Hepatitis B Virus (HBV) is a small DNA virus that replicates via an episomal covalently closed circular DNA (cccDNA) that serves as the transcriptional template for viral mRNAs. The host protein, CCCTC-binding factor (CTCF), is a key regulator of cellular transcription by maintaining epigenetic boundaries, nucleosome phasing, stabilisation of long-range chromatin loops and directing alternative exon splicing. We previously reported that CTCF binds two conserved motifs within Enhancer I of the HBV genome and represses viral transcription, however, the underlying mechanisms were not identified. We show that CTCF depletion in cells harbouring cccDNA-like HBV molecules and in de novo infected cells resulted in an increase in spliced transcripts, which was most notable in the abundant SP1 spliced transcript. In contrast, depletion of CTCF in cell lines with integrated HBV DNA had no effect on the abundance of viral transcripts and in line with this observation there was limited evidence for CTCF binding to viral integrants, suggesting that CTCF-regulation of HBV transcription is specific to episomal cccDNA. Analysis of HBV chromatin topology by Assay for Transposase Accessible Chromatin Sequencing (ATAC-Seq) revealed an accessible region spanning Enhancers I and II and the basal core promoter (BCP). Mutating the CTCF binding sites within Enhancer I resulted in a dramatic rearrangement of chromatin accessibility where the open chromatin region was no longer detected, indicating loss of the phased nucleosome up- and down-stream of the HBV enhancer/BCP. These data demonstrate that CTCF functions to regulate HBV chromatin conformation and nucleosomal positioning in episomal maintained cccDNA, which has important consequences for HBV transcription regulation.

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CTCF 调节乙型肝炎病毒 cccDNA 染色质拓扑

乙型肝炎病毒 (HBV) 是一种小型 DNA 病毒，通过附加型共价闭合环状 DNA (cccDNA) 进行复制，该 DNA 充当病毒 mRNA 的转录模板。宿主蛋白 CCCTC 结合因子 (CTCF) 是细胞转录的关键调节因子，可维持表观遗传边界、核小体定相、长程染色质环的稳定和指导选择性外显子剪接。我们之前报道过 CTCF 结合 HBV 基因组增强子 I 内的两个保守基序并抑制病毒转录，然而，其潜在机制尚未确定。我们发现，在含有 cccDNA 样 HBV 分子的细胞和从头感染的细胞中，CTCF 的消耗导致剪接转录本的增加，这在丰富的 SP1 剪接转录本中最为显着。相比之下，整合了 HBV DNA 的细胞系中 CTCF 的消耗对病毒转录本的丰度没有影响，并且与这一观察结果一致，CTCF 与病毒整合体结合的证据有限，表明 CTCF 对 HBV 转录的调节是特异性的。游离cccDNA。通过转座酶可及染色质测序 (ATAC-Seq) 分析 HBV 染色质拓扑，揭示了跨越增强子 I 和 II 以及基础核心启动子 (BCP) 的可及区域。增强子 I 内的 CTCF 结合位点突变导致染色质可及性发生显着重排，不再检测到开放染色质区域，表明 HBV 增强子/BCP 上游和下游的定相核小体丢失。这些数据表明 CTCF 具有调节 HBV 染色质构象和附加型 cccDNA 中核小体定位的功能，这对 HBV 转录调节具有重要影响。