Transcriptional regulation of CDKN2A/p16 by sirtuin 7 in senescence.

Cell senescence is a state of limited cell proliferation during a stress response or as part of a programmed process. When a senescent cell stops dividing, maintaining metabolic activity contributes to cellular homeostasis maintenance. In this process, the cell cycle is arrested at the G0/G1 phase. p16INK4A protein is a key regulator of this process via its cyclin­dependent kinase inhibitor (CDKI) function. CDKI 2A (CDKN2A)/p16 gene expression is regulated by DNA methylation and histone acetylation. Sirtuins (SIRTs) are nicotinamide dinucleotide (NAD+)­dependent deacetylases that have properties which prevent diseases and reverse certain aspects of aging (such as immune, metabolic and cardiovascular diseases). By performing quantitative PCR, Western blot, ChIP, and siRNAs assays, in this study it was demonstrated that CDKN2A/p16 gene transcriptional activation and repression were accompanied by selective deposition and elimination of histone acetylation during the senescence of MRC5 cells. Specifically, significant H3K9Ac and H3K18Ac enrichment in cells with a senescent phenotype concomitant with CDKN2A/p16 gene overexpression was demonstrated compared with the non­senescent phenotype. Furthermore, the presence of H3K18Ac in deacetyl­transferase SIRT7 knockdown MRC5 cells allowed CDKN2A/p16 promoter activation. These results suggested that SIRT7 served as a critical component of an epigenetic mechanism involved in senescence mediated by the CDKN2A/p16 gene.

RUNX family: Oncogenes or tumor suppressors (Review).

Runt­related transcription factor (RUNX) proteins belong to a transcription factors family known as master regulators of important embryonic developmental programs. In the last decade, the whole family has been implicated in the regulation of different oncogenic processes and signaling pathways associated with cancer. Furthermore, a suppressor tumor function has been also reported, suggesting the RUNX family serves key role in all different types of cancer. In this review, the known biological characteristics, specific regulatory abilities and experimental evidence of RUNX proteins will be analyzed to demonstrate their oncogenic potential and tumor suppressor abilities during oncogenic processes, suggesting their importance as biomarkers of cancer. Additionally, the importance of continuing with the molecular studies of RUNX proteins' and its dual functions in cancer will be underlined in order to apply it in the future development of specific diagnostic methods and therapies against different types of cancer.

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.

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.

Epigenetic Control of the Bone-master Runx2 Gene during Osteoblast-lineage Commitment by the Histone Demethylase JARID1B/KDM5B.

Transcription factor Runx2 controls bone development and osteoblast differentiation by regulating expression of a significant number of bone-related target genes. Here, we report that transcriptional activation and repression of the Runx2 gene via its osteoblast-specific P1 promoter (encoding mRNA for the Runx2/p57 isoform) is accompanied by selective deposition and elimination of histone marks during differentiation of mesenchymal cells to the osteogenic and myoblastic lineages. These epigenetic profiles are mediated by key components of the Trithorax/COMPASS-like and Polycomb group complexes together with histone arginine methylases like PRMT5 and lysine demethylases like JARID1B/KDM5B. Importantly, knockdown of the H3K4me2/3 demethylase JARID1B, but not of the demethylases UTX and NO66, prevents repression of the Runx2 P1 promoter during myogenic differentiation of mesenchymal cells. The epigenetically forced expression of Runx2/p57 and osteocalcin, a classical bone-related target gene, under myoblastic-differentiation is accompanied by enrichment of the H3K4me3 and H3K27ac marks at the Runx2 P1 promoter region. Our results identify JARID1B as a key component of a potent epigenetic switch that controls mesenchymal cell fate into myogenic and osteogenic lineages.

PSD95 suppresses dendritic arbor development in mature hippocampal neurons by occluding the clustering of NR2B-NMDA receptors.

Considerable evidence indicates that the NMDA receptor (NMDAR) subunits NR2A and NR2B are critical mediators of synaptic plasticity and dendritogenesis; however, how they differentially regulate these processes is unclear. Here we investigate the roles of the NR2A and NR2B subunits, and of their scaffolding proteins PSD-95 and SAP102, in remodeling the dendritic architecture of developing hippocampal neurons (2-25 DIV). Analysis of the dendritic architecture and the temporal and spatial expression patterns of the NMDARs and anchoring proteins in immature cultures revealed a strong positive correlation between synaptic expression of the NR2B subunit and dendritogenesis. With maturation, the pruning of dendritic branches was paralleled by a strong reduction in overall and synaptic expression of NR2B, and a significant elevation in synaptic expression of NR2A and PSD95. Using constructs that alter the synaptic composition, we found that either over-expression of NR2B or knock-down of PSD95 by shRNA-PSD95 augmented dendritogenesis in immature neurons. Reactivation of dendritogenesis could also be achieved in mature cultured neurons, but required both manipulations simultaneously, and was accompanied by increased dendritic clustering of NR2B. Our results indicate that the developmental increase in synaptic expression of PSD95 obstructs the synaptic clustering of NR2B-NMDARs, and thereby restricts reactivation of dendritic branching. Experiments with shRNA-PSD95 and chimeric NR2A/NR2B constructs further revealed that C-terminus of the NR2B subunit (tail) was sufficient to induce robust dendritic branching in mature hippocampal neurons, and suggest that the NR2B tail is important in recruiting calcium-dependent signaling proteins and scaffolding proteins necessary for dendritogenesis.

Ezh1 and Ezh2 differentially regulate PSD-95 gene transcription in developing hippocampal neurons.

Polycomb Repressive Complex 2 (PRC2) mediates transcriptional silencing by catalyzing histone H3 lysine 27 trimethylation (H3K27me3), but its role in the maturation of postmitotic mammalian neurons remains largely unknown. We report that the PRC2 paralogs Ezh1 and Ezh2 are differentially expressed during hippocampal development. We show that depletion of Ezh2 leads to increased expression of PSD-95, a critical plasticity gene, and that reduced PSD-95 gene transcription is correlated with enrichment of Ezh2 at the PSD-95 gene promoter; however, the H3K27me3 epigenetic mark is not present at the PSD-95 gene promoter, likely due to the antagonizing effects of the H3S28P and H3K27Ac marks and the activity of the H3K27 demethylases JMJD3 and UTX. In contrast, increased PSD-95 gene transcription is accompanied by the presence of Ezh1 and elongation-engaged RNA Polymerase II complexes at the PSD-95 gene promoter, while knock-down of Ezh1 reduces PSD-95 transcription. These results indicate that Ezh1 and Ezh2 have antagonistic roles in regulating PSD-95 transcription.

The Ric-8B gene is highly expressed in proliferating preosteoblastic cells and downregulated during osteoblast differentiation in a SWI/SNF- and C/EBPbeta-mediated manner.

The Ric-8 gene encodes a guanine exchange factor (GEF) that modulates G protein-mediated signaling, exhibiting a relevant role during regulation of cell division. In mammals, two Ric-8 homologues have been reported (Ric-8A and Ric-8B), and recent studies indicate equivalent roles for each protein. Here, we show that the Ric-8B gene is negatively regulated during osteoblast differentiation by the transcription factor C/EBPß. Only the larger C/EBPß isoform (C/EBPß-LAP*) downregulates Ric-8B gene promoter activity in osteoblastic cells. Accordingly, knockdown of C/EBPß expression by small intefering RNA in osteoblastic cells results in a significant increase of Ric-8B gene expression. Transient overexpression of Brg1 or Brm, the catalytic subunits of the SWI/SNF chromatin-remodeling complex, inhibits Ric-8B promoter activity. Also, the presence of inactive SWI/SNF complexes in osteoblastic cells results in increased endogenous Ric-8B transcription, indicating that SWI/SNF activity negatively regulates Ric-8B expression. During osteoblast differentiation, Ric-8B gene repression is accompanied by changes in nucleosome placement at the proximal Ric-8B gene promoter and reduced accessibility to regulatory sequences.

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.

Evolution of the interaction between Runx2 and VDR, two transcription factors involved in osteoblastogenesis.

BACKGROUND: The mineralized skeleton is a major evolutionary novelty that has contributed to the impressive morphological diversifications of the vertebrates. Essential to bone biology is the solidified extracellular matrix secreted by highly specialized cells, the osteoblasts. We now have a rather complete view of the events underlying osteogenesis, from a cellular, molecular, genetic, and epigenetic perspective. Because this knowledge is still largely restricted to mammals, it is difficult, if not impossible, to deduce the evolutionary history of the regulatory network involved in osteoblasts specification and differentiation. In this study, we focused on the transcriptional regulators Runx2 and VDR (the Vitamin D Receptor) that, in mammals, directly interact together and stabilize complexes of co-activators and chromatin remodellers, thereby allowing the transcriptional activation of target genes involved in extracellular matrix mineralization. Using a combination of functional, biochemical, and histological approaches, we have asked if the interaction observed between Runx2 and VDR represents a recent mammalian innovation, or if it results from more ancient changes that have occurred deep in the vertebrate lineage. RESULTS: Using immunohistochemistry and in situ hybridization in developing embryos of chick, frog and teleost fishes, we have revealed that the co-expression of Runx2 and VDR in skeletal elements has been particularly strengthened in the lineage leading to amniotes. We show that the teleost Runx2 orthologue as well as the three mammalian Runx1, Runx2 and Runx3 paralogues are able to co-immunoprecipitate with the VDR protein present in nuclear extracts of rat osteoblasts stimulated with 1alpha,25-dihydroxyvitamin D3. In addition, the teleost Runx2 can activate the transcription of the mammalian osteocalcin promoter in transfection experiments, and this response can be further enhanced by 1alpha,25-dihydroxyvitamin D3. Finally, using pull-down experiments between recombinant proteins, we show that the VDR homologue from teleosts, but not from ascidians, is able to directly interact with the mammalian Runx2 homologue. CONCLUSIONS: We propose an evolutionary scenario for the assembly of the molecular machinery involving Runx2 and VDR in vertebrates. In the last common ancestor of actinopterygians and sacropterygians, the three Runx paralogues possessed the potential to physically and functionally interact with the VDR protein. Therefore, 1alpha,25-dihydroxyvitamin D3 might have been able to modulate the transcriptional activity of Runx1, Runx2 or Runx3 in the tissues expressing VDR. After the split from amphibians, in the lineage leading to amniotes, Runx2 and VDR became robustly co-expressed in developing skeletal elements, and their regulatory interaction was incorporated in the genetic program involved in the specification and differentiation of osteoblasts.

Recruitment and subnuclear distribution of the regulatory machinery during 1alpha,25-dihydroxy vitamin D3-mediated transcriptional upregulation in osteoblasts.

The architectural organization of the genome and regulatory proteins within the nucleus supports gene expression in a physiologically regulated manner. In osteoblastic cells ligand activation induces a nuclear punctate distribution of the 1alpha,25-dihydroxy vitamin D3 (1alpha,25(OH)2D3) receptor (VDR) and promotes its interaction with transcriptional coactivators such as SRC-1, NCoA-62/Skip, and DRIP205. Here, we discuss evidence demonstrating that in osteoblastic cells VDR binds to the nuclear matrix fraction in a 1alpha,25(OH)2D3-dependent manner. This interaction occurs rapidly after exposure to 1alpha,25(OH)2D3 and does not require a functional VDR DNA binding domain. The nuclear matrix-bound VDR molecules colocalize with the also nuclear matrix-associated coactivator DRIP205. We propose a model where the rapid association of VDR with the nuclear matrix fraction represents an event that follows 1alpha,25(OH)2D3-dependent nuclear localization of VDR, but that precedes 1alpha,25(OH)2D3-dependent transcriptional upregulation at target genes.

SWI/SNF-independent nuclease hypersensitivity and an increased level of histone acetylation at the P1 promoter accompany active transcription of the bone master gene Runx2.

The Runx2 transcription factor is essential for skeletal development as it regulates expression of several key bone-related genes. Multiple lines of evidence indicate that expression of the Runx2/p57 isoform in osteoblasts is controlled by the distal P1 promoter. Alterations of chromatin structure are often associated with transcription and can be mediated by members of the SWI/SNF family of chromatin remodeling complexes, or by transcriptional coactivators that possess enzymatic activities that covalently modify structural components of the chromatin. Here, we report that a specific chromatin remodeling process at the proximal region (residues -400 to 35) of the Runx2 gene P1 promoter accompanies transcriptional activity in osteoblasts. This altered chromatin organization is reflected by the presence of two DNase I hypersensitive sites that span key regulatory elements for Runx2/p57 transcription. Chromatin remodeling and transcription of the Runx2 gene are associated with elevated levels of histone acetylation at the P1 promoter region and binding of active RNA polymerase II and are independent of the activity of the SWI/SNF chromatin remodeling complex. Changes in chromatin organization at the P1 promoter are stimulated during differentiation of C2C12 mesenchymal cells to the osteoblastic lineage by treatment with BMP2. Together, our results support a model in which changes in chromatin organization occur at very early stages of mesenchymal differentiation to facilitate subsequent expression of the Runx2/p57 isoform in osteoblastic cells.

1alpha,25-dihydroxy vitamin D3-enhanced expression of the osteocalcin gene involves increased promoter occupancy of basal transcription regulators and gradual recruitment of the 1alpha,25-dihydroxy vitamin D3 receptor-SRC-1 coactivator complex.

Binding of 1alpha,25-dihydroxy vitamin D(3) to the C-terminal ligand-binding domain (LBD) of its receptor (VDR) induces a conformational change that enables interaction of VDR with transcriptional coactivators such as members of the p160/SRC family or the DRIP (vitamin D receptor-interacting complex)/Mediator complex. These interactions are critical for VDR-mediated transcriptional enhancement of target genes. The p160/SRC members contain intrinsic histone acetyl transferase (HAT) activities that remodel chromatin at promoter regulatory regions, and the DRIP/Mediator complex may establish a molecular bridge between the VDR complex and the basal transcription machinery. Here, we have analyzed the rate of recruitment of these coactivators to the bone-specific osteocalcin (OC) gene in response to short and long exposures to 1alpha,25-dihydroxy vitamin D3. We report that in intact osteoblastic cells VDR, in association with SRC-1, rapidly binds to the OC promoter in response to the ligand. The recruitment of SRC-1 correlates with maximal transcriptional enhancement of the OC gene at 4 h and with increased histone acetylation at the OC promoter. In contrast to other 1alpha,25-dihydroxy vitamin D3-enhanced genes, binding of the DRIP205 subunit, which anchors the DRIP/Mediator complex to the VDR, is detected at the OC promoter only after several hours of incubation with 1alpha,25-dihydroxy vitamin D(3), concomitant with the release of SRC-1. Together, our results support a model where VDR preferentially recruits SRC-1 to enhance bone-specific OC gene transcription.

The 1alpha,25-dihydroxy Vitamin D3 receptor preferentially recruits the coactivator SRC-1 during up-regulation of the osteocalcin gene.

Binding of 1alpha,25-dihydroxy Vitamin D3 to the C-terminal domain (LBD) of its receptor (VDR), induces a conformational change that enables interaction of VDR with transcriptional coactivators such as the members of the p160/SRC family or the DRIP (Vitamin D interacting complex)/Mediator complex. These interactions are critical for VDR-mediated transcriptional enhancement of target genes. Recent reports indicate that nuclear receptors, including VDR, interact with p160/SRC members and the DRIP/Mediator complex in a sequential, cyclical, and mutually exclusive manner when bound to a target promoter, exhibiting also a high exchange rate. Here, we present an overview of how these coactivators are recruited to the bone-specific osteocalcin (OC) gene in response to short and long exposures to 1alpha,25-dihydroxy Vitamin D3. We find that in intact osteoblastic cells VDR and SRC-1 rapidly bind to the OC promoter in response to the ligand. This recruitment correlates with transcriptional enhancement of the OC gene and with increased histone acetylation at the OC promoter. In contrast, binding of the DRIP205 subunit, which anchors the DRIP/Mediator complex to the VDR, is detected at the OC promoter after several hours of incubation with 1alpha,25-dihydroxy Vitamin D3. Together, our results indicate that VDR preferentially recruits SRC-1 to enhance basal bone-specific OC gene transcription. We propose a model where specific protein-DNA and protein-protein interactions that occur within the context of the OC gene promoter in osteoblastic cells stabilize the preferential association of the VDR-SRC-1 complex.