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A cis-regulatory genetic element which targets gene expression to stem cells, termed stem cell enhancer, serves as a molecular handle for stem cell-specific genetic engineering. Here we show the generation and characterization of a tamoxifen-inducible CreER

Epstein-Barr virus nuclear antigens 2 (EBNA2) mediated super-enhancers, defined by in silico data, localize near genes associated with B cell transcription factors including RUNX3. However, the biological function of super-enhancer for RUNX3 gene (seR3) remains unclear. Here, we show that two seR3s, tandemly-located at 59- and 70-kb upstream of RUNX3 transcription start site, named seR3 –59h and seR3 –70h, are required for RUNX3 expression and cell proliferation in Epstein-Barr virus (EBV)-positive malignant B cells. A BET bromodomain inhibitor, JQ1, potently suppressed EBV-positive B cell growth through the reduction of RUNX3 and MYC expression. Excision of either or both seR3s by employing CRISPR/Cas9 system resulted in the decrease in RUNX3 expression and the subsequent suppression of cell proliferation and colony forming capability. The expression of MYC was also reduced when seR3s were deleted, probably due to the loss of trans effect of seR3s on the super-enhancers for MYC. These findings suggest that seR3s play a pivotal role in expression and biological function of both RUNX3 and MYC. seR3s would serve as a potential therapeutic target in EBV-related widespread tumors.

The Four-and-a-half LIM (FHL)-only protein is a subfamily of protein members under the LIM-only protein family. These proteins are identified by their characteristic four and a half cysteinerich LIM homeodomain. Five members have been categorized into the FHL subfamily, which are FHL1, FHL2, FHL3, FHL4 and activator of CREM in testis (ACT) in human. FHL2 is amongst the most examined members within the family. Fhl2, the gene that code for the protein, is transcriptionally regulated by diverse types of transcription factors, for example, p53, serum response factor (SRF), and specificity protein 1 (Sp1). The expression of FHL2 is found in different tissues and organs and has been reported as a critical participant influencing the wide types of cancer such as breast cancer, gastrointestinal (GI) cancers, liver cancer and prostate cancer. The expression profile of FHL2 appeared to have a significant functional role in the carcinogenesis of these cancers which are mediated by different types of transcription factor including both tumor suppressors and inducers. In this review, we will first describe the molecular network governing FHL2 expression, which focus on the transcription factors conveying FHL2-initiated responses. In the second part, FHL2-linked cancers and the underlying molecular machinery will be discussed. Factors other than transcriptional regulation which may involve the cancer progression such as mutations of fhl2 and posttranslational modifications of the protein will also be mentioned.

Physical exercise induces gene expression changes that trigger glucose metabolism pathways in organisms. In the present study, we monitored the expression levels of LDHA (lactate dehydrogenase) and GYS1 (glycogen synthase 1) in the blood, to confirm the roles of these genes in exercise physiology. LDHA and GYS1 are related to glucose metabolism and fatigue recovery, and these processes could elicit economically important traits in racehorses. We collected blood samples from three retired thoroughbred racehorses, pre-exercise and immediately after 30 min of exercise. We extracted total RNA and small RNA (≤ 200 nucleotide-long) from the blood, and assessed the expression levels of LDHA, GYS1, and microRNAs (miRNAs), by using qRT-PCR. We showed that LDHA and GYS1 were down-regulated, whereas eca-miR-33a and miR-17 were up-regulated, after exercise. We used sequences from the 3′ UTR of LDHA and GYS1, containing eca-miR-33a and miR-17 binding sites, to observe the down-regulation activity of each gene expression. We observed that the two miRNAs, namely, eca-miR-33a and miR-17, inhibited LDHA and GYS1 expression via binding to the 3′ UTR sequences of each gene. Our results indicate that eca-miR-33a and miR-17 play important roles in the glucose metabolism pathway. In addition, our findings provide a basis for further investigation of the exercise metabolism of racehorses.

The RUNX1/AML1 gene is among the most frequently mutated genes in human leukaemia. However, its association with T-cell acute lymphoblastic leukaemia (T-ALL) remains poorly understood. In order to examine RUNX1 point mutations in T-ALL, we conducted an amplicon-based deep sequencing in 65 Southeast Asian childhood patients and 20 T-ALL cell lines, and detected RUNX1 mutations in 6 patients (9.2%) and 5 cell lines (25%). Interestingly, RUNX1-mutated T-ALL cases seem to constitute a subset of early immature T-ALL that may originate from differentiated T-cells. This result provides a deeper insight into the mechanistic basis for leukaemogenesis.

The flagellated protozoan Giardia intestinalis is one of the most prevalent human-infective parasites with a worldwide distribution. This parasite must encyst to complete the life cycle and N-acetylgalactosamine is produced from endogenous glucose for cyst wall synthesis during the transformation. UDP-N-acetylglucosamine pyrophosphorylase in G. intestinalis (GiUAP, EC 2.7.7.23) is the fourth enzyme in the inducible pathway of N-acetylgalactosamine biosynthesis, catalysing the conversion of N-acetylglucosamine-1-P to UDP-N-acetylglucosamine. In this study the gene GiUAP was cloned and sequenced from the Portland 1 strain using PCR techniques. It has an ORF of approximately 1.3 kb and contains no introns. BLAST and ClustalW analysis of the deduced amino acid sequence revealed significant similarities to other eukaryotic UAPs with putative active sites identified. Southern hybridization showed that GiUAP exists as a single-copy gene and it was shown to have two transcripts by RT-PCR and Northern hybridization. RLM-RACE identified both 5' and 3' untranslated regions and suggested the transcripts exist as a 5'-capped mRNA, with the use of two tandem polyadenylation sites to generate two unusually long giardial 3' untranslated regions of approximately 522 bp and approximately 3 kb. Moreover, a recombinant protein (rGiUAP) was expressed in E. coli and subjected to physical characterizations. Surprisingly the results obtained in this study were significantly different from those reported for the GiUAP in MR4 strain, suggesting this gene is under different transcription control in different strains of G. intestinalis. This report describes the molecular characterization of GiUAP and provides an opportunity to explore the control of gene expression during encystation of the parasite.

MicroRNAs (miRNAs) generally pair with the 3'UTRs of their target mRNAs to repress gene expression. It has reported that miRNA targets (TGs) are longer and evolve more slowly than non-targets (NTGs). We confirmed the observation and also found novel structural and expressional characteristics of TGs. The length difference between TGs and NTGs was greatest for the 3'UTRs, although a difference was also observed for CDSs and introns. Widely expressed genes were shorter for both TGs and NTGs; however, TGs were significantly longer than NTGs in all ranges of expression. TGs were more likely than NTGs to be widely expressed, which might explain why TGs evolve more slowly than NTGs. Finally, we found that TG mRNAs have faster decay rates. In addition, the decay rate of a TG mRNA transcript was found to be positively correlated with the number or density of target sites located in that TG's mRNA transcript.

In the mitochondrial matrix, the OXCT1 gene catalyzes the reversible transfer of coenzyme A from succinyl-CoA to acetoacetate in a reaction related to energy production from ketone bodies. Here, horse OXCT1 gene containing coenzyme A transferase domain was identified in the transcriptome analysis of cDNAs derived from skeletal muscles. Horse OXCT1 gene consisted of 1761 [corrected] nucleotide sequences with an open reading frame of 1560 nucleotides encoding a protein of 520 putative amino acid residues.The number of non-synonymous substitutions was lower than the number of synonymous substitutions in the OXCT1 genes of other species, indicating that purifying selection occurred in the OXCT1 genes during evolutionary radiation. Quantitative real-time RT-RCR analysis showed a dominant expression pattern of horse OXCT1 gene in the cerebrum, heart, and skeletal muscle. Different expression levels of horse OXCT1 transcripts between before- and after-exercise samples were also measured in the skeletal muscles of six horses. These data could be of great use for further investigation of the relationship between energy products and horse OXCT1 gene.

A recombinant phage clone containing the 5' end of the gene HRS encoding human histidyl-tRNA synthetase (HRS) has been isolated. Primer extension analyses indicated that there are two types of HRS transcripts. The longer transcripts were initiated from a single transcription start point (tsp) located approximately 455 bp upstream and the shorter transcripts were initiated from multiple tsp located approximately 38 to 82 bp upstream from the HRS ATG start codon. Functionally, we have identified two regions (+1 to −122; −185 to −502), each of which when placed 5' of a promoterless cat construct can initiate transcription in both orientations after transfection into HeLa cells. A pair of imperfect inverted repeats (IIR) was located within the region +1 to −122. Using mobility shift assays, we have identified a nuclear factor that binds specifically to each half of the IIR. However, this pair of IIR (−73 to −110) was not sufficient for bidirectional transcription activity. At least one copy of a 27-bp oligodeoxyribonucleotide (oligo), which spans − 94 to−120, was required in order to facilitate bidirectional transcription activity. From mobility shift assays using HeLa cell nuclear extracts and this 27-bp oligo, we have identified two DNA-protein complexes, both of which are presumably required to initiate bidirectional transcription.

The 3′-terminal nucleotide (nt) sequence of the coat protein (CP)-encoding gene of the Korean isolate of cymbidium mosaic potexvirus (CyMV-K) has been determined. The sequence contained a full-length ORF coding for the viral CP. It comprises 663 nt and a polypeptide chain of 220 amino acids with a M r of 23 718. The stop codon (TGA) is followed by a 3′-untranslated region (3′-UTR) of 79 nt and a poly(A) tract. The putative polyadenylation signal, AATAAA motif, is found in the terminus of the 3′-UTR.