The role of macroH2A variants in cancer and senescence

描述（由申请人提供）：本提案的总体目标是了解macroH2A组蛋白变异在癌症和衰老的转录调控中所起的作用。MacroH2A是一种组蛋白变体，发现于基因组的异染色质区域，通常与基因抑制有关。然而，macroH2A通常不会在异染色质相关的转录抑制中起致病作用。相反，macroH2A是其靶标子集在异色环境中表达所必需的。人类基因组编码三种macroH2A变体。MacroH2A1.1和macroH2A1.2是H2AFY基因的备选剪接产物，而macroH2A2是由H2AFY2编码的。这些变体在功能上是不同的；而macroH2A1.1与NAD+利用PARP-1酶产生的聚adp -核糖（PAR）等信号分子相互作用，而macroH2A1.2和macroH2A2则不能。最近，降低macroH2A1.1表达的H2AFY选择性剪接的变化与几种癌症类型有关，甚至被证明可以预测治疗后的复发。MacroH2A还与一个重要的肿瘤抑制机制——细胞衰老有关，这使得这种组蛋白变体与癌症之间的联系更加引人注目。根据这些观察结果，我们的假设是macroH2A1.1在调节基因表达方面具有不同于其他最终发挥肿瘤抑制作用的macroH2A变异的功能。为了验证这一假设并阐明每个macroH2A变体的不同作用，提出了三个具体目标。第一个目的是确定macroH2A变异对癌症和细胞衰老中基因表达的作用。这一目标将通过使用全基因组方法来确定每个组蛋白变体的基因组定位模式来实现。此外，我们将在调节macroH2A变体表达的条件下进行表达分析。第二个目的是确定调节正常细胞和癌细胞中macroH2A1剪接的机制。为了实现这一目标，我们将使用生物信息学和“minigene”剪接实验来确定参与H2AFY选择性剪接调节的顺式和反式元件。第三个目的是确定PARP活性在macroh2a1.1特异性靶基因表达中的作用。PARP-1通过PAR结合与macroH2A1.1相互作用，提示macroH2A1.1和PARP-1可能在功能上协同调节基因表达。我们将通过药物抑制和RNAi调节PARP-1活性水平来验证这一假设，并评估其对macroH2A1.1靶基因的因子募集和转录活性的影响。PARP-1是目前癌症治疗中的一个治疗靶点，这突出了了解这两个癌症相关分子之间功能联系的必要性。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to understand the role macroH2A histone variants play in transcriptional regulation in cancer and senescence. MacroH2A is a histone variant found in heterochromatic regions of the genome, which are typically associated with gene repression. However, macroH2A does not generally play a causative role in heterochromatin-associated repression of transcription. Instead, macroH2A is required for a subset of its targets to be expressed from within a heterochromatic environment. The human genome encodes three macroH2A variants. MacroH2A1.1 and macroH2A1.2 are alternative splice products of the H2AFY gene, while macroH2A2 is encoded by H2AFY2. These variants are functionally distinct; while macroH2A1.1 interacts with signaling molecules such as poly(ADP-ribose) (PAR) generated by NAD+utilizing enzyme PARP-1, macroH2A1.2 and macroH2A2 cannot. Recently, changes in alternative splicing of H2AFY which reduce macroH2A1.1 expression have been associated with several cancer types and have even been shown to predict relapse after treatment. MacroH2A has also been implicated in an important tumor suppressive mechanism, cellular senescence, making the link between this histone variant and cancer even more compelling. As suggested by these observations, our broad hypothesis is that macroH2A1.1 has functions in regulating gene expression that are distinct from other macroH2A variants ultimately playing a role in tumor suppression. In order to test this hypothesis and to elucidate the distinct roles of each macroH2A variant, three specific aims are proposed. The first aim is to determine the role of macroH2A variants on gene expression in cancer and cellular senescence. This goal will be achieved by using genome-wide approaches to determine the pattern of genomic localization for each of the histone variants. In addition, we will perform expression analysis under conditions where we modulate the expression of macroH2A variants. The second aim is to determine the mechanisms that regulate macroH2A1 splicing in normal and cancer cells. To achieve this goal we will use both bioinformatics and a "minigene" splicing assay to determine both the cis- and trans-acting elements that contribute to the regulation of H2AFY alternative splicing. The third aim is to determine the role of PARP activity in macroH2A1.1-specific target gene expression. PARP-1 interacts with macroH2A1.1 through PAR binding, suggesting that macroH2A1.1 and PARP-1 may functionally collaborate to regulate gene expression. This hypothesis will be tested using both pharmacological inhibition and RNAi to modulate the level of PARP-1 activity, and assess the effect on factor recruitment to and transcriptional activity of macroH2A1.1 target genes. PARP-1 is a current therapeutic target in cancer treatment, highlighting the need to understand the functional connection between these two cancer-relevant molecules.