Analysis of Methylation Dynamics Reveals a Tissue-Specific, Age-Dependent Decline in 5-Methylcytosine Within the Genome of the Vertebrate Aging Model Nothobranchius furzeri.

Erosion of the epigenetic DNA methylation landscape is a widely recognized hallmark of aging. Emerging advances in high throughput sequencing techniques, in particular DNA methylation data analysis, have resulted in the establishment of precise human and murine age prediction tools. In vertebrates, methylation of cytosine at the C5 position of CpG dinucleotides is executed by DNA methyltransferases (DNMTs) whereas the process of enzymatic demethylation is highly dependent on the activity of the ten-eleven translocation methylcytosine dioxygenase (TET) family of enzymes. Here, we report the identification of the key players constituting the DNA methylation machinery in the short-lived teleost aging model Nothobranchius furzeri. We present a comprehensive spatio-temporal expression profile of the methylation-associated enzymes from embryogenesis into late adulthood, thereby covering the complete killifish life cycle. Data mining of the N. furzeri genome produced five dnmt gene family orthologues corresponding to the mammalian DNMTs (DNMT1, 2, 3A, and 3B). Comparable to other teleost species, N. furzeri harbors multiple genomic copies of the de novo DNA methylation subfamily. A related search for the DNMT1 recruitment factor UHRF1 and TET family members resulted in the identification of N. furzeri uhrf1, tet1, tet2, and tet3. Phylogenetic analysis revealed high cross-species similarity on the amino acid level of all individual dnmts, tets, and uhrf1, emphasizing a high degree of functional conservation. During early killifish development all analyzed dnmts and tets showed a similar expression profile characterized by a strong increase in transcript levels after fertilization, peaking either at embryonic day 6 or at the black eye stage of embryonic development. In adult N. furzeri, DNA methylation regulating enzymes showed a ubiquitous tissue distribution. Specifically, we observed an age-dependent downregulation of dnmts, and to some extent uhrf1, which correlated with a significant decrease in global DNA methylation levels in the aging killifish liver and muscle. The age-dependent DNA methylation profile and spatio-temporal expression characteristics of its enzymatic machinery reported here may serve as an essential platform for the identification of an epigenetic aging clock in the new vertebrate model system N. furzeri.

Spatio-temporal expression profile of sirtuins during aging of the annual fish Nothobranchius furzeri.

The founding member of the sirtuin family, yeast Sir2, was the first evolutionarily conserved gene to be identified as a regulator of longevity. Sirtuins constitute a protein family of metabolic sensors, translating changes in NAD + levels into adaptive responses, thereby acting as crucial regulators of the network that controls energy homeostasis and as such determines healthspan. In mammals the sirtuin family comprises seven proteins, SIRT1-SIRT7, which vary in tissue specificity, subcellular localization, enzymatic activity and targets. Here, we report the identification and a detailed spatio-temporal expression profile of sirtuin genes in the short-lived fish Nothobranchius furzeri, from embryogenesis to late adulthood, mapping its entire life cycle. Database exploration of the recently published N. furzeri genome revealed eight orthologues corresponding to the seven known mammalian sirtuins, including two copies of the sirt5 gene. Phylogenetic analysis showed high cross species similarity of individual sirtuins in both their overall amino acid sequence and catalytic domain, suggesting a high degree of functional conservation. Moreover, we show that N. furzeri sirtuins exhibit ubiquitous and wide tissue distribution with a unique spatial expression pattern for each individual member of this enzyme family. Specifically, we observed a transcriptional down-regulation of several sirtuin genes with age, most significantly sirt1, sirt5a, sirt6 and sirt7 in a wide range of functionally distinct tissues. Overall, this spatio-temporal expression analysis provides the foundation for future research, both into genetic and pharmacological manipulation of this important group of enzymes in Nothobranchius furzeri, an emerging model organism for aging research.

Phylogenetic analysis and expression profiling of the Klotho gene family in the short-lived African killifish Nothobranchius furzeri.

Members of the Klotho gene family have been identified as modulators of the aging process. Deletion of αklotho in the mouse results in a syndrome resembling rapid human aging. Conversely, overexpression of αklotho extends mammalian lifespan. Here, we identify klotho orthologs in the vertebrate aging model Nothobranchius furzeri and provide a detailed spatio-temporal expression profile of both paralogs, α and ßklotho, from embryogenesis until old age spanning the entire life cycle of the organism. Specifically, we observe low levels of expression of both paralogs during embryogenesis followed by a significant transcriptional induction as development proceeds. In adult killifish, αklotho is predominantly expressed in the liver, the kidney, and the developing pharyngeal teeth. Particularly high levels of αKlotho protein were identified in the kidney tubules, closely resembling mammalian expression patterns. Prominent ßklotho expression was detected in the killifish intestine and liver. Overall, qRT-PCR analysis of Klotho members as a function of age revealed steady transcript levels, except for ßklotho expression in the liver which was significantly downregulated with age. This spatio-temporal expression profiling may serve as a useful starting point to further investigate the distinct physiological roles of Klotho members during the aging process.

Histone deacetylase 1 expression is inversely correlated with age in the short-lived fish Nothobranchius furzeri.

Aging is associated with profound changes in the epigenome, resulting in alterations of gene expression, epigenetic landscape, and genome architecture. Class I Histone deacetylases (HDACs), consisting of HDAC1, HDAC2, HDAC3, and HDAC8, play a major role in epigenetic regulation of chromatin structure and transcriptional control, and have been implicated as key players in the pathogenesis of age-dependent diseases and disorders affecting health and longevity. Here, we report the identification of class I Hdac orthologs and their detailed spatio-temporal expression profile in the short-lived fish Nothobranchius furzeri from the onset of embryogenesis until old age covering the entire lifespan of the organism. Database search of the recently annotated N. furzeri genomes retrieved four distinct genes: two copies of hdac1 and one copy of each hdac3 and hdac8. However, no hdac2 ortholog could be identified. Phylogenetic analysis grouped the individual killifish class I Hdacs within the well-defined terminal clades. We find that upon aging, Hdac1 is significantly down-regulated in muscle, liver, and brain, and this age-dependent down-regulation in brain clearly correlates with increased mRNA levels of the cyclin-dependent kinase inhibitor cdkn1a (p21). Furthermore, this apparent reduction of class I HDACs in transcript and protein levels is mirrored in the mouse brain, highlighting an evolutionarily conserved role of class I HDACs during normal development and in the aging process.

Distinct chromatin signature of histone H3 variant H3.3 in human cells.

Actively transcribed regions of the genome have been found enriched for the histone H3 variant H3.3. This variant is incorporated into nucleosomes throughout the cell cycle whereas the canonical isoforms are predominately deposited in association with replication. In order to obtain a global picture of the deposition pattern at the single cell level we expressed H3.3 in both normal and malignant human cells and analyzed nuclei using conventional and structured illumination imaging (SIM). We found that the distribution pattern of H3.3 in interphase differs from that of the canonical histone H3 variants and this difference is conveyed to mitotic chromosomes which display a distinct H3.3 banding pattern. Histone H3.3 localization positively correlated with markers for transcriptionally active chromatin and, notably, H3.3 was almost completely absent from the inactive X chromosome. Collectively, our data show that histone variant H3.3 occupies distinct intranuclear chromatin domains and that these genomic loci are associated with gene expression.

Distinct and redundant functions of histone deacetylases HDAC1 and HDAC2 in proliferation and tumorigenesis.

Histone deacetylases (HDACs) are negative regulators of gene expression and have been implicated in tumorigenesis and tumor progression. Therefore, HDACs are promising targets for anti-tumor drugs. However, the relevant isoforms of the 18 members encompassing HDAC family have not been identified. Studies utilizing either gene targeting or knockdown approaches reveal both specific and redundant functions of the closely related class I deacetylases HDAC1 and HDAC2 in the control of proliferation and differentiation. Combined ablation of HDAC1 and HDAC2 in different cell types led to a severe proliferation defects or enhanced apoptosis supporting the idea that both enzymes are relevant targets for tumor therapy. In a recent study on the role of HDAC1 in teratoma formation we have reported a novel and surprising function of HDAC1 in tumorigenesis. In this tumor model HDAC1 attenuates proliferation during teratoma formation. In the present work we discuss new findings on redundant and unique functions of HDAC1 and HDAC2 as regulators of proliferation and tumorigenesis and potential implications for applications of HDAC inhibitors as therapeutic drugs.

A phosphorylation switch regulates the transcriptional activation of cell cycle regulator p21 by histone deacetylase inhibitors.

Histone deacetylase inhibitors induce cell cycle arrest and apoptosis in tumor cells and are, therefore, promising anti-cancer drugs. The cyclin-dependent kinase inhibitor p21 is activated in histone deacetylase (HDAC) inhibitor-treated tumor cells, and its growth-inhibitory function contributes to the anti-tumorigenic effect of HDAC inhibitors. We show here that induction of p21 by trichostatin A involves MAP kinase signaling. Activation of the MAP kinase signaling pathway by growth factors or stress signals results in histone H3 serine 10 phosphorylation at the p21 promoter and is crucial for acetylation of the neighboring lysine 14 and recruitment of activated RNA polymerase II in response to trichostatin A treatment. In non-induced cells, the protein phosphatase PP2A is associated with the p21 gene and counteracts its activation. Induction of p21 is linked to simultaneous acetylation and phosphorylation of histone H3. The dual modification mark H3S10phK14ac at the activated p21 promoter is recognized by the phospho-binding protein 14-3-3ζ, which protects the phosphoacetylation mark from being processed by PP2A. Taken together we have revealed a cross-talk of reversible phosphorylation and acetylation signals that controls the activation of p21 by HDAC inhibitors and identify the phosphatase PP2A as chromatin-associated transcriptional repressor in mammalian cells.

Conditional deletion of histone deacetylase 1 in T cells leads to enhanced airway inflammation and increased Th2 cytokine production.

Chromatin modifications, such as reversible histone acetylation, play a key role in the regulation of T cell development and function. However, the role of individual histone deacetylases (HDACs) in T cells is less well understood. In this article, we show by conditional gene targeting that T cell-specific loss of HDAC1 led to an increased inflammatory response in an in vivo allergic airway inflammation model. Mice with HDAC1-deficient T cells displayed an increase in all critical parameters in this Th2-type asthma model, such as eosinophil recruitment into the lung, mucus hypersecretion, parenchymal lung inflammation, and enhanced airway resistance. This correlated with enhanced Th2 cytokine production in HDAC1-deficient T cells isolated from diseased mice. In vitro-polarized HDAC1-deficient Th2 cells showed a similar enhancement of IL-4 expression, which was evident already at day 3 of Th2 differentiation cultures and restricted to T cell subsets that underwent several rounds of cell divisions. HDAC1 was recruited to the Il4 gene locus in ex vivo isolated nonstimulated CD4(+) T cells, indicating a direct control of the Il4 gene locus. Our data provide genetic evidence that HDAC1 is an essential HDAC that controls the magnitude of an inflammatory response by modulating cytokine expression in effector T cells.

The cyclin-dependent kinase inhibitor p21 is a crucial target for histone deacetylase 1 as a regulator of cellular proliferation.

Histone deacetylases (HDACs) are chromatin-modifying enzymes that are involved in the regulation of proliferation, differentiation and development. HDAC inhibitors induce cell cycle arrest, differentiation, or apoptosis in tumor cells and are therefore promising antitumor agents. Numerous genes were found to be deregulated upon HDAC inhibitor treatment; however, the relevant target enzymes are still unidentified. HDAC1 is required for mouse development and unrestricted proliferation of embryonic stem cells. We show here that HDAC1 reversibly regulates cellular proliferation and represses the cyclin-dependent kinase inhibitor p21 in embryonic stem cells. Disruption of the p21 gene rescues the proliferation phenotype of HDAC1(-/-) embryonic stem cells but not the embryonic lethality of HDAC1(-/-) mice. In the absence of HDAC1, mouse embryonic fibroblasts scarcely undergo spontaneous immortalization and display increased p21 expression. Chromatin immunoprecipitation assays demonstrate a direct regulation of the p21 gene by HDAC1 in mouse embryonic fibroblasts. Transformation with simian virus 40 large T antigen or ablation of p21 restores normal immortalization of primary HDAC1(-/-) fibroblasts. Our data demonstrate that repression of the p21 gene is crucial for HDAC1-mediated control of proliferation and immortalization. HDAC1 might therefore be one of the relevant targets for HDAC inhibitors as anticancer drugs.

Epigenetic regulation of dendritic cell differentiation and function by oxidized phospholipids.

Dendritic cells (DCs) are the key cell type in the regulation of an adaptive immune response. Under inflammatory conditions monocytes can give rise to immunostimulatory DCs, depending on microenvironmental stimuli. Here we show that oxidized phospholipids (Ox-Pls), which are generated during inflammatory reactions, dysregulate the differentiation of DCs. DCs generated in the presence of Ox-Pls up-regulated the typical DC marker DC-SIGN but did not express CD1a, CD1b, and CD1c. These DCs generated in the presence of Ox-Pls had a substantially diminished T cell-stimulating capacity after stimulation with Toll-like receptor ligands. Toll-like receptor ligand-induced production of interleukin-12 also was strongly diminished, whereas induction of CD83 was not altered. In addition, we found that Ox-Pls strongly inhibit inflammatory stimuli-induced phosphorylation of histone H3, a key step of interleukin-12 production, yet leaving activation of nuclear factor-kappaB unaltered. Taken together, Ox-Pls present during differentiation yielded DCs with a reduced capacity to become immunostimulatory mature DCs. Furthermore, the presence of Ox-Pls blocked histone modifications required for full activation of DCs. Therefore, inflammation-derived Ox-Pls control DC functions in part by epigenetic mechanisms.

14-3-3 proteins recognize a histone code at histone H3 and are required for transcriptional activation.

Interphase phosphorylation of S10 at histone H3 is linked to transcriptional activation of a specific subset of mammalian genes like HDAC1. Recently, 14-3-3 proteins have been described as detectors for this phosphorylated histone H3 form. Here, we report that 14-3-3 binding is modulated by combinatorial modifications of histone H3. S10 phosphorylation is necessary for an interaction, but additional H3K9 or H3K14 acetylation increases the affinity of 14-3-3 for histone H3. Histone H3 phosphoacetylation occurs concomitant with K9 methylation in vivo, suggesting that histone phosphorylation and acetylation can synergize to overcome repressive histone methylation. Chromatin immunoprecipitation experiments reveal recruitment of 14-3-3 proteins to the HDAC1 gene in an H3S10ph-dependent manner. Recruitment of 14-3-3 to the promoter is enhanced by additional histone H3 acetylation and correlates with dissociation of the repressive binding module HP1gamma. Finally, siRNA-mediated loss of 14-3-3 proteins abolishes the transcriptional activation of HDAC1. Together our data indicate that 14-3-3 proteins are crucial mediators of histone phosphoacetylation signals.

Distinct modes of action applied by transcription factors STAT1 and IRF1 to initiate transcription of the IFN-gamma-inducible gbp2 gene.

A subgroup of genes induced by IFN-gamma requires both STAT1 and IRF1 for transcriptional activation. Using WT, stat1(-/-), or irf1(-/-) cells, we analyzed the changes induced by IFN-gamma in gbp2 promoter chromatin. STAT1 associated with the promoter independently of IRF1 and played an essential role in the ordered recruitment of the coactivator/histone acetyl transferase CREB-binding protein (CBP) and the histone deacetylase HDAC1. Hyperacetylation of histone 4 also required STAT1. Phosphorylation at S727 in the transactivating domain increased transcriptional activity of STAT1. In cells expressing a STAT1S727A-mutant CBP recruitment, histone 4 hyperacetylation and RNA polymerase II association with the gbp2 promoter were strongly reduced. IRF1 association with the gbp2 promoter followed that of STAT1, but STAT1 association with DNA or histone hyperacetylation were not necessary for IRF1 binding. RNA polymerase II association with the gbp2 promoter required both STAT1 and IRF1, suggesting that both proteins mediate essential steps in transcriptional activation. IRF1, but not STAT1, was found to coimmunoprecipitate with RNA polymerase II. Together, the data support the assumption that the main role of STAT1 in activating gbp2 transcription is to provide transcriptionally competent chromatin, whereas the function of IRF1 may lie in directly contacting RNA polymerase II-containing transcriptional complexes.

Negative and positive regulation of gene expression by mouse histone deacetylase 1.

Histone deacetylases (HDACs) catalyze the removal of acetyl groups from core histones. Because of their capacity to induce local condensation of chromatin, HDACs are generally considered repressors of transcription. In this report, we analyzed the role of the class I histone deacetylase HDAC1 as a transcriptional regulator by comparing the expression profiles of wild-type and HDAC1-deficient embryonic stem cells. A specific subset of mouse genes (7%) was deregulated in the absence of HDAC1. We identified several putative tumor suppressors (JunB, Prss11, and Plagl1) and imprinted genes (Igf2, H19, and p57) as novel HDAC1 targets. The majority of HDAC1 target genes showed reduced expression accompanied by recruitment of HDAC1 and local reduction in histone acetylation at regulatory regions. At some target genes, the related deacetylase HDAC2 partially masks the loss of HDAC1. A second group of genes was found to be downregulated in HDAC1-deficient cells, predominantly by additional recruitment of HDAC2 in the absence of HDAC1. Finally, a small set of genes (Gja1, Irf1, and Gbp2) was found to require HDAC activity and recruitment of HDAC1 for their transcriptional activation. Our study reveals a regulatory cross talk between HDAC1 and HDAC2 and a novel function for HDAC1 as a transcriptional coactivator.

Optimizing the binding affinity of a carrier protein: a case study on the interaction between soluble ifnar2 and interferon beta.

Prolonging the circulatory half-life of low mass protein drugs can be achieved either by administration of a pro-drug or through co-injection with a carrier protein that will slowly release the active protein. The rate of release is concentration and affinity dependent. The optimal relationship between these two in prolonging the half-life of a pro-drug is the focus of this work. Interferon (IFN) beta is one of the most widely used protein drugs in the clinic. Here, we show that the circulatory half-life of IFNbeta can be significantly extended by co-administration with the extracellular domain of the IFN receptor ifnar2 (ifnar2-EC). To investigate the concentration/affinity relation, a range of tighter binding ifnar2-EC mutants was designed that bind IFNbeta, but not IFNalpha2, up to 50-fold tighter compared with the wild-type ifnar2-EC. This increased affinity is related to a slower dissociation rate, whereas the association of IFNbeta with ifnar2-EC is already near optimum. Using the wild-type and mutant receptors, we investigated their potential in occluding IFNbeta from circulation in a tissue culture assay, as well as in rats. To determine the potential of ifnar2-EC as a carrier protein, we co-administered a mixture of IFNbeta and ifnar2-EC to rats both intravenously and subcutaneously, and followed the blood plasma concentrations of IFNbeta over time. The tighter binding ifnar2-EC mutant had a clear advantage in prolonging the half-life of IFNbeta in circulation, even when lower protein concentrations were administered. A numerical simulation of the in vivo data demonstrates that the optimal binding affinity of a carrier protein is around the concentration needed to obtain optimal activity of the ligand.