A Pan-cancer Transcriptome Analysis Reveals Pervasive Regulation through Alternative Promoters.

Most human protein-coding genes are regulated by multiple, distinct promoters, suggesting that the choice of promoter is as important as its level of transcriptional activity. However, while a global change in transcription is recognized as a defining feature of cancer, the contribution of alternative promoters still remains largely unexplored. Here, we infer active promoters using RNA-seq data from 18,468 cancer and normal samples, demonstrating that alternative promoters are a major contributor to context-specific regulation of transcription. We find that promoters are deregulated across tissues, cancer types, and patients, affecting known cancer genes and novel candidates. For genes with independently regulated promoters, we demonstrate that promoter activity provides a more accurate predictor of patient survival than gene expression. Our study suggests that a dynamic landscape of active promoters shapes the cancer transcriptome, opening new diagnostic avenues and opportunities to further explore the interplay of regulatory mechanisms with transcriptional aberrations in cancer.

Activator protein-1 transactivation of the major immediate early locus is a determinant of cytomegalovirus reactivation from latency.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that latently infects hematopoietic cells and has the ability to reactivate when triggered by immunological stress. This reactivation causes significant morbidity and mortality in immune-deficient patients, who are unable to control viral dissemination. While a competent immune system helps prevent clinically detectable viremia, a portrait of the factors that induce reactivation following the proper cues remains incomplete. Our understanding of the complex molecular mechanisms underlying latency and reactivation continues to evolve. We previously showed the HCMV-encoded G protein-coupled receptor US28 is expressed during latency and facilitates latent infection by attenuating the activator protein-1 (AP-1) transcription factor subunit, c-fos, expression and activity. We now show AP-1 is a critical component for HCMV reactivation. Pharmacological inhibition of c-fos significantly attenuates viral reactivation. In agreement, infection with a virus in which we disrupted the proximal AP-1 binding site in the major immediate early (MIE) enhancer results in inefficient reactivation compared to WT. Concomitantly, AP-1 recruitment to the MIE enhancer is significantly decreased following reactivation of the mutant virus. Furthermore, AP-1 is critical for derepression of MIE-driven transcripts and downstream early and late genes, while immediate early genes from other loci remain unaffected. Our data also reveal MIE transcripts driven from the MIE promoter, the distal promoter, and the internal promoter, iP2, are dependent upon AP-1 recruitment, while iP1-driven transcripts are AP-1-independent. Collectively, our data demonstrate AP-1 binding to and activation of the MIE enhancer is a key molecular process controlling reactivation from latency.

[Reduced Expression of the Tissue-Specific Oct-IL Isoform Exerts an Antitumor Effect on Namalwa Burkitt's Lymphoma Cells].

Increased expression levels of the Oct-1 transcription factor is considered to be one of the key markers of poor cancer prognosis. In addition to the ubiquitous Oct-1A isoform, which is found in all cells, there also exists a tissue-specific Oct-1L isoform, which is expressed in hematopoietic cells. Oct-1L increases cell resistance to different stresses and also regulates the expression of genes controlling differentiation of hematopoietic and immune system cells. The tissue-specific Oct-1L isoform levels are significantly increased in the B-cell lymphoblastoma Namalwa and Raji lines and the T-cell lymphoblastoma Jurkat line compared to normal B and T cells. Apparently, aberrant Oct-1L overexpression not only enhances stress resistance but also leads to the disruption of developmental pathways in the cells promoting their malignant transformation. We report here that targeted suppression of the tissue-specific Oct-1L isoform expression reduces the proliferation rate of Namalwa B-lymphoblastic Burkitt's lymphoma cells, significantly increases cell death rate under hypoxic conditions, and makes cells more sensitive to chemotherapeutic agents such as docetaxel and doxorubicin. These results indicate that targeted therapy aimed at the suppression of the Oct-1 isoforms with increased expression levels in tumor cells rather than the total Oct-1, thus avoiding the traumatic effects of total Oct-1 knockdown, may be promising. Selective suppression of Oct-1 isoforms is a promising strategy in the treatment of lymphoid tumors and may contribute to mitigating the disease course and increasing survival rates in cancer patients.

5-Azacytidine Suppresses the Expression of Tissue-Specific Oct-1 Isoform in Namalwa Burkitt's Lymphoma Cell Culture.

Overexpression of the transcription factor POU2F1 (Oct-1) increases the malignant potential of the tumor and determines the unfavorable prognosis for both solid and hematological cases of the disease in human carcinogenesis. The Oct-1 level determines the rate of development of the disease in acute myelodysplastic leukemia (AML), and a decrease in its expression significantly delays the development of leukemia in mice; however, a complete knockout of Oct-1 leads to the death of the animals. POU2F1 (Oct-1) is expressed as several isoforms transcribed from alternative promoters. They include both ubiquitous and tissue-specific isoforms. It was shown that in Burkitt's lymphoma Namalwa cells 5-azacytidine specifically suppresses the expression of the tissue-specific isoform Oct-1L mRNA (level of Oct-1L is abnormally increased in these cells), while not causing changes in the amount of the ubiquitous isoform Oct-1A mRNA. These results show that it is possible to selectively reduce the transcription level of the Oct-1L isoform aberrantly expressed in human tumor cells.

Transcriptional Regulation of the Human IL5RA Gene through Alternative Promoter Usage during Eosinophil Development.

Regulation of the IL-5 receptor alpha (IL5RA) gene is complicated, with two known promoters (P1 and P2) driving transcription, and two known isoforms (transmembrane and soluble) dichotomously affecting the signaling potential of the protein products. Here, we sought to determine the patterns of P1 and P2 promoter usage and transcription factor occupancy during primary human eosinophil development from CD34+ hematopoietic stem cell progenitors. We found that during eosinophilopoiesis, both promoters were active but subject to distinct temporal regulation, coincident with combinatorial interactions of transcription factors, including GATA-1, PU.1, and C/EBP family members. P1 displayed a relatively constant level of activity throughout eosinophil development, while P2 activity peaked early and waned thereafter. The soluble IL-5Rα mRNA peaked early and showed the greatest magnitude fold-induction, while the signaling-competent transmembrane isoform peaked moderately. Two human eosinophilic cell lines whose relative use of P1 and P2 were similar to eosinophils differentiated in culture were used to functionally test putative transcription factor binding sites. Transcription factor occupancy was then validated in primary cultures by ChIP. We conclude that IL-5-dependent generation of eosinophils from CD34+ precursors involves complex and dynamic activity including both promoters, several interacting transcription factors, and both signaling and antagonistic protein products.

GSK3 Kinase Inhibitor, CHIR, Suppress Transcription of Tissue Specific POU2F1 Isoform in Burkitt Namalwa Lymphoma Cells.

POU2F1 (Oct-1) is a transcription factor, the overexpression of which is found in many human malignant tumors; a significant increase in its level in cells determines the malignant potential of the tumor. POU2F1 is represented in cells by several isoforms that are transcribed from alternative promoters. In Burkitt's B-cell lymphoma Namalwa, the concentration of tissue-specific isoform Oct-1L is several times higher than in normal B cells. We tested the potential to inhibit the transcription of individual Oct-1 isoforms using the GSK3 kinase inhibitor CHIR, an aminopyrimidine derivative. We have shown that CHIR specifically affects the expression of the tissue-specific isoform Oct-1L, significantly reducing the level of mRNA and Oct-1L protein. However, CHIR does not change the amount of mRNA and protein of the ubiquitous isoform Oct-1A in Namalwa tumor cells. The results obtained show that it is possible to develop a system for selective inhibition of Oct-1 transcription factor isoforms in human cells to suppress drug resistance of tumor cells with a high POU2F1 content.

Developmental stage-specific expression of genes for sphingomyelin synthase in rat brain.

Sphingomyelin synthase genes (Sgms1 and Sgms2) encode the vital enzymes that participate in the processes of membrane transport, cell proliferation and apoptosis. We previously determined the exon-intron structure of Sgms1 and some features of its expression in human and rodent tissues. The circular RNAs (circRNAs) emerging from exons of the 5'-untranslated region (5'-UTR) of Sgms1 were determined. These circRNAs are represented at a high level in the adult brain. Here, we demonstrate that, in contrast to Sgms1, Sgms2 does not contain the multi-exon 5'-UTR but encodes circRNAs, which are composed of the coding region of the gene and are expressed at a low level. We present a study of the expression of sphingomyelin synthase genes in rat brain at embryonic days 7, 9, 13, 17 and 21 and in adult rat brain. In contrast to Sgms1, Sgms2 is expressed at a significantly low level in adult brain. In embryonic rat brain, the mRNA expression of sphingomyelin synthase genes is varied in a developmental stage-specific manner. The level of Sgms1 mRNAs, differing by 5'-UTR-in the formation of which alternative promoters can participate-changes significantly during the process of embryonic development. The expression of circRNAs of Sgms1 was significantly raised during rat embryonic brain development. We assume that the circRNAs are involved in the regulation of sphingomyelin synthase activity in rat brain in different developmental stages.

Functional characterization of the ABCG2 5' non-coding exon variants: Stem cell specificity, translation efficiency and the influence of drug selection.

ABCG2 is a multidrug transporter with wide substrate specificity, and is believed to protect several cell types from various xenobiotics and endobiotics. This "guardian" function is important in numerous cell types and tissue barriers but becomes disadvantageous by being responsible for the multidrug resistance phenotype in certain tumor cells. ABCG2 regulation at the protein level has already been extensively studied, however, regulation at the mRNA level, especially the functional role of the various 5' untranslated exon variants (5' UTRs) has been elusive. In the present work, we describe a comprehensive characterization of four ABCG2 mRNA variants with different exon 1 sequences, investigate drug inducibility, stem cell specificity, mRNA stability, and translation efficiency. Although certain variants (E1B and E1C) are considered as "constitutive" mRNA isoforms, we show that chemotoxic drugs significantly alter the expression pattern of distinct ABCG2 mRNA isoforms. When examining human embryonic stem cell lines, we provide evidence that variant E1A has an expression pattern coupled to undifferentiated stem cell stage, as its transcript level is regulated parallel to mRNAs of Oct4 and Nanog pluripotency marker genes. When characterizing the four exon 1 variants we found no significant differences in terms of mRNA stabilities and half-lives of the isoforms. In contrast, variant E1U showed markedly lower translation efficiency both at the total protein level or regarding the functional presence in the plasma membrane. Taken together, these results indicate that the different 5' UTR variants play an important role in cell type specific regulation and fine tuning of ABCG2 expression.

Relatively frequent switching of transcription start sites during cerebellar development.

BACKGROUND: Alternative transcription start site (TSS) usage plays important roles in transcriptional control of mammalian gene expression. The growing interest in alternative TSSs and their role in genome diversification spawned many single-gene studies on differential usages of tissue-specific or temporal-specific alternative TSSs. However, exploration of the switching usage of alternative TSS usage on a genomic level, especially in the central nervous system, is largely lacking. RESULTS: In this study, We have prepared a unique set of time-course data for the developing cerebellum, as part of the FANTOM5 consortium ( http://fantom.gsc.riken.jp/5/ ) that uses their innovative capturing of 5' ends of all transcripts followed by Helicos next generation sequencing. We analyzed the usage of all transcription start sites (TSSs) at each time point during cerebellar development that provided information on multiple RNA isoforms that emerged from the same gene. We developed a mathematical method that systematically compares the expression of different TSSs of a gene to identify temporal crossover and non-crossover switching events. We identified 48,489 novel TSS switching events in 5433 genes during cerebellar development. This includes 9767 crossover TSS switching events in 1511 genes, where the dominant TSS shifts over time. CONCLUSIONS: We observed a relatively high prevalence of TSS switching in cerebellar development where the resulting temporally-specific gene transcripts and protein products can play important regulatory and functional roles.

Distinct isoform of FABP7 revealed by screening for retroelement-activated genes in diffuse large B-cell lymphoma.

Remnants of ancient transposable elements (TEs) are abundant in mammalian genomes. These sequences harbor multiple regulatory motifs and hence are capable of influencing expression of host genes. In response to environmental changes, TEs are known to be released from epigenetic repression and to become transcriptionally active. Such activation could also lead to lineage-inappropriate activation of oncogenes, as one study described in Hodgkin lymphoma. However, little further evidence for this mechanism in other cancers has been reported. Here, we reanalyzed whole transcriptome data from a large cohort of patients with diffuse large B-cell lymphoma (DLBCL) compared with normal B-cell centroblasts to detect genes ectopically expressed through activation of TE promoters. We have identified 98 such TE-gene chimeric transcripts that were exclusively expressed in primary DLBCL cases and confirmed several in DLBCL-derived cell lines. We further characterized a TE-gene chimeric transcript involving a fatty acid-binding protein gene (LTR2-FABP7), normally expressed in brain, that was ectopically expressed in a subset of DLBCL patients through the use of an endogenous retroviral LTR promoter of the LTR2 family. The LTR2-FABP7 chimeric transcript encodes a novel chimeric isoform of the protein with characteristics distinct from native FABP7. In vitro studies reveal a dependency for DLBCL cell line proliferation and growth on LTR2-FABP7 chimeric protein expression. Taken together, these data demonstrate the significance of TEs as regulators of aberrant gene expression in cancer and suggest that LTR2-FABP7 may contribute to the pathogenesis of DLBCL in a subgroup of patients.

Next-generation sequencing-based 5' rapid amplification of cDNA ends for alternative promoters.

Mammalian genomes contain many unknown alternative first exons and promoters. Thus, we have modified the existing 5'RACE (5' rapid amplification of cDNA ends) approach into a next-generation sequencing (NGS)-based new protocol that can identify these alternative promoters. This protocol has incorporated two main ideas: (i) 5'RACE starting from the known second exons of genes and (ii) NGS-based sequencing of the subsequent cDNA products. This protocol also provides a bioinformatics strategy that processes the sequence reads from NGS runs. This protocol has successfully identified several alternative promoters for an imprinted gene, PEG3. Overall, this NGS-based 5'RACE protocol is a sensitive and reliable method for detecting low-abundant transcripts and promoters.

Dual role of Tlx3 as modulator of Prrxl1 transcription and phosphorylation.

The proper establishment of the dorsal root ganglion/spinal cord nociceptive circuitry depends on a group of homeodomain transcription factors that includes Prrxl1, Brn3a and Tlx3. By the use of epistatic analysis, it was suggested that Tlx3 and Brn3a, which highly co-localize with Prrxl1 in these tissues, are required to maintain Prrxl1 expression. Here, we report two Tlx3-dependent transcriptional mechanisms acting on Prrxl1 alternative promoters, referred to as P3 and P1/P2 promoters. We demonstrate that (i) Tlx3 induces the transcriptional activity of the TATA-containing promoter P3 by directly binding to a bipartite DNA motif and (ii) it synergistically interacts with Prrxl1 by indirectly activating the Prrxl1 TATA-less promoters P1/P2 via the action of Brn3a. The Tlx3 N-terminal domain 1-38 was shown to have a major role on the overall Tlx3 transcriptional activity and the C-terminus domain (amino acids 256-291) to mediate the Tlx3 effect on promoters P1/P2. On the other hand, the 76-111 domain was shown to decrease Tlx3 activity on the TATA-promoter P3. In addition to its action on Prrxl1 alternative promoters, Tlx3 proved to have the ability to induce Prrxl1 phosphorylation. The Tlx3 domain responsible for Prrxl1 hyperphosphorylation was mapped and encompasses amino acid residues 76 to 111. Altogether, our results suggest that Tlx3 uses distinct mechanisms to tightly modulate Prrxl1 activity, either by controlling its transcriptional levels or by increasing Prrxl1 phosphorylation state.

Several cis-regulatory elements control mRNA stability, translation efficiency, and expression pattern of Prrxl1 (paired related homeobox protein-like 1).

The homeodomain transcription factor Prrxl1/DRG11 has emerged as a crucial molecule in the establishment of the pain circuitry, in particular spinal cord targeting of dorsal root ganglia (DRG) axons and differentiation of nociceptive glutamatergic spinal cord neurons. Despite Prrxl1 importance in the establishment of the DRG-spinal nociceptive circuit, the molecular mechanisms that regulate its expression along development remain largely unknown. Here, we show that Prrxl1 transcription is regulated by three alternative promoters (named P1, P2, and P3), which control the expression of three distinct Prrxl1 5'-UTR variants, named 5'-UTR-A, 5'-UTR-B, and 5'-UTR-C. These 5'-UTR sequences confer distinct mRNA stability and translation efficiency to the Prrxl1 transcript. The most conserved promoter (P3) contains a TATA-box and displays in vivo enhancer activity in a pattern that overlaps with the zebrafish Prrxl1 homologue, drgx. Regulatory modules present in this sequence were identified and characterized, including a binding site for Phox2b. Concomitantly, we demonstrate that zebrafish Phox2b is required for the expression of drgx in the facial, glossopharyngeal, and vagal cranial ganglia.

Genetic and epigenetic regulation of the brain-derived neurotrophic factor in the central nervous system.

The BDNF is required for the development and proper function of the central nervous system, where it is involved in a variety of neural and molecular events relevant to cognition, learning, and memory processes. Although only a functional mature protein is synthesized, the human BDNF gene possesses an extensive structural complexity, including the presence of multiple promoters, splicing events, and 3´UTR poly-adenylation sites, resulting in an intricate transcriptional regulation and numerous messengers RNA. Recent data support specific cellular roles of these transcripts. Moreover, a central role of epigenetic modifications on the regulation of BDNF gene transcription is also emerging. The present essay aims to summarize the published information on the matter, emphasizing their possible implications in health and disease or in the treatment of different neurologic and psychiatric disorders.

The prolactin receptor: A cross-species comparison of gene structure, transcriptional regulation, tissue-specificity, and genetic variation.

The conserved and multifaceted functions of prolactin (PRL) are coordinated through varied distribution and expression of its cell-surface receptor (PRLR) across a range of tissues and physiological states. The resultant heterogeneous expression of PRLR mRNA and protein across different organs and cell types supports a wide range of PRL-regulated processes including reproduction, lactation, development, and homeostasis. Genetic variation within the PRLR gene also accounts for several phenotypes impacting agricultural production and human pathology. The goal of this review is to highlight the many elements that control differential expression of the PRLR across tissues, and the various phenotypes that exist across species due to variation in the PRLR gene.

Corrigendum: A genome-wide investigation of effects of aberrant DNA methylation on the usage of alternative promoters in hepatocellular carcinoma.

[This corrects the article DOI: 10.3389/fonc.2021.780266.].

Mapping the splicing landscape of the human immune system.

Most human genes code for more than one transcript. Different ratios of transcripts of the same gene can be found in different cell types or states, indicating differential use of transcription start sites or differential splicing. Such differential transcript use (DTUs) events provide an additional layer of regulation and protein diversity. With the exceptions of PTPRC and CIITA, there are very few reported cases of DTU events in the immune system. To rigorously map DTUs between different human immune cell types, we leveraged four publicly available RNA sequencing datasets. We identified 282 DTU events between five human healthy immune cell types that appear in at least two datasets. The patterns of the DTU events were mostly cell-type-specific or lineage-specific, in the context of the five cell types tested. DTUs correlated with the expression pattern of potential regulators, namely, splicing factors and transcription factors. Of the several immune related conditions studied, only sepsis affected the splicing of more than a few genes and only in innate immune cells. Taken together, we map the DTUs landscape in human peripheral blood immune cell types, and present hundreds of genes whose transcript use changes between cell types or upon activation.

A Genome-Wide Investigation of Effects of Aberrant DNA Methylation on the Usage of Alternative Promoters in Hepatocellular Carcinoma.

BACKGROUND: The alternative usage of promoters provides a way to regulate gene expression, has a significant influence on the transcriptome, and contributes to the cellular transformation of cancer. However, the function of alternative promoters (APs) in hepatocellular carcinoma (HCC) has not been systematically studied yet. In addition, the potential mechanism of regulation to the usage of APs remains unclear. DNA methylation, one of the most aberrant epigenetic modifications in cancers, is known to regulate transcriptional activity. Whether DNA methylation regulates the usage of APs needs to be explored. Here, we aim to investigate the effects of DNA methylation on usage of APs in HCC. METHODS: Promoter activities were calculated based on RNA-seq data. Functional enrichment analysis was implemented to conduct GO terms. Correlation tests were used to detect the correlation between promoter activity and methylation status. The LASSO regression model was used to generate a diagnostic model. Kaplan-Meier analysis was used to compare the overall survival between high and low methylation groups. RNA-seq and whole-genome bisulfite sequencing (WGBS) in HCC samples were performed to validate the correlation of promoter activity and methylation. RESULTS: We identified 855 APs in total, which could be well used to distinguish cancer from normal samples. The correlation of promoter activity and DNA methylation in APs was observed, and the APs with negative correlation were defined as methylation-regulated APs (mrAPs). Six mrAPs were identified to generate a diagnostic model with good performance (AUC = 0.97). Notably, the majority of mrAPs had CpG sites that could be used to predict clinical outcomes by methylation status. Finally, we verified 85.6% of promoter activity variation and 92.3% of methylation changes in our paired RNA-seq and WGBS samples, respectively. The negative correlation between promoter activity and methylation status was further confirmed in our HCC samples. CONCLUSION: The aberrant methylation status plays a critical role in the precision usage of APs in HCC, which sheds light on the mechanism of cancer development and provides a new insight into cancer screening and treatment.

Widespread overexpression from the four DNA hypermethylated HOX clusters in aggressive (IDHwt) glioma is associated with H3K27me3 depletion and alternative promoter usage.

In human, the 39 coding HOX genes and 18 referenced noncoding antisense transcripts are arranged in four genomic clusters named HOXA, B, C, and D. This highly conserved family belongs to the homeobox class of genes that encode transcription factors required for normal development. Therefore, HOX gene deregulation might contribute to the development of many cancer types. Here, we study HOX gene deregulation in adult glioma, a common type of primary brain tumor. We performed extensive molecular analysis of tumor samples, classified according to their isocitrate dehydrogenase (IDH1) gene mutation status, and of glioma stem cells. We found widespread expression of sense and antisense HOX transcripts only in aggressive (IDHwt) glioma samples, although the four HOX clusters displayed DNA hypermethylation. Integrative analysis of expression, DNA methylation, and histone modification signatures along the clusters revealed that HOX gene upregulation relies on canonical and alternative bivalent CpG island promoters that escape hypermethylation. H3K27me3 loss at these promoters emerges as the main cause of widespread HOX gene upregulation in IDHwt glioma cell lines and tumors. Our study provides the first comprehensive description of the epigenetic changes at HOX clusters and their contribution to the transcriptional changes observed in adult glioma. It also identified putative 'master' HOX proteins that might contribute to the tumorigenic potential of glioma stem cells.

Rbfox1 Is Expressed in the Mouse Brain in the Form of Multiple Transcript Variants and Contains Functional E Boxes in Its Alternative Promoters.

The RNA-binding protein RBFOX1 is an important regulator of neuron development and neuronal excitability. Rbfox1 is a dosage-sensitive gene and in both mice and humans, decreased expression of Rbfox1 has been linked to neurodevelopmental disorders. Alternative promoters drive expression of Rbfox1 transcript isoforms that encode an identical protein. The tissue- and developmental stage-specific expression of these isoforms, as well as the underlying regulatory mechanisms, are, however, unclear. Here, we set out to capture all of the Rbfox1 transcript isoforms and identify transcriptional mechanisms that regulate brain-specific Rbfox1 expression. Isoform sequencing identified multiple alternative Rbfox1 transcript variants in the mouse cerebral cortex, including transcripts with novel first exons, alternatively spliced exons and 3'-truncations. Quantitative RT-PCR determined the expression of the alternative first exons in the developing cerebral cortex and different subregions of the juvenile brain. Alternative first exons were found to be highly stage- and subregion specific in their expression patterns suggesting that they fulfill specific functions during cortex development and in different brain regions. Using reporter assays we found that the promoter regions of the two first exons E1B and E1C/E1C.1 contain several functional E-boxes. Together, we provide an extensive picture of Rbfox1 isoform expression. We further identified important regulatory mechanisms that drive neuron-specific Rbfox1 expression. Thus, our study forms the basis for further research into the mechanisms that ensure physiological Rbfox1 expression in the brain. It also helps to understand why, in patients with neurodevelopmental disorders deletion of individual RBFOX1 transcript isoforms could affect brain function.