A 314-bp SINE insertion in the ZNF2 promoter region may act as a repressor related to regulation of fat deposition in pigs

Retrotransposons,a type of DNA fragment that can mobilize itself on genome,can generate genetic variations and develop for molecular markers based on the insertion polymorphism.Zinc finger proteins(ZNFs)are among the most abundant proteins in eukaryotic animals,and their functions are extraordinarily diverse and particularly important in gene regulation.In the current study,bioinformatic prediction was performed to screen for retrotransposon insertion polymorphisms(RIPs)in six ZNF genes(ZNF2,ZNF3,ZNF7,ZNF8,ZNF10 and ZNF12).Six RIPs in these ZNFs,including one short interspersed nuclear element(SINE)RIP in intron 1 and one long interspersed nuclear element 1(L1)RIP in intron 3 of ZNF2,one SINE RIP in 5′flanking region and one SINE RIP in intron 2 of ZNF3,one SINE RIP in 3′UTR of ZNF7 and one L1 RIP in intron 2 of ZNF12,were discovered and their presence was confirmed by PCR.The impact of the SINE RIP in the first intron of ZNF2,which is close to the core promoter of ZNF2,on the gene activity was investigated by dual-luciferase assay in three cell lines.Our results showed that the SINE insertion in the intron 1 of ZNF2 repressed the core promoter activity extremely significantly(P<0.01)in cervical cancer cells and porcine primary embryonic fibroblasts(HeLa and PEF),thus SINE may act as a repressor.This SINE RIP also significantly(P<0.05)affected the corrected back fat thickness in Yorkshire pigs.The corrected back fat thickness of individuals with SINE insertion in the first intron of ZNF2 was significantly(P<0.05)higher than that of individuals without SINE insertion.In summary,our data suggested that RIPs play important roles in the genetic variations of these ZNF genes and SINE RIP in the intron 1 of ZNF2 may provide a useful molecular marker for the screening of fat deposition in the pig breeding.

FHL2 Interacts with and Acts as a Functional Repressor of Id2 in Human Neuroblastoma Cells

Objective： Id2 is a natural inhibitor of the basic helix-loop-helix（bHLH） transcription factors. Although it is well known that active ld2 prevents differentiation and promotes cell cycle progression and tumorigenesis, the molecular events that regulate Id2 activity remain to be investigated. Methods： Yeast two-hybrid, mammalian two-hybrid, GST-pulldown and immunoprecipitation （ColP） assays were used to screen and identify novel Id2 interactors. Luciferase assays were used to detect E47-mediated transcription activity. Colony formation and BrdU incorporation assays were used to determine cellular proliferation abilities. Northorn blot, western blot and quantitative PCR methods were used to measure gene expression levels. Electrophoretic mobility shift assays （EMSAs） were performed to investigate protein/DNA binding. Results： The LIM-only protein FHL2 （four-and-a-half-LIM-only protein 2） was identified to be a novel Id2 interactor. The HLH domain within Id2 is not required for its interaction with FHL2. FHL2 antagonizes the inhibitory effect of Id2 on the basic helix-loop-helix protein E47-mediated transcription. FHL2 prevents the formation of Id2-E47 heterdimer, thus releasing E47 to its target DNA and restoring its transcriptional activity. FHL2 expression was remarkably up-regulated during retinoic acid-induced differentiation of neuroblastoma cells, during which the expression of Id2 is opposite to that. Ectopic FHL2 expression in neuroblastoma cells markedly reduces the transcriptional and cell-cycle promoting functions of Id2. Conclusion： These results indicate that FHL2 is an important repressor of the oncogenic activity of Id2 in neuroblastoma cells.

Expression of p27Kip1, A Cell Cycle Repressor Protein with Dual Roles for Both Cancer Prevention and Promotion, Is Regulated Primarily at the Level of Unusual p27Kip1 mRNA—A Short Concept Proposal

The p27Kip1 is a cell cycle repressor protein that regulates primarily the cell cycle transition from G1 to S phase and hence the DNA replication is in the S phase and cell division in the M phase. Expression of p27Kip1 protein has dual roles for both cancer prevention and promotion. For example, numerous nutritional and chemopreventive anti-cancer agents specifically increase the expression of p27Kip1 protein without directly affecting the expression of any other cell cycle regulatory proteins. On the other hand, pro-cancer agents (like glucose, insulin and other growth factors frequently seen in obesity and/or diabetes) specifically decrease the expression of p27Kip1 protein without directly affecting the expression of any other cell cycle regulatory proteins. Unlike expression of any other cell cycle regulatory proteins, expression of p27Kip1 protein is very unusual. The mRNA of p27Kip1 has a very long and unusual 5’-untranslated region (from -575 to -1 in human). It appears that the 5’-untranslated region of p27Kip1 mRNA forms two alternative secondary structures. One increases the expression of p27Kip1 protein when anti-cancer agents are added and another decrease the expression of p27K1p1 when pro-cancer agents are added. For this short concept proposal, Dr. Albert Einstein’s “visualized thought experiments (German: Gedanken experiment)” were used as a fundamental tool for understanding how either anti- or pro-cancer agents bring the primary structure of the 5’-untranslated region of p27Kip1 mRNA into two alternative secondary structures, thereby either increasing or decreasing, respectively, the translation initiation of p27Kip1 protein.

Lack of association between cellular repressor of E1A-stimulated genes(GREG)polymorphisms and coronary artery disease in the Han population of North China

Objectives Phenotypic switching of smooth muscle cells(SMCs) plays a critical role in the pathogenesis of atherosclerotic lesions such as coronary artery disease (CAD).Accumulating evidence demonstrates(hat a cellular repressor of E1A-stimulated genes(CREG) plays a role in the maintenance of the mature phenotype of vascular SMCs. The purpose of the present study was to assess the possible association between CREG and CAD in the Han population of North China.Methods The promoter region of CREG by direct sequencing was conducted in 48 subjects.Then SNP rs2995073 and another 4 tagSNPs(rs4657669,rs3767443, rsl6859185,rs3753921) were selected for the association study.All five selected SNPs were determined in 1161 patients with angiographically proven CAD and 960 controls with normal coronary angiograms to investigate the possible involvement of CREG in CAD.Results Genotype frequencies of the five examined polymorphisms were similarly distributed between CAD group and controls(P】0.05).Further haplotype analysis also found no significant differences in the distributions between CAD group and controls(P】0.05). Conclusions This study did not show an association between common variants of CREG and CAD in the northern Chinese Han population.

SB1 Encoding RING-Like Zinc-Finger Protein Regulates Branch Development as a Transcription Repressor

Inflorescence structure of rice,including the number and length of branches,and the density of the spikelet,can greatly affect the number of grains per panicle,which is one of the key factors in yield compositions.Here we identified five allelic mutants sb1-1/2/3/4/5 that related to branch development of rice.In these mutants,the branch meristem fate was prolonged sharply,resulting in delay of transition from branches to spikelets,and then increased the numbers of branches and spikelets per panicle.SB1 encodes a nuclear RING-like domain protein of SHI/LRP/SRS family and strongly expressed in branch meristems.The results of protein interaction and chromatin immunoprecipitation further suggested that SB1 directly repressed the expression of DEP1,TAW1,MOC1 and IPA1 by interacting with a co-repressor complex to affect acetylation level of histone H3 on target regions.Thus,we proposed that SB1 is a transcription repressor of branch meristem activity by widely and negatively regulating a series of genes that maintain branch meristem fate.

A Repressor in 5′-UTR of hKv4.3 Gene

The effect of repressors on ion channel gene expression was studied. The hKv4. 3 promoter and the sequence （ ＋ 2-- ＋ 160, S160） of 5＇-UTR of the hKv4. 3 gene was cloned into the pβ-gal-Basic vector. The transient expression of the pβ-gal vector and the analysis of the relative activity of β-galactosidase were carried out. The analysis of the mRNA level was carried out with the RT-PCR method. S160 could intensively repress the expression of the hKv4. 3 gene with position-specificity. The level of mRNA did not alter obviously. A repressor（ S160）, in 5＇-UTR of the hKv4. 3 gene was found and its repression to gene expression may play a role in the post-transcription process.

Lower Concentrations of Glucose or Insulin Decrease the Risk of Various Types of Cancer in the Long-Lived Ames Dwarf Mouse by Increasing the Expression of p27Kip1, a Cell-Cycle Repressor Protein

Introduction. The molecular biological mechanism of the increased incidence of the various types of cancer in obesity or type 2 diabetes in rodents or humans has largely been resolved in recent years. By contrast, the molecular biological mechanism of the decreased, not increased, incidence of the various types of cancer in the homozygous long-lived Ames dwarf mice still remains unresolved. Objective. The first objective of the present study was to investigate whether the decrease in the incidence of cancer in the homozygous long-lived Ames dwarf mice is due to the increase, not decrease, in the expression of p27Kip1, a cell cycle repressor protein. The second objective was to investigate whether the decrease in the incidence of cancer in the homozygous long-lived Ames dwarf mice is due to the decrease, not increase, in the levels of glucose or insulin. Methods. To achieve these objectives, we first performed western immunoblot analysis of the hepatic expression of p27Kip1 protein. We then performed, using a human breast cancer cell line in vitro, the luciferase reporter plasmid assay to determine whether the translation initiation activity of the p27Kip1 mRNA is increased when the concentrations of either glucose or insulin are decreased. Results and Conclusion. The results of the first objective indicated that the hepatic expression of p27Kip1 protein was up-regulated in the homozygous long-lived Ames dwarf mice as expected. We also found that the lower concentrations of glucose or insulin increased the translation initiation activity of the p27Kip1 mRNA.

Tissue expression and immunolocalization of cellular repressor of E1A-stimulated gene in postinfarction dysfunctional myocardium

Background Cellular Repressor of E1A-stimu-lated gene(CREG) is widely expressed in adult tissues such as the brain,heart,lung,liver,intestine and kidney in mice.It is not known whether tissue CREG is decreased in the common setting of myocardial infarction which may lead to heart failure.We studied the expression and protein localization of CREG and its main receptor(IFR2R) in a mouse model of myocardial infarction.Methods Male mice were randomized to proximal left anterior descending ligation.The animals were killed on day 1,3,7,14,and 28 after ligation to examine gene expression and protein production of CREG and IGF2R from the infarct,peri-infarct,and contralateral zones of infarcted heart.Results There was decreased CREG mRNA production throughout the myocardium at dav 1,and the expression gradually increased at day 28 after myocardial infarction.The decreased expression of this glycoprotein was not confined strictly to the infarct or peri-infarct zones but also expressed by cardiac myocytes within the myocardium in the contralateral normal zone.Levels of CREG protein in the infarct and peri-infarct zones declined to 1/3- to 1/2-fold of normal levels and declined to 1/2- to 2/3- fold in the contralateral zone.Finally,the expression of the IGF2R mRNA transcripts was downregulated at day 3 and 7 after ligation in the infarct and peri-infarct zones,suggesting that the signal transduction pathways necessary for CREG in the heart remain intact as CREG biosynthesis decreases. Conclusions CREG is constantly present in a model of large myocardial infarction and is decreased at the early stage within the myocardium.The decreased expression of this glycoprotein is not only confined strictly to the infarct or periinfarct zone but also is expressed by cardiac myocytes within the myocardium contralateral to the infarct.Therefore CREG production decreased due to myocardial stress response to injury.

Non-transmissible Sendai virus vector encoding c-myc suppressor FBP-interacting repressor for cancer therapy

AIM:To investigate a novel therapeutic strategy to target and suppress c-myc in human cancers using far up stream element(FUSE)-binding protein-interacting repressor(FIR).METHODS:Endogenous c-Myc suppression and apoptosis induction by a transient FIR-expressing vector was examined in vivo via a HA-tagged FIR(HA-FIR)expression vector.A fusion gene-deficient,non-transmissible,Sendai virus(SeV)vector encoding FIR cDNA,SeV/dF/FIR,was prepared.SeV/dF/FIR was examined for its gene transduction efficiency,viral dose dependency of antitumor effect and apoptosis induction in HeLa(cervical squamous cell carcinoma)cells and SW480(colon adenocarcinoma)cells.Antitumor efficacy in a mouse xenograft model was also examined.The molecular mechanism of the anti-tumor effect and c-Myc suppression by SeV/dF/FIR was examined using Spliceostatin A(SSA),a SAP155 inhibitor,or SAP155siRNA which induce c-Myc by increasing FIR△exon2 in HeLa cells.RESULTS:FIR was found to repress c-myc transcription and in turn the overexpression of FIR drove apoptosis through c-myc suppression.Thus,FIR expressing vectors are potentially applicable for cancer therapy.FIR is alternatively spliced by SAP155 in cancer cells lacking the transcriptional repression domain within exon 2(FIR△exon2),counteracting FIR for c-Myc protein expression.Furthermore,FIR forms a complex with SAP155 and inhibits mutual well-established functions.Thus,both the valuable effects and side effects of exogenous FIR stimuli should be tested for future clinical application.SeV/dF/FIR,a cytoplasmic RNA virus,was successfully prepared and showed highly efficient gene transduction in in vivo experiments.Furthermore,in nude mouse tumor xenograft models,SeV/dF/FIR displayed high antitumor efficiency against human cancer cells.SeV/dF/FIR suppressed SSA-activated c-Myc.SAP155 siRNA,potentially produces FIR△exon2,and led to c-Myc overexpression with phosphorylation at Ser62.HA-FIR suppressed endogenous c-Myc expression and induced apoptosis in HeLa and SW480 cells.A c-myc transcriptional suppressor FIR expressing SeV/dF/FIR showed high gene transduction efficiency with significant antitumor effects and apoptosis induction in HeLa and SW480 cells.CONCLUSION:SeV/dF/FIR showed strong tumor growth suppression with no significant side effects in an animal xenograft model,thus SeV/dF/FIR is potentially applicable for future clinical cancer treatment.

A critical role for the co-repressor N-CoR in erythroid differentiation and heme synthesis

合作抑压者 N 英国管(原子受体合作抑压者) 有重要角色冷淡的生物学过程包括增长，区别和开发。Mutantmice 缺乏 N 英国管是胚胎的联盟者 lethal 并且显得由于缺点从贫血症死非决定性红血球生成。然而， N-CoR-mediated erythroiddifferentiation 的内在的分子的机制大部分是未知的。化学上使用人的 erythroleukemic K562 房间线，它能是导致了系看情况区分进 erythroid 或 megakaryocytic 在 inducers 使用了，我们在 erythroid 区别调查了 N 英国管的角色。我们显示出那 N 英国管任何一个击倒短暂地(siRNA ) 或(shRNA ) 永久地损害 cytosinearabinoside (Ara-C )- 然而并非 K562 房间的导致 hemin 的 erythroid 区别。RT-PCR 分析表明 N 英国管被 5-aminolevulinate synthase (ALA-S2 ) 的 Ara-C 为正式就职要求，涉及的关键酶他我生合成。而且， N 英国管蛋白质的数量在 Ara-C-induced K562 区别期间显著地增加，显然通过 post-transcriptionalmechanism。与从 N-CoR-null 鼠标的数据一致， N 英国管没为 K562 房间的区别被要求进 megakaryocytic 系，由 phorbol 12-myristate13-acetate 导致了。因此，我们的试管内学习在 erythroid 区别为 N 英国管证实一个角色并且第一次表明 N 英国管为涉及 hemesynthesis 的关键酶的正式就职被要求。

Advances in the knowledge on the role of apoptosis repressor with caspase recruitment domain in hemorrhagic stroke

The apoptosis repressor with caspase recruitment domain(ARC)plays a critical role in extrinsic apoptosis initiation via death receptor ligands,physiological stress,infection response in a tissue-dependent manner,endoplasmic reticulum(ER)stress,genotoxic drugs,ionizing radiation,oxidative stress,and hypoxia.Recent studies have suggested that regulating apoptosis-related pathways can improve outcomes for patients with neurological diseases,such as hemorrhagic stroke.ARC expression is significantly correlated with acute cerebral hemorrhage.However,the mechanism by which it mediates the anti-apoptosis pathway remains poorly known.Here,we discuss the function of ARC in hemorrhagic stroke and argue that it could serve as an effective target for the treatment of hemorrhagic stroke.

Unraveling the regulatory network of flower coloration in soybean:Insights into roles of GmMYBA3 and GmMYBR1

Anthocyanins play crucial roles in pollen protection and pollinator attraction in flowering plants.However,the mechanisms underlying flower color determination and whether floral anthocyanin regulators participate in other processes remain largely unresolved in soybeans(Glycine max).In this study,we investigated the genetic components and mechanisms governing anthocyanin biosynthesis in soybean flowers.Molecular and genetic studies have characterized two antagonistic regulators,the positive activator GmMYBA3 and the negative repressor GmMYBR1,that modulate the gene expression of anthocyanin biosynthesis in soybean flowers.Further findings revealed a regulatory interplay between GmMYBA3 and GmMYBR1 bridged by GmTT8a,highlighting the complexity of anthocyanin regulation in different soybean organs.Exploration of additional soybean cultivars demonstrated the universality of GmMYBA3 and GmMYBR1 in regulating floral anthocyanin biosynthesis-related genes,with GmF3’5’H identified as a crucial determinant of white flower color.This study provides a molecular mechanism underlying soybean flower color determination,paving the way for the molecular modification of soybean flowers to probably enhance their resistance to abiotic stresses and attractiveness to pollinators.

Selection and design of high affinity DNA ligands for mutant single-chain derivatives of the bacteriophage 434 repressor

Single-chain repressor RRTRES is a derivative of bacteriophage 434 repressor, which contains covalently dimerized DNA-binding domains (amino acids 1-69) of the phage 434 repressor. In this single-chain molecule, the wild type domain R is connected to the mutant domain RTRES by a recombinant linker in a head-to-tail arrangement. The DNA-contacting amino acids of RTRES at the -1, 1, 2, and 5 positions of the a3 helix are T, R, E, S respectively. By using a randomized DNA pool containing the central sequence -CATACAAGAAAGNNNNNNTTT-, a cyclic, in vitro DNA-binding site selection was performed. The selected population was cloned and the individual members were characterized by determining their binding affinities to RRTRES. The results showed that the optimal operators contained the TTAC or TTCC sequences in the underlined positions as above, and that the Kd values were in the 1×10-12 mol/L-1×10-11mol/L concentration range. Since the affinity of the natural 434 repressor to its natural operator sites is in the 1×10-9 mol/L range, the observed binding affinity increase is remarkable. It was also found that binding affinity was strongly affected by the flanking bases of the optimal tetramer binding sites, especially by the base at the 5′ position. We constructed a new homodimeric single-chain repressor RTRESRTRES and its DNA-binding specificity was tested by using a series of new operators designed according to the recog-nition properties previously determined for the RTRES domain. These operators containing the con-sensus sequence GTAAGAAARNTTACN or GGAAGAAARNTTCCN (R is A or G) were recognized by RTRESRTRES specifically, and with high binding affinity. Thus, by using a combination of random selection and rational design principles, we have discovered novel, high affinity protein-DNA inter-actions with new specificity. This method can potentially be used to obtain new binding specificity for other DNA-binding proteins.

抑制蛋白在常见有遗传倾向疾病中的研究进展

抑制蛋白又称阻遏蛋白,是哺乳动物中细胞信号转导的调节因子,与许多疾病的发生密切相关。抑制蛋白可通过调控β-肾上腺素能受体和血管紧张素Ⅱ受体影响心血管舒张功能改善心血管疾病;上调视网膜色素上皮中的成纤维细胞维持视网膜稳态,以及在感光细胞中的增多对视网膜具有保护作用;降低多巴胺表达改善精神类疾病;参与小胶质细胞自噬过程改善神经系统疾病;增加肝细胞、脂肪细胞、β细胞的质量及增殖率改善胰岛素信号传导治疗代谢性相关疾病。本文综述抑制蛋白对以上具有遗传倾向常见疾病的发病机制及治疗等,旨在为临床治疗提供新的思路。

SOX9通过转化生长因子β信号通路调节角膜内皮损伤后内皮-间质转化过程

目的探究性别决定区Y框蛋白9(sex-determining region Y-box protein 9,SOX9)是否会调控角膜内皮细胞损伤后的角膜上皮-间质转化(endothelial-to-mesenchymal transition,EndMT)过程及具体机制。方法转染小干扰RNA(small interfering RNA,siRNA)敲低人角膜内皮细胞B4G12中SOX9的表达,使用甲萘醌构建体外B4G12细胞损伤模型,设4个分组:si-NC组(转染siRNA-阴性对照)、si-SOX9组(转染siRNA-SOX9)、si-NC+甲萘醌组(转染siRNA-阴性对照后添加外源性甲萘醌做损伤处理)、si-SOX9+甲萘醌组(转染siRNA-SOX9后添加外源性甲萘醌做损伤处理)。通过实时荧光逆转录聚合酶链反应(real-time reverse transcription polymerase chain reaction,RT-PCR)和Western blot检测各组细胞中EndMT关键因子Snail家族转录抑制因子2(snail family transcriptional repressor 2,SNAIL2)及相关信号通路关键因子表达变化,阐明SOX9调控EndMT过程的功能和机制。结果转染siRNA-SOX9敲低细胞SOX9表达后,给予甲萘醌细胞损伤处理,观察到细胞中SNAIL2的表达会随SOX9的敲低而降低,同时观察到转化生长因子β(transforming growth factor beta,TGF-β)信号通路中SNAIL2的上游关键因子Smad2/Smad3的表达也随着SOX9的敲低而降低。结论SOX9通过TGF-β信号通路调控角膜内皮损伤后EndMT过程。

SPOROCYTELESS Is a Novel Embryophyte-Specific Transcription Repressor that Interacts with TPL and TCP Proteins in Arabidopsis

Germlines in plants are formed de novo during post-embryonic development, while little is known about the mechanism that controls this process. In Arabidopsis, the earliest gene controlling this process is SPOROCYTELESS （SPL）. A decade ago, we showed that loss of SPL function abolished sporogenesis in both male and female organs of Arabidopsis. However, its function is unclear up to now. In this study, we showed that SPL belongs to a novel transcription repressor family specific in embryophyte, which consists of 173 members in the land plants so far. All of them contain a conserved SPL-motif in their N-terminal and an ethylene-responsive element binding factor-associated amphiphilic repression （EAR） motif in the C-terminal, therefore designated as SPL-like, EAR-containing proteins （SPEARs）. Consis- tently, SPL acts as a transcriptional repressor in yeast and tobacco cells, and SPEAR proteins are able to form homodimer and/or het- erodimer with each other in vitro. Furthermore, SPEARs interact with the TOPLESS （TPL） co-repressors via the EAR motif and TCP family transcription factors in yeast cells. Together, we propose that SPL and SPEARs most likely belong to a novel transcription repressor family in land plants which may play a variety of developmental roles in plants.

Repressor of GATA-3 can negatively regulate the expression of T cell cytokines through modulation on inducible costimulator

Background The transcription factor, repressor of GATA-3 （ROG）, can simultaneously suppress the expression of T helper cells （Thl and Th2） cytokines. Since the suppression of Th2 cytokines by GATA-3 is well understood, it is postulated that there are other molecular targets of ROG that can suppress the expression of the Thl cytokines. We hypothesized that ROG might suppress the stimulators of T lymphocyte cytokines such as CD3, CD28, and inducible costimulator （ICOS）, or indirectly enhance the expression of cytokine suppressors such as T lymphocyte-associated antigen-4 （CTLA-4） and CD45. The objective of this study was to clarify the molecular targets of ROG involved in suppressing Thl or Th2 cytokines. Methods Real-time quantitative PCR （RT-PCR） and Western blotting were performed to evaluate the mRNA and protein levels of CD3, CD28, ICOS, CTLA-4, and CD45 in Thl and Th2 cells during various levels of ROG expression. Enzyme-linked immunosorbent assay （ELISA） was used to measure the levels of interferon-y （IFN-y） and intedeukin （IL）-4 in culture media of Thl and Th2 cells. Results The results showed that the mRNA and protein levels of ROG were relatively low in Thl and Th2 cells （P 〈0.01）. After ROG-pcDNA3.1 transfection, the mRNA and protein level of ROG was significantly elevated, while the expression of ICOS, IFN-y, and IL-4 was markedly down-regulated （P 〈0.01）. Conversely, transfection of ROG-siRNA led to inhibition of ROG expression and up-regulation of ICOS, IFN-y and IL-4 （P 〈0.01）. However, the expression levels of CD3, CD28, CTLA-4 and CD45 did not change in either ROG-pcDNA3.1 or ROG-siRNA-transfected Thl and Th2 cells （P 〉0.05）. Conclusion It is concluded that ROG can inhibit the expression of Thl and Th2 cytokines by down-regulating the expression of ICOS, which might be a potential molecular target for asthma treatment.

COR27 and COR28 encode nighttime repressors integrating Arabidopsis circadian clock and cold response

Summary It was noted that circadian components function in plant adaptation to diurnal temperature cycles and freezing tolerance. Our genome-wide transcriptome analysis revealed that evening-phased COR27 and COR28 mainly repress the transcription of clockassociated evening genes PRRS, ELF4 and cold-responsive genes. Chromatin immunoprecipitation indicated that CCAI is recruited to the site containing EE elements of COR27 and COR28 promoters in a temperaturedependent way. Further genetic analysis shows COR28 is essential for the circadian function of PRR9 and PRRT. Together, our results support a role of COR27 and COR28 as nighttime repressors integrating circadian clock and plant cold stress responses.

A Single JAZ Repressor Controls the Jasmonate Pathway in Marchantia polymorpha

JAZ proteins are negative regulators of jasm onate responses,acting both as repressors of transcription factors and as co-receptors of JA-lle. The high redundancy of JAZ genes in angiosperms has hindered the characterization of a complete depletion of JAZ function. Moreover, the recent discovery that dn- OPDA is the jasmonate ligand in Marchantia polymorpha demonstrates that JA-lle is not the sole COI1/ JAZ ligand in land plants and highlights the importance of studying JAZ co-receptors in bryophytes? Here, we have exploited the low gene redundancy of the liverwort M. polymorpha to characterize the single MpJ4Z in this early diverging plant lineage. We clarify the phylogenetic history of the TIFY family, demonstrate that MpJAZ is the ortholog of AtJAZ with a conserved function, and characterize its repressor activity of dn-OPDA resp on ses. Our results show that, con sistent with previous findings in Arabidopsis, MpJAZ represses jasmonates biosynthesis, senescence, and plant defenses, and promotes cell growth and reproductive fitness, highlighting the power of studies in Marchantia.

A quantitative understanding of lac repressor＇s binding specificity and flexibility

Lac repressor, the first discovered transcriptional regulator, has been shown to confer multiple modes of binding to its operator sites depending on the central spacer length. Other homolog members in the LacI/GalR family （Purr and YcjW） cannot bind their operator sites with similar structural flexibility. To decipher the underlying mechanism for this unique property, we used Spec-seq approach combined with site-directed mutagenesis to quantify the DNA binding specificity of multiple hybrids of lacI and PurR. We find that lac repressor＇s recognition di-residues YQ and its hinge helix loop regions are both critical for its structural flexibility. Also, specificity profiling of the whole lac operator suggests that a simple additive model from single variants suffice to predict other multivariant sites＇ energy reasonably well, and the genome occupancy model based on this specificity data correlates well with in vivo iac repressor binding profile.