Cellular, biochemical and phylogenomic analyses of the mouse Jumonji domain containing 6 protein provide new evidence for functions as a 2-oxoglutarate dependent dioxygenase [Elektronische Ressource] / von Phillip Holgar Heinrich Hahn
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Cellular, biochemical and phylogenomic analyses of the mouse Jumonji domain containing 6 protein provide new evidence for functions as a 2-oxoglutarate dependent dioxygenase [Elektronische Ressource] / von Phillip Holgar Heinrich Hahn

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123 Pages
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Cellular, biochemical and phylogenomic analyses of the mouse Jumonji domain containing 6 protein provide new evidence for functions as a 2-oxoglutarate dependent dioxygenase Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Phillip Holgar Heinrich Hahn aus Georgsmarienhütte 1. Referentin oder Referent: Prof. Dr. Rudi Balling 2. Referentin oder Referent: Prof. Dr. Jürgen Wehland eingereicht am: 07.11.2007 mündliche Prüfung (Disputation) am: 20.12.2007 Druckjahr: 2008 Table of contents 1 Table of contents TABLE OF CONTENTS ............................................................................................. 1 SUMMARY ................................................................................................................. 4 1.  INTRODUCTION ................................................................................................ 5 1.1  The phosphatidylserine receptor ............................................................................................... 5 1.1.1  The phosphatidylserine receptor and its proposed function in apoptotic cell clearance ....... 5 1.1.

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Published 01 January 2008
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Cellular, biochemical and phylogenomic analyses of the
mouse Jumonji domain containing 6 protein provide new
evidence for functions as a 2-oxoglutarate dependent
dioxygenase






Von der Fakultät für Lebenswissenschaften

der Technischen Universität Carolo-Wilhelmina

zu Braunschweig

zur Erlangung des Grades eines

Doktors der Naturwissenschaften

(Dr. rer. nat.)

genehmigte

D i s s e r t a t i o n













von Phillip Holgar Heinrich Hahn
aus Georgsmarienhütte






















































1. Referentin oder Referent: Prof. Dr. Rudi Balling
2. Referentin oder Referent: Prof. Dr. Jürgen Wehland
eingereicht am: 07.11.2007
mündliche Prüfung (Disputation) am: 20.12.2007
Druckjahr: 2008 Table of contents 1

Table of contents
TABLE OF CONTENTS ............................................................................................. 1 
SUMMARY ................................................................................................................. 4 
1.  INTRODUCTION ................................................................................................ 5 
1.1  The phosphatidylserine receptor ............................................................................................... 5 
1.1.1  The phosphatidylserine receptor and its proposed function in apoptotic cell clearance ....... 5 
1.1.2  Phosphatidylserine receptor loss-of-function studies ............................................................ 6 
1.1.2.1  PSR-1 loss-of-function mutation in Caenorhabditis elegans ......................................... 6 
1.1.2.2  Knockdown of zfpsr in Danio rerio ................................................................................. 6 
1.1.2.3  Knockout and overexpression of dPSR in Drosophila melanogaster ........................... 7 
1.1.2.4  Knockout of Ptdsr in mice .............................................................................................. 8 
1.1.3  Biochemical properties, domains and motifs ....................................................................... 10 
1.1.3.1  Nuclear localization sites ............................................................................................. 11 
1.1.3.2  Putative AT-hook like DNA binding motif .................................................................... 11 
1.1.3.3  Poly-serine stretch ....................................................................................................... 12 
1.1.3.4  The Ptdsr JmjC domain ............................................................................................... 12 
1.1.4  Revision of Ptdsr gene nomenclature .................................................................................. 14 
1.2  The Histone code and histone lysine demethylation ............................................................. 14 
1.2.1  Lysine-specific demethylase 1 18 
1.2.2  JmjC domain-containing proteins ......................................................................................... 19 
1.2.2.1  Jontaining protein class of histone lysine demethylases ...................... 19 
1.2.2.1.1  Proposed catalytical mechanism ............................................................................ 20 
1.2.2.2  JmjC domain-containing proteins as transcription factors in development ................. 21 
1.2.2.3  Domain composition of JmjC domain-containing proteins .......................................... 22 
1.2.2.3.1  Complex JmjC domain-containing proteins ............................................................ 22 
1.2.2.3.2  JmjC-only domain-containing proteins ................................................................... 23 
1.2.2.4  JmjC domain-containing proteins in disease ............................................................... 24 
1.3  Nucleic acid methylation and oxidative repair ........................................................................ 24 
1.3.1  Demethylation of DNA and RNA by AlkB and homologues ................................................. 25 
1.4  Aim of the study ......................................................................................................................... 27 
2.  RESULTS ......................................................................................................... 28 
2.1  Genomic structure and expression of Jmjd6 and evolutionary analysis in the context of
related JmjC domain-containing proteins ............................................................................... 28 
2.1.1  Overview of bioinformatics analyses approaches ................................................................ 28 
2.1.2  Comparative analysis of Jmjd6 homologous loci in vertebrates .......................................... 31 
2.1.3  Phylogenetic analysis of the Jmjd6 protein .......................................................................... 42 
2.1.4  Comparative modelling and analysis of the putative catalytic core domain of Jmjd6 .......... 46 
2.1.5  Integration of Jmjd6 into the JmjC domain-containing protein family .................................. 51 Table of contents 2

2.2  A cellular and biochemical analysis indicates no function of Jmjd6 in histone
demethylation but provides evidence for RNA association .................................................. 58 
2.2.1  Overview on Jmjd6 protein analyses ................................................................................... 58 
2.2.2  Analysis of intracellular Jmjd6 localization using specific antibodies .................................. 59 
2.2.3  Characterization of Jmjd6 multimer formation ..................................................................... 65 
2.2.4  Jmjd6 is not involved in histone lysine demethylation ......................................................... 71 
2.2.5  Jmjd6 is associated with RNA but not with DNA in cell nuclei ............................................. 76 
3.  DISCUSSION ................................................................................................... 79 
3.1  Evolutional and structural features of the Jmjd6 protein ...................................................... 79 
3.1.1  Phylogeny of the Jmjd6 protein ............................................................................................ 79 
3.1.2  Functional 3D Jmjd6 conservation analysis a structural Jmjd6 model ................................ 80 
3.1.3  JmjC domain sequences are characteristic and allow protein family classification ............. 81 
3.2  Effects of intracellular Jmjd6 localization and Jmjd6 protein multimerization ................... 82 
3.2.1  Intracellular Jmjd6 localization ............................................................................................. 82 
3.2.2  Jmjd6 is linked to a ribonucleic matrix ................................................................................. 84 
3.2.3  Possible mechanism of Jmjd6 multimerization .................................................................... 84 
3.2.4  Identification of novel Jmjd6 transcripts and possible biological consequences ................. 86 
3.3  The biological functions of Jmjd6 ............................................................................................ 87 
3.3.1  Jmjd6 is not a phosphatidylserine receptor .......................................................................... 87 
3.3.2  Jmjd6 is not involved in histone lysine demethylation ......................................................... 88 
3.3.3  Jmjd6 is a H3R2 & H4R3 demethylase ................................................................................ 88 
3.4  Conclusive possible explanations for the Jmjd6 knockout phenotype ............................... 89 
4.  OUTLOOK ........................................................................................................ 90 
5.  MATERIAL AND METHODS ............................................................................ 92 
5.1.1  Chemicals, enzymes, medium and cells .............................................................................. 92 
5.1.2  Jmjd6 expression constructs and transfection ..................................................................... 92 
5.1.3  Monoclonal antibody generation and epitope mapping ....................................................... 93 
5.1.4  Immunofluorescence staining and imaging .......................................................................... 94 
5.1.5  Cell fractioning, immunoprecipitation and western blotting .................................................. 95 
5.1.6  Bioinformatic approaches ..................................................................................................... 96 
5.1.7  Identification of Jmjd6 splice variants .................................................................................. 97 
5.1.8  Analysis of the bidirectional transcriptional unit ................................................................... 98 
6.  REFERENCES ............................................................................................... 100 
7.  APPENDICES................................................................................................. 113 
7.1  Appendix A ............................................................................................................................... 113 Table of contents 3

7.2  Appendix B ............................................................................................................................... 115 
7.3  Appendix C ........... 117 
GRATIARUM ACTIO ............................................................................................. 119 
CURRICULUM VITAE ............................................................................................ 120 Summary 4

Summary
The “jumonji domain containing 6” protein (Jmjd6, formerly phosphatidylserine
receptor) was misleadingly annotated as transmembrane receptor for apoptotic cells.
Loss-of-function studies in mice revealed that Jmjd6 is involved in the differentiation of
multiple tissues during embryogenesis and cytokine release of activated macrophages.
Given the importance of jumonji C (JmjC) domain-containing proteins in oxidative DNA
and RNA repair, protein hydroxylation, and histone lysine demethylation, a comparative
analysis of Jmjd6 gene organisation, evolution, and protein function was carried out.
Sequences homologous to the mouse Jmjd6 protein were identified in 61 species
from all major living phyla using a reciprocal BLAST search. Phylogenetic analyses of
corresponding loci lead to the identification and characterisation of a bi-directional
transcriptional unit comprising Jmjd6 and the neighbouring 1110005A03Rik locus.
Expression studies verified the existence of an additional, previously not annotated
Jmjd6 exon and of two new splice variants in vivo. A structural model of the Jmjd6
protein was calculated and verified using a novel cross-validation approach. The amino
acid conservation grade during evolution from eubacteria to humans was automatically
extracted, translated into an indicative colour code, and applied to the structural model;
thereby showing conserved residues in their predicted spatial positions. A conserved
double stranded ß-helix (DSBH) fold, an HxDx H facial triad, and a 2-oxoglutarate co-n
ordination site were found as structural motifs. mAB328, a new monoclonal anti-Jmjd6
antibody, was identified by characterizing 384 hybridoma clones and allowed
demonstrating that endogenous Jmjd6 is a nuclear protein with occasional nucleolar
localisation. Jmjd6 shows no co-localisation with heterochromatic DNA and disappears
from the nucleus during the cell cycle. Western blot analyses showed that Jmjd6 forms
homo-multimers and reporter constructs identified a N-terminal protein multimerization
domain and demonstrated that homo-multimerization requires the full-length protein. An
analysis of H3K4, H3K9, H3K27, H3K36, and H4K20 histone methylation states in
-/- wildtype and Jmjd6 cells and overexpression of Jmjd6-reporter fusion constructs
excluded a function of Jmjd6 in the demethylation of these residues. Finally, nuclease
assays showed a likely association of Jmjd6 to a ribonucleic matrix.
In conclusion, this work presents novel evidence that Jmjd6 most likely functions
as a nonheme-Fe(II)-2-oxoglutarate-dependent dioxygenase as previously suggested
and provides novel insights into the evolution of Jmjd6 and JmjC domain-containing
proteins. The results suggest that enzymatic targets of Jmjd6 might be RNA or
RNAassociated proteins and not lysine residues in histone tails. Introduction 5

1. Introduction
1.1 The phosphatidylserine receptor
1.1.1 The phosphatidylserine receptor and its proposed function in apoptotic
cell clearance
Tissue homeostasis in multicellular organisms is a tight regulated process and
involves the generation of cells as well as their removal by cell death. Programmed
cell death, or apoptosis, is a physiological mechanism to remove unwanted
structures or abnormal cells and represents the fate of most dying cells. Removing
them is a rapid and efficient process and occurs, in contrast to e.g. the clearance of
microorganisms, without releasing inflammatory cytokines (Jacobson et al., 1997;
Savill et al., 2002; Lauber et al., 2004). In fact, upon recognition of apoptotic cells by
phagocytes, pro-inflammatory responses are strongly suppressed by release of
transforming growth factor ß (TGF-ß), interleukin-10 (IL-10), prostaglandin E2, and
platelet activating factor (PAF) (Savill et al., 2002). Professional phagocytes like
macrophages or unprofessional phagocytes like fibroblasts recognise pre-apoptotic
cells by appearing marks in their cell membrane, leading to their immediate
elimination through engulfment. Upon induction of apoptosis, phosphatidyl-L-serine
(phosphatidylserine, PS), a phospholipid normally present only on the inner leaflet, of
the cell membrane becomes exposed on the cell surface as a so-called
eat-mesignal (Fadok et al., 1992; Fadok et al., 1998).
The uptake of apoptotic cells is inhibited up to 50% by L-isomer PS containing
liposomes in TGF-ß/ß-glucan-treated human macrophages, but not by liposomes
containing other isomers or other anionic phospholipids (Fadok et al., 1998).
Therefore, Fadok et al. concluded that there must be a stereospecific PS receptor on
phagocytes for PS recognition, subsequently triggering induction of engulfment and
anti-inflammatory cytokine release (Fadok et al., 1992; Fadok et al., 1998).
To identify this receptor, Fadok et al. raised monoclonal antibodies against
TGFß1/ß-glucan-treated human macrophages. One such antibody, mAB217,
recognised stimulated PS-exposing macrophages significantly better than untreated
macrophages and this recognition could be blocked by liposomes containing PS.
Furthermore, blocking with mAB217 could partially prevent the uptake of apoptotic Introduction 6

cells by macrophages (Fadok et al., 2000). To identify the antigen of mAB217, Fadok
et al. used a phage display approach and identified cDNAs encoding a protein with a
corresponding, putative epitope. Using the isolated cDNAs, Jurkat T cells, normally
negative for mAB217, were transiently and permanently transfected to express the
antigen. These cells appear to bind to and engulf apoptotic cells. Therefore, Fadok et
al. named the antigen identified by mAB217 Phosphatidylserine receptor (PSR /
PTDSR) (Fadok et al., 2000).
1.1.2 Phosphatidylserine receptor loss-of-function studies
To confirm this important finding and to analyse the biological effects mediated
by the protein, several ablation studies for Ptdsr and its homologs were initiated. A
loss-of-function mutant was identified in C. elegans ( 1.1.2.1), morpholino
knockdown experiments were performed in D. rerio ( 1.1.2.2), a null-mutation
generated in D. melanogaster ( 1.1.2.3), and three groups independently created
Ptdsr knockouts in mice ( 1.1.2.4).
1.1.2.1 PSR-1 loss-of-function mutation in Caenorhabditis elegans
Analyzing PSR-1, the C. elegans homolog of human PTDSR, Wang et al.
identified a loss-of-function mutant (tm469), which carries a deletion in the psr-1
locus truncating the PSR-1 protein to its first 14 amino acids (Wang et al., 2003).
Analogous to the experiments performed by Fadok et al., transiently PSR-1
transfected human Jurkat T lymphocytes gain the ability to selectively bind PS
exposing cells. Furthermore, analyzing cell corpses during development, a delay in
corpse engulfment was found with cell corpses remaining 55% longer in mutant than
in wild-type embryos. This effect was rescued by expression of PSR-1 in the tm469
mutant strain (Wang et al., 2003).
They conclude from their results, that PSR-1 is involved in engulfment, but is
very unlikely the single important PS recognising receptor as other engulfment
related mutants show severe engulfment defects in contrast to the mild tm469
phenotype (Wang et al., 2003).
1.1.2.2 Knockdown of zfpsr in Danio rerio
Hong et al. identified the zebrafish homolog of Ptdsr, zfpsr, by screening a 24
hours post-fertilization cDNA library. They performed knockdown experiments using
????Introduction 7

morpholinos against zfpsr, revealing severe developmental defects in comparison to
wild-type embryos. Cell corpses accumulated in the brain and at somites boundaries,
embryos showed delayed heart development, and strong morphological defects
containing bent notochords were described (Hong et al., 2004). Furthermore, some
embryos with stronger phenotypes ranging from a tube-like heart without atria,
ventricles, and with drastically enlarged cavity, shrinkage of the brain and complete
loss of posterior somite development were found. The most severely affected
embryos died before three days post-fertilization (Hong et al., 2004).
1.1.2.3 Knockout and overexpression of dPSR in Drosophila melanogaster
Krieser and colleagues found a mutant containing a deletion of a locus
containing several genes including dPSR, the Drosophila homolog of the human
Ptdsr gene. This mutant showed defects indicating problems with phagocytic
clearance or macrophage development or macrophage migration capabilities. To
analyze a potential involvement, they generated a series of imprecise p-element
FM1mediated excisions of the dPSR locus. One mutant (dPSR ) contained a deletion
of the first 120 amino acids of the Drosophila homolog dPSR, resulting in a null
FM1mutation. Interestingly, homozygous dPSR are viable, fertile and show no obvious
morphological defects (Krieser et al., 2007). A comparison of this strain to the
wildtype showed no difference in apoptotic cell clusters and macrophages derived
FM1from dPSR were not impaired in apoptotic cell engulfment. Therefore, Krieser et
al. concluded that dPSR is not required for apoptotic cell clearance in D.
FM1 melanogaster (Krieser et al., 2007). Analyzing the dPSR mutant further, Krieser et
al. report increased apoptosis rates during eye development and about a protective
effect of dPSR overexpression by inhibiting the head of involution defective (hid)
gene product. Hid encodes a pro-apoptotic regulator that is activated by the JNK
pathway (Macias et al., 2004). Additional experiments using other mutants involved
in the JNK pathway suggested that dPSR inhibits the Hid pathway upstream of JNK.
Furthermore, they hypothesized that dPSR acts through the c-Jun-NH kinase 2
pathway to alter sensitivity to cell death (Krieser et al., 2007). Introduction 8

1.1.2.4 Knockout of Ptdsr in mice
Three research groups independently generated Ptdsr knockout mice (Ptdsr
tm1 Gbf tm1 Flv tm1 Ysfk: Böse, et al., 2004, Ptdsr : Li et al., 2003 and Ptdsr : Kunisaki et al.,
2004).

Figure 1.1: Published phenotypes of Ptdsr knockout mice
The figure shows Ptdsr expression (blue) in a healthy Ptdsr-lacZ reporter
gene trap embryo. The boxes describe the published phenotypes as
described by Böse et al., 2004 (B), Li et al., 2003 (L), and Kunisaki et al.,
2004 (K).
All groups describe different phenotypes in their mice (Figure 1.1), with an
overlap that all homozygous knockout mice show perinatal lethality, while
heterozygous mice have no obvious phenotype. In Ptdsr-deficient mice
erythropoiesis is disturbed resulting in a cyanotic appearance of the skin (Li et al.,
2003; Böse et al., 2004; Kunisaki et al., 2004). Böse et al. describe a continuous