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Brain-specific angiogenesis inhibitor 1 (BAI1) belongs to the adhesion G-protein-coupled receptors, which exhibit large multi-domain extracellular N termini that mediate cell-cell and cell-matrix interactions. To explore the existence of BAI1 isoforms, we queried genomic datasets for markers of active chromatin and new transcript variants in the ADGRB1 (adhesion G-protein-coupled receptor B1) gene. Two major types of mRNAs were identified in human/mouse brain, those with a start codon in exon 2 encoding a full-length protein of a predicted size of 173.5/173.3 kDa and shorter transcripts starting from alternative exons at the intron 17/exon 18 boundary with new or exon 19 start codons, predicting two shorter isoforms of 76.9/76.4 and 70.8/70.5 kDa, respectively. Immunoblots on wild-type and Adgrb1 exon 2-deleted mice, reverse transcription PCR, and promoter-luciferase reporter assay confirmed that the shorter isoforms originate from an alternative promoter in intron 17. The shorter BAI1 isoforms lack most of the N terminus and are very close in structure to the truncated BAI1 isoform generated through GPS processing from the full-length receptor. The cleaved BAI1 isoform has a 19 amino acid extracellular stalk that may serve as a receptor agonist, while the alternative transcripts generate BAI1 isoforms with extracellular N termini of 5 or 60 amino acids. Further studies are warranted to compare the functions of these isoforms and examine the distinct roles they play in different tissues and cell types., Competing Interests: Declarations. Ethical Approval: All animal procedures were performed under Institutional Animal Care and Use Committee (IACUC) approval. The human tissue was obtained with Institutional Review Board for Human Use (IRB) approval. Consent to Participate: Not applicable. Consent to Publication: Not applicable. Competing interests: EGVM is co-founder, Chief Scientific Officer, and shareholder of OncoSpherix, Inc. (not related to the current study). Other authors declare no competing interest., (© 2024. The Author(s).)

TEAD4 is a transcription factor that plays a crucial role in the Hippo pathway by regulating the expression of genes related to proliferation and apoptosis. It is also involved in the maintenance and differentiation of the trophectoderm during pre- and post-implantation embryonic development. An alternative promoter for the TEAD4 gene was identified through epigenetic profile analysis, and a new transcript from the intronic region of TEAD4 was discovered using the 5'RACE method. The transcript of the novel promoter encodes a TEAD4 isoform (TEAD4-ΔN) that lacks the DNA-binding domain but retains the C-terminal protein-protein interaction domain. Gene expression studies, including end-point PCR and Western blotting, showed that full-length TEAD4 was present in all investigated tissues. However, TEAD4-ΔN was only detectable in certain cell types. The TEAD4-ΔN promoter is conserved throughout evolution and demonstrates transcriptional activity in transient-expression experiments. Our study reveals that TEAD4 interacts with the alternative promoter and increases the expression of the truncated isoform. DNA methylation plays a crucial function in the restricted expression of the TEAD4-ΔN isoform in specific tissues, including the umbilical cord and the placenta. The data presented indicate that the DNA-methylation status of the TEAD4-ΔN promoter plays a critical role in regulating organ size, cancer development, and placenta differentiation.

The members of the IgLON superfamily of cell adhesion molecules facilitate fundamental cellular communication during brain development, maintain functional brain circuitry, and are associated with several neuropsychiatric disorders such as depression, autism, schizophrenia, and intellectual disabilities. Usage of alternative promoter-specific 1a and 1b mRNA isoforms in Lsamp , Opcml , Ntm , and the single promoter of Negr1 in the mouse and human brain has been previously described. To determine the precise spatiotemporal expression dynamics of Lsamp , Opcml , Ntm isoforms, and Negr1 , in the developing brain, we generated isoform-specific RNA probes and carried out in situ hybridization in the developing (embryonic, E10.5, E11.5, 13.5, 17; postnatal, P0) and adult mouse brains. We show that promoter-specific expression of IgLONs is established early during pallial development (at E10.5), where it remains throughout its differentiation through adulthood. In the diencephalon, midbrain, and hindbrain, strong expression patterns are initiated a few days later and begin fading after birth, being only faintly expressed during adulthood. Thus, the expression of specific IgLONs in the developing brain may provide the means for regionally specific functionality as well as for specific regional vulnerabilities. The current study will therefore improve the understanding of how IgLON genes are implicated in the development of neuropsychiatric disorders.

Human apolipoprotein A-I (ApoA-I) is a major structural and functional protein component of high-density lipoprotein (HDL). ApoA-I constitutes ~75% of the protein content of HDL. The main sites of ApoA-I synthesis in humans are the liver and the small intestine. The mechanisms that govern tissue-specific apoA-I transcription in tissues and organs other than the liver and the small intestine are poorly understood. It is known that the human apoA-I has two additional promoters, the proximal and the distal one. In this work these two alternative apoA-I promoters are characterized, their transcription start sites are mapped and their competition for apoA-Itranscription is demonstrated; the role of the alternative promoters in apoA-I expression in human cells and tissues other than hepatocytes and enterocytes is discussed.

SATB1 is a genome organizer protein that is expressed in a lineage specific manner in CD4 + T-cells. SATB1 plays a crucial role in expression of multiple genes throughout the thymic development and peripheral differentiation of T cells. Although SATB1 function has been subjected to intense investigation, regulation of SATB1 gene expression remains poorly understood. Analysis of RNA-seq data revealed multiple transcription start sites at the upstream regulatory region of SATB1 . We further demonstrated that SATB1 gene is expressed via alternative promoters during T-helper (Th) cell differentiation. The proximal promoter "P1" is used more by the naïve and activated CD4 + T-cells whereas the middle "P2" and the distal "P3" promoters are used at a significantly higher level by polarized T-helper cells. Cytokine and TCR signaling play crucial roles toward SATB1 alternative promoter usage. Under Th2 polarization conditions, transcription factor STAT6, which operates downstream of the cytokine signaling binds to the P2 and P3 promoters. Genetic perturbation by knockout and chemical inhibition of STAT6 activation resulted in the loss of P2 and P3 promoter activity. Moreover, chemical inhibition of activation of NF- κ B, a transcription factor that operates downstream of the TCR signaling, also resulted in reduced P2 and P3 promoter usage. Furthermore, usage of the P1 promoter correlated with lower SATB1 protein expression whereas P2 and P3 promoter usage correlated with higher SATB1 protein expression. Thus, the promoter switch might play a crucial role in fine-tuning of SATB1 protein expression in a cell type specific manner.

Hemocyanin (HMC) is a respiratory glycoprotein, which also plays multifunctional non-specific innate immune defense functions in shrimp. However, the transcriptional regulatory mechanisms of the hemocyanin gene expression have not been reported. In the present study, we cloned a 4324 bp fragment of small subunit hemocyanin (HMCs) gene of Litopenaeus vannamei including the 5'-flanking region, from upstream 2475 bp to downstream 1849 bp (exon 1-intron 1-exon 2) by genome walking method. Four deletion constructs were then generated and their promoter activity assessed using the luciferase reporter system. Interestingly, we identified an alternative promoter (+1516/+1849 bp) located in exon 2, which has stronger promoter activity than the full-length or the other constructs. Bioinformatics analyses revealed that the alternative promoter region contains two conserved binding sites of the transcription factor c-Jun. Mutational analysis and electrophoretic mobility shift assay showed that Litopenaeus vannamei c-Jun (Lvc-Jun) binds to the region +1582/+1589 bp and +1831/+1837 bp of the alternative promoter. Furthermore, overexpression of Lvc-Jun significantly increased the alternative promoter activity, while co-transfection with dsRNA-Lvc-Jun significantly reduced the alternative promoter activity of HMCs. Taken together, our present data indicate that the transcription factor Lvc-Jun is essential for the transcriptional regulation of the HMCs gene expression., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Promoter-specific expression of the PPARGC1A gene in untrained and trained human skeletal muscle was investigated. Ten untrained males performed a one-legged knee extension exercise (for 60 min) with the same relative intensity both before and after 8 weeks of cycling training. Samples from the m. vastus lateralis of each leg were taken before and after exercise. Postexercise PPARGC1A gene expression via the canonical promoter increased by ~100% ( P  < 0.05) in exercised and nonexercised untrained muscles, but did not change in either leg after training program. In untrained and trained exercised muscle, PPARGC1A gene expression via the alternative promoter increased by two orders of magnitude ( P  < 0.01). We found increases in postexercise content of dephosphorylated (activated) CRTC2, a coactivator of CREB1, in untrained exercised muscle and in expression of CREB1-related genes in untrained and trained exercised muscle ( P  < 0.01-0.05); this may partially explain the increased expression of PPARGC1A via the alternative promoter. In addition, comparison of the regulatory regions of both promoters revealed unique conserved motifs in the alternative promoter that were associated with transcriptional repressors SNAI1 and HIC1. In conclusion, in untrained muscle, exercise-induced expression of the PPARGC1A gene via the canonical promoter may be regulated by systemic factors, while in trained muscle the canonical promoter shows constitutive expression at rest and after exercise. Exercise-induced expression of PPARGC1A via the alternative promoter relates to intramuscular factors and associates with activation of CRTC2-CREB1. Apparently, expression via the alternative promoter is regulated by other transcription factors, particularly repressors., (© 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

New Findings: What is the central question of this study? This study was designed to investigate the role of AMPK in the regulation of PGC-1α gene expression via the alternative promoter through a cAMP response element-binding protein-1-dependent mechanism in human skeletal muscle. What is the main finding and its importance? Low-intensity exercise markedly increased the expression of PGC-1α mRNA via the alternative promoter, without increases in ACC Ser79/222 (a marker of AMPK activation) and AMPK Thr172 phosphorylation. A single dose of the AMPK activator metformin indicated that AMPK was not involved in regulating PGC-1α mRNA expression via the alternative promoter in endurance-trained human skeletal muscle. In human skeletal muscle, PGC-1α is constitutively expressed via the canonical promoter. In contrast, the expression of PGC-1α mRNA via the alternative promoter was found to be highly dependent on the intensity of exercise and to contribute largely to the postexercise increase of total PGC-1α mRNA. This study investigated the role of AMPK in regulating PGC-1α gene expression via the alternative promoter through a cAMP response element-binding protein-1-dependent mechanism in human skeletal muscle. AMPK activation and PGC-1α gene expression were assayed in skeletal muscle of nine endurance-trained men before and after low-intensity exercise (38% of maximal oxygen uptake) and with or without administration of a single dose (2 g) of the AMPK activator metformin. Low-intensity exercise markedly and significantly increased (∼100-fold, P < 0.05) the expression of PGC-1α mRNA via the alternative promoter, without increasing ACC Ser79/222 (a marker of AMPK activation) and AMPK Thr172 phosphorylation. Moreover, in contrast to placebo, metformin increased the level of ACC Ser79/222 phosphorylation immediately after exercise (2.6-fold, P < 0.05). However postexercise expression of PGC-1α gene via the alternative promoter was not affected. This study was unable to confirm that AMPK plays a role in regulating PGC-1α gene expression via the alternative promoter in endurance-trained human skeletal muscle., (© 2017 The Authors. Experimental Physiology © 2017 The Physiological Society.)

Transcription start-site (TSS) selection and alternative promoter (AP) usage contribute to gene expression complexity but little is known about their impact on translation. Here we performed TSS mapping of the translatome following energy stress. Assessing the contribution of cap-proximal TSS nucleotides, we found dramatic effect on translation only upon stress. As eIF4E levels were reduced, we determined its binding to capped-RNAs with different initiating nucleotides and found the lowest affinity to 5'cytidine in correlation with the translational stress-response. In addition, the number of differentially translated APs was elevated following stress. These include novel glucose starvation-induced downstream transcripts for the translation regulators eIF4A and Pabp, which are also translationally-induced despite general translational inhibition. The resultant eIF4A protein is N-terminally truncated and acts as eIF4A inhibitor. The induced Pabp isoform has shorter 5'UTR removing an auto-inhibitory element. Our findings uncovered several levels of coordination of transcription and translation responses to energy stress., Competing Interests: The authors declare that no competing interests exist.

DLC1 encodes GTPase-activating protein with a well-documented tumor suppressor activity. This gene is downregulated in various tumors through aberrant promoter hypermethylation. Five different DLC1 isoforms can be transcribed from alternative promoters. Tumor-related DNA methylation of the DLC1 isoform 1 alternative promoter was identified as being hypermethylated in meningiomas in genome-wide DNA methylation profiling. We determined the methylation pattern of this region in 50 meningioma FFPE samples and sections of 6 normal meninges, with targeted bisulfite sequencing. All histopathological subtypes of meningiomas showed similar and significant increase of DNA methylation levels. High DNA methylation was associated with lack of DLC1 protein expression in meningiomas as determined by immunohistochemistry. mRNA expression levels of 5 isoforms of DLC1 transcript were measured in an additional series of meningiomas and normal meninges. The DLC1 isoform 1 was found as the most expressed in normal control tissue and was significantly downregulated in meningiomas. Transfection of KT21 meningioma cell line with shRNA targeting DLC1 isoform 1 resulted in increased activation of RHO-GTPases assessed with pull-down assay, enhanced cell migration observed in scratch assay as well as slight increase of cell metabolism determind by MTT test. Results indicate that isoform 1 represents the main pool of DLC1 protein in meninges and its downregulation in meningiomas is associated with hypermethylation of CpG dinucleotides within the corresponding promoter region. This isoform is functional GAP protein and tumor suppressor and targeting of its expression results in the increase of DLC1 related cell processes: RHO activation and cell migration., Competing Interests: The authors report no potential conflicts of interest.

Alternative promoter usage has been proposed as a mechanism regulating transcriptional and translational diversity in highly elaborated systems like the immune system in humans. Here, we report that transcription of human glycoprotein A repetitions predominant (GARP) in regulatory CD4 T cells (Tregs) is tightly regulated by two alternative promoters. An intragenic promoter contains several CpGs and acts as a weak promoter that is demethylated and initiates transcription Treg-specifically. The strong up-stream promoter containing a CpG-island is, in contrast, fully demethylated throughout tissues. Transcriptional activity of the strong promoter was surprisingly down-regulated upon demethylation of the weak promoter. This demethylation-induced transcriptional attenuation regulated the magnitude of GARP expression and correlated with disease activity in rheumatoid arthritis. Treg-specific GARP transcription was initiated by synergistic interaction of forkhead box protein 3 (Foxp3) with nuclear factor of activated T cells (NFAT) and was underpinned by permissive chromatin remodeling caused by release of the H3K4 demethylase, PLU-1. Our findings describe a novel function of alternative promoters in regulating the extent of transcription. Moreover, since GARP functions as a transporter of transforming growth factor β (TGFβ), a cytokine with broad pleiotropic traits, GARP transcriptional attenuation by alternative promoters might provide a mechanism regulating peripheral TGFβ to avoid unwanted harmful effects., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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.

The goal of this study was to identify unknown transcription start sites of the PPARGC1A (PGC-1α) gene in human skeletal muscle and investigate the promoter-specific regulation of PGC-1α gene expression in human skeletal muscle. Ten amateur endurance-trained athletes performed high- and low-intensity exercise sessions (70  min, 70% or 50% o2max). High-throughput RNA sequencing and exon-exon junction mapping were applied to analyse muscle samples obtained at rest and after exercise. PGC-1α promoter-specific expression and activation of regulators of PGC-1α gene expression (AMPK, p38 MAPK, CaMKII, PKA and CREB1) after exercise were evaluated using qPCR and western blot. Our study has demonstrated that during post-exercise recovery, human skeletal muscle expresses the PGC-1α gene via two promoters only. As previously described, the additional exon 7a that contains a stop codon was found in all samples. Importantly, only minor levels of other splice site variants were found (and not in all samples). Constitutive expression PGC-1α gene occurs via the canonical promoter, independent of exercise intensity and exercise-induced increase of AMPK(Thr172) phosphorylation level. Expression of PGC-1α gene via the alternative promoter is increased of two orders after exercise. This post-exercise expression is highly dependent on the intensity of exercise. There is an apparent association between expression via the alternative promoter and activation of CREB1., (© 2015 Society for Endocrinology.)

Transcription factor GATA4 is required for the development and function of the mammalian gonads. We first reported that the GATA4 gene in both human and rodents is expressed as two major alternative transcripts that differ solely in their first untranslated exon (exon 1a vs. exon 1b). We had also showed by quantitative PCR that in mouse tissues, both Gata4 exon 1a- and 1b-containing transcripts are present in all sites that are normally positive for GATA4 protein. In adult tissues, exon 1a-containing transcripts generally predominate. A notable exception, however, is the testis where the Gata4 exon 1a and 1b transcripts exhibit a similar level of expression. We now confirm by in situ hybridization analysis that each transcript is also strongly expressed during gonad differentiation in both sexes in the rat. To gain further insights into how Gata4 gene expression is controlled, we characterized the mouse Gata4 promoter sequence located upstream of exon 1b. In vitro studies revealed that the Gata4 1b promoter is less active than the 1a promoter in several gonadal cell lines tested. Whereas we have previously shown that endogenous Gata4 transcription driven by the 1a promoter is dependent on a proximally located Ebox motif, we now show using complementary in vitro and in vivo approaches that Gata4 promoter 1b-directed expression is regulated by GATA4 itself. Thus, Gata4 transcription in the gonads and other tissues is ensured by distinct promoters that are regulated differentially and independently.