Jiayan Fan

Intrachromosomal Looping Is Required for Activation of Endogenous Pluripotency Genes during Reprogramming

Generation of induced pluripotent stem cells (iPSCs) by defined factors is an extremely inefficient process, because there is a strong epigenetic block preventing cells from achieving pluripotency. Here we report that virally expressed factors bound to the promoters of their target genes to the same extent in both iPSCs and unreprogrammed cells (URCs). However, expression of endogenous pluripotentcy genes was observed only in iPSCs. Comparison of local chromatin structure of the OCT4 locus revealed that there was a cohesin-complex-mediated intrachromosomal loop that juxtaposes a downstream enhancer to the genes promoter, enabling activation of endogenous stemness genes. None of these long-range interactions were observed in URCs. Knockdown of the cohesin-complex gene SMC1 by RNAi abolished the intrachromosomal interaction and affected pluripotency. These findings highlight the importance of the SMC1-orchestrated intrachromosomal loop as a critical epigenetic barrier to the induction of pluripotency.

Long non-coding RNA ROR decoys gene- specific histone methylation to promote tumorigenesis

Long non-coding RNAs (lncRNAs) are not translated into proteins and were initially considered to be part of the ‘dark matter’ of the genome. Recently, it has been shown that lncRNAs play a role in the recruitment of chromatin modifying complexes and can influence gene expression. However, it is unknown if lncRNAs function in a similar way in cancer. Here, we show that the lncRNA ROR occupies and activates the TESC promoter by repelling the histone G9A methyltransferase and promoting the release of histone H3K9 methylation. Suppression of ROR in tumors results in silencing of TESC expression, and G9A-mediated histone H3K9 methylation in the TESC promoter is restored, which significantly reduces tumor growth and metastasis. Without ROR silencing, TESC knockdown presents consistent and significant reductions in tumor progression. Our results reveal a novel mechanism by which ROR may serve as a decoy oncoRNA that blocks binding surfaces, preventing the recruitment of histone modifying enzymes, thereby specifying a new pattern of histone modifications that promote tumorigenesis.BackgroundLong non-coding RNAs (lncRNAs) are not translated into proteins and were initially considered to be part of the ‘dark matter’ of the genome. Recently, it has been shown that lncRNAs play a role in the recruitment of chromatin modifying complexes and can influence gene expression. However, it is unknown if lncRNAs function in a similar way in cancer.ResultsHere, we show that the lncRNA ROR occupies and activates the TESC promoter by repelling the histone G9A methyltransferase and promoting the release of histone H3K9 methylation. Suppression of ROR in tumors results in silencing of TESC expression, and G9A-mediated histone H3K9 methylation in the TESC promoter is restored, which significantly reduces tumor growth and metastasis. Without ROR silencing, TESC knockdown presents consistent and significant reductions in tumor progression.ConclusionsOur results reveal a novel mechanism by which ROR may serve as a decoy oncoRNA that blocks binding surfaces, preventing the recruitment of histone modifying enzymes, thereby specifying a new pattern of histone modifications that promote tumorigenesis.

Let-7b overexpression leads to increased radiosensitivity of uveal melanoma cells.

Uveal melanoma (UM) is an intraocular malignant tumor in adults that is characterized by rapid progression and recurrence. Irradiation has become the primary therapy for UM patients who are not candidates for surgery. However, after large-dose fraction irradiation treatment, some patients undergo subsequent enucleation because of radiotherapy-related complications. This situation has raised concerns on how to optimize the effectiveness of radiation treatment. Recent investigations of microRNAs are changing our understanding of UM tumor biology and are helping to identify novel targets for radiotherapy. The radioresistant UM cell lines OM431 and OCM1 were selected and exposed to irradiation, and let-7b was found to be downregulated after exposure. We then confirmed that let-7b mimics could inhibit UM growth both in vitro and in vivo. More specifically, transfection with let-7b mimics markedly resensitized OCM1 and OM431 cells to irradiation by reducing the population of S-phase cells. Cyclin D1 plays a vital role in cell cycle arrest, which is induced by let-7b overexpression. Cyclin D1 is also a target of let-7b and its expression is suppressed by upregulation of let-7b. Collectively, our results indicate that let-7b overexpression can in turn downregulate cyclin D1 expression and enhance the radiosensitivity of UM through cell cycle arrest. Let-7b could serve as a marker for radiosensitivity and could enhance the therapeutic benefit of UM cell irradiation.

The combination of polyalanine expansion mutation and a novel missense substitution in transcription factor FOXL2 leads to different ovarian phenotypes in blepharophimosis–ptosis–epicanthus inversus syndrome (BPES) patients

STUDY QUESTION What are the implications of multiple alterations of the forkhead box L2 (FOXL2) gene in blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) patients? SUMMARY ANSWER A multi-mutation of FOXL2, consisting of the expansion of the polyalanine tract from 14 to 24 residues (FOXL2-Ala24), an novel Y186C substitution from c.557A>G, and a synonymous variant (c.505G>A), had a cumulative effect on ovarian phenotypes in BPES patients. WHAT IS KNOWN ALREADY Mutations in FOXL2, a gene encoding a forkhead transcription factor cause BPES. Overall, the expansion of the polyalanine tract of FOXL2 from 14 to 24 residues (FOXL2-Ala24) accounts for 30% of intragenic mutations. STUDY DESIGN, SIZE, DURATION In this study, patients from seven BPES families and six sporadic cases were included. PARTICIPANTS/MATERIALS, SETTING, METHODS We conducted an extensive clinical, hormonal and functional study in 20 patients carrying the expansion of the polyalanine tract of FOXL2 associated with BPES. A multi-mutation of FOXL2 was detected in one BPES family that showed more severe BPES symptoms. Subcellular localization and transactivation studies were performed for the constructs of FOXL2-Ala24, Y186C and FOXL2-Ala24-Y186C. MAIN RESULTS We described the first multi-mutation of FOXL2 (c. [672_701dup30; 557A>G]) that leads to the polyalanine expansion of +10 residues (FOXL2-Ala24) combined with an Y186C substitution and a synonymous variant in a Chinese BPES family. This multi-mutation genotype was associated with more serious BPES clinical manifestations and the development of esotropia in the right eye. In in vitro studies, the multi-mutation affected the function of FOKL2 on the StAR promoter and DK3, and induced more aggressive aggregation and mislocalization of FOXL2 protein. The synonymous variant, while not affecting amino acid coding, causes a change in the RNA stem-loop structure. LIMITATIONS, REASONS FOR CAUTION The multi-mutation of FOXL2 was detected in one BPES family and it needs to be validated further by more BPES subjects. WIDER IMPLICATIONS OF THE FINDINGS The results of our study contribute new insights into the research field of BPES caused by the multi-mutation of FOXL2. STUDY FUNDING/COMPETING INTERESTS This study was supported by Shanghai Leading Academic Discipline Project (Grant number S30205) and Shanghai Jiao Tong University School of Medicine Doctor Innovation Fund (Grant number 201131). The authors have no competing interests to declare.

The Long Non-Coding RNA RHPN1-AS1 Promotes Uveal Melanoma Progression

Increasing evidence suggests that aberrant long non-coding RNAs (lncRNAs) are significantly correlated with the pathogenesis, development and metastasis of cancers. RHPN1 antisense RNA 1 (RHPN1-AS1) is a 2030-bp transcript originating from human chromosome 8q24. However, the role of RHPN1-AS1 in uveal melanoma (UM) remains to be clarified. In this study, we aimed to elucidate the molecular function of RHPN1-AS1 in UM. The RNA levels of RHPN1-AS1 in UM cell lines were examined using the quantitative real-time polymerase chain reaction (qRT-PCR). Short interfering RNAs (siRNAs) were designed to quench RHPN1-AS1 expression, and UM cells stably expressing short hairpin (sh) RHPN1-AS1 were established. Next, the cell proliferation and migration abilities were determined using a colony formation assay and a transwell migration/invasion assay. A tumor xenograft model in nude mice was established to confirm the function of RHPN1-AS1 in vivo. RHPN1-AS1 was significantly upregulated in a number of UM cell lines compared with the normal human retinal pigment epithelium (RPE) cell line. RHPN1-AS1 knockdown significantly inhibited UM cell proliferation and migration in vitro and in vivo. Our data suggest that RHPN1-AS1 could be an oncoRNA in UM, which may serve as a candidate prognostic biomarker and target for new therapies in malignant UM.

A Novel FOXL2 Mutation Implying Blepharophimosis-Ptosis-Epicanthus Inversus Syndrome Type I

Background/Aims: Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is a rare autosomal dominant disease caused by FOXL2 gene mutations, and it is clinically characterized by an eyelid malformation associated (type I) or not (type II) with premature ovarian failure (POF). Functional study of novel mutations is especially critical for female patients, as it may allow the prediction of infertility and early planning of an appropriate therapy. Methods: A clinical and molecular genetic investigation was performed in all members of a Chinese family with BPES. Genomic DNA was extracted, and the FOXL2 coding region was sequenced. Subcellular localization was performed by confocal microscopy. Transactivation studies were performed by real-time PCR, dual luciferase reporter assays and electrophoretic mobility shift assays. Results: A novel deletion mutation (C.634_641 del, CCCATGC) between the forkhead domain and the polyalanine domain was found, resulting in a frameshift mutation and a truncated protein. Functional studies showed a strong cytoplasmic mislocalization and abnormal transactivation activity, implying a type I kind mutation with a large chance of infertility. Conclusion: This study identifies that this mutation indicates the probability of developing into POF and shows the importance and necessity of early recognition of BPES type through mutation testing for female patients. Prompt personalized therapy and follow-up is of great clinical significance for female patients carrying this kind of mutation.

P2RX7-V3 is a novel oncogene that promotes tumorigenesis in uveal melanoma

Uveal melanoma (UM) has a high mortality rate for primary intraocular tumors. Approximately half of UM patients present with untreatable and fatal metastases. Long non-coding RNAs (lncRNAs) have emerged as potent regulatory RNAs that play key roles in various cellular processes and tumorigenesis. However, to date, their roles in UM are not well-known. Here, we identified a transcriptional variant transcribed from the P2RX7 gene locus, named P2RX7-V3 (P2RX7 variant 3), which was expressed at a high level in UM cells. P2RX7-V3 silencing revealed that this variant acts as a necessary UM oncoRNA. Knockdown of P2RX7-V3 expression significantly suppressed tumor growth in vitro and in vivo. A genome-wide cDNA array revealed that a variety of genes were dysregulated following P2RX7-V3 silencing. These observations identified P2RX7-V3 that plays a crucial role in UM tumorigenesis and may serve as a useful biomarker in the diagnosis and prognosis treatment of UM in the future.

Novel FOXC2 Mutation in Hereditary Distichiasis Impairs DNA-Binding Activity and Transcriptional Activation.

Distichiasis presents as double rows of eyelashes arising from aberrant differentiation of the meibomian glands of the eyelids, and it may be sporadic or hereditary. FOXC2 gene mutations in hereditary distichiasis are rarely reported. Here, we examined two generations of a Chinese family with hereditary distichiasis but without lymphedema or other features of LD syndrome. The FOXC2 gene was amplified and sequenced in all family members. Subcellular localization and luciferase assays were performed to assess the activity of the mutant FOXC2 protein. Clinical examinations showed distichiasis, lower eyelid ectropion, congenital ptosis and photophobia in all affected individuals. Sequence analysis revealed a novel frameshift mutation, c.964_965insG, in the coding region of the FOXC2 gene. This mutation caused protein truncation due to the presence of a premature stop codon. A fluorescence assay showed that this mutation did not change the nuclear localization of the protein. However, it impaired DNA-binding activity and decreased transcriptional activation. This is the first report of a FOXC2 mutation in hereditary distichiasis in the Chinese population. The findings of our study expand the FOXC2 mutation spectrum and contribute to the understanding of the genotype-phenotype correlation of this disease.

Novel mutations in the RB1 gene from Chinese families with a history of retinoblastoma

Retinoblastoma is an aggressive eye cancer that develops during infancy and is divided into two clinical types, sporadic and heritable. RB1 has been identified as the only pathological gene responsible for heritable retinoblastoma. Here, we identified 11 RB1 germline mutations in the Han pedigrees of 17 bilateral retinoblastoma patients from China. Four mutations were nonsense mutations, five were splice site mutations, and two resulted in a frame shift due to an insertion or a deletion. Three of the mutations had not been previously reported, and the p.Q344L mutation occurred in two generations of retinoblastoma patients. We investigated phenotypic–genotypic relationships for the novel mutations and showed that these mutations affected the expression, location, and function of the retinoblastoma protein. Abnormal protein localization was observed after transfection of the mutant genes. In addition, changes in the cell cycle distribution and apoptosis rates were observed when the Saos-2 cell line was transfected with plasmids encoding the mutant RB1 genes. Our findings expand the spectrum of known RB1 mutations and will benefit the investigation of RB1 mutation hotspots. Genetic counseling can be offered to families with heritable RB1 mutations.

An inherited FGFR2 mutation increased osteogenesis gene expression and result in Crouzon syndrome

BackgroundFGFR2 encodes a fibroblast growth factor receptor whose mutations are responsible for the Crouzon syndrome, involving craniosynostosis and facial dysostosis with shallow orbits. However, few reports are available quantifying the orbital volume of Crouzon syndrome and there was little direct evidence to show FGFR2 mutation actually influencing orbital morphology.MethodsTen Crouzon syndrome patients underwent a standard ophthalmologic assessment. Morphology study was carried out based on 3-dimensional computed tomography scan to calculate orbital volume. Genomic DNA was extracted from peripheral blood leukocytes of the patients and genomic screening of FGFR2. A three-dimensional computer model was used to analyse the structural positioning of the mutation site that was predicted possible impact on functional of FGFR2 protein. Real-time PCR was performed to analyse the expression of bone maker gene.ResultsWe describe a FGFR2 mutation (p.G338R, c.1012G > C) in a Chinese family with Crouzon syndrome. Computational analysis showed the mutate protein obviously changes in the local spatial structure compared with wild-type FGFR2. The expression of osteocalcin and alkaline phosphatase two osteoblast specific genes significantly increased in orbital bone directly from patient compared to normal individual, which may lead to facial dysostosis. This is compatible with the shallow and round orbits in our Crouzon syndrome patient.ConclusionsOur study further identified G338R FGFR2 mutation (c1012G > C) lead to inherited Crouzon syndrome. Thus, early intervention, both medically and surgically, as well as disciplined by a multiple interdisciplinary teams are crucial to the management of this disorder.