Expression of a SOX1 overlapping transcript in neural differentiation and cancer models

SOX1 is a member of the SOXB1 subgroup of transcription factors involved in early embryogenesis, CNS development and maintenance of neural stem cells. The structure and regulation of the human SOX1 locus has been less studied than that of SOX2, another member of the SOXB1 subgroup for which an overlapping transcript has been reported. Here we report that the SOX1 locus harbours a SOX1 overlapping transcript (SOX1-OT), and describe expression, splicing variants and detection of SOX1-OT in different stem and cancer cells. RT-PCR and RACE experiments were performed to detect and characterize the structure of SOX1-OT in neuroprogenitor cultures and across different cancer cell lines. SOX1-OT was found to present a complex structure including several unannotated exons, different transcript variants, and at least two potential transcription start sites. SOX1-OT was found to be highly expressed in differentiated neural stem cells across different time points of differentiation, and its expression correlated with SOX1 gene expression. Concomitant expression of SOX1 and SOX1-OT was further observed in several cancer cell models. While the function of this transcript is unknown, the regulatory role reported for other lncRNAs strongly suggests a possible role for SOX1-OT in regulating SOX1 expression, as previously observed for SOX2. The elucidation of the genetic and regulatory context governing SOX1 expression will contribute to clarifying its role in stem cell differentiation and tumorigenesis

Cannabidiol causes endothelium-dependent vasorelaxation of human mesenteric arteries via CB1 activation

AIMS: The protective effects of cannabidiol (CBD) have been widely shown in preclinical models and have translated into medicines for the treatment of multiple sclerosis and epilepsy. However, the direct vascular effects of CBD in humans are unknown. METHODS AND RESULTS: Using wire myography, the vascular effects of CBD were assessed in human mesenteric arteries, and the mechanisms of action probed pharmacologically. CBD-induced intracellular signalling was characterized using human aortic endothelial cells (HAECs). CBD caused acute, non-recoverable vasorelaxation of human mesenteric arteries with an R(max) of ∼40%. This was inhibited by cannabinoid receptor 1 (CB(1)) receptor antagonists, desensitization of transient receptor potential channels using capsaicin, removal of the endothelium, and inhibition of potassium efflux. There was no role for cannabinoid receptor-2 (CB(2)) receptor, peroxisome proliferator activated receptor (PPAR)γ, the novel endothelial cannabinoid receptor (CB(e)), or cyclooxygenase. CBD-induced vasorelaxation was blunted in males, and in patients with type 2 diabetes or hypercholesterolemia. In HAECs, CBD significantly reduced phosphorylated JNK, NFκB, p70s6 K and STAT5, and significantly increased phosphorylated CREB, ERK1/2, and Akt levels. CBD also increased phosphorylated eNOS (ser1177), which was correlated with increased levels of ERK1/2 and Akt levels. CB(1) receptor antagonism prevented the increase in eNOS phosphorylation. CONCLUSION: This study shows, for the first time, that CBD causes vasorelaxation of human mesenteric arteries via activation of CB(1) and TRP channels, and is endothelium- and nitric oxide-dependent

Decoupling of DNA methylation and activity of intergenic LINE-1 promoters in colorectal cancer

<p>Hypomethylation of LINE-1 repeats in cancer has been proposed as the main mechanism behind their activation; this assumption, however, was based on findings from early studies that were biased toward young and transpositionally active elements. Here, we investigate the relationship between methylation of 2 intergenic, transpositionally inactive LINE-1 elements and expression of the LINE-1 chimeric transcript (LCT) 13 and LCT14 driven by their antisense promoters (L1-ASP). Our data from DNA modification, expression, and 5′RACE analyses suggest that colorectal cancer methylation in the regions analyzed is not always associated with LCT repression. Consistent with this, in HCT116 colorectal cancer cells lacking DNA methyltransferases DNMT1 or DNMT3B, LCT13 expression decreases, while cells lacking both DNMTs or treated with the DNMT inhibitor 5-azacytidine (5-aza) show no change in LCT13 expression. Interestingly, levels of the H4K20me3 histone modification are inversely associated with LCT13 and LCT14 expression. Moreover, at these LINE-1s, H4K20me3 levels rather than DNA methylation seem to be good predictor of their sensitivity to 5-aza treatment. Therefore, by studying individual LINE-1 promoters we have shown that in some cases these promoters can be active without losing methylation; in addition, we provide evidence that other factors (e.g., H4K20me3 levels) play prominent roles in their regulation.</p

BRAF^V600E-mutated serrated colorectal neoplasia drives transcriptional activation of cholesterol metabolism

Abstract BRAF mutations occur early in serrated colorectal cancers, but their long-term influence on tissue homeostasis is poorly characterized. We investigated the impact of short-term (3 days) and long-term (6 months) expression of Braf V600E in the intestinal tissue of an inducible mouse model. We show that Braf V600E perturbs the homeostasis of intestinal epithelial cells, with impaired differentiation of enterocytes emerging after prolonged expression of the oncogene. Moreover, Braf V600E leads to a persistent transcriptional reprogramming with enrichment of numerous gene signatures indicative of proliferation and tumorigenesis, and signatures suggestive of metabolic rewiring. We focused on the top-ranking cholesterol biosynthesis signature and confirmed its increased expression in human serrated lesions. Functionally, the cholesterol lowering drug atorvastatin prevents the establishment of intestinal crypt hyperplasia in Braf V600E -mutant mice. Overall, our work unveils the long-term impact of Braf V600E expression in intestinal tissue and suggests that colorectal cancers with mutations in BRAF might be prevented by statins

DNA methylation of intragenic CpG islands depends on their transcriptional activity during differentiation and disease

The human genome contains ∼30,000 CpG islands (CGIs). While CGIs associated with promoters nearly always remain unmethylated, many of the ∼9,000 CGIs lying within gene bodies become methylated during development and differentiation. Both promoter and intragenic CGIs may also become abnormally methylated as a result of genome rearrangements and in malignancy. The epigenetic mechanisms by which some CGIs become methylated but others, in the same cell, remain unmethylated in these situations are poorly understood. Analyzing specific loci and using a genome-wide analysis, we show that transcription running across CGIs, associated with specific chromatin modifications, is required for DNA methyltransferase 3B (DNMT3B)-mediated DNA methylation of many naturally occurring intragenic CGIs. Importantly, we also show that a subgroup of intragenic CGIs is not sensitive to this process of transcription-mediated methylation and that this correlates with their individual intrinsic capacity to initiate transcription in vivo. We propose a general model of how transcription could act as a primary determinant of the patterns of CGI methylation in normal development and differentiation, and in human disease.</p

Differences in the pattern and regulation of mineral deposition in human cell lines of osteogenic and non-osteogenic origin

Bone marrow-derived mesenchymal stem cells (MSCs) are widely used as a cellular model of bone formation, and can mineralize in vitro in response to osteogenic medium (OM). It is unclear, however, whether this property is specific to cells of mesenchymal origin. We analysed the OM response in 3 non-osteogenic lines, HEK293, HeLa and NTera, compared to MSCs. Whereas HEK293 cells failed to respond to OM conditions, the 2 carcinoma-derived lines NTera and HeLa deposited a calcium phosphate mineral comparable to that present in MSC cultures. However, unlike MSCs, HeLa and NTera cultures did so in the absence of dexamethasone. This discrepancy was confirmed, as bone morphogenetic protein inhibition obliterated the OM response in MSCs but not in HeLa or NTera, indicating that these 2 models can deposit mineral through a mechanism independent of established dexamethasone or bone morphogenetic protein signalling

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Isolation of cancer-specific chimeric transcripts induced by hypomethylation of the LINE-1 antisense promoter

AbstractThe antisense promoter of human LINE-1 (L1) retroelements can direct transcription of adjacent unique genomic sequences generating chimeric RNAs, which can perturb transcription of neighbouring genes. As L1 elements constitute 17% of the human genome, chimeric transcription is potentially widespread, but the extent to which this occurs is largely unknown. Using a genome-wide screen we have isolated novel chimeric transcripts that are unique to breast cancer cell lines, primary tumours and colon cancer cells. Expression of the cancer-specific chimeric transcripts can be induced in non-malignant breast epithelial cells by the demethylating drug 5-azacytidine. These findings indicate that loss of L1 methylation in cancer cells is linked to the expression of L1-chimeric transcripts which may therefore constitute a useful set of markers of malignancy

Investigation of a developmentally linked transcriptional gene silencing mechanism involving an antisense RNA

An increasing number of RNA targeted gene repression mechanisms are being discovered operating in mammalian cells. Antisense-RNA mediated silencing of CpG island-associated genes is not limited to imprinting and X inactivation, but can occur at autosomal non-imprinted loci leading to disease. An antisense silencing phenomenon observed in a patient with an inherited form of anaemia was recently recreated in differentiating mouse embryonic stem cells, demonstrating that the silencing and methylation of the oppositely transcribed tissue specific alpha globin CpG island exclusively occurs in the presence of antisense RNA. The aim of this thesis is to use differentiating mouse embryonic stem cells to further characterise this silencing mechanism through analysis of the histone modifications associated with repression of the alpha globin gene upon differentiation, and to further define the developmental period in which repression is established. Also, the requirement for antisense RNA transcription through the alpha globin gene is examined through insertion of a transcriptional terminator to truncate antisense RNA transcription. The gene silencing mechanism is tested with other genes in place of the alpha globin gene to investigate whether repression is limited to the alpha globin gene or is also applicable to other tissue specific or ubiquitously expressed genes. Analysis of the tissue specific gene MYOD and the ubiquitously expressed gene UBC demonstrated that the silencing mechanism could effect other genes in a similar manner, though not all genes were susceptible to repression as the ubiquitously expressed gene ACTB remained unsilenced by antisense RNA expression. These observations establish that this silencing mechanism can play a more general role in repression, thereby suggesting its potential role as a constituent of the multivariate repressive pathways within the mammalian cell.EThOS - Electronic Theses Online ServiceMRCGBUnited Kingdo

Investigation of a developmentally linked transcriptional gene silencing mechanism involving an antisense RNA

An increasing number of RNA targeted gene repression mechanisms are being discovered operating in mammalian cells. Antisense-RNA mediated silencing of CpG island-associated genes is not limited to imprinting and X inactivation, but can occur at autosomal non-imprinted loci leading to disease. An antisense silencing phenomenon observed in a patient with an inherited form of anaemia was recently recreated in differentiating mouse embryonic stem cells, demonstrating that the silencing and methylation of the oppositely transcribed tissue specific alpha globin CpG island exclusively occurs in the presence of antisense RNA. The aim of this thesis is to use differentiating mouse embryonic stem cells to further characterise this silencing mechanism through analysis of the histone modifications associated with repression of the alpha globin gene upon differentiation, and to further define the developmental period in which repression is established. Also, the requirement for antisense RNA transcription through the alpha globin gene is examined through insertion of a transcriptional terminator to truncate antisense RNA transcription. The gene silencing mechanism is tested with other genes in place of the alpha globin gene to investigate whether repression is limited to the alpha globin gene or is also applicable to other tissue specific or ubiquitously expressed genes. Analysis of the tissue specific gene MYOD and the ubiquitously expressed gene UBC demonstrated that the silencing mechanism could effect other genes in a similar manner, though not all genes were susceptible to repression as the ubiquitously expressed gene ACTB remained unsilenced by antisense RNA expression. These observations establish that this silencing mechanism can play a more general role in repression, thereby suggesting its potential role as a constituent of the multivariate repressive pathways within the mammalian cell.EThOS - Electronic Theses Online ServiceMRCGBUnited Kingdo