Chromatin Immunoprecipitation Polymerase Chain Reaction Research Articles

The effect of Toxoplasma gondii infection on galectin-9 expression in decidual macrophages contributing to dysfunction of decidual NK cells during pregnancy

BackgroundToxoplasma gondii infection causes adverse pregnancy outcomes by affecting the expression of immunotolerant molecules in decidual immune cells. Galectin-9 (Gal-9) is widely expressed in decidual macrophages (dMφ) and is crucial for maintaining normal pregnancy by interacting with the immunomodulatory protein T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3). However, the effects of T. gondii infection on Gal-9 expression in dMφ, and the impact of altered Gal-9 expression levels on the maternal–fetal tolerance function of decidual natural killer (dNK) cells, are still unknown.MethodsPregnancy outcomes of T. gondii-infected C57BL/6 and Lgals9−/− pregnant mice models were recorded. Expression of Gal-9, c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), and Forkhead box protein O1 (FOXO1) was detected by western blotting, flow cytometry or immunofluorescence. The binding of FOXO1 to the promoter of Lgals9 was determined by chromatin immunoprecipitation–polymerase chain reaction (ChIP-PCR). The expression of extracellular signal-regulated kinase (ERK), phosphorylated ERK (p-ERK), cAMP-response element binding protein (CREB), phosphorylated CREB (p-CREB), T-box expressed in T cells (T-bet), interleukin 10 (IL-10), and interferon gamma (IFN-γ) in dNK cells was assayed by western blotting.ResultsToxoplasma gondii infection increased the expression of p-JNK and FOXO1 in dMφ, resulting in a reduction in Gal-9 due to the elevated binding of FOXO1 with Lgals9 promoter. Downregulation of Gal-9 enhanced the phosphorylation of ERK, inhibited the expression of p-CREB and IL-10, and promoted the expression of T-bet and IFN-γ in dNK cells. In the mice model, knockout of Lgals9 aggravated adverse pregnancy outcomes caused by T. gondii infection during pregnancy.ConclusionsToxoplasma gondii infection suppressed Gal-9 expression in dMφ by activating the JNK/FOXO1 signaling pathway, and reduction of Gal-9 contributed to dysfunction of dNK via Gal-9/Tim-3 interaction. This study provides new insights for the molecular mechanisms of the adverse pregnancy outcomes caused by T. gondii.Graphical

Effect of SIRT7 on inhibiting the epithelial-mesenchymal transformation in pancreatic cancer cells and related mechanism

Objective: To investigate the effect and mechanism of SIRT7 in epithelial mesenchymal transformation (EMT) of pancreatic cancer cells. Methods: The pancreatic cancer cells were divided into siControl, siSIRT7, over-expression SIRT7, siSIRT7+siCOL4A1, and siSIRT7+siSLUG groups using siRNA or plasmid transfection. The proliferation, migration and invasion of pancreatic cancer cells were detected by EdU, wound healing assay and Transwell experiments, respectively. The expression of EMT and cancer stem cell (CSC) markers were detected by quantitative real-time reverse transcription polymerase chain reaction assay (qRT-PCR) and western blot. RNA sequencing (RNA-seq) in SIRT7 knockdown PANC-1 cells was performed to explore the signaling pathways and target genes regulated by SIRT7. Then the target genes directly regulated by SIRT7 were identified with quantitative chromatin immunoprecipitation experiment (q-ChIP) and chromatin immunoprecipitation polymerase chain reaction (ChIP-PCR). The expressions of SIRT7 and target genes were detected by immunohistochemical (IHC) in pancreatic cancer tissues, and the correlation between SIRT7 and target gene expression was analyzed using TCGA dataset. The correlation between expression of SIRT7 or target genes and survival was analyzed on KM-plotter website. Finally, GeneMANIA, STRING and ENCORI were used to predict SIRT7-related proteins and miRNAs. Results: EdU assay showed that the cell proliferation rates in SIRT7-overexpressed PANC-1 [(19.33±0.35)%] and BxPC-3 cells [(17.00±1.89)%] were lower than those in the control group [(31.60±1.37)% and (24.33±0.78)%, respectively, P＜0.05]. The proliferation rates of SIRT7-knockdown PANC-1 [(23.94±1.00)% and (27.08±0.97)%] and BxPC-3 cells [(22.00±1.86)% and (25.96±1.61)%] were higher than those of the siControl group [(11.80±1.86)% and (13.42±1.39)%, respectively, P＜0.05]. In PANC-1 cells, the wound healing assay showed that the relative migration rate of SIRT7-overexpression cells [(76.67±2.74)%] was lower than that of control cells [(100.00±2.13)%, P＜0.05]; the relative migration rate of cells with SIRT7 knockdown [(134.22±4.08)% and (199.82±9.20)%, respectively] was higher than that of siControl group [(102.24±3.13)%, P＜0.05]. Compared with the control group, SIRT7 overexpression decreased the number of migrated BxPC-3 cells (45.66±1.69 vs 28.33±2.62, P＜0.05); while SIRT7 knockdown increased these numbers (65.66±2.86 and 82.00±2.94 versus 33.00±0.81, P＜0.01). Transwell experiment revealed that the number of invaded cells in SIRT7 overexpression groups (16.33±2.05 and 34.66±1.69) was lower than that control groups (54.33±4.64 and 58.66±5.90, P＜0.05); with SIRT7 knockdown, the numbers of invaded PANC-1 (63.66±2.49 and 69.33±3.29) and BxPC-3 cells (134.33±3.09 and 181.66±4.02) were higher than those in control groups (35.33±2.49 and 42.00±0.81, P˂0.05). Also, SIRT7 knockdown decreased the expressions of epithelial markers and increased the expressions of mesenchymal and CSC markers. RNA-seq analysis showed that SIRT7 was involved in regulating a variety of cancer-related signaling pathways, including the pancreatic cancer pathway and the EMT pathway. Furthermore, SIRT7 could directly bind to the promoter regions of target genes, such as COL4A1 and SLUG. SIRT7 was negatively correlated with the expression and function of COL4A1 and SLUG in pancreatic cancer cells. The expressions of SIRT7, COL4A1, SLUG and SOX2 were verified in pancreatic cancer tissues by IHC. Finally, SIRT7 was predicted to be associated with many proteins and miRNAs based on GeneMANIA, STRING, and ENCORI online tools. Conclusions: SIRT7 can inhibit the EMT of pancreatic cancer cells through transcriptionally inhibiting the expression of target genes, such as COL4A1 and SLUG. Thus, SIRT7 may serve as a potential tumor suppressor gene in pancreatic cancer.

Cdx1 and Gsc distinctly regulate the transcription of BMP4 target gene ventx3.2 by directly binding to the proximal promoter region in Xenopus gastrulae

A comprehensive regulatory network of transcription factors controls the dorsoventral patterning of the body axis in developing vertebrate embryos. Bone morphogenetic protein signaling is essential for activating the Ventx family of homeodomain transcription factors, which regulates embryonic patterning and germ layer identity during Xenopus gastrulation. Although Ventx1.1 and Ventx2.1 of the Xenopus Ventx family have been extensively investigated, Ventx3.2 remains largely understudied. Therefore, this study aimed to investigate the transcriptional regulation of ventx3.2 during the embryonic development of Xenopus. We used goosecoid (Gsc) genome-wide chromatin immunoprecipitation-sequencing data to isolate and replicate the promoter region of ventx3.2. Serial deletion and site-directed mutagenesis were used to identify the cis-acting elements for Gsc and caudal type homeobox 1 (Cdx1) within the ventx3.2 promoter. Cdx1 and Gsc differentially regulated ventx3.2 transcription in this study. Additionally, positive cis-acting and negative response elements were observed for Cdx1 and Gsc, respectively, within the 5' flanking region of the ventx3.2 promoter. This result was corroborated by mapping the active Cdx1 response element (CRE) and Gsc response element (GRE). Moreover, a point mutation within the CRE and GRE completely abolished the activator and repressive activities of Cdx1 and Gsc, respectively. Furthermore, the chromatin immunoprecipitation-polymerase chain reaction confirmed the direct binding of Cdx1 and Gsc to the CRE and GRE, respectively. Inhibition of Cdx1 and Gsc activities at their respective functional regions, namely, the ventral marginal zone and dorsal marginal zone, reversed their effects on ventx3.2 transcription. These results indicate that Cdx1 and Gsc modulate ventx3.2 transcription in the ventral marginal zone and dorsal marginal zone by directly binding to the promoter region during Xenopus gastrulation.

FLRT3 and TGF-β/SMAD4 signalling: Impacts on apoptosis, autophagy and ion channels in supraventricular tachycardia.

To explore the underlying molecular mechanisms of supraventricular tachycardia (SVT), this study aimed to analyse the complex relationship between FLRT3 and TGF-β/SMAD4 signalling pathway, which affects Na+ and K+ channels in cardiomyocytes. Bioinformatics analysis was performed on 85 SVT samples and 15 healthy controls to screen overlapping genes from the key module and differentially expressed genes (DEGs). Expression profiling of overlapping genes, coupled with Receiver Operating Characteristic (ROC) curve analyses, identified FLRT3 as a hub gene. Invitro studies utilizing Ang II-stimulated H9C2 cardiomyocytes were undertaken to elucidate the consequences of FLRT3 silencing on cardiomyocyte apoptosis and autophagic processes. Utilizing a combination of techniques such as quantitative reverse-transcription polymerase chain reaction (qRT-PCR), western blotting (WB), flow cytometry, dual-luciferase reporter assays and chromatin immunoprecipitation polymerase chain reaction (ChIP-PCR) assays were conducted to decipher the intricate interactions between FLRT3, the TGF-β/SMAD4 signalling cascade and ion channel gene expression. Six genes (AADAC, DSC3, FLRT3, SYT4, PRR9 and SERTM1) demonstrated reduced expression in SVT samples, each possessing significant clinical diagnostic potential. In H9C2 cardiomyocytes, FLRT3 silencing mitigated Ang II-induced apoptosis and modulated autophagy. With increasing TGF-β concentration, there was a dose-responsive decline in FLRT3 and SCN5A expression, while both KCNIP2 and KCND2 expressions were augmented. Moreover, a direct interaction between FLRT3 and SMAD4 was observed, and inhibition of SMAD4 expression resulted in increased FLRT3 expression. Our results demonstrated that the TGF-β/SMAD4 signalling pathway plays a critical role by regulating FLRT3 expression, with potential implications for ion channel function in SVT.

Regulation of Axon Guidance by Slit2 and Netrin-1 Signaling in the Lacrimal Gland of Aqp5 Knockout Mice.

Dry eye disease (DED) is multifactorial and associated with nerve abnormalities. We explored an Aquaporin 5 (AQP5)-deficiency-induced JunB activation mechanism, which causes abnormal lacrimal gland (LG) nerve distribution through Slit2 upregulation and Netrin-1 repression. Aqp5 knockout (Aqp5-/-) and wild-type (Aqp5+/+) mice were studied. LGs were permeabilized and stained with neuronal class III β-tubulin, tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), and calcitonin gene-related peptide (CGRP). Whole-mount images were acquired through tissue clearing and 3D fluorescence imaging. Mouse primary trigeminal ganglion (TG) neurons were treated with LG extracts and Netrin-1/Slit2 neutralizing antibody. Transcription factor (TF) prediction and chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) experiments verified the JunB binding and regulatory effect on Netrin-1 and Slit2. Three-dimensional tissue and section immunofluorescence showed reduced LG nerves in Aqp5-/- mice, with sympathetic and sensory nerves significantly decreased. Netrin-1 was reduced and Slit2 increased in Aqp5-/- mice LGs. Aqp5+/+ mice LG tissue extracts (TEs) promoted Aqp5-/- TG neurons axon growth, but Netrin-1 neutralizing antibody (NAb) could inhibit that promotion. Aqp5-/- mice LG TEs inhibited Aqp5+/+ TG axon growth, but Slit2 NAb alleviated that inhibition. Furthermore, JunB, a Netrin-1 and Slit2 TF, could bind them and regulate their expression. SR11302, meanwhile, reversed the Netrin-1 and Slit2 shifts caused by AQP5 deficiency. AQP5 deficiency causes LG nerve abnormalities. Persistent JunB activation, the common denominator for Netrin-1 suppression and Slit2 induction, was found in Aqp5-/- mice LG epithelial cells. This affected sensory and sympathetic nerve fibers' distribution in LGs. Our findings provide insights into preventing, reversing, and treating DED.

Mutation c.-379 C>T in DGAT1 affects intramyocellular lipid content by altering MYOD1 binding affinity.

Intramuscular fat (IMF) is one of the most important indexes of pork taste quality. Diacylglycerol acyltransferase 1 (DGAT1), belonging to the acyl-coenzyme A: DGAT enzymes family, is a rate-limiting enzyme responsible for the final step of triglyceride (TG) synthesis. It is involved in TG storage in skeletal muscle; however, the underlying mechanism is not well understood. This study aimed to uncover functional mutations that can influence DGAT1 expression and consequently affect IMF deposition in pork. Two experimental groups containing individuals with high and low IMF content (6.23 ± 0.20 vs. 1.25 ± 0.05, p < 0.01) were formed from 260 Duroc × Large White × Yorkshire (D × L × Y) cross-bred pigs. A novel SNP c.-379 C>T was uncovered in the DGAT1 gene using comparative sequencing with pool DNA of high- and low-IMF groups. The IMF content of CT genotype individuals (3.19 ± 0.11%) was higher than that of CC genotype individuals (2.86 ± 0.11%) when analyzing 260 D × L × Y pigs (p < 0.05). The DGAT1 expression levels revealed a significant positive correlation with IMF content (r=0.33, p < 0.01). Luciferase assay revealed that the DGAT1 promoter with the c.-379 T allele has a higher transcription activity than that bearing the C allele in C2C12 myoblast cells, but not in 3T3-L1 pre-adipocytes. Online prediction followed by chromatin immunoprecipitation-polymerase chain reaction assay confirmed that myogenic determination factor 1 (MYOD1) binds to the DGAT1 promoter with the c.-379 C allele but not the T allele. In vitro experiments demonstrated that MYOD1 represses DGAT1 transcription and lipogenesis. As a muscle-specific transcription factor, MYOD1 can inhibit the transcription of DGAT1 with the c.-379 C allele in muscle cells. However, in the absence of MYOD1 binding to the mutated DGAT1 promoter with the c.-379 T allele, DGAT1 expresses at a higher level in the muscle cells of the c.-379 T genotype, leading to more intramyocellular lipid accumulation than in the muscle cells of the c.-379 C genotype. The SNP c.-379 C>T in the promoter region of the DGAT1 gene provides a promising molecular marker for improving pork IMF content without affecting other fat depots.

NLRP3 Attenuates Intraocular Inflammation by Inhibiting AIM2-Mediated Pyroptosis Through the Phosphorylated Salt-Inducible Kinase 1/Sterol Regulatory Element Binding Transcription Factor 1 Pathway.

The NLRP3 inflammasome has been shown to be involved in the development of uveitis, but the exact mechanism remains elusive. This study was undertaken to explore the role of NLRP3 in the development of uveitis. First, Nlrp3-deficient mice were used to study the role of NLRP3 in experimental autoimmune diseases, such as experimental autoimmune uveitis (EAU) and experimental autoimmune encephalomyelitis (EAE). Next, the gathering of ASC, activation of caspase 1 and gasdermin D, and secretion of lactate dehydrogenase and interleukin-1β were detected to confirm macrophage pyroptosis and AIM2 activation in the Nlrp3-/- mice. Additionally, RNA sequencing and chromatin immunoprecipitation-polymerase chain reaction were used to investigate the phosphorylated salt-inducible kinase 1 (p-SIK1)/sterol regulatory element binding transcription factor 1 (SREBF1) pathway, which regulates the transcription of Aim2. Finally, overexpression of Nlrp3 was applied to treat EAU. Surprisingly, our findings show that NLRP3 plays an antiinflammatory role in 2 models of EAU and EAE. Additionally, macrophages show an increased M1 activation and pyroptosis in Nlrp3-/- mice. Further experiments indicate that this pyroptosis of macrophages was mediated by the up-regulated transcription of Aim2 as a result of Nlrp3 deficiency. In mechanistic studies, Nlrp3 deficiency was implicated in the down-regulation of p-SIK1 and subsequently the up-regulation of SREBF1, which binds to Aim2 and then promotes the latter's transcription. Finally, Aim2 deficiency, RNA silencing of Aim2 or Srebf1, and overexpression of Nlrp3 resulted in attenuated inflammation of EAU. Our data demonstrate that NLRP3 inhibits AIM2 inflammasome-mediated EAU by regulating the p-SIK1/SREBF1 pathway, highlighting the therapeutic potential of targeting Nlrp3.

The R2R3-MYB transcription factor ThRAX2 recognized a new element MYB-T (CTTCCA) to enhance cadmium tolerance in Tamarix hispida

R2R3-MYB transcription factors play an important role in plant development and response to various environmental stresses. In this study, a new R2R3-MYB gene, named ThRAX2, was isolated from T. hispida. ThRAX2 has an open reading frame (ORF) of 1191 bp and encodes a protein of 396 amino acids. ThRAX2 was localized in the nucleus. The overexpression of ThRAX2 in Arabidopsis and T. hispida significantly increased Cadmium (Cd) tolerance. Moreover, the accumulation of cadmium in roots and leaves was significantly reduced. The TF-centred Y1H and Y1H results showed that ThRAX2 was able to specifically bind a new cis-element (MYB-T, CTTCCA). The promoters of some Cd-responsive genes, such as ThSOS1, ThCKX3, ThCAX3A, ThMYB78, ThMIP2, ThTPS4, and ThSOD2, all contained 1–3 MYB-T sequences. Furthermore, chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) and ChIPquantitative (q)PCR showed that the ThRAX2 gene can bind to ThSOS1, ThCKX3, ThCAX3A and ThMYB78 promoter fragments, including the MYB-T motif. Meanwhile, the qRTPCR results also showed that the expression trends of ThSOS1, ThCKX3, ThCAX3A and ThMYB78 were similar to that of ThRAX2. This finding suggests that Cd tolerance of the ThRAX2 gene may regulate the expression of some downstream genes through specific recognition of the MYB-T motif and participate in regulating intracellular ion homeostasis, transport, and protein activity or enhance antioxidant enzyme activity. This study found a novel cis-acting element that binds ThRAX2 to regulate Cd tolerance, which lays the foundation for the ThRAX2 regulatory mechanism of Cd stress. This study provides a genetic and theoretical basis for the bioremediation of Cd-contaminated land by cultivating transgenic plants in the future.

Identification of a Novel GRHL3/HOPX/Wnt/β-Catenin Proto-oncogenic Axis in Squamous Cell Carcinoma of the Esophagus

Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with a poor long-term prognosis. The molecular mechanisms underlying the initiation and progression of this tumor are largely unknown. The transcription factor GRHL3 functions as a potent tumor suppressor in SCC of skin, head, and neck. This study aims to determine whether GRHL3 also plays a role in the homeostasis of the esophageal epithelium and in the development of ESCC. The effects of Grhl3 deletion on squamous epithelial homeostasis in embryos and adult mice were examined using immunohistochemistry, transmission electron microscopy, and real-time polymerase chain reaction. The conditionally deleted mice were subsequently used to determine susceptibility to ESCC. Whole-transcriptome sequencing (RNA-seq) was performed on ESCC in wild-type and Grhl3 deleted animals. To decipher the signaling pathways, real-time polymerase chain reaction, immunohistochemistry, analysis of chromatin immunoprecipitation sequencing, chromatin immunoprecipitation-polymerase chain reaction, and RNA seq datasets were used. Primary human samples were used to validate the findings in the mouse model. Loss of Grhl3 perturbs the proliferation-differentiation balance in the esophageal epithelium, thereby increasing the susceptibility to esophageal carcinogenesis in adult mice. Grhl3 imparts its tumor suppressor function by regulating the expression of HOPX. We have identified the Wnt/β-catenin pathway as the downstream effectors of GRHL3 and HOPX through our integrated approach using patient-derived ESCC samples and mouse models. GRHL3 conveys its tumor suppressor function in ESCC through regulating its target gene HOPX, which limits Wnt/β-catenin signaling. Targeted therapies to inhibit thispathway could be a potential treatment strategy for ESCC patients with reduced GRHL3 expression.

NF-κB/p65 Competes With Peroxisome Proliferator-Activated Receptor Gamma for Transient Receptor Potential Channel 6 in Hypoxia-Induced Human Pulmonary Arterial Smooth Muscle Cells.

Objective: Peroxisome proliferator-activated receptor gamma (PPARγ) has an anti-proliferation effect on pulmonary arterial smooth muscle cells (PASMCs) via the transient receptor potential channel (TRPC) and protects against pulmonary artery hypertension (PAH), whereas nuclear factor-kappa B (NF-κB) has pro-proliferation and pro-inflammation effects, which contributes to PAH. However, the association between them in PAH pathology remains unclear. Therefore, this study aimed to investigate this association and the mechanisms underlying TRPC1/6 signaling-mediated PAH.Methods: Human pulmonary arterial smooth muscle cells (hPASMCs) were transfected with p65 overexpressing (pcDNA-p65) and interfering plasmids (shp65) and incubated in normal and hypoxic conditions (4% O2 and 72 h). The effects of hypoxia and p65 expression on cell proliferation, invasion, apoptosis, [Ca2+]i, PPARγ, and TRPC1/6 expression were determined using Cell Counting Kit-8 (CCK-8), Transwell, Annexin V/PI, Fura-2/AM, and western blotting, respectively. In addition, the binding of p65 or PPARγ proteins to the TRPC6 promoter was validated using a dual-luciferase report assay, chromatin-immunoprecipitation-polymerase chain reaction (ChIP-PCR), and electrophoretic mobility shift assay (EMSA).Results: Hypoxia inhibited hPASMC apoptosis and promoted cell proliferation and invasion. Furthermore, it increased [Ca2+]i and the expression of TRPC1/6, p65, and Bcl-2 proteins. Moreover, pcDNA-p65 had similar effects on hypoxia treatment by increasing TRPC1/6 expression, [Ca2+]i, hPASMC proliferation, and invasion. The dual-luciferase report and ChIP-PCR assays revealed three p65 binding sites and two PPARγ binding sites on the promoter region of TRPC6. In addition, hypoxia treatment and shPPARγ promoted the binding of p65 to the TRPC6 promoter, whereas shp65 promoted the binding of PPARγ to the TRPC6 promoter.Conclusion: Competitive binding of NF-κB p65 and PPARγ to TRPC6 produced an anti-PAH effect.

Arsenic Trioxide Promotes Tumor Progression by Inducing the Formation of PGCCs and Embryonic Hemoglobin in Colon Cancer Cells.

Arsenic trioxide (ATO) has been used to treat acute promyelocytic leukemia. However, it is not effective in treating solid tumors such as colorectal cancer. We have previously reported that polyploid giant cancer cells (PGCCs) exhibiting the characteristics of cancer stem cells can be generated by various inducers. In this study, ATO was used to induce the formation of PGCCs in LoVo and Hct116 colon cancer cell lines. The migration, invasion, and proliferation abilities of colon cancer cells with and without ATO treatment were assessed by wound-healing, transwell, and plate colony formation assays. The expression of epithelial to mesenchymal transition-related proteins and erythroid differentiation-related proteins in colon cancer cells was further evaluated by western blot and immunocytochemical assays. LoVo and Hct116 cells were transfected with a eukaryotic expression vector for green fluorescent protein (GFP), red fluorescent protein (RFP), H2B-GFP, and H2B-mCherry to study PGCCs formation via cell fusion. WB and ICC assays were performed to assess the expression of cell fusion-related proteins. MG132, small interfering RNA-glial cell missing 1 (GCM1), and chromatin immunoprecipitation-polymerase chain reaction assays were performed to study the role of GCM1/syncytin-1-mediated cell fusion. Clinically, the significance of cell fusion-related proteins and erythroid differentiation-related proteins expression in human colorectal cancer tissues was evaluated. Results of our study showed that ATO induced the formation of PGCCs, and the daughter cells derived from PGCCs gained a mesenchymal phenotype and exhibited strong migration, invasion, and proliferation abilities. PGCCs also produced embryonic hemoglobin-delta and -zeta with strong oxygen-binding ability and erythroid differentiation-related proteins after ATO treatment. In addition, cell fusion was observed during the formation of PGCCs, indicated by the presence of yellow fluorescence via the GCM1/syncytin-1 signaling pathway. Clinically, the expression of cell fusion-related and erythroid differentiation-related proteins gradually increased with the progression of human colorectal cancer tissues. In conclusion, ATO can promote tumor progression by inducing the formation of PGCCs via GCM1/syncytin-1-mediated cell fusion. PGCCs can produce daughter cells with high invasion and migration abilities and embryonic hemoglobin with strong oxygen binding ability, promoting survival of tumor cells in a hypoxic microenvironment.

PagGRF12a interacts with PagGIF1b to regulate secondary xylem development through modulating PagXND1a expression in Populus alba × P. glandulosa.

Growth-regulating factors (GRFs) are important regulators of plant development and growth, but their possible roles in xylem development in woody plants remain unclear. Here, we report that Populus alba × Papulus glandulosa PagGRF12a negatively regulates xylem development in poplar. PagGRF12a is expressed in vascular tissues. Compared to non-transgenic control plants, transgenic poplar plants overexpressing PagGRF12a exhibited reduced xylem width and plants with repressed expression of PagGRF12a exhibited increased xylem width. Xylem NAC domain 1 (XND1) encodes a NAC domain transcription factor that regulates xylem development and transcriptional analyses revealed that PagXND1a is highly upregulated in PagGRF12a-overexpressing plants and downregulated in PagGRF12a-suppressed plants, indicating that PagGRF12a may regulate xylem development through PagXND1a. Transient transcriptional assays and chromatin immunoprecipitation-polymerase chain reaction assays confirmed that PagGRF12a directly upregulates PagXND1a. In addition, PagGRF12a interacts with the GRF-Interacting Factor (GIF) PagGIF1b, and this interaction enhances the effects of PagGRF12a on PagXND1a. Our results indicate that PagGRF12a inhibits xylem development by upregulating the expression of PagXND1a.

Ustilaginoidea virens modulates lysine 2-hydroxyisobutyrylation in rice flowers during infection.

The post-translational modification lysine 2-hydroxyisobutyrylation (Khib ) plays an important role in gene transcription, metabolism, and enzymatic activity. Khib sites have been identified in rice (Oryza sativa). However, the Khib status of proteins in rice flowers during pathogen infection remains unclear. Here, we report a comprehensive identification of Khib -modified proteins in rice flowers, and the changes in these proteins during infection with the fungal pathogen Ustilaginoidea virens. By using a tandem mass tag-based quantitative proteomics approach, we identified 2,891 Khib sites on 964 proteins in rice flowers. Our data demonstrated that 2-hydroxyisobutyrylated proteins are involved in diverse biological processes. Khib levels were substantially reduced upon infection with U. virens. Chromatin immunoprecipitation polymerase chain reaction (PCR) and reverse transcription quantitative PCR analyses revealed that histone Khib is involved in the expression of disease-resistance genes. More importantly, most quantified sites on core histones H3 were downregulated upon U. virens infection. In addition, the histone deacetylases HDA705, HDA716, SRT1, and SRT2 are involved in the removal of Khib marks in rice. HDA705 was further confirmed to negatively regulate rice disease resistance to pathogens U. virens, Magnaporthe oryzae, and Xanthomonas oryzae pv. oryzae (Xoo). Our data suggest that U. virens could modulate Khib in rice flowers during infection.

Smyd1 Orchestrates Early Heart Development Through Positive and Negative Gene Regulation.

SET and MYND domain-containing protein 1 (Smyd1) is a striated muscle-specific histone methyltransferase. Our previous work demonstrated that deletion of Smyd1 in either cardiomyocytes or the outflow tract (OFT) resulted in embryonic lethality at E9.5, with cardiac structural defects such as truncation of the OFT and right ventricle and impaired expansion and proliferation of the second heart field (SHF). The cardiac phenotype was accompanied by the downregulation of ISL LIM Homeobox 1 (Isl1) and upregulation of atrial natriuretic factor (ANF). However, the mechanisms of Smyd1 regulating Isl1 and ANF during embryonic heart development remain to be elucidated. Here, we employed various biochemical and molecular biological approaches including chromatin immunoprecipitation polymerase chain reaction (ChIP-PCR), pGL3 fluorescence reporter system, and co-immunoprecipitation (CoIP) and found that Smyd1 interacted with absent small homeotic-2-like protein (ASH2L) and activated the promoter of Isl1 by trimethylating H3K4. We also found that Smyd1 associated with HDAC to repress ANF expression using trichostatin A (TSA), a deacetylase inhibitor. In conclusion, Smyd1 participates in early heart development by upregulating the expression of Isl1 and downregulating the expression of ANF.

PspC domain-containing protein (PCP) determines Streptococcus mutans biofilm formation through bacterial extracellular DNA release and platelet adhesion in experimental endocarditis.

Bacterial extracellular DNA (eDNA) and activated platelets have been found to contribute to biofilm formation by Streptococcus mutans on injured heart valves to induce infective endocarditis (IE), yet the bacterial component directly responsible for biofilm formation or platelet adhesion remains unclear. Using in vivo survival assays coupled with microarray analysis, the present study identified a LiaR-regulated PspC domain-containing protein (PCP) in S. mutans that mediates bacterial biofilm formation in vivo. Reverse transcriptase- and chromatin immunoprecipitation-polymerase chain reaction assays confirmed the regulation of pcp by LiaR, while PCP is well-preserved among streptococcal pathogens. Deficiency of pcp reduced in vitro and in vivo biofilm formation and released the eDNA inside bacteria floe along with reduced bacterial platelet adhesion capacity in a fibrinogen-dependent manner. Therefore, LiaR-regulated PCP alone could determine release of bacterial eDNA and binding to platelets, thus contributing to biofilm formation in S. mutans-induced IE.

METTL3/METTL14 Transactivation and m6A-Dependent TGF-β1 Translation in Activated Kupffer Cells

Background and AimsTransforming growth factor β1 (TGF-β1) secreted from activated Kupffer cells (KC) promotes the progression of nonalcoholic steatohepatitis (NASH) to liver fibrosis. N6-methyladenosine (m6A) RNA modification participates in various cell stress responses, yet it remains unknown whether it plays a role in TGF-β1 upregulation in activated KCs.MethodsWestern blot, dot blot, and liquid chromatography with tandem mass spectrometry were used to determine the expression of m6A methyltransferase, METTL3, and METTL14, as well as global m6A modification. RNA-sequencing and m6A-seq were employed to screen differentially expressed genes and responsive m6A peaks. Nuclear factor κB (NF-κB)–mediated METTL3/METTL14 transactivation were validated with chromatin immunoprecipitation polymerase chain reaction and dual-luciferase reporter system, and the role of m6A in TGF-β1 upregulation was further verified in METTL3/METTL14-deficient KCs and myeloid lineage cell-specific METTL14 knockout mice.ResultsSerum lipopolysaccharide (LPS) concentration is increased in high-fat diet–induced NASH rats. TGF-β1 upregulation is closely associated with METTL3/METTL14 upregulation and global m6A hypermethylation, in both NASH rat liver and LPS-activated KCs. LPS-responsive m6A peaks are identified on the 5′ untranslated region (UTR) of TGF-β1 messenger RNA (mRNA). NF-κB directly transactivates METTL3 and METTL14 genes. LPS-stimulated TGF-β1 expression is abolished in METTL3/METTL14-deficient KCs and myeloid lineage cell-specific METTL14 knockout mice. Mutation of m6A sites on the 5′UTR of TGF-β1 mRNA blocks LPS-induced increase of luciferase reporter activity.ConclusionsNF-κB acts as transcription factor to transactivate METTL3/METTL14 genes upon LPS challenge, leading to global RNA m6A hypermethylation. Increased m6A on the 5′UTR of TGF-β1 mRNA results in m6A-dependent translation of TGF-β1 mRNA in a cap-independent manner. We identify a novel role of m6A modification in TGF-β1 upregulation, which helps to shed light on the molecular mechanism of NASH progression.

Identifying the p65-Dependent Effect of Sulforaphene on Esophageal Squamous Cell Carcinoma Progression via Bioinformatics Analysis.

Understanding the mechanism by which sulforaphene (SFE) affects esophageal squamous cell carcinoma (ESCC) contributes to the application of this isothiocyanate as a chemotherapeutic agent. Thus, we attempted to investigate SFE regulation of ESCC characteristics more deeply. We performed gene set enrichment analysis (GSEA) on microarray data of SFE-treated ESCC cells and found that differentially expressed genes are enriched in TNFα_Signaling_via_the_NFκB_Pathway. Coupled with the expression profile data from the GSE20347 and GSE75241 datasets, we narrowed the set to 8 genes, 4 of which (C-X-C motif chemokine ligand 10 (CXCL10), TNF alpha induced protein 3 (TNFAIP3), inhibin subunit beta A (INHBA), and plasminogen activator, urokinase (PLAU)) were verified as the targets of SFE. RNA-sequence (RNA-seq) data of 182 ESCC samples from The Cancer Genome Atlas (TCGA) were grouped into two phenotypes for GSEA according to the expression of CXCL10, TNFAIP3, INHBA, and PLAU. The enrichment results proved that they were all involved in the NFκB pathway. ChIP-seq analyses obtained from the Cistrome database indicated that NFκB-p65 is likely to control the transcription of CXCL10, TNFAIP3, INHBA, and PLAU, and considering TNFAIP3 and PLAU are the most significantly differentially expressed genes, we used chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) to verify the regulation of p65 on their expression. The results demonstrated that SFE suppresses ESCC progression by down-regulating TNFAIP3 and PLAU expression in a p65-dependent manner.

CAMTA3 negatively regulates disease resistance through modulating immune response and extensive transcriptional reprogramming in cassava.

As one of the important crops in the world, cassava production is seriously threatened by Xanthomonas axonopodis pv. manihotis (Xam) all year round. Calmodulin-binding transcription activators (CAMTAs) play key roles in biotic stress and abiotic stress in plants, however, their roles in cassava remain elusive. In this study, six MeCAMTAs were identified, and MeCAMTA3 with the highest induction upon Xam infection was confirmed as a transcription factor that binds to the vCGCGb motif. MeCAMTA3 negatively regulates plant disease resistance against Xam. On the one hand, MeCAMTA3 negatively regulated endogenous salicylic acid and reactive oxygen species accumulation, pathogenesis-related genes MePRs' transcripts and callose deposition during cassava-Xam interaction but not under control conditions. On the other hand, RNA sequencing showed extensive transcriptional reprogramming by MeCAMTA3, especially 18 genes with a vCGCGb motif in the promoter region in hormone signaling, antioxidant signaling and other disease resistance signaling. Notably, chromatin immunoprecipitation-polymerase chain reaction showed that eight of these genes might be directly regulated by MeCAMTA3 through transcriptional repression. In summary, MeCAMTA3 negatively regulates plant disease resistance against cassava bacterial blight through modulation of multiple immune responses during cassava-Xam interaction and extensive transcriptional reprogramming.

Epigenetic Changes During Human Thyroid Cell Differentiation.

Objective: It has been demonstrated that the transcription factors TAZ (transcriptional coactivator with PDZ-binding motif), paired box gene 8 (PAX8), and NK2 homeobox 1 (NKX2-1) are coexpressed in the nucleus of thyroid cells. Furthermore, TAZ is known to enhance the transcriptional activity of PAX8 and NKX2-1 as well as the key thyroid-specific gene, thyroglobulin (TG), suggesting a critical role for TAZ in the control of thyroid cell speciation. We previously reported that the small molecule ethacridine, identified as a TAZ activator, was able to induce thyroid-specific transcription in endodermal cells differentiated from human embryonic stem (hES) cells using activin A. Since transcription factors are epigenetically regulated in cell differentiation, we investigated the epigenetic changes in the promoter regions of these key transcription factors during in vitro differentiation of hES cells into thyrocytes. Methods: We initially profiled chromatin accessibility using the technique of Assay for Transposase Accessible Chromatin sequencing (ATAC-seq), and then examined DNA methylation and histone acetylation in the promoter regions of the three selected thyroid transcription factors and the thyroid-specific genes during hES cell differentiation. Results: ATAC-seq analysis showed enriched chromatin accessibility of TAZ, NKX2-1, and PAX8 after exposure to activin A and ethacridine. There were no methylation changes found in the NKX2-1, PAX8, and TAZ promoters by bisulfite sequencing. In contrast, acetylation of histone H4, specifically acetylation of lysine 16, was observed in each of the promoters when measured by chromatin immunoprecipitation polymerase chain reaction assays, which correlated with the activity and expression of NKX2-1 and PAX8 as well as sodium/iodide symporter, thyroid stimulating hormone receptor, and TG genes. Conclusions: These results indicate that ethacridine treatment of activin A-derived endodermal hES cells leads to enhanced chromatin accessibility, which, in turn, allows histone H4 acetylation in the regulation of active genes for speciation of thyroid follicular cells from hES cells.

Glyphosate influences cell proliferation in vitro

Glyphosate is the most widely used herbicide worldwide. However, since the International Agency for Research on Cancer (IARC) classified glyphosate as a Class 2A ‘probable carcinogen’ to humans, the safety of this synthetic chemical has been under debate and requires validation. Most studies on glyphosate have been conducted in animals, and the effects of glyphosate in humans are not fully understood. In the present study, we report that glyphosate increases the rate of cell growth in human embryonic kidney 293 (HEK293) cells. Interestingly, the concentrations of glyphosate ranging between 0.6 and 18 µM enhance cell proliferation, while lower or higher concentrations do not stimulate cell growth or interfere with it, respectively. At the molecular level, cell biology and biochemical data demonstrate that glyphosate promotes the transcription of EGR1, JUN, FOS, and MYC, which are critical transcription factors and immediate early genes for cell cycle progression, and cell cycle regulators including cyclin B1, cyclin D1, and p21. NEAT1, a long non-coding RNAs that is implicated in cancers and active gene transcription are increased in glyphosate-treated cells. In addition, the absorption efficiency of glyphosate to HEK293 cells estimates approximately 11 pM/cell/day, and glyphosate metabolism appears increased when incubated with HEK293 cells. Our findings provide valuable evidence on the effects of glyphosate on human cell growth and suggest that glyphosate promotes cell proliferation by activating gene expression of cell cycle regulators in humans in vitro. Abbreviations: IARC: International Agency for Research on Cancer; EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase; AMPA: aminomethylphosphonic acid; WHO: World Health Organization; EFSA: European Food Safety Authority; RT-qPCR: reverse transcription quantifying PCR; lncRNA: Long non-coding RNA; NEAT1: Nuclear paraspeckle assembly transcript 1; WST: water-soluble tetrazolium salt; ChIP-PCR: chromatin immunoprecipitation-polymerase chain reaction; CDKs: cyclin-dependent protein kinases; MALAT1: metastasis-associated lung adenocarcinoma transcription 1; LC-MS-MS: liquid chromatography-tandem mass spectrometry.