Publications by authors named "Junjiu Huang"

75 Publications

The Role of DNA Methylation Reprogramming during Sex Determination and Transition in Zebrafish.

Genomics Proteomics Bioinformatics 2021 Feb 18. Epub 2021 Feb 18.

CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; CAS Center for Excellence in Animal Evolution and Genetics, Chinese Academy of Sciences, Kunming 650223, China. Electronic address:

DNA methylation is a prevalent epigenetic modification in vertebrates, and it has been shown to be involved the regulation of gene expression and embryo development. However, it remains unclear how DNA methylation regulates sexual development, especially in species without sex chromosomes. To determine this, we utilized zebrafish to investigate DNA methylation reprogramming during juvenile germ cell development and adult female-to-male sex transition. We revealed that primordial germ cells (PGCs) undergo significant DNA methylation reprogramming during germline development and set to an oocyte/ovary-like pattern at 9 days post fertilization (9 dpf). When DNA methyltransferase (DNMT) activity in juveniles was blocked after 9 dpf, the zebrafish developed into females. We also show that Tet3 is involved in PGC development. Notably, we find that DNA methylome reprogramming during adult zebrafish sex transition is similar to the reprogramming during the sex differentiation from 9 dpf PGCs to sperm. Furthermore, inhibiting DNMTs activity can prevent the female-to-male sex transition, suggesting that methylation reprogramming is required for zebrafish sex transition. In summary, DNA methylation plays important roles in zebrafish germline development and sexual plasticity.
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http://dx.doi.org/10.1016/j.gpb.2020.10.004DOI Listing
February 2021

Effects of Cigarette Smoking on Preimplantation Embryo Development.

Adv Exp Med Biol 2021 ;1300:137-150

Key Laboratory of Reproductive Medicine of Guangdong Province, School of Life Sciences and the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.

In this chapter, we first gave a brief introduction to the detriments of cigarette smoking, with an emphasis on its adverse effects on female reproductive health. Then, we outlined recent advances about the impacts of cigarette smoke on preimplantation embryo development. Additionally, toxicities of cadmium and benzo(a)pyrene (BaP) at this specific developmental window were also discussed, to illustrate the potential mechanisms involved in cigarette smoke-associated embryotoxicity. Finally, we provide an overview of the issues to be solved in the future research. Further studies about the molecular mechanism of cigarette smoking-associated female infertility may provide vital insights into developing new interventions for the women smokers and thus improving their reproductive outcomes.
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http://dx.doi.org/10.1007/978-981-33-4187-6_6DOI Listing
February 2021

Methylation silencing and reactivation of exogenous genes in lentivirus-mediated transgenic mice.

Transgenic Res 2021 Feb 4;30(1):63-76. Epub 2021 Jan 4.

MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, China.

Taking advantage of their ability to integrate their genomes into the host genome, lentiviruses have been used to rapidly produce transgenic mice in biomedical research. In most cases, transgenes delivered by lentiviral vectors have resisted silencing mediated by epigenetic modifications in mice. However, some studies revealed that methylation caused decreased transgene expression in mice. Therefore, there is conflicting evidence regarding the methylation-induced silencing of transgenes delivered by lentiviral transduction in mice. In this study, we present evidence that the human TTR transgene was silenced by DNA methylation in the liver of a transgenic mouse model generated by lentiviral transduction. The density of methylation on the transgene was increased during reproduction, and the expression of the transgene was completely silenced in mice of the F2 generation. Interestingly, 5-azacytidine (5-AzaC), a methyltransferase inhibitor, potently reactivated the silenced genes in neonatal mice whose hepatocytes were actively proliferating and led to stable transgene expression during development. However, 5-AzaC did not rescue liver transgene expression when administered to adult mice. Moreover, 5-AzaC at the given dose had low developmental toxicity in the newborn mice. In summary, we demonstrate the methylation-induced silencing of an exogenous gene in the liver of a mouse model generated by lentiviral transduction and show that the silenced transgene can be safely and efficiently reactivated by 5-AzaC treatment, providing an alternative way to obtain progeny with stable transgene expression in the case of the methylation of exogenous genes in transgenic mice generated by lentiviral transduction.
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http://dx.doi.org/10.1007/s11248-020-00224-9DOI Listing
February 2021

USP38 Couples Histone Ubiquitination and Methylation via KDM5B to Resolve Inflammation.

Adv Sci (Weinh) 2020 Nov 11;7(22):2002680. Epub 2020 Oct 11.

MOE Key Laboratory of Gene Function and Regulation State Key Laboratory of Biocontrol School of Life Sciences Sun Yat-sen University Guangzhou Guangdong 510006 China.

Chromatin modifications, such as histone acetylation, ubiquitination, and methylation, play fundamental roles in maintaining chromatin architecture and regulating gene transcription. Although their crosstalk in chromatin remodeling has been gradually uncovered, the functional relationship between histone ubiquitination and methylation in regulating immunity and inflammation remains unclear. Here, it is reported that USP38 is a novel histone deubiquitinase that works together with the histone H3K4 modifier KDM5B to orchestrate inflammatory responses. USP38 specifically removes the monoubiquitin on H2B at lysine 120, which functions as a prerequisite for the subsequent recruitment of demethylase KDM5B to the promoters of proinflammatory cytokines and during LPS stimulation. KDM5B in turn inhibits the binding of NF-B transcription factors to the and promoters by reducing H3K4 trimethylation. Furthermore, USP38 can bind to KDM5B and prevent it from proteasomal degradation, which further enhances the function of KDM5B in the regulation of inflammation-related genes. Loss of in mice markedly enhances susceptibility to endotoxin shock and acute colitis, and these mice display a more severe inflammatory phenotype compared to wild-type mice. The studies identify USP38-KDM5B as a distinct chromatin modification complex that restrains inflammatory responses through manipulating the crosstalk of histone ubiquitination and methylation.
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http://dx.doi.org/10.1002/advs.202002680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7675183PMC
November 2020

USP19 suppresses inflammation and promotes M2-like macrophage polarization by manipulating NLRP3 function via autophagy.

Cell Mol Immunol 2020 Oct 23. Epub 2020 Oct 23.

MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, 510275, Guangzhou, Guangdong, People's Republic of China.

Macrophage polarization to proinflammatory M1-like or anti-inflammatory M2-like cells is critical to mount a host defense or repair tissue. The exact molecular mechanisms controlling this process are still elusive. Here, we report that ubiquitin-specific protease 19 (USP19) acts as an anti-inflammatory switch that inhibits inflammatory responses and promotes M2-like macrophage polarization. USP19 inhibited NLRP3 inflammasome activation by increasing autophagy flux and decreasing the generation of mitochondrial reactive oxygen species. In addition, USP19 inhibited the proteasomal degradation of inflammasome-independent NLRP3 by cleaving its polyubiquitin chains. USP19-stabilized NLRP3 promoted M2-like macrophage polarization by direct association with interferon regulatory factor 4, thereby preventing its p62-mediated selective autophagic degradation. Consistent with these observations, compared to wild-type mice, Usp19 mice had decreased M2-like macrophage polarization and increased interleukin-1β secretion, in response to alum and chitin injections. Thus, we have uncovered an unexpected mechanism by which USP19 switches the proinflammatory function of NLRP3 into an anti-inflammatory function, and suggest that USP19 is a potential therapeutic target for inflammatory interventions.
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http://dx.doi.org/10.1038/s41423-020-00567-7DOI Listing
October 2020

Cost-effective generation of A-to-G mutant mice by zygote electroporation of adenine base editor ribonucleoproteins.

J Genet Genomics 2020 06 20;47(6):337-340. Epub 2020 May 20.

MOE Key Laboratory of Gene Function and Regulation and Guangzhou Key Laboratory of Healthy Aging Research, SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China; Key Laboratory of Reproductive Medicine of Guangdong Province, School of Life Sciences, and the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510275, China; State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, China. Electronic address:

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http://dx.doi.org/10.1016/j.jgg.2020.05.005DOI Listing
June 2020

Ddx56 maintains proliferation of mouse embryonic stem cells via ribosome assembly and interaction with the Oct4/Sox2 complex.

Stem Cell Res Ther 2020 07 23;11(1):314. Epub 2020 Jul 23.

MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.

Background: Embryonic stem cells (ESCs) are important source of clinical stem cells for therapy, so dissecting the functional gene regulatory network involved in their self-renewal and proliferation is an urgent task. We previously reported that Ddx56 interacts with the core transcriptional factor Oct4 by mass spectrometry analysis in ESCs. However, the exact function of Ddx56 in ESCs remains unclear.

Methods: We investigated the role of Ddx56 in mouse ESCs (mESCs) through both gain- and loss-of-function strategies. The effect of Ddx56 on mESCs was determined based on morphological changes, involvement in the network of pluripotency markers (Nanog, Oct4, Sox2), and altered lineage marker expression. In addition, the role of Ddx56 in mESCs was evaluated by polysome fractionation, qRT-PCR, and co-immunoprecipitation (co-IP). Finally, RNA sequencing was applied to explore potential network regulation by Ddx56 in mESCs.

Result: We found that Ddx56 participated in ribosome assembly, as knockout or RNAi knockdown of Ddx56 led to ribosome dysfunction and cell lethality. Surprisingly, exogenous expression of C-terminal domain truncated Ddx56 (Ddx56 ΔC-ter) did not affect ribosome assembly, but decreased mESC proliferation by downregulation of proliferation-related genes and cell cycle changing. In terms of mechanism, Ddx56 interacted with the Oct4 and Sox2 complex by binding to Sox2, whereas Ddx56 ΔC-ter showed weaker interaction with Sox2 and led to retardation of mESC proliferation.

Conclusions: Ddx56 maintains ESC proliferation by conventional regulation of ribosome assembly and interaction with the Oct4 and Sox2 complex.
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http://dx.doi.org/10.1186/s13287-020-01800-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376950PMC
July 2020

MiR-185 targets POT1 to induce telomere dysfunction and cellular senescence.

Aging (Albany NY) 2020 07 18;12(14):14791-14807. Epub 2020 Jul 18.

MOE Key Laboratory of Gene Function and Regulation, Institute of Healthy Aging Research, School of Life Sciences, Sun Yat-sen University, Guangzhou, China.

Protection of telomere 1 (POT1), the telomeric single-stranded DNA (ssDNA)-binding protein in the shelterin complex, has been implicated in the DNA damage response, tumorigenesis and aging. Telomere dysfunction induced by telomere deprotection could accelerate cellular senescence in primary human cells. While previous work demonstrated the biological mechanism of POT1 in aging and cancer, how POT1 is posttranscriptionally regulated remains largely unknown. To better understand the POT1 regulatory axis, we performed bioinformatic prediction, and selected candidates were further confirmed by dual-luciferase reporter assay. Collectively, our results revealed that miR-185 can significantly reduce POT1 mRNA and protein levels by directly targeting the POT1 3'-untranslated region (3'-UTR). Overexpression of miR-185 increased telomere dysfunction-induced foci (TIF) signals in both cancer cells and primary human fibroblasts. Elevated miR-185 led to telomere elongation in the telomerase-positive cell line HTC75, which was phenotypically consistent with POT1 knocking down. Moreover, miR-185 accelerated the replicative senescence process in primary human fibroblasts in a POT1-dependent manner. Interestingly, increased serum miR-185 could represent a potential aging-related biomarker. Taken together, our findings reveal miR-185 as a novel aging-related miRNA that targets POT1 and provide insight into the telomere and senescence regulatory network at both the intracellular and extracellular levels.
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http://dx.doi.org/10.18632/aging.103541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7425516PMC
July 2020

Author Correction: Repurposing type I-F CRISPR-Cas system as a transcriptional activation tool in human cells.

Nat Commun 2020 07 9;11(1):3522. Epub 2020 Jul 9.

Sun Yat-Sen Memorial Hospital, Sun Yat-sen University; MOE Key Laboratory of Gene Function and Regulation and Guangzhou Key Laboratory of Healthy Aging Research, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-17379-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347611PMC
July 2020

Retrotransposons in pluripotent stem cells.

Cell Regen 2020 Jun 2;9(1). Epub 2020 Jun 2.

School of Life Sciences, SunYat-sen University, Guangzhou, 510275, P. R. China.

Transposable elements constitute about half of the mammalian genome, and can be divided into two classes: the class I (retrotransposons) and the class II (DNA transposons). A few hundred types of retrotransposons, which are dynamic and stage specific, have been annotated. The copy numbers and genomic locations are significantly varied in species. Retrotransposons are active in germ cells, early embryos and pluripotent stem cells (PSCs) correlated with low levels of DNA methylation in epigenetic regulation. Some key pluripotency transcriptional factors (such as OCT4, SOX2, and NANOG) bind retrotransposons and regulate their activities in PSCs, suggesting a vital role of retrotransposons in pluripotency maintenance and self-renewal. In response to retrotransposons transposition, cells employ a number of silencing mechanisms, such as DNA methylation and histone modification. This review summarizes expression patterns, functions, and regulation of retrotransposons in PSCs and early embryonic development.
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http://dx.doi.org/10.1186/s13619-020-00046-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7306833PMC
June 2020

Single AAV-Mediated CRISPR-SaCas9 Inhibits HSV-1 Replication by Editing ICP4 in Trigeminal Ganglion Neurons.

Mol Ther Methods Clin Dev 2020 Sep 22;18:33-43. Epub 2020 May 22.

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China.

Herpes simplex keratitis (HSK) is the most common cause of corneal blindness in developed nations, caused by primary or recurrent herpes simplex virus 1 (HSV-1) infection of the cornea. Latent infection of HSV-1, especially in the trigeminal ganglion (TG), causes recurrence of HSV-1 infection. As antiviral treatment is not effective on latent HSV-1, to test the possibility of inhibiting HSV-1 by SpCas9 ( Cas9) or SaCas9 ( Cas9), and , two important genes required for HSV-1 replication and reactivation, were chosen as targets. In Vero cells, SpCas9 and SaCas9 targeting or can effectively inhibit the proliferation of HSV-1 without affecting cell viability. No significant guide RNA (gRNA)-dependent off-targets were observed in the human genome by digenome sequencing and deep sequencing verification. Adeno-associated virus 1 (AAV1)-mediated delivery of SaCas9 inhibits HSV-1 replication by targeting in mouse primary TG neuronal cells. SpCas9 and SaCas9 are able to inhibit HSV-1 infection in Vero cells and mouse TG neuronal cultures with high efficiency and good biosafety. AAV1-mediated delivery of SaCas9 shows great potential in treating HSK and inhibiting HSV-1 in TG neurons. Further investigations may be needed to test the inhibition of latent infections, which may result in the development of novel methods for treating viral diseases.
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http://dx.doi.org/10.1016/j.omtm.2020.05.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7298336PMC
September 2020

Repurposing type I-F CRISPR-Cas system as a transcriptional activation tool in human cells.

Nat Commun 2020 06 19;11(1):3136. Epub 2020 Jun 19.

Sun Yat-Sen Memorial Hospital, Sun Yat-sen University; MOE Key Laboratory of Gene Function and Regulation and Guangzhou Key Laboratory of Healthy Aging Research, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.

Class 2 CRISPR-Cas proteins have been widely developed as genome editing and transcriptional regulating tools. Class 1 type I CRISPR-Cas constitutes ~60% of all the CRISPR-Cas systems. However, only type I-B and I-E systems have been used to control mammalian gene expression and for genome editing. Here we demonstrate the feasibility of using type I-F system to regulate human gene expression. By fusing transcription activation domain to Pseudomonas aeruginosa type I-F Cas proteins, we activate gene transcription in human cells. In most cases, type I-F system is more efficient than other CRISPR-based systems. Transcription activation is enhanced by elongating the crRNA. In addition, we achieve multiplexed gene activation with a crRNA array. Furthermore, type I-F system activates target genes specifically without off-target transcription activation. These data demonstrate the robustness and programmability of type I-F CRISPR-Cas in human cells.
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http://dx.doi.org/10.1038/s41467-020-16880-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305327PMC
June 2020

A critical role of telomere chromatin compaction in ALT tumor cell growth.

Nucleic Acids Res 2020 06;48(11):6019-6031

MOE Key Laboratory of Gene Function and Regulation, Guangzhou Key Laboratory of Healthy Aging Research and SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.

ALT tumor cells often contain abundant DNA damage foci at telomeres and rely on the alternative lengthening of telomeres (ALT) mechanism to maintain their telomeres. How the telomere chromatin is regulated and maintained in these cells remains largely unknown. In this study, we present evidence that heterochromatin protein 1 binding protein 3 (HP1BP3) can localize to telomeres and is particularly enriched on telomeres in ALT cells. HP1BP3 inhibition led to preferential growth inhibition of ALT cells, which was accompanied by telomere chromatin decompaction, increased presence of C-circles, more pronounced ALT-associated phenotypes and elongated telomeres. Furthermore, HP1BP3 appeared to participate in regulating telomere histone H3K9me3 epigenetic marks. Taken together, our data suggest that HP1BP3 functions on telomeres to maintain telomere chromatin and represents a novel target for inhibiting ALT cancer cells.
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http://dx.doi.org/10.1093/nar/gkaa224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293046PMC
June 2020

PF-127 hydrogel plus sodium ascorbyl phosphate improves Wharton's jelly mesenchymal stem cell-mediated skin wound healing in mice.

Stem Cell Res Ther 2020 04 3;11(1):143. Epub 2020 Apr 3.

Key Laboratory of Reproductive Medicine of Guangdong Province, The First Affiliated Hospital and School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.

Background: Factors such as poor engraftment, retention, and survival of the transplanted stem cells are deemed to limit their therapeutic efficacy for wound regeneration. Hence, it is necessary to explore these issues in order to resolve them. In this study, we aim to investigate the role of Pluronic F-127 (PF-127) hydrogel plus antioxidant sodium ascorbyl phosphate (SAP) in enhancing Wharton's jelly mesenchymal stem cell (WJMSC)-mediated effectiveness on full-thickness skin wound healing in mice.

Methods: First, the cytotoxicity of PF-127 and the biological effect of SAP on the survival of WJMSCs were tested in vitro using cell viability and proliferation assays. Next, a cell suspension containing WJMSCs, PF-127, and SAP was topically administered onto an 8-mm diameter excisional full-thickness wound bed. Eight days after transplantation, the mice were sacrificed and the skin tissue was excised for histological and immunohistochemical analysis. Finally, in vivo distribution of transplanted WJMSCs was traced to investigate cell engraftment and the potential therapeutic mechanism.

Results: PF-127 was found to be cytotoxic to WJMSCs while SAP significantly improved the survival of PF-127-embedded WJMSCs. When this combination was topically transplanted onto the wound bed, wound healing was facilitated and dermis regeneration was achieved on the 8th day after surgery, as evidenced by an increase in dermal thickness, newly developed hair follicles, and collagen fiber deposition accompanied by a reduction in scar width. Further, immunohistochemical analysis demonstrated a higher number of anti-inflammatory M2 macrophages, proliferating cells, and newly formed blood vessels in the WJMSCs/PF-127/SAP group relative to all other groups. In addition, in vivo tracking results revealed a highly enhanced engraftment of WJMSCs accumulated in the dermis in the WJMSCs/PF-127/SAP group.

Conclusions: SAP significantly improves the survival of WJMSCs in PF-127 encapsulation. Further, PF-127 plus SAP is an effective combination that enhances WJMSC engraftment in the dermis, which then promotes full-thickness wound healing through potential M2 macrophage formation and angiogenesis.
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http://dx.doi.org/10.1186/s13287-020-01638-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7119174PMC
April 2020

Effective generation of maternal genome point mutated porcine embryos by injection of cytosine base editor into germinal vesicle oocytes.

Sci China Life Sci 2020 Jul 21;63(7):996-1005. Epub 2020 Jan 21.

Key Laboratory of Reproductive Medicine of Guangdong Province, the First Affiliated Hospital and School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.

Cytosine and adenine base editors are promising new tools for introducing precise genetic modifications that are required to generate disease models and to improve traits in pigs. Base editors can catalyze the conversion of C→T (C>T) or A→G (A>G) in the target site through a single guide RNA. Injection of base editors into the zygote cytoplasm can result in the production of offspring with precise point mutations, but most F are mosaic, and breeding of F heterozygous pigs is time-intensive. Here, we developed a method called germinal vesicle oocyte base editing (GVBE) to produce point mutant F porcine embryos by editing the maternal alleles during the GV to MII transition. Injection of cytosine base editor 3 (BE3) mRNA and X-linked Dmd-specific guide RNAs into GVoocytes efficiently edited maternal Dmd during in vitro maturation and did not affect the maturation potential of the oocytes. The edited MII oocytes developed into blastocysts after parthenogenetic activation (PA) or in vitro fertilization (IVF). However, BE3 may reduce the developmental potential of IVF blastocysts from 31.5%±0.8% to 20.4% ±2.1%. There 40%-78.3% diploid PA blastocysts had no more than two different alleles, including up to 10% embryos that had only C>T mutation alleles. Genotyping of IVF blastocysts indicated that over 70% of the edited embryos had one allele or two different alleles of Dmd. Since the male embryos had only a copy of Dmd allele, all five (5/19) F male embryos are homozygous and three of them were Dmd precise C>T mutation. Nine (9/19) female IVF embryos had two different alleles including a WT and a C>T mutation. DNA sequencing showed that some of them might be heterozygous embryos. In conclusion, the GVBE method is a valuable method for generating F embryos with maternal point mutated alleles in a single step.
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http://dx.doi.org/10.1007/s11427-019-1611-1DOI Listing
July 2020

TRIM14 Promotes Noncanonical NF-κB Activation by Modulating p100/p52 Stability via Selective Autophagy.

Adv Sci (Weinh) 2020 Jan 11;7(1):1901261. Epub 2019 Nov 11.

State Key Laboratory of Oncology in South China MOE Key Laboratory of Gene Function and Regulation School of Life Sciences Sun Yat-Sen University Guangzhou Guangdong 510006 China.

The noncanonical NF-κB signaling pathway plays a critical role in a variety of biological functions including chronic inflammation and tumorigenesis. Activation of noncanonical NF-κB signaling largely relies on the abundance as well as the processing of the NF-κB family member p100/p52. Here, TRIM14 is identified as a novel positive regulator of the noncanonical NF-κB signaling pathway. TRIM14 promotes noncanonical NF-κB activation by targeting p100/p52 in vitro and in vivo. Furthermore, a mechanistic study shows that TRIM14 recruits deubiquitinase USP14 to cleave the K63-linked ubiquitin chains of p100/p52 at multiple sites, thereby preventing p100/p52 from cargo receptor p62-mediated autophagic degradation. TRIM14 deficiency in mice significantly impairs noncanonical NF-κB-mediated inflammatory responses as well as acute colitis and colitis-associated colon cancer development. Taken together, these findings establish the TRIM14-USP14 axis as a crucial checkpoint that controls noncanonical NF-κB signaling and highlight the crosstalk between autophagy and innate immunity.
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http://dx.doi.org/10.1002/advs.201901261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6947505PMC
January 2020

Off-target effects of cytidine base editor and adenine base editor: What can we do?

J Genet Genomics 2019 11 2;46(11):509-512. Epub 2019 Nov 2.

Key Laboratory of Reproductive Medicine of Guangdong Province, School of Life Sciences, and the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510275, China; MOE Key Laboratory of Gene Function and Regulation and Guangzhou Key Laboratory of Healthy Aging Research, SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China; Key Laboratory of Reproductive Medicine of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510150, China. Electronic address:

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http://dx.doi.org/10.1016/j.jgg.2019.09.004DOI Listing
November 2019

Production of non-mosaic genome edited porcine embryos by injection of CRISPR/Cas9 into germinal vesicle oocytes.

J Genet Genomics 2019 07 20;46(7):335-342. Epub 2019 Jul 20.

Key Laboratory of Reproductive Medicine of Guangdong Province, First Affiliated Hospital and School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China; MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China; Key Laboratory of Reproductive Medicine of Guangdong Province, Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510150, China. Electronic address:

Genetically modified pigs represent a great promise for generating models of human diseases and producing new breeds. Generation of genetically edited pigs using somatic cell nuclear transfer (SCNT) or zygote cytoplasmic microinjection is a tedious process due to the low developmental rate or mosaicism of the founder (F0). Herein, we developed a method termed germinal vesicle oocyte gene editing (GVGE) to produce non-mosaic porcine embryos by editing maternal alleles during the GV to MⅡ transition. Injection of Cas9 mRNA and X-linked Dmd gene-specific gRNA into GV oocytes did not affect their developmental potential. The MⅡ oocytes edited during in vitro maturation (IVM) could develop into blastocysts after parthenogenetic activation (PA) or in vitro fertilization (IVF). Genotyping results indicated that the maternal gene X-linked Dmd could be efficiently edited during oocyte maturation. Up to 81.3% of the edited IVF embryos were non-mosaic Dmd gene mutant embryos. In conclusion, GVGE might be a valuable method for the generation of non-mosaic maternal allele edited F0 embryos in a short simple step.
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http://dx.doi.org/10.1016/j.jgg.2019.07.002DOI Listing
July 2019

Correction to: Inhibition of p53 and/or AKT as a new therapeutic approach specifically targeting ALT cancers.

Protein Cell 2019 Dec;10(12):927-928

MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510006, China.

In the original publication the labels in figure 4C and D are incorrectly published. The correct labels for figure 4C and D is provided in this correction.
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http://dx.doi.org/10.1007/s13238-019-0640-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6881410PMC
December 2019

The long non-coding RNA Snhg3 is essential for mouse embryonic stem cell self-renewal and pluripotency.

Stem Cell Res Ther 2019 05 31;10(1):157. Epub 2019 May 31.

The State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, China.

Background: Small nucleolar RNA host gene 3 (Snhg3) is a long non-coding RNA (lncRNA) that was shown to participate in the tumorigenesis of certain cancers. However, little is known about its role in embryonic stem cells (ESCs).

Methods: Here, we investigated the role of Snhg3 in mouse ESCs (mESCs) through both loss-of-function (knockdown) and gain-of-function (overexpression) approaches. Alkaline phosphatase staining, secondary colony formation, propidium iodide staining, western blotting, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to access self-renewal capacity, whereas immunofluorescence, qRT-PCR, and embryoid body formation were performed to examine pluripotency. In addition, the effect of Snhg3 on mouse embryonic development was determined based on the morphological changes, blastocyst rate, and altered pluripotency marker (Nanog, Oct4) expression. Moreover, the relationship between Snhg3 and key pluripotency factors was evaluated by chromatin immunoprecipitation qPCR, qRT-PCR, subcellular fractionation, and RNA immunoprecipitation. Finally, RNA pull-down and mass spectrometry were applied to explore the potential interacting proteins of Snhg3 in mESCs.

Results: We demonstrated that Snhg3 is essential for self-renewal and pluripotency maintenance in mESCs. In addition, Snhg3 knockdown disrupted mouse early embryo development. Mechanistically, Snhg3 formed a positive feedback network with Nanog and Oct4, and 126 Snhg3-interacting proteins were identified in mESCs.

Conclusions: Snhg3 is essential for mESC self-renewal and pluripotency, as well as mouse early embryo development.
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http://dx.doi.org/10.1186/s13287-019-1270-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545032PMC
May 2019

Inhibition of p53 and/or AKT as a new therapeutic approach specifically targeting ALT cancers.

Protein Cell 2019 11 21;10(11):808-824. Epub 2019 May 21.

MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510006, China.

While the majority of all human cancers counteract telomere shortening by expressing telomerase, ~15% of all cancers maintain telomere length by a telomerase-independent mechanism known as alternative lengthening of telomeres (ALT). Here, we show that high load of intrinsic DNA damage is present in ALT cancer cells, leading to apoptosis stress by activating p53-independent, but JNK/c-Myc-dependent apoptotic pathway. Notably, ALT cells expressing wild-type p53 show much lower apoptosis than p53-deficient ALT cells. Mechanistically, we find that intrinsic DNA damage in ALT cells induces low level of p53 that is insufficient to initiate the transcription of apoptosis-related genes, but is sufficient to stimulate the expression of key components of mTORC2 (mTOR and Rictor), which in turn leads to phosphorylation of AKT. Activated AKT (p-AKT) thereby stimulates downstream anti-apoptotic events. Therefore, p53 and AKT are the key factors that suppress spontaneous apoptosis in ALT cells. Indeed, inhibition of p53 or AKT selectively induces rapid death of ALT cells in vitro, and p53 inhibitor severely suppresses the growth of ALT-cell xenograft tumors in mice. These findings reveal a previously unrecognized function of p53 in anti-apoptosis and identify that the inhibition of p53 or AKT has a potential as therapeutics for specifically targeting ALT cancers.
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http://dx.doi.org/10.1007/s13238-019-0634-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834538PMC
November 2019

HBB-deficient Macaca fascicularis monkey presents with human β-thalassemia.

Protein Cell 2019 07;10(7):538-542

Key Laboratory of Reproductive Medicine of Guangdong Province, The First Affiliated Hospital and School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.

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http://dx.doi.org/10.1007/s13238-019-0627-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588645PMC
July 2019

Off-target challenge for base editor-mediated genome editing.

Cell Biol Toxicol 2019 06 30;35(3):185-187. Epub 2019 Apr 30.

Key Laboratory of Reproductive Medicine of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University and School of Life Sciences of Sun Yat-sen University, Guangzhou, 510000, China.

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http://dx.doi.org/10.1007/s10565-019-09474-8DOI Listing
June 2019

Interaction Between Sympk and Oct4 Promotes Mouse Embryonic Stem Cell Proliferation.

Stem Cells 2019 06 9;37(6):743-753. Epub 2019 Mar 9.

MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, SunYat-sen University, Guangzhou, People's Republic of China.

The scaffold protein Symplekin (Sympk) is involved in cytoplasmic RNA polyadenylation, transcriptional modulation, and the regulation of epithelial differentiation and proliferation via tight junctions. It is highly expressed in embryonic stem cells (ESCs), in which its role remains unknown. In this study, we found Sympk overexpression in mouse ESCs significantly increased colony formation, and Sympk deletion via CRISPR/Cas9 decreased colony formation. Sympk promoted ESC growth and its overexpression sustained ESC pluripotency, as assessed by teratoma and chimeric mouse formation. Genomic stability was preserved in these cells after long-term passage. The domain of unknown function 3453 (DUF3453) in Sympk was required for its interaction with the key pluripotent factor Oct4, and its depletion led to impaired colony formation. Sympk activated proliferation-related genes and suppressed differentiation-related genes. Our results indicate that Sympk interacts with Oct4 to promote self-renewal and pluripotency in ESCs and preserves genome integrity; accordingly, it has potential value for stem cell therapies. Stem Cells 2019;37:743-753.
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http://dx.doi.org/10.1002/stem.2992DOI Listing
June 2019

Genome-wide profiling of adenine base editor specificity by EndoV-seq.

Nat Commun 2019 01 8;10(1):67. Epub 2019 Jan 8.

The First Affiliated Hospital, Sun Yat-sen University; MOE Key Laboratory of Gene Function and Regulation, Guangzhou Key Laboratory of Healthy Aging Research, SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, 510275, Guangzhou, China.

The adenine base editor (ABE), capable of catalyzing A•T to G•C conversions, is an important gene editing toolbox. Here, we systematically evaluate genome-wide off-target deamination by ABEs using the EndoV-seq platform we developed. EndoV-seq utilizes Endonuclease V to nick the inosine-containing DNA strand of genomic DNA deaminated by ABE in vitro. The treated DNA is then whole-genome sequenced to identify off-target sites. Of the eight gRNAs we tested with ABE, 2-19 (with an average of 8.0) off-target sites are found, significantly fewer than those found for canonical Cas9 nuclease (7-320, 160.7 on average). In vivo off-target deamination is further validated through target site deep sequencing. Moreover, we demonstrated that six different ABE-gRNA complexes could be examined in a single EndoV-seq assay. Our study presents the first detection method to evaluate genome-wide off-target effects of ABE, and reveals possible similarities and differences between ABE and canonical Cas9 nuclease.
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http://dx.doi.org/10.1038/s41467-018-07988-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325126PMC
January 2019

TOE1 acts as a 3' exonuclease for telomerase RNA and regulates telomere maintenance.

Nucleic Acids Res 2019 01;47(1):391-405

State Key Laboratory of Oncology in South China, Cancer Center, Collaborative Innovation Center for Cancer Medicine, MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China.

In human cells, telomeres are elongated by the telomerase complex that contains the reverse transcriptase hTERT and RNA template TERC/hTR. Poly(A)-specific ribonuclease (PARN) is known to trim hTR precursors by removing poly(A) tails. However, the precise mechanism of hTR 3' maturation remains largely unknown. Target of Egr1 (TOE1) is an Asp-Glu-Asp-Asp (DEDD) domain containing deadenylase that is mutated in the human disease Pontocerebella Hypoplasia Type 7 (PCH7) and implicated in snRNA and hTR processing. We have previously found TOE1 to localize specifically in Cajal bodies, where telomerase RNP complex assembly takes place. In this study, we showed that TOE1 could interact with hTR and the telomerase complex. TOE1-deficient cells accumulated hTR precursors, including oligoadenylated and 3'-extended forms, which was accompanied by impaired telomerase activity and shortened telomeres. Telomerase activity in TOE1-deficient cells could be rescued by wild-type TOE1 but not the catalytically inactive mutant. Our results suggest that hTR 3' end processing likely involves multiple exonucleases that work in parallel and/or sequentially, where TOE1 may function non-redundantly as a 3'-to-5' exonuclease in conjunction with PARN. Our study highlights a mechanistic link between TOE1 mutation, improper hTR processing and telomere dysfunction in diseases such as PCH7.
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http://dx.doi.org/10.1093/nar/gky1019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6326811PMC
January 2019

DeepNitro: Prediction of Protein Nitration and Nitrosylation Sites by Deep Learning.

Genomics Proteomics Bioinformatics 2018 08 27;16(4):294-306. Epub 2018 Sep 27.

State Key Laboratory of Oncology in South China, Cancer Center, Collaborative Innovation Center for Cancer Medicine, School of Life Sciences, Sun Yat-sen University, Guangzhou 510060, China. Electronic address:

Protein nitration and nitrosylation are essential post-translational modifications (PTMs) involved in many fundamental cellular processes. Recent studies have revealed that excessive levels of nitration and nitrosylation in some critical proteins are linked to numerous chronic diseases. Therefore, the identification of substrates that undergo such modifications in a site-specific manner is an important research topic in the community and will provide candidates for targeted therapy. In this study, we aimed to develop a computational tool for predicting nitration and nitrosylation sites in proteins. We first constructed four types of encoding features, including positional amino acid distributions, sequence contextual dependencies, physicochemical properties, and position-specific scoring features, to represent the modified residues. Based on these encoding features, we established a predictor called DeepNitro using deep learning methods for predicting protein nitration and nitrosylation. Using n-fold cross-validation, our evaluation shows great AUC values for DeepNitro, 0.65 for tyrosine nitration, 0.80 for tryptophan nitration, and 0.70 for cysteine nitrosylation, respectively, demonstrating the robustness and reliability of our tool. Also, when tested in the independent dataset, DeepNitro is substantially superior to other similar tools with a 7%-42% improvement in the prediction performance. Taken together, the application of deep learning method and novel encoding schemes, especially the position-specific scoring feature, greatly improves the accuracy of nitration and nitrosylation site prediction and may facilitate the prediction of other PTM sites. DeepNitro is implemented in JAVA and PHP and is freely available for academic research at http://deepnitro.renlab.org.
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http://dx.doi.org/10.1016/j.gpb.2018.04.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205083PMC
August 2018

Effective and precise adenine base editing in mouse zygotes.

Protein Cell 2018 09;9(9):808-813

Key Laboratory of Gene Engineering of the Ministry of Education, Guangzhou Key Laboratory of Healthy Aging Research and SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.

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http://dx.doi.org/10.1007/s13238-018-0566-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6107484PMC
September 2018

mA RNA modification controls autophagy through upregulating ULK1 protein abundance.

Cell Res 2018 09 25;28(9):955-957. Epub 2018 Jul 25.

State Key Laboratory of Oncology in South China, Cancer Center, Collaborative Innovation Center for Cancer Medicine, School of Life Sciences, Sun Yat-sen University, 510060, Guangzhou, China.

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http://dx.doi.org/10.1038/s41422-018-0069-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6123428PMC
September 2018

Gain-of-Function Mutation of Card14 Leads to Spontaneous Psoriasis-like Skin Inflammation through Enhanced Keratinocyte Response to IL-17A.

Immunity 2018 07 3;49(1):66-79.e5. Epub 2018 Jul 3.

Institute for Immunology, Tsinghua University School of Medicine, Tsinghua University-Peking University Jointed Center for Life Sciences, Beijing, 100084, China. Electronic address:

Genetic mutations of CARD14 (encoding CARMA2) are observed in psoriasis patients. Here we showed that Card14 and Card14 mice developed spontaneous psoriasis-like skin inflammation, which resulted from constitutively activated CARMA2 via self-aggregation leading to the enhanced activation of the IL-23-IL-17A cytokine axis. Card14 mice displayed attenuated skin inflammation in the imiquimod-induced psoriasis model due to impaired IL-17A signaling in keratinocytes. CARMA2, mainly expressed in keratinocytes, associates with the ACT1-TRAF6 signaling complex and mediates IL-17A-induced NF-κB and MAPK signaling pathway activation, which leads to expression of pro-inflammatory factors. Thus, CARMA2 serves as a key mediator of IL-17A signaling and its constitutive activation in keratinocytes leads to the onset of psoriasis, which indicates an important role of NF-κB activation in keratinocytes in psoriatic initiation.
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http://dx.doi.org/10.1016/j.immuni.2018.05.012DOI Listing
July 2018