NAD+ Analog-sensitive PARPs Reveal a Role for PARP-1 in Transcription Elongation. NAD+ Analog-sensitive PARPs Reveal a Role for PARP-1 in Transcription Elongation

The PARP family of proteins comprises 17 members, about two thirds of which are active mono- or poly(ADP-ribosyl)transferase enzymes that transfer the ADP-ribose moiety of NAD+ onto target proteins. In many cases, ADP-ribosylation, which plays critical roles in human diseases (e.g., cancer, heart disease, and neuropathies) is associated with abrogation of the molecular functions of the target. Discerning ADP-ribosylation events mediated by a specific PARP is challenging, since all PARPs use the same substrate (i.e., NAD+) and the available inhibitors lack the specificity needed to make such conclusions. In order to identify the direct and specific targets of individual PARP family members, we have developed a chemical and genetic approach known as analog sensitivity, in which alteration of a single conserved amino acid in the active site of the PARP protein creates a pocket that allows use of an unnatural NAD+ analog containing a steric moiety. We have functionalized the steric moiety with alkyne for use in click chemistry reactions. This approach, which is transferable to other PARP family members, creates substrate specificity where none previously existed, allowing PARP-specific post-translational modification followed by target visualization, or isolation of ADP-ribosylated targets using click chemistry techniques. We have used this technology in conjunction with mass spectrometry to identify hundreds of targets both unique to, as well as shared among, the nuclear PARP proteins, PARP-1, PARP-2, and PARP-3. We have also determined the genome-wide distribution of PARP-1-specific ADP-ribosylation by coupling this analog-sensitive PARP approach with chromatin cross-linking in method that we call “Click-ChIP-seq”. We observed that PARP-1-specific ADP-ribosylation is enriched at transcriptionally active promoters in proximity to sites of PARP-1 enrichment. In addition, we observed that NELF, an important regulator of RNA Polymerase II (Pol II) pausing, is not only a target of ADP-ribosylation by PARP-1 but also spatially correlated with chromatin-associated ADP-ribose and PARP-1 in Click-ChIP-seq and ChIP-seq assays, respectively. Given these observations, we hypothesized that ADP-ribosylation might modulate NELF function and result altered Pol II pausing. We have explored this possibility using global run-on coupled with deep sequencing (GRO-seq) in MCF-7 cells in which PARP-1 was depleted by RNAi-mediated knockdown. PARP-1 depletion caused an increase in Pol II pausing genome-wide. Taken together, these results suggest the intriguing possibility that ADP-ribosylation of NELF by PARP-1 may be an important and heretofore unknown step in the release of Pol II into productive elongation. Overall design: Using ChIP-seq (knockdown of Luciferase [control] with or without CDK inhibitor [DRB] treatment, knockdown of PARP-1, knockdown of NELF-E, and re-expression of GFP [control], wild-type NELF-E, mutant NELF-E, or viral NELF inhibitor HDAg-S) in MCF-7 human breast cancer cells and using ChIP-seq (PARP-1 and click-ChIP-seq) in mouse embryonic fibroblasts (MEFs)

聚ADP核糖聚合酶（PARP）家族包含17个成员，其中约三分之二为具有活性的单ADP核糖基转移酶或多ADP核糖基转移酶，可将烟酰胺腺嘌呤二核苷酸（NAD+）的ADP核糖基团转移至靶蛋白上。ADP核糖基化（ADP-ribosylation）在多数情况下与靶蛋白分子功能的丧失相关，且该修饰在人类疾病（如癌症、心脏病及神经病变）中发挥关键作用。由于所有PARP均使用相同的底物（即烟酰胺腺嘌呤二核苷酸，NAD+），且现有抑制剂缺乏实现此类研究所需的特异性，因此甄别特定PARP介导的ADP核糖基化事件极具挑战性。
为鉴定单个PARP家族成员的直接特异性靶标，我们开发了一种名为“类似物敏感性”的化学与遗传学方法：通过改变PARP蛋白活性位点内单个保守氨基酸，可形成一个口袋结构，从而能够使用带有空间位阻基团的非天然NAD+类似物。我们已将炔基修饰至该空间位阻基团上，以用于点击化学反应（click chemistry）。该方法可推广至其他PARP家族成员，能够赋予原本不存在的底物特异性，从而实现PARP特异性的翻译后修饰，后续可通过点击化学技术完成靶标的可视化成像或ADP核糖基化靶标的分离纯化。
我们将该技术与质谱（mass spectrometry）联用，已鉴定出核内PARP蛋白（PARP-1、PARP-2及PARP-3）特有的以及共有的数百个靶标。我们还通过将该类似物敏感性PARP方法与染色质交联相结合，建立了名为“Click-ChIP-seq”的技术，以此确定了PARP-1特异性ADP核糖基化的全基因组分布情况。我们发现，PARP-1特异性ADP核糖基化富集于转录活跃的启动子区域，且紧邻PARP-1富集位点。
此外我们观察到，作为RNA聚合酶II（Pol II）暂停的重要调控因子，NELF不仅是PARP-1介导ADP核糖基化的靶标，在Click-ChIP-seq与染色质免疫共沉淀测序（ChIP-seq）实验中，其分别与染色质结合的ADP核糖及PARP-1存在空间相关性。基于上述观察结果，我们提出假设：ADP核糖基化可能调控NELF的功能，进而改变Pol II的暂停状态。
我们通过RNA干扰（RNAi）介导的敲低技术在MCF-7细胞中沉默PARP-1，并结合全局转录延伸测序（GRO-seq）对此假设进行了验证。实验结果显示，PARP-1沉默会导致全基因组范围内Pol II暂停现象增加。综合以上结果，我们提出了一个颇具意义的可能性：PARP-1介导的NELF ADP核糖基化，可能是Pol II释放进入有效延伸过程中一个此前未被发现的关键步骤。
整体实验设计：在人乳腺癌MCF-7细胞中开展ChIP-seq实验，处理组包括：荧光素酶（Luciferase）敲低对照组（是否添加CDK抑制剂DRB处理）、PARP-1敲低组、NELF-E敲低组，以及绿色荧光蛋白（GFP）对照、野生型NELF-E、突变型NELF-E或病毒来源NELF抑制剂HDAg-S的重表达组；同时在小鼠胚胎成纤维细胞（MEFs）中开展ChIP-seq（PARP-1检测）与Click-ChIP-seq实验。