlated (Additional file 1: Figure S1C-S1D).. - Systematic differences between WT cells and CTCF-KD cells. - CTCF-KD cells were distinguishable on PCA projection and concentrated in two different clusters on PC1 (Fig. - 2b,c), implying a systematic difference of gene expression profiles between CTCF-KD and WT cells.. - We further calculated the differential gene expression be- tween WT and CTCF-KD cells using edgeR [26]. - In total, we identified 195 up-regulated and 107 down-regulated genes in CTCF-KD cells compared to WT cells (Additional file 2: Figure S2B). - Heatmap of the most differentially expressed genes between WT cells and CTCF-KD cells. - 2 Systematic difference between CTCF-KD and WT cells. - CTCF-KD cells were largely distinguishable from WT cells on PCA projection. - Distribution of individual WT cells and individual CTCF- KD cells on PC1. - Heatmap of differentially expressed genes (TOP 20) between WT cells and CTCF-KD cells. - In order to distinguish true signals of cellular variation from technical noise, we calculated the expression noise of each gene (σ 2 /μ . - We identified 602 cellular variation increased genes and 890 cellular variation decreased genes after CTCF knockdown by mean ± SD (Fig. - The significant enrichment of transcription factors in CTCF-KD-specific highly variable genes potentially indicates a high sensitivity of transcrip- tion factors to CTCF level. - Dysregulation of certain tran- scription factors possibly explains why knockdown of CTCF leads to a systematic change of gene expression. - The enrichment of housekeeping genes in WT-specific highly variable genes potentially in- dicates a higher cellular variation of cell activity in WT cells compared to CTCF-KD cells.. - CTCF knockdown simultaneously altered expression level and cellular variation of its regulated genes. - Venn diagram showed that 47 genes out of total 107 down-regulated genes exhibited increased cellular variation (Fig. - These results indicate CTCF knockdown simultaneously reduced the expression level and increased the gene expression noise. - Meanwhile, there were 96 genes out of the total 195 up- regulated genes exhibiting decreased cellular variation (Fig. - The 96 genes with decreased cellular variation and increased expression level were significantly enriched in poly(A) RNA binding, rRNA pro- cessing, WD40, purine nucleobase biosynthetic process- ing, rRNA methylation and RNA methyltransferase activity. - Taken together, our results clearly indicate that distortion of CTCF expression could simul- taneously change the gene expression level and cell-to-cell variation of its regulated genes.. - These results suggest that genes regulated by multiple CTCF binding sites tend to possess a higher cellular variation after CTCF knockdown.. - CTCF plays an important role in chromatin structure organization and regulation of gene expression [14–17]. - In this study, we used single cell RNA-seq to analyze genome-wide gene expression profiles of WT and CTCF- KD cells at single cell resolution. - Indeed, WT cell popula- tion and CTCF-KD cell population showed distinct con- centration on PC1, indicating that knockdown of CTCF resulted in a systematic impact on the genome-wide gene expression profile. - CV of gene expression in WT cells. - CV of gene expression in CTCF KD cells. - We generated the genome- wide landscape of cell-to-cell variation in both WT and CTCF-KD cells. - After comparing cell-to-cell variations be- tween WT and CTCF-KD cells, we identified those genes showing a significant change of cellular variation after CTCF knockdown. - Interestingly, the cellular variation- increased genes are significantly enriched in expression- decreased genes, suggesting that CTCF-medicated promoter-enhancer interaction did not only play an im- portant role in maintaining the expression of its regulated genes, but also reduced their expression noise.. - In this study, we identified numerous genes with an ob- vious change of cellular variation after CTCF knockdown, although the knockdown efficiency is moderate. - The increased cellular variation of zinc finger proteins potentially indicates a high sensitiv- ity of zinc finger proteins to CTCF expression level or cel- lular environmental change within the cell. - In fact, the majority of those zinc finger proteins were transcription factors that played an important role in the regulation of cell type-specific gene expression. - Taken together, our findings provide convincing evidence that CTCF serves as a key player in stabilizing the gene expression noise of zinc finger related genes.. - We conducted single cell RNA-seq on both wild type (WT) cells and CTCF-Knockdown (CTCF-KD) cells using Fluidigm C1 system. - Principal component analysis of sin- gle cell RNA-seq data showed that WT and CTCF-KD cells concentrated in two different clusters on PC1, indi- cating a systematic difference of gene expression profiles between WT and CTCF-KD cells. - Interestingly, GO terms including regulation of transcription, DNA binding, zinc finger and transcription factor binding were significantly enriched in CTCF-KD-specific highly variable genes, im- plying tissue-specific genes such as transcription factors were highly sensitive to CTCF level. - Dysregulation of tran- scription factors possibly explains why knockdown of CTCF leads to a systematic change of gene expression. - We further noticed that cellular variation-increased genes were significantly enriched in down-regulated genes, indicating CTCF knockdown simultaneously reduced the expression levels and increased the expression noise of its regulated genes.. - To our knowledge, this is the first attempt to explore the genome-wide landscape of cellular variation after CTCF knockdown. - Our study not only advances our understand- ing of CTCF function in maintaining gene expression and reducing expression noise, but also provides a framework for examining gene function.. - 3 Identification and analyses of genes showing cellular variation changes after CTCF KD. - Genes with low cellular variation was used for further analyses. - Scatter plot of cellular variation-changed genes after CTCF KD. - 4 Genes showing cellular variation change tend to be differentially expressed genes. - Genes with expression decrease and cellular variation increase were significantly over-represented ( P χ 2 test).. - Genes with decreased cellular variation and increased expression level were significantly over-represented ( P χ 2 test). - Knockdown of CTCF by shRNA. - Knockdown of CTCF was performed using Lentiviral- mediated short hairpin RNA (shRNA) in EL4 cells as de- scribed previously [18]. - The cell populations displaying efficient knockdown of CTCF were used for single cell RNA-seq.. - Single cell RNA sequencing. - Fluidigm C1™ Single-Cell Autoprep System (Fluidigm, South San Francisco, CA, USA) was used for single cell RNA-seq. - Transcripts per million (TPM) was used to normalize the gene expression level and log2 trans- formed. - μ denotes mean) to measure the noise of gene expression . - High reproducibility of single cell RNA-seq data. - showing the correlation of gene expression between shLuc #1 and shLuc#2. - (C) Scatter plot showing the correlation of gene expressions between pooled single cells and bulk data in CTCF-KD cells. - Analysis of differentially expressed genes between WT cells and CTCF KD cells. - (A) The expression of CTCF is correlated with the cell coordination on PC2. - Histogram showing that CV difference of gene expression between CTCF-KD cells and WT cells followed the normal distribution.. - CTCF-KD: CTCF-Knockdown. - The single cell RNA-seq data of EL4 cells have been deposited in the Gene Expression Omnibus database with the accession number GSE135769.. - Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene. - CTCF- mediated enhancer-promoter interaction is a critical regulator of cell-to-cell variation of gene expression. - Cohesin and CTCF differentially affect chromatin architecture and gene expression in human cells. - CRISPR inversion of CTCF sites alters genome topology and enhancer/. - edgeR: a bioconductor package for differential expression analysis of digital gene expression data.. - Single-cell proteomic analysis of S. - Accounting for technical noise in single-cell RNA-seq experiments
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