Allele-specific Manner Research Articles

A Comprehensive Structure-Function Study of Neurogenin3 Disease-Causing Alleles during Human Pancreas and Intestinal Organoid Development

Neurogenin3 (NEUROG3) is required for endocrine lineage formation of the pancreas and intestine. Patients with NEUROG3 mutations are born with congenital malabsorptive diarrhea due to complete loss of enteroendocrine cells, whereas endocrine pancreas development varies in an allele-specific manner. These findings suggest a context-dependent requirement for NEUROG3 in pancreas versus intestine. We utilized human tissue differentiated from NEUROG3-/- pluripotent stem cells for functional analyses. Most disease-associated alleles had hypomorphic or null phenotype in both tissues, whereas the S171fsX68 mutation had reduced activity in the pancreas but largely null in the intestine. Biochemical studies revealed NEUROG3 variants have distinct molecular defects with altered protein stability, DNA binding, and gene transcription. Moreover, NEUROG3 was highly unstable in the intestinal epithelium, explaining the enhanced sensitivity of intestinal defects relative to the pancreas. These studies emphasize that studies of human mutations in the endogenous tissue context may be required to assess structure-function relationships.

Genome-wide DNA methylation analysis in ankylosing spondylitis identifies HLA-B*27 dependent and independent DNA methylation changes in whole blood

Background and objectiveAnkylosing spondylitis is a chronic inflammatory disease characterized by inflammation of the sacroiliac joints and the spine that can lead to significant pain, immobility, and disability. The etiology and pathogenesis of ankylosing spondylitis are incompletely understood, though most patients carry the HLA-B*27 allele. The objective of this study was to evaluate DNA methylation changes in ankylosing spondylitis with the goal of revealing novel mechanistic insights into this disease. MethodsGenome-wide DNA methylation analysis was performed in whole blood DNA samples using the Infinium MethylationEPIC array in patients with ankylosing spondylitis compared to age, sex, and race matched patients with osteoarthritis as a non-inflammatory disease control. We studied 24 patients with ankylosing spondylitis, including 12 patients who carry HLA-B*27 and 12 patients who are HLA-B*27 negative. DNA methylation analysis was performed with adjustment for blood cell composition in each sample. ResultsWe identified a total of 67 differentially methylated sites between ankylosing spondylitis patients and osteoarthritis controls. Hypermethylated genes found included GTPase-related genes, while hypomethylated genes included HCP5, which encodes a lncRNA within the MHC region, previously associated with genetic risk for psoriasis and toxic epidermal necrolysis. Carrying HLA-B*27 was associated with robust hypomethylation of HCP5, tubulin folding cofactor A (TBCA) and phospholipase D Family Member 6 (PLD6) in ankylosing spondylitis patients. Hypomethylation within HCP5 involves a CpG site that contains a single nucleotide polymorphism in linkage disequilibrium with HLA-B*27 and that controls DNA methylation at this locus in an allele-specific manner. ConclusionsA genome-wide DNA methylation analysis in ankylosing spondylitis identified DNA methylation patterns that could provide potential novel insights into this disease. Our findings suggest that HLA-B*27 might play a role in ankylosing spondylitis in part through inducing epigenetic dysregulation.

Epigenetic modulation of a hardwired 3D chromatin landscape in two naive states of pluripotency.

The mechanisms underlying enhancer activation and the extent to which enhancer-promoter rewiring contributes to spatiotemporal gene expression are not well understood. Using integrative and time-resolved analyses we show that the extensive transcriptome and epigenome resetting during the conversion between 'serum' and '2i' states of mouse embryonic stem cells (ESCs) takes place with minimal enhancer-promoter rewiring that becomes more evident in primed-state pluripotency. Instead, differential gene expression is strongly linked to enhancer activation via H3K27ac. Conditional depletion of transcription factors and allele-specific enhancer analysis reveal an essential role for Esrrb in H3K27 acetylation and activation of 2i-specific enhancers. Restoration of a polymorphic ESRRB motif using CRISPR-Cas9 in a hybrid ESC line restores ESRRB binding and enhancer H3K27ac in an allele-specific manner but has no effect on chromatin interactions. Our study shows that enhancer activation in serum- and 2i-ESCs is largely driven by transcription factor binding and epigenetic marking in a hardwired network of chromatin interactions.

Functional Analysis of Promoter Variants in Genes Involved in Sex Steroid Action, DNA Repair and Cell Cycle Control.

Genetic variants affecting the regulation of gene expression are among the main causes of human diversity. The potential importance of regulatory polymorphisms is underscored by results from Genome Wide Association Studies, which have already implicated such polymorphisms in the susceptibility to complex diseases such as breast cancer. In this study, we re-sequenced the promoter regions of 24 genes involved in pathways related to breast cancer including sex steroid action, DNA repair, and cell cycle control in 60 unrelated Caucasian individuals. We constructed haplotypes and assessed the functional impact of promoter variants using gene reporter assays and electrophoretic mobility shift assays. We identified putative functional variants within the promoter regions of estrogen receptor 1 (ESR1), ESR2, forkhead box A1 (FOXA1), ubiquitin interaction motif containing 1 (UIMC1) and cell division cycle 7 (CDC7). The functional polymorphism on CDC7, rs13447455, influences CDC7 transcriptional activity in an allele-specific manner and alters DNA–protein complex formation in breast cancer cell lines. Moreover, results from the Breast Cancer Association Consortium show a marginal association between rs13447455 and breast cancer risk (p = 9.3 × 10−5), thus warranting further investigation. Furthermore, our study has helped provide methodological solutions to some technical difficulties that were encountered with gene reporter assays, particularly regarding inter-clone variability and statistical consistency.

Allele-Specific CRISPR/Cas9 Correction of a Heterozygous DNM2 Mutation Rescues Centronuclear Myopathy Cell Phenotypes

Genome editing with the CRISPR/Cas9 technology has emerged recently as a potential strategy for therapy in genetic diseases. For dominant mutations linked to gain-of-function effects, allele-specific correction may be the most suitable approach. In this study, we tested allele-specific inactivation or correction of a heterozygous mutation in the Dynamin 2 (DNM2) gene that causes the autosomal dominant form of centronuclear myopathies (CNMs), a rare muscle disorder belonging to the large group of congenital myopathies. Truncated single-guide RNAs targeting specifically the mutated allele were tested on cells derived from a mouse model and patients. The mutated allele was successfully targeted in patient fibroblasts and Dnm2R465W/+ mouse myoblasts, and clones were obtained with precise genome correction or inactivation. Dnm2R465W/+ myoblasts showed an alteration in transferrin uptake and autophagy. Specific inactivation or correction of the mutated allele rescued these phenotypes. These findings illustrate the potential of CRISPR/Cas9 to target and correct in an allele-specific manner heterozygous point mutations leading to a gain-of-function effect, and to rescue autosomal dominant CNM-related phenotypes. This strategy may be suitable for a large number of diseases caused by germline or somatic mutations resulting in a gain-of-function mechanism.

Three Distinct Contact-Dependent Growth Inhibition Systems Mediate Interbacterial Competition by the Cystic Fibrosis Pathogen Burkholderia dolosa.

The respiratory tracts of individuals afflicted with cystic fibrosis (CF) harbor complex polymicrobial communities. By an unknown mechanism, species of the Gram-negative Burkholderia cepacia complex, such as Burkholderia dolosa, can displace other bacteria in the CF lung, causing cepacia syndrome, which has a poor prognosis. The genome of Bdolosa strain AU0158 (BdAU0158) contains three loci that are predicted to encode contact-dependent growth inhibition (CDI) systems. CDI systems function by translocating the toxic C terminus of a large exoprotein directly into target cells, resulting in growth inhibition or death unless the target cells produce a cognate immunity protein. We demonstrate here that each of the three bcpAIOB loci in BdAU0158 encodes a distinct CDI system that mediates interbacterial competition in an allele-specific manner. While only two of the three bcpAIOB loci were expressed under the in vitro conditions tested, the third conferred immunity under these conditions due to the presence of an internal promoter driving expression of the bcpI gene. One BdAU0158 bcpAIOB allele is highly similar to bcpAIOB in Burkholderia thailandensis strain E264 (BtE264), and we showed that their BcpI proteins are functionally interchangeable, but contact-dependent signaling (CDS) phenotypes were not observed in BdAU0158. Our findings suggest that the CDI systems of BdAU0158 may provide this pathogen an ecological advantage during polymicrobial infections of the CF respiratory tract.IMPORTANCE Human-associated polymicrobial communities can promote health and disease, and interbacterial interactions influence the microbial ecology of such communities. Polymicrobial infections of the cystic fibrosis respiratory tract impair lung function and lead to the death of individuals suffering from this disorder; therefore, a greater understanding of these microbial communities is necessary for improving treatment strategies. Bacteria utilize contact-dependent growth inhibition systems to kill neighboring competitors and maintain their niche within multicellular communities. Several cystic fibrosis pathogens have the potential to gain an ecological advantage during infection via contact-dependent growth inhibition systems, including Burkholderia dolosa Our research is significant, as it has identified three functional contact-dependent growth inhibition systems in Bdolosa that may provide this pathogen a competitive advantage during polymicrobial infections.

Celiac Diasease-associated lncRNA Named HCG14 Regulates NOD1 Expression in Intestinal Cells.

The aim of the study is to identify additional celiac disease associated loci in the major histocompatibility complex (MHC) independent from classical HLA risk alleles (HLA-DR3-DQ2) and to characterize their potential functional impact in celiac disease pathogenesis at the intestinal level. We performed a high-resolution single-nucleotide polymorphism (SNP) genotyping of the MHC region, comparing HLA-DR3 homozygous celiac patients and non-celiac controls carrying a single copy of the B8-DR3-DQ2 conserved extended haplotype. Expression level of potential novel risk genes was determined by RT-PCR in intestinal biopsies and in intestinal and immune cells isolated from control and celiac individuals. Small interfering RNA-driven silencing of selected genes was performed in the intestinal cell line T84. MHC genotyping revealed 2 associated SNPs, one located in TRIM27 gene and another in the non-coding gene HCG14. After stratification analysis, only HCG14 showed significant association independent from HLA-DR-DQ loci. Expression of HCG14 was slightly downregulated in epithelial cells isolated from duodenal biopsies of celiac patients, and eQTL analysis revealed that polymorphisms in HCG14 region were associated with decreased NOD1 expression in duodenal intestinal cells. We have successfully employed a conserved extended haplotype-matching strategy and identified a novel additional celiac disease risk variant in the lncRNA HCG14. This lncRNA seems to regulate the expression of NOD1 in an allele-specific manner. Further functional studies are needed to clarify the role of HCG14 in the regulation of gene expression and to determine the molecular mechanisms by which the risk variant in HCG14 contributes to celiac disease pathogenesis.

The Dysregulation of the DLK1-MEG3 Locus in Islets From Patients With Type 2 Diabetes Is Mimicked by Targeted Epimutation of Its Promoter With TALE-DNMT Constructs.

Type 2 diabetes mellitus (T2DM) is characterized by the inability of the insulin-producing β-cells to overcome insulin resistance. We previously identified an imprinted region on chromosome 14, the DLK1-MEG3 locus, as being downregulated in islets from humans with T2DM. In this study, using targeted epigenetic modifiers, we prove that increased methylation at the promoter of Meg3 in mouse βTC6 β-cells results in decreased transcription of the maternal transcripts associated with this locus. As a result, the sensitivity of β-cells to cytokine-mediated oxidative stress was increased. Additionally, we demonstrate that an evolutionarily conserved intronic region at the MEG3 locus can function as an enhancer in βTC6 β-cells. Using circular chromosome conformation capture followed by high-throughput sequencing, we demonstrate that the promoter of MEG3 physically interacts with this novel enhancer and other putative regulatory elements in this imprinted region in human islets. Remarkably, this enhancer is bound in an allele-specific manner by the transcription factors FOXA2, PDX1, and NKX2.2. Overall, these data suggest that the intronic MEG3 enhancer plays an important role in the regulation of allele-specific expression at the imprinted DLK1-MEG3 locus in human β-cells, which in turn impacts the sensitivity of β-cells to cytokine-mediated oxidative stress.

Abstract 240: SNP-mediated repression of DCBLD1 by YY1 may explain a GWAS finding on lung cancer

Abstract Genome-wide association studies (GWAS) have identified many cancer-associated single nucleotide polymorphisms (SNP) with limited understanding of their real implication in cancer. To study the biological relevance of a GWAS-discovered SNP, we performed linkage disequilibrium (LD) analysis on rs9387478 and identified four potentially functional SNPs. We found that SNP rs17079281 C>T was associated with lung cancer in 766 Chinese cases and 773 matched controls, and the association was validated by another independent study (558 cases and 534 controls). Luciferase assay and CRISP/Cas9-generated genotype experiments showed that transcription factor YY1 suppressed DCBLD1 expression in an allele-specific manner, higher inhibition to T than to C alleles. In vitro and in vivo experiments on gain and loss of function further demonstrated that DCBLD1 behaved like an oncogene for lung cancer. These findings suggest that rs17079281 genotype may differentially affect YY1's suppression on DCBLD1 which influences the risk of lung cancer as an oncogene. Citation Format: Yu Wang, Rongna Ma, Hongyan Lin, Xiao Yu, Herbert Yu, Biyun Qian. SNP-mediated repression of DCBLD1 by YY1 may explain a GWAS finding on lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 240.

Convergent evolution of gene expression in two high-toothed stickleback populations.

Changes in developmental gene regulatory networks enable evolved changes in morphology. These changes can be in cis regulatory elements that act in an allele-specific manner, or changes to the overall trans regulatory environment that interacts with cis regulatory sequences. Here we address several questions about the evolution of gene expression accompanying a convergently evolved constructive morphological trait, increases in tooth number in two independently derived freshwater populations of threespine stickleback fish (Gasterosteus aculeatus). Are convergently evolved cis and/or trans changes in gene expression associated with convergently evolved morphological evolution? Do cis or trans regulatory changes contribute more to gene expression changes accompanying an evolved morphological gain trait? Transcriptome data from dental tissue of ancestral low-toothed and two independently derived high-toothed stickleback populations revealed significantly shared gene expression changes that have convergently evolved in the two high-toothed populations. Comparing cis and trans regulatory changes using phased gene expression data from F1 hybrids, we found that trans regulatory changes were predominant and more likely to be shared among both high-toothed populations. In contrast, while cis regulatory changes have evolved in both high-toothed populations, overall these changes were distinct and not shared among high-toothed populations. Together these data suggest that a convergently evolved trait can occur through genetically distinct regulatory changes that converge on similar trans regulatory environments.

Interaction of Nevirapine with the Peptide Binding Groove of HLA-DRB1*01:01 and Its Effect on the Conformation of HLA-Peptide Complex.

Human leukocyte antigen (HLA)-DRB1*01:01 has been shown to be involved in nevirapine-induced hepatic hypersensitivity reactions. In the present study, in silico docking simulations and molecular dynamics simulations were performed to predict the interaction mode of nevirapine with the peptide binding groove of HLA-DRB1*01:01 and its possible effect on the position and orientation of the ligand peptide derived from hemagglutinin (HA). In silico analyses suggested that nevirapine interacts with HLA-DRB1*01:01 around the P4 pocket within the peptide binding groove and the HA peptide stably binds on top of nevirapine at the groove. The analyses also showed that binding of nevirapine at the groove will significantly change the inter-helical distances of the groove. An in vitro competitive assay showed that nevirapine (1000 μM) increases the binding of the HA peptide to HLA-DRB1*01:01 in an allele-specific manner. These results indicate that nevirapine might interact directly with the P4 pocket and modifies its structure, which could change the orientation of loaded peptides and the conformation of HLA-DRB1*01:01; these changes could be distinctively recognized by T-cell receptors. Through this molecular mechanism, nevirapine might stimulate the immune system, resulting in hepatic hypersensitivity reactions.

A TAD boundary is preserved upon deletion of the CTCF-rich Firre locus

The binding of the transcriptional regulator CTCF to the genome has been implicated in the formation of topologically associated domains (TADs). However, the general mechanisms of folding the genome into TADs are not fully understood. Here we test the effects of deleting a CTCF-rich locus on TAD boundary formation. Using genome-wide chromosome conformation capture (Hi-C), we focus on one TAD boundary on chromosome X harboring ~ 15 CTCF binding sites and located at the long non-coding RNA (lncRNA) locus Firre. Specifically, this TAD boundary is invariant across evolution, tissues, and temporal dynamics of X-chromosome inactivation. We demonstrate that neither the deletion of this locus nor the ectopic insertion of Firre cDNA or its ectopic expression are sufficient to alter TADs in a sex-specific or allele-specific manner. In contrast, Firre’s deletion disrupts the chromatin super-loop formation of the inactive X-chromosome. Collectively, our findings suggest that apart from CTCF binding, additional mechanisms may play roles in establishing TAD boundary formation.

Host-mediated selection impacts the diversity of Plasmodium falciparum antigens within infections

Host immunity exerts strong selective pressure on pathogens. Population-level genetic analysis can identify signatures of this selection, but these signatures reflect the net selective effect of all hosts and vectors in a population. In contrast, analysis of pathogen diversity within hosts provides information on individual, host-specific selection pressures. Here, we combine these complementary approaches in an analysis of the malaria parasite Plasmodium falciparum using haplotype sequences from thousands of natural infections in sub-Saharan Africa. We find that parasite genotypes show preferential clustering within multi-strain infections in young children, and identify individual amino acid positions that may contribute to strain-specific immunity. Our results demonstrate that natural host defenses to P. falciparum act in an allele-specific manner to block specific parasite haplotypes from establishing blood-stage infections. This selection partially explains the extreme amino acid diversity of many parasite antigens and suggests that vaccines targeting such proteins should account for allele-specific immunity.

Cancer-associated rs6983267 SNP and its accompanying long noncoding RNA CCAT2 induce myeloid malignancies via unique SNP-specific RNA mutations.

The cancer-risk-associated rs6983267 single nucleotide polymorphism (SNP) and the accompanying long noncoding RNA CCAT2 in the highly amplified 8q24.21 region have been implicated in cancer predisposition, although causality has not been established. Here, using allele-specific CCAT2 transgenic mice, we demonstrate that CCAT2 overexpression leads to spontaneous myeloid malignancies. We further identified that CCAT2 is overexpressed in bone marrow and peripheral blood of myelodysplastic/myeloproliferative neoplasms (MDS/MPN) patients. CCAT2 induces global deregulation of gene expression by down-regulating EZH2 in vitro and in vivo in an allele-specific manner. We also identified a novel non-APOBEC, non-ADAR, RNA editing at the SNP locus in MDS/MPN patients and CCAT2-transgenic mice. The RNA transcribed from the SNP locus in malignant hematopoietic cells have different allelic composition from the corresponding genomic DNA, a phenomenon rarely observed in normal cells. Our findings provide fundamental insights into the functional role of rs6983267 SNP and CCAT2 in myeloid malignancies.

Allele-specific repression of Sox2 through the long non-coding RNA Sox2ot

The transcription factor Sox2 controls the fate of pluripotent stem cells and neural stem cells. This gatekeeper function requires well-regulated Sox2 levels. We postulated that Sox2 regulation is partially controlled by the Sox2 overlapping long non-coding RNA (lncRNA) gene Sox2ot. Here we show that the RNA levels of Sox2ot and Sox2 are inversely correlated during neural differentiation of mouse embryonic stem cells (ESCs). Through allele-specific enhanced transcription of Sox2ot in mouse Sox2eGFP knockin ESCs we demonstrate that increased Sox2ot transcriptional activity reduces Sox2 RNA levels in an allele-specific manner. Enhanced Sox2ot transcription, yielding lower Sox2 RNA levels, correlates with a decreased chromatin interaction of the upstream regulatory sequence of Sox2 and the ESC-specific Sox2 super enhancer. Our study indicates that, in addition to previously reported in trans mechanisms, Sox2ot can regulate Sox2 by an allele-specific mechanism, in particular during development.

Computational and functional characterization of four SNPs in the SOST locus associated with osteoporosis.

The SOST gene encodes sclerostin, a C-terminal cysteine knot-like domain containing key negative regulator of osteoblastic bone formation that inhibits LRP5/6-mediated canonical Wnt signaling. Numerous single nucleotide polymorphisms (SNPs) in the SOST locus are firmly associated with bone mineral density (BMD) and fracture in genome-wide association studies (GWAS) and candidate gene association studies. However, the validation and mechanistic elucidation of causal genetic variants, especially for SNPs located beyond the promoter-proximal region, remain largely unresolved. By employing computational and experimental approaches, here we identify four SNPs rs1230399, rs7220711, rs1107748 and rs75901553 as functional variants which display allelic variation in SOST gene expression. The osteoporosis associated SNP rs1230399 in the SOST distal upstream regulatory region shows FOXA1 binding activity with subsequent transinactivation in a T allele-specific manner. The BMD GWAS lead SNPs rs7220711 and rs1107748 both reside in the 52-kb regulatory element deletion 35-kb downstream of the SOST gene which leads to Van Buchem disease. The rs7220711-A has a higher affinity for the transcriptional repressors MAFF or MAFK homodimers than rs7220711-G, while rs1107748 confers C allele specific transcriptional enhancer activity via a CTCF binding element. The variant rs75901553 C>T located in a conserved site of the SOST 3' UTR abolishes a target binding site for miR-98-5p which is negatively responsive to parathyroid hormone or 17β-estradiol in osteoblastic cell lines. Our findings uncover the biological consequences of four independent genetic variants in the SOST region and their important roles in SOST expression via diverse mechanisms, providing new insights into the genetics and molecular pathogenesis of osteoporosis.

The role of SMAD3 in the genetic predisposition to papillary thyroid carcinoma.

To identify and characterize the functional variants, regulatory gene networks, and potential binding targets of SMAD3 in the 15q22 thyroid cancer risk locus. We performed linkage disequilibrium (LD) and haplotype analyses to fine map the 15q22 locus. Luciferase reporter assays were applied to evaluate the regulatory effects of the candidate variants. Knockdown by small interfering RNA, microarray analysis, chromatin immunoprecipitation (ChIP) and quantitative real-time polymerase chain reaction assays were performed to reveal the regulatory gene network and identify its binding targets. We report a 25.6-kb haplotype within SMAD3 containing numerous single-nucleotide polymorphisms (SNPs) in high LD. SNPs rs17293632 and rs4562997 were identified as functional variants of SMAD3 by luciferase assays within the LD region. These variants regulate SMAD3 transcription in an allele-specific manner through enhancer elements in introns of SMAD3. Knockdown of SMAD3 in thyroid cancer cell lines revealed its regulatory gene network including two upregulated genes, SPRY4 and SPRY4-IT1. Sequence analysis and ChIP assays validated the actual binding of SMAD3 protein to multiple SMAD binding element sites in the region upstream of SPRY4. Our data provide a functional annotation of the 15q22 thyroid cancer risk locus.

Abstract B211: Cellular zinc homeostatic mechanisms function as an off switch for zinc metallochaperone-mediated reactivation of mutant p53

Abstract The p53 transcription factor functions as one of cancer’s most potent tumor suppressors and is the most frequently mutated gene in human cancer. The majority of p53 mutations (>70%) are missense that generate a defective protein found at high levels in cells that is targetable. Restoration of wild type structure and function of mutant p53 with a small molecule (so-called reactivation) is a highly sought after goal in anticancer drug development. The p53 protein requires the binding of a single zinc ion to fold properly, and mutations that impair the protein’s ability to bind zinc (and cause it to misfold) are highly prevalent in cancer. We recently discovered a new class of small-molecule zinc chelators named zinc metallochaperones (ZMCs) that reactivate zinc-deficient mutant p53 through a novel mechanism involving both zinc ionophore activity to raise intracellular zinc concentrations and donation to restore zinc binding to mutant p53. This induces a wild type conformation change and a p53-mediated apoptotic program. The lead compound (ZMC1) displays a transient pharmacodynamics (p21 levels) in vitro. We hypothesized that the regulation of these pharmacodynamics is governed by cellular zinc homeostatic mechanisms that function to restore zinc to its physiologic picomolar levels. We examined the entire suite of zinc homeostatic genes in response to ZMC1 and manipulated several metallothionein genes by knockout and knockdown. The net effect of this was to increase the peak and duration of intracellular zinc levels that lead to a more potent and sustained duration of p21 expression. This translated to increased sensitivity to ZMC1. We further postulated that this pharmacodynamics would allow the drug to function with very minimal exposure, and colony formation studies in vitro indicated that a 2-hour exposure was as effective as a 72-hour exposure. We then sought to translate this mechanism in vivo using a genetically engineered murine model of KPC pancreatic cancer (Pdx-1Cre; KrasG12D) that expresses either the p53R172H (zinc deficient) allele or p53R270H (non-zinc deficient). Pharmacokinetic (PK) studies of the drug revealed a short half-life (15 minutes) indicating a minimal exposure. Despite this, daily, intermittent dosing at the maximum tolerated dose resulted in a statistically significant increase in the overall survival of the KPC-p53R172H mice while having no such effect in the KPC-p53R270H. We sought to improve the efficacy of ZMC1 by preloading it with zinc in a 2:1 molar ratio based on the crystal structure. The drug-zinc complex (Zn-1) increased the median survival of KPCp53-R172H mice from 26 days to 35 days (ZMC1 monomer versus Zn-1). These studies indicate that cellular zinc homeostatic mechanisms function as an “off” switch for ZMCs, which has important implications for the translation of ZMCs in humans. Principally, this allows the drug to function with minimal exposure, which minimizes potential zinc toxicity. ZMC1 as monotherapy improves survival in an allele-specific mutant p53 manner. Furthermore, ZMC1 can be optimized by synthesizing it complexed with zinc. Overall, this “off” switch is novel for a targeted molecular therapeutic and represents a significant departure from the traditional paradigm where the goal is to develop a compound that binds the target with a PK profile that provides maximal exposure. Citation Format: Xin Yu, Samuel Kogan, Ying Chen, Ashley T. Tsang, Tracy Withers, Hongxia Lin, Chunxia Chen, Dirk F. Moore, Joseph Bertino, Chang Chan, Darren R. Carpizo. Cellular zinc homeostatic mechanisms function as an off switch for zinc metallochaperone-mediated reactivation of mutant p53 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B211.

A functional SNP in the 3'-UTR of TAP2 gene interacts with microRNA hsa-miR-1270 to suppress the gene expression.

The transporter associated with antigen processing 2 (TAP2) is involved in the development of multidrug resistance and the etiology of immunological diseases. In this study, we investigated whether the expression of TAP2 can be perturbed by single nucleotide polymorphisms (SNPs) located in 3'-untranslated region (3'-UTR) of the gene via interactions with microRNAs. Using a series of in silico assays, we selected the candidate microRNAs (miRNAs) with the potential to interact with functional SNPs of TAP2. The SNP rs241456-located in the 3'-UTR of TAP2-resides in a potential binding site for hsa-miR-1270 and hsa-miR-620. HEK 293 cells, from a human kidney cell line, were used to characterize the extent of binding of miRNAs to each polymorphic allele of the SNP by a luciferase reporter gene assay. RNA electrophoretic mobility shift assays were used to evaluate the interaction between the miRNAs and each allele sequence of the SNP. We found that hsa-miR-1270 inhibited luciferase activity by binding to the T allele of the SNP in an allele-specific manner. A negative correlation was also found between the expression of hsa-miR-1270 and the T allele of the SNP in kidney tissues. Our findings support the hypothesis that hsa-miR-1270 suppresses the production of TAP2 by binding to this SNP in the 3'-UTR of this gene. Environ. Mol. Mutagen. 59:134-143, 2018. © 2017 Wiley Periodicals, Inc.

A type 2 diabetes-associated SNP in KCNQ1 (rs163184) modulates the binding activity of the locus for Sp3 and Lsd1/Kdm1a, potentially affecting CDKN1C expression.

Although genome-wide association studies have shown that potassium voltage-gated channel subfamily Q member 1 (KCNQ1) is one of the genes that is most significantly associated with type 2 diabetes mellitus (T2DM), functionally annotating disease-associated single nucleotide polymorphisms (SNPs) remains a challenge. Recently, our group described a novel strategy to identify proteins that bind to SNP-containing loci in an allele-specific manner. The present study successfully applied this strategy to investigate rs163184, a T2DM susceptibility SNP located in the intronic region of KCNQ1. Comparative analysis of DNA-binding proteins revealed that the binding activities for the genomic region containing SNP rs163184 differed between alleles for several proteins, including Sp3 and Lsd1/Kdm1a. Sp3 preferentially bound to the non-risk rs163184 allele and stimulated transcriptional activity in an artificial promoter containing this region. Lsd1/Kdm1a was identified to be preferentially recruited to the non-risk allele of the rs163184 region and reduced Sp3-dependent transcriptional activity in the artificial promoter. In addition, expression of the nearby cyclin-dependent kinase inhibitor 1C (CDKN1C) gene was revealed to be upregulated after SP3 knockdown in cells that possessed non-risk alleles. This suggests that CDKN1C is potentially one of the functional targets of SNP rs163184, which modulates the binding activity of the locus for Sp3 and Lsd1/Kdm1a.