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Several clinical and experimental studies have documented a compelling and critical role for the full-length matrix metalloproteinase-2 (FL-MMP-2) in ischemic renal injury, progressive renal fibrosis, and diabetic nephropathy. A novel N-terminal truncated isoform of MMP-2 (NTT-MMP-2) was recently discovered, which is induced by hypoxia and oxidative stress by the activation of a latent promoter located in the first intron of the MMP2 gene. This NTT-MMP-2 isoform is enzymatically active but remains intracellular in or near the mitochondria. In this perspective article, we first present the findings about the discovery of the NTT-MMP-2 isoform, and its functional and structural differences as compared with the FL-MMP-2 isoform. Based on publicly available epigenomics data from the Encyclopedia of DNA Elements (ENCODE) project, we provide insights into the epigenetic regulation of the latent promoter located in the first intron of the MMP2 gene, which support the activation of the NTT-MMP-2 isoform. We then focus on its functional assessment by covering the alterations found in the kidney of transgenic mice expressing the NTT-MMP-2 isoform. Next, we highlight recent findings regarding the presence of the NTT-MMP-2 isoform in renal dysfunction, in kidney and cardiac diseases, including damage observed in aging, acute ischemia-reperfusion injury (IRI), chronic kidney disease, diabetic nephropathy, and human renal transplants with delayed graft function. Finally, we briefly discuss how our insights may guide further experimental and clinical studies that are needed to elucidate the underlying mechanisms and the role of the NTT-MMP-2 isoform in renal dysfunction, which may help to establish it as a potential therapeutic target in kidney diseases.

Unrelated genes establish head-to-tail polarity in embryos of different fly species, raising the question of how they evolve this function. We show that in moth flies (Clogmia, Lutzomyia), a maternal transcript isoform of odd-paired (Zic) is localized in the anterior egg and adopted the role of anterior determinant without essential protein change. Additionally, Clogmia lost maternal germ plasm, which contributes to embryo polarity in fruit flies (Drosophila). In culicine (Culex, Aedes) and anopheline mosquitoes (Anopheles), embryo polarity rests on a previously unnamed zinc finger gene (cucoid), or pangolin (dTcf), respectively. These genes also localize an alternative transcript isoform at the anterior egg pole. Basal-branching crane flies (Nephrotoma) also enrich maternal pangolin transcript at the anterior egg pole, suggesting that pangolin functioned as ancestral axis determinant in flies. In conclusion, flies evolved an unexpected diversity of anterior determinants, and alternative transcript isoforms with distinct expression can adopt fundamentally distinct developmental roles., eLife digest With very few exceptions, animals have ‘head’ and ‘tail’ ends that develop when they are an embryo. The genes involved in specifying these ends vary between species and even closely-related animals may use different genes for the same roles. For example, the products of two unrelated genes called bicoid in fruit flies and panish in common midges accumulate at one end of their respective eggs to distinguish head from tail ends. It remained unclear how other fly species, which have neither a bicoid nor a panish gene, distinguish the head from the tail end, or how genes can evolve the specific function of bicoid and panish. Cells express genes by producing gene templates called messenger ribonucleic acids (or mRNAs for short). The central portions of mRNAs, known as protein-coding sequences, are then used to produce the protein. Proteins can play several distinct roles, which they acquire through evolution. This can happen in different ways, for example, genetic mutations in the part of a gene that codes for protein may alter the resulting protein, giving it a new activity. Alternatively, sequences at the beginning and the end of an mRNA molecule that do not code for protein, but regulate when and where proteins are made, can influence a protein’s role by changing its environment. Many genes produce mRNAs with alternative sequences at the beginning or the end, a process known as alternative transcription. Here, Yoon et al. identified three unrelated genes that perform similar roles to bicoid and panish in the embryos of several different moth flies and mosquitoes. These genes appear to have acquired their activity because one of their alternative transcripts accumulated at the future head end, rather than through mutations in the protein-coding sequences. Studying multiple species also made it clear that panish inherited its function from a localized alternative transcript of an old gene that duplicated and diverged. These findings suggest that alternative transcription may provide opportunities for genes to evolve new roles in fundamental processes in flies. Most animal genes use alternative start and stop sites for transcription, but the reasons for this remain largely obscure. This is especially the case in the human brain. The findings of Yoon et al., therefore, raise the question of whether alternative transcription has played an important role in the evolution of the human brain.

Cancer arises from a series of genetic and epigenetic changes, which result in abnormal expression or mutational activation of oncogenes, as well as suppression/inactivation of tumor suppressor genes. Aberrant expression of coding genes or long non-coding RNAs (lncRNAs) with oncogenic properties can be caused by translocations, gene amplifications, point mutations or other less characterized mechanisms. One such mechanism is the inappropriate usage of normally dormant, tissue-restricted or cryptic enhancers or promoters that serve to drive oncogenic gene expression. Dispersed across the human genome, endogenous retroviruses (ERVs) provide an enormous reservoir of autonomous gene regulatory modules, some of which have been co-opted by the host during evolution to play important roles in normal regulation of genes and gene networks. This review focuses on the “dark side” of such ERV regulatory capacity. Specifically, we discuss a growing number of examples of normally dormant or epigenetically repressed ERVs that have been harnessed to drive oncogenes in human cancer, a process we term onco-exaptation, and we propose potential mechanisms that may underlie this phenomenon.

Early studies suggested that most connexin genes share a relatively simple structure with a single intron of variable length interrupting the 5′ untranslated region (UTR). Here we report that zebrafish cx45.6 shows six isoforms of alternative 5′UTRs which are generated from multiple promoter usage and alternative pre-mRNA splicing. Interestingly, cx45.6 undergoes tandem alternative splicing, which produces transcripts only differing by 3 nucleotides. This is the first study that has demonstrated tandem alternative pre-mRNA splicing in the connexin gene family. Expression patterns of cx45.6 alternative transcripts were demonstrated by real-time RT-PCR during zebrafish embryonic development and in adult tissues. The complexity of 5′UTR diversity suggests complicated regulatory mechanisms for cx45.6 gene expression at both transcriptional and post-transcriptional levels, and we propose that tandem alternative splicing in cx45.6 5′UTRs could play a role in translational control. These results lay groundwork for further investigations on the regulation and function of cx45.6 gene expression.

The fact that promoters are essential for the function of all genes presents the basis of the general idea that retrotranspositions give rise to processed pseudogenes. However, recent studies have demonstrated that some retrotransposed genes are transcriptionally active. Because promoters are not thought to be retrotransposed along with exonic sequences, these transcriptionally active genes must have acquired a functional promoter by mechanisms that are yet to be determined. Hence, comparison between a retrotransposed gene and its source gene appears to provide a unique opportunity to investigate the promoter creation for a new gene. Here, we identified 29 gene pairs in the human genome, consisting of a functional retrotransposed gene and its parental gene, and compared their respective promoters. In more than half of these cases, we unexpectedly found that a large part of the core promoter had been transcribed, reverse transcribed, and then integrated to be operative at the transposed locus. This observation can be ascribed to the recent discovery that transcription start sites tend to be interspersed rather than situated at 1 specific site. This propensity could confer retrotransposability to promoters per se. Accordingly, the retrotransposability can explain the genesis of some alternative promoters.

Background Gender, genetic makeup, and prior experience interact to determine physiological responses to an external perceived stressor. Here, we investigated the contribution of both genetic variants and promoter methylation of the NR3C1 (glucocorticoid receptor) gene to the cardiovascular and hypothalamus-pituitary-adrenal (HPA) axis response to the socially evaluated cold pressor test (seCPT). Results Two hundred thirty-two healthy participants were recruited and underwent the experiment. They were randomly assigned to either the seCPT group (cold water) or a control group (warm water). The seCPT group had a clear stress reaction; salivary cortisol levels and peak systolic and diastolic blood pressure all increased significantly compared to the control group. GR genotype (TthIIII, NR3C1-I, 1H, E22E, R23K, BclI and 9beta) and methylation data were obtained from 218 participants. Haplotypes were built from the GR genotypes, and haplotype 2 (minor allele of BclI) carriers had a higher cortisol response to the seCPT in comparison to non-carriers (20.77 ± 13.22; 14.99 ± 8.42; p = 0.034), as well as independently of the experimental manipulation, higher baseline heart rate (72.44 ± 10.99; 68.74 ± 9.79; p = 0.022) and blood pressure (115.81 ± 10.47; 111.61 ± 10.74; p = 0.048). Average methylation levels throughout promoter 1F and 1H were low (2.76 and 1.69 %, respectively), but there was a strong correlation between individual CpGs and the distance separating them (Pearson’s correlation r = 0.725, p = 3.03 × 10−26). Higher promoter-wide methylation levels were associated with decreased baseline blood pressure, and when incorporated into a linear mixed effect model significantly predicted lower systolic and diastolic blood pressure evolution over time in response to the experimental manipulation. The underlying genotype significantly predicted methylation levels; particularly, the homozygous BclI minor allele was associated with higher methylation in promoter 1H (p = 0.042). Conclusions This is one of the first studies linking epigenetic modifications of the GR promoter, receptor genotype and physiological measures of the stress response. At baseline, there were clear genetic and epigenetic effects on blood pressure. The seCPT induced a strong cardiovascular and HPA axis response, and both systems were affected by the functional genetic variants, although methylation also predicted blood pressure reactivity. The return to baseline was predominantly influenced by the genomic sequence. Overall, the physiological response to the seCPT is controlled by an exquisite mix of genetic and epigenetic factors. Electronic supplementary material The online version of this article (doi:10.1186/s13148-016-0180-y) contains supplementary material, which is available to authorized users.

The Pax6 gene plays several roles in retinal development, including control of cell proliferation, maintenance of the retinogenic potential of progenitor cells, and cell fate specification. Emerging evidence suggests that these different aspects of Pax6 gene function are mediated by different isoforms of the Pax6 protein; however, relatively little is known about the spatiotemporal expression of Pax6 isoforms in the vertebrate retina. Using bacterial artificial chromosome (BAC) technology, we modified a zebrafish Pax6a BAC such that we could distinguish paired-containing Pax6a transcripts from paired-less Pax6a transcripts. In the zebrafish, the spatial and temporal onset of expression of these transcripts suggests that the paired-less isoform is involved in the cell fate decision leading to the generation of amacrine cells; however, because of limitations associated with transient transgenic analysis, it was not feasible to establish whether this promoter was active in all amacrine cells or in a specific population of amacrine cells. By making mice transgenic for the zebrafish Pax6a BAC reporter transgene, we were able to show that paired-containing and paired-less Pax6a transcripts were differentially expressed in amacrine subpopulations. Our study also directly demonstrates the functional conservation of the regulatory mechanisms governing Pax6 transcription in teleosts and mammals.

Highlight High expression of the FRD3 gene in the metal hyperaccumulator Arabidopsis halleri stems from complex zinc-regulated transcriptional and post-transcriptional mechanisms present in the non-hyperaccumulating Arabidopsis thaliana., In Arabidopsis thaliana, FRD3 (FERRIC CHELATE REDUCTASE DEFECTIVE 3) plays a central role in metal homeostasis. FRD3 is among a set of metal homeostasis genes that are constitutively highly expressed in roots and shoots of Arabidopsis halleri, a zinc hyperaccumulating and hypertolerant species. Here, we examined the regulation of FRD3 by zinc in both species to shed light on the evolutionary processes underlying the evolution of hyperaccumulation in A. halleri. We combined gene expression studies with the use of β-glucuronidase and green fluorescent protein reporter constructs to compare the expression profile and transcriptional and post-transcriptional regulation of FRD3 in both species. The AtFRD3 and AhFRD3 genes displayed a conserved expression profile. In A. thaliana, alternative transcription initiation sites from two promoters determined transcript variants that were differentially regulated by zinc supply in roots and shoots to favour the most highly translated variant under zinc-excess conditions. In A. halleri, a single transcript variant with higher transcript stability and enhanced translation has been maintained. The FRD3 gene thus undergoes complex transcriptional and post-transcriptional regulation in Arabidopsis relatives. Our study reveals that a diverse set of mechanisms underlie increased gene dosage in the A. halleri lineage and illustrates how an environmental challenge can alter gene regulation.

There are two classes of an intracellular ‘modulator protease’, calpain: ubiquitous and tissue-specific. p94/calpain 3 is an example of the latter, predominantly expressed in muscle. A defect in the p94 gene causes muscular dystrophy. Here we report that human and mouse p94 genes have a possible novel alternative promoter expressing p94 variants in all tissues examined including human lens epithelial cells. The possible promoter region and the following novel exons overlap the 3′ region of the neutral α-glucosidase C gene. Unlike p94, the novel p94 variants expressed in COS7 cells do not undergo rapid autolysis, suggesting basic functions different from p94.

The human clusterin (CLU) gene codes for several mRNAs characterized by different sequences at their 5’ end. We investigated the expression of two CLU mRNAs, called CLU 1 and CLU 2, in immortalized (PNT1a) and tumorigenic (PC3 and DU145) prostate epithelial cells, as well as in normal fetal fibroblasts (WI38) following the administration of the epigenetic drugs 5-aza-2'-deoxycytidine (AZDC) and trichostatin A (TSA) given either as single or combined treatment (AZDC-TSA). Our experimental evidences show that: a) CLU 1 is the most abundant transcript variant. b) CLU 2 is expressed at a low level in normal fibroblasts and virtually absent in prostate cancer cells. c) CLU 1, and to a greater extent CLU 2 expression, increased by AZDC-TSA treatment in prostate cancer cells. d) Both CLU 1 and CLU 2 encode for secreted CLU. e) P2, a novel promoter that overlaps the CLU 2 Transcription Start Site (TSS), drives CLU 2 expression. f) A CpG island, methylated in prostate cancer cells and not in normal fibroblasts, is responsible for long-term heritable regulation of CLU 1 expression. g) ChIP assay of histone tail modifications at CLU promoters (P1 and P2) shows that treatment of prostate cancer cells with AZDC-TSA causes enrichment of Histone3(Lys9)acetylated (H3K9ac) and reduction of Histone3(Lys27)trimethylated (H3K27me3), inducing active transcription of both CLU variants. In conclusion, we show for the first time that the expression of CLU 2 mRNA is driven by a novel promoter, P2, whose activity responds to epigenetic drugs treatment through changes in histone modifications.

Background: X-chromosome inactivation silences one X chromosome in females to achieve dosage compensation with the single X chromosome in males. While most genes are silenced on the inactive X chromosome, the gene for the long non-coding RNA XIST is silenced on the active X chromosome and expressed from the inactive X chromosome with which the XIST RNA associates, triggering silencing of the chromosome. In mouse, an alternative Xist promoter, P2 is also the site of YY1 binding, which has been shown to serve as a tether between the Xist RNA and the DNA of the chromosome. In humans there are many differences from the initial events of mouse Xist activation, including absence of a functional antisense regulator Tsix, and absence of strictly paternal inactivation in extraembryonic tissues, prompting us to examine regulatory regions for the human XIST gene. Results: We demonstrate that the female-specific DNase hypersensitivity site within XIST is specific to the inactive X chromosome and correlates with transcription from an internal P2 promoter. P2 is located within a CpG island that is differentially methylated between males and females and overlaps conserved YY1 binding sites that are only bound on the inactive X chromosome where the sites are unmethylated. However, YY1 binding is insufficient to drive P2 expression or establish the DHS, which may require a development-specific factor. Furthermore, reduction of YY1 reduces XIST transcription in addition to causing delocalization of XIST. Conclusions: The differentially methylated DNase hypersensitive site within XIST marks the location of an alternative promoter, P2, that generates a transcript of unknown function as it lacks the A repeats that are critical for silencing. In addition, this region binds YY1 on the unmethylated inactive X chromosome, and depletion of YY1 untethers the XIST RNA as well as decreasing transcription of XIST.

Cytochrome b5 and NADH-cytochrome b5, reductase are integral membrane proteins with cytosolic active domains and short membrane anchors, which are inserted post-translationally into their target membranes. Both are produced as different isoforms, with different localizations, in mammalian cells. In the rat, the reductase gene generates two transcripts by an alternative promoter mechanism: a ubiquitous mRNA coding for the myristylated membrane-bound form, and an erythroid mRNA which generates both the soluble form and a nonmyristylated membrane-binding form. The available evidence indicates that the ubiquitous myristylated form binds to the cytosolic face of both outer mitochondrial membranes and ER. In contrast, two genes code for two homologous forms of cytochrome b5, one of which is found on outer mitochondrial membranes, the other on the ER. The gene specifying the ER form probably also generates an erythroid-specific mRNA by alternative splicing, which codes for soluble cytochrome b5. Possible molecular mechanisms responsible for the observed localizations of these different enzyme isoforms are discussed.

Human ECE-1 is expressed in four isoforms with different tissue distribution and its mRNA and protein levels are altered under certain pathophysiological conditions. To investigate the transcriptional regulation of ECE-1, we studied the regulatory region of ECE-1c, the major ECE-1 isoform. A genomic clone comprising the complete human ECE-1 gene including the putative ECE-1c-specific promoter was obtained. Up to 968 bp upstream of the putative c-specific translation initiation start codon and several serial deletion mutants were subcloned into a reporter vector and transfected into endothelial (BAEC, EA.hy926, ECV304) and epithelial (MDA MB435S, MCF7) cells, showing very strong promoter activity in comparison to the SV40 promoter and to the previously described ECE-1a and 1b promoters. Transfection of serial deletion mutants indicated two positive regulatory regions within the promoter (−142/−240 and −240/490) likely involved in binding GATA and ETS transcription factors. RNase protection assay (RPA) and 5′-RACE revealed multiple transcriptional start sites located at about −110, −140 and −350 bp. Site-directed mutagenesis demonstrated a crucial role for the E2F cis-element for basal ECE-1c promoter activity. Additionally, we found a correlation between isoform-specific ECE-1 mRNA levels and corresponding ECE-1a, 1b, 1c promoter activities.

Interactions between various events are essential for complex and delicate transcriptional regulation. To delineate the features and potential roles of alternative promoters (APs) correlated with alternative splicing (AS), we have systematically analyzed 9908 putative alternative promoters (PAPs) from 3797 human genes. Our results showed that approximately 65% of AS events are associated with PAPs. Intriguingly, PAPs per human AS gene only averaged 2.6 for our dataset, which was significantly lower than previously reported. This seems to imply that the human genome contains a small pool of appropriable PAPs for AS genes. Exploration of the characteristics of PAPs such as CpG islands, TATA boxes, GC-content, transcription factor binding sites (TFBSs) and repetitive elements suggested that, respectively, 87% and 90% of PAPs of human AS genes are CpG- and TATA box-poor. The GC-content is significantly higher in the downstream of transcription start sites (TSSs) than upstream (58% vs. 53%), and there is a strong negative correlation between the GC-content and the number of PAPs. These suggested that GC-content around the TSSs plays an important role in the regulation of AS. Moreover, different APs contain distinct densities of repetitive elements and TFBSs, indicating that such sequences have an intrinsic role in the divergent regulation of PAPs and AS. Substantial difference was also found between human AS genes in terms of PAP numbers. A close connection between PAPs and AS may play a critical role in the choice of APs and regulation of AS genes. Furthermore, the distribution of AS genes on different human chromosomes also influences the numbers of PAPs and isoforms of AS genes. Our results may provide important clues for further studies on regulatory network of transcription-related events.

cDNAs for rat transthyretin mRNA were cloned from a brain cDNA library. Sequencing analyses showed the presence of an additional 5' sequence that had not been reported for the liver mRNA corresponding to the flanking promoter region of the gene. This additional sequence was expressed only in the brain, suggesting the presence of a brain-specific promoter.

Intersectin 1 (ITSN1) gene encodes an evolutionarily conserved adaptor protein that functions in clathrin-mediated endocytosis, cell signalling, apoptosis and cytoskeleton rearrangements. Its expression is characterized by multiple alternative splicing. Alternative promoter usage is an additional way to create diversity and flexibility in the regulation of gene expression. The aim of this study was to identify possible alternative promoters of ITSN1 gene. Methods. In silico prediction, 5' RACE, RT-PCR and reporter gene expression assay were used for identification and functional characterization of alternative promoter region. Results. We detected an alternative promoter of human ITSN1 gene which is located in intron 5 and generates 5' truncated transcripts containing in-frame ATG codon with strong Kozak sequence and could encode an N-terminally truncated isoforms lacking first EH domain. The region located 246–190 bp upstream of exon 6 is required for alternative promoter activity. ITSN1 transcripts generated from an alternative promoter were detected in human kidney, liver, lung and brain tissues. However, the level of their expression was significantly lower than that of major ITSN1 isoforms. Conclusion. The results obtained suggest that alternative promoter region located in intron 5 of ITSN1 gene functions as a weak promoter. Further experiments are required to clarify the role of 5' truncated ITSN1 transcripts. Ген інтерсектину 1 (ITSN1) кодує еволюційно консервативний адаптерний білок, причетний до клатрин-опосередкованого ендоцитозу, внутрішньоклітинної передачі сигналу, апоптозу та реорганізації цитоскелету. Його експресія пов‘язана з багатьмя подіями альтернативного сплайсингу. Додатковим способом досягнення різноманіття і тонкої регуляції експресії генів є пошук альтернативних промоторів. Мета роботи полягала у виявленні можливих альтернативних промоторів гена ITSN1. Методи. Застосоваано комп’ютерне передбачення, 5' RACE, RT-РCR і тест, оснований на аналізі експресії репортерного гена. Результати. Виявлено альтернативний промотор гена ITSN1 людини, локалізований у 5-му інтроні, внаслідок використання якого утворюються транскрипти з відкритою рамкою зчитування та консенсусною послідовністю Козак, здатні кодувати ITSN1-ізоформи без першого EH-домену. Визначено, що ділянка, розташована за 246–190 п. н. до початку 6-го екзона, необхідна для функціонування альтернативного промотору. Транскрипти ITSN1, що утворюються в результаті використання альтернативного промотору, знайдено в тканинах нирок, печінки, легень і мозку людини, проте рівень їхньої експресії значно нижчий порівняно з основними ізоформами ITSN1. Висновки. Отримані дані свідчать про те, що альтернативний промотор, локалізований у 5-му інтроні гена ITSN1 людини, функціонує як слабкий промотор. Подальші дослідження необхідні для з’ясування функції транскриптів ITSN1 з альтернативним 5'-кінцем. Ген интерсектина 1 (ITSN1) кодирует эволюционно консервативный адаптерный белок, участвующий в клатрин-опосредованном эндоцитозе, внутриклеточной передаче сигнала, апоптозе и реорганизации цитоскелета. Его экспрессия характеризуется многочисленными событиями альтернативного сплайсинга. Дополнительным способом достижения разнообразия и тонкой регуляции экспрессии генов является использование альтернативных промоторов. Цель данной работы состояла в обнаружении возможных альтернативных промоторов гена ITSN1. Методы. Применены компьютерное предсказание, 5' RACE, RT-PCR и тест, основанный на анализе экспрессии репортерного гена. Результаты. Нами выявлен альтернативный промотор гена ITSN1 человека, локализованный в 5-м интроне, в результате использования которого образуются транскрипты с открытой рамкой считывания и консенсусной последовательностью Козак, способные кодировать изоформы ITSN1 без первого ЕН-домена. Показано, что участок, находящийся за 246–190 п. н. до начала 6-го экзона, необходим для функционирования альтернативного промотора. Транскрипты ITSN1, полученные с альтернативного промотора, обнаружены в тканях почек, печени, легких и мозга человека, но уровень их экспрессии значительно ниже по сравнению с основными изоформами ITSN1. Выводы. Полученные результаты свидетельствуют о том, что альтернативный промотор, локализованный в 5-м интроне гена ITSN1, функционирует как слабый промотор. Дальнейшие исследования необходимы для выяснения функции транскриптов ITSN1 с альтернативным 5'-концом.