世界生命科学前沿动态周报（六十七）

（11.21-11.27/2011）
美宝国际集团:陶国新 

　　主要内容：指（趾）的形成显示了“垃圾基因”的作用；糖尿病药降低患癌风险；细胞移植受体的免疫系统控制移植干细胞的再生；线虫研究揭示伤口愈合反应的秘密；维持Sip2的乙酰化延长酵母寿命；发现端粒酶延长端粒的关键分子开关。

　　焦点动态：发现端粒酶延长端粒的关键分子开关。

1. 指（趾）的形成显示了“垃圾基因”的作用
【动态】
　　指（趾）头的进化是四足动物成功的必要步骤，在指（趾）头发育的关键因素中，Hoxd基因受到协调控制，帮助管理生长和模式。瑞士、荷兰和德国的科学家通过探查发育中的四肢的三位构造确定了与这些基因有关的肢端调节位点。该方法结合体内删除特定的调节区，显示基因簇的活性部位与数个增强子样的序列接触。这些序列分散在临近的所谓垃圾基因序列中，各自对预期的指（趾）的Hox基因转录做出定量或定性的贡献。他们提出名为“调节列岛”的基因体系，提供一种遗传灵活性可能部分支持四足动物中指（趾）数目和形态的多样性以及顺应剧烈变化的能力。

【点评】
　　该研究表明在曾经错以为没有任何作用的垃圾基因序列中的一些序列能够相互结合，调节负责形成指（趾）头的基因的作用。这样的结果表明目前人类对基因的研究仍未成熟，尤其是对功能基因的作用机制还有很多工作要做。

【参考论文】
Cell, 2011; 147 (5): 1132 DOI: 10.1016/j.cell.2011.10.023 
A Regulatory Archipelago Controls Hox Genes Transcription in Digits
Thomas Montavon, Natalia Soshnikova, Bénédicte Mascrez, et al.
Highlights
Hox genes active in digits integrate the input of multiple regulatory elements
The global regulatory architecture of the HoxD locus involves flanking gene deserts
Alterations in this regulatory structure may fine tune digital morphology in tetrapods
Summary
The evolution of digits was an essential step in the success of tetrapods. Among the key players, Hoxd genes are coordinately regulated in developing digits, where they help organize growth and patterns. We identified the distal regulatory sites associated with these genes by probing the three-dimensional architecture of this regulatory unit in developing limbs. This approach, combined with in vivo deletions of distinct regulatory regions, revealed that the active part of the gene cluster contacts several enhancer-like sequences. These elements are dispersed throughout the nearby gene desert, and each contributes either quantitatively or qualitatively to Hox gene transcription in presumptive digits. We propose that this genetic system, which we call a regulatory archipelago, provides an inherent flexibility that may partly underlie the diversity in number and morphology of digits across tetrapods, as well as their resilience to drastic variations.
 
2. 糖尿病药降低患癌风险
【动态】
　　一种廉价的2型糖尿病治疗药物，抑制糖异生基因转录的二甲双胍证明能够预防很多天然或人工合成的化合物刺激乳腺癌细胞生长，降低糖尿病相关肿瘤的风险。最近，肿瘤干细胞被认为是负责维持肿瘤生长，抵抗治疗。韩国和美国科学家为了验证二甲双胍可能减少乳腺癌风险的假设，将生长于代表人体乳腺癌干细胞群的三维乳球体中的MCF-7人体乳腺癌细胞系在有/无非细胞毒性浓度的二甲双胍存在下用各种已知的和可能的乳腺癌诱发剂处理，OCT4表达作为肿瘤干细胞的标志，测定这些细胞的数目和大小。结果表明100nM的TCDD和10µM的双酚A像10µM的雌激素一样增加了三维乳球体的数目和大小。通过检测标志物OCT4，这些致癌物的刺激与OCT4表达增多相关。另一方面，1mM和10mM的二甲双胍能够大大减少三维乳球体的数目和大小。结果还显示二甲双胍能够减少雌激素和TCDD诱生的三维乳球体中OCT4的表达，但在双酚A诱生的三维乳球体中则不能，表明双酚A对人体乳腺癌细胞有不同的作用机制。另外，这些结果支持使用三维人体乳腺癌干细胞作为一种手段筛选潜在的人体乳腺癌致癌物以及化学预防和治疗剂。

【点评】
　　该研究发现了廉价的糖尿病药物二甲双胍能够通过抑制雌激素受体介导的癌基因OCT4的表达，减少人体乳腺癌干细胞的自我复制。同时为乳腺癌的防治和二甲双胍的老药新用提供了新的可能。

【参考论文】
PLoS ONE, 2011; 6 (11): e28068 DOI: 10.1371/journal.pone.0028068 
Metformin Represses Self-Renewal of the Human Breast Carcinoma Stem Cells via Inhibition of Estrogen Receptor-Mediated OCT4 Expression
Ji-Won Jung, Sang-Bum Park, Soo-Jin Lee, et al.
Metformin, a Type II diabetic treatment drug, which inhibits transcription of gluconeogenesis genes, has recently been shown to lower the risk of some diabetes-related tumors, including breast cancer. Recently, “cancer stem cells” have been demonstrated to sustain the growth of tumors and are resistant to therapy. To test the hypothesis that metformin might be reducing the risk to breast cancers, the human breast carcinoma cell line, MCF-7, grown in 3-dimensional mammospheres which represent human breast cancer stem cell population, were treated with various known and suspected breast cancer chemicals with and without non-cytotoxic concentrations of metformin. Using OCT4 expression as a marker for the cancer stem cells, the number and size were measured in these cells. Results demonstrated that TCDD (100 nM) and bisphenol A (10 µM) increased the number and size of the mammospheres, as did estrogen (10 nM E2). By monitoring a cancer stem cell marker, OCT4, the stimulation by these chemicals was correlated with the increased expression of OCT4. On the other hand, metformin at 1 and 10 mM concentration dramatically reduced the size and number of mammospheres. Results also demonstrated the metformin reduced the expression of OCT4 in E2 & TCDD mammospheres but not in the bisphenol A mammospheres, suggesting different mechanisms of action of the bisphenol A on human breast carcinoma cells. In addition, these results support the use of 3-dimensional human breast cancer stem cells as a means to screen for potential human breast tumor promoters and breast chemopreventive and chemotherapeutic agents.

3. 细胞移植受体的免疫系统控制移植干细胞的再生
【动态】
　　受体的T淋巴细胞通过IFN-γ和TNF-α控制基于间充质干细胞的组织再生。在组织重建中基于干细胞的再生医学是一种很有希望的方法。南加大的美国科学家最近报道了促炎T细胞抑制外源骨髓间充质干细胞（BMMSCs）引导骨头修复的能力。这一抑制作用来自干扰素γ（IFN-γ）诱导的干细胞中runt相关转录因子2（Runx-2）途径下调和肿瘤坏死因子α（TNF-α）信号的增强。他们还发现，通过抑制核因子κB (NF-κB)，TNF-α将BMMSCs中IFN-γ激活的，非凋亡形态的TNF受体超家族成员6（Fas）信号转变为细胞凋亡蛋白酶3和8相关的促凋亡级联反应信号，导致这些细胞凋亡。相反地，通过全身输入Foxp3(+)调节性T细胞或局部给药阿司匹林来减少IFN-γ 和 TNF-α的浓度，显著地促进了C57BL/6老鼠基于BMMSC的骨骼再生和颅骨缺损的修复。这些数据合起来表明前所未知的受体T细胞在基于BMMSC的组织工程中的作用。

【点评】
　　该研究表明基于干细胞的再生医学研究需要关注机体自身的免疫系统对组织再生的调控，干细胞研究和再生医学的突破离不开干细胞所处机体环境的研究。

【参考论文】
Nature Medicine, 2011 DOI: 10.1038/nm.254 
Mesenchymal stem cell-based tissue regeneration is governed by recipient T lymphocytes via IFN-γ and TNF-α
Yi Liu, Lei Wang, Takashi Kikuiri, et al.
Stem cell-based regenerative medicine is a promising approach in tissue reconstruction. Here we show that proinflammatory T cells inhibit the ability of exogenously added bone marrow mesenchymal stem cells (BMMSCs) to mediate bone repair. This inhibition is due to interferon γ (IFN-γ)-induced downregulation of the runt-related transcription factor 2 (Runx-2) pathway and enhancement of tumor necrosis factor α (TNF-α) signaling in the stem cells. We also found that, through inhibition of nuclear factor κB (NF-κB), TNF-α converts the signaling of the IFN-γ-activated, nonapoptotic form of TNF receptor superfamily member 6 (Fas) in BMMSCs to a caspase 3- and caspase 8-associated proapoptotic cascade, resulting in the apoptosis of these cells. Conversely, reduction of IFN-γ and TNF-α concentrations by systemic infusion of Foxp3(+) regulatory T cells, or by local administration of aspirin, markedly improved BMMSC-based bone regeneration and calvarial defect repair in C57BL/6 mice. These data collectively show a previously unrecognized role of recipient T cells in BMMSC-based tissue engineering.

4. 线虫研究揭示伤口愈合反应的秘密
【动态】
　　在严酷的环境中生存，动物必须能够修复皮肤创伤，然而体内启动创伤修复的信号途径还不明了。在线虫中，p38分裂素激活的蛋白激酶（MAPK）级联反应促进应对创伤的先天免疫反应，但不是创伤愈合其他方面所需的。美国科学家为此在线虫表皮中探查了其他的创伤反应信号途径。结果表明线虫表皮创伤引发迅速而持续的表皮中钙离子浓度的上升，这是创伤后存活的关键。创伤触发的钙离子增多需要表皮瞬时受体电压通道、melastatin家族（TRPM）通道GTL-2和IP3R刺激的内部储存池的释放。他们确立了一个表皮信号传导途径，包括Gαq EGL-30 及其效应器 PLCβ EGL-8。该途径失效的话会导致损害创伤后存活。Gαq-Ca2+途径对已知的应对创伤的先天免疫反应不是必须的 ，但会促进肌动蛋白依赖的伤口闭合。伤口闭合需要Cdc42小GTP酶和Arp2/3-依赖的肌动蛋白聚合，并被Rho 和非肌肉的肌球蛋白负向调节。他们还发现死亡相关蛋白激酶DAPK-1负向调节伤口闭合。

【点评】
　　该研究表明线虫皮肤创伤会触发钙离子依赖的信号级联反应促进伤口闭合，同时伴有损伤引起的先天免疫反应。对于认识创伤早期的生化事件和促进创伤愈合研究有帮助。

【参考论文】
Current Biology, 2011; DOI: 10.1016/j.cub.2011.10.050 
A Gαq-Ca2 Signaling Pathway Promotes Actin-Mediated Epidermal Wound Closure in C. elegans
Suhong Xu, Andrew D. Chisholm.
Background
Repair of skin wounds is essential for animals to survive in a harsh environment, yet the signaling pathways initiating wound repair in vivo remain little understood. In Caenorhabditis elegans, a p38 mitogen-activated protein kinase (MAPK) cascade promotes innate immune responses to wounding but is not required for other aspects of wound healing. We therefore set out to identify additional wound response pathways in C. elegans epidermis.
Results
We show here that wounding the adult C. elegans skin triggers a rapid and sustained rise in epidermal Ca2+ that is critical for survival after wounding. The wound-triggered rise in Ca2+ requires the epidermal transient receptor potential channel, melastatin family (TRPM) channel GTL-2 and IP3R-stimulated release from internal stores. We identify an epidermal signal transduction pathway that includes the Gαq EGL-30 and its effector PLCβ EGL-8. Loss of function in this pathway impairs survival after wounding. The Gαq-Ca2+ pathway is not required for known innate immune responses to wounding but instead promotes actin-dependent wound closure. Wound closure requires the Cdc42 small GTPase and Arp2/3-dependent actin polymerization and is negatively regulated by Rho and nonmuscle myosin. Finally, we show that the death-associated protein kinase DAPK-1 acts as a negative regulator of wound closure.
Conclusions
Skin wounding in C. elegans triggers a Ca2+-dependent signaling cascade that promotes wound closure, in parallel to the innate immune response to damage. Wound closure requires actin polymerization and is negatively regulated by nonmuscle myosin.

5. 维持Sip2的乙酰化延长酵母寿命
【动态】
　　蛋白乙酰化是影响很多细胞生命过程的重要的翻译后修饰。美国和台湾的科学家最近发现随着细胞变老，单细胞酵母中AMP激活的蛋白激酶Snf1复合物的β调节亚基Sip2的NuA4乙酰化减少。通过拮抗NuA4乙酰基转移酶和Rpd3脱乙酰基酶控制的Sip2乙酰化增强了与Snf1复合物的催化亚基Snf1的相互作用。Sip2-Snf1相互作用抑制Snf1的活性，减少下游靶点Sch9（Akt/S6K同源物）的磷酸化，最终导致生长变慢但是延长了细胞复制的寿命（复制次数）。Sip2乙酰化模拟物更能抵抗氧化压力。他们还发现Sip2乙酰化的抗衰老作用不依赖外来营养和TORC1活性。他们提出蛋白乙酰化-磷酸化连锁反应调节Sch9活性，控制本身的衰老，延长酵母繁殖的寿命（繁殖更多代）。天然酵母的正常繁殖寿命是25代，而通过基因工程改造恢复乙酰化化学修饰的酵母繁殖寿命达到38代，延长了50%。

【点评】
　　在酵母中维持Snf1复合物β调节亚基Sip2的乙酰化，具有抗衰老作用。文中实验了通过基因改造模拟这一乙酰化维持过程，达到了延长酵母寿命达50%的效果。如果不用改造基因，通过其他更自然的方法也能维持Sip2的乙酰化，可能是抗衰老的重大突破。

【参考论文】
Cell, 2011; 146 (6): 969 DOI: 10.1016/j.cell.2011.07.044
Acetylation of Yeast AMPK Controls Intrinsic Aging Independently of Caloric Restriction
Jin-Ying Lu, Yu-Yi Lin, Jin-Chuan Sheu, et al. 
Acetylation of histone and nonhistone proteins is an important posttranslational modification affecting many cellular processes. Here, we report that NuA4 acetylation of Sip2, a regulatory β subunit of the Snf1 complex (yeast AMP-activated protein kinase), decreases as cells age. Sip2 acetylation, controlled by antagonizing NuA4 acetyltransferase and Rpd3 deacetylase, enhances interaction with Snf1, the catalytic subunit of Snf1 complex. Sip2-Snf1 interaction inhibits Snf1 activity, thus decreasing phosphorylation of a downstream target, Sch9 (homolog of Akt/S6K), and ultimately leading to slower growth but extended replicative life span. Sip2 acetylation mimetics are more resistant to oxidative stress. We further demonstrate that the anti-aging effect of Sip2 acetylation is independent of extrinsic nutrient availability and TORC1 activity. We propose a protein acetylation-phosphorylation cascade that regulates Sch9 activity, controls intrinsic aging, and extends replicative life span in yeast.

6. 发现端粒酶延长端粒的关键分子开关
【动态】
　　进化上保守的端粒庇护蛋白复合物在哺乳动物和裂殖酵母的端粒酶调节中既有正面作用也有负面作用。尽管哺乳动物细胞的庇护蛋白防止检查点激酶ATM和ATR在端粒处充分激活DNA损伤反应，这些激酶也促进端粒的维护。缺少Tel1（ATM直接同源物）和Rad3（ATR直接同源物）的裂殖酵母细胞无法招募端粒酶到端粒上，无法通过环形染色体存活。但是，还不知道Tel1ATM和Rad3ATR对应的端粒底物。美国科学家最近的研究表明Tel1ATM和/或Rad3ATR调节的庇护蛋白亚基Ccq1在93号苏氨酸上的磷酸化是端粒酶联系端粒所必需的。另外，端粒酶亚基Est1直接与Ccq1的磷酸化的93号苏氨酸相互作用以保证端粒的维护。庇护蛋白亚基Taz1, Rap1 和 Poz1 (以前已确认的端粒酶抑制剂) 也负向调节 Ccq1磷酸化 。所有这些发现证明Tel1ATM/Rad3ATR依赖的 Ccq1 的93号苏氨酸磷酸化是裂殖酵母维护端粒的关键调节器。

【点评】
　　该研究进一步阐释了端粒酶途径维护端粒长度的机制，对于认识各细胞组分如何协调一致维持端粒的正常功能和寻找预防癌症的方法有重要意义。

【参考论文】
Nature Structural & Molecular Biology, 2011; DOI: 10.1038/nsmb.2187 
Tel1ATM and Rad3ATR kinases promote Ccq1-Est1 interaction to maintain telomeres in fission yeast
Bettina A Moser, Ya-Ting Chang, Jorgena Kosti, et al.
The evolutionarily conserved shelterin complex has been shown to play both positive and negative roles in telomerase regulation in mammals and fission yeast. Although shelterin prevents the checkpoint kinases ATM and ATR from fully activating DNA damage responses at telomeres in mammalian cells, those kinases also promote telomere maintenance. In fission yeast, cells lacking both Tel1 (ATM ortholog) and Rad3 (ATR ortholog) fail to recruit telomerase to telomeres and survive by circularizing chromosomes. However, the critical telomere substrate(s) of Tel1ATM and Rad3ATR was unknown. Here we show that phosphorylation of the shelterin subunit Ccq1 on Thr93, redundantly mediated by Tel1ATM and/or Rad3ATR, is essential for telomerase association with telomeres. In addition, we show that the telomerase subunit Est1 interacts directly with the phosphorylated Thr93 of Ccq1 to ensure telomere maintenance. The shelterin subunits Taz1, Rap1 and Poz1 (previously established inhibitors of telomerase) were also found to negatively regulate Ccq1 phosphorylation. These findings establish Tel1ATM/Rad3ATR-dependent Ccq1 Thr93 phosphorylation as a critical regulator of telomere maintenance in fission yeast.