CBIO-05. SOX2 CHROMATIN ARCHITECTURE AND ENHANCER ACTIVITY IN THE MAINTENANCE AND DEVELOPMENT OF NEURAL STEM CELLS

Gain of function mutations in isocitrate dehydrogenase I (IDH1) result in the formation of the oncometabolite 2-hydroxyglutarate (2HG) in adult lower grade gliomas. To gain insight into mechanisms of gliomagenesis, our lab previously created a tractable human cellular model of low grade astrocytoma (LGA) using the putative cell-of-origin, human neural stem cells (NSCs), engineered to express mutant IDH1 and knockdown constructs against TP53 and ATRX, the two other genetic changes that accompany the IDH mutation in these tumors. We found that transcription factor (sex determining region Y)-box 2 SOX2, which is essential to NSC multipotency, the ability to differentiate to neuroglial lineages, behaves as a tumor suppressor during glioma initiation. In this context, we showed SOX2 is transcriptionally downregulated to impair NSC multipotency, thus locking NSCs in an undifferentiated state to initiate gliomagenesis. This downregulation occurs secondary to dynamic reorganization of the topologically associating domain (TAD) of SOX2 and the loss of contact with several genomic loci with histone modifications and chromatin accessibility suggestive of being enhancers. Here we show that those putative enhancers acquire enhancer-like features simultaneous to tje TAD organizing in a way that facilitates interaction with the SOX2 promoter during the process of pluripotent stem cell differentiation into neuroectodermal lineages, suggesting a developmental role. Preliminary data suggests that disruption of the SOX2 TAD by preventing binding of the genome organizer CTCF downregulates SOX2 expression in NSCs. Targeted silencing of several regions of a putative enhancer with CRISPRi also downregulates SOX2. In human embryonic stem cells (hESCs), interfering with these CTCF binding sites biases their differentiation away from the neuroectoderm. We are currently performing CRISPRi screen against all putative enhancer loci, teratoma formation assays on hESCs lacking relevant CTCF binding, and CRISPR mediated deletion of putative enhancers. Understanding this developmental process may reveal underlying vulnerabilities in LGA.

Identification of a Putative Enhancer RNA for EGFR in Hyper-Accessible Regions in Esophageal Squamous Cell Carcinoma Cells by Analysis of Chromatin Accessibility Landscapes

Abnormal genetic and epigenetic modifications play a key role in esophageal cancer. By Assay for Transposase-Accessible Chromatin by sequencing (ATAC-seq), this study compared chromatin accessibility landscapes among two esophageal squamous cell carcinoma (ESCC) cell lines, KYSE-30 and KYSE-150, and a non-cancerous esophageal epithelial cell line, HET-1A. Data showed that hyper-accessible regions in ESCC cells contained genes related with cancer hallmarks, such as epidermal growth factor receptor (EGFR). Multi-omics analysis and digital-droplet PCR results demonstrated that several non-coding RNAs in EGFR upstream were upregulated in ESCC cells. Among them, one appeared to act as an enhancer RNA responsible for EGFR overexpression. Further motif analysis and pharmacological data suggested that AP-1 family transcription factors were able to bind the hyper-accessible regions and thus to regulate cancer cell proliferation and migration. This study discovered a putative enhancer RNA for EGFR gene and the reliance of ESCC on AP-1 transcription factor.

Targeting MYC-inducing enhancer-associated noncoding (MYC-IEANC) RNAs inhibits the proliferation of HCC cells

Background MYC, a critical oncogene, encodes the c-MYC transcription factor (TF) and plays an essential role in hepatocellular carcinoma (HCC) development. Recent studies have identified numerous tissue-specific enhancers of MYC in various cancers, but an HCC-specific enhancer of MYC remains elusive. Methods We analyzed enhancer markers, including H3K27ac enrichment and enhancer RNA (eRNA) expression, to determine putative enhancer regions of MYC in HCC cells. Enhancer activity was detected using a dual-luciferase reporter assay. We used the CRISPR-Cas9 system to edit the enhancer regions and performed antisense oligonucleotide (ASO) to inhibit eRNA. The functions of enhancers and eRNAs on HCC cells were confirmed by cell proliferation assay and sphere formation assay. Results We choose two active enhancers R2 and R3, with high activity among six putative enhancer regions by analyzing enhancer markers. Enhancer R2 and R3 are present approximately 800 kb downstream from the MYC gene. We confirmed eRNA activities in the enhancer regions. Depletion of these enhancer regions inhibited eRNAs significantly reduced MYC expression. In addition, MYC enhancers and eRNAs regulated HCC cell proliferation and progression. Conclusion In this study, we present MYC enhancers in HCC and elucidate the molecular functions of MYC-inducing enhancer-associated noncoding (MYC-IEANC) RNAs in the proliferation of HCC cells. Furthermore, our results suggest that MYC-IEANC RNAs, which play an oncogenic role in HCC cells, can be a target for HCC treatment.

An Insertion Mutation Located on Putative Enhancer Regions of the MYB26-Like Gene Induce Inhibition of Anther Dehiscence Resulting in Novel Genic Male Sterility in Radish (Raphanus Sativus L.)

A novel male-sterility trait was identified in a radish (Raphanus sativus L.) population. Although the size of male-sterile anthers was comparable to that of normal flowers, no pollen grain was observed during anther dehiscence. However, dissection of male-sterile anthers revealed an abundance of normal pollen grains. Analysis of segregating populations showed that a single recessive locus, designated RsMs1 conferred male sterility. Based on two radish draft genome sequences, molecular markers were developed to delimit the genomic region harboring the RsMs1. The region was narrowed down to approximately 27 kb after analyzing recombinants selected from 7,511 individuals of a segregating population. Sequencing of the delimited region yielded six putative genes including four genes expressed in the floral tissue, and one gene with significant differential expression between male-fertile and male-sterile individuals of a segregating population. This differentially expressed gene was orthologous to the Arabidopsis MYB26 gene, which played a critical role in anther dehiscence. Excluding a synonymous single nucleotide polymorphism in exon3, no polymorphism involving coding and putative promoter regions was detected between alleles. A 955-bp insertion was identified 7.5 kb upstream of the recessive allele. Highly conserved motifs among four Brassicaceae species were identified around this insertion site, suggesting the presence of putative enhancer sequences. A functional marker was developed for genotyping of the RsMs1 based on the 955-bp insertion. A total of 120 PI accessions were analyzed using this marker, and 11 accessions were shown to carry the recessive RsMs1 allele.

Variants associated with HHIP expression have sex-differential effects on lung function

Background: Lung function is highly heritable and differs between the sexes throughout life. However, little is known about sex-differential genetic effects on lung function. We aimed to conduct the first genome-wide genotype-by-sex interaction study on lung function to identify genetic effects that differ between males and females. Methods: We tested for interactions between 7,745,864 variants and sex on spirometry-based measures of lung function in UK Biobank (N=303,612), and sought replication in 75,696 independent individuals from the SpiroMeta consortium. Results: Five independent single-nucleotide polymorphisms (SNPs) showed genome-wide significant (P<5x10-8) interactions with sex on lung function, and 21 showed suggestive interactions (P<1x10-6). The strongest signal, from rs7697189 (chr4:145436894) on forced expiratory volume in 1 second (FEV1) (P=3.15x10-15), was replicated (P=0.016) in SpiroMeta. The C allele increased FEV1 more in males (untransformed FEV1 β=0.028 [SE 0.0022] litres) than females (β=0.009 [SE 0.0014] litres), and this effect was not accounted for by differential effects on height, smoking or pubertal age. rs7697189 resides upstream of the hedgehog-interacting protein (HHIP) gene and was previously associated with lung function and HHIP lung expression. We found HHIP expression was significantly different between the sexes (P=6.90x10-6), but we could not detect sex differential effects of rs7697189 on expression. Conclusions: We identified a novel genotype-by-sex interaction at a putative enhancer region upstream of the HHIP gene. Establishing the mechanism by which HHIP SNPs have different effects on lung function in males and females will be important for our understanding of lung health and diseases in both sexes.

A transcription-centric model of SNP-age interaction

Complex age-associated phenotypes are caused, in part, by an interaction between an individual’s genotype and age. The mechanisms governing such interactions are however not entirely understood. Here, we provide a novel transcriptional mechanism-based framework–SNiPage, to investigate such interactions, whereby a transcription factor (TF) whose expression changes with age (age-associated TF), binds to a polymorphic regulatory element in an allele-dependent fashion, rendering the target gene’s expression dependent on both, the age and the genotype. Applying SNiPage to GTEx, we detected ~637 significant TF-SNP-Gene triplets on average across 25 tissues, where the TF binds to a regulatory SNP in the gene’s promoter or putative enhancer and potentially regulates its expression in an age- and allele-dependent fashion. The detected SNPs are enriched for epigenomic marks indicative of regulatory activity, exhibit allele-specific chromatin accessibility, and spatial proximity to their putative gene targets. Furthermore, the TF-SNP interaction-dependent target genes have established links to aging and to age-associated diseases. In six hypertension-implicated tissues, detected interactions significantly inform hypertension state of an individual. Lastly, the age-interacting SNPs exhibit a greater proximity to the reported phenotype/diseases-associated SNPs than eSNPs identified in an interaction-independent fashion. Overall, we present a novel mechanism-based model, and a novel framework SNiPage, to identify functionally relevant SNP-age interactions in transcriptional control and illustrate their potential utility in understanding complex age-associated phenotypes.

Correction to: Analyzing a putative enhancer of optic disc morphology

An amendment to this paper has been published and can be accessed via the original article.

Analyzing a putative enhancer of optic disc morphology

Background Genome-wide association studies have identified the CDC7-TGFBR3 intergenic region on chromosome 1 to be strongly associated with optic disc area size. The mechanism of its function remained unclear until new data on eQTL markers emerged from the Genotype-Tissue Expression project. The target region was found to contain a strong silencer of the distal (800 kb) Transcription Factor (TF) gene GFI1 (Growth Factor Independent Transcription Repressor 1) specifically in neuroendocrine cells (pituitary gland). GFI1 has also been reported to be involved in the development of sensory neurons and hematopoiesis. Therefore, GFI1, being a developmental gene, is likely to affect optic disc area size by altering the expression of the associated genes via long-range interactions. Results Distribution of haplotypes in the putative enhancer region has been assessed using the data on four continental supergroups generated by the 1000 Genomes Project. The East Asian (EAS) populations were shown to manifest a highly homogenous unimodal haplotype distribution pattern within the region with the major haplotype occurring with the frequency of 0.9. Another European specific haplotype was observed with the frequency of 0.21. The major haplotype appears to be involved in silencing GFI1repressor gene expression, which might be the cause of increased optic disc area characteristic of the EAS populations. The enhancer/eQTL region overlaps AluJo element, which implies that this particular regulatory element is primate-specific and confined to few tissues. Conclusion Population specific distribution of GFI1 enhancer alleles may predispose certain ethnic groups to glaucoma.

Abstract MP171: Transcription Start Site Profiling Defines Promoter and Enhancer Regions for Cardiomyopathy Genes

Background: Mutations in more than 100 genes lead to dilated, hypertrophic and other forms of cardiomyopathy. Autosomal dominant mutations in the MYH7 and LMNA genes cause autosomal dominant hypertrophic and dilated cardiomyopathy, respectively. Individual mutations display a range of clinical expression from severe early onset disease to minimal or no symptoms. Genetic variation in noncoding gene regulatory regions including enhancers is expected to modify expression of cardiomyopathy genes and disease expressivity. In addition, heart failure is associated a fetal gene re-expression program, mediated by genetic regulatory regions. The contribution of noncoding genetic variation to cardiomyopathy and heart failure has been hampered by limited genome wide descriptions of human cardiac regulatory regions. Methods and Results: We used C ap A nalysis of G ene E xpression by sequencing (CAGE-seq) to profile the transcriptional start sites in healthy and failed human hearts. CAGE-seq detects the unidirectional signals of gene promoters and the bidirectional signal of transcribed enhancer regions. We identified ~17,000 transcriptional start sites associated with gene promoters and ~1,500 putative enhancer regions active in cardiac tissue. These CAGE-defined regulatory regions carried histone modifications and transcription factor binding properties characteristic of enhancers or promoters. We specifically identified promoter switching and differential enhancer usage between healthy and failed hearts. We intersected CAGE-defined enhancers with additional epigenomic datasets to identify regulatory regions for MYH7 and LMNA genes. We identified 13 putative enhancer regions and validated the functionality of a subset of these regulatory regions using reporter assays and gene editing. Conclusions: This CAGE-seq dataset defines the regulatory environment for heart failure. These promoter and enhancer regions could be used to target heart-failure associated gene expression changes. Additionally, this data can be used to identify enhancer regions regulating cardiomyopathy genes.