Intron Research Articles

Introns: Guardians of the genome

Genome Stability Introns are intragenic regions that are removed by RNA splicing. Bonnet et al. have discovered previously unknown functions of introns in counteracting transcription-associated genetic instability in yeast and human cells. Transcription naturally introduces DNA-RNA hybrids called R-

Methamphetamine Induces TET1- and TET3-Dependent DNA Hydroxymethylation of Crh and Avp Genes in the Rat Nucleus Accumbens

Methamphetamine (METH) addiction is a biopsychosocial disorder that is accompanied by multiple relapses even after prolonged abstinence, suggesting the possibilities of long-lasting maladaptive epigenetic changes in the brain. Here, we show that METH administration produced time-dependent increases in the expression of corticotropin-releasing hormone (Crh/Crf), arginine vasopressin (Avp), and cocaine- and amphetamine-regulated transcript prepropeptide (Cartpt) mRNAs in the rat nucleus accumbens (NAc). Chromatin immunoprecipitation (ChIP) assays revealed that METH increased the abundance of phosphorylated CREB (pCREB) at the promoter of Cartpt but not at Avp or Crh DNA sequences. In contrast, METH produced DNA hypomethylation at sites near the Crh transcription start site (TSS) and at intragenic Avp sequences. METH also increased DNA hydroxymethylation at the Crh TSS and at intragenic Avp sites. In addition, METH increased the protein expression of ten-eleven-translocation enzymes that catalyze DNA hydroxymethylation. Importantly, METH increased TET1 binding at the Crh promoter and increased TET3 binding at Avp intragenic regions. We further tested the role of TET enzymes in METH-induced changes in gene expression by using the TET inhibitor, 1,5-isoquinolinediol (IQD), and found that IQD blocked METH-induced increases in Crh and Avp mRNA expression. Together, these results indicate that METH produced changes in neuropeptide transcription by both activation of the cAMP/CREB pathway and stimulation of TET-dependent DNA hydroxymethylation. These results provide molecular evidence for epigenetic controls of METH-induced changes in the expression of neuropeptides.

Update on mutations of precursor mRNA splicing factor genes linked to retinitis pigmentosa

Retinitis pigmentosa (RP), one of the common forms of hereditary retinal dystrophies (HRD), is typified by significant genetic heterogeneities.Executed by the spliceosome, precursor mRNA (pre-mRNA) splicing is a highly regulated process by which introns are removed and exons are ligated together.To date, more than 80 genes have been involved in RP etiology.Specially, 8 of these genes (PRPF3, PRPF8, PRPF31, PRPF6, PRPF4, SNRNP200, RP9 and DHX38) encode proteins essential for pre-mRNA splicing and are expressed ubiquitously.However, mutations of these RP causative pre-mRNA splicing genes exclusively result in only retinal phenotypes, and the mechanism remains unknown.In this review, we recapitulate splicing process, summarize the mutations identified in pre-mRNA splicing genes related to RP and discuss conceivable hypothesis explaining for the consequent retina-specific phenotypes. Key words: Retinitis pigmentosa; Spliceosome; Precursor mRNA; Splicing factor

Simple Expression Domains Are Regulated by Discrete CRMs During Drosophila Oogenesis.

Eggshell patterning has been extensively studied in Drosophila melanogaster. However, the cis-regulatory modules (CRMs), which control spatiotemporal expression of these patterns, are vastly unexplored. The FlyLight collection contains >7000 intergenic and intronic DNA fragments that, if containing CRMs, can drive the transcription factor GAL4. We cross-listed the 84 genes known to be expressed during D. melanogaster oogenesis with the ∼1200 listed genes of the FlyLight collection, and found 22 common genes that are represented by 281 FlyLight fly lines. Of these lines, 54 show expression patterns during oogenesis when crossed to an UAS-GFP reporter. Of the 54 lines, 16 recapitulate the full or partial pattern of the associated gene pattern. Interestingly, while the average DNA fragment size is ∼3 kb in length, the vast majority of fragments show one type of spatiotemporal pattern in oogenesis. Mapping the distribution of all 54 lines, we found a significant enrichment of CRMs in the first intron of the associated genes’ model. In addition, we demonstrate the use of different anteriorly active FlyLight lines as tools to disrupt eggshell patterning in a targeted manner. Our screen provides further evidence that complex gene patterns are assembled combinatorially by different CRMs controlling the expression of genes in simple domains.

Abstract LB-003: Racial differences of intron retention and DNA methylation in breast cancer

Abstract Regulation of gene expression by DNA methylation in gene promoter regions is well-studied; however, the effects of methylation in the gene body (i.e., exons and introns) on gene expression are comparatively understudied. Recently, hyper-methylation has been implicated in the inclusion of alternatively spliced exons; moreover, exon recognition can be enhanced by recruiting the methyl-CpG-binding protein (MeCP2) to hyper-methylated sites. In this study, we examined whether or not the level of methylation of an intron is correlated with how frequently that intron is retained during splicing using DNA methylation and RNA sequencing data from breast cancer tissue samples in The Cancer Genome Atlas (TCGA). We found that hypo-methylation of introns is correlated with higher levels of intron expression in mRNA and that most novel cancer-specific mRNA isoforms are due to intron retention. The methylation level of an intron is inversely correlated with its retention in mRNA transcripts from the gene in which it is located. Furthermore, we observed significant racial differences in the methylation level of retained introns: In samples from African-American donors, retained introns were not only less methylated compared to Caucasian donors, but also were more highly expressed. This underscores the need for Understanding racial epigenetic differences and their correlation with breast cancer is an important step toward achieving personalized cancer care. Citation Format: Dongwook Kim, Manu Shivakumar, Youngji Lee, Olufunmilayo Olopade, Dokyoon Kim, Younghee Lee. Racial differences of intron retention and DNA methylation in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-003. doi:10.1158/1538-7445.AM2017-LB-003

Development of EPIC-PCR Markers for Lutjanus purpureus (Lutjanidae-Perciformes) and their Potential Applicability in Population Analyses.

In the present study, a novel set of eight EPIC primers were developed for Lutjanus purpureus and assayed in five other marine teleosts including three lutjanids, one scianid and one anablepid. Most of the genomic regions used in this study presented genetic diversity indexes equal or greater than the intragenic regions commonly used in population genetics studies. Moreover, six out of eight markers showed cross-amplification with other taxa. Thus, the primers described here may be used to elucidate questions at the intraspecific level for a large number of taxa.

Tumor antigen PRAME is up-regulated by MZF1 in cooperation with DNA hypomethylation in melanoma cells

Elevated expression of preferentially expressed antigen in melanoma (PRAME) has been implicated in disease progression in a variety of cancers. However, the mechanisms underlying the transcriptional regulation of PRAME remain largely unexplored. Initially, we observed that PRAME was elevated in proportion to the malignant potential of melanoma cells. From the in silico prediction of PRAME gene structure, we identified the putative myeloid zinc finger 1 (MZF1) binding sites, which overlap with a CpG-rich region located in the first intron. The transcription factor MZF1 increased PRAME expression via its direct binding to the intron DNA. Upon treatment with a DNA methylation inhibitor, 5-aza-2′-deoxycitidine (5-azaC), together with ectopic expression of MZF1, PRAME expression was significantly enhanced at both the protein and mRNA levels. More pronounced MZF1 binding to the PRAME DNA was observed in the presence of 5-azaC. DNA methylation was inversely correlated with PRAME expression in melanoma cells. Finally, we observed that MZF1, like PRAME, promotes the colony-forming ability in melanoma cells. Overall, our findings suggest that MZF1, via stimulation of PRAME expression, may be a potential prognostic and therapeutic target in melanoma.

Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells

Single-cell epigenome sequencing techniques have recently been developed. However, the combination of different layers of epigenome sequencing in an individual cell has not yet been achieved. Here, we developed a single-cell multi-omics sequencing technology (single-cell COOL-seq) that can analyze the chromatin state/nucleosome positioning, DNA methylation, copy number variation and ploidy simultaneously from the same individual mammalian cell. We used this method to analyze the reprogramming of the chromatin state and DNA methylation in mouse preimplantation embryos. We found that within < 12 h of fertilization, each individual cell undergoes global genome demethylation together with the rapid and global reprogramming of both maternal and paternal genomes to a highly opened chromatin state. This was followed by decreased openness after the late zygote stage. Furthermore, from the late zygote to the 4-cell stage, the residual DNA methylation is preferentially preserved on intergenic regions of the paternal alleles and intragenic regions of maternal alleles in each individual blastomere. However, chromatin accessibility is similar between paternal and maternal alleles in each individual cell from the late zygote to the blastocyst stage. The binding motifs of several pluripotency regulators are enriched at distal nucleosome depleted regions from as early as the 2-cell stage. This indicates that the cis-regulatory elements of such target genes have been primed to an open state from the 2-cell stage onward, long before pluripotency is eventually established in the ICM of the blastocyst. Genes may be classified into homogeneously open, homogeneously closed and divergent states based on the chromatin accessibility of their promoter regions among individual cells. This can be traced to step-wise transitions during preimplantation development. Our study offers the first single-cell and parental allele-specific analysis of the genome-scale chromatin state and DNA methylation dynamics at single-base resolution in early mouse embryos and provides new insights into the heterogeneous yet highly ordered features of epigenomic reprogramming during this process.

Distinctive features and differential regulation of the DRTS genes of Arabidopsis thaliana.

In plants and protists, dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are part of a bifunctional enzyme (DRTS) that allows efficient recycling of the dihydrofolate resulting from TS activity. Arabidopsis thaliana possesses three DRTS genes, called AtDRTS1, AtDRTS2 and AtDRTS3, that are located downstream of three members of the sec14-like SFH gene family. In this study, a characterization of the AtDRTS genes identified alternatively spliced transcripts coding for AtDRTS isoforms which may account for monofunctional DHFR enzymes supporting pathways unrelated to DNA synthesis. Moreover, we discovered a complex differential regulation of the AtDRTS genes that confirms the expected involvement of the AtDRTS genes in cell proliferation and endoreduplication, but indicates also functions related to other cellular activities. AtDRTS1 is widely expressed in both meristematic and differentiated tissues, whereas AtDRTS2 expression is almost exclusively limited to the apical meristems and AtDRTS3 is preferentially expressed in the shoot apex, in stipules and in root cap cells. The differential regulation of the AtDRTS genes is associated to distinctive promoter architectures and the expression of AtDRTS1 in the apical meristems is strictly dependent on the presence of an intragenic region that includes the second intron of the gene. Upon activation of cell proliferation in germinating seeds, the activity of the AtDRTS1 and AtDRTS2 promoters in meristematic cells appears to be maximal at the G1/S phase of the cell cycle. In addition, the promoters of AtDRTS2 and AtDRTS3 are negatively regulated through E2F cis-acting elements and both genes, but not AtDRTS1, are downregulated in plants overexpressing the AtE2Fa factor. Our study provides new information concerning the function and the regulation of plant DRTS genes and opens the way to further investigations addressing the importance of folate synthesis with respect to specific cellular activities.

A misannotated locus positively influencing Arabidopsis seed germination is deconvoluted using multiple methods, including surrogate splicing

A screen of activation tagged lines of Arabidopsis thaliana retrieved COLD TEMPERATURE GERMINATING10-D(tag) (CTG10-D(tag)) seeds, capable of radicle protrusion in advance of wild type (WT) at suboptimal- and optimal-temperatures. Genomic walking revealed T-DNA in the intragenic region that upregulates expression of the At4g19330 locus previously predicted to encode an F-BOX protein. A combination of surrogate splicing, primer scanning, RACE, and Illumina PolyAdenylation Tag (PAT) sequencing in petunia (Petunia X hybrida) and Arabidopsis were required to demonstrate that the region around At4g19330 was misannotated and is actually compromised of two separate genes. Even though homologous regions nearby and elsewhere on chromosome 4 are confounding elements in the molecular characterization of the locus, we could determine that the 5′ entity encodes a ribonucleoprotein of unknown function whereas the 3′ gene includes the promoter and full coding region of an F-BOX protein. Although both genes were upregulated, only independently-transformed lines over-expressing the F-Box exhibited enhanced completion of seed germination. We named it CTG10, and a single, poorly penetrant, mutant line of ctg10 manifested the expected reduced completion of seed germination. Whereas CTG10-OE lines are hyposensitive to the gibberellin biosynthetic inhibitor paclobutrazol, the ctg10 mutant line is hypersensitive; a phenotype which could be alleviated when transgenically rescued with CTG10. The F-Box moiety promoted association of CTG10 with ASK proteins in yeast two hybrid assays, indicating that it likely assembles into an SCF-type ubiquitin ligase to promote the ubiquitination of one or more substrates. In this capacity CTG10 might target a protein repressing seed germination for polyubiquitination and subsequent proteasomal degradation.

Analysis of clinical phenotype and genotype of two hereditary coagulation factor XII deficiency

Objective Two Chinese pedigrees with congenital factor Ⅻ (FⅫ) deficiency were enrolled in the present study, and studies on the clinical manifestations, family survey, biochemical examinations and gene diagnosis of these pedigrees were performed. Methods In October 2014-2015 March, two cases of hereditary FⅫ deficiency patients were included in Xinhua hospital . Activated partial thromboplastin time(APTT), FⅫ procoagulant activity (FⅫ: C), FⅫ antigen(FⅫ: Ag)and other parameters of coagulant were detected. The FⅫ deficiency pedigree members, exons 1-14, boundary introns including the splice junctions of the F12 gene were amplified with polymerase chain reaction (PCR). Direct sequencing was exerted to purified PCR product to detect the gene mutation. If the gene mutations were found, polymorphism should be ruled out by directing sequence. One hundred and three healthy persons as normal controls. Results The two probands were manifested prolonged APTT (101 s and 143 s). They showed lower FⅫ activity and FⅫ antigen (2% and 6%, 0.4% and 4%, respectively). FⅡ: C, FⅦ: C, FⅧ: C, FⅨ: C, FⅩ: C and Fg are normal in the two probands. LAC is negative. Proband 1 has c. 1285C>T (p. Q429 stop) mutation. His parents and son have the heterozygous mutation in the same position. Proband 2 has c. 1556T>C(p.L519P)mutation. Her two sons have the heterozygous mutation in the same position. In the promoter regions of F12 gene, there were common 46C/T and 619 G/C polymorphisms in two pedigrees. Conclusion c. 1285C>T(p.Q429 stop)and c. 1556T>C(p.L519P)are the cause of FⅫ deficiency.(Chin J Lab Med, 2017, 40: 378-382) Key words: Factor Ⅻ deficiency; Factor Ⅻ; Mutation; Phenotype; Genotype; Pedigree

Crebbp loss cooperates with Bcl2 overexpression to promote lymphoma in mice

AbstractCREBBP is targeted by inactivating mutations in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). Here, we provide evidence from transgenic mouse models that Crebbp deletion results in deficits in B-cell development and can cooperate with Bcl2 overexpression to promote B-cell lymphoma. Through transcriptional and epigenetic profiling of these B cells, we found that Crebbp inactivation was associated with broad transcriptional alterations, but no changes in the patterns of histone acetylation at the proximal regulatory regions of these genes. However, B cells with Crebbp inactivation showed high expression of Myc and patterns of altered histone acetylation that were localized to intragenic regions, enriched for Myc DNA binding motifs, and showed Myc binding. Through the analysis of CREBBP mutations from a large cohort of primary human FL and DLBCL, we show a significant difference in the spectrum of CREBBP mutations in these 2 diseases, with higher frequencies of nonsense/frameshift mutations in DLBCL compared with FL. Together, our data therefore provide important links between Crebbp inactivation and Bcl2 dependence and show a role for Crebbp inactivation in the induction of Myc expression. We suggest this may parallel the role of CREBBP frameshift/nonsense mutations in DLBCL that result in loss of the protein, but may contrast the role of missense mutations in the lysine acetyltransferase domain that are more frequently observed in FL and yield an inactive protein.

Divergence of paralogous growth-hormone-encoding genes and their promoters in Salmonidae

In many fish species, including salmonids, the growth-hormone is encoded by two duplicated paralogous genes, gh1 and gh2. Both genes were already in place at the time of divergence of species in this group. A comparison of the entire sequence of these genes of salmonids has shown that their conserved regions are associated with exons, while their most variable regions correspond to introns. Introns C and D include putative regulatory elements (sites Pit-1, CRE, and ERE), that are also conserved. In chars, the degree of polymorphism of gh2 gene is 2-3 times as large as that in gh1 gene. However, a comparison across all Salmonidae species would not extent this observation to other species. In both these chars' genes, the promoters are conserved mainly because they correspond to putative regulatory sequences (TATA box, binding sites for the pituitary transcription factor Pit-1 (F1-F4), CRE, GRE and RAR/RXR elements). The promoter of gh2 gene has a greater degree of polymorphism compared with gh1 gene promoter in all investigated species of salmonids. The observed differences in the rates of accumulation of changes in growth hormone encoding paralogs could be explained by differences in the intensity of selection.

Loss and Gain of Group I Introns in the Mitochondrial Cox1 Gene of the Scleractinia (Cnidaria; Anthozoa).

Yaoyang Chuang, Marcelo Kitahara, Hironobu Fukami, Dianne Tracey, David J. Miller, and Chaolun Allen Chen (2017) Group I introns encoding a homing endonuclease gene (HEG) that is potentially capable of sponsoring mobility are present in the cytochrome oxidase subunit 1 (cox1) gene of some Hexacorallia, including a number of scleractinians assigned to the "robust" coral clade. In an e ort to infer the evolutionary history of this cox1 group I intron, DNA sequences were determined for 12 representative "basal" and "complex" corals and for 11 members of the Corallimorpharia, a sister order of the Scleractinia. Comparisons of insertion sites, secondary structures, and amino acid sequences of the HEG implied a common origin for cox1 introns of corallimorpharians, and basal and complex corals, but cox1 introns of robust corals were highly divergent, most likely reflecting independent acquisition. Phylogenetic analyses with a calibrated molecular clock suggested that cox1 introns of scleractinians and corallimorpharians have persisted at the same insertion site as that in the common ancestor 552 million years ago (mya). This ancestral intron was probably lost in complex corals around 213 to 190 mya at the junction between the Trassic and Jurassic. The coral cox1 gene remained intronless until new introns, probably from sponges or fungi, reinvaded different positions of the cox1 gene in robust corals around 135 mya in the Cretaceous, and then it subsequently began to lose them around 65.5 mya in some robust coral lineages coincident with the later Maastrichtian extinction at the Cretaceous-Tertiary boundary.

Intron DNA Sequences Can Be More Important Than the Proximal Promoter in Determining the Site of Transcript Initiation.

To more precisely define the positions from which certain intronic regulatory sequences increase mRNA accumulation, the effect of a UBIQUITIN intron on gene expression was tested from six different positions surrounding the transcription start site (TSS) of a reporter gene fusion in Arabidopsis thaliana The intron increased expression from all transcribed positions but had no effect when upstream of the 5'-most TSS. While this implies that the intron must be transcribed to increase expression, the TSS changed when the intron was located in the 5'-untranslated region (UTR), suggesting that the intron affects transcription initiation. Remarkably, deleting 303 nucleotides of the promoter including all known TSSs and all but 18 nucleotides of the 5'-UTR had virtually no effect on the level of gene expression as long as an intron containing stimulatory sequences was included. Instead, transcription was initiated in normally untranscribed sequences the same distance upstream of the intron as when the promoter was intact. These results suggest that certain intronic DNA sequences play unexpectedly large roles in directing transcription initiation and constitute a previously unrecognized type of downstream regulatory element for genes transcribed by RNA polymerase II.

NFAT5 and SLC4A10 Loci Associate with Plasma Osmolality.

Disorders of water balance, an excess or deficit of total body water relative to body electrolyte content, are common and ascertained by plasma hypo- or hypernatremia, respectively. We performed a two-stage genome-wide association study meta-analysis on plasma sodium concentration in 45,889 individuals of European descent (stage 1 discovery) and 17,637 additional individuals of European descent (stage 2 replication), and a transethnic meta-analysis of replicated single-nucleotide polymorphisms in 79,506 individuals (63,526 individuals of European descent, 8765 individuals of Asian Indian descent, and 7215 individuals of African descent). In stage 1, we identified eight loci associated with plasma sodium concentration at P<5.0 × 10-6 Of these, rs9980 at NFAT5 replicated in stage 2 meta-analysis (P=3.1 × 10-5), with combined stages 1 and 2 genome-wide significance of P=5.6 × 10-10 Transethnic meta-analysis further supported the association at rs9980 (P=5.9 × 10-12). Additionally, rs16846053 at SLC4A10 showed nominally, but not genome-wide, significant association in combined stages 1 and 2 meta-analysis (P=6.7 × 10-8). NFAT5 encodes a ubiquitously expressed transcription factor that coordinates the intracellular response to hypertonic stress but was not previously implicated in the regulation of systemic water balance. SLC4A10 encodes a sodium bicarbonate transporter with a brain-restricted expression pattern, and variant rs16846053 affects a putative intronic NFAT5 DNA binding motif. The lead variants for NFAT5 and SLC4A10 are cis expression quantitative trait loci in tissues of the central nervous system and relevant to transcriptional regulation. Thus, genetic variation in NFAT5 and SLC4A10 expression and function in the central nervous system may affect the regulation of systemic water balance.

BMPR1B mutation causes Pierre Robin sequence.

BackgroundWe investigated a large family with Pierre Robin sequence (PRS).Aim of the studyThis study aims to determine the genetic cause of PRS.ResultsThe reciprocal translocation t(4;6)(q22;p21) was identified to be segregated with PRS in a three-generation family. Whole-genome sequencing and Sanger sequencing successfully detected breakpoints in the intragenic regions of BMRP1B and GRM4. We hypothesized that PRS in this family was caused by (i) haploinsufficiency for BMPR1B or (ii) a gain of function mechanism mediated by the BMPR1B-GRM4 fusion gene. In an unrelated family, we identified another BMPR1B-splicing mutation that co-segregated with PRS.ConclusionWe detected two BMPR1B mutations in two unrelated PRS families, suggesting that BMPR1B disruption is probably a cause of human PRS.MethodsGTG banding, comparative genomic hybridization, whole-genome sequencing, and Sanger sequencing were performed to identify the gene causing PRS.

Identification of LRP5 mutations in families with familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is a hereditary eye disease characterized by defects in the development of periphery retinal vessels. However, the clinical phenotypes of FEVR vary widely from asymptomatic to complete blindness. We analyzed patients from three Chinese families and one sporadic patient with FEVR to investigate the clinical features and disease-causing mutations. Ocular phenotypes included increased ramification of the peripheral retinal vessels, a peripheral avascular zone, inferotemporal dragging of the optic disc and macula, and retinal folds. Peripheral blood DNA samples were obtained from patients with FEVR and their family members. Primers were designed to amplify the coding exons and adjacent intronic regions of the FEVR-causing genes FZD4, LRP5, NDP and TSPAN12. By polymerase chain reactions, each amplicon was subjected to direct Sanger sequencing analysis. Potential pathogenic changes of the sequence variants were analyzed by the orthologous protein sequence alignment and computational prediction software. We identified five LRP5 mutations: three novel heterozygous mutations-p.M181R, p.R399S and p.G503R and two known mutations that were never reported in FEVR patients: p.R494Q and p.G876S. All five mutations involved highly conserved residues and were predicted to be damaging by SIFT and PolyPhen-2. None was present in 500 normal individuals. To assess the pathogenesis of these mutations, wild-type and all five mutant LRP5 proteins were assayed for the ability to activate the Norrin/β-catenin pathway by established luciferase reporter assays, and all mutants failed to activate the pathway. This study extends the genetic database of the FEVR disease in China and provides a basis for molecular diagnosis of the disease.

Silencing of ATP4B of ATPase H+/K+ Transporting Beta Subunit by Intragenic Epigenetic Alteration in Human Gastric Cancer Cells.

The ATPase H+/K+ Transporting Beta Subunit (ATP4B) encodes the β subunit of the gastric H+, K+-ATPase, which controls gastric acid secretion and is therefore a target for acid reduction. Downregulation of ATP4B was recently observed in human gastric cancer (GC) without known mechanisms. In the present study, we demonstrated that ATP4B expression was decreased in human GC tissues and cell lines associated with DNA hypermethylation and histone hypoacetylation of histone H3 lysine 9 at its intragenic region close to the transcriptional start site. The expression of ATP4B was restored in GC cell lines by treatment with the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-AZA), or histone deacetylase inhibitor, trichostatin A (TSA), with further enhancement by combined treatment with both drugs. In contrast, 5-AZA had no effect on ATP4B expression in human hepatocellular carcinoma (HCC) and pancreatic cancer cell lines, in which ATP4B was silenced and accompanied by intragenic methylation. Chromatin immunoprecipitation (ChIP) showed that, in BGC823 GC cells, histone H3 lysine 9 acetylation (H3K9ac) was enhanced in the intragenic region of ATP4B upon TSA treatment, whereas 5-AZA showed a minimal effect. Additionally, ATP4B expression enhanced the inhibitory effects of chemotherapeutic mediation docetaxel on GC cell growth. Thus, as opposed to HCC and pancreatic cancer cells, the silencing of ATP4B in GC cells is attributable to the interplay between intragenic DNA methylation and histone acetylation of ATP4B, the restoration of which is associated with a favorable anticancer effect of docetaxel. These results have implications for targeting epigenetic alteration at the intragenic region of ATP4B in GC cells to benefit diagnosis and treatment of GC.

SMN deficiency in severe models of spinal muscular atrophy causes widespread intron retention and DNA damage

Spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disease, is the leading monogenic cause of infant mortality. Homozygous loss of the gene survival of motor neuron 1 (SMN1) causes the selective degeneration of lower motor neurons and subsequent atrophy of proximal skeletal muscles. The SMN1 protein product, survival of motor neuron (SMN), is ubiquitously expressed and is a key factor in the assembly of the core splicing machinery. The molecular mechanisms by which disruption of the broad functions of SMN leads to neurodegeneration remain unclear. We used an antisense oligonucleotide (ASO)-based inducible mouse model of SMA to investigate the SMN-specific transcriptome changes associated with neurodegeneration. We found evidence of widespread intron retention, particularly of minor U12 introns, in the spinal cord of mice 30 d after SMA induction, which was then rescued by a therapeutic ASO. Intron retention was concomitant with a strong induction of the p53 pathway and DNA damage response, manifesting as γ-H2A.X positivity in neurons of the spinal cord and brain. Widespread intron retention and markers of the DNA damage response were also observed with SMN depletion in human SH-SY5Y neuroblastoma cells and human induced pluripotent stem cell-derived motor neurons. We also found that retained introns, high in GC content, served as substrates for the formation of transcriptional R-loops. We propose that defects in intron removal in SMA promote DNA damage in part through the formation of RNA:DNA hybrid structures, leading to motor neuron death.