Angelika Böttger

The dynamic genome of Hydra

The freshwater cnidarian Hydra was first described in 1702 and has been the object of study for 300 years. Experimental studies of Hydra between 1736 and 1744 culminated in the discovery of asexual reproduction of an animal by budding, the first description of regeneration in an animal, and successful transplantation of tissue between animals. Today, Hydra is an important model for studies of axial patterning, stem cell biology and regeneration. Here we report the genome of Hydra magnipapillata and compare it to the genomes of the anthozoan Nematostella vectensis and other animals. The Hydra genome has been shaped by bursts of transposable element expansion, horizontal gene transfer, trans-splicing, and simplification of gene structure and gene content that parallel simplification of the Hydra life cycle. We also report the sequence of the genome of a novel bacterium stably associated with H. magnipapillata. Comparisons of the Hydra genome to the genomes of other animals shed light on the evolution of epithelia, contractile tissues, developmentally regulated transcription factors, the Spemann–Mangold organizer, pluripotency genes and the neuromuscular junction.

Jmjd6 Catalyses Lysyl-Hydroxylation of U2AF65, a Protein Associated with RNA Splicing

Modifying the Modifier Covalent modification of proteins provides an important means whereby their function is regulated. Hydroxylation, catalyzed by oxygenase enzymes, plays an important role in the response to hypoxia, for example. The human protein Jmjd6 has been thought to act as an oxygenase, catalyzing the demethylation of histone H3 at arginine-2 and histone H4 at arginine-3. Webby et al. (p. 90) now show that Jmjd6 interacts with the messenger RNA splicing factor U2AF65 and acts to hydroxylate this protein at lysine residues, modifications also seen in vivo. Furthermore, Jmjd6 modulates the alternative splicing of both an endogenous gene and an introduced mini-gene. An oxygenase with an important role in vertebrate development hydroxylates a messenger RNA splicing factor. The finding that the metazoan hypoxic response is regulated by oxygen-dependent posttranslational hydroxylations, which regulate the activity and lifetime of hypoxia-inducible factor (HIF), has raised the question of whether other hydroxylases are involved in the regulation of gene expression. We reveal that the splicing factor U2 small nuclear ribonucleoprotein auxiliary factor 65-kilodalton subunit (U2AF65) undergoes posttranslational lysyl-5-hydroxylation catalyzed by the Fe(II) and 2-oxoglutarate–dependent dioxygenase Jumonji domain-6 protein (Jmjd6). Jmjd6 is a nuclear protein that has an important role in vertebrate development and is a human homolog of the HIF asparaginyl-hydroxylase. Jmjd6 is shown to change alternative RNA splicing of some, but not all, of the endogenous and reporter genes, supporting a specific role for Jmjd6 in the regulation of RNA splicing.

The phosphatidylserine receptor from Hydra is a nuclear protein with potential Fe(II) dependent oxygenase activity

BackgroundApoptotic cell death plays an essential part in embryogenesis, development and maintenance of tissue homeostasis in metazoan animals. The culmination of apoptosis in vivo is the phagocytosis of cellular corpses. One morphological characteristic of cells undergoing apoptosis is loss of plasma membrane phospholipid asymmetry and exposure of phosphatidylserine on the outer leaflet. Surface exposure of phosphatidylserine is recognised by a specific receptor (phosphatidylserine receptor, PSR) and is required for phagocytosis of apoptotic cells by macrophages and fibroblasts.ResultsWe have cloned the PSR receptor from Hydra in order to investigate its function in this early metazoan. Bioinformatic analysis of the Hydra PSR protein structure revealed the presence of three nuclear localisation signals, an AT-hook like DNA binding motif and a putative 2-oxoglutarate (2OG)-and Fe(II)-dependent oxygenase activity. All of these features are conserved from human PSR to Hydra PSR. Expression of GFP tagged Hydra PSR in hydra cells revealed clear nuclear localisation. Deletion of one of the three NLS sequences strongly diminished nuclear localisation of the protein. Membrane localisation was never detected.ConclusionsOur results suggest that Hydra PSR is a nuclear 2-oxoglutarate (2OG)-and Fe(II)-dependent oxygenase. This is in contrast with the proposed function of Hydra PSR as a cell surface receptor involved in the recognition of apoptotic cells displaying phosphatidylserine on their surface. The conservation of the protein from Hydra to human infers that our results also apply to PSR from higher animals.

Evolution of gap junctions: the missing link?

Connexin molecules form gap-junction channels in vertebrates and there are at least 20 of them in humans [1]. Intuitively, one would imagine that cardinal features of the cellular machinery, such as gap-junctions, would be highly conserved. Paradoxically, however, Drosophila and Caenorhabditis elegans do not have connexin genes, but instead use innexins for gap-junctional communication, a protein family with the same 4-transmembrane topology but no sequence similarity to the connexins [2,3]. In this paper we show that the simple diploblastic organism Hydra appears to possess only innexins. We conclude that innexins are the primordial gap-junction molecules, while connexins evolved more recently in the deuterostomes. The major question is whether the connexin–innexin dichotomy is an extreme case of sequence divergence from a common ancestor or a convergent solution to the problem of intercellular communication. A critical experiment was to identify gap-junction proteins in diploblastic organisms, e.g cnidaria. These organisms have functional gap junctions [4] and represent an evolutionary grade before the deuterostome–protostome divergence. We focused on the hydrozoan Hydra because the cells that comprise its body have large gap-junction plaques and are electrically and dye coupled [4,5]. During a signal peptide screen, we recovered a fragment, which matched a set of 14 overlapping ESTs (contig number C_CD267995, available at http://mpc.uci.edu/ hampson/public_html/blast/jf), which we identified as a true innexin. The original EST collection had 3500 distinct sequences derived from 13,000 ESTs. The novel sequence has 396 amino acids and a predicted molecular weight of 44.9 kDa. A structural prediction using the Kyte-Doolittle algorithm showed that this molecule, named Hydra innexin-1 (Hv-inx1), has a typical innexin topology with 4-transmembrane (TM) domains and amino-and carboxy-terminal domains on the cytoplasmic face of the membrane. The extracellular loops contain pairs of invariant cysteine residues and the transmembrane domains (TM) contain signature residues Y, Q, W, P (second TM) and W, F (fourth TM) at conserved positions ([6]; Figure 1). This strongly supports the identification of Hv-inx1 as a true innexin despite low overall sequence identity. Expression of a Hv-inx1-GFP fusion protein in Hydra revealed a punctate pattern of GFP fluorescence along the basal lateral membrane of epithelial cells (Figure 2), corresponding to known sites of gap junctions [5]. revealed four more innexin homologs in addition to innexin-1. The enlarged EST collection was also searched for connexins using the BLAST algorithm but no statistically significant hits (e-value < 1) were recorded. This suggests that an earlier report of …

Analysis of Jmjd6 Cellular Localization and Testing for Its Involvement in Histone Demethylation

Background Methylation of residues in histone tails is part of a network that regulates gene expression. JmjC domain containing proteins catalyze the oxidative removal of methyl groups on histone lysine residues. Here, we report studies to test the involvement of Jumonji domain-containing protein 6 (Jmjd6) in histone lysine demethylation. Jmjd6 has recently been shown to hydroxylate RNA splicing factors and is known to be essential for the differentiation of multiple tissues and cells during embryogenesis. However, there have been conflicting reports as to whether Jmjd6 is a histone-modifying enzyme. Methodology/Principal Findings Immunolocalization studies reveal that Jmjd6 is distributed throughout the nucleoplasm outside of regions containing heterochromatic DNA, with occasional localization in nucleoli. During mitosis, Jmjd6 is excluded from the nucleus and reappears in the telophase of the cell cycle. Western blot analyses confirmed that Jmjd6 forms homo-multimers of different molecular weights in the nucleus and cytoplasm. A comparison of mono-, di-, and tri-methylation states of H3K4, H3K9, H3K27, H3K36, and H4K20 histone residues in wildtype and Jmjd6-knockout cells indicate that Jmjd6 is not involved in the demethylation of these histone lysine residues. This is further supported by overexpression of enzymatically active and inactive forms of Jmjd6 and subsequent analysis of histone methylation patterns by immunocytochemistry and western blot analysis. Finally, treatment of cells with RNase A and DNase I indicate that Jmjd6 may preferentially associate with RNA/RNA complexes and less likely with chromatin. Conclusions/Significance Taken together, our results provide further evidence that Jmjd6 is unlikely to be involved in histone lysine demethylation. We confirmed that Jmjd6 forms multimers and showed that nuclear localization of the protein involves association with a nucleic acid matrix.

The molecular cell death machinery in the simple cnidarian Hydra includes an expanded caspase family and pro- and anti-apoptotic Bcl-2 proteins

The fresh water polyp Hydra belongs to the phylum Cnidaria, which diverged from the metazoan lineage before the appearance of bilaterians. In order to understand the evolution of apoptosis in metazoans, we have begun to elucidate the molecular cell death machinery in this model organism. Based on ESTs and the whole Hydra genome assembly, we have identified 15 caspases. We show that one is activated during apoptosis, four have characteristics of initiator caspases with N-terminal DED, CARD or DD domain and two undergo autoprocessing in vitro. In addition, we describe seven Bcl-2-like and two Bak-like proteins. For most of the Bcl-2 family proteins, we have observed mitochondrial localization. When expressed in mammalian cells, HyBak-like 1 and 2 strongly induced apoptosis. Six of the Bcl-2 family members inhibited apoptosis induced by camptothecin in mammalian cells with HyBcl-2-like 4 showing an especially strong protective effect. This protein also interacted with HyBak-like 1 in a yeast two-hybrid assay. Mutation of the conserved leucine in its BH3 domain abolished both the interaction with HyBak-like 1 and the anti-apoptotic effect. Moreover, we describe novel Hydra BH-3-only proteins. One of these interacted with Bcl-2-like 4 and induced apoptosis in mammalian cells. Our data indicate that the evolution of a complex network for cell death regulation arose at the earliest and simplest level of multicellular organization, where it exhibited a substantially higher level of complexity than in the protostome model organisms Caenorhabditis and Drosophila.

Apoptosis in pre-Bilaterians: Hydra as a model

Hydra is a member of the ancient metazoan phylum Cnidaria and is an especially well investigated model organism for questions of the evolutionary origin of metazoan processes. Apoptosis in Hydra is important for the regulation of cellular homeostasis under different conditions of nutrient supply. The molecular mechanisms leading to apoptosis in Hydra are surprisingly extensive and comparable to those in mammals. Genome wide sequence analysis has revealed the presence of large caspase and Bcl-2 families, the apoptotic protease activating factor (APAF-1), inhibitors of apoptotic proteases (IAPs) and components of a putative death receptor pathway. Regulation of apoptosis in Hydra may involve BH-3 only proteins and survival pathways, possibly including insulin signalling.

GFP expression in Hydra: lessons from the particle gun.

Abstract. The cnidarian Hydra is an important model organism to study pattern formation and stem cell differentiation. In the past, however, it has been difficult to study gene function in Hydra because the animals have not been accessible to gene transfection studies. We have now developed a method to transiently express GFP-tagged proteins in Hydra using a green fluorescent protein (GFP) expression plasmid under the control of the Hydra actin promoter and a particle gun to introduce it into Hydra cell nuclei. We achieve strong transient GFP expression in a small but reproducible number of epithelial and interstitial cells. Implications for the use of this method to carry out single cell assays with GFP-tagged Hydra proteins are discussed.

The oxygenase Jmjd6–a case study in conflicting assignments

The Jumonji domain-containing protein 6 (Jmjd6) is a member of the superfamily of non-haem iron(II) and 2-oxoglutarate (2OG)-dependent oxygenases; it plays an important developmental role in higher animals. Jmjd6 was initially assigned a role as the phosphatidylserine receptor responsible for engulfment of apoptotic cells but this now seems unlikely. Jmjd6 has been shown to be a nuclear localized protein with a JmjC domain comprising a distorted double-stranded β-helical structure characteristic of the 2OG-dependent oxygenases. Jmjd6 was subsequently assigned a role in catalysing N-methyl-arginine residue demethylation on the N-terminus of the human histones H3 and H4; however, this function is also subject to conflicting reports. Jmjd6 does catalyse 2OG-dependent C-5 hydroxylation of lysine residues in mRNA splicing-regulatory proteins and histones; there is also accumulating evidence that Jmjd6 plays a role in splicing (potentially in an iron- and oxygen-dependent manner) as well as in other processes regulating gene expression, including transcriptional pause release. Moreover, a link with tumour progression has been suggested. In the present review we look at biochemical, structural and cellular work on Jmjd6, highlighting areas of controversy and consensus.

Hydra and the Evolution of Apoptosis

Abstract Programmed cell death occurs in most, if not all life forms. It is used to sculpt tissue during embryogenesis, to remove damaged cells, to protect against pathogen infection and to regulate cell numbers and tissue homeostasis. In animals cell death often occurs by a morphologically and biochemically conserved process called apoptosis. A novel group of cysteine proteases, referred to as caspases, constitute the central component of this process. Caspases are activated following the induction of apoptosis and cleave a variety of cellular substrates, thus giving rise to the characteristic morphological events of apoptosis. Apoptosis is rapid and cell corpses are removed by phagocytosis. Recent work has shown that apoptosis also occurs in Cnidaria and Porifera, thus extending the origin of this evolutionary innovation down to the first metazoan animal phyla. Here, we review several examples of the role of apoptosis in cnidarians and then summarize new results on the subcellular localization of caspases and the control of apoptosis in Hydra. We show by immuncytochemistry that caspases in Hydra are localized in mitochondria. Following induction of apoptosis caspases are released from mitochondria as proenzymes and then activated by proteolytic cleavage in the cytoplasm. We also present evidence that apoptosis in Hydra is dramatically stimulated by inhibitors of PI3-kinase. Since PI3-kinase is a central component of growth factor signaling cascades in higher metazoans, this result suggests that control of apoptosis by growth factors is also evolutionarily conserved. We speculate on the role of growth factors in the evolution of apoptosis.