DNA methylation remodeling and the functional implication during male gametogenesis in rice.

BACKGROUND: Epigenetic marks are reprogrammed during sexual reproduction. In flowering plants, DNA methylation is only partially remodeled in the gametes and the zygote. However, the timing and functional significance of the remodeling during plant gametogenesis remain obscure. RESULTS: Here we show that DNA methylation remodeling starts after male meiosis in rice, with non-CG methylation, particularly at CHG sites, being first enhanced in the microspore and subsequently decreased in sperm. Functional analysis of rice CHG methyltransferase genes CMT3a and CMT3b indicates that CMT3a functions as the major CHG methyltransferase in rice meiocyte, while CMT3b is responsible for the increase of CHG methylation in microspore. The function of the two histone demethylases JMJ706 and JMJ707 that remove H3K9me2 may contribute to the decreased CHG methylation in sperm. During male gametogenesis CMT3a mainly silences TE and TE-related genes while CMT3b is required for repression of genes encoding factors involved in transcriptional and translational activities. In addition, CMT3b functions to repress zygotic gene expression in egg and participates in establishing the zygotic epigenome upon fertilization. CONCLUSION: Collectively, the results indicate that DNA methylation is dynamically remodeled during male gametogenesis, distinguish the function of CMT3a and CMT3b in sex cells, and underpin the functional significance of DNA methylation remodeling during rice reproduction.

DNMT1 determines osteosarcoma cell resistance to apoptosis by associatively modulating DNA and mRNA cytosine-5 methylation.

Cellular apoptosis is a central mechanism leveraged by chemotherapy to treat human cancers. 5-Methylcytosine (m5C) modifications installed on both DNA and mRNA are documented to regulate apoptosis independently. However, the interplay or crosstalk between them in cellular apoptosis has not yet been explored. Here, we reported that promoter methylation by DNMT1 coordinated with mRNA methylation by NSun2 to regulate osteosarcoma cell apoptosis. DNMT1 was induced during osteosarcoma cell apoptosis triggered by chemotherapeutic drugs, whereas NSun2 expression was suppressed. DNMT1 was found to repress NSun2 expression by methylating the NSun2 promoter. Moreover, DNMT1 and NSun2 regulate the anti-apoptotic genes AXL, NOTCH2, and YAP1 through DNA and mRNA methylation, respectively. Upon exposure to cisplatin or doxorubicin, DNMT1 elevation drastically reduced the expression of these anti-apoptotic genes via enhanced promoter methylation coupled with NSun2 ablation-mediated attenuation of mRNA methylation, thus rendering osteosarcoma cells to apoptosis. Collectively, our findings establish crosstalk of importance between DNA and RNA cytosine methylations in determining osteosarcoma resistance to apoptosis during chemotherapy, shedding new light on future treatment of osteosarcoma, and adding additional layers to the control of gene expression at different epigenetic levels.

3D organization of regulatory elements for transcriptional regulation in Arabidopsis.

BACKGROUND: Although spatial organization of compartments and topologically associating domains at large scale is relatively well studied, the spatial organization of regulatory elements at fine scale is poorly understood in plants. RESULTS: Here we perform high-resolution chromatin interaction analysis using paired-end tag sequencing approach. We map chromatin interactions tethered with RNA polymerase II and associated with heterochromatic, transcriptionally active, and Polycomb-repressive histone modifications in Arabidopsis. Analysis of the regulatory repertoire shows that distal active cis-regulatory elements are linked to their target genes through long-range chromatin interactions with increased expression of the target genes, while poised cis-regulatory elements are linked to their target genes through long-range chromatin interactions with depressed expression of the target genes. Furthermore, we demonstrate that transcription factor MYC2 is critical for chromatin spatial organization, and propose that MYC2 occupancy and MYC2-mediated chromatin interactions coordinately facilitate transcription within the framework of 3D chromatin architecture. Analysis of functionally related gene-defined chromatin connectivity networks reveals that genes implicated in flowering-time control are functionally compartmentalized into separate subdomains via their spatial activity in the leaf or shoot apical meristem, linking active mark- or Polycomb-repressive mark-associated chromatin conformation to coordinated gene expression. CONCLUSION: The results reveal that the regulation of gene transcription in Arabidopsis is not only by linear juxtaposition, but also by long-range chromatin interactions. Our study uncovers the fine scale genome organization of Arabidopsis and the potential roles of such organization in orchestrating transcription and development.

Exploring Idiopathic Pulmonary Fibrosis Biomarker by Simultaneous Two-Photon Fluorescence Imaging of Cysteine and Peroxynitrite.

Idiopathic pulmonary fibrosis (IPF) has been characterized as a chronic inflammatory disease that leads to irreversible damage to pulmonary function. However, there is no specific IPF biomarker that can be used to distinguish IPF and not pneumonia. Endoplasmic reticulum (ER) stress is prominent in IPF. To search for a specific biomarker of IPF, we developed two ER-targeting two-photon (TP) fluorescent probes, TPER-ONOO and TPER-Cys, for peroxynitrite (ONOO-) and cysteine (Cys) imaging, respectively. A significant increase in Cys levels in the lungs was discovered only in mice with IPF, which implied that Cys might be an IPF biomarker candidate. Furthermore, we uncovered the mechanism of glutathione (GSH) deficiency in IPF, which was not due to Cys shortage but instead was attributable to impaired glutamate cysteine ligase and glutathione synthetase activities via ONOO--induced post-transcriptional modification. This work has potential to provide a new method for IPF early diagnosis and drug efficacy evaluation.

The G4 resolvase RHAU modulates mRNA translation and stability to sustain postnatal heart function and regeneration.

Post-transcriptional regulation of mRNA translation and stability is primarily achieved by RNA-binding proteins, which are of increasing importance for heart function. Furthermore, G-quadruplex (G4) and G4 resolvase activity are involved in a variety of biological processes. However, the role of G4 resolvase activity in heart function remains unknown. The present study aims to investigate the role of RNA helicase associated with adenylate- and uridylate-rich element (RHAU), an RNA-binding protein with G4 resolvase activity in postnatal heart function through deletion of Rhau in the cardiomyocytes of postnatal mice. RHAU-deficient mice displayed progressive pathological remodeling leading to heart failure and mortality and impaired neonatal heart regeneration. RHAU ablation reduced the protein levels but enhanced mRNA levels of Yap1 and Hexim1 that are important regulators for heart development and postnatal heart function. Furthermore, RHAU was found to associate with both the 5' and 3' UTRs of these genes to destabilize mRNA and enhance translation. Thus, we have demonstrated the important functions of RHAU in the dual regulation of mRNA translation and stability, which is vital for heart physiology.

Dynamics of euphotic zone depth in the Bohai Sea and Yellow Sea.

Euphotic zone depth (Zeu) plays an important role in studies of marine biogeochemical processes and ecosystems. Remote sensing techniques are ideal tools to investigate Zeu distributions because of their advanced observation ability with broad spatial coverage and frequent observation intervals. This study aims to develop a new approach that derives Zeu directly from remote sensing reflectance (Rrs(λ)) values rather than by using other intermediate variables and then reveals the dynamic characteristics of Zeu in the Bohai Sea (BS) and Yellow Sea (YS). To do this, in situ data collected from various seasons were first used to assess the ability of several spectral indicators of Rrs(λ) for deriving Zeu and the optimal spectral indicator was determined to build a Zeu retrieval model. This model was further applied to Geostationary Ocean Color Imager (GOCI) data to study the spatial and temporal variations in Zeu. The results showed that the new Zeu retrieval model performed well with R2, RMSE and MAPE values of 0.843, 4.42 m and 17.9%, respectively. High Zeu levels were generally observed during summer for both coastal and offshore waters while the lowest Zeu values were observed during winter. Changing concentrations of total suspended matter, which are often modulated by sediment resuspension and transportation, are probably the main factor responsible for the spatial and temporal variability of Zeu. These findings provide crucial information for modeling primary production, carbon flux, and heat transfer, etc., in the BS and YS, as well as contribute a useful alternative approach that will be easily implemented to study Zeu from satellite data for other water environments.

The landscape of RNA Pol II binding reveals a stepwise transition during ZGA.

Zygotic genome activation (ZGA) is the first transcription event in life1. However, it is unclear how RNA polymerase is engaged in initiating ZGA in mammals. Here, by developing small-scale Tn5-assisted chromatin cleavage with sequencing (Stacc-seq), we investigated the landscapes of RNA polymerase II (Pol II) binding in mouse embryos. We found that Pol II undergoes 'loading', 'pre-configuration', and 'production' during the transition from minor ZGA to major ZGA. After fertilization, Pol II is preferentially loaded to CG-rich promoters and accessible distal regions in one-cell embryos (loading), in part shaped by the inherited parental epigenome. Pol II then initiates relocation to future gene targets before genome activation (pre-configuration), where it later engages in full transcription elongation upon major ZGA (production). Pol II also maintains low poising at inactive promoters after major ZGA until the blastocyst stage, coinciding with the loss of promoter epigenetic silencing factors. Notably, inhibition of minor ZGA impairs the Pol II pre-configuration and embryonic development, accompanied by aberrant retention of Pol II and ectopic expression of one-cell targets upon major ZGA. Hence, stepwise transition of Pol II occurs when mammalian life begins, and minor ZGA has a key role in the pre-configuration of transcription machinery and chromatin for genome activation.

Post-transcriptional Regulation of Nkx2-5 by RHAU in Heart Development.

RNA G-quadruplexes (G4s) play important roles in RNA biology. However, the function and regulation of mRNA G-quadruplexes in embryonic development remain elusive. Previously, we identified RHAU (DHX36, G4R1) as an RNA helicase that resolves mRNA G-quadruplexes. Here, we find that cardiac deletion of Rhau leads to heart defects and embryonic lethality in mice. Gene expression profiling identified Nkx2-5 mRNA as a target of RHAU that associates with its 5' and 3' UTRs and modulates its stability and translation. The 5' UTR of Nkx2-5 mRNA contains a G-quadruplex that requires RHAU for protein translation, while the 3' UTR of Nkx2-5 mRNA possesses an AU-rich element (ARE) that facilitates RHAU-mediated mRNA decay. Thus, we uncovered the mechanisms underlying Nkx2-5 post-transcriptional regulation during heart development. Meanwhile, this study demonstrates the function of mRNA 5' UTR G-quadruplex-mediated protein translation in organogenesis.

A cardiomyocyte-specific Wdr1 knockout demonstrates essential functional roles for actin disassembly during myocardial growth and maintenance in mice.

Actin dynamics are critical for muscle development and function, and mutations leading to deregulation of actin dynamics cause various forms of heritable muscle diseases. AIP1 is a major cofactor of the actin depolymerizing factor/cofilin in eukaryotes, promoting actin depolymerizing factor/cofilin-mediated actin disassembly. Its function in vertebrate muscle has been unknown. To investigate functional roles of AIP1 in myocardium, we generated conditional knockout (cKO) mice with cardiomyocyte-specific deletion of Wdr1, the mammalian homolog of yeast AIP1. Wdr1 cKO mice began to die at postnatal day 13 (P13), and none survived past P24. At P12, cKO mice exhibited cardiac hypertrophy and impaired contraction of the left ventricle. Electrocardiography revealed reduced heart rate, abnormal P wave, and abnormal T wave at P10 and prolonged QT interval at P12. Actin filament (F-actin) accumulations began at P10 and became prominent at P12 in the myocardium of cKO mice. Within regions of F-actin accumulation in myofibrils, the sarcomeric components α-actinin and tropomodulin-1 exhibited disrupted patterns, indicating that F-actin accumulations caused by Wdr1 deletion result in disruption of sarcomeric structure. Ectopic cofilin colocalized with F-actin aggregates. In adult mice, Wdr1 deletion resulted in similar but much milder phenotypes of heart hypertrophy, F-actin accumulations within myofibrils, and lethality. Taken together, these results demonstrate that AIP1-regulated actin dynamics play essential roles in heart function in mice.

Phosphoinositide-dependent kinase 1 and mTORC2 synergistically maintain postnatal heart growth and heart function in mice.

The protein kinase Akt plays a critical role in heart function and is activated by phosphorylation of threonine 308 (T308) and serine 473 (S473). While phosphoinositide-dependent kinase 1 (PDK1) is responsible for Akt T308 phosphorylation, the identities of the kinases for Akt S473 phosphorylation in the heart remain controversial. Here, we disrupted mTOR complex 2 (mTORC2) through deletion of Rictor in the heart and found normal heart growth and function. Rictor deletion caused significant reduction of Akt S473 phosphorylation but enhanced Akt T308 phosphorylation, suggesting that a high level of Akt T308 phosphorylation maintains Akt activity and heart function. Deletion of Pdk1 in the heart caused significantly enhanced Akt S473 phosphorylation that was suppressed by removal of Rictor, leading to worsened dilated cardiomyopathy (DCM) and accelerated heart failure in Pdk1-deficient mice. In addition, we found that increasing Akt S473 phosphorylation through deletion of Pten or chemical inhibition of PTEN reversed DCM and heart failure in Pdk1-deficient mice. Investigation of heart samples from human DCM patients revealed changes similar to those in the mouse models. These results demonstrated that PDK1 and mTORC2 synergistically promote postnatal heart growth and maintain heart function in postnatal mice.

Cardiac ablation of Rheb1 induces impaired heart growth, endoplasmic reticulum-associated apoptosis and heart failure in infant mice.

Ras homologue enriched in brain 1 (Rheb1) plays an important role in a variety of cellular processes. In this study, we investigate the role of Rheb1 in the post-natal heart. We found that deletion of the gene responsible for production of Rheb1 from cardiomyocytes of post-natal mice resulted in malignant arrhythmias, heart failure, and premature death of these mice. In addition, heart growth impairment, aberrant metabolism relative gene expression, and increased cardiomyocyte apoptosis were observed in Rheb1-knockout mice prior to the development of heart failure and arrhythmias. Also, protein kinase B (PKB/Akt) signaling was enhanced in Rheb1-knockout mice, and removal of phosphatase and tensin homolog (Pten) significantly prolonged the survival of Rheb1-knockouts. Furthermore, signaling via the mammalian target of rapamycin complex 1 (mTORC1) was abolished and C/EBP homologous protein (CHOP) and phosphorylation levels of c-Jun N-terminal kinase (JNK) were increased in Rheb1 mutant mice. In conclusion, this study demonstrates that Rheb1 is important for maintaining cardiac function in post-natal mice via regulation of mTORC1 activity and stress on the endoplasmic reticulum. Moreover, activation of Akt signaling helps to improve the survival of mice with advanced heart failure. Thus, this study provides direct evidence that Rheb1 performs multiple important functions in the heart of the post-natal mouse. Enhancing Akt activity improves the survival of infant mice with advanced heart failure.

Compared analysis of LncRNA expression profiling in pdk1 gene knockout mice at two time points.

BACKGROUND/AIMS: Previous studies have indicated that long non-coding RNAs (lncRNA) are related to the occurrence and development of many human diseases, such as cancer and the HELLP and the brachydactyly syndromes. However, studies of LncRNA in heart failure have not yet been reported. Here, we investigated cardiac lncRNA expression profiles in the myocardial-specific knockout pdk1 gene (KO) mouse model of heart failure. METHODS: Cardiac samples were obtained from PDK1 KO and WT mice on postnatal (P) day 8 (P8) and day 40 (P40), and lncRNA expression profiles were analyzed by sequencing and screening using the Arraystar mouse lncRNA microarray. Quantitative real-time PCR analysis of these lncRNAs confirmed the identity of some genes. RESULTS: Comparisons of the KO and control groups showed fold changes of >1.5 in the expression levels of 2,024 lncRNAs at P8, while fold changes of >2 in the expression levels of 4,095 lncRNAs were detected at P40. Nineteen lncRNAs were validated by RT-PCR. Bioinformatic and pathway analyses indicated that mkk7, a sense overlap lncRNA, may be involved in the pathological processes of heart failure through the MAPK signaling pathway. CONCLUSION: These data reveal differentially expressed lncRNA in mice with a myocardial-specific deletion of the pdk1 gene, which may provide new insights into the mechanism of heart failure in PDK1 knockout mice.

Genetic and pharmacological inhibition of Rheb1-mTORC1 signaling exerts cardioprotection against adverse cardiac remodeling in mice.

A previous study indicated that Rheb1 is required for mammalian target of TOR complex 1 (mTORC1) signaling in the brain. However, the function of Rheb1 in the heart is still elusive. In the present study, we deleted Rheb1 specifically in cardiomyocytes and found that reduced Rheb1 levels conferred cardioprotection against pathologic remodeling in myocardial infarction (MI) and pressure overload (transverse aortic constriction) mouse models. Cardiomyocyte apoptosis was reduced and mTORC1 activity was suppressed in cardiomyocyte Rheb1-deletion mice, suggesting that Rheb1 regulates mTORC1 activation in myocardium. Furthermore, we demonstrated that astragaloside IV (As-IV) could inhibit mTORC1, and As-IV treatment displayed similar protection against MI and transverse aortic constriction as Rheb1 genetic inhibition. This study indicates that Rheb1 is essential for mTORC1 activation in cardiomyocytes and suggests that targeting Rheb1-mTORC1 signaling, such as by As-IV treatment, may be an effective therapeutic method for treating patients with adverse cardiac remodeling after MI and hypertrophy.

Phosphorylation of the Twist1-family basic helix-loop-helix transcription factors is involved in pathological cardiac remodeling.

BACKGROUND: The Twist1-family basic helix-loop-helix (bHLH) transcription factors including Twist1, Hand1 and Hand2, play an essential role in heart development and are implicated in pathological heart remodeling. Previously, it was reported that these bHLH transcription factors can be regulated by phosphorylation within the basic-helix I domain, which is involved in developmental processes such as limb formation and trophoblast differentiation. However, how phosphorylation of Twist1 family functions in post-natal heart is elusive. PRINCIPAL FINDINGS: Here, we generated transgenic mice with over-expression of Hand1 and Twist1 mutants (to mimic or to abolish phosphorylation) in cardiomyocytes and found pathological cardiac remodeling leading to heart failure and sudden death. Gene expression profile analysis revealed up-regulation of growth-promoting genes and down-regulation of metabolic genes. It is well known that aberrant activation of Akt signaling causes pathological cardiac remodeling and results in heart failure. The basic-helix I domain of Twist1 family members contain Akt substrate consensus motif and may be downstream targets of Akt signaling. Using biochemical analysis, we demonstrated that Hand1 and Twist1 were phosphorylated by Akt in the basic-helix I domain. Phosphorylation of Hand1 regulated its transcriptional activation of luciferase reporter genes and DNA binding ability. CONCLUSIONS: This study provides novel insights into the regulation of Twist1 family in cardiac remodeling and suggests that the Twist1 family can be regulated by Akt signaling.

[Infrequent X chromosome abnormality and X-linked syndromic deafness].

OBJECTIVE: To make clear the relationship between the X chromosome abnormality and sydromic deafness through genetic analysis of a pedigree with X-linked syndromic deafness. METHODS: The chromosome number and structure of the family members were analyzed by the standard and high resolution banding with Giemsa, and fluorescent in situ hybridization. The allelic number of the DNA segment in X chromosome was studied with genetic markers. RESULTS: The 2 probands, their mothers and grandmother with normal phenotype all had X(p22-pter) duplication. The whole X chromosome of both the proband III-3 and his mother could be stained with X chromosome staining probe. The proband III-3 had 2 copies of DXS7108. CONCLUSION: The abnormal X chromosome occurring in this pedigree of X-linked syndromic deafness derives from partial Xp duplication, which will guide further research to identify the breakpoint of this abnormal chromosome.