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p73, a p53 family member is a transcription factor that plays a role in cell cycle, differentiation and apoptosis. p73 is regulated through post translational modifications and protein interactions. c-Abl is the only known tyrosine kinase that phosphorylates and activates p73. Here we have analyzed the role of Src family kinases, which are involved in diverse signaling pathways, in regulating p73. Exogenously expressed as well as cellular Hck and p73 interact in vivo. In vitro binding assays show that SH3 domain of Hck interacts with p73. Co-expression of p73 with Hck or c-Src in mammalian cells resulted in tyrosine phosphorylation of p73. Using site directed mutational analysis, we determined that Tyr-28 was the major site of phosphorylation by Hck and c-Src, unlike c-Abl which phosphorylates Tyr-99. In a kinase dependent manner, Hck co-expression resulted in stabilization of p73 protein in the cytoplasm. Activation of Hck in HL-60 cells resulted in tyrosine phosphorylation of endogenous p73. Both exogenous and endogenous Hck localize to the nuclear as well as cytoplasmic compartment, just as does p73. Ectopically expressed Hck repressed the transcriptional activity of p73 as determined by promoter assays and semi-quantitative RT-PCR analysis of the p73 target, Ipaf and MDM2. SH3 domain- dependent function of Hck was required for its effect on p73 activity, which was also reflected in its ability to inhibit p73-mediated apoptosis. We also show that Hck interacts with Yes associated protein (YAP), a transcriptional co-activator of p73, and shRNA mediated knockdown of YAP protein reduces p73 induced Ipaf promoter activation. We have identified p73 as a novel substrate and interacting partner of Hck and show that it regulates p73 through mechanisms that are dependent on either catalytic activity or protein interaction domains. Hck-SH3 domain-mediated interactions play an important role in the inhibition of p73-dependent transcriptional activation of a target gene, Ipaf, as well as apoptosis.

Coupling of alternative splicing with nonsense-mediated mRNA decay (NMD) may regulate gene expression. We report here the identification of a nonsense alternative transcript of the fumarylacetoacetate hydrolase (fah) gene, which produces a protein despite the fact that it is subject to NMD. During the characterization of the effects of the W262X nonsense mutation on FAH mRNA metabolism, two alternative transcripts (del100 and del231) of the fah gene were identified. Del100 lacks exon 8 and as a consequence, the reading frame is shifted and a premature termination codon appears at the 3'end of exon 10. Exons 8 and 9 are skipped in del231, without any disruption of the reading frame. Specific amplification of these transcripts demonstrate that they are produced through minor alternative splicing pathways, and that they are not caused by the W262X mutation per se. As shown with an antiserum raised against the C-terminal part of the putative DEL100 protein, the del100 transcript produces a protein, expressed at different levels in various human tissues. Interestingly, the del100 transcript seems to be subjected to nonsense-mediated mRNA decay, as its level was stabilized following a cycloheximide treatment. The del100 and del231 transcripts arise due to minor alternative splicing pathways and del100 is likely subjected to nonsense-mediated mRNA decay. However the remaining amount of transcript seems sufficient to produce a protein in different human tissues. This suggests that NMD has a broader role than simply eliminating aberrant transcripts and when coupled to alternative splicing, may act to modulate gene expression, by allowing the production of low amounts of protein.

Pifithrin-α is a small molecule inhibitor of p53 transcriptional activity. It has been proposed that the use of pifithrin-α in conjunction with chemotherapeutic and radiation therapies for cancer will reduce the side effects of these treatments in normal tissue that still contains wild type p53. In addition, pifithrin-α provides a useful tool in the laboratory to investigate the function of p53 in model systems. While investigating the effects of pifithrin-α on the transcriptional activity of NF-κB, we observed a strong inhibition of reporter plasmids containing the firefly luciferase gene. Firefly luciferase is one of the most commonly used enzymes in reporter gene assays. In contrast, no inhibition of reporter plasmids containing Renilla luciferase or chloramphenicol acetyltransferase was observed. The inhibition of firefly luciferase activity by pifithrin-α was observed both in vivo and in vitro. Pifithrin-α did not inhibit firefly luciferase protein expression, but rather suppressed light production/emission, since addition of exogenous pifithrin-α to active extracts inhibited this activity. Furthermore, pifithrin-α also inhibited recombinant firefly luciferase protein activity. Among its other biological activities, pifithrin-α is an inhibitor of firefly luciferase activity. Caution must therefore be taken when using this compound, which has been characterised as an inhibitor of p53 transcriptional activity, to investigate effects on gene expression using transiently transfected reporter plasmids. Furthermore, these results demonstrate that when using novel compounds, the choice of vectors used in the experimental procedures might be of great importance for the correct conclusions to be made.

In functional genomics, transcript measurement is of fundamental importance. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays are the most popular technology and depend on the initial molecular step, the reverse transcription (RT). This study provides a complex overview of the influence of elements such as RT systems, amount of background RNA, and transcript abundance on the efficiency of qRT-PCR. Using qRT-PCR, we compared the efficiency of some commonly used RT systems and measured the production of PCR-amplifiable products and the influence of PCR inhibitor contents. The qRT-PCR assays were conducted using the TaqMan system, although we also tested the SYBR Green I chemistry, which is not compatible with all the RT systems. When dealing with low-abundance transcripts, the SuperScript II system generated more detectable molecules than the four other systems tested: Sensiscript, Omniscript, SuperScript III and PowerScript (P < 0.05). However, the Sensiscript and PowerScript systems were more efficient for detecting high-abundance transcripts in the presence of 1 to 2 μg background RNA (P < 0.05). The most striking aspect was the influence of the dilution of the RT reaction on the subsequent PCR. Indeed, some inhibition was released when diluted RT reactions were used for the quantitative PCR measurements. Furthermore, the amount of background RNA in the RT reaction was also a major component influencing a downstream step in qRT-PCR, the PCR reaction. Whereas Sensiscript was less biased, the other systems contained an important source of PCR inhibitors, interfering as much as 70% with the qRT-PCR. This study provides a complex overview of the influence of elements such as RT systems, qRTPCR chemistry, amount of background RNA, and transcript abundance on the efficiency of qRT-PCR. Whereas the most significant influencing factor is the presence of inhibitors in the RT systems, total background RNA is also a major influencing component that affects the PCR reaction. Whenever the aim of a study is to obtain a precise gene expression measurement or to profile the global transcriptome (e.g. microarray), the RT step is critical and should be examined with care.

Trypanosomes mostly control gene expression by post-transcriptional events such as modulation of mRNA stability and translational efficiency. These mechanisms involve RNA-binding proteins (RBPs), which associate with transcripts to form messenger ribonucleoprotein (mRNP) complexes. In this study, we report the identification of mRNA targets for Trypanosoma cruzi U-rich RBP 1 (Tc UBP1) and T. cruzi RBP 3 (Tc RBP3), two phylogenetically conserved proteins among Kinetoplastids. Co-immunoprecipitated RBP-associated RNAs were extracted from mRNP complexes and binding of RBPs to several targets was confirmed by independent experimental assays. Analysis of target transcript sequences allowed the identification of different signature RNA motifs for each protein. Cis-elements for RBP binding have a stem-loop structure of 30–35 bases and are more frequently represented in the 3'-untranslated region (UTR) of mRNAs. Insertion of the correctly folded RNA elements to a non-specific mRNA rendered it into a target transcript, whereas substitution of the RNA elements abolished RBP interaction. In addition, RBPs competed for RNA-binding sites in accordance with the distribution of different and overlapping motifs in the 3'-UTRs of common mRNAs. Functionally related transcripts were preferentially associated with a given RBP; Tc UBP1 targets were enriched in genes encoding proteins involved in metabolism, whereas ribosomal protein-encoding transcripts were the largest group within Tc RBP3 targets. Together, these results suggest coordinated control of different mRNA subsets at the post-transcriptional level by specific RBPs.

The Enabled/Vasodilator stimulated phosphoprotein (Ena/VASP) gene family comprises three genes in vertebrates: Vasp, Enabled homologue (Enah) and Ena-VASP like (Evl). Enah has the most complex gene structure. It has extra alternatively included exons compared to Vasp and Evl, and possibly one alternatively excluded intron S. The aim of this mapping study was to probe the occurrence of combinations of exon usage in Enah thereby identifying possible vertebrate ENAH splice variants. We investigated this via an in silico analysis and by performing a reverse transcription-polymerase chain reaction (RT-PCR) screen on mouse samples. We further probed the expression pattern of mouse Enah splice variants during development and in a selection of mouse adult tissues and mouse cell lines. In silico analysis of the vertebrate Ena/VASP gene family reveals that birds do not have Vasp, while fish have two Evl genes. Analysis of expressed sequence tags of vertebrate Enah splice variants confirms that an Enah transcript without alternative exons is ubiquitously expressed, but yields only limited information about the existence of other possible alternatively spliced Enah transcripts. Via a RT-PCR screen, we provide evidence that during mouse development and in adult mice at least eight and maximally sixteen different Enah transcripts are expressed. We also show that tissues and cell lines display specific expression profiles of these different transcripts. Exons previously associated with neuronal expression of Enah splice variants are also present in other tissues, in particular in heart. We propose a more uniform nomenclature for alternative exons in Enah. We provide an overview of distinct expression profiles of mouse Enah splice variants during mouse development, in adult mouse tissues and in a subset of mouse cell lines.

Ranh giới cromatin, còn được gọi là bộ cách, điều chỉnh hoạt động gen bằng cách tổ chức các miền cromatin hoạt động và ức chế và điều chỉnh các tương tác giữa tăng cường và promoter. Tuy nhiên, cơ chế hoạt động của các ranh giới vẫn chưa được hiểu rõ, một phần là do kiến thức hạn chế của chúng ta về các protein bộ cách và sự thiếu thốn các xét nghiệm chuẩn mà có thể so sánh các ranh giới khác nhau. Chúng tôi báo cáo ở đây sự phát triển của một xét nghiệm chặn tăng cường để nghiên cứu hoạt động của bộ cách trong các tế bào Drosophila nuôi cấy. Chúng tôi cho thấy rằng hoạt động của các bộ cách Drosophila khác nhau, bao gồm suHw, SF1, SF1b, Fab7 và Fab8, được hỗ trợ trong các tế bào này. Chúng tôi cũng chỉ ra rằng sự giảm mức thuốc của các yếu tố SuHw và dCTCF thông qua RNA chuỗi đôi (dsRNA) làm gián đoạn chức năng ngăn chặn tăng cường của suHw và Fab8, từ đó xác lập hiệu quả của việc sử dụng can thiệp RNA trong xét nghiệm dựa trên tế bào của chúng tôi để điều tra chức năng của bộ cách. Xét nghiệm ranh giới mới cung cấp một phương pháp định lượng và hiệu quả để phân tích cơ chế bộ cách và có thể được khai thác thêm trong các sàng lọc RNAi trên quy mô toàn bộ gen cho các thành phần bộ cách. Nó cung cấp một công cụ hữu ích bổ sung cho các phương pháp chuyển gen và di truyền để nghiên cứu loại yếu tố điều tiết quan trọng này.

The Gateway recombinatorial cloning system allows easy and rapid joining of DNA fragments. Here we report the construction and evaluation of three different Gram-positive vectors that can be used with the Multisite Gateway cloning system to rapidly produce new gene arrangements in plasmid constructs for use in a variety of Gram-positive bacteria. Comparison of patterns of reporter gene expression with conventionally constructed clones show that the presence of residual recombination (att) sites does not have an effect on patterns of gene expression, although overall levels of gene expression may vary. Rapid construction of these new vectors allowed vector/gene combinations to be optimized following evaluation of plasmid constructs in different bacterial cells and demonstrates the benefits of plasmid construction using Gateway cloning. The residual att sites present after Gateway cloning did not affect patterns of promoter induction in Gram-positive bacteria and there was no evidence of differences in mRNA stability of transcripts. However overall levels of gene expression may be reduced, possibly due to some post-transcriptional event. The new vectors described here allow faster, more efficient cloning in range of Gram-positive bacteria.

Circulating CD4+ T helper cells are activated through interactions with antigen presenting cells and undergo differentiation into specific T helper cell subsets depending on the type of antigen encountered. In addition, the relative composition of the circulating CD4+ T cell population changes as animals mature with an increased percentage of the population being memory/effector type cells. Here, we report on the highly plastic nature of DNA methylation at the genome-wide level as T cells undergo activation, differentiation and aging. Of particular note were the findings that DNA demethylation occurred rapidly following T cell activation and that all differentiated T cell populations displayed lower levels of global methylation than the non-differentiated population. In addition, T cells from older mice had a reduced level of DNA methylation, most likely explained by the increase in the memory/effector cell fraction. Although significant genome-wide changes were observed, changes in DNA methylation at individual genes were restricted to specific cell types. Changes in the expression of enzymes involved in DNA methylation and demethylation reflect in most cases the changes observed in the genome-wide DNA methylation status. We have demonstrated that DNA methylation is dynamic and flexible in CD4+ T cells and changes rapidly both in a genome-wide and in a targeted manner during T cell activation, differentiation. These changes are accompanied by parallel changes in the enzymatic complexes that have been implicated in DNA methylation and demethylation implying that the balance between these opposing activities may play a role in the maintaining the methylation profile of a given cell type but also allow flexibility in a cell population that needs to respond rapidly to environmental signals.

The circadian rhythm of about 24 hours is a fundamental physiological function observed in almost all organisms from prokaryotes to humans. Identification of clock genes has allowed us to study the molecular bases for circadian behaviors and temporal physiological processes such as hormonal secretion, and has prompted the idea that molecular clocks reside not only in a central pacemaker, the suprachiasmatic nuclei (SCN) of hypothalamus in mammals, but also in peripheral tissues, even in immortalized cells. Furthermore, previous molecular dissection revealed that the mechanism of circadian oscillation at a molecular level is based on transcriptional regulation of clock and clock-controlled genes. We systematically analyzed the mRNA expression of clock and clock-controlled genes in mouse peripheral tissues. Eight genes (mBmal1, mNpas2, mRev-erbα, mDbp, mRev-erbβ, mPer3, mPer1 and mPer2; given in the temporal order of the rhythm peak) showed robust circadian expressions of mRNAs in all tissues except testis, suggesting that these genes are core molecules of the molecular biological clock. The bioinformatics analysis revealed that these genes have one or a combination of 3 transcriptional elements (RORE, DBPE, and E-box), which are conserved among human, mouse, and rat genome sequences, and indicated that these 3 elements may be responsible for the biological timing of expression of canonical clock genes. The observation of oscillatory profiles of canonical clock genes is not only useful for physiological and pathological examination of the circadian clock in various organs but also important for systematic understanding of transcriptional regulation on a genome-wide basis. Our finding of the oscillatory expression of canonical clock genes with a temporal order provides us an interesting hypothesis, that cyclic timing of all clock and clock-controlled genes may be dependent on several transcriptional elements including 3 known elements, E-box, RORE, and DBPE.