OGT controls mammalian cell viability by regulating the proteasome/mTOR/ mitochondrial axis.

O-GlcNAc transferase (OGT) modifies serine and threonine residues on nuclear and cytosolic proteins with O-linked N-acetylglucosamine (GlcNAc). OGT is essential for mammalian cell viability, but the underlying mechanisms are still enigmatic. We performed a genome-wide CRISPR-Cas9 screen in mouse embryonic stem cells (mESCs) to identify candidates whose depletion rescued the block in cell proliferation induced by OGT deficiency. We show that the block in cell proliferation in OGT-deficient cells stems from mitochondrial dysfunction secondary to mTOR (mechanistic target of rapamycin) hyperactivation. In normal cells, OGT maintains low mTOR activity and mitochondrial fitness through suppression of proteasome activity; in the absence of OGT, increased proteasome activity results in increased steady-state amino acid levels, which in turn promote mTOR lysosomal translocation and activation, and increased oxidative phosphorylation. mTOR activation in OGT-deficient mESCs was confirmed by an independent phospho-proteomic screen. Our study highlights a unique series of events whereby OGT regulates the proteasome/ mTOR/ mitochondrial axis in a manner that maintains homeostasis of intracellular amino acid levels, mitochondrial fitness, and cell viability. A similar mechanism operates in CD8+ T cells, indicating its generality across mammalian cell types. Manipulating OGT activity may have therapeutic potential in diseases in which this signaling pathway is impaired.

Mll-COMPASS complexes mediate H3K4me3 enrichment and transcription of the osteoblast master gene Runx2/p57 in osteoblasts.

Expression of Runx2/p57 is a hallmark of the osteoblast-lineage identity. Although several regulators that control the expression of Runx2/p57 during osteoblast-lineage commitment have been identified, the epigenetic mechanisms that sustain this expression in differentiated osteoblasts remain to be completely determined. Here, we assess epigenetic mechanisms associated with Runx2/p57 gene transcription in differentiating MC3T3 mouse osteoblasts. Our results show that an enrichment of activating histone marks at the Runx2/p57 P1 promoter is accompanied by the simultaneous interaction of Wdr5 and Utx proteins, both are components of COMPASS complexes. Knockdown of Wdr5 and Utx expression confirms the activating role of both proteins at the Runx2-P1 promoter. Other chromatin modifiers that were previously described to regulate Runx2/p57 transcription in mesenchymal precursor cells (Ezh2, Prmt5, and Jarid1b proteins) were not found to contribute to Runx2/p57 transcription in full-committed osteoblasts. We also determined the presence of additional components of COMPASS complexes at the Runx2/p57 promoter, evidencing that the Mll2/COMPASS- and Mll3/COMPASS-like complexes bind to the P1 promoter in osteoblastic cells expressing Runx2/p57 to modulate the H3K4me1 to H3K4me3 transition.

Variable transcriptional responsiveness of the P2X3 receptor gene during CFA-induced inflammatory hyperalgesia.

The purinergic receptor P2X3 (P2X3-R) plays important roles in molecular pathways of pain, and reduction of its activity or expression effectively reduces chronic inflammatory and neuropathic pain sensation. Inflammation, nerve injury, and cancer-induced pain can increase P2X3-R mRNA and/or protein levels in dorsal root ganglia (DRG). However, P2X3-R expression is unaltered or even reduced in other pain studies. The reasons for these discrepancies are unknown and might depend on the applied traumatic intervention or on intrinsic factors such as age, gender, genetic background, and/or epigenetics. In this study, we sought to get insights into the molecular mechanisms responsible for inflammatory hyperalgesia by determining P2X3-R expression in DRG neurons of juvenile male rats that received a Complete Freund's Adjuvant (CFA) bilateral paw injection. We demonstrate that all CFA-treated rats showed inflammatory hyperalgesia, however, only a fraction (14-20%) displayed increased P2X3-R mRNA levels, reproducible across both sides. Immunostaining assays did not reveal significant increases in the percentage of P2X3-positive neurons, indicating that increased P2X3-R at DRG somas is not critical for inducing inflammatory hyperalgesia in CFA-treated rats. Chromatin immunoprecipitation (ChIP) assays showed a correlated (R2 = 0.671) enrichment of the transcription factor Runx1 and the epigenetic active mark histone H3 acetylation (H3Ac) at the P2X3-R gene promoter in a fraction of the CFA-treated rats. These results suggest that animal-specific increases in P2X3-R mRNA levels are likely associated with the genetic/epigenetic context of the P2X3-R locus that controls P2X3-R gene transcription by recruiting Runx1 and epigenetic co-regulators that mediate histone acetylation.

Expression of the ectodomain-releasing protease ADAM17 is directly regulated by the osteosarcoma and bone-related transcription factor RUNX2.

Osteoblast differentiation is controlled by transcription factor RUNX2 which temporally activates or represses several bone-related genes, including those encoding extracellular matrix proteins or factors that control cell-cell, and cell-matrix interactions. Cell-cell communication in the many skeletal pericellular micro-niches is critical for bone development and involves paracrine secretion of growth factors and morphogens. This paracrine signaling is in part regulated by "A Disintegrin And Metalloproteinase" (ADAM) proteins. These cell membrane-associated metalloproteinases support proteolytic release ("shedding") of protein ectodomains residing at the cell surface. We analyzed microarray and RNA-sequencing data for Adam genes and show that Adam17, Adam10, and Adam9 are stimulated during BMP2 mediated induction of osteogenic differentiation and are robustly expressed in human osteoblastic cells. ADAM17, which was initially identified as a tumor necrosis factor alpha (TNFα) converting enzyme also called (TACE), regulates TNFα-signaling pathway, which inhibits osteoblast differentiation. We demonstrate that Adam17 expression is suppressed by RUNX2 during osteoblast differentiation through the proximal Adam17 promoter region (-0.4 kb) containing two functional RUNX2 binding motifs. Adam17 downregulation during osteoblast differentiation is paralleled by increased RUNX2 expression, cytoplasmic-nuclear translocation and enhanced binding to the Adam17 proximal promoter. Forced expression of Adam17 reduces Runx2 and Alpl expression, indicating that Adam17 may negatively modulate osteoblast differentiation. These findings suggest a novel regulatory mechanism involving a reciprocal Runx2-Adam17 negative feedback loop to regulate progression through osteoblast differentiation. Our results suggest that RUNX2 may control paracrine signaling through regulation of ectodomain shedding at the cell surface of osteoblasts by directly suppressing Adam17 expression.

Runt-Related Transcription Factor 2 Induction During Differentiation of Wharton's Jelly Mesenchymal Stem Cells to Osteoblasts Is Regulated by Jumonji AT-Rich Interactive Domain 1B Histone Demethylase.

Novel bone regeneration approaches aim to obtain immature osteoblasts from somatic stem cells. Umbilical cord Wharton's jelly mesenchymal stem cells (WJ-MSCs) are an ideal source for cell therapy. Hence, the study of mechanisms involved in WJ-MSC osteoblastic differentiation is crucial to exploit their developmental capacity. Here, we have assessed epigenetic control of the Runt-related transcription factor 2 (RUNX2) osteogenic master regulator gene in WJ-MSC. We present evidence indicating that modulation of RUNX2 expression through preventing Jumonji AT-rich interactive domain 1B (JARID1B) histone demethylase activity is relevant to enhance WJ-MSC osteoblastic potential. Hence, JARID1B loss of function in WJ-MSC results in increased RUNX2/p57 expression. Our data highlight JARID1B activity as a novel target to modulate WJ-MSC osteoblastic differentiation with potential applications in bone tissue engineering. Stem Cells 2017;35:2430-2441.

Application of a JA-Ile Biosynthesis Inhibitor to Methyl Jasmonate-Treated Strawberry Fruit Induces Upregulation of Specific MBW Complex-Related Genes and Accumulation of Proanthocyanidins.

Fleshy fruits are an important source of anthocyanins and proanthocyanidins (PAs), which protect plants against stress, and their consumption provides beneficial effects for human health. In strawberry fruit, the application of exogenous methyl jasmonate (MeJA) upregulates anthocyanin accumulation, although the relationship between the jasmonate pathway and anthocyanin and PA biosynthesis in fruits remains to be understood. Anthocyanin and PA accumulation is mainly regulated at the transcriptional level through R2R3-MYB and bHLH transcription factors in different plant species and organs. Here, the effect of jarin-1, a specific inhibitor of bioactive JA (jasmonoyl-isoleucine, JA-Ile) biosynthesis, on anthocyanin and PA accumulation was evaluated during strawberry (Fragaria × ananassa) fruit development using an in vitro ripening system for 48 h. Also, we observed the effects of MeJA and the application of jarin-1 to MeJA-treated fruits (MeJA + jarin-1 treatment). We assessed changes of expression levels for the JA-Ile and MeJA biosynthetic (FaJAR1.2 and FaJMT), JA signaling-related (FaMYC2 and FaJAZ1), MYB-bHLH-WD40 (MBW) complex-related (FabHLH3/33, FaMYB9/10/11, and repressor FaMYB1), and anthocyanin and PA biosynthetic (FaANS, FaUFGT, FaANR, and FaLAR) genes. In addition, the promoter region of MBW complex-related MYB genes was isolated and sequenced. We found a higher redness of strawberry fruit skin and anthocyanin content in MeJA-treated fruits with respect to jarin-1-treated ones concomitant with an upregulation of FaANS and FaUFGT genes. Inversely, the PA content was higher in jarin-1- and MeJA + jarin-1-treated than in MeJA-treated fruits. MeJA + jarin-1 treatment resulted in an upregulation of FaANR and associated transcription factors such as FabHLH33 and FaMYB9/11 along with FaJMT and FaJAR1.2. Finally, we found JA-responsive elements in the promoter regions of FaMYB1/9/10/11 genes. It is proposed that PA biosynthesis-related genes can be upregulated by the application of jarin-1 to MeJA-treated fruit, thus increasing PA accumulation in strawberry.

Epigenetic Signatures at the RUNX2-P1 and Sp7 Gene Promoters Control Osteogenic Lineage Commitment of Umbilical Cord-Derived Mesenchymal Stem Cells.

Wharton's Jelly mesenchymal stem cells (WJ-MSCs) are an attractive potential source of multipotent stem cells for bone tissue replacement therapies. However, the molecular mechanisms involved in their osteogenic conversion are poorly understood. Particularly, epigenetic control operating at the promoter regions of the two master regulators of the osteogenic program, RUNX2/P57 and SP7 has not yet been described in WJ-MSCs. Via quantitative PCR profiling and chromatin immunoprecipitation (ChIP) studies, here we analyze the ability of WJ-MSCs to engage osteoblast lineage. In undifferentiated WJ-MSCs, RUNX2/P57 P1, and SP7 promoters are found deprived of significant levels of the histone post-translational marks that are normally associated with transcriptionally active genes (H3ac, H3K27ac, and H3K4me3). Moreover, the RUNX2 P1 promoter lacks two relevant histone repressive marks (H3K9me3 and H3K27me3). Importantly, RUNX2 P1 promoter is found highly enriched in the H3K4me1 mark, which has been shown recently to mediate gene repression of key regulatory genes. Upon induction of WJ-MSCs osteogenic differentiation, we found that RUNX2/P57, but not SP7 gene expression is strongly activated, in a process that is accompanied by enrichment of activating histone marks (H3K4me3, H3ac, and H3K27ac) at the P1 promoter region. Histone mark analysis showed that SP7 gene promoter is robustly enriched in epigenetic repressive marks that may explain its poor transcriptional response to osteoblast differentiating media. Together, these results point to critical regulatory steps during epigenetic control of WJ-MSCs osteogenic lineage commitment that are relevant for future applications in regenerative medicine. J. Cell. Physiol. 232: 2519-2527, 2017. © 2016 Wiley Periodicals, Inc.

The Specification of Cortical Subcerebral Projection Neurons Depends on the Direct Repression of TBR1 by CTIP1/BCL11a.

The acquisition of distinct neuronal fates is fundamental for the function of the cerebral cortex. We find that the development of subcerebral projections from layer 5 neurons in the mouse neocortex depends on the high levels of expression of the transcription factor CTIP1; CTIP1 is coexpressed with CTIP2 in neurons that project to subcerebral targets and with SATB2 in those that project to the contralateral cortex. CTIP1 directly represses Tbr1 in layer 5, which appears as a critical step for the acquisition of the subcerebral fate. In contrast, lower levels of CTIP1 in layer 6 are required for TBR1 expression, which directs the corticothalamic fate. CTIP1 does not appear to play a critical role in the acquisition of the callosal projection fate in layer 5. These findings unravel a key step in the acquisition of cell fate for closely related corticofugal neurons and indicate that differential dosages of transcriptions factors are critical to specify different neuronal identities.

Mitotic Inheritance of mRNA Facilitates Translational Activation of the Osteogenic-Lineage Commitment Factor Runx2 in Progeny of Osteoblastic Cells.

Epigenetic mechanisms mediate the acquisition of specialized cellular phenotypes during tissue development, maintenance and repair. When phenotype-committed cells transit through mitosis, chromosomal condensation counteracts epigenetic activation of gene expression. Subsequent post-mitotic re-activation of transcription depends on epigenetic DNA and histone modifications, as well as other architecturally bound proteins that "bookmark" the genome. Osteogenic lineage commitment, differentiation and progenitor proliferation require the bone-related runt-related transcription factor Runx2. Here, we characterized a non-genomic mRNA mediated mechanism by which osteoblast precursors retain their phenotype during self-renewal. We show that osteoblasts produce maximal levels of Runx2 mRNA, but not protein, prior to mitotic cell division. Runx2 mRNA partitions symmetrically between daughter cells in a non-chromosomal tubulin-containing compartment. Subsequently, transcription-independent de novo synthesis of Runx2 protein in early G1 phase results in increased functional interactions of Runx2 with a representative osteoblast-specific target gene (osteocalcin/BGLAP2) in chromatin. Somatic transmission of Runx2 mRNAs in osteoblasts and osteosarcoma cells represents a versatile mechanism for translational rather than transcriptional induction of this principal gene regulator to maintain osteoblast phenotype identity after mitosis.

A functional N-terminal domain in C/EBPß-LAP* is required for interacting with SWI/SNF and to repress Ric-8B gene transcription in osteoblasts.

The chromatin remodeling complex SWI/SNF and the transcription factor C/EBPß play critical roles in osteoblastic cells as they jointly control transcription of a number of bone-related target genes. The largest C/EBPß isoform, LAP*, possesses a short additional N-terminal domain that has been proposed to mediate the interaction of this factor with SWI/SNF in myeloid cells. Here we examine the requirement of a functional N-terminus in C/EBPß-LAP* for binding SWI/SNF and for recruiting this complex to the Ric-8B gene to mediate transcriptional repression. We find that both C/EBPß-LAP* and SWI/SNF simultaneously bind to the Ric-8B promoter in differentiating osteoblasts that repress Ric-8B expression. This decreased expression of Ric-8B is not accompanied by significant changes in histone acetylation at the Ric-8B gene promoter sequence. A single aminoacid change at the C/EBPß-LAP* N-terminus (R3L) that inhibits C/EBPß-LAP*-SWI/SNF interaction, also prevents SWI/SNF recruitment to the Ric-8B promoter as well as C/EBPß-LAP*-dependent repression of the Ric-8B gene. Inducible expression of the C/EBPß-LAP*R3L protein in stably transfected osteoblastic cells demonstrates that this mutant protein binds to C/EBPß-LAP*-target promoters and competes with the endogenous C/EBPß factor. Together our results indicate that a functional N-terminus in C/EBPß-LAP* is required for interacting with SWI/SNF and for Ric-8B gene repression in osteoblasts.

The Role of Fatty Acid Synthase in the Vascular Smooth Muscle Cell to Foam Cell Transition.

Vascular smooth muscle cells (VSMCs), in their contractile and differentiated state, are fundamental for maintaining vascular function. Upon exposure to cholesterol (CHO), VSMCs undergo dedifferentiation, adopting characteristics of foam cells-lipid-laden, macrophage-like cells pivotal in atherosclerotic plaque formation. CHO uptake by VSMCs leads to two primary pathways: ABCA1-mediated efflux or storage in lipid droplets as cholesterol esters (CEs). CE formation, involving the condensation of free CHO and fatty acids, is catalyzed by sterol O-acyltransferase 1 (SOAT1). The necessary fatty acids are synthesized by the lipogenic enzyme fatty acid synthase (FASN), which we found to be upregulated in atherosclerotic human coronary arteries. This observation led us to hypothesize that FASN-mediated fatty acid biosynthesis is crucial in the transformation of VSMCs into foam cells. Our study reveals that CHO treatment upregulates FASN in human aortic SMCs, concurrent with increased expression of CD68 and upregulation of KLF4, markers associated with the foam cell transition. Crucially, downregulation of FASN inhibits the CHO-induced upregulation of CD68 and KLF4 in VSMCs. Additionally, FASN-deficient VSMCs exhibit hindered lipid accumulation and an impaired transition to the foam cell phenotype following CHO exposure, while the addition of the fatty acid palmitate, the main FASN product, exacerbates this transition. FASN-deficient cells also show decreased SOAT1 expression and elevated ABCA1. Notably, similar effects are observed in KLF4-deficient cells. Our findings demonstrate that FASN plays an essential role in the CHO-induced upregulation of KLF4 and the VSMC to foam cell transition and suggest that targeting FASN could be a novel therapeutic strategy to regulate VSMC phenotypic modulation.

OGT prevents DNA demethylation and suppresses the expression of transposable elements in heterochromatin by restraining TET activity genome-wide.

The O- GlcNAc transferase OGT interacts robustly with all three mammalian TET methylcytosine dioxygenases. We show here that deletion of the Ogt gene in mouse embryonic stem cells (mESC) results in a widespread increase in the TET product 5-hydroxymethylcytosine (5hmC) in both euchromatic and heterochromatic compartments, with concomitant reduction of the TET substrate 5-methylcytosine (5mC) at the same genomic regions. mESC engineered to abolish the TET1-OGT interaction likewise displayed a genome-wide decrease of 5mC. DNA hypomethylation in OGT-deficient cells was accompanied by de-repression of transposable elements (TEs) predominantly located in heterochromatin, and this increase in TE expression was sometimes accompanied by increased cis -expression of genes and exons located 3' of the expressed TE. Thus, the TET-OGT interaction prevents DNA demethylation and TE expression in heterochromatin by restraining TET activity genome-wide. We suggest that OGT protects the genome against DNA hypomethylation and impaired heterochromatin integrity, preventing the aberrant increase in TE expression observed in cancer, autoimmune-inflammatory diseases, cellular senescence and ageing.

Acute deletion of TET enzymes results in aneuploidy in mouse embryonic stem cells through decreased expression of Khdc3.

TET (Ten-Eleven Translocation) dioxygenases effect DNA demethylation through successive oxidation of the methyl group of 5-methylcytosine (5mC) in DNA. In humans and in mouse models, TET loss-of-function has been linked to DNA damage, genome instability and oncogenesis. Here we show that acute deletion of all three Tet genes, after brief exposure of triple-floxed, Cre-ERT2-expressing mouse embryonic stem cells (mESC) to 4-hydroxytamoxifen, results in chromosome mis-segregation and aneuploidy; moreover, embryos lacking all three TET proteins showed striking variation in blastomere numbers and nuclear morphology at the 8-cell stage. Transcriptional profiling revealed that mRNA encoding a KH-domain protein, Khdc3 (Filia), was downregulated in triple TET-deficient mESC, concomitantly with increased methylation of CpG dinucleotides in the vicinity of the Khdc3 gene. Restoring KHDC3 levels in triple Tet-deficient mESC prevented aneuploidy. Thus, TET proteins regulate Khdc3 gene expression, and TET deficiency results in mitotic infidelity and genome instability in mESC at least partly through decreased expression of KHDC3.

C/EBPß binds the P1 promoter of the Runx2 gene and up-regulates Runx2 transcription in osteoblastic cells.

The Runx2 factor is an essential component of the regulatory mechanisms that control transcription during skeletogenesis. Runx2/p57 expression in osteoblastic cells is controlled by the P1 promoter, which is recognized by key regulators of osteoblast differentiation including homeodomain factors and Wnt- and BMP-signaling mediators. Here, we report that the transcription factor C/EBPß up-regulates Runx2/p57 expression by directly binding to the Runx2 P1 promoter in mesenchymal, pre-osteoblastic, and osteoblastic cells. This C/EBPß-mediated up-regulation is principally dependent on C/EBP site II that is located within the first 180 bp of the proximal P1 promoter region and is highly conserved among mouse, rat, and human Runx2 genes. Our studies reveal how the C/EBPß factor, known to have a key role during osteogenesis, contributes to regulating the expression of Runx2, the master regulator of osteoblast differentiation.

Whole-Genome Transcript Expression Profiling Reveals Novel Insights into Transposon Genes and Non-Coding RNAs during Atlantic Salmon Seawater Adaptation.

The growing amount of genome information and transcriptomes data available allows for a better understanding of biological processes. However, analysis of complex transcriptomic experimental designs involving different conditions, tissues, or times is relevant. This study proposes a novel approach to analyze complex data sets combining transcriptomes and miRNAs at the chromosome-level genome. Atlantic salmon smolts were transferred to seawater under two strategies: (i) fish group exposed to gradual salinity changes (GSC) and (ii) fish group exposed to a salinity shock (SS). Gills, intestine, and head kidney samples were used for total RNA extraction, followed by mRNA and small RNA illumina sequencing. Different expression patterns among the tissues and treatments were observed through a whole-genome transcriptomic approach. Chromosome regions highly expressed between experimental conditions included a great abundance of transposable elements. In addition, differential expression analysis showed a greater number of transcripts modulated in response to SS in gills and head kidney. miRNA expression analysis suggested a small number of miRNAs involved in the smoltification process. However, target analysis of these miRNAs showed a regulatory role in growth, stress response, and immunity. This study is the first to evidence the interplaying among mRNAs and miRNAs and the structural relationship at the genome level during Atlantic salmon smoltification.

Chromosome-scale genome assembly of the sea louse Caligus rogercresseyi by SMRT sequencing and Hi-C analysis.

Caligus rogercresseyi, commonly known as sea louse, is an ectoparasite copepod that impacts the salmon aquaculture in Chile, causing losses of hundreds of million dollars per year. In this study, we report a chromosome-scale assembly of the sea louse (C. rogercresseyi) genome based on single-molecule real-time sequencing (SMRT) and proximity ligation (Hi-C) analysis. Coding RNAs and non-coding RNAs, and specifically long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) were identified through whole transcriptome sequencing from different life stages. A total of 23,686 protein-coding genes and 12,558 non-coding RNAs were annotated. In addition, 6,308 lncRNAs and 5,774 miRNAs were found to be transcriptionally active from larvae to adult stages. Taken together, this genomic resource for C. rogercresseyi represents a valuable tool to develop sustainable control strategies in the salmon aquaculture industry.

The RabGEF ALS2 is a hypoxia inducible target associated with the acquisition of aggressive traits in tumor cells.

Tumor hypoxia and the hypoxia inducible factor-1, HIF-1, play critical roles in cancer progression and metastasis. We previously showed that hypoxia activates the endosomal GTPase Rab5, leading to tumor cell migration and invasion, and that these events do not involve changes in Rab protein expression, suggesting the participation of intermediate activators. Here, we identified ALS2, a guanine nucleotide exchange factor that is upregulated in cancer, as responsible for increased Rab5-GTP loading, cell migration and metastasis in hypoxia. Specifically, hypoxia augmented ALS2 mRNA and protein levels, and these events involved HIF-1α-dependent transcription, as shown by RNAi, pharmacological inhibition, chromatin immunoprecipitation and bioinformatics analyses, which identified a functional HIF-1α-binding site in the proximal promoter region of ALS2. Moreover, ALS2 and Rab5 activity were elevated both in a model of endogenous HIF-1α stabilization (renal cell carcinoma) and by following expression of stable non-hydroxylatable HIF-1α. Strikingly, ALS2 upregulation in hypoxia was required for Rab5 activation, tumor cell migration and invasion, as well as experimental metastasis in C57BL/6 mice. Finally, immunohistochemical analyses in patient biopsies with renal cell carcinoma showed that elevated HIF-1α correlates with increased ALS2 expression. Hence, this study identifies ALS2 as a novel hypoxia-inducible gene associated with tumor progression and metastasis.

Ski Is Required for Tri-Methylation of H3K9 in Major Satellite and for Repression of Pericentromeric Genes: Mmp3, Mmp10 and Mmp13, in Mouse Fibroblasts.

Several mechanisms directing a rapid transcriptional reactivation of genes immediately after mitosis have been described. However, little is known about the maintenance of repressive signals during mitosis. In this work, we address the role of Ski in the repression of gene expression during M/G1 transition in mouse embryonic fibroblasts (MEFs). We found that Ski localises as a distinct pair of dots at the pericentromeric region of mitotic chromosomes, and the absence of the protein is related to high acetylation and low tri-methylation of H3K9 in pericentromeric major satellite. Moreover, differential expression assays in early G1 cells showed that the presence of Ski is significantly associated with repression of genes localised nearby to pericentromeric DNA. In mitotic cells, chromatin immunoprecipitation assays confirmed the association of Ski to major satellite and the promoters of the most repressed genes: Mmp3, Mmp10 and Mmp13. These genes are at pericentromeric region of chromosome 9. In these promoters, the presence of Ski resulted in increased H3K9 tri-methylation levels. This Ski-dependent regulation is also observed during interphase. Consequently, Mmp activity is augmented in Ski-/- MEFs. Altogether, these data indicate that association of Ski with the pericentromeric region of chromosomes during mitosis is required to maintain the silencing bookmarks of underlying chromatin.

Silicon flow from root to shoot in pepper: a comprehensive in silico analysis reveals a potential linkage between gene expression and hormone signaling that stimulates plant growth and metabolism.

BACKGROUND: Silicon (Si) is categorized as a quasi-essential element for plants thanks to the benefits on growth, development and metabolism in a hormetic manner. Si uptake is cooperatively mediated by Lsi1 and Lsi2. Nevertheless, Lsi channels have not yet been identified and characterized in pepper (Capsicum annuum), while genes involved in major physiological processes in pepper are Si-regulated. Furthermore, Si and phytohormones may act together in regulating plant growth, metabolism and tolerance against stress. Our aim was to identify potential synergies between Si and phytohormones stimulating growth and metabolism in pepper, based on in silico data. METHODS: We established a hydroponic system to test the effect of Si (0, 60, 125 and 250 mg L-1 Si) on the concentrations of this element in different pepper plant tissues. We also performed an in silico analysis of putative Lsi genes from pepper and other species, including tomato (Solanum lycopersicum), potato (Solanum tuberosum) and Arabidopsis thaliana, to look for cis-acting elements responsive to phytohormones in their promoter regions. With the Lsi1 and Lsi2 protein sequences from various plant species, we performed a phylogenetic analysis. Taking into consideration the Lsi genes retrieved from tomato, potato and Arabidopsis, an expression profiling analysis in different plant tissues was carried out. Expression of Si-regulated genes was also analyzed in response to phytohormones and different plant tissues and developmental stages in Arabidopsis. RESULTS: Si concentrations in plant tissues exhibited the following gradient: roots > stems > leaves. We were able to identify 16 Lsi1 and three Lsi2 genes in silico in the pepper genome, while putative Lsi homologs were also found in other plant species. They were mainly expressed in root tissues in the genomes analyzed. Both Lsi and Si-regulated genes displayed cis-acting elements responsive to diverse phytohormones. In Arabidopsis, Si-regulated genes were transcriptionally active in most tissues analyzed, though at different expressed levels. From the set of Si-responsive genes, the NOCS2 gene was highly expressed in germinated seeds, whereas RABH1B, and RBCS-1A, were moderately expressed in developed flowers. All genes analyzed showed responsiveness to phytohormones and phytohormone precursors. CONCLUSION: Pepper root cells are capable of absorbing Si, but small amounts of this element are transported to the upper parts of the plant. We could identify putative Si influx (Lsi1) and efflux (Lsi2) channels that potentially participate in the absorption and transport of Si, since they are mainly expressed in roots. Both Lsi and Si-regulated genes exhibit cis-regulatory elements in their promoter regions, which are involved in phytohormone responses, pointing to a potential connection among Si, phytohormones, plant growth, and other vital physiological processes triggered by Si in pepper.

Silicon induces hormetic dose-response effects on growth and concentrations of chlorophylls, amino acids and sugars in pepper plants during the early developmental stage.

BACKGROUND: Silicon (Si) is a beneficial element that has been proven to influence plant responses including growth, development and metabolism in a hormetic manner. METHODS: In the present study, we evaluated the effect of Si on the growth and concentrations of chlorophylls, total amino acids, and total sugars of pepper plants (Capsicum annuum L.) during the early developmental stage in a hydroponic system under conventional (unstressed) conditions. We tested four Si concentrations (applied as calcium silicate): 0, 60, 125 and 250 mg L-1, and growth variables were measured 7, 14, 21 and 28 days after treatment (dat), while biochemical variables were recorded at the end of the experiment, 28 dat. RESULTS: The application of 125 mg L-1 Si improved leaf area, fresh and dry biomass weight in leaves and stems, total soluble sugars, and concentrations of chlorophylls a and b in both leaves and stems. The amino acids concentration in leaves and roots, as well as the stem diameter were the highest in plants treated with 60 mg L-1 Si. Nevertheless, Si applications reduced root length, stem diameter and total free amino acids in leaves and stems, especially when applied at the highest concentration (i.e., 250 mg L-1 Si). CONCLUSION: The application of Si has positive effects on pepper plants during the early developmental stage, including stimulation of growth, as well as increased concentrations of chlorophylls, total free amino acids and total soluble sugars. In general, most benefits from Si applications were observed in the range of 60-125 mg L-1 Si, while some negative effects were observed at the highest concentration applied (i.e., 250 mg L-1 Si). Therefore, pepper is a good candidate crop to benefit from Si application during the early developmental stage under unstressed conditions.