The Cation Diffusion Facilitator (CDF) family is a ubiquitous family of heavy metal transporters. Much interest in this family has focused on implications for human health and bioremediation. In this ...work a broad phylogenetic study has been undertaken which, considered in the context of the functional characteristics of some fully characterised CDF transporters, has aimed at identifying molecular determinants of substrate selectivity and at suggesting metal specificity for newly identified CDF transporters.
Representative CDF members from all three kingdoms of life (Archaea, Eubacteria, Eukaryotes) were retrieved from genomic databases. Protein sequence alignment has allowed detection of a modified signature that can be used to identify new hypothetical CDF members. Phylogenetic reconstruction has classified the majority of CDF family members into three groups, each containing characterised members that share the same specificity towards the principally-transported metal, i.e. Zn, Fe/Zn or Mn. The metal selectivity of newly identified CDF transporters can be inferred by their position in one of these groups. The function of some conserved amino acids was assessed by site-directed mutagenesis in the poplar Zn2+ transporter PtdMTP1 and compared with similar experiments performed in prokaryotic members. An essential structural role can be assigned to a widely conserved glycine residue, while aspartate and histidine residues, highly conserved in putative transmembrane domains, might be involved in metal transport. The potential role of group-conserved amino acid residues in metal specificity is discussed.
In the present study phylogenetic and functional analyses have allowed the identification of three major substrate-specific CDF groups. The metal selectivity of newly identified CDF transporters can be inferred by their position in one of these groups. The modified signature sequence proposed in this work can be used to identify new hypothetical CDF members.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Methylation of cytosines (5(me)C) is a widespread heritable DNA modification. During mammalian development, two global demethylation events are followed by waves of de novo DNA methylation. In vivo ...mechanisms of DNA methylation establishment are largely uncharacterized. Here, we use Saccharomyces cerevisiae as a system lacking DNA methylation to define the chromatin features influencing the activity of the murine DNMT3B. Our data demonstrate that DNMT3B and H3K4 methylation are mutually exclusive and that DNMT3B is co-localized with H3K36 methylated regions. In support of this observation, DNA methylation analysis in yeast strains without Set1 and Set2 shows an increase of relative 5(me)C levels at the transcription start site and a decrease in the gene-body, respectively. We extend our observation to the murine male germline, where H3K4me3 is strongly anti-correlated while H3K36me3 correlates with accelerated DNA methylation. These results show the importance of H3K36 methylation for gene-body DNA methylation in vivo.
Hepatitis C virus infection (HCV) represents a unique model to characterize, from early to late stages of infection, the T cell differentiation process leading to exhaustion of human CD8+ T cells. ...Here we show that in early HCV infection, exhaustion-committed virus-specific CD8+ T cells display a marked upregulation of transcription associated with impaired glycolytic and mitochondrial functions, that are linked to enhanced ataxia-telangiectasia mutated (ATM) and p53 signaling. After evolution to chronic infection, exhaustion of HCV-specific T cell responses is instead characterized by a broad gene downregulation associated with a wide metabolic and anti-viral function impairment, which can be rescued by histone methyltransferase inhibitors. These results have implications not only for treatment of HCV-positive patients not responding to last-generation antivirals, but also for other chronic pathologies associated with T cell dysfunction, including cancer.
retroelements, whose retrotransposition requires prior transcription by RNA polymerase III to generate
RNAs, represent the most numerous non-coding RNA (ncRNA) gene family in the human genome.
...transcription is generally kept to extremely low levels by tight epigenetic silencing, but it has been reported to increase under different types of cell perturbation, such as viral infection and cancer.
RNAs, being able to act as gene expression modulators, may be directly involved in the mechanisms determining cellular behavior in such perturbed states. To directly address the regulatory potential of
RNAs, we generated IMR90 fibroblasts and HeLa cell lines stably overexpressing two slightly different
RNAs, and analyzed genome-wide the expression changes of protein-coding genes through RNA-sequencing. Among the genes that were upregulated or downregulated in response to
overexpression in IMR90, but not in HeLa cells, we found a highly significant enrichment of pathways involved in cell cycle progression and mitotic entry. Accordingly,
overexpression was found to promote transition from G1 to S phase, as revealed by flow cytometry. Therefore, increased
RNA may contribute to sustained cell proliferation, which is an important factor of cancer development and progression.
Macrophages are a heterogeneous cell population which in response to the cytokine milieu polarize in either classically activated macrophages (M1) or alternatively activated macrophages (M2). This ...plasticity makes macrophages essential in regulating inflammation, immune response and tissue remodeling and a novel therapeutic target in inflammatory diseases such as atherosclerosis. The aim of the study was to describe the transcriptomic profiles of differently polarized human macrophages to generate new hypotheses on the biological function of the different macrophage subtypes.
Polarization of circulating monocytes/macrophages of blood donors was induced in vitro by IFN-γ and LPS (M1), by IL-4 (M2a), and by IL-10 (M2c). Unstimulated cells (RM) served as time controls. Gene expression profile of M1, M2a, M2c and RM was assessed at 6, 12 and 24h after polarization with Whole Human Genome Agilent Microarray technique. When compared to RM, M1 significantly upregulated pathways involved in immunity and inflammation, whereas M2a did the opposite. Conversely, decreased and increased expression of mitochondrial metabolism, consistent with insulin resistant and insulin sensitive patterns, was seen in M1 and M2a, respectively. The time sequence in the expression of some pathways appeared to have some specific bearing on M1 function. Finally, canonical and non-canonical Wnt genes and gene groups, promoting inflammation and tissue remodeling, were upregulated in M2a compared to RM.
Our data in in vitro polarized human macrophages: 1. confirm and extend known inflammatory and anti-inflammatory gene expression patterns; 2. demonstrate changes in mitochondrial metabolism associated to insulin resistance and insulin sensitivity in M1 and M2a, respectively; 3. highlight the potential relevance of gene expression timing in M1 function; 4. unveil enhanced expression of Wnt pathways in M2a suggesting a potential dual (pro-inflammatory and anti-inflammatory) role of M2a in inflammatory diseases.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Ammonium and nitrate are the prevalent nitrogen sources for growth and development of higher plants. Here, we report on the characterization of the ammonium transporter (AMT) family in the perennial ...species Populus trichocarpa. In silico analysis and expression analysis of AMT genes from poplar was performed. In addition, AMT1;2 and AMT1;6 function was studied in detail by heterologous expression in yeast. The P. trichocarpa genome contains 14 putative AMTs, which is more than twice the number of AMTs in Arabidopsis. In roots, the high-affinity AMT1;2 strongly increased upon mycorrhiza formation and might be partly responsible for the high-affinity ammonium uptake component measured in poplar. Transcript level for the high-affinity AMT1;6 was strongly affected by the diurnal cycle. AMT3;1 was exclusively expressed in senescing poplar leaves. Remarkably AMT2;1 was highly expressed in leaves while AMT2;2 was mostly expressed in petioles. Specific expression of AMT1;5 in stamen and of AMT1;6 in female flower indicate that they have key functions in reproductive organ development in poplar. The present study provides basic genomic and transcriptomic information for the poplar AMT family and will pave the way for deciphering the precise role of AMTs in poplar physiology.
Regulation of histone acetylation dictates patterns of gene expression and hence cell identity. Due to their clinical relevance in cancer biology, understanding how human embryonic stem cells (hESCs) ...regulate their genomic patterns of histone acetylation is critical, but it remains largely to be investigated. Here, we provide evidence that acetylation of histone H3 lysine-18 (H3K18ac) and lysine-27 (H3K27ac) is only partially established by p300 in stem cells, while it represents the main histone acetyltransferase (HAT) for these marks in somatic cells. Our analysis reveals that whereas p300 marginally associated with H3K18ac and H3K27ac in hESCs, it largely overlapped with these histone marks upon differentiation. Interestingly, we show that H3K18ac is found at "stemness" genes enriched in RNA polymerase III transcription factor C (TFIIIC) in hESCs, whilst lacking p300. Moreover, TFIIIC was also found in the vicinity of genes involved in neuronal biology, although devoid of H3K18ac. Our data suggest a more complex pattern of HATs responsible for histone acetylations in hESCs than previously considered, suggesting a putative role for H3K18ac and TFIIIC in regulating "stemness" genes as well as genes associated with neuronal differentiation of hESCs. The results break ground for possible new paradigms for genome acetylation in hESCs that could lead to new avenues for therapeutic intervention in cancer and developmental diseases.
Manganese toxicity is a major problem for plant growth in acidic soils, but cellular mechanisms that facilitate growth in such conditions have not been clearly delineated. Established mechanisms that ...counter metal toxicity in plants involve chelation and cytoplasmic export of the metal across the plasma or vacuolar membranes out of the cell or sequestered into a large organelle, respectively. We report here that expression of the Arabidopsis and poplar MTP11 cation diffusion facilitators in a manganese-hypersensitive yeast mutant restores manganese tolerance to wild-type levels. Microsomes from yeast expressing AtMTP11 exhibit enhanced manganese uptake. In accord with a presumed function of MTP11 in manganese tolerance, Arabidopsis mtp11 mutants are hypersensitive to elevated levels of manganese, whereas plants overexpressing MTP11 are hypertolerant. In contrast, sensitivity to manganese deficiency is slightly decreased in mutants and increased in overexpressing lines. Promoter-GUS studies showed that AtMTP11 is most highly expressed in root tips, shoot margins, and hydathodes, but not in epidermal cells and trichomes, which are generally associated with manganese accumulation. Surprisingly, imaging of MTP11-EYFP fusions demonstrated that MTP11 localizes neither to the plasma membrane nor to the vacuole, but to a punctate endomembrane compartment that largely coincides with the distribution of the trans-Golgi marker sialyl transferase. Golgi-based manganese accumulation might therefore result in manganese tolerance through vesicular trafficking and exocytosis. In accord with this proposal, Arabidopsis mtp11 mutants exhibit enhanced manganese concentrations in shoots and roots. We propose that Golgi-mediated exocytosis comprises a conserved mechanism for heavy metal tolerance in plants.
Transcription factors (TFs) bind DNA in a sequence-specific manner and are generally cell type-specific factors and/or developmental master regulators. In contrast, general TFs (GTFs) are part of ...very large protein complexes and serve for RNA polymerases' recruitment to promoter sequences, generally in a cell type-independent manner. Whereas, several TFs have been proven to serve as anchors for the 3D genome organization, the role of GTFs in genome architecture have not been carefully explored. Here, we used ChIP-seq and Hi-C data to depict the role of TFIIIC, one of the RNA polymerase III GTFs, in 3D genome organization. We find that TFIIIC genome occupancy mainly occurs at specific regions, which largely correspond to Alu elements; other characteristic classes of repetitive elements (REs) such as MIR, FLAM-C and ALR/alpha are also found depending on the cell's developmental origin. The analysis also shows that TFIIIC-enriched regions are involved in cell type-specific DNA looping, which does not depend on colocalization with the master architectural protein CTCF. This work extends previous knowledge on the role of TFIIIC as a bona fide genome organizer whose action participates in cell type-dependent 3D genome looping via binding to REs.
Premature birth, perinatal inflammation, and life-saving therapies such as postnatal oxygen and mechanical ventilation are strongly associated with the development of bronchopulmonary dysplasia ...(BPD); these risk factors, alone or combined, cause lung inflammation and alter programmed molecular patterns of normal lung development. The current knowledge on the molecular regulation of lung development mainly derives from mechanistic studies conducted in newborn rodents exposed to postnatal hyperoxia, which have been proven useful but have some limitations.
Here, we used the rabbit model of BPD as a cost-effective alternative model that mirrors human lung development and, in addition, enables investigating the impact of premature birth per se on the pathophysiology of BPD without further perinatal insults (e.g., hyperoxia, LPS-induced inflammation). First, we characterized the rabbit's normal lung development along the distinct stages (i.e., pseudoglandular, canalicular, saccular, and alveolar phases) using histological, transcriptomic and proteomic analyses. Then, the impact of premature birth was investigated, comparing the sequential transcriptomic profiles of preterm rabbits obtained at different time intervals during their first week of postnatal life with those from age-matched term pups.
Histological findings showed stage-specific morphological features of the developing rabbit's lung and validated the selected time intervals for the transcriptomic profiling. Cell cycle and embryo development, oxidative phosphorylation, and WNT signaling, among others, showed high gene expression in the pseudoglandular phase. Autophagy, epithelial morphogenesis, response to transforming growth factor β, angiogenesis, epithelium/endothelial cells development, and epithelium/endothelial cells migration pathways appeared upregulated from the 28th day of gestation (early saccular phase), which represents the starting point of the premature rabbit model. Premature birth caused a significant dysregulation of the inflammatory response. TNF-responsive, NF-κB regulated genes were significantly upregulated at premature delivery and triggered downstream inflammatory pathways such as leukocyte activation and cytokine signaling, which persisted upregulated during the first week of life. Preterm birth also dysregulated relevant pathways for normal lung development, such as blood vessel morphogenesis and epithelial-mesenchymal transition.
These findings establish the 28-day gestation premature rabbit as a suitable model for mechanistic and pharmacological studies in the context of BPD.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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