The expression of Igf2 in mammals shows a complex regulation involving multiple promoters and epigenetic mechanisms. We previously identified a novel regulatory mechanism based on the interaction ...between the transcriptional factor ZBED6 and Igf2 intron. Disruption of the ZBED6-Igf2 interaction leads to a dramatic up-regulation of IGF2 expression postnatally. In the current study we characterize an additional layer of regulation involving miR483 encoded by another Igf2 intron. We found a highly significant up-regulation of miR483 expression when the ZBED6-Igf2 axis is disrupted in transgenic mice. Furthermore, CRISPR/Cas9 mediated knock-out of miR483 in C2C12 myoblast cells, both wild-type and cells with disrupted ZBED6-Igf2 axis (Igf2
), resulted in down-regulation of Igf2 expression and a reduced proliferation rate. This was further validated using miR483 mimics and inhibitors. RNA-seq analysis revealed a significant enrichment of genes involved in the PI3K-Akt signaling pathway among genes down-regulated in miR483
cells, including Igf2 down-regulation. The opposite pattern was observed in Igf2
cells, where Igf2 is up-regulated. Our data suggest a positive feedback between miR483 and Igf2 promoter activity, strongly affecting how ZBED6 controls Igf2 expression in various cell types.
Aims/hypothesis
ZBED6 (zinc finger, BED-type containing 6) is known to regulate muscle mass by suppression of
Igf2
gene transcription. In insulin-producing cell lines, ZBED6 maintains proliferative ...capacity at the expense of differentiation and beta cell function. The aim was to study the impact of
Zbed6
knockout on beta cell function and glucose tolerance in C57BL/6 mice.
Methods
Beta cell area and proliferation were determined in
Zbed6
knockout mice using immunohistochemical analysis. Muscle and fat distribution were assessed using micro-computed tomography. Islet gene expression was assessed by RNA sequencing. Effects of a high-fat diet were analysed by glucose tolerance and insulin tolerance tests. ZBED6 was overexpressed in EndoC-βH1 cells and human islet cells using an adenoviral vector. Beta cell cell-cycle analysis, insulin release and mitochondrial function were studied in vitro using propidium iodide staining and flow cytometry, ELISA, the Seahorse technique, and the fluorescent probes JC-1 and MitoSox.
Results
Islets from
Zbed6
knockout mice showed lowered expression of the cell cycle gene
Pttg1
, decreased beta cell proliferation and decreased beta cell area, which occurred independently from ZBED6 effects on
Igf2
gene expression.
Zbed6
knockout mice, but not wild-type mice, developed glucose intolerance when given a high-fat diet. The high-fat diet
Zbed6
knockout islets displayed upregulated expression of oxidative phosphorylation genes and genes associated with beta cell differentiation. In vitro
,
ZBED6 overexpression resulted in increased EndoC-βH1 cell proliferation and a reduced glucose-stimulated insulin release in human islets. ZBED6 also reduced mitochondrial JC-1 J-aggregate formation, mitochondrial oxygen consumption rates (OCR) and mitochondrial reactive oxygen species (ROS) production, both at basal and palmitate + high glucose-stimulated conditions. ZBED6-induced inhibition of OCR was not rescued by IGF2 addition. ZBED6 reduced levels of the mitochondrial regulator PPAR-γ related coactivator 1 protein (PRC) and bound its promoter/enhancer region. Knockdown of PRC resulted in a lowered OCR.
Conclusions/interpretation
It is concluded that ZBED6 is required for normal beta cell replication and also limits excessive beta cell mitochondrial activation in response to an increased functional demand. ZBED6 may act, at least in part, by restricting PRC-mediated mitochondrial activation/ROS production, which may lead to protection against beta cell dysfunction and glucose intolerance in vivo.
Graphical abstract
The immune mechanisms that mediate synovitis and joint destruction in rheumatoid arthritis (RA) remain poorly defined. Although increased levels of CD8
T cells have been described in RA, their ...function in pathogenesis remains unclear. Here we perform single cell transcriptome and T cell receptor (TCR) sequencing of CD8
T cells derived from anti-citrullinated protein antibodies (ACPA)+ RA blood. We identify GZMB
CD8
subpopulations containing large clonal lineage expansions that express cytotoxic and tissue homing transcriptional programs, while a GZMK
CD8
memory subpopulation comprises smaller clonal expansions that express effector T cell transcriptional programs. We demonstrate RA citrullinated autoantigens presented by MHC class I activate RA blood-derived GZMB
CD8
T cells to expand, express cytotoxic mediators, and mediate killing of target cells. We also demonstrate that these clonally expanded GZMB
CD8
cells are present in RA synovium. These findings suggest that cytotoxic CD8
T cells targeting citrullinated antigens contribute to synovitis and joint tissue destruction in ACPA+ RA.
The zinc finger CCCH-type containing 11A (ZC3H11A) gene encodes a well-conserved zinc finger protein that may function in mRNA export as it has been shown to associate with the transcription export ...(TREX) complex in proteomic screens. Here, we report that ZC3H11A is a stress-induced nuclear protein with RNA-binding capacity that localizes to nuclear splicing speckles. During an adenovirus infection, the ZC3H11A protein and splicing factor SRSF2 relocalize to nuclear regions where viral DNA replication and transcription take place. Knockout (KO) of ZC3H11A in HeLa cells demonstrated that several nuclear-replicating viruses are dependent on ZC3H11A for efficient growth (HIV, influenza virus, herpes simplex virus, and adenovirus), whereas cytoplasmic replicating viruses are not (vaccinia virus and Semliki Forest virus). High-throughput sequencing of ZC3H11A–cross-linked RNA showed that ZC3H11A binds to short purine-rich ribonucleotide stretches in cellular and adenoviral transcripts. We show that the RNA-binding property of ZC3H11A is crucial for its function and localization. In ZC3H11A KO cells, the adenovirus fiber mRNA accumulates in the cell nucleus. Our results suggest that ZC3H11A is important for maintaining nuclear export of mRNAs during stress and that several nuclear-replicating viruses take advantage of this mechanism to facilitate their replication.
ZC3H11A is a cellular protein associated with the transcription export (TREX) complex that is induced during heat-shock. Several nuclear-replicating viruses exploit the mRNA export mechanism of ...ZC3H11A protein for their efficient replication. Here we show that ZC3H11A protein plays a role in regulation of NF-κB signal transduction. Depletion of ZC3H11A resulted in enhanced NF-κB mediated signaling, with upregulation of numerous innate immune related mRNAs, including IL-6 and a large group of interferon-stimulated genes. IL-6 upregulation in the absence of the ZC3H11A protein correlated with an increased NF-κB transcription factor binding to the IL-6 promoter and decreased IL-6 mRNA decay. The enhanced NF-κB signaling pathway in ZC3H11A deficient cells correlated with a defect in IκBα inhibitory mRNA and protein accumulation. Upon ZC3H11A depletion The IκBα mRNA was retained in the cell nucleus resulting in failure to maintain normal levels of the cytoplasmic IκBα mRNA and protein that is essential for its inhibitory feedback loop on NF-κB activity. These findings indicate towards a previously unknown mechanism of ZC3H11A in regulating the NF-κB pathway at the level of IkBα mRNA export.
Significance The ZBED6 (zinc finger, BED-type containing 6) transcription factor is unique to placental mammals. Its high degree of sequence conservation among placental mammals indicates that it has ...an essential function. Using two colorectal cancer cell lines we have, for the first time to our knowledge, completely inactivated ZBED6 by genome editing. Our results demonstrate that ZBED6 is not required for cell survival, but its ablation led to consistent changes in cell growth within cell lines but opposite trends between cell lines. The results are in line with the hypothesis that ZBED6 is a transcriptional modulator that does not determine whether or not its target genes are active but fine-tunes their expression. Thus, its effect on tumorigenesis will depend on the transcriptional state of the cell.
The transcription factor ZBED6 (zinc finger, BED-type containing 6) is a repressor of IGF2 whose action impacts development, cell proliferation, and growth in placental mammals. In human colorectal cancers, IGF2 overexpression is mutually exclusive with somatic mutations in PI3K signaling components, providing genetic evidence for a role in the PI3K pathway. To understand the role of ZBED6 in tumorigenesis, we engineered and validated somatic cell ZBED6 knock-outs in the human colorectal cancer cell lines RKO and HCT116. Ablation of ZBED6 affected the cell cycle and led to increased growth rate in RKO cells but reduced growth in HCT116 cells. This striking difference was reflected in the transcriptome analyses, which revealed enrichment of cell-cycleârelated processes among differentially expressed genes in both cell lines, but the direction of change often differed between the cell lines. ChIP sequencing analyses displayed enrichment of ZBED6 binding at genes up-regulated in ZBED6 -knockout clones, consistent with the view that ZBED6 modulates gene expression primarily by repressing transcription. Ten differentially expressed genes were identified as putative direct gene targets, and their down-regulation by ZBED6 was validated experimentally. Eight of these genes were linked to the Wnt, Hippo, TGF-β, EGF receptor, or PI3K pathways, all involved in colorectal cancer development. The results of this study show that the effect of ZBED6 on tumor development depends on the genetic background and the transcriptional state of its target genes.
Transforming growth factor β (TGFβ) induces epithelial–mesenchymal transition (EMT), which correlates with stemness and invasiveness. Mesenchymal–epithelial transition (MET) is induced by TGFβ ...withdrawal and correlates with metastatic colonization. Whether TGFβ promotes stemness and invasiveness simultaneously via EMT remains unclear. We established a breast cancer cell model expressing red fluorescent protein (RFP) under the E‐cadherin promoter. In 2D cultures, TGFβ induced EMT, generating RFPlow cells with a mesenchymal transcriptome, and regained RFP, with an epithelial transcriptome, after MET induced by TGFβ withdrawal. RFPlow cells generated robust mammospheres, with epithelio‐mesenchymal cell surface features. Mammospheres that were forced to adhere generated migratory cells, devoid of RFP, a phenotype which was inhibited by a TGFβ receptor kinase inhibitor. Further stimulation of RFPlow mammospheres with TGFβ suppressed the generation of motile cells, but enhanced mammosphere growth. Accordingly, mammary fat‐pad‐transplanted mammospheres, in the absence of exogenous TGFβ treatment, established lung metastases with evident MET (RFPhigh cells). In contrast, TGFβ‐treated mammospheres revealed high tumour‐initiating capacity, but limited metastatic potential. Thus, the biological context of partial EMT and MET allows TGFβ to differentiate between pro‐stemness and pro‐invasive phenotypes.
Whether TGFβ promotes stemness and invasiveness simultaneously via epithelial‐mesenchymal transition (EMT) still remains unclear. Using a breast cancer cell model expressing red fluorescent protein under the E‐cadherin promoter, we demonstrate that the biological context of partial mesenchymal–epithelial transition (pMET) enriches mammospheres with invasive and pro‐metastatic potential. Sustained partial EMT under TGFβ enriches mammospheres with a pro‐stemness phenotype with higher tumour‐initiating potential.
Abstract
The underlying molecular mechanisms that determine long day versus short day breeders remain unknown in any organism. Atlantic herring provides a unique opportunity to examine the molecular ...mechanisms involved in reproduction timing, because both spring and autumn spawners exist within the same species. Although our previous whole genome comparisons revealed a strong association of
TSHR
alleles with spawning seasons, the functional consequences of these variants remain unknown. Here we examined the functional significance of six candidate
TSHR
mutations strongly associated with herring reproductive seasonality. We show that the L471M missense mutation in the spring-allele causes enhanced cAMP signaling. The best candidate non-coding mutation is a 5.2 kb retrotransposon insertion upstream of the
TSHR
transcription start site, near an open chromatin region, which is likely to affect
TSHR
expression. The insertion occurred prior to the split between Pacific and Atlantic herring and was lost in the autumn-allele. Our study shows that strongly associated coding and non-coding variants at the
TSHR
locus may both contribute to the regulation of seasonal reproduction in herring.
ZBED6 is a recently discovered transcription factor, unique to placental mammals, that has evolved from a domesticated DNA transposon. It acts as a repressor at the IGF2 locus. Here we show that ...ZBED6 acts as a transcriptional modulator in mouse myoblast cells, where more than 700 genes were differentially expressed after Zbed6-silencing. The most significantly enriched GO term was muscle protein and contractile fiber, which was consistent with increased myotube formation. Twenty small nucleolar RNAs all showed increased expression after Zbed6-silencing. The co-localization of histone marks and ZBED6 binding sites and the effect of Zbed6-silencing on distribution of histone marks was evaluated by ChIP-seq analysis. There was a strong association between ZBED6 binding sites and the H3K4me3, H3K4me2 and H3K27ac modifications, which are usually found at active promoters, but no association with the repressive mark H3K27me3. Zbed6-silencing led to increased enrichment of active marks at myogenic genes, in agreement with the RNA-seq findings. We propose that ZBED6 preferentially binds to active promoters and modulates transcriptional activity without recruiting repressive histone modifications.
ZC3H11A (zinc finger CCCH domain-containing protein 11A) is a stress-induced mRNA-binding protein required for efficient growth of nuclear-replicating viruses. The cellular functions of ZC3H11A ...during embryonic development are unknown. Here, we report the generation and phenotypic characterization of
knockout (KO) mice. Heterozygous null
mice were born at the expected frequency without distinguishable phenotypic differences compared with wild-type mice. In contrast, homozygous null
mice were missing, indicating that
is crucial for embryonic viability and survival.
embryos were detected at the expected Mendelian ratios up to late preimplantation stage (E4.5). However, phenotypic characterization at E6.5 revealed degeneration of
embryos, indicating developmental defects around the time of implantation. Transcriptomic analyses documented a dysregulation of glycolysis and fatty acid metabolic pathways in
embryos at E4.5. Proteomic analysis indicated a tight interaction between ZC3H11A and mRNA-export proteins in embryonic stem cells. CLIP-seq analysis demonstrated that ZC3H11A binds a subset of mRNA transcripts that are critical for metabolic regulation of embryonic cells. Furthermore, embryonic stem cells with an induced deletion of
display an impaired differentiation toward epiblast-like cells and impaired mitochondrial membrane potential. Altogether, the results show that ZC3H11A is participating in export and posttranscriptional regulation of selected mRNA transcripts required to maintain metabolic processes in embryonic cells. While ZC3H11A is essential for the viability of the early mouse embryo, inactivation of
expression in adult tissues using a conditional KO did not lead to obvious phenotypic defects.
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