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

BACKGROUND: The jumonji C (JmjC) domain containing gene 6 (Jmjd6, 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 Jmjd6 gene organisation, evolution, and protein function. RESULTS: 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 Jmjd6 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 Jmjd6 and 1110005A03Rik genes and to the recognition of a new, before overseen Jmjd6 exon in mammals. Using expression studies, two novel Jmjd6 splice variants were identified and validated in vivo. Analysis of the Jmjd6 neighbouring gene 1110005A03Rik revealed an incident deletion of this gene in two out of three earlier reported Jmjd6 knockout mice, which might affect previously described conflicting phenotypes. To determine potentially important residues for Jmjd6 function a structural model of the Jmjd6 protein was calculated based on sequence conservation. This approach identified a conserved double-stranded beta-helix (DSBH) fold and a HxDxnH 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. CONCLUSION: We give further evidence that Jmjd6 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 Jmjd6 and 1110005A03Rik in an antagonistic biochemical pathway.

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.

The role of the MHC on resistance to group a streptococci in mice.

The severity of infection with Streptococcus pyogenes is strongly influenced by the host's genetics. This observation extends to the murine model of streptococcal infection, where the background of the mouse strain determines the infection outcome (BALB/c are resistant, whereas C3H/HeN are susceptible). To determine the extent to which the MHC complex (H2) contributed to diseases susceptibility, the response to S. pyogenes of congenic BALB mice from a resistant background (BALB/c), but carrying the H2(k) region of susceptible C3H/HeN mice (BALB/k), was examined. BALB/k were as susceptible as the H2 donor strain (C3H/HeN). Linkage analysis performed in F(2) backcross ([BALB/c x C3H/HeN] x BALB/c) mice confirmed the presence of a susceptibility locus within the H2 region on proximal chromosome 17. The possibility that modulation of T cell responses to streptococcal superantigens (GAS-SAgs) by different H2 haplotypes may influence disease severity was examined. BALB/k exhibited a significantly stronger response at the level of cell proliferation and cytokine production to GAS-SAgs than did BALB/c mice. However, the fact that T cell-deficient SCID-C3H/HeN mice also exhibited a susceptible phenotype suggests a more important contribution of innate effector cells to disease susceptibility. Lower transcriptional levels of certain inflammation-related regulatory genes located on chromosome 17 were detected in macrophages from susceptible than in those from resistant mice in response to infection. These results suggest that susceptibility to S. pyogenes may be associated with an altered transcription of specific genes that may compromise the endogenous regulatory processes controlling the inflammatory cascade and favor the progression to sepsis.

1alpha,25-Dihydroxyvitamin D3 is a potent suppressor of interferon gamma-mediated macrophage activation.

1alpha,25-Dihydroxyvitamin D3 (1alpha,25(OH)2D3), the activated vitamin D3 hormone, is a key regulator of calcium homeostasis and thereby indispensable for bone metabolism. In addition, 1alpha,25(OH)2D3 is known to mediate predominantly immunosuppressive responses in vitro and in vivo. It has been demonstrated that macrophages can produce 1alpha,25(OH)2D3 on activation with interferon gamma (IFN-gamma), although little is understood about the biologic significance of this response. We show here that 1alpha,25(OH)2D3 can selectively suppress key effector functions of IFN-gamma-activated macrophages. Among these are the suppression of listericidal activity, the inhibition of phagocyte oxidase-mediated oxidative burst, and the suppression of important IFN-gamma-induced genes, including Ccl5, Cxcl10, Cxcl9, Irf2, Fcgr1, Fcgr3, and Tlr2. The deactivation of IFN-gamma-stimulated macrophages is dependent on a functional vitamin D receptor and 1alpha,25(OH)2D3 acts specifically on IFN-gamma-activated macrophages, whereas the steroid has no effects on resting macrophages. Therefore, the 1alpha,25(OH)2D3-mediated suppression of macrophage functions is distinct from previously described macrophage deactivation mechanisms. In conclusion, our data indicate that the production of 1alpha,25(OH)2D3 by IFN-gamma-stimulated macrophages might be an important negative feedback mechanism to control innate and inflammatory responses of activated macrophages.

Pallister-Hall syndrome phenotype in mice mutant for Gli3.

Mutations in the GLI3 gene have been identified in several human malformation syndromes. One of these autosomal dominant developmental disorders is Pallister-Hall syndrome (PHS; MIM146510), which is associated with central polydactyly and other malformations. Interestingly, the mutations in the GLI3 transcription factor gene identified in patients with PHS are restricted to the region 3' of the zinc finger-encoding domain, leaving this DNA-binding domain intact. We have investigated the consequences of this mutation on the development of multiple organ systems by introducing a targeted mutation in mice. We found that mice homozygous for the mutation showed a central polydactyly, thus modeling one of the major abnormalities of the human syndrome. Moreover, Gli3-mutant mice displayed a wide range of developmental abnormalities encompassing almost all of the common PHS features, including imperforate anus, gastrointestinal, epiglottis and larynx defects, abnormal kidney development, and absence of adrenal glands. Thus, our Gli3-mutant mice provide an excellent model for studies of both the pathogenesis of PHS and Gli3 functions in the development of the affected organ systems.

Dorsal-ventral patterning of the spinal cord requires Gli3 transcriptional repressor activity.

Sonic hedgehog (Shh) plays a critical role in organizing cell pattern in the developing spinal cord. Gli proteins are thought to mediate Shh signaling, but their role in directing neural tube patterning remains unclear. Here we identify a role for Gli3 transcriptional repressor activity in patterning the intermediate region of the spinal cord that complements the requirement for Gli2 in ventral regions. Moreover, blocking all Gli responses results in a complete dorsalization of ventral spinal cord, indicating that in addition to the specific roles of Gli2 and Gli3 in the neural tube, there is functional redundancy between Gli proteins. Finally, analysis of Shh/Gli3 compound mutant mice substantiates the idea that ventral patterning may involve a mechanism independent, or parallel, to graded Shh signaling. However, even in the absence of graded Shh signaling, Gli3 is required for the dorsal-ventral patterning of the intermediate neural tube. Together these data raise the possibility that Gli proteins act as common mediators integrating Shh signals, and other sources of positional information, to control patterning throughout the ventral neural tube.

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.