Klf4的功能研究进展

Klf4作为真核生物转录因子,参与调控细胞增殖、分化、胚胎发育等重要生命过程,是体细胞重编程为诱导多能干细胞（iPS细胞）的重要诱导因子之一。本文综述了Klf4在细胞增殖、分化、肿瘤发生、皮肤屏障、热休克及iPS细胞诱导等方面的研究进展,为深入研究Klf4在重编程中的作用机制和功能提供参考。     

小鼠Klf4基因的克隆及原核表达分析

目的克隆Klf4基因并对其重组蛋白进行原核表达及分析。方法提取胎鼠皮肤mRNA后反转录为cDNA序列,用一对两端引入特定酶切位点引物,从该cDNA中扩增出Klf4基因编码区序列,将其克隆到pEasy-T3载体上。对质粒双酶切并回收其中Klf4基因片段后,克隆入pET-52b（＋）载体后转化Origmai B（DE3）型大肠杆菌,用IPTG诱导表达,最后采用SDS-PAGE对重组蛋白进行鉴定及分析。结果对所克隆的Klf4mRNA蛋白编码区的DNA序列分析表明,klf4CDS区包括终止密码子在内为1452 bp,与参照序列对比仅有四处存在差异,不仅其同源性达到99.72%,且其氨基酸序列同源性为100%;在IPTG诱导下pET-52b（＋）-Klf4重组质粒可表达与预期相符的约为57×103的蛋白质;经IPTG刺激后重组蛋白表达明显上调,其中IPTG为0.4 mol/L时效果最佳。结论从胎鼠皮肤中克隆的Klf4基因可在原核中表达。     

HDAC2 phosphorylation-dependent Klf5 deacetylation and RARα acetylation induced by RAR agonist switch the transcription regulatory programs of p21 in VSMCs

Abnormal proliferation of vascular smooth muscle cells (VSMCs) occurs in hypertension, atherosclerosis and restenosis after angioplasty, leading to pathophysiological vascular remodeling. As an important growth arrest gene, p21 plays critical roles in vascular remodeling. Regulation of p21 expression by retinoic acid receptor (RAR) and its ligand has important implications for control of pathological vascular remodeling. Nevertheless, the mechanism of RAR-mediated p21 expression in VSMCs remains poorly understood. Here, we show that, under basal conditions, RARα forms a complex with histone deacetylase 2 (HDAC2) and Krüppel-like factor 5 (Klf5) at the p21 promoter to inhibit its expression. Upon RARα agonist stimulation, HDAC2 is phosphorylated by CK2α. Phosphorylation of HDAC2, on the one hand, promotes its dissociation from RARα, thus allowing the liganded-RARα to interact with co-activators; on the other hand, it increases its interaction with Klf5, thus leading to deacetylation of Klf5. Deacetylation of Klf5 facilitates its dissociation from the p21 promoter, relieving its repressive effect on the p21 promoter. Interference with HDAC2 phosphorylation by either CK2α knockdown or the use of phosphorylation-deficient mutant of HDAC2 prevents the dissociation of Klf5 from the p21 promoter and impairs RAR agonist-induced p21 activation. Our results reveal a novel mechanism involving a phosphorylation-deacetylation cascade that functions to remove the basal repression complex from the p21 promoter upon RAR agonist treatment, allowing for optimum agonist-induced p21 expression.     

The magic of four： induction of pluripotent stem cells from somatic cells by Oct4, Sox2, Myc and Klf4

The developmental process from a fertilized egg to agrown adult is programmed with remarkable accuracy.While the genetic information of the fertilized egg and itsdescendent somatic cells are the same, it is the selectiveexpressions of the same genome that give rise to the 200or so different cell types in an adult. The differentiatedstates of these adult cells are maintained epigenetically,presumably through the modification of chromatins and theassociated histones. In higher mammals, it was thought thatthe differentiation process is irreversible until the successfulcloning of Dolly [1]. By transferring a nucleus from a fullydifferentiated cell in the mammary gland, Wilmut and col-leagues were able to generate an exact replica of a highermammal, the Doily [1]. This work not only demonstratedthat the genome of differentiated cells can be reprogrammedinto an embryonic state and then to resume a full-fledgeddevelopmental process to generate a normal adult, but alsorejuvenated the field of animal cloning. The prospect thata somatic cell from a patient may be reprogrammed to the     

PRMT5沉默与TSA处理对Klf4表达的影响

[目的]旨在研究PRMT5沉默与TSA处理抑制组蛋白去乙酰化酶(HDAC)的活性对人肝癌Hep G2细胞中Klf4基因表达的影响。[方法]采取siRNA转染HepG2细胞的方法沉默PRMT5,TSA处理抑制HDAC,通过RT-PCR、luciferase assay、Western Blotting检测PRMT5沉默和TSA处理对Klf4表达的影响。[结果]在HepG2细胞中转染的三组PRMT5 siRNA均能有效干扰PRMT5的表达,其中si PRMT5-1对于PRMT5 mRNA和蛋白的干扰效果最好。TSA处理细胞后,组蛋白乙酰化修饰H3K9ac增加,HDAC3 mRNA和PRMT5 mRNA表达下降,PRMT5和HDAC1蛋白表达下降。RT-PCR、luciferase assay、Western Blotting结果显示单独沉默PRMT5或用TSA处理细胞,Klf4的转录被激活,而沉默PRMT5的同时用TSA处理细胞对Klf4转录的促进作用更为明显。[结论]PRMT5沉默或TSA处理都能促进Klf4的转录,同时沉默PRMT5和用TSA处理对Klf4的转录激活更强烈,说明PRMT...     

非洲爪蛙Klf4基因新转录本的克隆及功能研究

[目的]克隆非洲爪蛙Klf4基因的新转录本,并研究其在早期胚胎发育中的功能。[方法]提取胚胎总RNA,反转录构建c DNA文库。从Xenbase数据库中获得Klf4的基因组序列,分析序列发现新的ATG位点,设计引物、PCR扩增,将扩增的目的片段插入p CS2+,插入位点为EcoRⅠ和XhoⅠ。经双酶切和测序鉴定,发现载体中插入的目的片段为编码483个氨基酸的Klf4的新转录本。体外转录获得mRNA,显微注射入胚胎,进行表型分析以及原位杂交检测。[结果]成功克隆出了Klf4基因的新转录本;过表达该转录本,表型分析发现胚胎发育异常,原位杂交检测发现Xag2和Dkk1的表达上升。[结论]成功克隆编码483个氨基酸的Klf4基因的新转录本,过表达该转录本胚胎发育异常,Xag2和Dkk1的表达被激活。     

大熊猫转录因子Klf4在间充质干细胞中的转录调控作用研究

大熊猫Ailuropoda melanoleuca是我国特有的濒危物种之一,对其分子及基因功能的研究可以加深对大熊猫的了解以及提供更加有效的保护。Krüppel样因子4(KLF4)是一个重要的锌指转录因子,在细胞周期调控、细胞生长、迁移、分化、凋亡以及正常组织稳态维持等生理过程中发挥重要作用。此外,KLF4也是体细胞重编程为诱导多能干细胞的重要诱导因子之一。为了深入了解大熊猫Klf4基因的结构和功能,以大熊猫间充质干细胞(MSCs)为材料,通过快速扩增cDNA末端的方法克隆得到转录因子Klf4的cDNA全长序列,分析了其结构特点,并利用RNA-seq技术探索KLF4在大熊猫MSCs中的调控作用。结果表明,大熊猫KLF4在进化过程中比较保守,并在细胞增殖、细胞凋亡、RNA转运和蛋白质生物合成等生物过程中发挥着重要的生理作用。本研究结果将为后续大熊猫的分子水平研究及基因资源保护奠定研究基础。     

过表达鸡Klf2促进klf7转录抑制脂肪细胞分化

过表达Krüppel样因子2(Krüppel like factor 2,Klf2)或Klf7可抑制脂肪细胞形成,但是在脂肪组织中Klf2是否调控klf7表达还不清楚。本研究采用油红O染色和蛋白质印迹法技术研究了过表达Klf2对鸡前脂肪细胞分化的影响,结果显示过表达Klf2抑制油酸诱导的前脂肪细胞分化和pparγ表达,同时促进klf7表达(P<0.05)。利用Spearman相关性分析研究人和鸡脂肪组织中klf2和klf7的表达数据之间的关系,发现klf2和klf7的表达数据存在明显的正相关(r>0.1)。荧光素酶报告基因分析显示,在鸡前脂肪细胞中过表达Klf2促进鸡klf7启动子(–241/–91、–521/–91、–1845/–91、–2286/–91和–1215/–91)的活性(P<0.05);此外,在鸡前脂肪细胞中,klf7启动子(–241/–91)报告基因活性与共转染的klf2过表达质粒的浓度正相关(Tau=0.91766,P=1.074×10–7),过表达Klf2显著促进klf7的mRNA表达水平。综上所述,促进klf7表达可能是Klf2抑制鸡脂肪细胞形成的作用途径之一,鸡klf7翻译起始位点上游–241 bp/–91 bp序列可能介导转录因子Klf2对klf7表达的转录调控作用。     

MiR-301b抑制转录因子Klf4影响肝癌细胞迁移

目的:探讨mi R-301b对肝癌细胞迁移能力的影响及其分子机制,为肝癌的分子靶向治疗研究提供新线索。方法:体外培养人肝癌细胞株SK-Hep-1、HCC-LM3和人永生化肝细胞株L02,采用RT-PCR方法检测mi R-301b表达。通过生物信息学软件Targetscan及mi Randa预测mi R-301b的靶基因,筛选出转录因子Klf4基因为mi R-301b的下游靶基因,通过双荧光素酶报告基因实验和Western Blot实验证明其调控作用。通过划痕和Transwell实验探究mi R-301b靶向Klf4基因对肝癌细胞迁移性的影响,Western Blot检测mi R-301b对上皮间质转化标记物E-cadherin、N-cadherin蛋白表达的影响。结果:与正常肝细胞相比,肝癌细胞株中mi R-301b表达水平明显升高。瞬时转染mi R-301b mimic后,实验组mi R-301b的表达显著高于对照组;瞬时转染mi R-301b inhibitor后,实验组mi R-301b的表达显著低于对照组。双荧光素酶报告基因实验显示:mi R-301b直接作用于Klf4基因的3'UTR区,并下调Klf4蛋白的表达,与软件预测结果相符合。划痕实验及Transwell迁移实验显示:mi R-301b通过下调Klf4基因,促进肝癌细胞的迁移。进一步实验显示:过表达mi R-301b显著下调E-cadherin的表达,而上调N-cadherin的表达。结论:mi R-301b在肝癌细胞SK-Hep-1、HCC-LM3中高表达,可能通过抑制靶基因Klf4的表达,促进肝癌的迁移,mi R-301b可能参与了肝癌细胞的上皮间质转化过程。     

山羊Klf4基因克隆、原核表达和His-Klf4融合蛋白纯化北大核心CSCD

旨在克隆山羊Klf4基因,构建重组质粒pET30a-Klf4,通过原核表达技术获得纯化的His-Klf4融合蛋白。从山羊的生殖脊中提取总RNA,用RT-PCR的方法扩增Klf4基因编码区序列,通过TA克隆构建pMD18-T-Klf4重组质粒。酶切鉴定与测序分析后将Klf4的cDNA亚克隆至pET-30a载体,构建重组质粒pET30a-Klf4。酶切鉴定后将其导入大肠杆菌BL21中,用IPTG诱导表达,SDS-PAGE和Western blotting检测确认,镍离子金属螯合亲和层析法纯化His-Klf4融合蛋白。结果表明:(1)从山羊生殖脊中克隆了Klf4基因,其开放阅读框由1 434个核苷酸组成,编码478个氨基酸,而且该序列与绵羊的同源性最高,达到98.5%;(2)经过SignalP 4.0在线分析,Klf4的编码蛋白不存在信号肽;(3)经SDS-PAGE和Western blotting分析表明,重组质粒pET30a-Klf4在大肠杆菌中得以表达;在变性条件下纯化,获得了His-Klf4融合蛋白。     

Feeling by sight or seeing by touch?

We have addressed the role of occipital and somatosensory cortex in a tactile discrimination task. Sight-ed and congenitally blind subjects rated the roughness and distance spacing for a series of raised dot patterns. When judging roughness, intermediate dot spacings were perceived as being the most rough, while distance judgments generated a linear relation. Low-frequency rTMS applied to somatosensory cortex disrupted roughness without affecting distance judgments, while rTMS to occipital cortex disrupted distance but not roughness judgments. We also tested an early blind patient with bilateral occipital cortex damage. Her performance on the roughness determination task was normal; however, she was greatly impaired with distance judgments. The findings suggest a double-dissociation effect in which roughness and distance are primarily processed in somatosensory and occipital cortex, respectively. The differential effect of rTMS on task performance and corroborative clinical evidence suggest that occipital cortex is engaged in tactile tasks requiring fine spatial discrimination.     

Neuronal death induced by nanomolar amyloid β is mediated by primary phagocytosis of neurons by microglia

Alzheimer disease is characterized by neuronal loss and brain plaques of extracellular amyloid β (Aβ), but the means by which Aβ may induce neuronal loss is not entirely clear. Although high concentrations of Aβ (μM) can induce direct toxicity to neurons, we find that low concentration (nM) induce neuronal loss through a microglia-mediated mechanism. In mixed neuronal-glial cultures from rat cerebellum, 250 nM Aβ1-42 (added as monomers, oligomers or fibers) induced about 30% loss of neurons between 2 and 3 days. This neuronal loss occurred without any increase in neuronal apoptosis or necrosis, and no neuronal loss occurred with Aβ42-1. Aβ greatly increased the phagocytic capacity of microglia and induced phosphatidylserine exposure (an "eat-me" signal) on neuronal processes. Blocking exposed phosphatidylserine by adding annexin V or an antibody to phosphatidylserine or inhibiting microglial phagocytosis by adding either cytochalasin D (to block actin polymerization) or cyclo(RGDfV) (to block vitronectin receptors) significantly prevented neuronal loss. Loss of neuronal synapses occurred in parallel with loss of cell bodies and was also prevented by blocking phagocytosis. Inhibition of phagocytosis prevented neuronal loss with no increase in neuronal death, even after 7 days, suggesting that microglial phagocytosis was the primary cause of neuronal death induced by nanomolar Aβ.     

Some properties of auditory neuron’s model trained by firing caused by tones modulated by low-frequency noise

In many sensory systems the formation of burst firing can be observed along a way from the periphery to the central nuclei. We investigate the putative transformation of spontaneous activity in the auditory pathway using a neuron model trained by real firing recorded in the auditory nuclei of the frog. The model has 200 separate inputs (neuronal spines). It is supposed that every spine is a coincidence detector. Its output (synaptic potential) sharply increases at emergence of the precisely certain interpulse interval in an input pulse sequence. If the total synaptic potentials excess a threshold, the model generates output spike, which changes weight of all spines according to the simplified Hebb principle. The model was trained by real firing caused in the auditory nuclei of the frog by tones modulated by low-frequency noise in the frequency ranges of 0–15 Hz, 0–50 Hz or 0–150 Hz. After that training the synaptic weights of every spine essentially changed. Thus, along with some increase of weights of spines tuned to boundary frequencies of modulating noise, the most characteristic change was the emphasizing weights of spines tuned to short interpulse intervals. As a result the spontaneous activity passed through the trained model became much more bursting. Efficiency of a signal transmission in model was higher when input spontaneous activity of real cells contains bursts of spikes. Results of modeling are discussed in connection with modern physiological data demonstrating the functional advantage of bursting.     

Formation of Three New Trisaccharide-azoxyglycosides by Transglucosylation by Cycad β-Glucosidase

transglucosylation by a β-d-glucosidase from cycad seeds. These azoxyglycosides, named neocycasin H, I, and J, were identified as O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl-(l→3)-O-β-d-glucopyranoside of methylazoxymethanol (MAM), O-β-d-glucopyranosyl-(1→3)-[O-β-d-glucopyranosyl-(1→6)]-O-β-d-glucopyranoside of MAM, and O-β-d-glucopyranosyl-(1→3)-[O-β-d-xylopyranosyl-(1→6)]-O-β-d-glucopyranoside of MAM, respectively. On the basis of their structures, the mechanism of the formation of these neocycasins is also discussed.     

BAFfled by Poxviruses?

Successful viruses engage in a dynamic interplay with their hosts, where both utilize diverse strategies to impose their supremacy. In this issue of Cell Host & Microbe, Wiebe and Traktman describe a novel interaction between vaccinia virus and mammalian cells. A host protein called BAF can bind ectopic cytoplasmic DNA and block viral DNA replication, whereas vaccinia in turn counteracts this inhibition with a virus-encoded serine threonine kinase that inactivates BAF.