Location Of Introns Research Articles

CRISPR editing of the GLI1 first intron abrogates GLI1 expression and differentially alters lineage commitment.

GLI1 is one of three GLI family transcription factors that mediate Sonic Hedgehog signaling, which plays a role in development and cell differentiation. GLI1 forms a positive feedback loop with GLI2 and likely with itself. To determine the impact of GLI1 and its intronic regulatory locus on this transcriptional loop and human stem cell differentiation, we deleted the region containing six GLI binding sites in the human GLI1 intron using CRISPR/Cas9 editing to produce H1 human embryonic stem cell (hESC) GLI1‐edited clones. Editing out this intronic region, without removing the entire GLI1 gene, allowed us to study the effects of this highly complex region, which binds transcription factors in a variety of cells. The roles of GLI1 in human ESC differentiation were investigated by comparing RNA sequencing, quantitative‐real time PCR (q‐rtPCR), and functional assays. Editing this region resulted in GLI1 transcriptional knockdown, delayed neural commitment, and inhibition of endodermal and mesodermal differentiation during spontaneous and directed differentiation experiments. We found a delay in the onset of early osteogenic markers, a reduction in the hematopoietic potential to form granulocyte units, and a decrease in cancer‐related gene expression. Furthermore, inhibition of GLI1 via antagonist GANT‐61 had similar in vitro effects. These results indicate that the GLI1 intronic region is critical for the feedback loop and that GLI1 has lineage‐specific effects on hESC differentiation. Our work is the first study to document the extent of GLI1 abrogation on early stages of human development and to show that GLI1 transcription can be altered in a therapeutically useful way.

Gender bias in the genetic vulnerability towards type 2 diabetes and diabetic nephropathy: Role of forkhead box Protein3 transcription factor gene variants.

Forkhead Box Protein3 Transcription Factor (FOXP3) gene is an essential role player in the function and differentiation of regulatory T cells. Polymorphisms/mutations in FOXP3 gene cause Treg cell dysfunction, promote autoimmunity and inflammation. Based on this presumption, we screened 600 subjects from south India (equal number of diabetic (T2DM), diabetic nephropathy (T2DN) and healthy controls) for promoter and intronic (rs3761548C/A and rs2294021C/T) polymorphisms of FOXP3 gene. PCR-RFLP method used for genotyping, revealed an association of promoter SNP for both T2DM (OR=2.41, 95% C.I=1.67-3.49; p<0.0001) and T2DN (OR=2.16, 95% C.I=1.45-3.24; p<0.005). While intronic polymorphism with T2DN (OR=1.91, 95% C.I=1.28-2.84; p<0.05). Further, in females rs3761548C/A showed 2.6 and 5.5-fold; rs2294021C/T showed 2.2- and 2.5-fold predisposition towards T2DM and T2DN respectively. Males exhibited a twofold risk (OR=2.01, 95% C.I=1.22-3.30; p<0.05) towards T2DM with promoter and no association with intronic polymorphism. The combined genotypes in females with AA-CC; AA-TT predisposed and CA-CC; CA-CT protected heading towards T2DM and T2DN respectively, suggesting irrespective of type of allele at intronic locus AA and CA at promoter locus promote or protect the individual for diabetes and diabetic nephropathy, further confirmed by MLR. To our knowledge, the current study is the first of its kind that revealed an association of these polymorphisms of FOXP3 gene and gender influence on T2DM and T2DN among South Indians. Functional and cell-based studies on Treg cells are warranted to confirm our results that help to develop FOXP3/Treg based therapeutic interventions. Lack of data on Treg cells is the limitation of this study.

Bacterial Group II Intron Genomic Neighborhoods Reflect Survival Strategies: Hiding and Hijacking.

Group II (gII) introns are mobile retroelements that can spread to new DNA sites through retrotransposition, which can be influenced by a variety of host factors. To determine if these host factors bear any relationship to the genomic location of gII introns, we developed a bioinformatic pipeline wherein we focused on the genomic neighborhoods of bacterial gII introns within their native contexts and sought to determine global relationships between introns and their surrounding genes. We found that, although gII introns inhabit diverse regions, these neighborhoods are often functionally enriched for genes that could promote gII intron retention or proliferation. On one hand, we observe that gII introns are frequently found hiding in mobile elements or after transcription terminators. On the other hand, gII introns are enriched in locations in which they could hijack host functions for their movement, potentially timing expression of the intron with genes that produce favorable conditions for retrotransposition. Thus, we propose that gII intron distributions have been shaped by relationships with their surrounding genomic neighbors.

Investigating L1 promoter activity during mouse development using LacZ transgenes

Long interspersed elements type 1 (L1s) are highly abundant retrotransposons in the human genome. To fully understand the role of L1s during development and disease, it is important to track L1 expression and epigenetic regulation at individual L1 loci throughout development at the cellular level. However, it is extremely technically challenging to monitor transcriptional activities of individual endogenous L1s due to high sequence homology. Toward this goal, we created independent single‐copy LacZ transgenic mouse lines, in which the LacZ reporter is regulated by a copy of the endogenous mouse L1 promoter. To control for the effect of gene‐body DNA methylation on L1 promoter activity, we also generated independent mouse lines carrying the CpG‐less version of the LacZ, termed LacG. After mapping of the transgene locus in each line, mouse tissues were stained with X‐gal to visualize L1 promoter activity at single cell level. QuPath was used to quantify X‐gal positive cells in whole‐slide scanned brain sections. We found that the LacG reporter supported robust expression as compared to the LacZ reporter. However, there were substantial variations among the LacG reporter lines, in which the transgene is integrated in random genomic loci. Intraline variations of expression in the same organ might be dictated by generation and age, which is yet to be established statistically. Orientation dependent regulation of L1 promoter was also studied by targeting the LacG transgene in a specific intronic locus in either sense or antisense orientation. Interestingly, antisense orientation showed augmented expression than its counterpart. Ongoing effort aims to reveal the pattern of these signals in different anatomical regions of brain and also in other major organs, which might support L1 retrotransposition in different developmental period in these mouse models. The goal is to provide a comprehensive mapping of L1 promoter activity in a cell dependent and locus dependent manner. Such information will be instrumental in our understanding of L1’s involvement in physiological and pathological conditions, including embryogenesis, gametogenesis, and under environmental stress.Support or Funding InformationSupported by NIH R21HD080143 and R21OD017965.

Estimating the Allele-Specific Expression of SNVs From 10× Genomics Single-Cell RNA-Sequencing Data.

With the recent advances in single-cell RNA-sequencing (scRNA-seq) technologies, the estimation of allele expression from single cells is becoming increasingly reliable. Allele expression is both quantitative and dynamic and is an essential component of the genomic interactome. Here, we systematically estimate the allele expression from heterozygous single nucleotide variant (SNV) loci using scRNA-seq data generated on the 10×Genomics Chromium platform. We analyzed 26,640 human adipose-derived mesenchymal stem cells (from three healthy donors), sequenced to an average of 150K sequencing reads per cell (more than 4 billion scRNA-seq reads in total). High-quality SNV calls assessed in our study contained approximately 15% exonic and >50% intronic loci. To analyze the allele expression, we estimated the expressed variant allele fraction (VAFRNA) from SNV-aware alignments and analyzed its variance and distribution (mono- and bi-allelic) at different minimum sequencing read thresholds. Our analysis shows that when assessing positions covered by a minimum of three unique sequencing reads, over 50% of the heterozygous SNVs show bi-allelic expression, while at a threshold of 10 reads, nearly 90% of the SNVs are bi-allelic. In addition, our analysis demonstrates the feasibility of scVAFRNA estimation from current scRNA-seq datasets and shows that the 3′-based library generation protocol of 10×Genomics scRNA-seq data can be informative in SNV-based studies, including analyses of transcriptional kinetics.

Abstract P4-05-19: Genome wide cell specification of R-loops in BRCA1 mutation carriers without malignancy

Abstract An estimated 35,000 patients with breast cancer (BC) have pathogenic BRCA1/2 variants. Unfortunately, in the current state of medical practice, 50-80% of pathogenic mutation carriers remain undetected and undiagnosed. These are largely women with moderate penetrance mutations, including women with BRCA1 mutations, which may not have been identified. There is an unmet challenge to improve the identification and risks of patients who have an inherited increased lifetime risk of BC. BRCA1 mutation carriers (BRCA1-mc) consist ~8% of woman suffering from BCs. A transcriptional R-loop is a guanine-rich 3-stranded nucleic acid structure that plays key roles in (dis)regulation of transcription events, DNA damage, and genome (in)stability. R-loops can be involved in cancer cell precursor development in BRCA1-mc, but it is not clear how they affect luminal precursor differentiation to mature basal-like BCs associated with BRCA1-mc. The role of R-loops in basal-like cells in normal-like tissue of BRCA1-mc have not been studied, limiting the biological interpretation of previous observations. At a genome wide scale, accuracy and resolution of experimentally defined R-loop boundaries is still a challenge. Integrative statistical and genomic approaches have demonstrated that it is possible to computationally predict R-loop forming sequences (RLFSs) in the human genome at an accuracy of 84-92%. In these regards, integrative analysis of available datasets could be useful. We hypothesize that such analyses of RLFS-positive RNA:DNA hybrids/R-loops and gene expression profiles of BRCA1 wild-type and BRCA1 mutation non-cancer epithelial cells could distinguish normal individuals and pre-malignant risks in BRCA1-mc. To test this hypothesis, we used publicly available datasets and carried out a comprehensive genome-wide analysis for BRCA1-mc and non-carriers without BC malignancy. We identified differentially expressed genes (DEGs) for non-cancer breast epithelium cells from publicly available 10 non-cancer individuals with and 10 without BRCA1 mutations. Our QmRLFS finder, DRIP-seq dataset (GEO:GSE96672) and our R-loop database (http://rloop.bii.a-star.edu.sg/) were used to identify RLFS-positive R-loops genome wide. A majority of DEGs, 1813, in non-cancer epithelial cells of BRCA1-mc are downregulated; Gene Ontology terms include metabolism, immune system, cell signaling, cell adhesion, transcription, and stress response. But, 597 DEGs were upregulated; splicing events. 662 downregulated and 185 upregulated DEGs contained RLFS-positive R-loops. In BRCA1 mutation carrier and non-carrier genomes, RLFS and R-loops were commonly located upstream and downstream promoter regions of many genes regardless of breast cell types. Interestingly, RLFS-positive R-loops were observed most often in luminal cells, however they were also found in mature basal-like cells. In particular, RLFS-positive R-loops were found only in BRCA1 locus (in 1st intron) of mature basal-like cells of non-BRCA1 mutation breast tissue samples, but not in luminal cells. However, for BRCA1-mc, we observed R-loops in BRCA1 locus of luminal precursors and mature luminal cells, but not in basal-like cells. Also, in BRCA1 pathway BACH1 promoter region, the RLFS-positive R-loops were observed in all breast cell types for both BRCA1-mc and non-carriers. This study suggests strong association of RLFS-positive R-loop formation and its enrichment variations within mature basal-like breast cells. We highlight the potential clinical significance of early detection of RLFS-positive R-loops in non-cancer breast Citation Format: Andre Grageda, Debasree Sarkar, Vladimir A Kuznetsov. Genome wide cell specification of R-loops in BRCA1 mutation carriers without malignancy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-05-19.

The fungal mitochondrial Nad5 pan-genic intron landscape

An intron landscape was prepared for the fungal mitochondrial nad5 gene. A hundred and eighty-eight fungal species were examined and a total of 265 introns were noted to be located in 29 intron insertion sites within the examined nad5 genes. Two hundred and sixty-three introns could be classified as group I types and two group II introns were noted. One additional group II intron module was identified nested within a composite group I intron. Based on features related to RNA secondary structures, introns can be classified into different subtypes and it was observed that intron insertion-sites are biased towards phase 0 and they appear to be specific to an intron type. Intron landscapes could be used as a guide map to predict the location of fungal mtDNA mobile introns, which are composite elements that include a ribozyme component and in some instances open reading frames encoding homing endonucleases or reverse transcriptases and all of these have applications in biotechnology.

Phylogenetic Signal of Indels and the Neoavian Radiation

The early radiation of Neoaves has been hypothesized to be an intractable “hard polytomy”. We explore the fundamental properties of insertion/deletion alleles (indels), an under-utilized form of genomic data with the potential to help solve this. We scored &gt;5 million indels from &gt;7000 pan-genomic intronic and ultraconserved element (UCE) loci in 48 representatives of all neoavian orders. We found that intronic and UCE indels exhibited less homoplasy than nucleotide (nt) data. Gene trees estimated using indel data were less resolved than those estimated using nt data. Nevertheless, Accurate Species TRee Algorithm (ASTRAL) species trees estimated using indels were generally similar to nt-based ASTRAL trees, albeit with lower support. However, the power of indel gene trees became clear when we combined them with nt gene trees, including a striking result for UCEs. The individual UCE indel and nt ASTRAL trees were incongruent with each other and with the intron ASTRAL trees; however, the combined indel+nt ASTRAL tree was much more congruent with the intronic trees. Finally, combining indel and nt data for both introns and UCEs provided sufficient power to reduce the scope of the polytomy that was previously proposed for several supraordinal lineages of Neoaves.

Alleles associated with physical activity levels are estimated to be older than anatomically modern humans.

The purpose of this study was to determine the estimated mutation age and conservation of single-nucleotide polymorphisms (SNPs) associated with physical activity (PA) in humans. All human SNPs found to be significantly associated with PA levels in the literature were cross-referenced with the National Heart, Lung, and Blood Institute’s Grand Opportunity Exome Sequencing Project to find estimated African-American (AA) and European-American (EA) mutation age. As a secondary measure of mutation age, SNPs were searched for in Hawk’s mutation age prediction database which utilizes linkage equilibrium. To determine conservation among hominids, all SNPs were searched in the University of California, Santa Cruz Genome Browser, which contains Neanderthal and chimpanzee reference genomes. Six of the 104 SNPs associated with PA regulation were exon-located missense variants found in IFNAR2, PPARGC1A, PML, CTBP2, IL5RA, and APOE genes. The remaining 98 SNPs were located in non-protein coding regions. Average AA and EA estimated mutation age of the exon-located SNPs were 478.4 ± 327.5 kya and 542.1 ± 369.4 kya, respectively. There were four selective sweeps (suggestive of strong positive selection) of SNPs in humans when compared to Neanderthal or chimpanzee genomes. Exon-located PA candidate SNPs are older than the hypothesized emergence of anatomically modern humans. However, 95% of PA associated SNPs are found in intron and intergenic location. Across all SNPs, there seems to be a high level of conservation of alleles between humans, Neanderthals, and chimpanzees. However, the presence of four selective sweeps suggests there were selection pressures or drift unique to Homo sapiens that influenced the development of mutations associated with PA regulation.

The complete mitochondrial genome of the Chan-hua fungus Isaria cicadae: a tale of intron evolution in Cordycipitaceae.

Isaria cicadae is an entomogenous fungus of great medicinal value. Its nuclear genome has been reported, while its mitogenome remains unknown. Herein, we first described its mitogenome and then inferred intron evolution from both intraspecific and interspecific perspectives. The fungus represented the largest mitogenome (56.6 kb in strain CCAD02) known in Cordycipitaceae due to the presence of 25 introns interrupting nine genes. Comparison of three I. cicadae strains revealed intron presence/absence dynamics at six intron loci plus a few indels and single nucleotide polymorphisms. Phylogenetic analyses confirmed the placement of I. cicadae in Cordycipitaceae. Comparison of 10 Cordycipitaceae species revealed a high degree of synteny and conserved genetic content. They, however, varied in intron numbers (1-25 per species) with overall 34 intron loci identified, which resulted in more than twofold variations in mitogenome sizes (24.5-56.6 kb). An rnl intron encoding ribosomal protein S3 was present in all species, suggesting its early invasion in Cordycipitaceae, while further divergence occurred for this intron. The other introns identified in this study were present in some, but not all of the species and have undergone multiple gains and losses in Cordycipitaceae. This study greatly enhanced our understanding of intron evolution in Cordycipitaceae.

M66 - INVESTIGATING SEX DIFFERENCES IN GENETIC EFFECTS ON SUBCORTICAL BRAIN STRUCTURES AND OVERLAP WITH PSYCHIATRIC DISORDERS

Background Brain morphometry is both heritable and correlated with psychiatric disorders. Notable structural differences include reduced volume in subcortical structures, some are observed in several disorders while others are specific to one or two disorders. The prevalence of most psychiatric disorders differs markedly by sex and sex differences are also observed in the volume of subcortical structures. Moreover, brain structure and psychiatric disorders are both highly heritable. Here we assess if genetic variants influencing subcortical brain structures differ between females and males and if the genetic overlap between brain structure and risk of illness differs by sex. Methods We present preliminary results from sex-stratified GWAS meta-analyses on seven subcortical structures derived from T1-weighted magnetic resonance imaging scans from ~6000 females and ~5400 males from 22 cohorts from the ENIGMA consortium. This sample is a subset of the larger ENIGMA consortium analyses on variation in subcortical structures among ~30,000 individuals. The subcortical structures analysed were the nucleus accumbens, amygdala, caudate nucleus, hippocampus, globus pallidus, putamen, and thalamus. The volume of these structures was calculated with validated and freely available software, and mean volume calculated from the left and right hemispheres. Genotypic data were imputed to the 1000 genome reference panel. Results Preliminary results in this discovery meta-analysis identified four genome-wide significant loci, three were novel. One intergenic locus (14q22.3), which we have previously reported as significant in a meta-analysis that combined females and males, was also associated with putamen volume in females and in males in these stratified analyses. Of the novel loci, one was an intronic locus (11q15.2) within SPON1 associated with nucleus accumbens volume in females. The second was an intronic locus (2q34) within ERBB4 associated with amygdala volume in males. The third was an intergenic locus (13q21.31) associated with putamen volume in males. In this discovery sample SNP heritability was .18 (se=.08) for the nucleus accumbens in females, was .16 (se=.08) for the putamen in females, and was .14 (se=.09) for the thalamus in males. Genetic correlations were between each of these traits and GWAS results from the psychiatric genetics consortium on schizophrenia, bipolar, major depressive disorder, anorexia, and autism were not significant. Heritability in all other structures was close to zero, thus genetic correlations were not calculated on this discovery sample. Discussion These preliminary results point to heterogeneity between females and males in genetic influence on some subcortical structures. We plan to extend these findings with data from the UK Biobank. We will present SNP heritability and genetic correlation with psychiatric and cognitive traits using the results from the second round of meta-analysis.

A Role of U12 Intron in Proper Pre-mRNA Splicing of Plant Cap Binding Protein 20 Genes.

The nuclear cap-binding complex (CBC) is composed of two cap-binding proteins: CBP20 and CBP80. The CBP20 gene structure is highly conserved across land plant species. All studied CBP20 genes contain eight exons and seven introns, with the fourth intron belonging to the U12 class. This highly conserved U12 intron always divides the plant CBP20 gene into two parts: one part encodes the core domain containing the RNA binding domain (RBD), and the second part encodes the tail domain with a nuclear localization signal (NLS). In this study, we investigate the importance of the U12 intron in the Arabidopsis thaliana CBP20 gene by moving it to different intron locations of the gene. Relocation of the U12 intron resulted in a significant decrease in the U12 intron splicing efficiency and the accumulation of wrongly processed transcripts. These results suggest that moving the U12 intron to any other position of the A. thaliana CBP20 gene disturbs splicing, leading to substantial downregulation of the level of properly spliced mRNA and CBP20 protein. Moreover, the replacement of the U12 intron with a U2 intron leads to undesired alternative splicing events, indicating that the proper localization of the U12 intron in the CBP20 gene secures correct CBP20 pre-mRNA maturation and CBP20 protein levels in a plant. Surprisingly, our results also show that the efficiency of U12 splicing depends on intron length. In conclusion, our study emphasizes the importance of proper U12 intron localization in plant CBP20 genes for correct pre-mRNA processing.

An intronic mutation in Chd7 creates a cryptic splice site, causing aberrant splicing in a mouse model of CHARGE syndrome

Alternate splicing is a critical regulator of gene expression in eukaryotes, however genetic mutations can cause erroneous splicing and disease. Most recorded splicing disorders are caused by mutations of splice donor/acceptor sites, however intronic mutations can affect splicing. Clinical exome analyses largely ignore intronic sequence, limiting the detection of mutations to within coding regions. We describe ‘Trooper’, a novel mouse model of CHARGE syndrome harbouring a pathogenic point mutation in Chd7. The mutation is 18 nucleotides upstream of exon 10 and creates a cryptic acceptor site, causing exon skipping and partial intron retention. This mutation, though detectable in exome sequence, was initially dismissed by computational filtering due to its intronic location. The Trooper strain exhibited many of the previously described CHARGE-like anomalies of CHD7 deficient mouse lines; including hearing impairment, vestibular hypoplasia and growth retardation. However, more common features such as facial asymmetry and circling were rarely observed. Recognition of these characteristic features prompted manual reexamination of Chd7 sequence and subsequent validation of the intronic mutation, highlighting the importance of phenotyping alongside exome analyses. The Trooper mouse serves as a valuable model of atypical CHARGE syndrome and reveals a molecular mechanism that may underpin milder clinical presentation of the syndrome.

A Genome-Wide Association Study of Idiopathic Dilated Cardiomyopathy in African Americans.

Idiopathic dilated cardiomyopathy (IDC) is the most common form of non-ischemic chronic heart failure. Despite the higher prevalence of IDC in African Americans, the genetics of IDC have been relatively understudied in this ethnic group. We performed a genome-wide association study to identify susceptibility genes for IDC in African Americans recruited from five sites in the U.S. (662 unrelated cases and 1167 controls). The heritability of IDC was calculated to be 33% (95% confidence interval: 19–47%; p = 6.4 × 10−7). We detected association of a variant in a novel intronic locus in the CACNB4 gene meeting genome-wide levels of significance (p = 4.1 × 10−8). The CACNB4 gene encodes a calcium channel subunit expressed in the heart that is important for cardiac muscle contraction. This variant has not previously been associated with IDC in any racial group. Pathway analysis, based on the 1000 genes most strongly associated with IDC, showed an enrichment for genes related to calcium signaling, growth factor signaling, neuronal/neuromuscular signaling, and various types of cellular level signaling, including gap junction and cAMP signaling. Our results suggest a novel locus for IDC in African Americans and provide additional insights into the genetic architecture and etiology.

Mutation in an alternative transcript of CDKL5 in a boy with early-onset seizures.

Infantile-onset epilepsies are a set of severe, heterogeneous disorders for which clinical genetic testing yields causative mutations in ∼20%–50% of affected individuals. We report the case of a boy presenting with intractable seizures at 2 wk of age, for whom gene panel testing was unrevealing. Research-based whole-genome sequencing of the proband and four unaffected family members identified a de novo mutation, NM_001323289.1:c.2828_2829delGA in CDKL5, a gene associated with X-linked early infantile epileptic encephalopathy 2. CDKL5 has multiple alternative transcripts, and the mutation lies in an exon in the brain-expressed forms. The mutation was undetected by gene panel sequencing because of its intronic location in the CDKL5 transcript typically used to define the exons of this gene for clinical exon-based tests (NM_003159). This is the first report of a patient with a mutation in an alternative transcript of CDKL5. This finding suggests that incorporating alternative transcripts into the design and variant interpretation of exon-based tests, including gene panel and exome sequencing, could improve the diagnostic yield.

Abstract WMP56: Genetics of Acute Ischemic Lesion Volume: the MRI-Genetics Interface Exploration (MRI-GENIE) Study

Introduction: Genome-wide association (GWA) studies have contributed substantially to the understanding of complex vascular traits including ischemic stroke; however, genetic determinants of acute cerebral ischemia remain to be elucidated. Objective: To investigate the genetic architecture of cerebral infarct lesion burden in patients with acute ischemic stroke (AIS). Methods: Twelve international sites contributed 3,301 AIS subjects with acute MRI and genome-wide genotyping to the MRI-GENIE study. In a preliminary analysis of 657 subjects of European ancestry, acute cerebral infarct lesions were outlined on diffusion-weighted images (DWI) using a semi-automated volumetric method. Standard quality control measures were performed per single nucleotide polymorphism (SNP) and per subject. SNPs were imputed to the Haplotype Reference Consortium v1.1 panel. Natural log-transformed DWI volume was used for GWA analysis with allelic dosage per SNP, age, sex and principal components 1-5 of genetic ancestry as covariates. Results: GWA testing for DWI volume in 7.7 million SNPs yielded no signal crossing the Bonferroni-corrected genome-wide significance threshold of p &lt;5*10 -8 . However, several loci passed the nominal significance threshold of p &lt;1*10 -6 (Figure 1), including a locus on chromosome 2 in the SPATA3-AS1 (spermatogenesis associated 3 antisense RNA 1) gene (lead SNP: rs2368999, MAF=36%, p =8.4*10 -7 ) and an intronic locus on chromosome 10 in the ATRNL1 (Attractin like 1) gene (lead SNP: rs592284, MAF=10%, p =9.1*10 -7 ). Conclusion: In this first-to-date, preliminary GWAS of acute cerebral infarct volume in AIS patients, we identified several new, nominally significant loci including a locus in ATRNL1, a gene previously linked to carotid plaque burden. Further analyses of the MRI-GENIE cohort to replicate these findings, to include stroke subtype specific analyses, and to increase the overall statistical power of the study are ongoing.

Transcriptional Plasticity Compensates for Ikaros and Aiolos Proteasomal Degradation and Mediates Resistance to IMiDs in Multiple Myeloma (MM)

Background: Immunoglobulin (IGH, IGL, IGK) and non-immunoglobulin (PVT1, TXNDC5, FAM46C, DUSP22, etc.) enhancers hijacking by variable genes (MYC, MAF, MAFB, CCND1/2/3, MMSET, IRF4) is a recognized oncogenic driver event in MM. However, the identity of the transcription factors (TFs) or transcriptional regulatory complexes binding and regulating the activity of these enhancers remains to be fully elucidated and may yield valuable therapeutic targets. As such the discovery of the BET family member BRD4 as the master histone acetyl mark reader at enhancers loci regulating MYC lead to promising therapeutic developments in MM and numerous other cancers. Immunomodulatory drugs (IMiDs) promote the proteasomal degradation of IKAROS (IKZF1) and AIOLOs (IKZF3) leading to the transcriptional repression of MYC and the suppression of MM cells survival and proliferation. However, acquired resistance to IMIDs and the loss of the transcriptional repression of MYC are nearly universal and occur in spite of sustained IKZF1/3 degradation suggesting that transcriptional rewiring may be sustaining hijacked enhancers activity and transcription of driver oncogenes.Methods and Results: In order to define how IMiDs repress MYC transcription, we first defined IKZF1, BRD4, the lysine acetyl transferase P300 and the mediator complex subunit MED1 mapping within the MM genome using ChIPseq. In MM cell lines (MM1S, RPMI8226, ARP1 and AMO1), IKZF1 predominantly mapped to intronic and intergenic loci which are typically enriched with enhancer and superenhancer elements. Indeed, IKZF1 mapping to the genome nearly completely (96.5%) overlapped that of P300, MED1 and BRD4 co-occupied enhancer and superenhancer loci. We also confirmed that in the MM1S sensitive cell lines IMiDs (lenalidomide 10 μM, 24h) exposure efficiently depleted IKZF1, BRD4, P300 and MED1 at enhancer loci with ensuing MYC (and MAF) downregulation. In contrast, in resistant cell lines (RPMI8226) and in spite efficient IKZF1 displacement, BRD4, P300 and MED1 were retained at the oncogenic enhancer (IGLL5) driving MYC (and MAF). These findings lead us to postulate that in IMiDs resistant cells retention of BRD4 and MED1 at oncogenic enhancers in the absence of IKZF1 likely results from rewiring of the TFs regulating MYC. To identify TFs that may co-localize with BRD4 and IKZF1, we analyzed the enrichment of DNA motifs at IKZF1and BRD4 co-occupied enhancers using the MEME suite motif-finding algorithms. This computational analysis revealed a strong enrichment at these MM enhancers of the GGAA motif recognized by the ETS family of TFs (P= 3.2 e-743) and other motifs boxes for the RUNX (P= 9.6 e-725), MYC/MYB (P= 8.8 e-52) and interferon regulatory (IRF) (P= 3.1 e-293) TFs. We next confirmed that the ETS family TF ETV4 was indeed expressed in IMiDs resistant, but not sensitive, MM cell lines. ChiPseq occupancy profiles in IMiDs resistant RPMI8226 cell line revealed co-localization of ETV4 with IKZF1, P300 and BRD4. As predicted, lenalidomide treatment induced global depletion of IKZF1 but not ETV4 at BRD4 occupied enhancers in resistant cell lines (RPMI8226 and ARP1). Importantly, Cas9-mediated knock out of ETV4 in RPMI8226 cells sensitized them to lenalidomide with ensuing MYC downregulation and cell death.Confirming its role in MM, ETV4 transcript was indeed detectable in primary patients' samples in the CoMMpass data repository (ETV4 FPKM >1.0 in 112/724) and its expression was associated with significantly reduced survival outcomes (HR 0.64; P=0.0008). Similarly, high expression (top quartiles) of RUNX2 or MYB, TFs with enriched motifs at IKZF1 co-occupied enhancer loci, was also associated with decreased survival. Of note RNAseq analysis of paired patient samples pre- and post-IMiDs treatment (n=14 pairs) revealed significant upregulation of ETV4 at the time of acquired IMiDs resistance (7/14). Lastly transcriptome analysis of 101 patients enrolled in the RD arm (lenalidomide and dexamethasone) of the POLLUX trial (NCT02076009) confirmed the reduced survival of patients with top quartiles expression of ETV4 as well as MYB and RUNX2 (Fig.1)Conclusion: Transcriptional plasticity with expression of extra-lineage TFs such as the ETS family member ETV4 sustains oncogenic enhancers in MM overcoming IKAROS and AIOLOS dependency and promoting IMiDs resistance. [Display omitted] DisclosuresNeri:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Soong:Jannsen: Employment. Chiu:Janssen: Employment. Bahlis:Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

A workflow of massive identification and application of intron markers using snakes as a model.

Relative to the commonly used mitochondrial and nuclear protein‐coding genes, the noncoding intron sequences are a promising source of informative markers that have the potential to resolve difficult phylogenetic nodes such as rapid radiations and recent divergences. Yet many issues exist in the use of intron markers, which prevent their extensive application as conventional markers. We used the diverse group of snakes as an example to try paving the way for massive identification and application of intron markers. We performed a series of bioinformatics screenings which identified appropriate introns between single‐copy and conserved exons from two snake genomes, adding particular constraints on sequence length variability and sequence variability. A total of 1,273 candidate intron loci were retrieved. Primers for nested polymerase chain reaction (PCR) were designed for over a hundred candidates and tested in 16 snake representatives. 96 intron markers were developed that could be amplified across a broad range of snake taxa with high PCR successful rates. The markers were then applied to 49 snake samples. The large number of amplicons was subjected to next‐generation sequencing (NGS). An analytic strategy was developed to accurately recover the amplicon sequences, and approximately, 76% of the marker sequences were recovered. The average p‐distances of the intron markers at interfamily, intergenus, interspecies, and intraspecies levels were .168, .052, .015, and .004, respectively, suggesting that they were useful to study snake relationships of different evolutionary depths. A snake phylogeny was constructed with the intron markers, which produced concordant results with robust support at both interfamily and intragenus levels. The intron markers provide a convenient way to explore the signals in the noncoding regions to address the controversies on the snake tree. Our improved strategy of genome screening is effective and can be applied to other animal groups. NGS coupled with appropriate sequence processing can greatly facilitate the extensive application of molecular markers.

Transcription elongation rate has a tissue-specific impact on alternative cleavage and polyadenylation in Drosophila melanogaster.

Alternative polyadenylation (APA) is a mechanism that generates multiple mRNA isoforms with different 3′UTRs and/or coding sequences from a single gene. Here, using 3′ region extraction and deep sequencing (3′READS), we have systematically mapped cleavage and polyadenylation sites (PASs) in Drosophila melanogaster, expanding the total repertoire of PASs previously identified for the species, especially those located in A-rich genomic sequences. Cis-element analysis revealed distinct sequence motifs around fly PASs when compared to mammalian ones, including the greater enrichment of upstream UAUA elements and the less prominent presence of downstream UGUG elements. We found that over 75% of mRNA genes in Drosophila melanogaster undergo APA. The head tissue tends to use distal PASs when compared to the body, leading to preferential expression of APA isoforms with long 3′UTRs as well as with distal terminal exons. The distance between the APA sites and intron location of PAS are important parameters for APA difference between body and head, suggesting distinct PAS selection contexts. APA analysis of the RpII215C4 mutant strain, which harbors a mutant RNA polymerase II (RNAPII) with a slower elongation rate, revealed that a 50% decrease in transcriptional elongation rate leads to a mild trend of more usage of proximal, weaker PASs, both in 3′UTRs and in introns, consistent with the “first come, first served” model of APA regulation. However, this trend was not observed in the head, suggesting a different regulatory context in neuronal cells. Together, our data expand the PAS collection for Drosophila melanogaster and reveal a tissue-specific effect of APA regulation by RNAPII elongation rate.

Identification and characterization of a FOXA2-regulated transcriptional enhancer at a type 2 diabetes intronic locus that controls GCKR expression in liver cells

BackgroundGenome-wide association studies (GWAS) have identified more than 100 genetic loci associated with type 2 diabetes (T2D). However, the underlying biological mechanisms for many of these associations remain unknown. GWAS signals close to the glucokinase regulatory protein gene (GCKR) have been reported for lipid and glucose metabolism traits and the risk of T2D. We investigated the regulatory function of an intronic locus at GCKR represented by the lead single nucleotide polymorphism (SNP) rs780094.MethodsWe used ENCODE project histone modification and transcription factor binding data to determine the regulatory features of a GCKR intronic locus formed by the high linkage disequilibrium rs780094(C/T), rs780095(G/A), and rs780096(G/C) SNPs. Characterization of the transcriptional activity of this region was assessed by luciferase reporter assays in HepG2 cells and mouse primary hepatocytes. ChIP-qPCR was used to determine the levels of haplotype specific transcription factor binding and histone marks. A CRISPR-dCas9 transcriptional activator system and qPCR were used to activate the locus and measure GCKR expression, respectively. Differential haplotype expression was measured from human liver biopsies.ResultsThe ENCODE data suggest the existence of a liver-specific intragenic enhancer at the locus represented by s780094. We observed that FOXA2 increased the transcriptional activity of this region in a haplotype specific way (CGG > TAC; rs780094, rs780095, and rs780096). In addition, the CGG haplotype showed higher binding to FOXA2 and higher levels of the H3K27Ac histone mark. The epigenetic activation of this locus increased the expression of endogenous GCKR in HepG2 cells, confirming that GCKR is the direct target gene of the enhancer. Finally, we confirmed that the CGG haplotype exhibits higher levels of transcription in human liver.ConclusionsOur results demonstrate the existence of a liver-specific FOXA2-regulated transcriptional enhancer at an intronic T2D locus represented by rs780094, rs780095, and rs780096 SNPs that increases GCKR expression. Differential haplotype regulation suggests the existence of cis regulatory effects that may contribute to the associated traits at this locus.