Genomic studies controvert the existence of the CUX1 p75 isoform

CUX1, encoding a homeodomain-containing transcription factor, is recurrently deleted or mutated in multiple tumor types. In myeloid neoplasms, CUX1 deletion or mutation carries a poor prognosis. We have previously established that CUX1 functions as a tumor suppressor in hematopoietic cells across multiple organisms. Others, however, have described oncogenic functions of CUX1 in solid tumors, often attributed to truncated CUX1 isoforms, p75 and p110, generated by an alternative transcriptional start site or post-translational cleavage, respectively. Given the clinical relevance, it is imperative to clarify these discrepant activities. Herein, we sought to determine the CUX1 isoforms expressed in hematopoietic cells and find that they express the full-length p200 isoform. Through the course of this analysis, we found no evidence of the p75 alternative transcript in any cell type examined. Using an array of orthogonal approaches, including biochemistry, proteomics, CRISPR/Cas9 genomic editing, and analysis of functional genomics datasets across a spectrum of normal and malignant tissue types, we found no data to support the existence of the CUX1 p75 isoform as previously described. Based on these results, prior studies of p75 require reevaluation, including the interpretation of oncogenic roles attributed to CUX1.

Targeted Long-Read Sequencing Decodes the Transcriptional Atlas of the Founding RAS Gene Family Members

The complicity of human RAS proteins in cancer is a well-documented fact, both due to the mutational hyperactivation of these GTPases and the overexpression of the genes encoding these proteins. Thus, it can be easily assumed that the study of RAS genes at the transcriptional and post-transcriptional level is of the utmost importance. Although previous research has shed some light on the basic mechanisms by which GTPases are involved in tumorigenesis, limited information is known regarding the transcriptional profile of the genes encoding these proteins. The present study highlights for the first time the wide spectrum of the mRNAs generated by the three most significant RAS genes (KRAS, NRAS and HRAS), providing an in-depth analysis of the splicing events and exon/intron boundaries. The implementation of a versatile, targeted nanopore-sequencing approach led to the identification of 39 novel RAS mRNA transcript variants and to the elucidation of their expression profiles in a broad panel of human cell lines. Although the present work unveiled multiple hidden aspects of the RAS gene family, further study is required to unravel the biological function of all the novel alternative transcript variants, as well as the putative protein isoforms.

A single-cell genomic strategy for alternative transcript start sites identification

Alternative transcription start sites (TSSs) usage plays a critical role in gene transcription regulation in mammals. However, precisely identifying alternative TSSs remains challenging at the genome-wide level. Here, we report a single-cell genomic technology for alternative TSSs annotation and cell heterogeneity detection. In the method, we utilize Fluidigm C1 system to capture individual cells of interest, SMARTer cDNA synthesis kit to recover full-length cDNAs, then dual priming oligonucleotide system to specifically enrich TSSs for genomic analysis. We apply this method to a genome-wide study of alternative TSSs identification in two different IFN-β stimulated mouse embryonic fibroblasts (MEFs). We quantify the performance of our method and find it as accurate as other single cell methods for the detection of TSSs. Our data are also clearly discriminate two IFN-β stimulated MEFs. Moreover, our results indicate 82% expressed genes in these two cell types containing multiple TSSs, which is much higher than previous predictions based on CAGE (58%) or empirical determination (54%) in various cell types. This indicates that alternative TSSs are more pervasive than expected and implies our strategy could position them at an unprecedented sensitivity. It would be helpful for elucidating their biological insights in future.

Comprehensive characterization of single-cell full-length isoforms in human and mouse with long-read sequencing

A modified Chromium 10x droplet-based protocol that subsamples cells for both short-read and long-read (nanopore) sequencing together with a new computational pipeline (FLAMES) is developed to enable isoform discovery, splicing analysis, and mutation detection in single cells. We identify thousands of unannotated isoforms and find conserved functional modules that are enriched for alternative transcript usage in different cell types and species, including ribosome biogenesis and mRNA splicing. Analysis at the transcript level allows data integration with scATAC-seq on individual promoters, improved correlation with protein expression data, and linked mutations known to confer drug resistance to transcriptome heterogeneity.

Nanopore Sequencing Unveils Diverse Transcript Variants of the Epithelial Cell-Specific Transcription Factor Elf-3 in Human Malignancies

The human E74-like ETS transcription factor 3 (Elf-3) is an epithelium-specific member of the ETS family, all members of which are characterized by a highly conserved DNA-binding domain. Elf-3 plays a crucial role in epithelial cell differentiation by participating in morphogenesis and terminal differentiation of the murine small intestinal epithelium, and also acts as an indispensable regulator of mesenchymal to epithelial transition, underlying its significant involvement in development and in pathological states, such as cancer. Although previous research works have deciphered the functional role of Elf-3 in normal physiology as well as in tumorigenesis, the present study highlights for the first time the wide spectrum of ELF3 mRNAs that are transcribed, providing an in-depth analysis of splicing events and exon/intron boundaries in a broad panel of human cell lines. The implementation of a versatile targeted nanopore sequencing approach led to the identification of 25 novel ELF3 mRNA transcript variants (ELF3 v.3–v.27) with new alternative splicing events, as well as two novel exons. Although the current study provides a qualitative transcriptional profile regarding ELF3, further studies must be conducted, so the biological function of all novel alternative transcript variants as well as the putative protein isoforms are elucidated.

The Detection and Bioinformatic Analysis of Alternative 3′ UTR Isoforms as Potential Cancer Biomarkers

Alternative transcript cleavage and polyadenylation is linked to cancer cell transformation, proliferation and outcome. This has led researchers to develop methods to detect and bioinformatically analyse alternative polyadenylation as potential cancer biomarkers. If incorporated into standard prognostic measures such as gene expression and clinical parameters, these could advance cancer prognostic testing and possibly guide therapy. In this review, we focus on the existing methodologies, both experimental and computational, that have been applied to support the use of alternative polyadenylation as cancer biomarkers.

Nuclear ErbB-2-Induced Transcriptome Drives Triple Negative Breast Cancer Growth

Triple negative breast cancer (TNBC) refers to tumors that do not express clinically significant levels of estrogen and progesterone receptors, and lack membrane overexpression or gene amplification of ErbB-2 tyrosine kinase receptor. Transcriptome and proteome heterogeneity of TNBC poses a major challenge to precision medicine. Gene expression analyses have categorized TNBC into distinct molecular subtypes. Up to 78% of clinical TNBCs belong to the basal-like (BL) subtype. Here we found ErbB-2 in an unanticipated scenario: the nucleus of TNBC (NErbB-2). Our study on ErbB-2 alternative splicing, using a PCR-sequencing approach combined with RNA interference, revealed that BL TNBC cells express the canonical ErbB-2 (WTErbB-2), encoded by transcript 1, and the non-canonical isoform c, encoded by alternative transcript 3 (T3). The latter was not previously reported in normal or malignant cells. To characterize the isoform c we designed siRNAs targeting T3 (T3 siRNAs), which silenced up to 93% of said isoform. Transfection of T3 siRNAs into BL cells expressing only isoform c or both isoform c and WTErbB-2 was sufficient to decrease cell proliferation. Intratumoral injections of T3 siRNAs into mice bearing BL TN tumors also blocked in vivo growth. To explore whether isoform c growth-promoting effect is due to its functions as a transcriptional regulator, we performed RNA-seq in BL cells expressing only this isoform. We identified a set of genes differentially regulated in BL cells where we evicted isoform c from the nucleus, as compared to control cells. In the up-regulated group, we found enrichment of pro-apoptotic and tumor suppressor genes and in the down-regulated one, genes involved in proliferation and stemness. We used gene set enrichment analysis (GSEA) to identify the biological processes associated with these isoform c-regulated genes. We found a pronounced enrichment of gene sets related to apoptosis, activation of DNA damage pathways and cell cycle arrest in response to eviction of nuclear isoform c. GSEA also revealed negative regulation of gene sets involved in cell motility, cellular differentiation and growth pathways in BL cells lacking nuclear isoform c expression. These results suggest that NErbB-2 function modulates tumor growth and promotes a metastatic phenotype in TNBC. Furthermore, our clinical findings identified NErbB-2 as an independent predictor of shorter OS (HR 2.54; 95% CI 1.22-5.28; P = 0.013), DFS (HR 2.91; 95% CI 1.44-5.87; P = 0.003), and DMFS (HR 2.59; 95% CI 1.20-5.60; P = 0.015) in 99 TN primary tumors. Our discoveries challenge the present scenario of drug development for personalized BC medicine that focuses on wild-type proteins, which conserve the canonical domains and are located in their classical cellular compartments, highlighting the potential of NErbB-2 isoforms as novel therapeutic targets and clinical biomarkers in TNBC.

Breast Cancer Patient Prognosis Is Determined by the Interplay between TP53 Mutation and Alternative Transcript Expression: Insights from TP53 Long Amplicon Digital PCR Assays

The TP53 gene locus is capable of producing multiple RNA transcripts encoding the different p53 protein isoforms. We recently described multiplex long amplicon droplet digital PCR (ddPCR) assays to quantify seven of eight TP53 reference transcripts in human tumors. Here, we describe a new long amplicon ddPCR assay to quantify expression of the eighth TP53 reference transcript encoding ∆40p53α. We then applied these assays, alongside DNA sequencing of the TP53 gene locus, to tumors from a cohort of New Zealand (NZ) breast cancer patients. We found a high prevalence of mutations at TP53 splice sites in the NZ breast cancer cohort. Mutations at TP53 intron 4 splice sites were associated with overexpression of ∆133TP53 transcripts. Cox proportional hazards survival analysis showed that interplay between TP53 mutation status and expression of TP53 transcript variants was significantly associated with patient outcome, over and above standard clinical and pathological information. In particular, patients with no TP53 mutation and a low ratio of TP53 transcripts t2 to t1, which derive from alternative intron 1 acceptor splice sites, had a remarkably good outcome. We suggest that this type of analysis, integrating mutation and transcript expression, provides a step-change in our understanding of TP53 in cancer.

Genetic, epigenetic, and environmental factors controlling oxytocin receptor gene expression

Background The neuropeptide oxytocin regulates mammalian social behavior. Disruptions in oxytocin signaling are a feature of many psychopathologies. One commonly studied biomarker for oxytocin involvement in psychiatric diseases is DNA methylation at the oxytocin receptor gene (OXTR). Such studies focus on DNA methylation in two regions of OXTR, exon 3 and a region termed MT2 which overlaps exon 1 and intron 1. However, the relative contribution of exon 3 and MT2 in regulating OXTR gene expression in the brain is currently unknown. Results Here, we use the prairie vole as a translational animal model to investigate genetic, epigenetic, and environmental factors affecting Oxtr gene expression in a region of the brain that has been shown to drive Oxtr related behavior in the vole, the nucleus accumbens. We show that the genetic structure of Oxtr in prairie voles resembles human OXTR. We then studied the effects of early life experience on DNA methylation in two regions of a CpG island surrounding the Oxtr promoter: MT2 and exon 3. We show that early nurture in the form of parental care results in DNA hypomethylation of Oxtr in both MT2 and exon 3, but only DNA methylation in MT2 is associated with Oxtr gene expression. Network analyses indicate that CpG sites in the 3′ portion of MT2 are most highly associated with Oxtr gene expression. We also identify two novel SNPs in exon 3 of Oxtr in prairie voles and a novel alternative transcript originating from the third intron of the gene. Expression of the novel alternative transcript is associated with genotype at SNP KLW2. Conclusions These results identify putative regulatory features of Oxtr in prairie voles which inform future studies examining OXTR in human social behaviors and disorders. These studies indicate that in prairie voles, DNA methylation in MT2, particularly in the 3′ portion, is more predictive of Oxtr gene expression than DNA methylation in exon 3. Similarly, in human temporal cortex, we find that DNA methylation in the 3′ portion of MT2 is associated with OXTR expression. Together, these results suggest that among the CpG sites studied, DNA methylation of MT2 may be the most reliable indicator of OXTR gene expression. We also identify novel features of prairie vole Oxtr, including SNPs and an alternative transcript, which further develop the prairie vole as a translational model for studies of OXTR.