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Uveal melanoma (UM) remains the most common intraocular malignancy among diseases affecting the adult eye. The primary tumor disseminates to the liver in half of patients and leads to a 6 to 12-month survival rate, making UM a particularly aggressive type of cancer. Genomic analyses have led to the development of gene-expression profiles that can efficiently predict metastatic progression. Among these genes, that encoding the serotonin receptor 2B (HTR2B) represents the most discriminant from this molecular signature, its aberrant expression being the hallmark of UM metastatic progression. Recent evidence suggests that expression of HTR2B might be regulated through the Janus kinase/Signal Transducer and Activator of Transcription proteins (JAK/STAT) intracellular signalization pathway. However, little is actually known about the molecular mechanisms involved in the abnormally elevated expression of the HTR2B gene in metastatic UM and whether activated STAT proteins participates to this mechanism. In this study, we determined the pattern of STAT family members expressed in both primary tumors and UM cell-lines, and evaluated their contribution to HTR2B gene expression. Examination of the HTR2B promoter sequence revealed the presence of a STAT putative target site (5'-TTC (N)3 GAA3') located 280 bp upstream of the mRNA start site that is completely identical to the high affinity binding site recognized by these TFs. Gene profiling on microarrays provided evidence that metastatic UM cell lines with high levels of HTR2B also express high levels of STAT proteins whereas low levels of these TFs are observed in non-metastatic UM cells with low levels of HTR2B, suggesting that STAT proteins contribute to HTR2B gene expression in UM cells. All UM cell lines tested were found to express their own pattern of STAT proteins in Western blot analyses. Furthermore, T142 and T143 UM cells responded to interleukins IL-4 and IL-6 by increasing the phosphorylation status of STAT1. Most of all, expression of HTR2B also considerably increased in response to both IL-4 and IL-6 therefore providing evidence that HTR2B gene expression is modulated by STAT proteins in UM cells. The binding of STAT proteins to the -280 HTR2B/STAT site was also demonstrated by electrophoretic mobility shift assay (EMSA) analyses and site-directed mutation of that STAT site also abolished both IL-4 and IL-6 responsiveness in in vitro transfection analyses. The results of this study therefore demonstrate that members from the STAT family of TFs positively contribute to the expression of HTR2B in uveal melanoma.

Background: Human organic cation transporter 3 (OCT3,SLC22A3) mediates the uptake of many important endogenous substances and basic drugs, and has been identified as one of the transporters that are highly expressed in human skin. However, the mechanisms responsible for variability in mRNA expression, and the role of SLC22A3 in human skin is not clear., Objective: We examined the effects of the single nucleotide polymorphisms ofSLC22A3 on the variability in SLC22A3 expression and sebum levels in humans., Methods: Immunostaining of OCT3 in human skin was performed. We analyzed the association of promoter variants with the SLC22A3 mRNA expression levels in human skins. Luciferase, knockdown, chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay were employed to investigate transcriptional regulation of SLC22A3 expression. Effects of the identified variant on sebum levels were evaluated in healthy volunteers., Results: Immunohistochemistry revealed marked expressions of OCT3 in the basal epidermis, sebaceous glands, hair follicles, and sweat glands of human skin. SLC22A3 mRNA levels were significantly lower in skin samples with homozygotes for -1603A/A than in those for -1603 G/G. The analysis of p53 binding to -1603 G > A in the promoter ofSLC22A3 suggested that -1603 G > A down-regulates SLC22A3 gene expression by decreased p53 binding in the vicinity of the -1603 site. In humans, squalene levels in samples from the back at the baseline were significantly lower in homozygotes for -1603A/A than in those for -1603 G/G., Conclusion: These results suggest that the genetic variant contributes to the variability of expression and activities of OCT3 in human skin., (Copyright © 2020 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

We previously reported that blue eggshell color in chickens is associated with a partial endogenous retroviral (EAV-HP) insertion in the promoter region of the solute carrier organic anion transporter family member 1B3 (SLCO1B3) gene. The EAV-HP sequence includes numerous regulatory elements, which may modulate the expression of adjacent genes. To determine whether this insertion influences the expression of neighboring genes, we screened the expression of solute carrier organic anion transporter family members 1C1, 1B1 (SLCO1C1, SLCO1B1), and SLCO1B3 in 13 and 10 tissues from female and male Yimeng chickens, respectively. We observed that the insertion only significantly modulated the expression of SLCO1B3 and did not majorly affect that of SLCO1C1 and SLCO1B1. High expression of SLCO1B3 was detected in the shell gland, magnum, isthmus, and vagina of the oviduct in female blue-eggshell chickens. We also observed ectopic expression of SLCO1B3 in the testes of male chickens. SLCO1B3 is typically highly expressed in the liver; however, the EAV-HP insertion significantly reduces SLCO1B3 expression. As a liver-specific transporter, a reduction in the expression of SLCO1B3 may affect liver metabolism, particularly that of bile acids. We also detected higher ectopic expression of SLCO1B3 in the lungs of birds heterozygous for the EAV-HP insertion than in homozygous genotypes. In conclusion, we confirmed that the EAV-HP insertion modifies SLCO1B3 expression, and showed, for the first time, similar expression profile of this gene in all parts of the oviduct in females and testis in males. We also observed different levels of SLCO1B3 expression in the liver, which were associated with the EAV-HP insertion, and significantly higher expression in the lungs of birds with heterozygous genotype. The effects of these changes in the SLCO1B3 expression pattern on the function of the tissues warrant further investigation., (Copyright © 2020. Published by Elsevier Inc.)

Here, we report a rapid CRISPR-Cas9-mediated gene knock-in strategy that uses Cas9 ribonucleoprotein and 5'-modified double-stranded DNA donors with 50-base-pair homology arms and achieved unprecedented 65/40% knock-in rates for 0.7/2.5 kilobase inserts, respectively, in human embryonic kidney 293T cells. The identified 5'-end modification led to up to a fivefold increase in gene knock-in rates at various genomic loci in human cancer and stem cells.

Somatic DNMT3A mutations at R882 are frequently observed in AML patients including the very abundant R882H, but also R882C, R882P and R882S. Using deep enzymology, we show here that DNMT3A-R882H has more than 70-fold altered flanking sequence preferences when compared with wildtype DNMT3A. The R882H flanking sequence preferences mainly differ on the 3' side of the CpG site, where they resemble DNMT3B, while 5' flanking sequence preferences resemble wildtype DNMT3A, indicating that R882H behaves like a DNMT3A/DNMT3B chimera. Investigation of the activity and flanking sequence preferences of other mutations of R882 revealed that they cause similar effects. Bioinformatic analyses of genomic methylation patterns focusing on flanking sequence effects after expression of wildtype DNMT3A and R882H in human cells revealed that genomic methylation patterns reflect the details of the altered flanking sequence preferences of R882H. Concordantly, R882H specific hypermethylation in AML patients was strongly correlated with the R882H flanking sequence preferences. R882H specific DNA hypermethylation events in AML patients were accompanied by R882H specific mis-regulation of several genes with strong cancer connection, which are potential downstream targets of R882H. In conclusion, our data provide novel and detailed mechanistic understanding of the pathogenic mechanism of the DNMT3A R882H somatic cancer mutation., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In order to apply a useful STR system in DNA database construction, we performed a population study in China. Allele and genotype frequencies for STR SE33 were obtained for a sample of 213 random Chinese in view of application in personal identification. And we observed a new structural variation of 21.2 allele at SE33 locus which is described here for the first time., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Changes of Selenoprotein F (SELENOF) protein levels have been reported during selenium supplementation, stressful, and pathological conditions. However, the mechanisms of how these external factors regulate SELENOF gene expression are largely unknown. In this study, HEK293T cells were chosen as an in vitro model. The 5'-flanking regions of SELENOF were analyzed for promoter features. Dual-Glo Luciferase assays were used to detect promoter activities. Putative binding sites of Heat Shock Factor 1 (HSF1) were predicted in silico and the associations were further proved by chromatin immunoprecipitation (ChIP) assay. Selenate and tunicamycin (Tm) treatment were used to induce SELENOF up-regulation. The fold changes in SELENOF expression and other relative proteins were analyzed by Q-PCR and western blot. Our results showed that selenate and Tm treatment up-regulated SELENOF at mRNA and protein levels. SELENOF 5'-flanking regions from -818 to -248 were identified as core positive regulatory element regions. Four putative HSF1 binding sites were predicted in regions from -1430 to -248, and six out of seven primers detected positive results in ChIP assay. HSF1 over-expression and heat shock activation increased the promoter activities, and mRNA and protein levels of SELENOF. Over-expression and knockdown of HSF1 showed transcriptional regulation effects on SELENOF during selenate and Tm treatment. In conclusion, HSF1 was discovered as one of the transcription factors that were associated with SELENOF 5'-flanking regions and mediated the up-regulation of SELENOF during selenate and Tm treatment. Our work has provided experimental data for the molecular mechanism of SELENOF gene regulation, as well as uncovered the involvement of HSF1 in selenotranscriptomic for the first time.

The maize (Zea mays) enzyme β-carotene hydroxylase 2 (ZmBCH2) controls key steps in the conversion of β-carotene to zeaxanthin in the endosperm. The ZmBCH2 gene has an endosperm-preferred and developmentally regulated expression profile, but the detailed regulatory mechanism is unknown. To gain insight into the regulation of ZmBCH2, we isolated 2036 bp of the 5'-flanking region containing the 263 bp 5'-untranslated region (5'-UTR) including the first intron. We linked this to the β-glucuronidase reporter gene gusA. We found that high-level expression of gusA in rice seeds requires the 5'-UTR for enhanced activation. Truncated variants of the ZmBCH2 promoter retained their seed-preferred expression profile as long as a prolamin box and AACA motif were present. We identified candidate genes encoding the corresponding transcription factors (ZmPBF and ZmGAMYB) and confirmed that their spatiotemporal expression profiles are similar to ZmBCH2. Both ZmPBF and ZmGAMYB can transactivate ZmBCH2 expression in maize endosperm. To eliminate potential confounding effects in maize, we characterized the regulation of the minimal promoter region of ZmBCH2 in transgenic rice. This revealed that ZmPBF and ZmGAMYB independently transactivate the ZmBCH2 promoter. The mechanism that underpins our data provides an exciting new strategy for the control of target gene expression in engineered plants., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Lysophosphatidic acid (LPA) is an endogenous lysophospholipid with signaling properties outside of the cell and it signals through specific G protein-coupled receptors, known as LPA 1-6 . For one of its receptors, LPA 1 (gene name Lpar1) , details on the cis-acting elements for transcriptional control have not been defined. Using 5'RACE analysis, we report the identification of an alternative transcription start site of mouse Lpar1 and characterize approximately 3,500 bp of non-coding flanking sequence 5' of mouse Lpar1 gene for promoter activity. Transient transfection of cells derived from mouse neocortical neuroblasts with constructs from the 5' regions of mouse Lpar1 gene revealed the region between -248 to +225 serving as the basal promoter for Lpar1 . This region also lacks a TATA box. For the region between -761 to -248, a negative regulatory element affected the basal expression of Lpar1 . This region has three E-box sequences and mutagenesis of these E-boxes, followed by transient expression, demonstrated that two of the E-boxes act as negative modulators of Lpar1 . One of these E-box sequences bound the HeLa E-box binding protein (HEB), and modulation of HEB levels in the transfected cells regulated the transcription of the reporter gene. Based on our data, we propose that HEB may be required for a proper regulation of Lpar1 expression in the embryonic neocortical neuroblast cells and to affect its function in both normal brain development and disease settings.

The objective of this study was to evaluate the association between the cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) gene and piglet diarrhea. In this study, the mRNA expression of the CTLA4 gene increased significantly in IPEC-J2 cells after Escherichia coli K88 infection. Single nucleotide polymorphisms (SNPs) located in the 5' flanking region (SNPs g.107281989C>T) and 3'-untranslated region (3'-UTR; SNPs g.107288753C>A) were identified, and they were in linkage disequilibrium in both Min pigs and the Landrace population. Association analysis showed that Landrace piglets with a TT or AA genotype had a lower diarrhea index, and AA animals had higher average daily gain when compared to CC pigs, respectively (p < 0.05). However, the relationship between SNPs and diarrhea and performance traits in the Min population was not significant. Haplotype analysis indicated that the TC haplotype had the lowest diarrhea index. The 5' flanking deletion assay suggested that SNP g.107281989C>T was a molecular marker instead of the functional marker. This research demonstrated that genetic variances in the CTLA4 gene had significant effects on Landrace piglet diarrhea resistance.

Bisphenol A (BPA), a representative endocrine disrupting compound, exists ubiquitously in the aquatic environment. Several studies on fish have validated the role of BPA in the lipid metabolism. However, the action mechanisms of BPA on lipid metabolism have been little studied. To clarify how BPA regulates lipid metabolism, Gobiocypris rarus were exposed to 15 μg/L BPA for 3 and 6 weeks. Results showed that BPA altered lipid content by regulating some metabolism-related genes. The BPA's inhibiting effect on fatty acid β-oxidation might be stronger than on lipid synthesis. BPA disturbed the expression of acaca (acetyl-CoA carboxylase), fasn (fatty acid synthase) and cpt1α (carnitine palmitoyltransferase 1α) by altering the sterol regulatory element binding protein 1 (SREBP-1) binding to their sterol regulatory elements (SREs). Our result also revealed that DNA methylation in the 5' flanking regions of cpt1α could perturb the SREBP-1 binding adjacent to its SRE in females under BPA exposure. Besides, BPA exposure led to gender-specific effect on fatty acid β-oxidation in G. rarus. This will contribute to our understanding of the regulation mechanisms of BPA on lipid metabolism in fish., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Bisphenol A (BPA), an ubiquitous environmental endocrine disruptor chemical, disturbs the mRNA expressions of steroidogenic genes and subsequently steroid hormone synthesis in mammals and aquatic species. However, the underlying regulation mechanisms are barely understood, especially in fish. To explore the regulation mechanism, we exposed female rare minnow Gobiocypris rarus (G. rarus) to BPA at a nominal concentration of 15 μg/L for 7 and 14 days in the present study. Results showed significant increase of gonad somatic index (GSI) and serum estradiol (E2) levels in response to BPA at day 14. The 7-day BPA exposure notably repressed the expression of two ovarian steroidogenic genes (star and hsd11b2) and suppressed their capacity of estrogen response elements (ERE) to recruit estrogen receptor (ER), while the 14-day BPA treatment remarkably induced transcript of hsd3b and enhanced the capacity of ERE to recruitment ER in ovaries. Furthermore, the 7-day BPA exposure caused DNA hypermethylation of star (CpGs: -742 bp and -719 bp) and hsd11b2 (CpG: -1788 bp). However, 14-day BPA exposure resulted in DNA hypomethylation of hsd3b (CpG: -181 bp). Correlation analysis revealed that the DNA methylation levels at specific CpGs in star, hsd3b and hsd11b2 were significantly correlated to their mRNA levels and ER-EREs interactions. These findings suggest that the disturbed steroidogenesis and the transcripts of ovarian steroidogenic genes might attribute to the altered DNA methylation status of these ovarian steroidogenic genes in response to BPA., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background/aim: This study aimed to determine the effect of different BRCA1 exonal mutations on the clinical course of epithelial ovarian cancer (EOC)., Patients and Methods: Clinicopathological variables and survival outcomes were compared among 53 primary EOC patients with pathogenic BRCA1 mutations in exons 1-11 (5' mutations) and in exons 12-24 (3' mutations)., Results: BRCA1 5' exonal mutations were found in 35 (66.0%) patients. The median follow-up period was 40 months. Clinicopathological variables remained unchanged between the two groups. Patients with 5' mutations had a significantly longer progression-free survival than those with C-terminal mutations (p=0.034), better predicting progression-free survival [2.923 (1.402-6.093), p=0.004], but not overall survival in cases of multiple relapses (p=0.497)., Conclusion: N-terminal BRCA1 mutations in EOC patients are associated with favourable primary progression-free survival, a trend observed only in primary progression-free survival, not in overall survival., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Melting-flesh peaches produce large amounts of ethylene, resulting in rapid fruit softening at the late-ripening stage. In contrast, stony hard peaches do not soften and produce little ethylene. The indole-3-acetic acid (IAA) level in stony hard peaches is low at the late-ripening stage, resulting in low ethylene production and inhibition of fruit softening. To elucidate the mechanism of low IAA concentration in stony hard peaches, endogenous levels of IAA and IAA intermediates or metabolites were analysed by ultra-performance liquid chromatography-tandem mass spectrometry. Although the IAA level was low, the indole-3-pyruvic acid (IPyA) level was high in stony hard peaches at the ripening stage. These results indicate that YUCCA activity is reduced in ripening stony hard peaches. The expression of one of the YUCCA isogenes in peach, PpYUC11, was suppressed in ripening stony hard peaches. Furthermore, an insertion of a transposon-like sequence was found upstream of the PpYUC11 gene in the 5'-flanking region. Analyses of the segregation ratio of the stony hard phenotype and genotype in F1 progenies indicated that the transposon-inserted allele of PpYUC11, hd-t, correlated with the stony hard phenotype. On the basis of the above findings, we propose that the IPyA pathway (YUCCA pathway) is the main auxin biosynthetic pathway in ripening peaches of 'Akatsuki' and 'Manami' cultivars. Because IAA is not supplied from storage forms, IAAde novo synthesis via the IPyA pathway (YUCCA pathway) in mesocarp tissues is responsible for auxin generation to support fruit softening, and its disruption can lead to the stony hard phenotype., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

The rabbitfish Siganus canaliculatus is the first marine teleost shown to be able to biosynthesize long-chain polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors catalyzed by two fatty acyl desaturases (fad) including Δ4 Fad and Δ6/Δ5 Fad as well as two elongases (Elovl4 and Elovl5). Previously, hepatocyte nuclear factor 4α (Hnf4α) was demonstrated to be predominant in the transcriptional regulation of two fads . To clarify the regulatory mechanisms involved in rabbitfish lipogenesis, the present study focused on the regulatory role of Hnf4α to elovl5 expression and LC-PUFA biosynthesis. Bioinformatics analysis predicted two potential Hnf4α elements in elovl5 promoter, one binding site was confirmed to interact with Hnf4α by gel shift assays. Moreover, overexpression of hnf4α caused a remarkable increase both in elovl5 promoter activity and mRNA contents, while knock-down of hnf4α in S. canaliculatus hepatocyte line (SCHL) resulted in a significant decrease of elovl5 gene expression. Meanwhile, hnf4α overexpression enhanced LC-PUFA biosynthesis in SCHL cell, and intraperitoneal injection to rabbitfish juveniles with Hnf4α agonists (Alverine and Benfluorex) increased the expression of hnf4α , elvol5 and Δ4 fad , coupled with an increased proportion of total LC-PUFA in liver. The results demonstrated that Hnf4α is involved in LC-PUFA biosynthesis by up-regulating the transcription of the elovl5 gene in rabbitfish, which is the first report of Hnf4α as a transcription factor of the elovl5 gene in vertebrates.

The human riboflavin transporter-3 (encoded by SLC52A3) plays a prominent role in riboflavin absorption. Interestingly, abnormal expression patterns of SLC52A3 in multiple types of human cancers have been recently noted. However, the molecular mechanisms underlying its dysregulation remain unclear. In this study, we find that SLC52A3 has two transcript variants that differ in the transcriptional start site, and encode different proteins: SLC52A3a and SLC52A3b. Importantly, aberrant expressions of SLC52A3 are associated with stepwise development of esophageal squamous cell carcinoma (ESCC) as well as the survival rates of ESCC patients. Functionally, SLC52A3a, but not SLC52A3b, strongly promotes the proliferation and colony formation of ESCC cells. Furthermore, SLC52A3 5'-flanking regions contain NF-κB p65/Rel-B-binding sites, which are crucial for mediating SLC52A3 transcriptional activity in ESCC cells. Chromatin immunoprecipitation and electrophoretic mobility shift assay reveal that p65/Rel-B bind to 5'-flanking regions of SLC52A3. Accordingly, NF-κB signaling upregulates SLC52A3 transcription upon TNFα stimulation. Taken together, these results elucidate the mechanisms underlying SLC52A3 overexpression in ESCC. More importantly, our findings identify SLC52A3 as both a predictive and prognostic biomarker for this deadly cancer.

Key Message: Data obtained from Illumina resequencing of 63 apple cultivars were used to obtain full-length S-RNase sequences using a strategy based on both alignment and de novo assembly of reads. The reproductive biology of apple is regulated by the S-RNase-based gametophytic self-incompatibility system, that is genetically controlled by the single, multi-genic and multi-allelic S locus. Resequencing of apple cultivars provided a huge amount of genetic data, that can be aligned to the reference genome in order to characterize variation to a genome-wide level. However, this approach is not immediately adaptable to the S-locus, due to some peculiar features such as the high degree of polymorphism, lack of colinearity between haplotypes and extensive presence of repetitive elements. In this study we describe a dedicated procedure aimed at characterizing S-RNase alleles from resequenced cultivars. The S-genotype of 63 apple accessions is reported; the full length coding sequence was determined for the 25 S-RNase alleles present in the 63 resequenced cultivars; these included 10 previously incomplete sequences (S 5 , S 6a , S 6b , S 8 , S 11 , S 23 , S 39 , S 46 , S 50 and S 58 ). Moreover, sequence divergence clearly suggests that alleles S 6a and S 6b , proposed to be neutral variants of the same alleles, should be instead considered different specificities. The promoter sequences have also been analyzed, highlighting regions of homology conserved among all the alleles.

We aimed to analyze the associations of single nucleotide polymorphisms (SNP) in the 5' regulatory region of the human organic anion transporter 1 (OAT1) gene with chronic kidney disease (CKD). A case-control study including age- and sex-matched groups of normal subjects and patients with CKD (n = 162 each) was designed. Direct sequencing of the 5' regulatory region (+88 to -1196 region) showed that patients with CKD had a higher frequency of the -475 SNP (T > T/G) than normal subjects (14/162 vs. 2/162). The luciferase activity assay results indicated that the -475G SNP had a higher promoter efficiency than the -475T SNP. Chromatin immunoprecipitation (ChIP) and LC/MS/MS analyses showed that the -475G SNP up-regulated 26 proteins and down-regulated 74 proteins. The Southwestern blot assay results revealed that the -475G SNP decreased the binding of Hepatoma-derived growth factor (HDGF), a transcription repressor, compared to the -475T SNP. Overexpression of HDGF significantly down-regulated OAT1 in renal tubular cells. Moreover, a zebrafish animal model showed that HDGF-knockdown zebrafish embryos had higher rates of kidney malformation than wild-type controls [18/78 (23.1%) vs. 1/30 (3.3%)]. In conclusion, our results suggest that an OAT1 SNP might be clinically associated with CKD. Renal tubular cells with the -475 SNP had increased OAT1 expression, which resulted in increased transportation of organic anion toxins into cells. Cellular accumulation of organic anion toxins caused cytotoxicity and resulted in CKD.

The introduction of a 5'-tailed duplex (5'-TD) fragment into cells corrects a base-substitution mutation in a target DNA. We previously reported that the gene correction efficiency was improved when a frameshift type of second mismatch was present ∼330 bases distant from the target position, between the target DNA and the 5'-TD fragment. In this study, the effects of the second mismatches on the gene correction were further examined. Base-base mismatches 332 bases distant from the target position slightly enhanced gene correction, but less efficiently than the previously studied frameshift mismatches. The gene correction efficiency was also increased when the distance between the target position and the second frameshift mismatch was changed to ∼270 bases. These results suggested that the introduction of an appropriate second frameshift mismatch into the 5'-TD fragment improves the gene correction efficiency., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Heat shock cognate 70 (HSC70) is a class of highly conserved proteins which functions as a molecular chaperon, participates in tolerance processes, and is involved in protein folding, degradation, targeting, translocation, and protein complex remodeling. In this study, the mRNA expression level of the Haliotis diversicolor HSC70 (HdHSC70) gene was detected by quantitative real-time PCR in different tissues and under different stresses. The results showed that the HdHSC70 gene was ubiquitously expressed in seven selected tissues. The highest expression level was detected in gills (P < 0.05). The expression level of the HdHSC70 gene was significantly upregulated by thermal stress, hypoxia stress, Vibrio parahaemolyticus infection, and combined thermal and hypoxia stress. The upregulation occurred at the early stage of stress. These results indicated that the HdHSC70 is an important component in the immune system of H. diversicolor and is involved in the early stress response. Meanwhile, 5'-flanking region sequence (2013 bp) of the HdHSC70 gene was cloned; it contains a putative core promoter region, heat shock element, CpG, and transcription elements including NF-1, Sp1, Oct-1, interferon consensus sequence binding protein (ICSBP), etc. In HEK 293T cells, the 5'-flanking region sequence is able to drive expression of the enhanced green fluorescent protein (EGFP), proving its promoter function. The promoter activity increased after high-temperature treatment, which may be the immediate reason why the expression of the HdHSC70 gene was significantly upregulated by thermal stress. After the ICSBP-binding site was mutated, we found the luciferase activity significantly reduced, which suggested that the ICSBP-binding site has a certain enhancement effect on the activity of the HdHSC70 promoter.

Early endosperm development presents a unique system in which to uncover epigenetic regulatory mechanisms because the contributing maternal and paternal genomes possess differential epigenetic modifications. In Arabidopsis ( Arabidopsis thaliana ), the initiation of endosperm coenocytic growth upon fertilization and the transition to endosperm cellularization are regulated by the FERTILIZATION-INDEPENDENT SEED (FIS)-Polycomb Repressive Complex 2 (PRC2), a putative H3K27 methyltransferase. Here, we address the possible role of the FIS-PRC2 complex in regulating the type I MADS-box gene family, which has been shown previously to regulate early endosperm development. We show that a subclass of type I MADS-box genes (C2 genes) was expressed in distinct domains of the coenocytic endosperm in wild-type seeds. Furthermore, the C2 genes were mostly up-regulated biallelically during the extended coenocytic phase of endosperm development in the FIS-PRC2 mutant background. Using allele-specific expression analysis, we also identified a small subset of C2 genes subjected to FIS-PRC2-dependent maternal or FIS-PRC2-independent paternal imprinting. Our data support a dual role for the FIS-PRC2 complex in the regulation of C2 type I MADS-box genes, as evidenced by a generalized role in the repression of gene expression at both alleles associated with endosperm cellularization and a specialized role in silencing the maternal allele of imprinted genes., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

Glutamate dehydrogenase (Gdh) plays a central role in ammonia detoxification by catalysing reversible oxidative deamination of l-glutamate into α-ketoglutarate using NAD + or NADP + as cofactor. To gain insight into transcriptional regulation of glud , the gene that codes for Gdh, we isolated and characterised the 5' flanking region of glud from gilthead sea bream ( Sparus aurata ). In addition, tissue distribution, the effect of starvation as well as short- and long-term refeeding on Gdh mRNA levels in the liver of S. aurata were also addressed. 5'-Deletion analysis of glud promoter in transiently transfected HepG2 cells, electrophoretic mobility shift assays, chromatin immunoprecipitation (ChIP) and site-directed mutagenesis allowed us to identify upstream stimulatory factor 2 (Usf2) as a novel factor involved in the transcriptional regulation of glud Analysis of tissue distribution of Gdh and Usf2 mRNA levels by reverse transcriptase-coupled quantitative real-time PCR (RT-qPCR) showed that Gdh is mainly expressed in the liver of S. aurata , while Usf2 displayed ubiquitous distribution. RT-qPCR and ChIP assays revealed that long-term starvation down-regulated the hepatic expression of Gdh and Usf2 to similar levels and reduced Usf2 binding to glud promoter, while refeeding resulted in a slow but gradual restoration of both Gdh and Usf2 mRNA abundance. Herein, we demonstrate that Usf2 transactivates S. aurata glud by binding to an E-box located in the proximal region of glud promoter. In addition, our findings provide evidence for a new regulatory mechanism involving Usf2 as a key factor in the nutritional regulation of glud transcription in the fish liver., (© 2018 Society for Endocrinology.)

Using 5' rapid amplification of cDNA ends, Illumina MiSeq, and basic flow cytometry, we systematically analyzed the expressed B cell receptor (BCR) repertoire in 14 healthy adult PBMCs, 5 HIV-1+ adult PBMCs, 5 cord blood samples, and 3 HIS-CD4/B mice, examining the full-length variable region of μ, γ, α, κ, and λ chains for V-gene usage, somatic hypermutation (SHM), and CDR3 length. Adding to the known repertoire of healthy adults, Illumina MiSeq consistently detected small fractions of reads with high mutation frequencies including hypermutated μ reads, and reads with long CDR3s. Additionally, the less studied IgA repertoire displayed similar characteristics to that of IgG. Compared to healthy adults, the five HIV-1 chronically infected adults displayed elevated mutation frequencies for all μ, γ, α, κ, and λ chains examined and slightly longer CDR3 lengths for γ, α, and λ. To evaluate the reconstituted human BCR sequences in a humanized mouse model, we analyzed cord blood and HIS-CD4/B mice, which all lacked the typical SHM seen in the adult reference. Furthermore, MiSeq revealed identical unmutated IgM sequences derived from separate cell aliquots, thus for the first time demonstrating rare clonal members of unmutated IgM B cells by sequencing.

Interferon (IFN)-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that is heavily involved in immune response elicitation. As an important member of interferon regulatory factor (IRF) family, IRF1 can activate the expression of multiple genes, including the human optineurin gene (Sudhakar et al., 2013). In this study, a sequence in the promoter region of the optineurin gene was compared to the 5' flanking region of the porcine isg15 gene. Porcine IRF1 also possesses antiviral activity against several swine viruses (Li et al., 2015), but the mechanism is not well understood. Herein, we report that porcine IRF1 and ISG15 were up-regulated in porcine kidney (PK-15) cells following stimulation with double-stranded RNA (dsRNA) or classical swine fever virus (CSFV) infection. We also found that siRNA-mediated knockdown of IRF1 expression resulted in lower ISG15 expression in response to polyinosinic:polycytidylic acid [poly(I:C)] or CSFV infection. The overexpression of IRF1 resulted in ISG15 up-regulation. IRF1 was shown to translocate to the nucleus in response to dsRNA stimulation. To further identify the functional domain of the isg15 gene that promotes IRF1 transcriptional activity, firefly luciferase and ISG15 reporter systems were constructed. The results of the firefly luciferase and ISG15 reporter assay suggested that IRF1 mediates the up-regulation of ISG15. Nucleotides -487 to -325, located in the 5' flanking region of the isg15 gene, constituted the promoter region of IRF1. ChIP assay indicated that IRF1 protein was able to interact with the DNA in the 5'fr of isg15 gene in cells. As an innate immune response protein with broad-spectrum antiviral activity, the up-regulation of ISG15 mediated by IRF1 in porcine cells is reported for the first time. These results warrant further investigation into the antiviral activity of porcine IRF1 against reported swine viruses., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Vitellogenesis-inhibiting hormone (VIH) is known to regulate ovarian maturation by suppressing the synthesis of vitellogenin (Vtg) in crustaceans, which belongs to a member of crustacean hyperglycemic hormone (CHH) family synthesized and secreted from the X-organ/sinus gland complex of eyestalks. In this study, the cDNA, genomic DNA (gDNA) and the 5'-upstream regulatory (promoter region) sequences of VIH gene were obtained by conventional PCR, genome walker and tail-PCR techniques according to our transcriptomic database of Scylla paramamosain. The full-length cDNA of SpVIH is 634bp including 105bp 5'UTR, 151bp 3'UTR and 378bp ORF that encodes a peptide of 125 amino acids. The full length gDNA of SpVIH is 790bp containing two exons and one intron. The 5'-flanking promoter regions of SpVIH we isolated are 3070bp from the translation initiation (ATG) and 2398bp from the predicted transcription initiation (A), which consists of putative core promoter region and multiple potential transcription factor binding sites. SpVIH was only expressed in eyestalk. The expression level of SpVIH in eyestalk of female crab decreased gradually along with the development of ovary. As there is not cell line of crabs available, we chose the mature transfection system HEK293FT cell lines to explore the mechanism of transcription regulation of SpVIH in crabs. Sequential deletion assays using luciferase reporter gene in HEK293FT cells revealed that the possible promoter activity regions (including positive and negative transcription factors binding sites simultaneously) presented between pSpVIH-4 and pSpVIH-6. In order to further identify the crucial transcription factors binding site in this region, the site-directed mutagenesis of Sox9/Oct4/Oct1 binding site of pSpVIH-4 was created. The results demonstrated that the transcriptional activity of pSpVIH-4△ decreased significantly (p<0.05). Thus, it is reasonable to deduce that the Sox9/Oct4/Oct1 may be the essential positive transcription factors which regulate the expression of SpVIH., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Chlamydomonas reinhardtii is a unicellular green alga that has attracted interest due to its potential biotechnological applications, and as a model for algal biofuel and energy metabolism. Despite all the advantages that this unicellular alga offers, poor and inconsistent expression of nuclear transgenes remains an obstacle for basic and applied research. We used a data-mining strategy to identify highly expressed genes in Chlamydomonas whose flanking sequences were tested for the ability to drive heterologous nuclear transgene expression. Candidates identified in this search included two ribosomal protein genes, RPL35a and RPL23, and ferredoxin, FDX1, whose flanking regions including promoters, terminators and untranslated sequences could drive stable luciferase transgene expression to significantly higher levels than the commonly used Hsp70A-RBCS2 (AR) hybrid promoter/terminator sequences. The RPL23 flanking sequences were further tested using the zeocin resistance gene sh-ble as a reporter in monocistronic and dicistronic constructs, and consistently yielded higher numbers of zeocin-resistant transformants and higher levels of resistance than AR- or PSAD-based vectors. Chlamydomonas RPL23 sequences also enabled transgene expression in Volvox carteri. Our study provides an additional benchmark for strong constitutive expression of transgenes in Chlamydomonas, and develops a general approach for identifying flanking sequences that can be used to drive transgene expression for any organism where transcriptome data are available., (© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.)

Belted cattle have a circular belt of unpigmented hair and skin around their midsection. The belt is inherited as a monogenic autosomal dominant trait. We mapped the causative variant to a 37 kb segment on bovine chromosome 3. Whole genome sequence data of 2 belted and 130 control cattle yielded only one private genetic variant in the critical interval in the two belted animals. The belt-associated variant was a copy number variant (CNV) involving the quadruplication of a 6 kb non-coding sequence located approximately 16 kb upstream of the TWIST2 gene. Increased copy numbers at this CNV were strongly associated with the belt phenotype in a cohort of 333 cases and 1322 controls. We hypothesized that the CNV causes aberrant expression of TWIST2 during neural crest development, which might negatively affect melanoblasts. Functional studies showed that ectopic expression of bovine TWIST2 in neural crest in transgenic zebrafish led to a decrease in melanocyte numbers. Our results thus implicate an unsuspected involvement of TWIST2 in regulating pigmentation and reveal a non-coding CNV underlying a captivating Mendelian character.

Coronary artery disease (CAD) is a multifactorial disease with a genetic component. Pigment epithelium-derived factor (PEDF) exerts anti-inflammatory, anti-oxidant, anti-thrombotic, and anti-angiogenic effects and thus has received increasing attention as a sensitive biomarker of atherosclerosis and CAD. To explore the potential association between PEDF single nucleotide polymorphisms (SNPs) and CAD, we performed this case-control study of consecutive elderly Chinese Han male patients (n = 416) and age-matched male controls (n = 528) without a history of CAD or electrocardiographic signs of CAD. The enrolled CAD patients (age ≥ 60 years) are not biologically related. A tag approach was used to examine 100% of common variations in the PEDF gene (r 2 ≥ 0.8, minor allele frequency > 0.1). PEDF tag SNPs (tSNPs) were selected using the HapMap Data-CHB which describes the common patterns of human DNA sequence variation and Tagger program. SNPs were genotyped using ligase detection reaction (LDR). Seven tSNPs (rs8075977, rs11658342, rs1136287, rs12603825, rs12453107, rs6828 and rs11078634) were selected. Among them, only one SNP, rs8075977 (C/T) located in the 5'-flanking region, showed the significant effect on the susceptibility to CAD. The frequency of its T allele was significantly higher in the controls (52.7%) than that in the CAD group (46.2%) (adjusted OR = 0.88, 95% CI: 0.80-0.96; P = 0.005). In conclusion, the T allele of rs8075977 in the 5'-flanking region of the PEDF gene may be protective for CAD. Conversely, the C allele at this variation site is associated with CAD in elderly Chinese Han men.

DNA double-strand break repair by homologous recombination entails nucleolytic resection of the 5' strand at break ends. Dna2, a flap endonuclease with 5'-3' helicase activity, is involved in the resection process. The Dna2 helicase activity has been implicated in Okazaki fragment processing during DNA replication but is thought to be dispensable for DNA end resection. Unexpectedly, we found a requirement for the helicase function of Dna2 in end resection in budding yeast cells lacking exonuclease 1. Biochemical analysis reveals that ATP hydrolysis-fueled translocation of Dna2 on ssDNA facilitates 5' flap cleavage near a single-strand-double strand junction while attenuating 3' flap incision. Accordingly, the ATP hydrolysis-defective dna2-K1080E mutant is less able to generate long products in a reconstituted resection system. Our study thus reveals a previously unrecognized role of the Dna2 translocase activity in DNA break end resection and in the imposition of the 5' strand specificity of end resection., (© 2017 Miller et al.; Published by Cold Spring Harbor Laboratory Press.)

Ulcerative colitis (UC) is a chronic inflammatory disorder of unknown etiology and a polygenic disease. IL-27 encodes p28, a subunit of IL-12 family cytokines, and has been implicated in the pathogenesis of UC. The aims of the present study were to evaluate the genetic association of a variant of the IL-27 gene with UC and to further characterize the functional variant in the IL-27 gene that influences the risk for UC. Our data demonstrated that the genetic variant rs153109 in the 5' upstream region of IL-27 is significantly associated with UC in Chinese Han individuals. Analysis of IL-27 transcripts demonstrated that individuals carrying the risk allele of rs153109 display reduced transcription of IL-27 in PBMCs. Luciferase activity assays demonstrated that the risk allele rs153109 results in decreased promoter activity compared to a non-risk allele in a tissue specific manner. Mechanistic characterization of histone modifications in the promoter region revealed that the risk haplotype tagged by the risk allele of rs153109 reduces the levels of H3K3me3 and H3K27ac., (Copyright © 2017 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Potato (Solanum tuberosum L.) vacuolar invertase (β-fructofuranosidase; EC 3.2.1.26) inhibitor 2 (StInvInh2) plays an important role in cold-induced sweetening (CIS) of potato tubers. The transcript levels of StInvInh2 were increased by prolonged cold in potato tubers with CIS-resistance but decreased in potato tubers with CIS-sensitivity. However, the transcript regulation mechanisms of StInvInh2 responding to prolonged cold are largely unclear in CIS-resistant and CIS-sensitive genotypes. In the present study, the 5'-flanking sequence of the StInvInh2 was cloned, and cis-acting elements were predicted. No informative differences in StInvInh2 promoter structure between resistant and sensitive-CIS potato genotypes were observed. Histochemical assay showed that the promoter of StInvInh2 mainly governed β-glucuronidase (GUS) expression in potato microtubers. Quantitative analysis of GUS expression suggested that StInvInh2 promoter activity was enhanced by prolonged cold in CIS-resistant genotype tubers but suppressed in CIS-sensitive tubers. These findings provide essential information regarding transcriptional regulatory mechanisms of StInvInh2 in cold-stored tubers contrasting CIS capacity., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Octamer-binding transcription factor 4 (Oct4) is a member of POU (Pit-Oct-Unc) transcription factor family Class V that plays a crucial role in maintaining the pluripotency and self-renewal of stem cells. Though it has been deeply investigated in mammals, its lower vertebrate homologue, especially in the marine fish, is poorly studied. In this study, we isolated the full-length sequence of Paralichthys olivaceus pou5f3 ( Popou5f3 ), and we found that it is homologous to mammalian Oct4 . We identified two transcript variants with different lengths of 3'-untranslated regions (UTRs) generated by alternative polyadenylation (APA). Quantitative real-time RT-PCR (qRT-PCR), in situ hybridization (ISH) and immunohistochemistry (IHC) were implemented to characterize the spatial and temporal expression pattern of Popou5f3 during early development and in adult tissues. Our results show that Popou5f3 is maternally inherited, abundantly expressed at the blastula and early gastrula stages, then greatly diminishes at the end of gastrulation. It is hardly detectable from the heart-beating stage onward. We found that Popou5f3 expression is restricted to the adult gonads, and continuously expresses during oogenesis while its dynamics are downregulated during spermatogenesis. Additionally, numerous cis -regulatory elements (CRE) on both sides of the flanking regions show potential roles in regulating the expression of Popou5f3 . Taken together, these findings could further our understanding of the functions and evolution of pou5f3 in lower vertebrates, and also provides fundamental information for stem cell tracing and genetic manipulation in Paralichthys olivaceus ., Competing Interests: The authors declare no conflict of interest.

Drosophila has three types of visual organs, the larval eyes or Bolwig's organs (BO), the ocelli (OC) and the compound eyes (CE). In all, the bHLH protein Atonal (Ato) functions as the proneural factor for photoreceptors and effects the transition from progenitor cells to differentiating neurons. In this work, we investigate the regulation of ato expression in the BO primordium (BOP). Surprisingly, we find that ato transcription in the BOP is entirely independent of the shared regulatory DNA for the developing CE and OC. The core enhancer for BOP expression, ato BO , lies ~6kb upstream of the ato gene, in contrast to the downstream location of CE and OC regulatory elements. Moreover, maintenance of ato expression in the neuronal precursors through autoregulation-a common and ancient feature of ato expression that is well-documented in eyes, ocelli and chordotonal organs-does not occur in the BO. We also show that the ato BO enhancer contains two binding sites for the transcription factor Sine oculis (So), a core component of the progenitor specification network in all three visual organs. These binding sites function in vivo and are specifically bound by So in vitro. Taken together, our findings reveal that the control of ato transcription in the evolutionarily derived BO has diverged considerably from ato regulation in the more ancestral compound eyes and ocelli, to the extent of acquiring what appears to be a distinct and evolutionarily novel cis-regulatory module., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objectives: In the present study, we examined whether DNA methylation of the sortilin-related receptor 1 (SORL1) 5'-flanking region is associated with the manifestation and clinical presentation of mild cognitive impairment in type 2 diabetes mellitus (T2DM)., Methods: Of 84 diabetic patients with mild cognitive impairment (MCI group), 78 diabetic patients without mild cognitive impairment (NMCI group) and 80 age-matched normal controls (NC group), the DNA methylation of the SORL1 5'-flanking region was completely analyzed. The SORL1 methylation ratios of the above three groups were compared statistically. Next, we investigated the correlation between the DNA methylation status and the clinical presentation of diabetes with or without cognitive impairment (MCI and NMCI groups)., Results: The methylation ratio (86.9%) of MCI patients was significantly higher than that in the NMCI patients (35.9%, P<0.05) and in the NC group (11.3%, P<0.05). Moreover, the diabetic patients with methylation alleles had greater ages, longer diabetes duration, lower MOCA scores and higher plasma amyloid Aβ 1-42 levels than those with unmethylation alleles (P<0.05)., Conclusion: These results suggested that the DNA methylation of the SORL1 5'-flanking region may significantly influence the manifestation of mild cognitive impairment in T2DM, and might be associated with its neurocognitive presentation., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Cytosolic sulfotransferase 1C3 (SULT1C3) is the least characterized of the three human SULT1C subfamily members. Originally identified as an orphan SULT by computational analysis of the human genome, we recently reported that SULT1C3 is expressed in human intestine and LS180 colorectal adenocarcinoma cells and is upregulated by agonists of peroxisome proliferator-activated receptor (PPAR) α and γ To determine the mechanism responsible for PPAR-mediated upregulation, we prepared reporter plasmids containing fragments of the SULT1C3 5'-flanking region. During initial attempts to amplify a 2.8-kb fragment from different sources of human genomic DNA, a 1.9-kb fragment was sometimes coamplified with the expected 2.8-kb fragment. Comparison of the 1.9-kb fragment sequence to the published SULT1C3 5'-flanking sequence revealed an 863-nt deletion (nt -146 to -1008 relative to the transcription start site). Transfection analysis in LS180 cells demonstrated that PPARα, δ, and γ agonist treatments induced luciferase expression from a reporter plasmid containing the 2.8-kb but not the 1.9-kb fragment. The PPAR agonists also activated a 1-kb reporter containing the 863-nt deletion region. Computational analysis identified three peroxisome proliferator response elements (PPREs) within the 863-nt region and serial deletions and site-directed mutations indicated that the most distal PPRE (at nt -769) was essential for obtaining PPAR-mediated transcriptional activation. Although agonists of all three PPARs could activate SULT1C3 transcription, RNA interference analysis indicated the predominance of PPARγ These data demonstrate that the PPARγ regulatory network includes SULT1C3 and imply that this enzyme contributes to the control of such PPARγ-regulated intestinal processes as growth, differentiation, and metabolism., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

In this research, we firstly demonstrated that physcion, an anthraquinone derivative, specifically increased the expression of the human α2,8-sialyltransferase (hST8Sia VI) gene in SK-N-BE(2)-C human neuroblastoma cells. To establish the mechanism responsible for the up-regulation of hST8Sia VI gene expression in physcion-treated SK-N-BE(2)-C cells, the putative promoter region of the hST8Sia VI gene was functionally characterized. Promoter analysis with serially truncated fragments of the 5'-flanking region showed that the region between -320 and -240 is crucial for physcion-induced transcription of hST8Sia VI in SK-N-BE(2)-C cells. Putative binding sites for transcription factors Pax-5 and NF-Y are located at this region. The Pax-5 binding site at -262 to -256 was essential for the expression of the hST8Sia VI gene by physcion in SK-N-BE(2)-C cells. Moreover, the transcription of hST8Sia VI induced by physcion in SK-N-BE(2)-C cells was inhibited by extracellular signal-regulated protein kinase (ERK) inhibitor U0126 and p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, but not c-Jun N-terminal kinase (JNK) inhibitor SP600125. These results suggest that physcion upregulates hST8Sia VI gene expression via ERK and p38 MAPK pathways in SK-N-BE(2)-C cells.

Peroxisome proliferator-activated receptor γ (PPARγ) is a key transcription factor in adipogenesis and can be regulated by adipogenesis-related factors. However, little information is available regarding its regulation by myogenic factors. In this study, we found that over-expression of MyoD enhanced porcine adipocyte differentiation and up-regulated PPARγ expression, whereas small interfering RNA against MyoD significantly attenuated porcine adipocyte differentiation and inhibited PPARγ expression. The MyoD-binding sites in the PPARγ promoter region at -412 to -396 and -155 to -150 were identified by promoter deletion analysis and site-directed mutagenesis. Electrophoretic mobility shift assays and chromatin immunoprecipitation further showed that these two regions are MyoD-binding sites, both in vitro and in vivo, indicating that MyoD directly interacts with the porcine PPARγ promoter. Thus, our results demonstrate that an Enhancer box and a binding site for a cooperative co-activator of MyoD are present in the promoter region of porcine PPARγ; furthermore, MyoD up-regulates PPARγ expression and promotes porcine adipocyte differentiation.

The pyruvate dehydrogenase beta subunit (PDHB) is a subunit of pyruvate dehydrogenase (E1), which catalyzes pyruvate into acetyl-CoA and provides a linkage between the tricarboxylic acid cycle (TCA) and the glycolysis pathway. Previous studies demonstrated PDHB to be positively related to the intramuscular fat (IMF) content. However, the transcriptional regulation of PDHB remains unclear. In our present study, the cDNA of bovine PDHB was cloned and the genomic structure was analyzed. The phylogenetic tree showed bovine PDHB to be closely related to goat and sheep, and least related to chicken. Spatial expression pattern analysis revealed the products of bovine PDHB to be widely expressed with the highest level in the fat of testis. To understand the transcriptional regulation of bovine PDHB, 1899 base pairs (bp) of the 5'-regulatory region was cloned. Sequence analysis neither found consensus TATA-box nor CCAAT-box in the 5'-flanking region of bovine PDHB. However, a CpG island was predicted from nucleotides -284 to +117. Serial deletion constructs of the 5'-flanking region, evaluated in dual-luciferase reporter assay, revealed the core promoter to be located 490bp upstream from the transcription initiation site (+1). Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP) in combination with asite-directed mutation experiment indicated both myogenin (MYOG) and the CCAAT/enhancer-binding protein beta (C/EBPß) to be important transcription factors for bovine PDHB in skeletal muscle cells and adipocytes. Our results provide an important basis for further investigation of the bovine PDHB function and regulation in cattle.

The Breast cancer 1, early onset gene (BRCA1) is known to be significantly associated with human familial breast cancer and is identified to play an important role in canine mammary tumors. Here, genetic variations in the coding region and DNA methylation in the 5' flanking region of BRCA1 in canine mammary tumor samples, 15 each of benign and malignant against 10 normal canine mammary tissue samples, were analyzed using the direct sequencing method. The results indicated two point mutations each in the coding region of canine BRCA1 in one benign mammary tumor sample (4702G >T and 4765G >T) and in one malignant canine mammary tumor sample (3619A >G and 4006G >A). No mutations were detected in the normal canine mammary tissue samples. The 4702G >T mutation was found to terminate further translation. The physical effect of the 4765G >T mutation was found to be the repalacement of the glutamate residue with glutamine. The physical effect of the 3619A >G mutation was found to be the replacement of the threonine residue with alanine, and that of mutation 4006G >A was the replacement of the valine residue with isoleucine in the BRCA1 protein. Bisulfite sequencing detected methylated CpG sites in one canine malignant mammary tumor sample. In conclusion, the present study elucidated the mutational status of the BRCA1 coding region and methylation status of the 5' flanking region of BRCA1 in canine mammary tumors.

TBX15 is a T-box transcription factor essential for development, also proposed as a marker in prostate cancer; and, recently, its antiapoptotic function indicates a role in carcinogenesis. Regulation of TBX15 is uncovered. In this study, we investigated the regulation of TBX15 expression in human cancer cells, by analyzing the regulatory function of a 5'-distal conserved region of TBX15. Bisulfite sequencing showed high methylation of the CpG island contained in this region that was not correlated with TBX15 mRNA levels, in the cancer cell lines analyzed; however, after 5-aza-dC treatment of TPC-1 cells an increase of TBX15 expression was observed. We also found a significant response of TBX15 to TNF-α activation of the NF-κB pathway using five cancer cell lines, and similar results were obtained when NF-κB was activated with PMA/ionomycin. Next, by luciferase reporter assays, we identified the TBX15 regulatory region containing two functional NF-κB binding sites with response to NF-κBp65, mapping on the -3302 and -3059 positions of the TBX15 gene. Moreover, a direct interaction of NF-κBp65 with one of the two NF-κB binding sites was indicated by ChIP assays. In summary, we provide novel data showing that NF-κB signaling up-regulates TBX15 expression in cancer cells. Furthermore, the link between TBX15 and NF-κB found in this study may be important to understand cancer and development processes.

Oestrogens influence the pathology and development of hormone-sensitive breast cancers. Tissue factor pathway inhibitor (TFPI) has been shown to be associated with breast cancer pathogenesis. Recently, we found TFPI mRNA levels to be significantly reduced by oestrogens in a breast cancer cell line (MCF7), a process mediated through the oestrogen receptor alpha (ERα). The aim of the present study was to investigate the mechanism(s) by which oestrogens may regulate TFPI at the transcriptional level. The TFPI 5'-flanking region contains three oestrogen response element (ERE) half-sites at positions -845, -769 and -50. Constructs containing the wild type or mutated ERE half-sites of the TFPI 5'-flanking region were generated in a luciferase reporter gene vector and transiently co-transfected with an ERα expression vector into HEK293 cells and subsequently treated with oestrogens. We found that luciferase activity was significantly downregulated after oestrogen stimulation in cells transfected with the wild type construct, an effect that was abolished by mutating either ERE half-sites. Electrophoretic mobility shift assay suggested direct and specific interaction of ERα with the ERE half-sites in the TFPI 5'-flanking region. Chromatin immunoprecipitation showed that ERα was recruited to the region -899 to -578 of the TFPI 5'-flanking region in vivo, where the ERE half-sites -845 and -769 are located. Our results indicate that ERα can interact with all three ERE half-sites in the TFPI 5'-flanking region and thus participate in the repression of oestrogen mediated TFPI transcription in breast cancer cells.

ELT-2 is the major regulator of genes involved in differentiation, maintenance and function of C. elegans intestine from the early embryo to mature adult. elt-2 responds to overexpression of the GATA transcription factors END-1 and END-3, which specify the intestine, as well as to overexpression of the two GATA factors that are normally involved in intestinal differentiation, ELT-7 and ELT-2 itself. Little is known about the molecular mechanisms underlying these interactions, how ELT-2 levels are maintained throughout development or how such systems respond to developmental perturbations. Here, we analyse elt-2 gene regulation through transgenic reporter assays, ELT-2 ChIP and characterisation of in vitro DNA-protein interactions. Our results indicate that elt-2 is controlled by three discrete regulatory regions conserved between C. elegans and C. briggsae that span >4 kb of 5' flanking sequence. These regions are superficially interchangeable but have quantitatively different enhancer properties, and their combined activities indicate inter-region synergies. Their regulatory activity is mediated by a small number of conserved TGATAA sites that are largely interchangeable and interact with different endodermal GATA factors with only modest differences in affinity. The redundant molecular mechanism that forms the elt-2 regulatory network is robust and flexible, as loss of end-3 halves ELT-2 levels in the early embryo but levels fully recover by the time of hatching. When ELT-2 is expressed under the control of end-1 regulatory elements, in addition to its own endogenous promoter, it can replace the complete set of endoderm-specific GATA factors: END-1, END-3, ELT-7 and (the probably non-functional) ELT-4. Thus, in addition to controlling gene expression during differentiation, ELT-2 is capable of specifying the entire C. elegans endoderm., (© 2016. Published by The Company of Biologists Ltd.)

In zebrafish, fast muscle-specific myosin heavy chain genes have their unique expression patterns in a well-defined and restricted region of the skeletal muscle. However, the transcriptional regulatory mechanisms involved have remained unclear. Here, we examined the regulation of spatio-temporal expression patterns of myhz1 (myhz1.1, myhz1.2 and myhz1.3) and myhz2 during their development by using transient gene and stable transgenic techniques. Embryos microinjected with different length 5'-flanking sequences of myhz1 conjugated with the enhanced green fluorescent protein (EGFP) gene showed EGFP expression in the anterior and medial subsections of somites, but not in the tail somite region. In contrast, embryos microinjected with different length 5'-flanking sequences of myhz2 showed EGFP expression exclusively at the posterior tail somite domain. Promoter deletion analyses demonstrated that reduced EGFP fluorescence typically is correlated with smaller 5'-flanking sequences. The immunohistochemical observation revealed that zebrafish larvae provided with the transient gene and those from stable transgenic lines consistently expressed EGFP in the fast muscle fibers. r-VISTA plot identified one common conserved region of about 140°bp among myhz1.1, myhz1.2 and myhz1.3. Deletion of this conserved region from the 5'-flanking sequence of each myhz1 markedly reduced EGFP expression in its unique spatial somite region. Deletion mutation analysis demonstrated that myhz2 expression in the tail somite region might be mediated by Tbx (family of transcription factors having a common DNA-binding sequence known as T-box) binding elements. In summary, 5'-flanking sequences of myhz1 and myhz2 regulate their unique expression patterns in a well-defined and restricted somite region of the skeletal muscle in zebrafish., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Diverse changes in coloration across distant taxa are mediated through alterations in certain highly conserved pigmentation genes. Among these genes, Mc1r is a frequent target for mutation, and many documented alterations involve coding sequence changes. We investigated whether regulatory mutations in Mc1r may also contribute to pigmentation loss in the blind Mexican cavefish, Astyanax mexicanus. This species comprises multiple independent cave populations that have evolved reduced (or absent) melanic pigmentation as a consequence of living in darkness for millions of generations. Among the most salient cave-associated traits, complete absence (albinism) or reduced levels of pigmentation (brown) have long been the focus of degenerative pigmentation research in Astyanax. These two Mendelian traits have been linked to specific coding mutations in Oca2 (albinism) and Mc1r (brown). However, four of the seven caves harboring the brown phenotype exhibit unaffected coding sequences compared to surface fish. Thus, diverse genetic changes involving the same genes likely impact reduced pigmentation among cavefish populations. Using both sequence and expression analyses, we show that certain cave-dwelling populations harboring the brown mutation have substantial alterations to the putative Mc1r cis-regulatory region. Several of these sequence mutations in the Mc1r 5' region were present across multiple, independent cave populations. This study suggests that pigmentation reduction in Astyanax cavefish evolves through a combination of both coding and cis-regulatory mutations. Moreover, this study represents one of the first attempts to identify regulatory alterations linked to regressive changes in cave-dwelling populations of A. mexicanus.

LEAFY COTYLEDON1 (LEC1) is a B subunit of Nuclear Factor Y (NF-YB) transcription factor that mainly accumulates during embryo development. We cloned the 5' flanking regulatory sequence of AhLEC1B gene, a homolog of Arabidopsis LEC1, and analyzed its regulatory elements using online software. To identify the crucial regulatory region, we generated a series of GUS expression frameworks driven by different length promoters with 5' terminal and/or 3' terminal deletion. We further characterized the GUS expression patterns in the transgenic Arabidopsis lines. Our results show that both the 65 bp proximal promoter region and the 52 bp 5' UTR of AhLEC1B contain the key motifs required for the essential promoting activity. Moreover, AhLEC1B is preferentially expressed in the embryo and is co-regulated by binding of its upstream genes with both positive and negative corresponding cis-regulatory elements.

Background: Adipose tissue is recognized as a highly active metabolic and endocrine organ. The hormones secreted by this tissue play an important role in many biochemical processes. It is known that dysfunction of adipocytes can cause insulin resistance, type 2 diabetes or hyperlipidemia. One of the important factors produced in fat tissue is resistin (Retn). It has been postulated that this hormone is involved in glucose homeostasis and insulin resistance. In the present study, the impact of five diet types (ad libitum normal, restricted, high-carbohydrate, high-fat and high-protein) on the Retn gene transcription and methylation profile was evaluated in rats of different ages., Results: Transcript levels and methylation status of the Retn gene were studied in three tissues (muscle, subcutaneous and abdominal fat) in rats at 30, 60 and 120 days of age. We found an effect of tissue type on the Retn transcription in all diet types, as well as an effect of feeding type and age on the mRNA levels for high-fat and high-protein diets. The DNA methylation levels depended only on tissue type., Conclusions: The obtained results demonstrate a tissue-specific expression pattern and a characteristic DNA methylation profile of the Retn gene in rats. Retn expression seems to be sensitive to nutritional changes, but only in the case of high-fat and high-protein diets. Moreover, an effect of age on Retn mRNA content was observed in these diets. Because no correlation between the transcript level and methylation status was found, we assumed that the transcription control of this gene by DNA methylation of the promoter seems to be unlikely.

Pseudoautosomal region 1 (PAR1) contains SHOX, in addition to seven highly conserved non-coding DNA elements (CNEs) with cis-regulatory activity. Microdeletions involving SHOX exons 1-6a and/or the CNEs result in idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis (LWD). Here, we report six rare copy-number variations (CNVs) in PAR1 identified through copy-number analyzes of 245 ISS/LWD patients and 15 unaffected individuals. The six CNVs consisted of three microduplications encompassing SHOX and some of the CNEs, two microduplications in the SHOX 3'-region affecting one or four of the downstream CNEs, and a microdeletion involving SHOX exon 6b and its neighboring CNE. The amplified DNA fragments of two SHOX-containing duplications were detected at chromosomal regions adjacent to the original positions. The breakpoints of a SHOX-containing duplication resided within Alu repeats. A microduplication encompassing four downstream CNEs was identified in an unaffected father-daughter pair, whereas the other five CNVs were detected in ISS patients. These results suggest that microduplications involving SHOX cause ISS by disrupting the cis-regulatory machinery of this gene and that at least some of microduplications in PAR1 arise from Alu-mediated non-allelic homologous recombination. The pathogenicity of other rare PAR1-linked CNVs, such as CNE-containing microduplications and exon 6b-flanking microdeletions, merits further investigation.

During cholestasis, the bile acid-conjugating enzymes, SULT2A1 and UGT2B4, work in concert to prevent the accumulation of toxic bile acids. To understand the impact of sulfotransferase deficiency on human hepatic gene expression, we knocked down 3'-phosphoadenosine-5'-phosphosulfate synthases (PAPSS) 1 and 2, which catalyze synthesis of the obligate sulfotransferase cofactor, in HepG2 cells. PAPSS knockdown caused no change in SULT2A1 expression; however, UGT2B4 expression increased markedly (∼41-fold increase in UGT2B4 mRNA content). Knockdown of SULT2A1 in HepG2 cells also increased UGT2B4 expression. To investigate the underlying mechanism, we transfected PAPSS-deficient HepG2 cells with a luciferase reporter plasmid containing ∼2 Kb of the UGT2B4 5'-flanking region, which included a response element for the bile acid-sensing nuclear receptor, farnesoid X receptor (FXR). FXR activation or overexpression increased UGT2B4 promoter activity; however, knocking down FXR or mutating or deleting the FXR response element did not significantly decrease UGT2B4 promoter activity. Further evaluation of the UGT2B4 5'-flanking region indicated the presence of distal regulatory elements between nucleotides -10090 and -10037 that negatively and positively regulated UGT2B4 transcription. Pulse-chase analysis showed that increased UGT2B4 expression in PAPSS-deficient cells was attributable to both increased mRNA synthesis and stability. Transfection analysis demonstrated that the UGT2B4 3'-untranslated region decreased luciferase reporter expression less in PAPSS-deficient cells than in control cells. These data indicate that knocking down PAPSS increases UGT2B4 transcription and mRNA stability as a compensatory response to the loss of SULT2A1 activity, presumably to maintain bile acid-conjugating activity., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) share high genetic and antigenic similarities, but exhibit marked differences in tissue tropism and neurovirulence. The amino-terminal region of glycoprotein C (gC), which is markedly different in each of the viruses, is involved in virus binding to cellular receptors and in interactions with the immune system. This study investigated the genetic and antigenic differences of the 5' region of the gC (5' gC) gene (amino-terminal) of South American BoHV-1 (n=19) and BoHV-5 (n=25) isolates. Sequence alignments of 374 nucleotides (104 amino acids) revealed mean similarity levels of 97.3 and 94.2% among BoHV-1 gC (gC1), respectively, 96.8 and 95.6% among BoHV-5 gC (gC5), and 62 and 53.3% between gC1 and gC5. Differences included the absence of 40 amino acid residues (27 encompassing predicted linear epitopes) scattered throughout 5' gC1 compared to 5' gC5. Virus neutralizing assays testing BoHV-1 and BoHV-5 antisera against each isolate revealed a high degree of cross-neutralization between the viruses, yet some isolates were neutralized at very low titers by heterologous sera, and a few BoHV-5 isolates reacted weakly with either sera. The virus neutralization differences observed within the same viral species, and more pronounced between BoHV-1 and BoHV-5, likely reflect sequence differences in neutralizing epitopes. These results demonstrate that the 5' gC region is well conserved within each viral species but is divergent between BoHV-1 and BoHV-5, likely contributing to their biological and antigenic differences.