Widening of narrow urethral plates with lateral skin in TIP hypospadias repair: single center series.

BACKGROUND: To compare the outcomes of hypospadias repair using tubularized incised plate (TIP) urethroplasty and modified TIP with lateral skin to widen the urethral plate (WTIP). MATERIALS AND METHODS: Data were obtained from pre-pubertal boys who underwent primary hypospadias repair between May 2018 and July 2023. The cases were divided into two groups; one group underwent TIP with urethral plate ≥ 6 mm width and the other group with urethral plate width < 6 mm underwent WTIP. WTIP urethroplasty was performed by widening incisions on the outer margins of the urethral plate to incorporate penile and glandular skin lateral to the urethral plate to facilitate tubularization. Complication rates and urinary functions were compared. RESULTS: A total of 157 patients were enrolled in this study. Eighty-eight cases with narrow urethral plate were subjected to WTIP urethroplasty, and the rest were subjected to TIP urethroplasty. The preoperative glans width in WTIP group was less than that in TIP group (P < 0.001), and 44.3% had midshaft meatus in WTIP group compared to 17.4% in TIP group (P < 0.001). However, the incidences of postoperative complications (17.6% vs. 21.6%, P = 0.550) were not statistically different between the TIP and WTIP groups. In addition, both groups did not differ significantly in postoperative uroflowmetry assessment. CONCLUSIONS: The described technique helps to create an adequately caliber aesthetic neomeatus and facilitates tubularization, especially in hypospadias with a narrow urethral plate. Our data suggest that augmentation of a narrow urethral plate with WTIP has a similar surgical outcome to that of the TIP procedure in patients with a wide urethral plate.

Foxp and Skor family proteins control differentiation of Purkinje cells from Ptf1a- and Neurog1-expressing progenitors in zebrafish.

Cerebellar neurons, such as GABAergic Purkinje cells (PCs), interneurons (INs) and glutamatergic granule cells (GCs) are differentiated from neural progenitors expressing proneural genes, including ptf1a, neurog1 and atoh1a/b/c. Studies in mammals previously suggested that these genes determine cerebellar neuron cell fate. However, our studies on ptf1a;neurog1 zebrafish mutants and lineage tracing of ptf1a-expressing progenitors have revealed that the ptf1a/neurog1-expressing progenitors can generate diverse cerebellar neurons, including PCs, INs and a subset of GCs in zebrafish. The precise mechanisms of how each cerebellar neuron type is specified remains elusive. We found that genes encoding the transcriptional regulators Foxp1b, Foxp4, Skor1b and Skor2, which are reportedly expressed in PCs, were absent in ptf1a;neurog1 mutants. foxp1b;foxp4 mutants showed a strong reduction in PCs, whereas skor1b;skor2 mutants completely lacked PCs, and displayed an increase in immature GCs. Misexpression of skor2 in GC progenitors expressing atoh1c suppressed GC fate. These data indicate that Foxp1b/4 and Skor1b/2 function as key transcriptional regulators in the initial step of PC differentiation from ptf1a/neurog1-expressing neural progenitors, and that Skor1b and Skor2 control PC differentiation by suppressing their differentiation into GCs.

SMXL5 attenuates strigolactone signaling in Arabidopsis thaliana by inhibiting SMXL7 degradation.

Hormone-activated proteolysis is a recurring theme of plant hormone signaling mechanisms. In strigolactone signaling, the enzyme receptor DWARF14 (D14) and an F-box protein, MORE AXILLARY GROWTH2 (MAX2), mark SUPPRESSOR OF MAX2 1-LIKE (SMXL) family proteins SMXL6, SMXL7, and SMXL8 for rapid degradation. Removal of these transcriptional corepressors initiates downstream growth responses. The homologous proteins SMXL3, SMXL4, and SMXL5, however, are resistant to MAX2-mediated degradation. We discovered that the smxl4 smxl5 mutant has enhanced responses to strigolactone. SMXL5 attenuates strigolactone signaling by interfering with AtD14-SMXL7 interactions. SMXL5 interacts with AtD14 and SMXL7, providing two possible ways to inhibit SMXL7 degradation. SMXL5 function is partially dependent on an ethylene-responsive-element binding-factor-associated amphiphilic repression (EAR) motif, which typically mediates interactions with the TOPLESS family of transcriptional corepressors. However, we found that loss of the EAR motif reduces SMXL5-SMXL7 interactions and the attenuation of strigolactone signaling by SMXL5. We hypothesize that integration of SMXL5 into heteromeric SMXL complexes reduces the susceptibility of SMXL6/7/8 proteins to strigolactone-activated degradation and that the EAR motif promotes the formation or stability of these complexes. This mechanism may provide a way to spatially or temporally fine-tune strigolactone signaling through the regulation of SMXL5 expression or translation.

SOX4 regulates proliferation and apoptosis of human ovarian granulosa-like tumor cell line KGN through the Hippo pathway.

The proliferation and apoptosis of ovarian granulosa cells are important for folliculogenesis. As a transcription factor, SRY-box transcription factor 4 (SOX4) has important roles in regulating cellular proliferation and apoptosis. Nonetheless, the regulatory mechanisms of SOX4 on proliferation and apoptosis of granulosa cells remain elusive. Therefore, a stably overexpressed SOX4 ovarian granulosa cell line KGN was generated by lentivirus encapsulation. We observed that overexpression of SOX4 inhibits apoptosis, promotes proliferation and migration of KGN cells. Comparative analysis of the transcriptome revealed 868 upregulated and 696 downregulated DEGs in LV-SOX4 in comparison with LV-CON KGN cell lines. Afterward, further assessments were performed to explore the possible functions about these DEGs. The data showed their involvement in many biological processes, particularly the Hippo signaling pathway. Moreover, the expression levels of YAP1, WWTR1, WTIP, DLG3, CCN2, and AMOT, which were associated with the Hippo signaling pathway, were further validated by qRT-PCR. In addition, the protein expression levels of YAP1 were markedly elevated, while p-YAP1 were notably reduced after overexpression of SOX4 in KGN cells. Thus, these results suggested that SOX4 regulates apoptosis, proliferation and migration of KGN cells, at least partly, through activation of the Hippo signaling pathway, which might be implicated in mammalian follicle development.

Further Delineation of Clinical Phenotype of ZMYND11 Variants in Patients with Neurodevelopmental Dysmorphic Syndrome.

Intellectual disability with speech delay and behavioural abnormalities, as well as hypotonia, seizures, feeding difficulties and craniofacial dysmorphism, are the main symptoms associated with pathogenic variants of the ZMYND11 gene. The range of clinical manifestations of the ZMYND phenotype is constantly being expanded by new cases described in the literature. Here, we present two previously unreported paediatric patients with neurodevelopmental challenges, who were diagnosed with missense variants in the ZMYND11 gene. It should be noted that one of the individuals manifested with hyperinsulinaemic hypoglycaemia (HH), a symptom that was not described before in published works. The reason for the occurrence of HH in our proband is not clear, so we try to explain the origin of this symptom in the context of the ZMYND11 syndrome. Thus, this paper contributes to knowledge on the range of possible manifestations of the ZMYND disease and provides further evidence supporting its association with neurodevelopmental challenges.

The CoREST repressor complex mediates phenotype switching and therapy resistance in melanoma.

Virtually all patients with BRAF-mutant melanoma develop resistance to MAPK inhibitors largely through nonmutational events. Although the epigenetic landscape is shown to be altered in therapy-resistant melanomas and other cancers, a specific targetable epigenetic mechanism has not been validated. Here, we evaluated the corepressor for element 1-silencing transcription factor (CoREST) epigenetic repressor complex and the recently developed bivalent inhibitor corin within the context of melanoma phenotype plasticity and therapeutic resistance. We found that CoREST was a critical mediator of the major distinct melanoma phenotypes and that corin treatment of melanoma cells led to phenotype reprogramming. Global assessment of transcript and chromatin changes conferred by corin revealed specific effects on histone marks connected to epithelial-mesenchymal transition-associated (EMT-associated) transcription factors and the dual-specificity phosphatases (DUSPs). Remarkably, treatment of BRAF inhibitor-resistant (BRAFi-R) melanomas with corin promoted resensitization to BRAFi therapy. DUSP1 was consistently downregulated in BRAFi-R melanomas, which was reversed by corin treatment and associated with inhibition of p38 MAPK activity and resensitization to BRAFi therapies. Moreover, this activity was recapitulated by the p38 MAPK inhibitor BIRB 796. These findings identify the CoREST repressor complex as a central mediator of melanoma phenotype plasticity and resistance to targeted therapy and suggest that CoREST inhibitors may prove beneficial for patients with BRAFi-resistant melanoma.

Targeting nuclear receptor corepressors for reversible male contraception.

Despite numerous female contraceptive options, nearly half of all pregnancies are unintended. Family planning choices for men are currently limited to unreliable condoms and invasive vasectomies with questionable reversibility. Here, we report the development of an oral contraceptive approach based on transcriptional disruption of cyclical gene expression patterns during spermatogenesis. Spermatogenesis involves a continuous series of self-renewal and differentiation programs of spermatogonial stem cells (SSCs) that is regulated by retinoic acid (RA)-dependent activation of receptors (RARs), which control target gene expression through association with corepressor proteins. We have found that the interaction between RAR and the corepressor silencing mediator of retinoid and thyroid hormone receptors (SMRT) is essential for spermatogenesis. In a genetically engineered mouse model that negates SMRT-RAR binding (SMRTmRID mice), the synchronized, cyclic expression of RAR-dependent genes along the seminiferous tubules is disrupted. Notably, the presence of an RA-resistant SSC population that survives RAR de-repression suggests that the infertility attributed to the loss of SMRT-mediated repression is reversible. Supporting this notion, we show that inhibiting the action of the SMRT complex with chronic, low-dose oral administration of a histone deacetylase inhibitor reversibly blocks spermatogenesis and fertility without affecting libido. This demonstration validates pharmacologic targeting of the SMRT repressor complex for non-hormonal male contraception.

NCoR1 limits angiogenic capacity by altering Notch signaling.

Corepressors negatively regulate gene expression by chromatin compaction. Targeted regulation of gene expression could provide a means to control endothelial cell phenotype. We hypothesize that by targeting corepressor proteins, endothelial angiogenic function can be improved. To study this, the expression and function of nuclear corepressors in human umbilical vein endothelial cells (HUVEC) and in murine organ culture was studied. RNA-seq revealed that nuclear receptor corepressor 1 (NCoR1), silencing mediator of retinoid and thyroid hormone receptors (SMRT) and repressor element-1 silencing transcription factor (REST) are the highest expressed corepressors in HUVECs. Knockout and knockdown strategies demonstrated that the depletion of NCoR1 increased the angiogenic capacity of endothelial cells, whereas depletion of SMRT or REST did not. Interestingly, the effect was VEGF signaling independent. NCoR1 depletion significantly upregulated angiogenesis-associated genes, especially tip cell genes, including ESM1, DLL4 and NOTCH4, as observed by RNA- and ATAC-seq. Confrontation assays comparing cells with and without NCoR1-deficiency revealed that loss of NCoR1 promotes a tip-cell position during spheroid sprouting. Moreover, a proximity ligation assay identified NCoR1 as a direct binding partner of the Notch-signaling-related transcription factor RBPJk. Luciferase assays showed that siRNA-mediated knockdown of NCOR1 promotes RBPJk activity. Furthermore, NCoR1 depletion prompts upregulation of several elements in the Notch signaling cascade. Downregulation of NOTCH4, but not NOTCH1, prevented the positive effect of NCOR1 knockdown on spheroid outgrowth. Collectively, these data indicate that decreasing NCOR1 expression is an attractive approach to promote angiogenic function.

BTB domain mutations perturbing KCTD15 oligomerisation cause a distinctive frontonasal dysplasia syndrome.

INTRODUCTION: KCTD15 encodes an oligomeric BTB domain protein reported to inhibit neural crest formation through repression of Wnt/beta-catenin signalling, as well as transactivation by TFAP2. Heterozygous missense variants in the closely related paralogue KCTD1 cause scalp-ear-nipple syndrome. METHODS: Exome sequencing was performed on a two-generation family affected by a distinctive phenotype comprising a lipomatous frontonasal malformation, anosmia, cutis aplasia of the scalp and/or sparse hair, and congenital heart disease. Identification of a de novo missense substitution within KCTD15 led to targeted sequencing of DNA from a similarly affected sporadic patient, revealing a different missense mutation. Structural and biophysical analyses were performed to assess the effects of both amino acid substitutions on the KCTD15 protein. RESULTS: A heterozygous c.310G>C variant encoding p.(Asp104His) within the BTB domain of KCTD15 was identified in an affected father and daughter and segregated with the phenotype. In the sporadically affected patient, a de novo heterozygous c.263G>A variant encoding p.(Gly88Asp) was present in KCTD15. Both substitutions were found to perturb the pentameric assembly of the BTB domain. A crystal structure of the BTB domain variant p.(Gly88Asp) revealed a closed hexameric assembly, whereas biophysical analyses showed that the p.(Asp104His) substitution resulted in a monomeric BTB domain likely to be partially unfolded at physiological temperatures. CONCLUSION: BTB domain substitutions in KCTD1 and KCTD15 cause clinically overlapping phenotypes involving craniofacial abnormalities and cutis aplasia. The structural analyses demonstrate that missense substitutions act through a dominant negative mechanism by disrupting the higher order structure of the KCTD15 protein complex.

TOPLESS Corepressors as an Emerging Hub of Plant Pathogen Effectors.

Transcriptional corepressors form an ancient and essential layer of gene expression control in eukaryotes. TOPLESS and TOPLESS-RELATED (TPL/TPR) proteins constitute a conserved family of Groucho (Gro)/thymidine uptake 1 (Tup1)-type transcriptional corepressors and control diverse growth, developmental, and stress signaling responses in plants. Because of their central and versatile regulatory roles, they act as a signaling hub to integrate various input signaling pathways in the transcriptional responses. Recently, increasing pieces of evidence indicate the roles of TPL/TPR family proteins in the modulation of plant immunity. This is supported by studies on effectors of distantly related pathogens that target TPL/TPR proteins in planta. In this short review, we will summarize the latest findings concerning pathogens targeting plant TPL/TPR proteins to manipulate plant signaling responses for the successful invasion of their hosts. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The Wnt-pathway corepressor TLE3 interacts with the histone methyltransferase KMT1A to inhibit differentiation in Rhabdomyosarcoma.

Rhabdomyosarcoma tumor cells resemble differentiating skeletal muscle cells, which unlike normal muscle cells, fail to undergo terminal differentiation, underlying their proliferative and metastatic properties. We identify the corepressor TLE3 as a key regulator of rhabdomyosarcoma tumorigenesis by inhibiting the Wnt-pathway. Loss of TLE3 function leads to Wnt-pathway activation, reduced proliferation, decreased migration, and enhanced differentiation in rhabdomyosarcoma cells. Muscle-specific TLE3-knockout results in enhanced expression of terminal myogenic differentiation markers during normal mouse development. TLE3-knockout rhabdomyosarcoma cell xenografts result in significantly smaller tumors characterized by reduced proliferation, increased apoptosis and enhanced differentiation. We demonstrate that TLE3 interacts with and recruits the histone methyltransferase KMT1A, leading to repression of target gene activation and inhibition of differentiation in rhabdomyosarcoma. A combination drug therapy regime to promote Wnt-pathway activation by the small molecule BIO and inhibit KMT1A by the drug chaetocin led to significantly reduced tumor volume, decreased proliferation, increased expression of differentiation markers and increased survival in rhabdomyosarcoma tumor-bearing mice. Thus, TLE3, the Wnt-pathway and KMT1A are excellent drug targets which can be exploited for treating rhabdomyosarcoma tumors.

A TLR4/TRAF6-dependent signaling pathway mediates NCoR coactivator complex formation for inflammatory gene activation.

The nuclear receptor corepressor (NCoR) forms a complex with histone deacetylase 3 (HDAC3) that mediates repressive functions of unliganded nuclear receptors and other transcriptional repressors by deacetylation of histone substrates. Recent studies provide evidence that NCoR/HDAC3 complexes can also exert coactivator functions in brown adipocytes by deacetylating and activating PPARγ coactivator 1α (PGC1α) and that signaling via receptor activator of nuclear factor kappa-B (RANK) promotes the formation of a stable NCoR/HDAC3/PGC1ß complex that coactivates nuclear factor kappa-B (NFκB)- and activator protein 1 (AP-1)-dependent genes required for osteoclast differentiation. Here, we demonstrate that activation of Toll-like receptor (TLR) 4, but not TLR3, the interleukin 4 (IL4) receptor nor the Type I interferon receptor, also promotes assembly of an NCoR/HDAC3/PGC1ß coactivator complex. Receptor-specific utilization of TNF receptor-associated factor 6 (TRAF6) and downstream activation of extracellular signal-regulated kinase 1 (ERK1) and TANK-binding kinase 1 (TBK1) accounts for the common ability of RANK and TLR4 to drive assembly of an NCoR/HDAC3/PGC1ß complex in macrophages. ERK1, the p65 component of NFκB, and the p300 histone acetyltransferase (HAT) are also components of the induced complex and are associated with local histone acetylation and transcriptional activation of TLR4-dependent enhancers and promoters. These observations identify a TLR4/TRAF6-dependent signaling pathway that converts NCoR from a corepressor of nuclear receptors to a coactivator of NFκB and AP-1 that may be relevant to functions of NCoR in other developmental and homeostatic processes.

HIRA vs. DAXX: the two axes shaping the histone H3.3 landscape.

H3.3, the most common replacement variant for histone H3, has emerged as an important player in chromatin dynamics for controlling gene expression and genome integrity. While replicative variants H3.1 and H3.2 are primarily incorporated into nucleosomes during DNA synthesis, H3.3 is under the control of H3.3-specific histone chaperones for spatiotemporal incorporation throughout the cell cycle. Over the years, there has been progress in understanding the mechanisms by which H3.3 affects domain structure and function. Furthermore, H3.3 distribution and relative abundance profoundly impact cellular identity and plasticity during normal development and pathogenesis. Recurrent mutations in H3.3 and its chaperones have been identified in neoplastic transformation and developmental disorders, providing new insights into chromatin biology and disease. Here, we review recent findings emphasizing how two distinct histone chaperones, HIRA and DAXX, take part in the spatial and temporal distribution of H3.3 in different chromatin domains and ultimately achieve dynamic control of chromatin organization and function. Elucidating the H3.3 deposition pathways from the available histone pool will open new avenues for understanding the mechanisms by which H3.3 epigenetically regulates gene expression and its impact on cellular integrity and pathogenesis.

Microglia nuclear receptor corepressor 1 deficiency alleviates neuroinflammation in mice.

Given the established role of nuclear receptor corepressor 1 (NCoR1) in sensing environmental cues and the importance of inflammation in neurodegenerative diseases, elucidation of NCoR1 involvement in neuroinflammation has notable implications. Yet, its regulatory mechanism remains largely unclear. Under in vitro conditions, NCoR1 expression peaked and then decreased at 12 h after lipopolysaccharides (LPS) stimulation in BV2 cells, However, NCoR1 knockdown using si-RNA attenuated microglial inflammation, evident by reduced the levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX2), phosphorylated-JNK and high mobility group box-1 (HMGB1). Furthermore, NCoR1 suppression could counteract the decline in mitochondrial membrane potential while simultaneously enhancing the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). Under in vivo conditions, microglia-specific NCoR1 knockout (MNKO) mice after LPS injections alleviated the symptoms of anhedonia, diminished autonomic activity and cognitive impairment. Additionally, MNKO mice showed attenuation of microglial activation, downregulated HMGB1 and COX2, and upregulated PGC-1α expression in the cortex. In conclusion, these findings suggest that NCoR1 deficiency leads to a modest reduction in neuroinflammation, possibly attributed to the increased expression of PGC-1α.

DAXX promotes centromeric stability independently of ATRX by preventing the accumulation of R-loop-induced DNA double-stranded breaks.

Maintaining chromatin integrity at the repetitive non-coding DNA sequences underlying centromeres is crucial to prevent replicative stress, DNA breaks and genomic instability. The concerted action of transcriptional repressors, chromatin remodelling complexes and epigenetic factors controls transcription and chromatin structure in these regions. The histone chaperone complex ATRX/DAXX is involved in the establishment and maintenance of centromeric chromatin through the deposition of the histone variant H3.3. ATRX and DAXX have also evolved mutually-independent functions in transcription and chromatin dynamics. Here, using paediatric glioma and pancreatic neuroendocrine tumor cell lines, we identify a novel ATRX-independent function for DAXX in promoting genome stability by preventing transcription-associated R-loop accumulation and DNA double-strand break formation at centromeres. This function of DAXX required its interaction with histone H3.3 but was independent of H3.3 deposition and did not reflect a role in the repression of centromeric transcription. DAXX depletion mobilized BRCA1 at centromeres, in line with BRCA1 role in counteracting centromeric R-loop accumulation. Our results provide novel insights into the mechanisms protecting the human genome from chromosomal instability, as well as potential perspectives in the treatment of cancers with DAXX alterations.

Integrative multi-omics analysis reveals the critical role of the PBXIP1 gene in Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder, and its strongest risk factor is aging. A few studies have explored the relationship between aging and AD, while the underlying mechanism remains unclear. We assembled data across multi-omics (i.e., epigenetics, transcriptomics, and proteomics, based on frozen tissues from the dorsolateral prefrontal cortex) and neuropathological and clinical traits from the Religious Orders Study and Rush Memory and Aging Project (ROSMAP). Aging was assessed using six DNA methylation clocks (including the Horvath clock, Hannum clock, Levine clock, HorvathSkin clock, Lin clock, and Cortical clock) that capture mortality risk in literature. After accounting for age, we first identified a gene module (including 263 genes) that was related to the integrated aging measure of six clocks, as well as three neuropathological traits of AD (i.e., ß-amyloid, Tau tangles, and tangle density). Interestingly, among 20 key genes with top intramodular connectivity of the module, PBXIP1 was the only one that was significantly associated with all three neuropathological traits of AD at the protein level after Bonferroni correction. Furthermore, PBXIP1 was associated with the clinical diagnosis of AD in both ROSMAP and three independent datasets. Moreover, PBXIP1 may be related to AD through its role in astrocytes and hippocampal neurons, and the mTOR pathway. The results suggest the critical role of PBXIP1 in AD and support the potential and feasibility of using multi-omics data to investigate mechanisms of complex diseases. However, more validations in different populations and experiments in vitro and in vivo are required in the future.

LRP5 competes for SPOP binding to enhance tumorigenesis mediated by Daxx and PD-L1 in prostate cancer.

Genetic factors coordinate with environmental factors to drive the pathogenesis of prostate adenocarcinoma (PRAD). SPOP is one of the most mutated genes and LRP5 mediates lipid metabolism that is abnormally altered in PRAD. Here, we investigated the potential cross-talk between SPOP and LRP5 in PRAD. We find a negative correlation between SPOP and LRP5 proteins in PRAD. SPOP knockdown increased LRP5 protein while SPOP overexpression resulted in LRP5 reduction that was fully rescued by proteasome inhibitors. LRP5 intracellular tail has SPOP binding site and the direct interaction between LRP5 and SPOP was confirmed by Co-IP and GST-pulldown. Moreover, LRP5 competed with Daxx for SPOP-mediated degradation, establishing a dynamic balance among SPOP, LRP5 and Daxx. Overexpression of LRP5 tail could shift this balance to enhance Daxx-mediated transcriptional inhibition, and inhibit T cell activity in a co-culture system. Further, we generated human and mouse prostate cancer cell lines expressing SPOP variants (F133V, A227V, R368H). SPOP-F133V and SPOP-A227V have specific effects in up-regulating the protein levels of PD-1 and PD-L1. Consistently, SPOP-F133V and SPOP-A227V show robust inhibitory effects on T cells compared to WT SPOP in co-culture. This is further supported by the mouse syngeneic model showing that SPOP-F133V and SPOP-A227V enhance tumorigenesis of prostate cancer in in-vivo condition. Taken together, our study provides evidence that SPOP-LRP5 crosstalk plays an essential role, and the genetic variants of SPOP differentially modulate the expression and activity of immune checkpoints in prostate cancer.

Phenethyl isothiocyanate inhibits metastasis potential of non-small cell lung cancer cells through FTO mediated TLE1 m6A modification.

N6-methyladenosine (m6A) modification is a prevalent RNA epigenetic modification, which plays a crucial role in tumor progression including metastasis. Isothiocyanates (ITCs) are natural compounds and inhibit the tumorigenesis of various cancers. Our previous studies show that ITCs inhibit the proliferation and metastasis of non-small cell lung cancer (NSCLC) cells, and have synergistic effects with chemotherapy drugs. In this study, we investigated the molecular mechanisms underlying the inhibitory effects of ITCs on cancer cell metastasis. We showed that phenethyl isothiocyanate (PEITC) dose-dependently inhibited the cell viability of both NSCLC cell lines H1299 and H226 with IC50 values of 17.6 and 15.2 µM, respectively. Furthermore, PEITC dose-dependently inhibited the invasion and migration of H1299 and H226 cells. We demonstrated that PEITC treatment dose-dependently increased m6A methylation levels and inhibited the expression of the m6A demethylase fat mass and obesity-associated protein (FTO) in H1299 and H226 cells. Knockdown of FTO significantly increased m6A methylation in H1299 and H226 cells, impaired their abilities of invasion and migration in vitro, and enhanced the inhibition of PEITC on tumor growth in vivo. Overexpression of FTO promoted the migration of NSCLC cells, and also mitigated the inhibitory effect of PEITC on migration of NSCLC cells. Furthermore, we found that FTO regulated the mRNA m6A modification of a transcriptional co-repressor Transducin-Like Enhancer of split-1 (TLE1) and further affected its stability and expression. TCGA database analysis revealed TLE1 was upregulated in NSCLC tissues compared to normal tissues, which might be correlated with the metastasis status. Moreover, we showed that PEITC suppressed the migration of NSCLC cells by inhibiting TLE1 expression and downstream Akt/NF-κB pathway. This study reveals a novel mechanism underlying ITC's inhibitory effect on metastasis of lung cancer cells, and provided valuable information for developing new therapeutics for lung cancer by targeting m6A methylation.

Daxx knockdown promoted rDNA transcription without impairing H3.3 expression in mouse preimplantation embryos.

During fertilization, DAXX (death domain-associated protein) mediates histone variant H3.3 incorporation into heterochromatin, which plays an important role in the maintenance of genomic integrity. rDNA, the ribosomal gene, is included in the first wave of gene activation after fertilization. Our and other studies indicated that loss of Daxx disturbs rDNA heterochromatinization and promotes rDNA transcription without change in protein expression of H3.3. However, maternal and zygotic deletion of Daxx impairs blastocyst development. Whether Daxx knockdown affects H3.3 expression and improves the rDNA transcription in preimplantation development has not been reported. In the present study, we injected HA-labelled H3.3 (H3.3-HA) into oocytes during ICSI procedure, and detected H3.3 and DAXX by immunofluorescent staining. Then, we knockdowned Daxx and detected the gene expression levels of Daxx, H3.3, 18s and 47s rRNA. We also performed immunofluorescent staining of B23, γH2A and EdU incorporation to demonstrate nuclear structure, DNA damage and replication. We found injection of H3.3-HA did not impair preimplantation development. Daxx siRNA did not change expression of H3.3 mRNA, and the development of two-cell embryos and blastocysts, but the overall replication and expression levels of rRNA were increased compared with that in the control group. Finally, knockdown of DAXX did not aggravate the DNA damage but loosened the nucleolus. We concluded that Daxx knockdown promoted DNA replication and rDNA transcription, but did not affect H3.3 expression and subsequent preimplantation development.