Genomic structure and expression of Jmjd6 and evolutionary analysis in the context of related JmjC domain containing proteins

<p>Abstract</p> <p>Background</p> <p>The <it>jumonji C (JmjC) domain containing gene 6 </it>(<it>Jmjd6</it>, previously known as phosphatidylserine receptor) has misleadingly been annotated to encode a transmembrane receptor for the engulfment of apoptotic cells. Given the importance of JmjC domain containing proteins in controlling a wide range of diverse biological functions, we undertook a comparative genomic analysis to gain further insights in <it>Jmjd6 </it>gene organisation, evolution, and protein function.</p> <p>Results</p> <p>We describe here a semiautomated computational pipeline to identify and annotate JmjC domain containing proteins. Using a sequence segment N-terminal of the Jmjd6 JmjC domain as query for a reciprocal BLAST search, we identified homologous sequences in 62 species across all major phyla. Retrieved <it>Jmjd6 </it>sequences were used to phylogenetically analyse corresponding loci and their genomic neighbourhood. This analysis let to the identification and characterisation of a bi-directional transcriptional unit compromising the <it>Jmjd6 </it>and <it>1110005A03Rik </it>genes and to the recognition of a new, before overseen <it>Jmjd6 </it>exon in mammals. Using expression studies, two novel <it>Jmjd6 </it>splice variants were identified and validated <it>in vivo</it>. Analysis of the <it>Jmjd6 </it>neighbouring gene <it>1110005A03Rik </it>revealed an incident deletion of this gene in two out of three earlier reported <it>Jmjd6 </it>knockout mice, which might affect previously described conflicting phenotypes. To determine potentially important residues for <it>Jmjd6 </it>function a structural model of the Jmjd6 protein was calculated based on sequence conservation. This approach identified a conserved double-stranded β^-helix (DSBH) fold and a HxDx_nH facial triad as structural motifs. Moreover, our systematic annotation in nine species identified 313 DSBH fold-containing proteins that split into 25 highly conserved subgroups.</p> <p>Conclusion</p> <p>We give further evidence that <it>Jmjd6 </it>most likely has a function as a nonheme-Fe(II)-2-oxoglutarate-dependent dioxygenase as previously suggested. Further, we provide novel insights into the evolution of Jmjd6 and other related members of the superfamily of JmjC domain containing proteins. Finally, we discuss possibilities of the involvement of <it>Jmjd6 </it>and <it>1110005A03Rik </it>in an antagonistic biochemical pathway.</p

Genomic structure and expression of Jmjd6 and evolutionary analysis in the context of related JmjC domain containing proteins

<p>Abstract</p> <p>Background</p> <p>The <it>jumonji C (JmjC) domain containing gene 6 </it>(<it>Jmjd6</it>, previously known as phosphatidylserine receptor) has misleadingly been annotated to encode a transmembrane receptor for the engulfment of apoptotic cells. Given the importance of JmjC domain containing proteins in controlling a wide range of diverse biological functions, we undertook a comparative genomic analysis to gain further insights in <it>Jmjd6 </it>gene organisation, evolution, and protein function.</p> <p>Results</p> <p>We describe here a semiautomated computational pipeline to identify and annotate JmjC domain containing proteins. Using a sequence segment N-terminal of the Jmjd6 JmjC domain as query for a reciprocal BLAST search, we identified homologous sequences in 62 species across all major phyla. Retrieved <it>Jmjd6 </it>sequences were used to phylogenetically analyse corresponding loci and their genomic neighbourhood. This analysis let to the identification and characterisation of a bi-directional transcriptional unit compromising the <it>Jmjd6 </it>and <it>1110005A03Rik </it>genes and to the recognition of a new, before overseen <it>Jmjd6 </it>exon in mammals. Using expression studies, two novel <it>Jmjd6 </it>splice variants were identified and validated <it>in vivo</it>. Analysis of the <it>Jmjd6 </it>neighbouring gene <it>1110005A03Rik </it>revealed an incident deletion of this gene in two out of three earlier reported <it>Jmjd6 </it>knockout mice, which might affect previously described conflicting phenotypes. To determine potentially important residues for <it>Jmjd6 </it>function a structural model of the Jmjd6 protein was calculated based on sequence conservation. This approach identified a conserved double-stranded β^-helix (DSBH) fold and a HxDx_nH facial triad as structural motifs. Moreover, our systematic annotation in nine species identified 313 DSBH fold-containing proteins that split into 25 highly conserved subgroups.</p> <p>Conclusion</p> <p>We give further evidence that <it>Jmjd6 </it>most likely has a function as a nonheme-Fe(II)-2-oxoglutarate-dependent dioxygenase as previously suggested. Further, we provide novel insights into the evolution of Jmjd6 and other related members of the superfamily of JmjC domain containing proteins. Finally, we discuss possibilities of the involvement of <it>Jmjd6 </it>and <it>1110005A03Rik </it>in an antagonistic biochemical pathway.</p

The phosphatidylserine receptor has essential functions during embryogenesis but not in apoptotic cell removal

BACKGROUND: Phagocytosis of apoptotic cells is fundamental to animal development, immune function and cellular homeostasis. The phosphatidylserine receptor (Ptdsr) on phagocytes has been implicated in the recognition and engulfment of apoptotic cells and in anti-inflammatory signaling. To determine the biological function of the phosphatidylserine receptor in vivo, we inactivated the Ptdsr gene in the mouse. RESULTS: Ablation of Ptdsr function in mice causes perinatal lethality, growth retardation and a delay in terminal differentiation of the kidney, intestine, liver and lungs during embryogenesis. Moreover, eye development can be severely disturbed, ranging from defects in retinal differentiation to complete unilateral or bilateral absence of eyes. Ptdsr (-/-) mice with anophthalmia develop novel lesions, with induction of ectopic retinal-pigmented epithelium in nasal cavities. A comprehensive investigation of apoptotic cell clearance in vivo and in vitro demonstrated that engulfment of apoptotic cells was normal in Ptdsr knockout mice, but Ptdsr-deficient macrophages were impaired in pro- and anti-inflammatory cytokine signaling after stimulation with apoptotic cells or with lipopolysaccharide. CONCLUSION: Ptdsr is essential for the development and differentiation of multiple organs during embryogenesis but not for apoptotic cell removal. Ptdsr may thus have a novel, unexpected developmental function as an important differentiation-promoting gene. Moreover, Ptdsr is not required for apoptotic cell clearance by macrophages but seems to be necessary for the regulation of macrophage cytokine responses. These results clearly contradict the current view that the phosphatidylserine receptor primarily functions in apoptotic cell clearance

Identification of cardiac malformations in mice lacking Ptdsr using a novel high-throughput magnetic resonance imaging technique

BACKGROUND: Congenital heart defects are the leading non-infectious cause of death in children. Genetic studies in the mouse have been crucial to uncover new genes and signaling pathways associated with heart development and congenital heart disease. The identification of murine models of congenital cardiac malformations in high-throughput mutagenesis screens and in gene-targeted models is hindered by the opacity of the mouse embryo. RESULTS: We developed and optimized a novel method for high-throughput multi-embryo magnetic resonance imaging (MRI). Using this approach we identified cardiac malformations in phosphatidylserine receptor (Ptdsr) deficient embryos. These included ventricular septal defects, double-outlet right ventricle, and hypoplasia of the pulmonary artery and thymus. These results indicate that Ptdsr plays a key role in cardiac development. CONCLUSIONS: Our novel multi-embryo MRI technique enables high-throughput identification of murine models for human congenital cardiopulmonary malformations at high spatial resolution. The technique can be easily adapted for mouse mutagenesis screens and, thus provides an important new tool for identifying new mouse models for human congenital heart diseases

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

Genomic structure and expression of and evolutionary analysis in the context of related JmjC domain containing proteins-7

of 25 identified DSBH fold containing protein subgroups as indicated with protein family names on the right side. Scale bar represents the relative phylogenetic distance as determined with PHYLIP. Bootstrap values are shown for values <p><b>Copyright information:</b></p><p>Taken from "Genomic structure and expression of and evolutionary analysis in the context of related JmjC domain containing proteins"</p><p>http://www.biomedcentral.com/1471-2164/9/293</p><p>BMC Genomics 2008;9():293-293.</p><p>Published online 18 Jun 2008</p><p>PMCID:PMC2453528.</p><p></p

Genomic structure and expression of and evolutionary analysis in the context of related JmjC domain containing proteins-7

of 25 identified DSBH fold containing protein subgroups as indicated with protein family names on the right side. Scale bar represents the relative phylogenetic distance as determined with PHYLIP. Bootstrap values are shown for values <p><b>Copyright information:</b></p><p>Taken from "Genomic structure and expression of and evolutionary analysis in the context of related JmjC domain containing proteins"</p><p>http://www.biomedcentral.com/1471-2164/9/293</p><p>BMC Genomics 2008;9():293-293.</p><p>Published online 18 Jun 2008</p><p>PMCID:PMC2453528.</p><p></p

Genomic structure and expression of and evolutionary analysis in the context of related JmjC domain containing proteins-5

Ve asparaginyl hydroxylase – 2636534 () and Jmjd2a (), respectively. Stereoview of the predicted structure of Jmjd6 presented as a ribbon model. The conserved eight-stranded DSBH core found in all Fe(II) and 2-oxoglutarate (2OG)-dependent dioxygenases is coloured in blue. Additional β-strands attached to the major β-sheet are shown in orange. Helices are depicted in green. View of the predicted active site of the Jmjd6-Fe(II)-2OG complex showing coordination of Fe(II) to 2OG, His187, Asp189, and His273. 2OG also ligates to Trp174, Asn197 and Lys204 with Thr285 stabilising Asn197 and Trp174. Additional important residues for putative interactions are shown in cyan and include hydrophobic interactions from Phe133 and Val275 as well as Thr184, which is involved in 2OG-binding in Hif1an. Interacting residues along with the 2OG co-substrate are shown as stick presentations, putative H-bond interactions as dotted lines. Fe(II) is depicted as an orange ball.<p><b>Copyright information:</b></p><p>Taken from "Genomic structure and expression of and evolutionary analysis in the context of related JmjC domain containing proteins"</p><p>http://www.biomedcentral.com/1471-2164/9/293</p><p>BMC Genomics 2008;9():293-293.</p><p>Published online 18 Jun 2008</p><p>PMCID:PMC2453528.</p><p></p

Genomic structure and expression of and evolutionary analysis in the context of related JmjC domain containing proteins-1

S and its neighbouring genes using PipMaker. Green, blue and yellow overlays highlight the conservation of the annotated exons/coding sequences of the individual genes found in the reference sequence. The orange overlay indicates conserved sequences of the first exons of the and genes and sequences outside of the coding elements. The red overlay marks a high-scoring segment in all analysed mammalian species supporting the presence of at least one additional exon (No. 5) that is not included in the current Ensembl gene annotation (build 44). Transcriptional orientations of the genes, their exons, UTRs, repetitive elements, and CpG islands are in the symbol key at the figure bottom.<p><b>Copyright information:</b></p><p>Taken from "Genomic structure and expression of and evolutionary analysis in the context of related JmjC domain containing proteins"</p><p>http://www.biomedcentral.com/1471-2164/9/293</p><p>BMC Genomics 2008;9():293-293.</p><p>Published online 18 Jun 2008</p><p>PMCID:PMC2453528.</p><p></p