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大分子组蛋白变异体在癌症和衰老的转录调控中所起的作用。MacroH2A是一种组蛋白变异体，发现于基因组的异染色区，通常与基因抑制有关。然而，在异染色质相关的转录抑制中，宏H2A通常并不起到致病作用。相反，需要在异色环境中表达其靶标的子集。人类基因组编码三种大分子H_2A变种。宏H_2A1.1和宏H_2A1.2是H_2AFY基因的选择性剪接产物，而H_2AFY2编码的是宏H_2A2。这些变体在功能上是不同的；而宏H_2A1.1与信号分子相互作用，如由NAD+利用PARP-1酶产生的聚(ADP-核糖)(PAR)，而宏H_2A1.2和宏H_2A2则不能。最近，H_2AFY选择性剪接的变化降低了宏观H_2A1.1的表达，这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还与一种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变得更加引人注目。这些观察表明，我们的广泛假设是，宏H_2A1.1具有调控基因表达的功能，不同于最终在肿瘤抑制中发挥作用的其他宏H_2A变异体。为了验证这一假说并阐明每个大分子H2A变异体的不同作用，提出了三个特定的目标。第一个目标是确定大分子H_2A变异在癌症和细胞衰老中对基因表达的作用。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，我们将在调控大分子H2A变异体表达的条件下进行表达分析。第二个目标是确定调节正常细胞和癌细胞中大分子H_2A1剪接的机制。为了实现这一目标，我们将使用生物信息学和“微型基因”剪接试验来确定有助于调节H2AFY选择性剪接的顺式和反式作用元件。第三个目的是确定PARP活性在宏H_2A1.1特异性靶基因表达中的作用。PARP-1通过PAR结合与宏H_2A1.1相互作用，提示宏H_2A1.1和PARP-1可能在功能上协同调节基因表达。这一假说将通过药理抑制和RNAi来调节PARP-1的活性水平，并评估对因子募集和宏H2A1.1靶基因转录活性的影响。PARP-1是目前癌症治疗的治疗靶点，强调了了解这两个与癌症相关的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.