Cis-acting Regulatory Mutations Research Articles

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.

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.

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.

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.

GATA1 Activity Governed by Configurations of cis-Acting Elements

The transcription factor GATA1 regulates the expression of essential erythroid and megakaryocytic differentiation genes through binding to the DNA consensus sequence WGATAR. The GATA1 protein has four functional domains, including two centrally located zinc-finger domains and two transactivation domains at the N- and C-termini. These functional domains play characteristic roles in the elaborate regulation of diversified GATA1 target genes, each of which exhibits a unique expression profile. Three types of GATA1-related hematological malignancies have been reported. One is a structural mutation in the GATA1 gene, resulting in the production of a short form of GATA1 that lacks the N-terminal transactivation domain and is found in Down syndrome-related acute megakaryocytic leukemia. The other two are cis-acting regulatory mutations affecting expression of the Gata1 gene, which have been shown to cause acute erythroblastic leukemia and myelofibrosis in mice. Therefore, imbalanced gene regulation caused by qualitative and quantitative changes in GATA1 is thought to be involved in specific hematological disease pathogenesis. In the present review, we discuss recent advances in understanding the mechanisms of differential transcriptional regulation by GATA1 during erythroid differentiation, with special reference to the binding kinetics of GATA1 at conformation-specific binding sites.

The Crest Phenotype in Chicken Is Associated with Ectopic Expression of HOXC8 in Cranial Skin

The Crest phenotype is characterised by a tuft of elongated feathers atop the head. A similar phenotype is also seen in several wild bird species. Crest shows an autosomal incompletely dominant mode of inheritance and is associated with cerebral hernia. Here we show, using linkage analysis and genome-wide association, that Crest is located on the E22C19W28 linkage group and that it shows complete association to the HOXC-cluster on this chromosome. Expression analysis of tissues from Crested and non-crested chickens, representing 26 different breeds, revealed that HOXC8, but not HOXC12 or HOXC13, showed ectopic expression in cranial skin during embryonic development. We propose that Crest is caused by a cis-acting regulatory mutation underlying the ectopic expression of HOXC8. However, the identification of the causative mutation(s) has to await until a method becomes available for assembling this chromosomal region. Crest is unfortunately located in a genomic region that has so far defied all attempts to establish a contiguous sequence.

How selective sweeps in domestic animals provide new insight into biological mechanisms

Genetic studies of domestic animals are of general interest because there is more phenotypic diversity to explore in these species than in any experimental organism. Some mutations with favourable phenotypic effects have been highly enriched and gone through selective sweeps during the process of domestication and selective breeding. Three such selective sweeps are described in this review. All three mutations are intronic and constitute cis-acting regulatory mutations. Two of the mutations constitute structural changes (one duplication and one copy number expansion). These examples illustrate a general trend that noncoding mutations and structural changes have both contributed significantly to the evolution of phenotypic diversity in domestic animals. How the molecular characterization of trait loci in domestic animals can provide new basic knowledge of relevance for human medicine is discussed.

A cis-acting regulatory mutation causes premature hair graying and susceptibility to melanoma in the horse

In horses, graying with age is an autosomal dominant trait associated with a high incidence of melanoma and vitiligo-like depigmentation. Here we show that the Gray phenotype is caused by a 4.6-kb duplication in intron 6 of STX17 (syntaxin-17) that constitutes a cis-acting regulatory mutation. Both STX17 and the neighboring NR4A3 gene are overexpressed in melanomas from Gray horses. Gray horses carrying a loss-of-function mutation in ASIP (agouti signaling protein) had a higher incidence of melanoma, implying that increased melanocortin-1 receptor signaling promotes melanoma development in Gray horses. The Gray horse provides a notable example of how humans have cherry-picked mutations with favorable phenotypic effects in domestic animals.

Roles of manganese and iron in the regulation of the biosynthesis of manganese-superoxide dismutase inEscherichia coli

Aerobic life-style offers both benefits and risks to living cells. The major risk comes from the formation of reactive oxygen intermediates (i.e. superoxide radical, O2-; hydrogen peroxide, H2O2; and hydroxyl radical, OH.) during normal oxygen metabolism. However, living cells are able to cope with oxygen toxicity by virtue of a unique set of antioxidant enzymes that scavenge O2- and H2O2, and prevent the formation OH.. Superoxide dismutases (SODs; EC 1.15.1.1) are metalloenzymes essential for aerobic survival. Escherichia coli contains two forms of this enzyme: an iron-containing enzyme (FeSOD) and a manganese-containing enzyme (MnSOD). In E. coli, MnSOD biosynthesis is under rigorous control. The enzyme is induced in response to a variety of environmental stress conditions including exposure to oxygen, redox cycling compounds such as paraquat which exacerbate the level of intracellular superoxide radicals, iron chelation (i.e. iron deprivation), and oxidants. A model for the regulation of the MnSOD has been proposed in which the MnSOD gene (sodA) is negatively regulated at the level of transcription by an iron-containing redox-sensitive repressor protein. The effect of iron-chelation most probably results in removal of the iron necessary for repressor activity. Recent studies have shown that sodA expression is regulated by three iron-dependent regulatory proteins, Fur (ferric uptake regulation), Fnr (fumarate nitrate regulation) and SoxR (superoxide regulon), and by the ArcA/ArcB (aerobic respiration control) system. The potential Fur-, Fnr- and ArcA-binding sites in the sodA promoter region have been identified by using different cis-acting regulatory mutations that caused anaerobic derepression of the gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of cis-Acting Regulatory Mutations Causing Anaerobic Expression of the sodA Gene in Escherichia coli

The biosynthesis of Mn-containing superoxide dismutase is regulated in response to stimuli that affect the redox potential of the cell. To further investigate the mode of regulation of the gene ( sodA) encoding this enzyme, cis-acting regulatory mutations in a strain containing a sodA::lacZ gene fusion were studied. The mutant strains expressed β-galactosidase under anaerobic conditions, whereas the wild-type did not. Furthermore, the mutants were not induced in response to the presence of iron chelator, 2,2′-dipyridyl, or to the redox cycling compound, paraquat. The wild-type, however, did respond to these effectors. In vivo cloning was used to isolate the cis-acting regulatory elements from the mutants (NC4 and NC5). Replacement of the wild-type 5′-regulatory region with either of the mutants′ cis-acting regulatory element resulted in the anaerobic expression of active Mn-superoxide dismutase. Sequence and restriction analysis revealed the presence of an IS 2 insertion element in the promoter region of one of the mutants (NC5). This insertion caused the displacement of the 5′-regulatory region of sodA and the formation of a functional hybrid promoter consisting of the resident −10 region from sodA and −35 from IS 2. The second mutation (from NC4) was similarly analyzed, and an IS 5 element was identified. The insertion site of IS 5 (in NC4) was 6 bp (5′-TTAATT-3′) upstream from the IS 2 site (in NC5). Anaerobic expression of sodA in NC4 was lower than in NC5. This difference was almost eliminated in an arc − background, suggesting that the sequence 5′-TTAATT-3′ might be essential for negative regulation by ArcA.

Isolation and characterization of trans-acting mutations involved in oxygen regulation of puc operon transcription in Rhodobacter sphaeroides

Transcriptional expression of the puc operon in Rhodobacter sphaeroides 2.4.1 is dependent on the partial pressure of oxygen. By using transcriptional fusions in trans of a promoterless fragment derived from the aminoglycoside-3'-phosphotransferase gene of Tn903 to puc operon-specific DNA containing a 629-bp 5' cis-acting regulatory region involved in the expression of puc-specific mRNA, we selected Kmr colonies under aerobic conditions. Two broad classes of mutations, trans and cis, which are involved in O2 control of puc operon transcription, fall into several distinct phenotypic classes. The cis-acting regulatory mutations are characterized in detail elsewhere (J.K. Lee and S. Kaplan, J. Bacteriol. 174:1146-1157, 1992). Two trans-acting regulatory mutants, CL1a and T1a, which are B800-850- Car- and apparently B875-, respectively, were shown to derepress puc operon transcription in the presence of oxygen. The mutation giving rise to CL1a has been shown to act at the puc operon-specific cis-acting upstream regulatory region (-629 to -92). On the other hand, the mutation giving rise to T1a, identifying a second trans-acting regulatory factor(s), appears to act at both the upstream (-629 to -92) and the downstream (-92 to -1) regulatory regions of the puc operon as well as at the level(s) of bacteriochlorophyll and carotenoid biosyntheses, as revealed by the presence of the B800-850 complex under chemoheterotrophic growth conditions. Both the B800-850- Car- phenotype and the trans-acting effect on puc operon expression in mutant CL1a were complemented with a 2.2-kb DNA fragment located within the carotenoid gene cluster. Mutant T1a was complemented with a 7.0-kb EcoRI restriction fragment containing the puhA gene and its flanking DNA (6.3 kb) to restore expression of the B875 complex and to suppress the trans-acting effect resulting in the loss of 02 control. Under chemoheterotrophic conditions, mutant T1a was highly unstable, segregating into a PS- mutant designated T4.

Genetic definition of the translational operator of the threonine-tRNA ligase gene in Escherichia coli.

The Escherichia coli gene thrS that codes for threonine-tRNA ligase (tRNAThr ligase, formerly threonine-tRNA synthetase, EC 6.1.1.3) has previously been shown to be negatively autoregulated at the level of translation. Here we describe the use of several thrS-lac gene fusions to isolate cis-acting regulatory mutations that increase the translation but not the transcription of the thrS gene. These mutations lead to a total loss of control of repression and derepression of thrS. DNA sequence analysis locates the mutations between 10 and 40 base pairs upstream of the translation initiation codon of thrS and more than 100 base pairs downstream of the transcription initiation site. The mRNA region where these mutations are located shares primary and secondary structure homologies with specific parts of several isoacceptor tRNAThr species. These findings suggest that the ligase regulates its translation by binding to its mRNA at a place that shares some homology with its natural substrate.

Characterization of a cis-acting regulatory mutation that maps at the distal end of the Escherichia coli glyA gene.

We have isolated a phage Mu cts-generated glyA mutant with only 30% of the normal level of serine hydroxymethyltransferase activity. Genetic and physical mapping studies show that Mu cts has inserted between the end of the glyA structural gene and its proposed transcription termination site. The mutation is cis acting and shows that sequences distal to the glyA structural gene play an important role in the expression of this gene.

Regulation of Proline Transport in Aspergillus nidulans

Aspergillus nidulans has at least two permeases for l-proline. The prnB gene of the prn gene cluster specifies the major proline permease, which is inducible by proline. Synthesis of the prnB permease is subject to repression by ammonium at 37 °C but not at 25 °C. A genetically unidentified minor proline permease(s) does not respond to proline induction or ammonium repression but is inhibited by ammonium. areAr mutants are unable to utilize nitrogen sources other than ammonium because they lack a positive-acting regulatory product required for expression of ammonium-repressible activities. However, there are very few cases in which the lack of growth of areAr mutants on a particular nitrogen source can be attributed to a reduction in the level of a particular enzyme activity or permease. Reduced expression of the prnB permease can account for the inability of areAr mutants to utilize proline. This is demonstrated by the ability of cis-acting regulatory mutations designated prnd , which derepress synthesis of the prnB permease, to suppress areAr mutations for proline utilization. The apparent ability of prnd mutations to derepress synthesis of proline oxidase and l-pyrroline-5-carboxylate dehydrogenase is probably an indirect consequence of their ability to derepress synthesis of the prnB permease, preventing inducer exclusion. There is presently no evidence that prnd mutations directly affect expression of the prnA, prnC or prnD genes, but this possibility has not been definitively eliminated.

The nature of an initiator constitutive mutation in Aspergillus nidulans

ONLY a few cis-acting regulatory mutations have been described in eukaryotes. One of these, uap-100, affects the expression of uapA, the adjacent putative structural gene for a uric acid-xanthine permease in the ascomycete Aspergillus nidulans1. Only one protein coded by a formally defined regulatory gene has even been tentatively identified from an eukaryote. This is the positively acting product of the uaY gene of A. nidulans (D. Philippides and C.S., in preparation). As the uapA gene is under the control of uaY (ref. 2 and this paper) the interactions between uap-100 and wild type and mutant uaY products are of considerable interest. Here we show that the constitutivity conferred by uap-100 is strictly dependent on a functional uaY product. This is compelling evidence for the direct involvement of the uaY product in the regulation of uapA and, by implication, of other genes under uaY control, and might indicate that the uaY product exists as at least two conformers in equilibrium.

Integrator gene in Aspergillus nidulans.

ONE of the most intriguing problems in genetics is how the regulation of a particular structural gene can be programmed in such a way that its expression can be elicited in a variety of different contexts. Stated differently, how can the concomitant expression of two or more non-contiguous structural genes capable of independent expression be achieved? Britten and Davidson1–3 have proposed a model for gene regulation which gives considerable insight into this problem. One feature of their model is the existence of positively acting regulatory genes, designated integrator genes, whose role is to achieve precisely this concomitant expression of non-contiguous structural genes. Although the model was developed to account for cell differentiation in higher eukaryotes, a similar, although possibly less elaborate, form of regulatory machinery might be applicable in lower eukaryotes. In the fungus Aspergillus nidulans, as in higher eukaryotes, functionally related genes are seldom clustered4. Given the considerable metabolic versatility of this organism, instances of inducible enzymes participating in more than one metabolic pathway should not be unduly rare. Evidence for the existence of integrator genes could come from the study of regulatory mutations affecting such enzymes. The model also requires the existence of receptor sites for integrator gene products, a not implausible requirement for A. nidulans, where several cis-acting regulatory mutations tightly linked to structural genes under their control have been identified (refs 5–8 and unpublished results of C. R. Bailey and H. N. A.). Here I report that the intAgene of A.nidulans, previously designated amdR and shown by Hynes and coworkers9–11 to be a positive regulatory gene in linkage group II involved in acetamidase synthesis, is an integrator gene of the type described by Britten and Davidson. Moreover, intA is possibly especially interesting because one of the compounds whose metabolism it controls is γ-amino-n-butyric acid (GABA), whose metabolism has been extensively investigated in a wide variety of organisms because of its role in the regulation of neuronal activity.