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macroH 2A组蛋白变体在癌症和衰老的转录调控中的作用。MacroH 2A是在基因组的异染色质区域中发现的组蛋白变体，其通常与基因阻遏相关。然而，macroH 2A一般不发挥致病作用，在异染色质相关的转录抑制。相反，macroH 2A需要从异染色质环境中表达其靶标的子集。人类基因组编码三种macroH 2A变体。macroH2A1.1和macroH2A1.2是H2 AFY基因的选择性剪接产物，而macroH 2A 2由H2 AFY 2编码。这些变体在功能上是不同的;而macroH2A1.1与信号分子相互作用，如利用酶PARP-1由NAD+产生的聚（ADP-核糖）（PAR），macroH2A1.2和macroH 2A 2不能。最近，减少macroH2A1.1表达的H2 AFY选择性剪接的变化与几种癌症类型相关，甚至显示出可预测治疗后的复发。MacroH 2A还涉及一种重要的肿瘤抑制机制，即细胞衰老，这使得这种组蛋白变体与癌症之间的联系更加引人注目。正如这些观察所表明的，我们的广泛假设是macroH2A1.1在调节基因表达方面具有功能，这些功能与最终在肿瘤抑制中发挥作用的其他macroH 2A变体不同。 为了检验这一假设，并阐明每个macroH 2A变体的不同作用，提出了三个具体的目标。第一个目的是确定macroH 2A变体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macroH 2A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和癌细胞中macroH 2A 1剪接的机制。为了实现这一目标，我们将使用生物信息学和“小基因”剪接测定，以确定顺式和反式作用元件，有助于调节H2 AFY选择性剪接。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，表明macroH2A1.1和PARP-1可能在功能上协同调节基因表达。将使用药理学抑制和RNAi来检测这一假设，以调节PARP-1活性水平，并评估对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，强调了了解这两种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.