Genomes of the Orestias pupfish from the Andean Altiplano shed light on their evolutionary history and phylogenetic relationships within Cyprinodontiformes.

BACKGROUND: To unravel the evolutionary history of a complex group, a comprehensive reconstruction of its phylogenetic relationships is crucial. This requires meticulous taxon sampling and careful consideration of multiple characters to ensure a complete and accurate reconstruction. The phylogenetic position of the Orestias genus has been estimated partly on unavailable or incomplete information. As a consequence, it was assigned to the family Cyprindontidae, relating this Andean fish to other geographically distant genera distributed in the Mediterranean, Middle East and North and Central America. In this study, using complete genome sequencing, we aim to clarify the phylogenetic position of Orestias within the Cyprinodontiformes order. RESULTS: We sequenced the genome of three Orestias species from the Andean Altiplano. Our analysis revealed that the small genome size in this genus (~ 0.7 Gb) was caused by a contraction in transposable element (TE) content, particularly in DNA elements and short interspersed nuclear elements (SINEs). Using predicted gene sequences, we generated a phylogenetic tree of Cyprinodontiformes using 902 orthologs extracted from all 32 available genomes as well as three outgroup species. We complemented this analysis with a phylogenetic reconstruction and time calibration considering 12 molecular markers (eight nuclear and four mitochondrial genes) and a stratified taxon sampling to consider 198 species of nearly all families and genera of this order. Overall, our results show that phylogenetic closeness is directly related to geographical distance. Importantly, we found that Orestias is not part of the Cyprinodontidae family, and that it is more closely related to the South American fish fauna, being the Fluviphylacidae the closest sister group. CONCLUSIONS: The evolutionary history of the Orestias genus is linked to the South American ichthyofauna and it should no longer be considered a member of the Cyprinodontidae family. Instead, we submit that Orestias belongs to the Orestiidae family, as suggested by Freyhof et al. (2017), and that it is the sister group of the Fluviphylacidae family, distributed in the Amazonian and Orinoco basins. These two groups likely diverged during the Late Eocene concomitant with hydrogeological changes in the South American landscape.

Inorganic polyphosphate: from basic research to diagnostic and therapeutic opportunities in ALS/FTD.

Inorganic polyphosphate (polyP) is a simple, negatively charged biopolymer with chain lengths ranging from just a few to over a thousand ortho-phosphate (Pi) residues. polyP is detected in every cell type across all organisms in nature thus far analyzed. Despite its structural simplicity, polyP has been shown to play important roles in a remarkably broad spectrum of biological processes, including blood coagulation, bone mineralization and inflammation. Furthermore, polyP has been implicated in brain function and the neurodegenerative diseases amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer's disease and Parkinson's disease. In this review, we first address the challenges associated with identifying mammalian polyP metabolizing enzymes, such as Nudt3, and quantifying polyP levels in brain tissue, cultured neural cells and cerebrospinal fluid. Subsequently, we focus on recent studies that unveil how the excessive release of polyP by human and mouse ALS/FTD astrocytes contributes to these devastating diseases by inducing hyperexcitability, leading to motoneuron death. Potential implications of elevated polyP levels in ALS/FTD patients for innovative diagnostic and therapeutic approaches are explored. It is emphasized, however, that caution is required in targeting polyP in the brain due to its diverse physiological functions, serving as an energy source, a chelator for divalent cations and a scaffold for amyloidogenic proteins. Reducing polyP levels, especially in neurons, might thus have adverse effects in brain functioning. Finally, we discuss how activated mast cells and platelets also can significantly contribute to ALS progression, as they can massively release polyP.

Plant ecological genomics at the limits of life in the Atacama Desert.

The Atacama Desert in Chile-hyperarid and with high-ultraviolet irradiance levels-is one of the harshest environments on Earth. Yet, dozens of species grow there, including Atacama-endemic plants. Herein, we establish the Talabre-Lejía transect (TLT) in the Atacama as an unparalleled natural laboratory to study plant adaptation to extreme environmental conditions. We characterized climate, soil, plant, and soil-microbe diversity at 22 sites (every 100 m of altitude) along the TLT over a 10-y period. We quantified drought, nutrient deficiencies, large diurnal temperature oscillations, and pH gradients that define three distinct vegetational belts along the altitudinal cline. We deep-sequenced transcriptomes of 32 dominant plant species spanning the major plant clades, and assessed soil microbes by metabarcoding sequencing. The top-expressed genes in the 32 Atacama species are enriched in stress responses, metabolism, and energy production. Moreover, their root-associated soils are enriched in growth-promoting bacteria, including nitrogen fixers. To identify genes associated with plant adaptation to harsh environments, we compared 32 Atacama species with the 32 closest sequenced species, comprising 70 taxa and 1,686,950 proteins. To perform phylogenomic reconstruction, we concatenated 15,972 ortholog groups into a supermatrix of 8,599,764 amino acids. Using two codon-based methods, we identified 265 candidate positively selected genes (PSGs) in the Atacama plants, 64% of which are located in Pfam domains, supporting their functional relevance. For 59/184 PSGs with an Arabidopsis ortholog, we uncovered functional evidence linking them to plant resilience. As some Atacama plants are closely related to staple crops, these candidate PSGs are a "genetic goldmine" to engineer crop resilience to face climate change.

Selective Concurrence of the Long Non-Coding RNA MALAT1 and the Polycomb Repressive Complex 2 to Promoter Regions of Active Genes in MCF7 Breast Cancer Cells.

In cancer cells, the long non-coding RNA (lncRNA) MALAT1 has arisen as a key partner for the Polycomb Repressive Complex 2 (PRC2), an epigenetic modifier. However, it is unknown whether this partnership occurs genome-wide at the chromatin level, as most of the studies focus on single genes that are usually repressed. Due to the genomic binding properties of both macromolecules, we wondered whether there are binding sites shared by PRC2 and MALAT1. Using public genome-binding datasets for PRC2 and MALAT1 derived from independent ChIP- and CHART-seq experiments performed with the breast cancer cell line MCF7, we searched for regions containing PRC2 and MALAT1 overlapping peaks. Peak calls for each molecule were performed using MACS2 and then overlapping peaks were identified by bedtools intersect. Using this approach, we identified 1293 genomic sites where PRC2 and MALAT1 concur. Interestingly, 54.75% of those sites are within gene promoter regions (<3000 bases from the TSS). These analyses were also linked with the transcription profiles of MCF7 cells, obtained from public RNA-seq data. Hence, it is suggested that MALAT1 and PRC2 can concomitantly bind to promoters of actively-transcribed genes in MCF7 cells. Gene ontology analyses revealed an enrichment of genes related to categories including cancer malignancy and epigenetic regulation. Thus, by re-visiting occupancy and transcriptomic data, we identified a key gene subset controlled by the collaboration of MALAT1 and PRC2.

Long Noncoding RNA TALAM1 Is a Transcriptional Target of the RUNX2 Transcription Factor in Lung Adenocarcinoma.

BACKGROUND: Lung cancer is the leading cause of cancer death worldwide. It has been reported that genetic and epigenetic factors play a crucial role in the onset and evolution of lung cancer. Previous reports have shown that essential transcription factors in embryonic development contribute to this pathology. Runt-related transcription factor (RUNX) proteins belong to a family of master regulators of embryonic developmental programs. Specifically, RUNX2 is the master transcription factor (TF) of osteoblastic differentiation, and it can be involved in pathological conditions such as prostate, thyroid, and lung cancer by regulating apoptosis and mesenchymal-epithelial transition processes. In this paper, we identified TALAM1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) as a genetic target of the RUNX2 TF in lung cancer and then performed functional validation of the main findings. METHODS: We performed ChIP-seq analysis of tumor samples from a patient diagnosed with lung adenocarcinoma to evaluate the target genes of the RUNX2 TF. In addition, we performed shRNA-mediated knockdown of RUNX2 in this lung adenocarcinoma cell line to confirm the regulatory role of RUNX2 in TALAM1 expression. RESULTS: We observed RUNX2 overexpression in cell lines and primary cultured lung cancer cells. Interestingly, we found that lncRNA TALAM1 was a target of RUNX2 and that RUNX2 exerted a negative regulatory effect on TALAM1 transcription.

DNA sequencing in the classroom: complete genome sequence of two earwig (Dermaptera; Insecta) species.

BACKGROUND: Despite representing the largest fraction of animal life, the number of insect species whose genome has been sequenced is barely in the hundreds. The order Dermaptera (the earwigs) suffers from a lack of genomic information despite its unique position as one of the basally derived insect groups and its importance in agroecosystems. As part of a national educational and outreach program in genomics, a plan was formulated to engage the participation of high school students in a genome sequencing project. Students from twelve schools across Chile were instructed to capture earwig specimens in their geographical area, to identify them and to provide material for genome sequencing to be carried out by themselves in their schools. RESULTS: The school students collected specimens from two cosmopolitan earwig species: Euborellia annulipes (Fam. Anisolabididae) and Forficula auricularia (Fam. Forficulidae). Genomic DNA was extracted and, with the help of scientific teams that traveled to the schools, was sequenced using nanopore sequencers. The sequence data obtained for both species was assembled and annotated. We obtained genome sizes of 1.18 Gb (F. auricularia) and 0.94 Gb (E. annulipes) with the number of predicted protein coding genes being 31,800 and 40,000, respectively. Our analysis showed that we were able to capture a high percentage (≥ 93%) of conserved proteins indicating genomes that are useful for comparative and functional analysis. We were also able to characterize structural elements such as repetitive sequences and non-coding RNA genes. Finally, functional categories of genes that are overrepresented in each species suggest important differences in the process underlying the formation of germ cells, and modes of reproduction between them, features that are one of the distinguishing biological properties that characterize these two distant families of Dermaptera. CONCLUSIONS: This work represents an unprecedented instance where the scientific and lay community have come together to collaborate in a genome sequencing project. The versatility and accessibility of nanopore sequencers was key to the success of the initiative. We were able to obtain full genome sequences of two important and widely distributed species of insects which had not been analyzed at this level previously. The data made available by the project should illuminate future studies on the Dermaptera.

Genome sequencing and transcriptomic analysis of the Andean killifish Orestias ascotanensis reveals adaptation to high-altitude aquatic life.

Orestias ascotanensis (Cyprinodontidae) is a teleost pupfish endemic to springs feeding into the Ascotan saltpan in the Chilean Altiplano (3,700 m.a.s.l.) and represents an opportunity to study adaptations to high-altitude aquatic environments. We have de novo assembled the genome of O. ascotanensis at high coverage. Comparative analysis of the O. ascotanensis genome showed an overall process of contraction, including loss of genes related to G-protein signaling, chemotaxis and signal transduction, while there was expansion of gene families associated with microtubule-based movement and protein ubiquitination. We identified 818 genes under positive selection, many of which are involved in DNA repair. Additionally, we identified novel and conserved microRNAs expressed in O. ascotanensis and its closely-related species, Orestias gloriae. Our analysis suggests that positive selection and expansion of genes that preserve genome stability are a potential adaptive mechanism to cope with the increased solar UV radiation to which high-altitude animals are exposed to.

Epigenetic Changes and Chromatin Reorganization in Brain Function: Lessons from Fear Memory Ensemble and Alzheimer's Disease.

Healthy brain functioning in mammals requires a continuous fine-tuning of gene expression. Accumulating evidence over the last three decades demonstrates that epigenetic mechanisms and dynamic changes in chromatin organization are critical components during the control of gene transcription in neural cells. Recent genome-wide analyses show that the regulation of brain genes requires the contribution of both promoter and long-distance enhancer elements, which must functionally interact with upregulated gene expression in response to physiological cues. Hence, a deep comprehension of the mechanisms mediating these enhancer-promoter interactions (EPIs) is critical if we are to understand the processes associated with learning, memory and recall. Moreover, the onset and progression of several neurodegenerative diseases and neurological alterations are found to be strongly associated with changes in the components that support and/or modulate the dynamics of these EPIs. Here, we overview relevant discoveries in the field supporting the role of the chromatin organization and of specific epigenetic mechanisms during the control of gene transcription in neural cells from healthy mice subjected to the fear conditioning paradigm, a relevant model to study memory ensemble. Additionally, special consideration is dedicated to revising recent results generated by investigators working with animal models and human postmortem brain tissue to address how changes in the epigenome and chromatin architecture contribute to transcriptional dysregulation in Alzheimer's disease, a widely studied neurodegenerative disease. We also discuss recent developments of potential new therapeutic strategies involving epigenetic editing and small chromatin-modifying molecules (or epidrugs).

Epigenetic silencing of the osteoblast-lineage gene program during hippocampal maturation.

Accumulating evidence indicates that epigenetic control of gene expression plays a significant role during cell lineage commitment and subsequent cell fate maintenance. Here, we assess epigenetic mechanisms operating in the rat brain that mediate silencing of genes that are expressed during early and late stages of osteogenesis. We report that repression of the osteoblast master regulator Sp7 in embryonic (E18) hippocampus is mainly mediated through the Polycomb complex PRC2 and its enzymatic product H3K27me3. During early postnatal (P10), juvenile (P30), and adult (P90) hippocampal stages, the repressive H3K27me3 mark is progressively replaced by nucleosome enrichment and increased CpG DNA methylation at the Sp7 gene promoter. In contrast, silencing of the late bone phenotypic Bglap gene in the hippocampus is PRC2-independent and accompanied by strong CpG methylation from E18 through postnatal and adult stages. Forced ectopic expression of the primary master regulator of osteogenesis Runx2 in embryonic hippocampal neurons activates the expression of its downstream target Sp7 gene. Moreover, transcriptomic analyses show that several genes associated with the mesenchymal-osteogenic lineages are transcriptionally activated in these hippocampal cells that express Runx2 and Sp7. This effect is accompanied by a loss in neuronal properties, including a significant reduction in secondary processes at the dendritic arbor and reduced expression of critical postsynaptic genes like PSD95. Together, our results reveal a developmental progression in epigenetic control mechanisms that repress the expression of the osteogenic program in hippocampal neurons at embryonic, postnatal, and adult stages.

Ezh2-dependent H3K27me3 modification dynamically regulates vitamin D3-dependent epigenetic control of CYP24A1 gene expression in osteoblastic cells.

Epigenetic control is critical for the regulation of gene transcription in mammalian cells. Among the most important epigenetic mechanisms are those associated with posttranslational modifications of chromosomal histone proteins, which modulate chromatin structure and increased accessibility of promoter regulatory elements for competency to support transcription. A critical histone mark is trimethylation of histone H3 at lysine residue 27 (H3K27me3), which is mediated by Ezh2, the catalytic subunit of the polycomb group complex PRC2 to repress transcription. Treatment of cells with the active vitamin D metabolite 1,25(OH)2 D3 , results in transcriptional activation of the CYP24A1 gene, which encodes a 24-hydroxylase enzyme, that is, essential for physiological control of vitamin D3 levels. We report that the Ezh2-mediated deposition of H3K27me3 at the CYP24A1 gene promoter is a requisite regulatory component during transcriptional silencing of this gene in osteoblastic cells in the absence of 1,25(OH)2 D3 . 1,25(OH)2 D3 dependent transcriptional activation of the CYP24A1 gene is accompanied by a rapid release of Ezh2 from the promoter, together with the binding of the H3K27me3-specific demethylase Utx/Kdm6a and thereby subsequent erasing of the H3K27me3 mark. Importantly, we find that these changes in H3K27me3 enrichment at the CYP24A1 gene promoter are highly dynamic, as this modification is rapidly reacquired following the withdrawal of 1,25(OH)2 D3 .

Switches in histone modifications epigenetically control vitamin D3-dependent transcriptional upregulation of the CYP24A1 gene in osteoblastic cells.

In bone cells vitamin D dependent regulation of gene expression principally occurs through modulation of gene transcription. Binding of the active vitamin D metabolite, 1,25-dihydroxy vitamin D3 (1,25(OH)2 D3 ) to the vitamin D receptor (VDR) induces conformational changes in its C-terminal domain enabling competency for interaction with physiologically relevant coactivators, including SRC-1. Consequently, regulatory complexes can be assembled that support intrinsic enzymatic activities with competency to posttranslationally modify chromatin histones at target genomic sequences to epigenetically alter transcription. Here we examine specific transitions in representation and/or enrichment of epigenetic histone marks during 1,25(OH)2 D3 mediated upregulation of CYP24A1 gene expression in osteoblastic cells. This gene encodes the 24-hydroxylase enzyme, essential for biological control of vitamin D levels. We demonstrate that as the CYP24A1 gene promoter remains transcriptionally silent, there is enrichment of H4R3me2s together with its "writing" enzyme PRMT5 and decreased abundance of the istone H3 and H4 acetylation, H3R17me2a, and H4R3me2a marks as well as of their corresponding "writers." Exposure of osteoblastic cells to 1,25(OH)2 D3 stimulates the recruitment of a VDR/SRC-1 containing complex to the CYP24A1 promoter to mediate increased H3/H4 acetylation. VDR/SRC-1 binding occurs concomitant with the release of PRMT5 and the recruitment of the arginine methyltransferases CARM1 and PRMT1 to catalyze the deposition of the H3R17me2a and H4R3me2a marks, respectively. Our results indicate that these dynamic transitions of histone marks at the CYP24A1 promoter, provide a "chromatin context" that is transcriptionally competent for activation of the CYP24A1 gene in osteoblastic cells in response to 1,25(OH)2 D3 .

Downregulation of the Polycomb-Associated Methyltransferase Ezh2 during Maturation of Hippocampal Neurons Is Mediated by MicroRNAs Let-7 and miR-124.

Ezh2 is a catalytic subunit of the polycomb repressive complex 2 (PRC2) which mediates epigenetic gene silencing through depositing the mark histone H3 lysine 27 trimethylation (H3K27me3) at target genomic sequences. Previous studies have demonstrated that Enhancer of Zeste Homolog 2 (Ezh2) was differentially expressed during maturation of hippocampal neurons; in immature neurons, Ezh2 was abundantly expressed, whereas in mature neurons the expression Ezh2 was significantly reduced. Here, we report that Ezh2 is downregulated by microRNAs (miRs) that are expressed during the hippocampal maturation process. We show that, in mature hippocampal neurons, lethal-7 (let-7) and microRNA-124 (miR-124) are robustly expressed and can target cognate motifs at the 3'-UTR of the Ezh2 gene sequence to downregulate Ezh2 expression. Together, these data demonstrate that the PRC2 repressive activity during hippocampal maturation is controlled through a post-transcriptional mechanism that mediates Ezh2 downregulation in mature neurons.

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.

Wnt/ß-catenin signaling enhances transcription of the CX43 gene in murine Sertoli cells.

Sertoli cells provide the nutritional and metabolic support for germ cells. Wnt/ß-catenin signaling is important for the development of the seminiferous epithelium during embryonic age, although after birth this pathway is downregulated. Cx43 gene codes for a protein that is critical during testicular development. The Cx43 promoter contains TCF/ß-catenin binding elements (TBEs) that contribute CX43 expression in different cell types and which may also be regulating the expression of this gene in Sertoli cells. In this study, we demonstrate that 42GPA9 Sertoli cells respond to treatments that result in accumulation of ß-catenin within the nucleus and in upregulation of CX43 gene transcription. ß-Catenin binds to TBEs located both upstream and downstream of the transcriptional start site (TSS). Luciferase reporter experiments revealed that TBEs located upstream of the TSS are necessary for ß-catenin-mediated upregulation. Our results also indicate that the Wnt/ß-catenin-dependent upregulation of the Cx43 gene in Sertoli cells is accompanied by changes in epigenetic parameters that may be directly contributing to generating a chromatin environment that facilitates the establishment of the transcriptional machinery at this promoter.

Role of RUNX2 transcription factor in epithelial mesenchymal transition in non-small cell lung cancer lung cancer: Epigenetic control of the RUNX2 P1 promoter.

Lung cancer has a high mortality rate in men and women worldwide. Approximately 15% of diagnosed patients with this type of cancer do not exceed the 5-year survival rate. Unfortunately, diagnosis is established in advanced stages, where other tissues or organs can be affected. In recent years, lineage-specific transcription factors have been associated with a variety of cancers. One such transcription factor possibly regulating cancer is RUNX2, the master gene of early and late osteogenesis. In thyroid and prostate cancer, it has been reported that RUNX2 regulates expression of genes important in tumor cell migration and invasion. In this study, we report on RUNX2/ p57 overexpression in 16 patients with primary non-small cell lung cancer and/or metastatic lung cancer associated with H3K27Ac at P1 gene promoter region. In some patients, H3K4Me3 enrichment was also detected, in addition to WDR5, MLL2, MLL4, and UTX enzyme recruitment, members of the COMPASS-LIKE complex. Moreover, transforming growth factor-ß induced RUNX2/ p57 overexpression and specific RUNX2 knockdown supported a role for RUNX2 in epithelial mesenchymal transition, which was demonstrated through loss of function assays in adenocarcinoma A549 lung cancer cell line. Furthermore, RUNX2 increased expression of epithelial mesenchymal transition genes VIMENTIN, TWIST1, and SNAIL1, which reflected increased migratory capacity in lung adenocarcinoma cells.

Identification of a novel long noncoding RNA that promotes osteoblast differentiation.

Long noncoding RNAs (lncRNAs) are a heterogeneous class of transcripts, longer than 200 nucleotides, 5'-capped, polyadenylated, and poorly conserved among mammalian species. Several studies have shown the contribution of lncRNAs to different cellular processes, including regulation of the chromatin structure, control of messenger RNA translation, regulation of gene transcription, regulation of embryonic pluripotency, and differentiation. Although limited numbers of functional lncRNAs have been identified so far, the immense regulatory potential of these RNAs is already evident, indicating that a functional characterization of lncRNAs is needed. In this study, mouse preosteoblastic cells were induced to differentiate into osteoblasts. At 3 sequential differentiation stages, total RNA was isolated and libraries were constructed for Illumina sequencing. The resulting sequences were aligned and transcript abundances were determined. New lncRNA candidates that displayed differential expression patterns during osteoblast differentiation were identified by combining bioinformatics and reverse transcription polymerase chain reaction analyses. Among these, lncRNA-1 that exhibited increased expression during osteogenesis and was downregulated during myogenesis. Importantly, knockdown of lncRNA-1 expression in primary mouse preosteoblasts was found to inhibit osteogenic differentiation, reflected by a reduced transcription of the Runx2/p57 and Sp7 bone master genes. Together, our results indicate that lncRNA-1 represents a new regulatory RNA that plays a relevant role during the early stages of osteogenesis.

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.

Epigenetic editing of the Dlg4/PSD95 gene improves cognition in aged and Alzheimer's disease mice.

The Dlg4 gene encodes for post-synaptic density protein 95 (PSD95), a major synaptic protein that clusters glutamate receptors and is critical for plasticity. PSD95 levels are diminished in ageing and neurodegenerative disorders, including Alzheimer's disease and Huntington's disease. The epigenetic mechanisms that (dys)regulate transcription of Dlg4/PSD95, or other plasticity genes, are largely unknown, limiting the development of targeted epigenome therapy. We analysed the Dlg4/PSD95 epigenetic landscape in hippocampal tissue and designed a Dlg4/PSD95 gene-targeting strategy: a Dlg4/PSD95 zinc finger DNA-binding domain was engineered and fused to effector domains to either repress (G9a, Suvdel76, SKD) or activate (VP64) transcription, generating artificial transcription factors or epigenetic editors (methylating H3K9). These epi-editors altered critical histone marks and subsequently Dlg4/PSD95 expression, which, importantly, impacted several hippocampal neuron plasticity processes. Intriguingly, transduction of the artificial transcription factor PSD95-VP64 rescued memory deficits in aged and Alzheimer's disease mice. Conclusively, this work validates PSD95 as a key player in memory and establishes epigenetic editing as a potential therapy to treat human neurological disorders.