Cis-acting Mutations Research Articles

Identification and functional marker development of SbPLSH1 conferring purple leaf sheath in sorghum.

We identified a SbPLSH1gene conferring purple leaf sheath in sorghum (sorghumbicolor(L.) Moench)and developed a functional markerfor it. The purple leaf sheath of sorghum, a trait mostly related to anthocyanin deposition, is a visually distinguishable morphological marker widely used to evaluate the purity of crop hybrids. We aimed to dissect the genetic mechanism for leaf sheath color to mine the genes regulating this trait. In this study, two F2 populations were constructed by crossing a purple leaf sheath inbred line (Gaoliangzhe) with two green leaf sheath inbred lines (BTx623 and Silimei). Based on the results of bulked-segregant analysis sequencing, bulk-segregant RNA sequencing, and map-based cloning, SbPLSH1 (Sobic.006G175700), which encodes a bHLH transcription factor on chromosome 6, was identified as the candidate gene for purple leaf sheath in sorghum. Genetic analysis demonstrated that overexpression of SbPLSH1 in Arabidopsis resulted in anthocyanin deposition and purple petiole, while two single-nucleotide polymorphism (SNP) variants on the exon 6 resulted in loss of function. Further haplotype analysis revealed that there were two missense mutations and one cis-acting element mutation in SbPLSH1, which are closely associated with leaf sheath color in sorghum. Based on the variations, a functional marker (LSC4-2) for marker-assisted selection was developed, which has a broad-spectrum capability of distinguishing leaf sheath color in natural variants. In summary, this study lays a foundation for analyzing the genetic mechanism for sorghum leaf sheath color.

The Effects of De Novo Mutation on Gene Expression and the Consequences for Fitness in Chlamydomonas reinhardtii.

Mutation is the ultimate source of genetic variation, the bedrock of evolution. Yet, predicting the consequences of new mutations remains a challenge in biology. Gene expression provides a potential link between a genotype and its phenotype. But the variation in gene expression created by de novo mutation and the fitness consequences of mutational changes to expression remain relatively unexplored. Here, we investigate the effects of >2,600 de novo mutations on gene expression across the transcriptome of 28 mutation accumulation lines derived from 2 independent wild-type genotypes of the green algae Chlamydomonas reinhardtii. We observed that the amount of genetic variance in gene expression created by mutation (Vm) was similar to the variance that mutation generates in typical polygenic phenotypic traits and approximately 15-fold the variance seen in the limited species where Vm in gene expression has been estimated. Despite the clear effect of mutation on expression, we did not observe a simple additive effect of mutation on expression change, with no linear correlation between the total expression change and mutation count of individual MA lines. We therefore inferred the distribution of expression effects of new mutations to connect the number of mutations to the number of differentially expressed genes (DEGs). Our inferred DEE is highly L-shaped with 95% of mutations causing 0-1 DEG while the remaining 5% are spread over a long tail of large effect mutations that cause multiple genes to change expression. The distribution is consistent with many cis-acting mutation targets that affect the expression of only 1 gene and a large target of trans-acting targets that have the potential to affect tens or hundreds of genes. Further evidence for cis-acting mutations can be seen in the overabundance of mutations in or near differentially expressed genes. Supporting evidence for trans-acting mutations comes from a 15:1 ratio of DEGs to mutations and the clusters of DEGs in the co-expression network, indicative of shared regulatory architecture. Lastly, we show that there is a negative correlation with the extent of expression divergence from the ancestor and fitness, providing direct evidence of the deleterious effects of perturbing gene expression.

Identification of the Teopod1, Teopod2, and Early Phase Change genes in maize.

Teopod1 (Tp1), Teopod2 (Tp2), and Early phase change (Epc) have profound effects on the timing of vegetative phase change in maize. Gain-of-function mutations in Tp1 and Tp2 delay all known phase-specific vegetative traits, whereas loss-of-function mutations in Epc accelerate vegetative phase change and cause shoot abortion in some genetic backgrounds. Here, we show that Tp1 and Tp2 likely represent cis-acting mutations that cause the overexpression of Zma-miR156j and Zma-miR156h, respectively. Epc is the maize ortholog of HASTY, an Arabidopsis gene that stabilizes miRNAs and promotes their intercellular movement. Consistent with its pleiotropic phenotype and epistatic interaction with Tp1 and Tp2, epc reduces the levels of miR156 and several other miRNAs.

Network Topology Can Explain Differences in Pleiotropy Between Cis- and Trans-regulatory Mutations.

A mutation's degree of pleiotropy (i.e., the number of traits it alters) is predicted to impact the probability of the mutation being detrimental to fitness. For mutations that impact gene expression, mutations acting in cis have been hypothesized to generally be less pleiotropic than mutations affecting the same gene's expression in trans, suggesting that cis-regulatory mutations should be less deleterious and more likely to fix over evolutionary time. Here, we use expression and fitness data from Saccharomyces cerevisiae gene deletion strains to test these hypotheses. By treating deletion of each gene as a cis-regulatory mutation affecting its own expression and deletions of other genes affecting expression of this focal gene as trans-regulatory mutations, we find that cis-acting mutations do indeed tend to be less pleiotropic than trans-acting mutations affecting expression of the same gene. This pattern was observed for the vast majority of genes in the data set and could be explained by the topology of the regulatory network controlling gene expression. Comparing the fitness of cis- and trans-acting mutations affecting expression of the same gene also confirmed that trans-acting deletions tend to be more deleterious. These findings provide strong support for pleiotropy playing a role in the preferential fixation of cis-regulatory alleles over evolutionary time.

111. pVACsplice: Predicting neoantigens from tumor-specific alternative splicing events derived from regulatory mutations

Neoantigens are tumor-specific peptides presented on the cell surface by MHC that can be recognized by the adaptive immune system. Personalized immunotherapies, such as cancer vaccines, rely on neoantigen prediction to identify sequences that can activate T cells to recognize and destroy the tumor. The majority of cancer vaccine trials have utilized neoantigens derived from missense mutations and small insertions and deletions. However, other mutation types contribute to the overall neoantigen landscape, including aberrantly spliced transcripts arising from cis-acting regulatory mutations. In this study, we explore the potential immunogenicity of alternative splicing events and present pVACsplice, a tool to expand the capability of pVACtools, a suite of tools for neoantigen prediction (http://www.pvactools.org). pVACsplice assembles alternative transcripts from tumor-specific splicing patterns, identifies sequence changes by comparison to a reference, and predicts neoantigens from the novel peptide sequences. Matched whole exome sequencing and RNA sequencing datasets from glioblastoma, melanoma, and colorectal cancer cohorts will be analyzed with pVACsplice to obtain binding affinity estimates. We will compare these results to neoantigen predictions from other mutation sources and across cancer types to discover the prevalence of immunogenic splicing events. Finally, we will perform immunogenicity testing with a set of high quality candidates to validate our predictions. We hope to increase the number of candidates for personalized vaccines by adding this functionality to our standard neoantigen prediction workflow. This tool could help generate a more accurate portrait of the neoantigen landscape in tumors, and in turn, enhance responses to personalized immunotherapies. Neoantigens are tumor-specific peptides presented on the cell surface by MHC that can be recognized by the adaptive immune system. Personalized immunotherapies, such as cancer vaccines, rely on neoantigen prediction to identify sequences that can activate T cells to recognize and destroy the tumor. The majority of cancer vaccine trials have utilized neoantigens derived from missense mutations and small insertions and deletions. However, other mutation types contribute to the overall neoantigen landscape, including aberrantly spliced transcripts arising from cis-acting regulatory mutations. In this study, we explore the potential immunogenicity of alternative splicing events and present pVACsplice, a tool to expand the capability of pVACtools, a suite of tools for neoantigen prediction (http://www.pvactools.org). pVACsplice assembles alternative transcripts from tumor-specific splicing patterns, identifies sequence changes by comparison to a reference, and predicts neoantigens from the novel peptide sequences. Matched whole exome sequencing and RNA sequencing datasets from glioblastoma, melanoma, and colorectal cancer cohorts will be analyzed with pVACsplice to obtain binding affinity estimates. We will compare these results to neoantigen predictions from other mutation sources and across cancer types to discover the prevalence of immunogenic splicing events. Finally, we will perform immunogenicity testing with a set of high quality candidates to validate our predictions. We hope to increase the number of candidates for personalized vaccines by adding this functionality to our standard neoantigen prediction workflow. This tool could help generate a more accurate portrait of the neoantigen landscape in tumors, and in turn, enhance responses to personalized immunotherapies.

Abstract 1215: pVACsplice: Predicting neoantigens from tumor-specific alternative splicing events derived from cis-acting regulatory mutations using whole exome and RNA sequencing data

Abstract Neoantigens are tumor-specific peptides on the cell surface that can be recognized by the adaptive immune system. Personalized immunotherapies, such as cancer vaccines, rely on neoantigen prediction to identify sequences that can activate T cells to recognize and destroy the tumor. The majority of cancer vaccine trials have utilized neoantigens derived from missense mutations and small insertions and deletions. However, other mutation types could contribute to the overall neoantigen landscape, such as aberrantly spliced transcripts arising from cis-acting regulatory mutations. In this study, we explore the potential immunogenicity of alternative splicing events by creating pVACsplice, a tool to expand the capability of pVACtools, a suite of tools for neoantigen prediction (http://www.pvactools.org). pVACsplice assembles alternative transcripts from tumor-specific splicing patterns, identifies sequence changes by comparison to a reference, and predicts neoantigens from the novel regions. To verify the accuracy of alternative transcript assembly, we ran pVACsplice with HCC1395 cell line samples and performed long-read sequencing to detect the transcripts in vitro. Matched whole exome sequencing and RNA sequencing datasets from glioblastoma, melanoma, and colorectal cancer cohorts will also be analyzed with pVACsplice to obtain binding affinity estimates. We will compare these results to neoantigen predictions from other mutation sources and across cancer types to discover the prevalence of immunogenic splicing events. Finally, we will perform immunogenicity testing with a set of high quality candidates to validate our predictions. We hope to increase the number of candidates for patients’ vaccines by adding this functionality to our standard neoantigen prediction workflow. This tool could help generate a more accurate portrait of the neoantigen landscape in tumors, and in turn, enhance responses to personalized immunotherapies. Citation Format: Megan M. Richters, Kelsy C. Cotto, Susanna Kiwala, Huiming Xia, Beatriz M. Carreno, Gavin P. Dunn, Antoni Ribas, Obi L. Griffith, Malachi Griffith. pVACsplice: Predicting neoantigens from tumor-specific alternative splicing events derived from cis-acting regulatory mutations using whole exome and RNA sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1215.

Allele-specific expression reveals genes with recurrent cis-regulatory alterations in high-risk neuroblastoma

BackgroundNeuroblastoma is a pediatric malignancy with a high frequency of metastatic disease at initial diagnosis. Neuroblastoma tumors have few recurrent protein-coding mutations but contain extensive somatic copy number alterations (SCNAs) suggesting that mutations that alter gene dosage are important drivers of tumorigenesis. Here, we analyze allele-specific expression in 96 high-risk neuroblastoma tumors to discover genes impacted by cis-acting mutations that alter dosage.ResultsWe identify 1043 genes with recurrent, neuroblastoma-specific allele-specific expression. While most of these genes lie within common SCNA regions, many of them exhibit allele-specific expression in copy neutral samples and these samples are enriched for mutations that are predicted to cause nonsense-mediated decay. Thus, both SCNA and non-SCNA mutations frequently alter gene expression in neuroblastoma. We focus on genes with neuroblastoma-specific allele-specific expression in the absence of SCNAs and find 26 such genes that have reduced expression in stage 4 disease. At least two of these genes have evidence for tumor suppressor activity including the transcription factor TFAP2B and the protein tyrosine phosphatase PTPRH.ConclusionsIn summary, our allele-specific expression analysis discovers genes that are recurrently dysregulated by both large SCNAs and other cis-acting mutations in high-risk neuroblastoma.

Genome-wide association mapping of transcriptome variation in Mimulus guttatus indicates differing patterns of selection on cis- versus trans-acting mutations.

We measured the floral bud transcriptome of 151 fully sequenced lines of Mimulus guttatus from one natural population. Thousands of single nucleotide polymorphisms (SNPs) are implicated as transcription regulators, but there is a striking difference in the allele frequency spectrum of cis-acting and trans-acting mutations. Cis-SNPs have intermediate frequencies (consistent with balancing selection) while trans-SNPs exhibit a rare-alleles model (consistent with purifying selection). This pattern only becomes clear when transcript variation is normalized on a gene-to-gene basis. If a global normalization is applied, as is typically in RNAseq experiments, asymmetric transcript distributions combined with "rarity disequilibrium" produce a superabundance of false positives for trans-acting SNPs. To explore the cause of purifying selection on trans-acting mutations, we identified gene expression modules as sets of coexpressed genes. The extent to which trans-acting mutations influence modules is a strong predictor of allele frequency. Mutations altering expression of genes with high "connectedness" (those that are highly predictive of the representative module expression value) have the lowest allele frequency. The expression modules can also predict whole-plant traits such as flower size. We find that a substantial portion of the genetic (co)variance among traits can be described as an emergent property of genetic effects on expression modules.

Cis-acting mutation affecting GJA5 transcription is underlying the Melanotic within-feather pigmentation pattern in chickens

Significance The molecular mechanisms underlying pigmentation patterns in animals is to a large extent an unresolved mystery in biology. For example, compared with mammals, birds show a stunning diversity in pigmentation patterns. This study advances the knowledge concerning the mechanisms creating periodic pigmentation patterns in individual feathers. We show that a mutation upstream of GJA5 encoding a gap-junction protein is causing the Melanotic phenotype in domestic chickens. Melanotic affects within-feather pigmentation patterns by enhancing the contrast between dark- and light-colored regions in the feather. The result implies that cell–cell communications between melanocytes and other cells in the feather follicle play a critical role for pattern formation.

A cis-Acting Mutation in the PxABCG1 Promoter Is Associated with Cry1Ac Resistance in Plutella xylostella (L.).

The molecular mechanisms of insect resistance to Cry toxins generated from the bacterium Bacillus thuringiensis (Bt) urgently need to be elucidated to enable the improvement and sustainability of Bt-based products. Although downregulation of the expression of midgut receptor genes is a pivotal mechanism of insect resistance to Bt Cry toxins, the underlying transcriptional regulation of these genes remains elusive. Herein, we unraveled the regulatory mechanism of the downregulation of the ABC transporter gene PxABCG1 (also called Pxwhite), a functional midgut receptor of the Bt Cry1Ac toxin in Plutella xylostella. The PxABCG1 promoters of Cry1Ac-susceptible and Cry1Ac-resistant strains were cloned and analyzed, and they showed clear differences in activity. Subsequently, a dual-luciferase reporter assay, a yeast one-hybrid (Y1H) assay, and RNA interference (RNAi) experiments demonstrated that a cis-mutation in a binding site of the Hox transcription factor Antennapedia (Antp) decreased the promoter activity of the resistant strain and eliminated the binding and regulation of Antp, thereby enhancing the resistance of P. xylostella to the Cry1Ac toxin. These results advance our knowledge of the roles of cis- and trans-regulatory variations in the regulation of midgut Cry receptor genes and the evolution of Bt resistance, contributing to a more complete understanding of the Bt resistance mechanism.

Zebrafish models of skeletal dysplasia induced by cholesterol biosynthesis deficiency.

ABSTRACTHuman disorders of the post-squalene cholesterol biosynthesis pathway frequently result in skeletal abnormalities, yet our understanding of the mechanisms involved is limited. In a forward-genetic approach, we have found that a late-onset skeletal mutant, named kolibernu7, is the result of a cis-acting regulatory mutation leading to loss of methylsterol monooxygenase 1 (msmo1) expression within pre-hypertrophic chondrocytes. Generated msmo1nu81 knockdown mutation resulted in lethality at larval stage. We demonstrated that this is a result of both cholesterol deprivation and sterol intermediate accumulation by creating a mutation eliminating activity of Lanosterol synthase (Lss). Our results indicate that double lssnu60;msmo1nu81 and single lssnu60 mutants survive significantly longer than msmo1nu81 homozygotes. Liver-specific restoration of either Msmo1 or Lss in corresponding mutant backgrounds suppresses larval lethality. Rescued mutants develop dramatic skeletal abnormalities, with a loss of Msmo1 activity resulting in a more-severe patterning defect of a near-complete loss of hypertrophic chondrocytes marked by col10a1a expression. Our analysis suggests that hypertrophic chondrocytes depend on endogenous cholesterol synthesis, and blocking C4 demethylation exacerbates the cholesterol deficiency phenotype. Our findings offer new insight into the genetic control of bone development and provide new zebrafish models for human disorders of the cholesterol biosynthesis pathway.

Phase and context shape the function of composite oncogenic mutations.

Cancers develop as a result of driver mutations1,2 that lead to clonal outgrowth and disease evolution3,4. The discovery and functional characterization of individual driver mutations is a central aim of cancer research and has elucidated myriad phenotypes5 and therapeutic vulnerabilities6. Serial genetic evolution of mutant cancer genes7,8 and the allelic context in which they arise, however, is poorly understood in both common and rare cancer genes and tumor types. Here, we find that nearly 1 in 4 human tumors harbor a composite mutation of a cancer-associated gene, defined as two nonsynonymous somatic mutations in the same gene and tumor. Composite mutations are enriched in specific genes, have an elevated rate of utilization of less common hotspot mutations acquired in a chronology driven in part by oncogenic fitness, and arise in an allelic configuration that reflects context-specific selective pressures. Cis-acting composite mutations are hypermorphic in some genes, such as TERT, where dosage effects predominate, while they lead to selection of function in others such as TP53. Collectively, composite mutations are driver alterations that arise from context- and allele-specific selective pressures dependent in part on gene and mutation function leading to complex, often neomorphic functions of biological and therapeutic significance.

Abstract 4345: A large-scale RNA-seq screen to identify regulators of alternative splicing in cancer

Abstract RNA splicing is dysregulated in a widespread manner in cancers including lung adenocarcinoma. In some cases, splicing changes can be attributed to cis-acting splice site mutations or trans-acting mutations in splicing factors. However, in most cases, the underlying causes of splicing changes are unknown. We hypothesized that upstream signaling inputs to alternative splicing regulation can explain some of these unknowns. Specifically, we studied how oncogenic lung cancer signaling pathways (EGFR/Ras, KEAP1/NRF2, MYC, and others) regulate alternative splicing and the expression and activity of splicing factors. We focus on signaling pathways because they can be readily therapeutically modulated with small molecule inhibitors (e.g. tyrosine kinase inhibitors), offering opportunities for therapeutic suppression of downstream splicing effects. To experimentally determine how signaling pathway perturbation affects alternative splicing, we perturbed A549 lung cancer cells with each of 82 alleles of 27 genes (n = 4 to 8 biological replicates per allele). Genes and variants were selected based on the occurrence of the variants in lung adenocarcinoma tumors. In total, 417 whole transcriptome profiles were generated using the Smart-Seq v4 method (Clontech) and Nextera XT library preparation (Illumina). We performed differential expression analysis using edgeR and differential splicing analysis using MISO. Using this approach, we identified a high-confidence set of 2430 alternative splicing events differentially spliced in the perturbed samples compared to controls. Of these alternative splicing events, 1219 are skipped exons, 469 are mutually exclusive exons, 235 are alternative 5’ splice sites, 255 are alternative 3’ splice sites, and 252 are retained introns. Among the perturbations tested, overexpression of the RBM45 wild-type allele and the RBM45 D434Y variant allele resulted in the greatest number of alternatively spliced events, with 367 and 323 events respectively. A closer look at these events reveals that when the RBM45 wild-type allele is overexpressed, exon 3 of the cyclin gene CCNG1 is skipped. Interestingly, when RBM45 variant alleles (D434Y, M126I) are overexpressed instead of the wild-type, the exon is not skipped, suggesting loss or change of function. These results suggest that, among other roles, RBM45 may regulate cell cycle patterns by regulating alternative splicing. In sum, our screen identifies splicing events which are regulated by oncogenic signaling pathways. With this information, it will be possible to propose therapeutic options that mitigate aberrant splicing driven by signaling pathway components. Such therapy may already exist in the form of drugs for other targeted uses but could be repurposed to address aberrant splicing. Importantly, therapy of this type has the potential to be more tolerable compared to treatments directly aimed at splicing factors and splice sites. Citation Format: April Lo, Maria McSharry, Alice Berger. A large-scale RNA-seq screen to identify regulators of alternative splicing in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4345.

Discovery and Functional Annotation of Quantitative Trait Loci Affecting Resistance to Sea Lice in Atlantic Salmon.

Sea lice (Caligus rogercresseyi) are ectoparasitic copepods which have a large negative economic and welfare impact in Atlantic salmon (Salmo salar) aquaculture, particularly in Chile. A multi-faceted prevention and control strategy is required to tackle lice, and selective breeding contributes via cumulative improvement of host resistance to the parasite. While host resistance has been shown to be heritable, little is yet known about the individual loci that contribute to this resistance, the potential underlying genes, and their mechanisms of action. In this study we took a multifaceted approach to identify and characterize quantitative trait loci (QTL) affecting host resistance in a population of 2,688 Caligus-challenged Atlantic salmon post-smolts from a commercial breeding program. We used low and medium density genotyping with imputation to collect genome-wide SNP marker data for all animals. Moderate heritability estimates of 0.28 and 0.24 were obtained for lice density (as a measure of host resistance) and growth during infestation, respectively. Three QTL explaining between 7 and 13% of the genetic variation in resistance to sea lice (as represented by the traits of lice density) were detected on chromosomes 3, 18, and 21. Characterisation of these QTL regions was undertaken using RNA sequencing and pooled whole genome sequencing data. This resulted in the identification of a shortlist of potential underlying causative genes, and candidate functional mutations for further study. For example, candidates within the chromosome 3 QTL include a putative premature stop mutation in TOB1 (an anti-proliferative transcription factor involved in T cell regulation) and an uncharacterized protein which showed significant differential allelic expression (implying the existence of a cis-acting regulatory mutation). While host resistance to sea lice is polygenic in nature, the results of this study highlight significant QTL regions together explaining between 7 and 13 % of the heritability of the trait. Future investigation of these QTL may enable improved knowledge of the functional mechanisms of host resistance to sea lice, and incorporation of functional variants to improve genomic selection accuracy.

Transcription factor ASCL2 is required for development of the glycogen trophoblast cell lineage.

The basic helix-loop-helix (bHLH) transcription factor ASCL2 plays essential roles in diploid multipotent trophoblast progenitors, intestinal stem cells, follicular T-helper cells, as well as during epidermal development and myogenesis. During early development, Ascl2 expression is regulated by genomic imprinting and only the maternally inherited allele is transcriptionally active in trophoblast. The paternal allele-specific silencing of Ascl2 requires expression of the long non-coding RNA Kcnq1ot1 in cis and the deposition of repressive histone marks. Here we show that Del7AI, a 280-kb deletion allele neighboring Ascl2, interferes with this process in cis and leads to a partial loss of silencing at Ascl2. Genetic rescue experiments show that the low level of Ascl2 expression from the paternal Del7AI allele can rescue the embryonic lethality associated with maternally inherited Ascl2 mutations, in a level-dependent manner. Despite their ability to support development to term, the rescued placentae have a pronounced phenotype characterized by severe hypoplasia of the junctional zone, expansion of the parietal trophoblast giant cell layer, and complete absence of invasive glycogen trophoblast cells. Transcriptome analysis of ectoplacental cones at E7.5 and differentiation assays of Ascl2 mutant trophoblast stem cells show that ASCL2 is required for the emergence or early maintenance of glycogen trophoblast cells during development. Our work identifies a new cis-acting mutation interfering with Kcnq1ot1 silencing function and establishes a novel critical developmental role for the transcription factor ASCL2.

RNA Polymerase II Transcription Attenuation at the Yeast DNA Repair Gene, DEF1, Involves Sen1-Dependent and Polyadenylation Site-Dependent Termination.

Termination of RNA Polymerase II (Pol II) activity serves a vital cellular role by separating ubiquitous transcription units and influencing RNA fate and function. In the yeast Saccharomyces cerevisiae, Pol II termination is carried out by cleavage and polyadenylation factor (CPF-CF) and Nrd1-Nab3-Sen1 (NNS) complexes, which operate primarily at mRNA and non-coding RNA genes, respectively. Premature Pol II termination (attenuation) contributes to gene regulation, but there is limited knowledge of its prevalence and biological significance. In particular, it is unclear how much crosstalk occurs between CPF-CF and NNS complexes and how Pol II attenuation is modulated during stress adaptation. In this study, we have identified an attenuator in the DEF1 DNA repair gene, which includes a portion of the 5′-untranslated region (UTR) and upstream open reading frame (ORF). Using a plasmid-based reporter gene system, we conducted a genetic screen of 14 termination mutants and their ability to confer Pol II read-through defects. The DEF1 attenuator behaved as a hybrid terminator, relying heavily on CPF-CF and Sen1 but without Nrd1 and Nab3 involvement. Our genetic selection identified 22 cis-acting point mutations that clustered into four regions, including a polyadenylation site efficiency element that genetically interacts with its cognate binding-protein Hrp1. Outside of the reporter gene context, a DEF1 attenuator mutant increased mRNA and protein expression, exacerbating the toxicity of a constitutively active Def1 protein. Overall, our data support a biologically significant role for transcription attenuation in regulating DEF1 expression, which can be modulated during the DNA damage response.

The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box Gene VviAGL11.

Seedlessness is greatly prized by consumers of fresh grapes. While stenospermocarpic seed abortion determined by the SEED DEVELOPMENT INHIBITOR (SDI) locus is the usual source of seedlessness in commercial grapevine (Vitis vinifera) cultivars, the underlying sdi mutation remains unknown. Here, we undertook an integrative approach to identify the causal mutation. Quantitative genetics and fine-mapping in two 'Crimson Seedless'-derived F1 mapping populations confirmed the major effect of the SDI locus and delimited the sdi mutation to a 323-kb region on chromosome 18. RNA-sequencing comparing seed traces of seedless and seeds of seeded F1 individuals identified processes triggered during sdi-determined seed abortion, including the activation of salicylic acid-dependent autoimmunity. The RNA-sequencing data set was investigated for candidate genes, and while no evidence for causal cis-acting regulatory mutations was detected, deleterious nucleotide changes in coding sequences of the seedless haplotype were predicted in two genes within the sdi fine-mapping interval. Targeted resequencing of the two genes in a collection of 124 grapevine cultivars showed that only the point variation causing the arginine-197-to-leucine substitution in the seed morphogenesis regulator gene AGAMOUS-LIKE11 (VviAGL11) was fully linked with stenospermocarpy. The concurrent postzygotic variation identified for this missense polymorphism and seedlessness phenotype in seeded somatic variants of the original stenospermocarpic cultivar supports a causal effect. We postulate that seed abortion caused by this amino acid substitution in VviAGL11 is the major cause of seedlessness in cultivated grapevine. This information can be exploited to boost seedless grape breeding.

Abstract 4995: Premature polyadenylation causes oncogenic truncations of the tumor suppressor genes BRCA1, LATS1 and MAGI3 in breast cancer

Abstract Cleavage and polyadenylation is a fundamental process in the control of gene expression, yet how cancer cells deregulate this process to generate cancer-causing alterations is only beginning to be appreciated. We previously showed that in human breast cancer, premature polyadenylation of the tumor suppressor gene MAGI3 causes the expression of a truncated protein that dominantly promotes malignant growth via the Hippo pathway. This established a precedent for premature polyadenylation as an aberrant process causing transformation in vitro and in vivo. However, little is known about the molecular mechanism that causes premature polyadenylation, or what types of genes it affects in cancer. Despite the absence of cis-acting mutations, premature polyadenylation of MAGI3 occurs specifically following the gene’s large internal exon. We therefore asked whether an epigenetic mechanism might underlie the phenomenon of premature polyadenylation in cancer. Here, we report that genes with large internal exons are susceptible to truncation by premature polyadenylation. In addition to MAGI3, premature polyadenylation truncates the gene products of two tumor suppressor genes with large internal exons, BRCA1 and LATS1. These truncated proteins are upregulated in breast cancer cells compared to non-transformed mammary epithelial cells and enable downstream oncogenic events instead of providing tumor suppressive functions. Large internal exons are infrequently found in the genome as the mechanics of efficient splice site recognition favor small exons. Interestingly in transcripts, large internal exons are conspicuously enriched in N6-methyladenosine (m6A), though the functional significance of these epitranscriptomic marks remains unknown. Molecularly, we find that methylated levels of N6-adenosine in the large internal exons of BRCA1, LATS1 and MAGI3 transcripts are significantly reduced in breast cancer cells that upregulate the truncated gene products compared to non-transformed mammary epithelial cells. These results raise the intriguing possibility that m6A marks in large internal exons are mechanistically involved in repressing premature polyadenylation. We are currently investigating this hypothesis by perturbing N6-adenosine methylation in cells and examining the functional consequences. In summary, our study suggests that premature polyadenylation may be a more pervasive alteration mechanism than previously appreciated in cancer and begins to illuminate a gene region-specific mechanism responsible for repressing premature polyadenylation. Citation Format: Thomas K. Ni, Charlotte Kuperwasser. Premature polyadenylation causes oncogenic truncations of the tumor suppressor genes BRCA1, LATS1 and MAGI3 in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4995. doi:10.1158/1538-7445.AM2017-4995

Epigenetic and Genetic Contributions to Adaptation in Chlamydomonas.

Epigenetic modifications, such as DNA methylation or histone modifications, can be transmitted between cellular or organismal generations. However, there are no experiments measuring their role in adaptation, so here we use experimental evolution to investigate how epigenetic variation can contribute to adaptation. We manipulated DNA methylation and histone acetylation in the unicellular green alga Chlamydomonas reinhardtii both genetically and chemically to change the amount of epigenetic variation generated or transmitted in adapting populations in three different environments (salt stress, phosphate starvation, and high CO2) for two hundred asexual generations. We find that reducing the amount of epigenetic variation available to populations can reduce adaptation in environments where it otherwise happens. From genomic and epigenomic sequences from a subset of the populations, we see changes in methylation patterns between the evolved populations over-represented in some functional categories of genes, which is consistent with some of these differences being adaptive. Based on whole genome sequencing of evolved clones, the majority of DNA methylation changes do not appear to be linked to cis-acting genetic mutations. Our results show that transgenerational epigenetic effects play a role in adaptive evolution, and suggest that the relationship between changes in methylation patterns and differences in evolutionary outcomes, at least for quantitative traits such as cell division rates, is complex.

The ETS family of oncogenic transcription factors in solid tumours.

Findings over the past decade have identified aberrant activation of the ETS transcription factor family throughout all stages of tumorigenesis. Specifically in solid tumours, gene rearrangement and amplification, feed-forward growth factor signalling loops, formation of gain-of-function co-regulatory complexes and novel cis-acting mutations in ETS target gene promoters can result in increased ETS activity. In turn, pro-oncogenic ETS signalling enhances tumorigenesis through a broad mechanistic toolbox that includes lineage specification and self-renewal, DNA damage and genome instability, epigenetics and metabolism. This Review discusses these different mechanisms of ETS activation and subsequent oncogenic implications, as well as the clinical utility of ETS factors.