DNA损伤反应诱导炎症和衰老通过抑制GATA4自噬

The DNA damage response induces inflammation and senescence by

inhibiting autophagy of GATA4

DNA损伤反应通过抑制GATA4自噬诱导炎症和老化

introduction：细胞衰老是一个由多重应激导致基因表达变异和扩增的过程。虽然这也是一种潜在的肿瘤抑制机制，但衰老机制也参与了一些病理过程，包括老化，年龄相关的疾病，甚至与致瘤机制有关。衰老细胞能分泌一些衰老相关分泌表型SASP影响自身的微环境，这些SASP包括前炎性细胞因子，趋化因子，生长因子和蛋白酶。SASP启动和维持的机制以转录因子NF-κB和C / EBPb为特征的经典炎症调节机制。

rational：P53和p16INK4a/ RB是衰老过程的两个重要核心调控通道。 不依赖于p53或p16INK4a的另一种独立衰老调控通路能调节SASP。根据miR-146a的启动子片段开发了一种绿色荧光蛋白标记的衰老信号，可以检测到人类成纤维细胞衰老过程中的miR-146a。 衰老诱导刺激物能激活SASP，包括复制疲惫，DNA损伤，致癌RAS激活。 RESULTS:

通过对miR-146a启动子的分析，我们定位衰老诱导活化的关键区域并确定了转录调控因子GATA4在衰老调控中的作用。正常情况下， GATA4 与P62自噬体结合发生选择性自噬而被降解。在衰老反应中，GATA4和P62反应减弱可以抑制这种自噬反应。GATA4通过诱导能激活NF-kB通路的因子启动和维持SASP，从而可以促进衰老过程，这些因子包括肿瘤坏死因子受体相关蛋白2和IL-1A。GATA4通路的活化同p53 和p16INK4a通路活化机制相似，需要DDR激酶ATM和ATR的活化。不同的是，GATA4通路是不同于p53和p16INK4a的另外的独立通路。GATA4蛋白大量存在与衰老刺激物诱导的老鼠、正常老化的老鼠及人的多个组织中，包括脑细胞，这些发现均提示GATA4通路参与年龄相关性炎症反应的过程。

CONCLUSION: 我们的结果说明GATA4通过TRAF3IP2和IL-1A激活的NF-κB通路参与自噬反应、DDR所致的衰老及炎症过程。GATA4通过DDR调控衰老是不同于P53和p16INK4a通路的独立通路的关键。我们认为衰老细胞中大量

积聚的GATA4能通过炎症反应促进老化和疾病，而抑制GATA4通路可能为疾病的治疗提供了一个新方向。细胞老化是应激反应的最终阶段，由P53和p16INK4a肿瘤抑制蛋白调控。老化的显著特征是一种与肿瘤生长和老化相关的前炎性反应，SASP。已经证实转录因子GATA4可以调控老化和SASP。正常情况下，GATA4能通过P62介导的选择性自噬作用降解，而衰老反应则可使GATA4保持恒定。 Text：

在应激状态下，细胞老化是一种防止异常细胞进一步扩增的机制。

细胞老化抑制细胞和组织的再生能力，衰老细胞的消除有助于诱导老化模型鼠的老化相关表型的表达。而如何接收衰老信号启动老化反应的具体调节机制并不清楚。多种衰老刺激信号能引起哺乳动物细胞进入不可逆的生长阻滞期，这些信号包括复发扩散导致的端粒的缩短、DNA损伤以及活化癌基因的表达。 衰老细胞除了能引起细胞生长阻滞外，在基因表达中也有很大的作用，包括SASP的表达。在生理状态下，SASP因子可以通过自分泌和旁分泌的方式加强衰老细胞的生长阻滞，老化细胞能刺激癌前细胞或癌变细胞扩增形成肿瘤。

另一方面，SASP激活免疫系统可以抑制肿瘤，而当衰老细胞在发挥作用被移除后可以促进受损组织的修复。SASP可以直接或间接地促进与一些年龄相关性疾病的慢性炎性因子的分泌。尽管SASP由很广泛的生物学活性，但是对于SASP产生的NF-kB经典途径以外的作用却是有限的，比如CCAAT/ 增强子结合蛋白 b (C/EBPb)，IL-1a和P38MAPK。在老化刺激下NF-kB炎性反应被激活的机制亦是未知的。与老化生长停滞相比，P53和p16INK4a/Rb肿瘤抑制途径有重要的作用，SASP并不依赖于P53和p16INK4a/Rb肿瘤抑制途起作用，而是有自己独立的衰老调控通路。与急剧的正常炎性反应相比，SASP反应很缓慢，到老化细胞生长阻滞通常需要几天的时间，这将提供一个衰老特异性的激活机制。 除了转录调控外，自噬通过target of rapamycin(TOR) autophagy spatial coupling compartment(TASCC)影响SASP的形成。TASCC通过mTOR与自噬溶酶体氨基酸残基结合而起到促进分泌蛋白质合成的作用。也有研究表明抑制自噬在某些条件下能促进老化。因此，自噬和老化之间的关系暂时还不清楚。

GATA4, a novel senescence regulator

我们分析了microRNA在非老化细胞和衰老人成纤维细胞的表达，通过复制疲惫诱导老化并发现miR-146a在老化细胞中高度表达。我们猜测miR-146a调控主要发生在转录水平的初始阶段。我们将1.5-kb miR-146a启动子片段融合如绿色荧光蛋白中PmiR-146a-GFP。miR-146a在一些老化诱导因子作用下表达增加，包括复制疲惫、电离辐射及致癌基因RAS 的表达。通过ECB浏览器，我们发现miR-146a有两个高度进化的保守域，ECR1和ECRU2。ECR2对基因活性有重要的作用，因为ECR2有NF-κB结合部位，而NF-κB能调控miR-146a，我们从药理学和遗传学角度抑制了完全表达SASP的衰老细胞的NF-κB通路。而衰老细胞中只有部分NF-κB被抑制，这个结果表明其它的转录因子也作用于激活miR-146a关联的细胞老化。为了确定其它转录因子的活性，我们搜查了那些被预测能结合ECR2的转录因子。我们逐一将搜查13个转录因子过度表达并检测miR-146a的活性，只有GATA4符合条件。在衰老细胞中，siRNA GATA4被耗尽。ChIP-qPCR显示GATA4表达与miR-146a ECR2直接连接。

GATA4是一种锌指转录因子，在各种器官的发育中是必不可少的，包括心脏，睾丸，前肠，肝，和腹侧胰腺。为了检测GATA4的功能，我们研究了异位表达或缺乏在人二倍体成纤维细胞老化反应中的影响。GATA4异位表达诱导人包皮成纤维细胞和IMR-90成纤维细胞的老化，通过增加SA-b-Gal的活性及减少BrdU的合成。更重要的是，通过稳定表达shRNA导致GATA4耗尽能部分下调IR诱导的SA-B-Gal的活性和延迟复制性衰老。这是由于 GATA4发生CRISPR突变。这些数据表明GATA4对老化有正性调节作用。在衰老中GATA4有调控作用，而在肺，结肠癌，前列腺癌，卵巢癌和乳腺癌中，则保持沉默。

Selective autophagy suppresses GATA4 and senescence

在检测GATA4 siRNA的效率时，我们发现GATA4的量在衰老细胞中增加。事实上，丰富的GATA4蛋白，而不是mRNA，能增加IR-和癌基因诱导的衰老和复制老化。这是由于蛋白质稳定性提高，通过放线菌酮和全球蛋白质稳定性分析检测蛋白质的稳定性。在真核细胞中有两个主要蛋白质降解途径：泛素蛋白酶体和自噬溶酶体途径。用MG-132抑制蛋白酶体，MG-132是一种蛋白酶体抑制剂，

对GATA4没有影响，而GATA4蛋白在溶酶体抑制剂（溶酶素A、E64d、胃蛋白酶抑制剂）存在的细胞中稳定表达。这些发现提示自噬溶酶体途径能调控GATA4。自噬部件ATG5或ATG7同样能上调GATA4蛋白。自噬可以通过特异性自噬受体介导途径选择性地降低某些基物。事实上，自噬受体P62的耗尽能增加GATA4蛋白。衰老过程中外源性表达和内源性GATA4与p62相互作用减弱。因此，在正常条件下，GATA4与P62发生特异性作用发生自噬，而在老化中，可能与P62的相互作用减弱，GATA4变得稳定了。衰老与自噬之间的关系目前还不清楚。自噬在老化或抑制老化过程中是必不可少的。我们结果可以解释这些矛盾。选择性自噬可能会通过抑制衰老调控因子如GATA4来防止老化。然而，衰老诱导刺激物可以引起GATA4逃脱选择性自噬。随后，非选择性自噬可能激活有助于老化。因此，GATA4的选择性自噬可能对老化的起到负性调节作用。如果是这样，瞬时抑制自噬可引起衰老。为了验证这一点，我们利用多西环素（阿霉素）诱导的shRNA耗尽ATG5或ATG7和瞬时抑制自噬，使GATA4增加，然后通过去除多西环素恢复自噬，使细胞达到一个可老化的状态。短暂抑制自噬（高浓度GATA4，自噬on）诱导的衰老比持续性抑制自噬（高浓度GATA4，自噬off）更有效，这种效果，至少在一部分取决于GATA4。连续和短暂抑制自噬使获得同样的GATA4浓度，但连续抑制未能诱导衰老。在正常状态下，P62缺乏比自噬调节受体ATG7或ATG5的缺乏更有效率的诱导老化。因此，选择性自噬可以成为GATA4的抗衰老机制，而非选择性自噬则是促衰老机制。

GATA4 regulates the SASP

为了确定GATA4调节老化的机制，我们探究了GATA4是如何影响人成纤维细胞基因表达的。GATA4异位表达时可以诱导老化，我们利用阿霉素诱导GATA4载体在GATA4表达之前和之后对RNA转录普进行测序。我们根据基因本体论系统描述了GATA4是如何影响细胞进程的。基因表达GATA4的增加对一些应答是有意义的，如免疫应答、炎症反应、创伤反应，而GATA4的表达减少则与细胞周期的生理过程有关。我们比较了GATA4基因集和与增殖老化基因集的区别，无论基因是上调还是下调均有意义，而基因的上调更具统计学意义，说明GATA4有转录激活因子的作用。结果说明GATA4可能激活一部分老化相关基因。

在GATA4调节的老化相关基因中我们发现了很多SAPA基因，这些基因能编码IL6, IL8, CXCL1, 粒-巨细胞集落刺激因子 (GM-CSF), 细胞外基质蛋白激酶和抑制剂。炎症应答和免疫应答中的细胞因子和趋化因子由老化细胞分泌，可以改变细胞内微环境，增强老化阻滞，GATA4通过SASP可能直接调节其他的老化表型，尤其是生长阻滞。GATA4的异位表达通过Rt-qPCR可以诱导SASP相关的基因表达。更为重要的是，在建立老化过程中，GATA4的过度消耗可以抑制很多SASP基因的表达，说明GATA4控制了很多SASP基因而GATA家族的另一成员GATA3并不能增加SASP相关基因的表达，即使预测说它是一种很强的肿瘤抑制因子。同样的，GATA3的表达不能增加肿瘤坏死因子相关受体蛋白2 TRAF3IP2的表达。

GATA4 regulates NF-kB

NF-kB在调控SASP上有至关重要的作用，但对于NF-kB是如何激活衰老过程的人们知道得很少。 为了检测GATA4与NF-κB在调节SASP的关系，我们测试了当NF-kB的重要成分RELA受到抑制时，是如何影响GATA4诱导SASP。 RELA耗尽能够抑制GATA4调节的SASP相关基因表达。GATA4的表达触发NF-kB的激活，而GATA4缺乏在老化过程中抑制NF-KB的活化; 这些研究结果表明，GATA4在调节SASP过程中作用于NF-κB的上游。为了了解GATA4是如何激活NF-κB的，我们收索了在基因组芯片实验中发现的约束GATA4的启动子，据此发现并检验与激活NF-kB具有同样功能的基因是如何被GATA4调控的。GATA4减少TRAF3IP2的表达，一种TRAF6的泛素连接酶和TRAF3IP2的缺乏能部分阻滞GATA4激活NF-kB，可以通过SASP基因的表达进行评估。TRAF3IP2异位表达能部分缓解在IR诱导老化细胞中由于GATA4缺乏导致的SASP减少。SASP因子能抵抗TRAF3IP2消耗，如IL6 和CXCL3，结果表明GATA4的下游基因通过TRAF3IP2通路对SASP起到调控作用。RELA消耗能完全阻滞GATA4调控的SASP的活化，除了IL1A外。IL1A在SASP调控中，对NF-KB有正反馈调控作用。因此，SASP在GATA4依赖的途径中，IL1A可能与TRAF3IP2有协同作用。通过评估SASP基因的表达，IL1A缺乏能减少GATA活化的NF-KB途径。此外，将SASP相关的活化基因转移到缺乏GATA4反应的细胞中，由

GATA4诱导的细胞通过条件培养液获得。因此，GATA4通过TRAF3IP2 and IL1A激活NF-KB途径作用于SASP。TRAF3IP2缺失也部分阻滞GATA4诱导的衰老，SA-B-Gal的活性。不同于GATA4的表达，TRAF3IP2表达不足以诱导的SA-B-Gal的活化，却能激活SASP相关的基因表达。此外，TRAF3IP2异位表达与IL1A一起仍然不足以诱导SA-B-Gal活化，却能激活GATA4。因此，该SASP可能不是GATA4调控衰老的唯一机制。事实上，此前确定的老化调控基因，如早幼粒白血病蛋白（PML）和YPEL3都属于GATA4诱导的基因。

GATA4, a new branch of the senescence regulatory pathway

为了更全面地了解GATA4调控衰老的机制，我们研究了两个核心衰老的调节通路中，p53和p16INK4a通路。GATA4对老化的影响是独立于 p53和p16INK4a/ Rb途径的。GATA4激活产生能诱导老化的IR剂量，能保持完整的细胞中缺乏的p53或p53基因和Rb。此外，没有活化的P53和p16INK4a中，GATA4同样被激活。因此，GATA4是独立于 p53-和p16INK4a的途径。DNA损伤应答（DDR）作用于该途径的上游。事实上，抑制DDR调控因子ATM和ATR能抑制老化的GATA4途径。因此，GATA4途径诱导的SASP似乎是在DDR的一个独立分支。DDR是如何抑制自噬基础上GATA4降解仍然是今后研究的核心问题。

GATA4’s role in senescence in vivo

为了测试GATA4活性和调控作用在体内同样发生，我们检测了存在氧生理条件下小鼠胚胎成纤维细胞通过IR诱导的老化的GATA4表达。Gata4增加能引起衰老诱导的IR增加，需要几个GATA4靶基因的作用，包括那些编码Traf3ip2和SASP因子的基因，如IL6，IL1A，和CXCL1因子。因此，GATA4调节老化表型的途径也能存在与小鼠中。为了确定GATA4在小鼠体内对老化应答是否有作用，我们检测了多种组织均老化的辐射鼠的GATA4水平。在皮肤可肝脏中，GATA4增加引起老化诱导的IR增加，因此，GATA4在体内积聚能通过DNA损伤诱导老化。衰老细胞随着小鼠和人年龄的增长而堆积。我们研究了年轻和老的小鼠（分别是6月龄和22月）并发现年龄大的小鼠的肾脏和肝脏中GATA4积聚增加：这种积累与p16INK4a基因相关。而且，老化小鼠肝脏表明NF-KB通过

RELAX磷酸化被活化，这个结果与我们培养的细胞一致。我们也研究了人脑中的GATA4，老年人的额叶皮质中，GATA4和p16INK4a均增加。为了证实这些结果，我们通过免疫荧光微镜研究了年轻人和老年人额叶皮质GATA4和p16INK4a基因之间的空间关系。GATA4和p16INK4a在老年人大脑中的表达更多。此外，我们在少突胶质细胞、椎体神经元、星形细胞中发现了GATA4和p16INK4a的一个显著空间相关性，进一步支持GATA4在人衰老过程中的老化作用。 因此，在鼠和人衰老过程中，GATA4可能促进细胞衰老和炎症反应。我们的研究结果表明，自噬对衰老有正性和负性调节作用。In response to IR，这些衰老表型依赖于DDR信号激酶ATM和ATR，和衰老相关基因p53和p16INK4a的表达类似。然而，GATA4途径是独立于p53和p16INK4a通路的，从而建立了一个新的DDR途径。 GATA4调节细胞衰老一部分是通过影响SASP起作用的。同样，GATA4可以作为TRAF3IP2 and IL1A 介导的NF-κB的上游调节因子启动和维持NF-κB活性的调节因子。GATA4也激活的miR-146a的表达，从而降低NF-KB的活性。 GATA4-MIR-146A-NF-kB通路形成了一个无规律的正反馈途径，在SASP基因表达风暴形成之后，能抑制炎症反应。 然而，这并不终止炎症反应，尽管在高水平的miR-146A存在下，衰老细胞也能维持SASP的水平。miR-146a可能防止意外激活该途径以至于GATA4或其它因子在正常状态下随机波动。 总之，我们的结果表明，GATA4是一衰老表型的关键条件因子，并通过IL1A和TRAF3IP2激活NF-κB途径参与自噬和DDR所致的老化过程。细胞老化被证明是把双刃剑。这个途径可以促进正常的发育和伤口的愈合。然而，衰老细胞的积累可作为识别生物体年龄，能促进老化相关的表型与衰老相关的疾病，包括癌症和神经退行性变。因此，了解潜在的对老化的控制途径对人体健康有重要意义。调节GATA4途径可能为治疗年龄相关性疾病提供了一个新思路。

GATA4, a novel senescence regulator

In an effort to develop new markers for senescence, we analyzed microRNA expression in non senescent and senescent human fibroblasts (strain IMR-90 from fetal lung). We induced senescence by replicative exhaustion and found miR-146a to be highly expressed by senescent but not non senescent cells (fig.S1A),a result also reported for human foreskin fibroblasts (HCA2)(34). Given its abundant expression,

we suspected that miR-146a regulation occurs primarily at the level of transcription. We therefore generated a 1.5-kb miR-146a promoter fragment fused to green fluorescent protein (PmiR-146a-GFP). Expression of this reporter construct was significantly increased in response to several senescence-inducing stimuli, including replicative exhaustion, ionizing radiation (IR; 12 Gy), and expression of oncogenic RAS (fig. S1B). Using the Evolutionarily Conserved Regions browser (35), we found that the miR-146a promoter contains two evolutionarily conserved regions, ECR1 and ECR2. ECR2 was critical for reporter activity (fig. S1C). Because ECR2 has a putative NF-kB binding site and NF-kB is known to regulate miR-146a (36), we inhibited NF-kB either pharmacologically (with an inhibitor of IkB kinase, Bay11-7082) or genetically [with short interfering RNAs (siRNAs) targeting the NF-kB family member RELA(p65)] in fully senescent SASP-expressing cells. NF-kB inhibition only partially decreased reporter activity in senescent cells (fig. S1D). These results indicate that other transcription factors also contribute to senescence dependent activation of the miR-146a reporter. To identify additional transcription factors responsible for the reporter activity, we searched for ones that were predicted to bind to ECR2(see Materials and Methods). We individually overexpressed each candidate and examined reporter activity. Of 13 transcription factors selected, only GATA4, but not other GATA family members, activated the reporter (Fig. 1A and fig. S2, A and B). Depletion of GATA4 with siRNAs showed that it was required for reporter activation during senescence (Fig. 1B). Chromatin immunoprecipitation combined with quantitative polymerase chainreaction(ChIP-qPCR) revealed that exogenously expressed GATA4 bound directly the miR-146a ECR2 region(fig.S2C). These results indicate that GATA4 contributes to activation of the miR-146a promoter in senescent cells. GATA4 is a zinc finger transcription factor essential for the development of various organs, including heart, testis, foregut, liver, and ventral pancreas (37). To examine whether GATA4 functions in senescence, we examined the effects of ectopic expression or depletion during the senescence response of human diploid fibroblasts. Ectopic expression of GATA4 induced senescence in human foreskin fibroblasts (strain BJ;Fig.1C)and IMR-90 fibroblasts(fig.S3,Aand B), as

shown by increased senescence-associated b-galactosidase (SA-b-Gal) activity and decreased 5-bromo-2’-deoxyuridine (BrdU) in corporation. More important, depletion of GATA4 with stably expressed short hair pinRNAs (shRNAs) partially decreased IR-induced SA-b-Gal activity (Fig. 1D and fig. S3C) and delayed replicative senescence (Fig. 1E). We confirmed these results by CRISPR mutation of GATA4 (fig. S3D). These data indicate that GATA4 is a positive regulator of senescence. Consistent with a regulatory role in senescence, GATA4 is frequently silenced in lung, colon,prostate,ovarian,and breast cancer (38,39). Selective autophagy suppresses GATA4 and senescence While examining the efficiency of GATA4 siRNAs (Fig. 1B), we noticed that the amount of GATA4 increased in senescent cells. Indeed, abundance of the GATA4 protein, but not mRNA, increased during IR- and oncogene-induced senescence and replicative senescence (Fig. 2A). This increase was primarily due to increased protein stability, as indicated by the measurement of protein stability in the presence of cycloheximide (Fig. 2B) and by global protein stability profiling (40) (fig. S4A). Two major pathways in eukaryotic cells mediate protein degradation: the ubiquitin-proteasome and autophagy-lysosome pathways.Inhibition of the proteasome by MG-132, a proteasome inhibitor,had no effect on GATA4 abundance, whereas GATA4 protein was stabilized in cells treated with distinct lysosomal inhibitors known to block autophagy: bafilomycin A1 (a selective inhibitor of the vacuolar-type H+–adenosine triphosphatase) or E64dandpepstatin(lysosomalproteaseinhib-itors)(Fig.2,CandD).Thesefindings suggest the

autophagy-lysosome

pathway,

not

the

ubiquitin

proteasome

pathway,regulatesGATA4.Consistent with this interpretation, depletion of the autophagy components ATG5 or ATG7 also increased the abundance of GATA4 protein (Fig. 2E). Autophagy can selectively degrade certain substrates, mediated by specific autophagic adaptors (41–43). Indeed, depletion of the autophagic adaptor p62 increased the abundance of GATA4 protein(Fig.2F). Exogenously expressed and endogenous GATA4 physically interacted with p62, and the interaction was reduced during senescence (Fig. 2G and fig. S4B). Thus, GATA4 appears to be targeted for autophagic degradation through its association with p62 under normal conditions but

becomes stabilized during senescence, possibly through reduced association with p62. The relationship between senescence and autophagy is unclear. Autophagy is reported to be required for the establishment of senescence (31, 32, 44) or to inhibit senescence (33). Our results can reconcile these conflicting reports. Selective autophagy might prevent senescence by suppressing positive senescence regulators such as GATA4. However, senescence-inducing stimuli cause GATA4 to escape selective autophagy. Subsequently, general autophagy may be activated, which then contributes to establishment of senescence. Hence, selective autophagy for GATA4 may be a negative senescence regulator, whereas general autophagy is a positive regulator of senescence. If so, transiently inhibiting autophagy should induce senescence. To test this, we used doxycycline(Dox)–inducible shRNAs to deplete ATG5 or ATG7 and transiently inhibit autophagy (45), allowing GATA4 to accumulate, and then restored autophagy by removing Dox, returning the cell to a senescence permissive state. Transient inhibition of autophagy (GATA4 abundance high, autophagy on) induced senescence more effectively than did continuous inhibition (GATA4 abundance high, autophagy off); this effect, at least in part, depended on GATA4 (Fig. 2, H and I, and fig.S4C). Continuous and transient inhibition of autophagy increased GATA4 abundance to similar extents, but continuous inhibition failed to induce senescence (Fig. 2I). Consistent with this finding, p62 depletion induced senescence even more effectively than did depletion of the core autophagy regulators ATG7 or ATG5 under normal conditions(fig.S4D). Thus,selective autophagy for GATA4 appears to function as an antisenescence mechanism, whereas general autophagy functions as a pro-senescence mechanism. GATA4 regulates the SASP

To investigate the mechanisms through which GATA4 regulates senescence, we explored how GATA4 influences gene expression in human fibroblasts. Because GATA4 induces senescence when ectopically expressed, we performed transcriptional profiling with RNA sequencing(RNA-seq) before and after GATA4 expression using a Dox inducible GATA4 vector. We used Gene Ontology(GO) analysis to systematically characterize the cellular processes affected by GATA4 (46). Genes showing increased

expression in response to GATA4 showed significant enrichment for the

terms ―immune response,‖ ―inflammatory response,‖ and ―response to wounding,‖ whereas genes with decreased expression were mostly enriched for biological processes related to the cell cycle, which correlated well with terms previously linked to senescence(Fig.3AandtableS1). We compared the GATA4-regulated gene set (GATA4-regulated set) with a gene set differentially regulated during replicative senescence(Senescent set). Both up-regulated and down-regulated genes overlapped significantly, with greater statistical significance for the up-regulated genes (P =2.46 × 10?40), consistent with the fact that GATA4 acts mostly as a transcriptional activator (Fig. 3B). These results suggest that GATA4 might activate a major portion of senescence-associated genes. Among the GATA4-regulated, senescence-associated genes, we found several SASP genes, including those encoding IL6, IL8, C-X-C motif ligand 1 (CXCL1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and extracellular matrix(ECM) proteases and inhibitors(7).Because inflammatory and immune-modulatory cytokines and chemokines secreted by senescent cells can reinforce senescence arrest and alter the microenvironment (1, 2, 10), GATA4 might indirectly regulate other senescent phenotypes, notably growth arrest, through the SASP. We confirmed that ectopic expression of GATA4 induces the expression of genes associated with the SASP by reverse transcription qPCR (RT-qPCR) (Fig. 3C). More important, depletion of GATA4 suppressed the expression of several SASP genes during the establishment of senescence (Fig.3D), indicating that GATA4 indeed controls many SASP genes. Ectopic expression of GATA3—another GATAfamily member predicted to be a strong tumor suppressor(47,48) —did not increase expression of genes associated with the SASP. Likewise, ectopic expression of GATA3 did not increase expression of TRAF3IP2 [tumor necrosis factor receptor associated factor(TRAF)interactingprotein2],a key GATA4 downstream target (see below), although hit is functionally active, as shown by its ability to activate its well-known target IL13 (fig. S5A). These results support a specific role for GATA4 in SASP regulation. However, we cannot rule out the possibility that other GATA factors including GATA3 may have a similar role in other cell types.

GATA4 regulates NF-kB

NF-kB has a crucial role in controlling the SASP (18, 19, 49) (Fig. 3D), yet little is known about how NF-kB is activated during senescence. To examine the relationship between GATA4 and NF-kB in regulating the SASP, we tested how suppression of the essential NF-kB component RELA affected the GATA4-induced SASP. RELA depletion inhibited the expression of genes associated with the SASP in response to GATA4(Fig. 4A). GATA4 expression triggered NF-kB activation, and GATA4 depletion inhibited NF-kB activation during senescence (Fig. 4B); these findings suggest that GATA4 acts upstream of NF-kB in regulating the SASP. To understand how GATA4 activates NF-kB, we searched promoters bound by GATA4 in genome wide ChIP experiments (50) to find associated genes that function as NF-kB activators, andexamined their regulation by GATA4. GATA4 induced the expression of TRAF3IP2,an E3 ubiquitin ligase for TRAF6 (51) (Fig.4C), and TRAF3IP2 depletion partially blocked GATA4 activation of NF-kB, as assessed by the expression of SASP genes(Fig.4D). Furthermore, ectopic expression of TRAF3IP2 partially rescued the reduced SASP caused by GATA4 depletion in IR-induced senescent cells (Fig. 4E). Note that certain SASP factors, such as IL6 and CXCL3,were resistant toTRAF3IP2 depletion; this result suggests that GATA4 regulates them in a TRAF3IP2-independent manner, either directly or indirectly through its other downstream targets. RELA depletion almost completely blocked the activation of SASP genes by GATA4, with the exception of IL1A (Fig. 4A). IL1A, a SASP component, is thought to form a feed forward activation loop with NF-kB in SASP regulation (13, 52). Thus, IL1A might cooperate with TRAF3IP2 during GATA4-dependent production of the SASP. Consistent with this idea, depletion of IL1A reduced GATA4 activation of NF-kB, as assessed by the expression of SASP genes (fig. S5B). Furthermore, activation of SASP-associated genes was transmitted to cells lacking GATA4 induction by conditioned medium from GATA4-induced cells (fig.S5C). Thus, GATA4 appears to act, at least in part, through TRAF3IP2 and IL1A to activate NF-kB in activating the SASP. TRAF3IP2 depletion also partially blocked GATA4-induced senescence, as determined from SA-b-Gal activity (Fig. 4F). Unlike

GATA4 expression, however, TRAF3IP2 expression alone was not sufficient to induce SA-b-Gal activity (fig. S6A), despite activating expression of a number of SASP associated genes (fig. S6C). Moreover, ectopic expression of TRAF3IP2 together with IL1A was still not sufficient to induce SA-b-Gal activity, in contrast to that of GATA4 (fig. S6B). Thus, the SASP may not be the only mechanism through which GATA4 regulates senescence. Indeed, previously identified regulators of senescence, such as promyelocytic leukemia protein (PML) and yippee like 3 (YPEL3), are among GATA4-induced genes(fig. S6D) (53).

GATA4 also activates expression of miR-146a, which dampens the activation of NF-kB(17).This GATA4–miR-146a–NF-kB circuit forms an incoherent type 1 feedforward loop[60]

Materials and methods Cell culture, viral transduction, and senescence induction Human diploid fibroblasts (IMR90 and BJ) were obtained from the American Tissue Type Collection. Human and mouse cells were maintained in 3% O2 and cultured in Dulbecco’s modified Eagle’s medium supplemented with 15% fetal bovine serum, penicillin-streptomycin, 2 mM glutamine, and 0.1 mM nonessential amino acids (Invitrogen). Retroviral and lentiviral gene delivery was performed as described (63). Infected cells were selected using puromycin (2 mg/ml; Clontech) for 3 days, neomycin (100 mg/ml; Invitrogen) for 7 days, or blasticidin (10 mg/ml; Invitrogen) for 4 days. For co-infection, cells were sequentially selected. Senescence was induced

by replicative exhaustion, ionizing radiation (12 Gy), or retrovirally delivered oncogenic RAS.

Growth curves and SA-b-gal assays

Population doublings weremonitored using a Coulter Counter. SA-b-gal activity was determined using the Senescent Cell Staining Kit (Sigma) according to manufacturer instructions, with 4′,6-diamidino- 2-phenylindole (DAPI) staining to identify nuclei.

BrdU incorporation assays

Cells were plated on Lab-Tek II chamber slides and labeled with BrdU (100 mg/ml, BD Science) for 4 hours. Nuclei incorporating BrdUwere visualized by immunofluorescence using an antibody to BrdU (BD Science) according tomanufacturer instructions.

Gene expression profiling

For the senescent set, total RNA from proliferating (PD = 30) and senescent (PD = 70) IMR90 cells was isolated using the acidic phenol extractionmethod. RibosomalRNAwas depleted using theRibo-Minus kit (Invitrogen). Ribo-depleted RNA was then fragmented, adaptor-ligated, reverse-transcribed, PCRamplified, and sequenced on an Illumina GA2 sequencer. For the GATA4-regulated set, cells expressing Tet (Dox)–inducible GATA4were culturedwith or without doxycycline (Dox; 1 mg/ml) for 2.5 days. Three independent experiments were performed, giving a total of six samples for mRNA-seq analysis. Total RNA was isolated using the RNAeasy MiniKit (Qiagen). mRNA-seq was performed at the HarvardMedical School Biopolymer Facility. Briefly,poly(A) mRNA was isolated from total RNA and fragmented, followed by adapter ligation, cDNA synthesis, and library preparation using the Apollo 324 system (IntegenX) for Illumina sequencing. MRNAseq reads were mapped to the humangenome (NCBI v36, hg18) and gene expression profiling was analyzed using SeqMonk (Babraham Bioinformatics). In silico transcription factor binding sites analysis

The miR-146a promoter region (1.5 kb upstream from its transcriptional start site)

was analyzedby the ECR browser (ecrbrowser.dcode.org) to identify potential transcription factor binding sites according to the program’s instructions. Briefly, evolutionary conserved regions between human and opossum were selected and transcription factor binding sites were analyzed by rVista 2.0 using TRANSFAC professional V10.2 library with matrix similarity predefined as 0.8. Gene Ontology (GO) analysis

The Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 was used for GO term analysis (http://david.abcc.ncifcrf.gov). Comparison of gene expression profiles

To compare gene expression profiles between the GATA4-regulated set (GATA4-induced/noninduced, mRNA-seq) and Senescent set (senescent/proliferating, total RNA-seq), genes with a total count less than 10 under both conditions were discarded and gene counts were normalized by the total number of mapped reads. After normalization, only genes sequenced between the two sets were selected, followed by calculation of the relative change in log2 units between the two samples (GATA4- induced versus GATA4-noninduced and senescent versus proliferating) within each set. Finally, the relative change in log2 units was compared between the GATA4-regulated and Senescent sets.

Quantitative RT-qPCR

Total RNA was isolated using the RNAeasy Mini Kit (Qiagen), and cDNA was synthesized using SuperScript III (Invitrogen) according to manufacturer instructions. Quantitative RT-qPCR was performed in triplicate using the Gene Expression Assay (Applied Biosystems) on an Applied Biosystems Fast 7500 machine with GAPDH as an endogenous normalization control. Primer sequences are available upon request.

Antibodies

Antibodies used were anti-GATA4 (Santa Cruz and Abcam), anti-p53 (Calbiochem), anti-p21 (Calbiochem), anti-p62 (BD Biosciences), anti-ATG7 (Cell Signaling),

anti-ATG5 (Cell Signaling), anti-LC3B (Cell Signaling), anti-GAPDH (Santa Cruz),anti-phospho p65 (Cell Signaling), anti-p65 (Santa Cruz), anti-phospho IkB (Cell Signaling), anti-IkB (Cell Signaling), anti-TRAF3IP2 (Santa Cruz), antip16 (Santa Cruz), anti-RB (BD Biosciences), and anti-FLAG-peroxidase (Sigma).

Retroviral vectors and siRNAs

Retroviral vectors used in this study were shRNAs targeting GATA4, ATG5, and ATG7 subcloned from the GIPZ shRNA library (Open Biosystems) into either pMSCV-PM or pInducer 10 vectors (45, 63). The shRNA targeting sequences are as follows: GATA4 shRNA1, CCAACATCTCTCAAAATAA;

GATA4 shRNA2, GGAAAGAAGACGACTGCTA; mouse Gata4 shRNA, GGCCTCTATCACAAGATGA; ATG5 shRNA1, TGAAAGAAGCTGATGCTTT; ATG5

shRNA2,TGGAATATCCTGCAGAAGA;ATG5

shRNA3, shRNA1,

CCCATCTTTCCTTAACGAA;ATG7

AGCATCATCTTCGAAGTGA;ATG7 shRNA2, AAGAGAAAGCTGGT Retroviral vectors used in this study were shRNAs targeting GATA4, ATG5, and ATG7 subcloned from the GIPZ shRNA library (Open Biosystems) into either pMSCV-PM or pInducer 10 vectors (45, 63). The shRNA targeting sequences are as follows: GATA4

shRNA1,

CCAACATCTCTCAAAATAA;

mouse

GATA4 Gata4

shRNA2, shRNA,

GGAAAGAAGACGACTGCTA;

GGCCTCTATCACAAGATGA; ATG5 shRNA1, TGAAAGAAGCTGATGCTTT; ATG5

shRNA2,TGGAATATCCTGCAGAAGA;

ATG5

shRNA3, shRNA1,

CCCATCTTTCCTTAACGAA;ATG7

AGCATCATCTTCGAAGTGA;ATG7 shRNA2, AAGAGAAAGCTGGTCATCA;p53 shRNA, AATGTCAGTCTGAGTCAGGCCC;firefly luciferase (used as a control) shRNA,CCCGCCTGAAGTCTCTGATTAA. The GATA4 and p53 cDNAs were obtained from Open Biosystems and subcloned into either pInducer 20 or pMSCV vectors. cDNAs for other open reading frames (ORFs) used in this study were from the human ORFeome library V8.1 (64). siRNAs were transfected into cells at 20 nM for the individual siRNA and 50 nM for pools using Lipofectamine RNAiMAX

transfection reagents (Invitrogen) according to manufacturer instructions. The following

siRNAs

were

used:

firefly

luciferase siRNA siRNA siRNA siRNA siRNA siRNA siRNA

siRNA

#3,

siRNA,

#1, #2, #1, #2, #3, #1, #2, CAAC

CGUACGCGGAAUACUUCGAUU;GATA4 CGACUUCUCAGAAGGCAGAtt;GATA4 CGAUAUGUUUGACGACUUC;ATG5 CAUCUGAGCUACCCGGAUA;ATG5 GACAAGAAGACAUUAGUGA;ATG5 CAAUUGGUUUGCUAUUUGA;ATG7 GAUCAAAGGUUUUCACUAA;ATG7 GAAGAUAACAAUUGGUGUA;ATG7

CAACAUCCCUGGUUACAAG;NBR1 siGENOME siRNA pool, Dharmacon, MU-010522-01-0002; NDP52 siGENOME siRNA pool, Dharmacon, MU-010637- 01-0002; BNIP3L siGENOME siRNA pool, Dharmacon,MU-011815-01-0002; WDFY3 siGENOME siRNA pool, Dharmacon, MU-012924-01-0002; p62 siRNA #1,GAUCUGCGAUGGCUGCAAU;

p62

siRNA

#2,

GCAUUGAAGUUGAUAUCGA; p62 siRNA #3,GAAGUGGACCCGUCUACAG; TRAF3IP2 siRNA #1,GAGCAUGGCUUACAUACUA; RELA siRNA #1, GAUUGAGGAGAAACGUAAA;IL1A

siRNA

#1,

GAUCAUCUGUCUCUGAAUC;IL1A siRNA #2,GAAAUCCUUCUAUCAUGUA.

CRISPR targeting

A guide RNA (gRNA) targeting the sequence GCTGTGGCGCCGCAATGCGGAGG in exon 4 of human GATA4 was cloned into lentiCRISPR puro (Addgene plasmid #49535) by annealing the following oligos and ligating into the BsmBI sites: Fwd, CACCGCTGTGGCGCCGCAATGCGG;Rev,AAACCCGCATTGCGGCGCCACAGC. A control gRNA targeting the AAVS1 locus GTCCCCTCCACCCCACAGTGGGG (65) was cloned into lentiCRISPR puro via the BsmBI sites by annealing and ligating the following annealed oligos: Fwd, CACCGTCCCCTCCACCCCACAGTG;Rev, AAACCACTGTGGGGTGGAGGGGAC. GATA4 CRISPR targeting was confirmed by PCR and Sanger sequencing of targeting genomic region with following primers:

Fwd, AGCCCCGGTCAGTTCTCCTCTCAGGAGAA; Rev,

TAAGGTAGGAGGTAGAGGTCATGCTTTCC.

Chromatin immunoprecipitation (ChIP)

Chromatin immunoprecipitation experiments were performed with the ChiP-IT Express kit (Active Motif) according to manufacturer instructions. Briefly, cells expressing HA/FLAG-tagged GATA4 were fixed in 1%formaldehyde for 10min at room temperature. The cross-linking reaction was quenched with Glycine Stop-Fix Solution. The pellet was resuspended in ice-cold lysis buffer

and dounced on ice with ~10 to 15 strokes to aid in nuclei release. The nuclei were resuspended in shearing buffer and the chromatin sheared by sonication. The sheared chromatin was centrifuged for 10 min at 15,000 rpm at 4°C and supernatants incubated with Flag-M2-magnetic beads for 3 hours at 4°C. The beads were washed three times with ChIP buffer and the DNA was eluted and reverse cross-linked. The DNA was subjected to the QIAquick PCR purification kit (Qiagen) before real-time PCR.

Brain sample procurement and Westernblotting

Postmortem human brain tissue was procured from the Rush University Medical Center, University of Maryland, Duke University, Brigham and Women’s Hospital, and Massachusetts General Hospital in accordance with institutional guidelines governed by approved protocols. Frozen specimens used in this study were from the prefrontal cortical gray matter (Brodmann areas 9 and 10) and were snap-frozen and stored at –140°C. Samples included tissue from young adults without neurological abnormalities and aged subjects without a diagnosis of Alzheimer’s disease or other neurodegenerative disease and neuropathological results within the normal range for age. Tissues were homogenized using dounce homogenizer, and cellswere lysed at 4°C using RIPA-DOC buffer supplementedwith protease and phosphatase inhibitors (Complete and Phosphostop, Roche). Sonication was performed prior to centrifugation at 10,000 rpm for 10 min at 4°C. The supernatant was removed and the

protein concentration determined (BioRad protein assay). SDS sample buffer containing b-mercaptoethanol was added and 20 mg of protein was loaded per lane.

Immunohistochemistry analysis of human brain

Immunofluorescence analysis using paraffinembedded brain sections was performed in the prefrontal cortex. Paraffin-embedded tissue sections were first deparaffinized in xylene, then rehydrated with decreasing concentrations of ethanol and placed in water. Sections then underwent antigen retrieval using the Diva decloaker (BioCare, USA). They were then washed and blocked with 3% bovine serum albumin (BSA), 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 1 hour at room temperature. Primary antibodieswere diluted in 2% BSA, 0.1% Triton in PBS. The following primary antibodies were used: GATA4 (mouse, Abcam); GATA4 (rabbit, Novus); GATA4 (goat, R&D systems); p16 (rabbit, Abcam); p16 (mouse, ThermoScientific); p16 (rabbit, Bethyl);GFAP (goat, Abcam). Analysis ofGATA4 and p16 expression by immunofluorescence with antibodies obtained from these sources led to similar conclusions. After overnight incubations, sections were washed three times with PBS. Secondary antibodies, diluted in 2% BSA, 0.1% Triton in PBS, were coupled to Alexa fluorophores (1:300, Invitrogen). After washes in PBS, sections were incubated with 1% Sudan Black in 70% ethanol for 10 min to suppress lipofuscin autofluorescence. Sections were mounted using ProLong anti-fade mounting medium containing DAPI (Life Technologies) and imaged using confocal microscopy. Images were randomly acquired in the prefrontal cortex for quantification of immunofluorescence. Oligodendrocytes were distinguished from the white matter where they are the predominant cell type by their location (densely distributed in a linear pattern along axonal tracts) and small nuclei size. Pyramidal neurons were identified fromthe graymatter by their characteristic morphology (pyramid shape), size (the largest cells in the gray matter), and distinctive pattern of DAPI staining (diffuse). Astrocytes were identified by costaining with the astrocyticmarker GFAP. Nuclei were selected using the Metamorph image analysis system and the average signal intensity measured. Values were corrected by subtracting the average slide background intensity

(measured outside of cells). The investigator was blinded to sample origin or diagnosis. Aged mice

C57BL/6J mice of 6 and 22 months of age were obtained from the National Institute on Aging mouse aging colony. Mice were acclimated for at least 1 week before killing. All animal studies followed the guidelines of and were approved by institutional animal care and use committees.Protein extractswere obtained by lysis in ice-cold lysis buffer (Cell Signaling) supplemented with a cocktail of protease and phosphatase inhibitors (Roche). Protein content was determined by the BCA protein assay kit (Pierce), and 20 to 30 mg of proteins were subjected to SDS–polyacrylamide gel electrophoresis.

Mouse senescence model experiments

For IR-induced senescence, C57BL/6J mice (8 weeks of age)weremock-irradiated or exposed to IR (7 Gy) and tissueswere collected 90 days later. Mice were maintained in the AALAC-accredited Buck Institute for Research on Aging (Novato, CA) animal facility. All procedures were approved by the institutional animal care and use committee.

Statistical analysis

Statistical significance was calculated by twotailed Student’s t test unless otherwise indicated.The c2 test was used to compare gene expression profiling between the GATA4-regulated set and Senescent set. Prism 6 software was used to generate graphs and statistical analyses.

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