G-quadruplex DNA suppresses transcription during DNA replication

In eukaryotic cells, although the B-form double helix is the predominant structure of DNA, various non-B-form DNA structures, including G-quadruplex DNA (G4-DNA), are also widely present. For a long time, G4-DNA has been thought to impede DNA replication due to its unique spatial conformation. However, this study demonstrates that S phase G4-DNA accumulation, far from being a passive byproduct, actively functions as a critical safeguard for genomic stability. Contrary to the traditional view of G4-DNA as a replication barrier, its physiological increase during Early S phase minimizes impact on replication itself. Instead, S phase G4-DNA specifically suppresses transcription by stabilizing the negative elongation factor (NELF) complex, thereby preventing transcription-replication conflicts (TRCs) by spatiotemporally segregating transcription and replication. Replication Factor C3 (RFC3), a component of the replication machinery, directly binds and stabilizes G4-DNA to promote its S phase accumulation. Disruption of RFC3 function or G4-DNA unwinding during S phase releases transcriptional suppression, exacerbating TRCs. Overall design: [Cut-tag Experimental Design] The objective of this study is to investigate changes in G4-DNA structures and transcription-associated proteins across different cell cycle phases, as well as to examine the impact of RFC3 knockdown on these elements, using the HCCLM3 human hepatocellular carcinoma cell line. The experiment includes one biological replicate for each condition to capture the CUT&Tag data. In order to achieve these goals, HCCLM3 cells were subjected to thymidine synchronization, allowing us to obtain samples representing various stages of the cell cycle. This synchronization process facilitates the analysis of chromatin features specific to distinct cell cycle phases. Additionally, an shRNA-mediated knockdown of RFC3 was performed on synchronized cells to assess how reduced RFC3 expression affects G4-DNA structures and transcription-related proteins. The focus of this investigation is twofold: first, to elucidate the variations in G4-DNA and associated transcription factors as cells progress through different phases of the cell cycle; second, to determine the effect of RFC3 knockdown on G4-DNA formation and the levels or activity of transcription-related proteins. Unmanipulated HCCLM3 cells, which have not undergone synchronization or RFC3 knockdown, serve as control samples, providing a baseline for comparison. By conducting this study, we aim to gain deeper insights into the dynamics of G4-DNA and transcriptional regulation during the cell cycle and under conditions of RFC3 depletion. [scRNA-seq]The experimental design aims to investigate the alterations in various cell types within mouse hepatocellular carcinoma (HCC) tissue following the knockout of the RFC3 gene. For this purpose, two biological groups were established: one group consists of HCC tissues from mice treated with a control guide RNA (sgCtrl), while the other group comprises HCC tissues from mice subjected to RFC3 knockdown using a specific guide RNA (sgRFC3). By comparing the scRNA-seq profiles between these two groups, we seek to uncover the differential expression patterns and potential functional shifts across distinct cell populations in response to RFC3 depletion. This analysis will provide insights into the role of RFC3 in modulating cellular heterogeneity and tumor microenvironment in liver cancer.[RNA-Seq] The experimental design investigates the changes in gene expression following the knockdown of RFC3 using shRNA in the hepatocellular carcinoma cell line HCCLM3. The primary objective of this experiment is to assess how reducing RFC3 expression affects the transcriptome of these cells. To achieve this, HCCLM3 cells were transfected with shRNA specifically targeting RFC3 to induce gene knockdown. As a control, HCCLM3 cells were also transfected with non-targeting shRNA. Biological triplicates were included for both the experimental and control conditions to ensure the reliability and reproducibility of the results. Each replicate represents an independent biological sample. All samples are derived from the HCCLM3 human liver cancer cell line. RNA was isolated from each replicate, and RNA sequencing (RNA-seq) was performed to quantify gene expression levels before and after RFC3 knockdown. This comprehensive approach aims to identify differentially expressed genes resulting from the reduced RFC3 expression.