LSD1和Set1A与病毒蛋白HBx的合作酶建立一个活动性乙肝病毒染

StephenW

Article | Open

The enzymes LSD1 and Set1A cooperate with the viral protein HBx to establish an active hepatitis B viral chromatin state

• 
  Valentina Alarcon
• , Sergio Hernández
• , Lorena Rubio
• , Francisca Alvarez
• , Yvo Flores
• , Manuel Varas-Godoy
• , Giancarlo V. De Ferrari
• , Michael Kann
• , Rodrigo A. Villanueva
• & Alejandra Loyola
  

• Scientific Reports 6, Article number: 25901 (2016)
• doi:10.1038/srep25901
• Download Citation
• 
  
  • Gene regulation
  • Methylation
    

Received:27 October 2015Accepted:25 April 2016Published online:13 May 2016


AbstractWith about 350 million people chronically infected around the world hepatitis B is a major health problem. Template for progeny HBV synthesis is the viral genome, organized as a minichromosome (cccDNA) inside the hepatocyte nucleus. How viral cccDNA gene expression is regulated by its chromatin structure; more importantly, how the modulation of this structure impacts on viral gene expression remains elusive. Here, we found that the enzyme SetDB1 contributes to setting up a repressed cccDNA chromatin state. This repressive state is activated by the histone lysine demethylase-1 (LSD1). Consistently, inhibiting or reducing LSD1 levels led to repression of viral gene expression. This correlates with the transcriptionally repressive mark H3K9 methylation and reduction on the activating marks H3 acetylation and H3K4 methylation on viral promoters. Investigating the importance of viral proteins we found that LSD1 recruitment to viral promoters was dependent on the viral transactivator protein HBx. Moreover, the histone methyltransferase Set1A and HBx are simultaneously bound to the core promoter, and Set1A expression correlates with cccDNA H3K4 methylation. Our results shed light on the mechanisms of HBV regulation mediated by the cccDNA chromatin structure, offering new therapeutic targets to develop drugs for the treatment of chronically infected HBV patients.

DiscussionIn chronically infected patients the HBV genome remains in the nucleus of the hepatocyte as a minichromosome, structured similarly to the cellular chromatin. Despite the knowledge that has been gained about the impact of chromatin structure on gene expression, only a few reports have investigated how histone modification changes on the cccDNA minichromosome influence HBV gene expression11,14,15,19,20. In this study, we have shown that the enzyme SetDB1 contributes to establishing a repressed HBV cccDNA chromatin state, consistent with a recent report20. This repressed cccDNA chromatin state is activated by the enzymes LSD1 and Set1A. Our results suggest that LSD1 mediates its activation by demethylating H3K9, whereas Set1A by methylating H3K4. In addition, we showed that both LSD1 and Set1A are recruited to viral promoters in an HBx dependent manner.
We found that 24 h after HBV genome transfection, histones H3 bound to HBV promoters are hypoacetylated and methylated at residue K9, both marks associated with transcriptional repression (Fig. 1). It has been suggested that the establishment of these marks is part of a cellular mechanism aimed at silencing the virus20. Therefore, in order for active HBV transcription and replication to occur, the repressed modifications need to be removed and active marks imposed on the HBV minichromosome. The viral HBx protein plays a key role in this activating mechanism by recruiting chromatin modifying enzymes, such as the histone acetyltransferase p30011,14,19,20. In its absence, histone deacetylases are recruited to the cccDNA instead11. Our results indicate that HBx is necessary for the recruitment of the enzymes LSD1 and Set1A to participate in the activation of the repressed HBV cccDNA chromatin state. Intriguingly, LSD1 can demethylate histone H3 on lysines 4 and 9, depending on the proteins that it interacts with. When LSD1 associates with the Co-REST complex it contributes to the establishment of transcriptional repression by removing H3K4 methylation. In contrast, when LSD1 associates with the androgen receptor, it establishes a transcriptionally active state by removing H3K9 methylation28. We showed that LSD1 is required for a reduction of the methylation levels of H3K9, but not H3K4, bound to the HBV cccDNA minichromosome; however, how LSD1 activity is regulated in the HBV context remains unknown. Further experiments should investigate whether androgen receptor and the HBx protein work together to regulate the association of LSD1 to viral promoters and its activity. Our results support the model proposed by the Levrero’s group11, in which the viral HBx protein regulates the cccDNA chromatin state by recruiting chromatin modifiers to viral promoters (Fig. 7), including the histone acetyltransferase p300, the histone demethylase LSD1, and the histone methyltransferase Set1A, as suggested in here. As a consequence, an active HBV cccDNA chromatin state is established that promotes HBV expression and production of viral progeny.
Figure 7: HBx contributes to establish an active cccDNA chromatin state.The viral HBx protein plays a key role in the establishment of an active cccDNA chromatin state by regulating the recruitment of chromatin modifying enzymes, including LSD1 and Set1A, as demonstrated here.


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For the treatment of chronically infected patients there are currently two types of antivirals: interferon-α, which stimulates the immune system and can eventually clear HBV29, and nucleotide/nucleoside analogs, which interfere with the viral replication by inhibiting the viral polymerase/reverse-transcriptase activity. However, only 20–30% of the treated patients eradicate the infection, due to the persistence of the cccDNA in infected hepatocytes30. In addition, HBV has developed resistance to these type of drugs31. Therefore, it is necessary to develop new therapeutic targets and compounds. We and others have shown that the cccDNA minichromosome structure is dynamic and viral gene expression adapts its outcome according to changes on the chromatin including histone modifications. For instance, upon treatment with deacetylase inhibitors, the histones bound to the cccDNA become hyperacetylated, correlating with activation of viral gene expression9,9. Our results indicate that modulating the cccDNA chromatin structure towards a repressed chromatin state is a potential therapeutic approach for chronically infected HBV patients. We have shown that by inhibiting LSD1 demethylase with pargyline, an irreversible non-selective monoamine oxidase inhibitor, viral gene expression is repressed. Interestingly, pargyline is used in the clinic by patients suffering from hypertension; thus, it would be interestingly to explore its effect in an in vivo HBV animal model as a potential drug for chronically infected HBV patients. However, the use of pargyline in HBV chronically infected patients has the limitation that it is not a specific LSD1 inhibitor. Giving that LSD1 is overexpressed in many types of cancers and it is believed that its overexpression contributes to carcinogenesis, screening for LSD1 inhibitors has been intently pursued, thus specific LSD1 inhibitors have been developed32.
However, it should be pointed out that therapies based on the inhibition of cellular enzymes, such as LSD1, will most likely have side effects. Therefore, ways of blocking the recruitment of chromatin modifying enzymes, such as LSD1, specifically to the cccDNA minichromosome rather than inhibiting the enzyme would be a safer approach for patient treatment. Given the evidence that the viral protein HBx is required for the recruitment of many chromatin modifying enzymes to the cccDNA11,14,19, HBx protein would be a target to explore. Thus, our results highlight the importance of understanding how the cccDNA minichromosome is structured and how this structure as well as changes in it can control HBV gene expression and replication.

StephenW

LSD1和Set1A与病毒蛋白HBx的合作酶建立一个活动性乙肝病毒染色质状态

    瓦伦蒂娜·阿拉尔孔，塞尔吉奥·埃尔南德斯，洛雷娜卢比奥，弗朗西斯·阿尔瓦雷斯，伊沃·弗洛雷斯，曼努埃尔·巴拉斯 - 戈多伊，吉安卡洛·五德法拉利，迈克尔·卡恩，罗德里戈A.维拉纽瓦和亚历杭罗耀拉

    科学报告6，商品编号：25901（2016）
    DOI：10.1038 / srep25901
    下载文献
        基因regulationMethylation

收稿日期：
    2015年10月27日
公认：
    2016年4月25日
网络发布时间：
    2016年5月13日

抽象

约3.5亿人长期全球乙肝感染周围是一个主要的健康问题。模板子代HBV合成是病毒基因组，组织成一个微染色体（cccDNA的）肝细胞细胞核内。如何病毒cccDNA的基因的表达是由它的染色质结构调节;更重要的是，如何对病毒基因的表达这种结构影响调制仍然遥遥无期。在这里，我们发现，这种酶有助于SETDB1设立一个压抑的cccDNA的染色质状态。此压抑状态由组蛋白赖氨酸脱甲基酶-1（LSD1）激活。一致，抑制或降低LSD1水平导致病毒基因表达的抑制。这与转录镇压标志H3K9甲基化和减少的激活相关标志着病毒启动子H3乙酰化和H3K4甲基。调查病毒蛋白的重要性，我们发现，LSD1招募病毒启动子是依赖于病毒反蛋白的HBx。此外，组蛋白甲基Set1A和HBx的同时结合到核心启动子，和Set1A表达的cccDNA H3K4甲基相关。我们的研究结果阐明HBV调控的cccDNA的染色质结构调节机制的光，提供了新的治疗靶点，开发药物为慢性乙肝病毒感染患者的治疗。

讨论

在慢性感染患者的HBV基因组保留在肝细胞作为微染色体的核，结构类似于蜂窝染色质。尽管已经获得了有关的染色质结构的对基因表达的影响的知识，只有少数报道已经调查修改对cccDNA的微染色体影响HBV基因expression11,14,15,19,20如何组蛋白的变化。在这项研究中，我们已经表明，这种酶SETDB1有助于建立压抑的HBV的cccDNA染色质状态，与最近report20一致。此压抑的cccDNA染色质状态是由酶LSD1和Set1A激活。我们的研究结果表明，LSD1去甲基化所H3K9，而通过甲基H3K4 Set1A介导其激活。此外，我们发现，无论LSD1和Set1A被招募到病毒启动子在HBx蛋白依赖性。

我们发现，HBV基因组转染后24小时，势必HBV启动子的组蛋白H3的是低乙酰化与在残基K9甲基化，与转录抑制有关的两个标记（图1）。已经提出，建立这些标记是旨在沉默virus20蜂窝机制的一部分。因此，为了使活性的HBV转录和复制发生，压抑的修改需要拆除和活性标记施加于HBV的微染色体。病毒的HBx蛋白起着通过招募染色质修饰酶，如组蛋白乙酰转移p30011,14,19,20在此作动机构的关键作用。在缺席聆讯下，组蛋白脱乙酰被招募到cccDNA的instead11。我们的研究结果表明，HBx蛋白是必要的酶LSD1和Set1A参加压抑HBV cccDNA的染色质状态的激活的招聘。有趣的是，LSD1可以脱甲基化上赖氨酸4和9组蛋白H3，取决于它与交互的蛋白质。当LSD1联营公司与联席REST复杂，它通过去除H3K4甲基有助于建立转录抑制的。相反，当LSD1同伙与雄激素受体，它通过去除H3K9 methylation28建立转录活性状态。我们发现，LSD1需要减少H3K9的甲基化水平的，但不是H3K4，结合于HBV cccDNA的微染色体;然而，LSD1活性是如何在HBV的上下文调节仍然不明。进一步的实验应该调查雄激素受体和HBx基因蛋白一起工作是否LSD1协会下调至病毒启动子和其活性。我们的研究结果支持由Levrero的组11提出的模型，其中该病毒的HBx蛋白招募染色质改性剂对病毒启动子调节的cccDNA染色质状态（图7），包括组蛋白乙酰P300中，组蛋白脱甲基LSD1，并且组蛋白甲基Set1A，所建议的在这里。作为结果，有源HBV cccDNA的染色质状态被建立促进HBV表达和生产的病毒后代。
图7：HB​​x蛋白有助于建立一个活跃的cccDNA的染色质状态。
图7

病毒的HBx蛋白通过调节染色质修饰酶，包括LSD1和Set1A招聘起着建立一个活跃的cccDNA的染色质状态的关键作用，因为这里演示。
全尺寸图片

用于治疗慢性感染的患者目前有两种类型的抗病毒药物的：α干扰素，它是通过抑制病毒聚合酶/反向刺激免疫系统，并能最终清晰HBV29，和核苷酸/核苷类似物，其与病毒复制干扰酶的活性。然而，只有20-30％的治疗的患者的根除感染，由于cccDNA的的感染hepatocytes30的持久性。此外，乙肝病毒已经发展阻力这些类型drugs31的。因此，有必要开发新的治疗靶点和化合物。我们和其他人已经表明，cccDNA的微染色体的结构是根据对染色质的变化，包括组蛋白修饰活力和病毒基因的表达适应它的结果。例如，在用脱乙酰酶抑制剂治疗，结合到cccDNA的组蛋白成为超乙酰，与病毒基因expression9,9的活化相关联。我们的研究结果表明，调节cccDNA的染色质结构朝着压抑染色质状态，是慢性乙肝病毒感染患者潜在的治疗方法。我们已经通过抑制LSD1脱甲基酶与帕吉林，一种不可逆的非选择性单胺氧化酶抑制剂，病毒基因的表达受到抑制表明。有趣的是，优降宁是在由高血压患者门诊使用;因此，这将是有趣的，探索在体内HBV动物模型的作用为慢性感染HBV的患者的潜在的药物。然而，在HBV慢性感染患者使用帕吉林的优点在于它是不是一个特定LSD1抑制剂的限制。赋予该LSD1在许多类型的癌症中过表达并且据信其过度表达有助于癌变，筛选LSD1抑制剂已专心追求的，从而特定LSD1抑制剂已经developed32。

然而，应该指出的是，根据细胞酶，如LSD1抑制疗法，将最有可能有副作用。因此，阻断染色质修饰酶，如LSD1招募，具体到cccDNA的微染色体，而不是抑制酶的方法将是对病人的治疗更安全的方法。鉴于病毒蛋白HBx蛋白是必需的许多染色质修饰酶的cccDNA11,14,19招聘证据，HBx蛋白蛋白质是探索的目标。因此，我们的结果突出的理解cccDNA的微染色体是如何构成的，以及如何这种结构以及变化它可以控制的HBV基因表达和复制的重要性。