Abstract
BackgroundCCCTC-Binding Factor (CTCF), also known as 11-zinc finger protein, participates in many cellular processes, including insulator activity, transcriptional regulation and organization of chromatin architecture. 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.ResultsWe 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.ConclusionsTo 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.
Highlights
CCCTC-Binding Factor (CTCF), known as 11-zinc finger protein, participates in many cellular processes, including insulator activity, transcriptional regulation and organization of chromatin architecture
We found that cellular variation-increased genes were significantly enriched in down-regulated genes, indicating knockdown of CTCF simultaneously reduced the expression level and increased the expression noises of its regulated genes
Efficient CTCF knockdown and single cell RNA-seq We knocked down CTCF in EL4 cells by short hairpin RNA
Summary
CCCTC-Binding Factor (CTCF), known as 11-zinc finger protein, participates in many cellular processes, including insulator activity, transcriptional regulation and organization of chromatin architecture. 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. CTCF has been reported being involved in multiple cellular processes, such as transcriptional regulation, insulator activity, epigenetic regulation, organization of chromatin architecture and X chromosome. 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. GO terms including regulation of transcription, DNA binding and Zinc finger were significantly enriched in CTCF-KD specific highly variable genes. We found that cellular variation-increased genes were significantly enriched in down-regulated genes, indicating knockdown of CTCF simultaneously reduced the expression level and increased the expression noises of its regulated genes
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