Dissecting the biological roles of Kdm3b and Kdm3a lysine demethylases

Lysine demethylases are a newly discovered group of enzymes that have rapidly expanded over evolutionary time by the acquisition of multiple functional domains, in addition to the unifying catalytic JmjC domain. There are thirty members of the JmjC-domain family in humans. A proportion of lysine demethylases catalyse the removal of methyl modifications from lysine residues of histones and non-histone proteins. The discovery of mutations in histone demethylase genes, in a number of human syndromes, stresses the functional importance of these enzymes in development and disease. Therefore, the phenotypic dissection of animal models of histone lysine demethylases will provide invaluable insights into the molecular mechanisms that underlie human disease. In mammals, the Kdm3 family of histone demethylases includes Kdm3a, Kdm3b, Jmjd1c and Hairless. However, in zebrafish, there are two kdm3 genes, one of which encodes a protein similar to both the mammalian Kdm3a and Kdm3b. Morpholino knock-down of the kdm3 gene in zebrafish faithfully recapitulates classical ciliary phenotypes, although the underlying causalities are still unclear. In recent years, Kdm3a function has been extensively dissected through the use of mouse models and cell culture studies, focusing on the nuclear histone demethylation function. Kdm3a gene-trap and knock-out mouse models present with obesity, infertility, sex reversal and predisposal to diabetes, reminiscent of a human ciliopathy syndrome. No mouse models for Kdm3b have been characterised yet. In this study, I hypothesized that the murine Kdm3a and Kdm3b histone demethylases have diverged biological roles and that the zebrafish kdm3 fulfils the functions of both. The aims of my thesis were: 1) to compare the evolutionary conservation of the zebrafish kdm3 and murine Kdm3b in function and check their spatial expression, 2) to dissect the phenotype of Kdm3b gene-trapped mice and 3) to characterise an alternative murine Kdm3a isoform. Protein sequence comparison studies show that the zebrafish kdm3 protein is closer in sequence to the mammalian Kdm3b. Both the zebrafish kdm3 and murine Kdm3b are di-methyl lysine 9 (H3K9me2) demethylases, however, they have diverged spatial expression during embryogenesis. In agreement with the phenotype of kdm3 morphants, over-expression of the zebrafish kdm3 reduces ciliation efficiency when transfected into animal cells. Notably, the phenotype analysis of Kdm3b gene-trapped mice does not resemble classical ciliary phenotypes, as one would expect from the zebrafish data. Homozygous Kdm3b gene-trapped mice are postnatally growth retarded, with plausible defects in thymus organ development. Interestingly, an alternative murine Kdm3a isoform (Kdm3a-i2) shows both nuclear and cytoplasmic localisation. Over-expression studies revealed that Kdm3ai2 retains its histone demethylation function, and a proportion of the over-expressed construct localises to the centrosome. In addition, over-expression of Kdm3a-i2 reduces ciliation efficiency. Overall, the data from my studies suggests that: 1) the zebrafish kdm3 is more similar in sequence to the murine Kdm3b than Kdm3a, is a histone demethylase and has a distinct spatial expression during embryogenesis. However, the phenotype of kdm3 zebrafish morphants is more closely related to the Kdm3a-than Kdm3bdeficient mice, 2) the murine Kdm3a and Kdm3b have distinct biological roles, as evidenced by the mouse models, 3) the Kdm3a-i2 isoform shares the same nuclear demethylation function as the full length Kdm3a and has a plausible centrosomal function

Genome defence in hypomethylated developmental contexts

Retrotransposons constitute around 40% of the mammalian genome and their aberrant activation can have wide ranging detrimental consequences, both throughout development and into somatic lineages. DNA methylation is one of the major epigenetic mechanisms in mammals, and is essential in repressing retrotransposons throughout mammalian development. Yet during normal mouse embryonic development some cell lineages become extensively DNA hypomethylated and it is not clear how these cells maintain retrotransposon silencing in a globally hypomethylated genomic context. In this thesis I determine that hypomethylation in multiple contexts results in the consistent activation of only one gene in the mouse genome - Tex19.1. Thus if a generic compensatory mechanism for loss of DNA methylation exists in mice, it must function through this gene. Tex19.1-/- mice de-repress retrotransposons in the hypomethylated component of the placenta and in the mouse germline, and have developmental defects in these tissues. In this thesis I examine the mechanism of TEX19.1 mediated genome defence and the developmental consequences upon its removal. I show that TEX19.1 functions in repressing retrotransposons, at least in part, through physically interacting with the transcriptional co-repressor, KAP1. Tex19.1-/- ES cells have reduced levels of KAP1 bound retrotransposon chromatin and reduced levels of the repressive H3K9me3 modification at these loci. Furthermore, these subsets of retrotransposon loci are de-repressed in Tex19.1-/- placentas. Thus, my data indicates that mouse cells respond to hypomethylation by activating expression of Tex19.1, which in turn augments compensatory, repressive histone modifications at retrotransposon sequences, thereby helping developmentally hypomethylated cells to maintain genome stability. I next aimed to further elucidate the role of Tex19.1 in the developing hypomethylated placenta. I determine that Tex19.1-/- placental defects precede intrauterine growth restriction of the embryo and that alterations in mRNA abundance in E12.5 Tex19.1-/- placentas is likely in part due to genic transcriptional changes. De-repression of LINE- 1 is evident in these placentas and elements of the de-repressed subfamily are associated with significantly downregulated genes. If retrotransposon de-repression is contributing to developmental defects by interfering with gene expression remains to be determined, however I identify a further possible mechanism leading to placental developmental defects. I determine that Tex19.1-/- placentas have an increased innate immune response and I propose that this is contributing to the developmental defects observed. Developmental defects and retrotransposon de-repression are also observed in spermatogenesis in Tex19.1-/- testes, the molecular basis for which is unclear. I therefore investigate the possibility that the TEX19.1 interacting partners, the E3 ubiquitin ligase proteins, may be contributing to the phenotypes observed in Tex19.1- /- testes. I show that repression of MMERVK10C in the testes is dependent on UBR2, alongside TEX19.1. Furthermore, I have identified a novel role for the TEX19.1 interacting partner, UBR5, in spermatogenesis, whose roles are distinct from those of TEX19.1. The work carried out during the course of this thesis provides mechanistic insights into TEX19.1 mediated genome defence and highlights the importance of protecting the genome from aberrant retrotransposon expression

Pleiotropic Effects of Sox2 during the Development of the Zebrafish Epithalamus

The zebrafish epithalamus is part of the diencephalon and encompasses three major components: the pineal, the parapineal and the habenular nuclei. Using sox2 knockdown, we show here that this key transcriptional regulator has pleiotropic effects during the development of these structures. Sox2 negatively regulates pineal neurogenesis. Also, Sox2 is identified as the unknown factor responsible for pineal photoreceptor prepatterning and performs this function independently of the BMP signaling. The correct levels of sox2 are critical for the functionally important asymmetrical positioning of the parapineal organ and for the migration of parapineal cells as a coherent structure. Deviations from this strict control result in defects associated with abnormal habenular laterality, which we have documented and quantified in sox2 morphants

Kdm3a lysine demethylase is an Hsp90 client required for cytoskeletal rearrangements during spermatogenesis

The lysine demethylase Kdm3a (Jhdm2a, Jmjd1a) is required for male fertility, sex determination, and metabolic homeostasis through its nuclear role in chromatin remodeling. Many histone-modifying enzymes have additional nonhistone substrates, as well as nonenzymatic functions, contributing to the full spectrum of events underlying their biological roles. We present two Kdm3a mouse models that exhibit cytoplasmic defects that may account in part for the globozoospermia phenotype reported previously. Electron microscopy revealed abnormal acrosome and manchette and the absence of implantation fossa at the caudal end of the nucleus in mice without Kdm3a demethylase activity, which affected cytoplasmic structures required to elongate the sperm head. We describe an enzymatically active new Kdm3a isoform and show that subcellular distribution, protein levels, and lysine demethylation activity of Kdm3a depended on Hsp90. We show that Kdm3a localizes to cytoplasmic structures of maturing spermatids affected in Kdm3a mutant mice, which in turn display altered fractionation of beta-actin and gamma-tubulin. Kdm3a is therefore a multifunctional Hsp90 client protein that participates directly in the regulation of cytoskeletal components.Publisher PDFPeer reviewe

The Histone Deacetylase 9 Stroke-Risk Variant Promotes Apoptosis and Inflammation in a Human iPSC-Derived Smooth Muscle Cells Model.

A common variant in the Histone Deacetylase 9 (HDAC9) gene is the strongest genetic risk for large-vessel stroke, and HDAC9 offers a novel target for therapeutic modulation. However, the mechanisms linking the HDAC9 variant with increased stroke risk is still unclear due to the lack of relevant models to study the underlying molecular mechanisms. We generated vascular smooth muscle cells using human induced pluripotent stem cells with the HDAC9 stroke risk variant to assess HDAC9-mediated phenotypic changes in a relevant cells model and test the efficacy of HDAC inhibitors for potential therapeutic strategies. Our human induced pluripotent stem cells derived vascular smooth muscle cells show enhanced HDAC9 expression and allow us to assess HDAC9-mediated effects on promoting smooth muscle cell dysfunction, including proliferation, migration, apoptosis and response to inflammation. These phenotypes could be reverted by treatment with HDAC inhibitors, including sodium valproate and small molecules inhibitors. By demonstrating the relevance of the model and the efficacy of HDAC inhibitors, our model provides a robust phenotypic screening platform, which could be applied to other stroke-associated genetic variants

The parapineal and habenular defects are coupled in <i>sox2</i> morphants.

<p>(<b>A–C</b>) In control embryos, the left-sided parapineal projects towards the left habenula. As a result, the left habenula has denser neuropils than the right, as judged by phalloidin staining. (<b>D–F</b>) In <i>sox2</i> morphants with left-sided parapineal projections, the left habenula is larger than the right. (<b>G–I</b>) Morphants with right-sided parapineal organs display reverse habenular asymmetries, whereas (<b>J–L</b>) morphants with bilateral parapineal projections have symmetric habenulae. (<b>M</b>) The average volume of the left (blue bars) and right (red bars) habenular neuropils, as judged by the volume of phalloidin-positive areas within the habenulae. y-axis show volume in µm³. (<b>N</b>) Average asymmetry index in control (purple bar) and <i>sox2</i> morphants (orange bars). 3D reconstructions of confocal images at 4 dpf, arrows show parapineal projections and blue lines surround the habenular neuropils, error bars represent ± standard error, (<b>M</b>) * = p-value <0.05 and ** = p-value <0.001 (Wilcoxon test).</p

Knockdown of <i>sox2</i> results in abnormal parapineal development and disruption of the habenular asymmetries.

<p>(<b>A</b>) <i>gfi1ab</i> is expressed in parapineal cells on the left side of the brain in control embryos. (<b>B–D</b>) <i>sox2</i> morphants are categorized into three groups according to <i>gfi1ab</i> expression: left-sided expression (<b>B</b>), right-sided (<b>C</b>) and embryos with scattered <i>gfi1ab</i> cells (<b>D</b>). (<b>E</b>) <i>kctd12.1</i> is asymmetrically expressed in the habenulae, with a broader expression domain in the left than the right habenula. (<b>F–H</b>) In <i>sox2</i> morphants <i>kctd12.1</i> expression can be: asymmetric with more on the left side similar to control embryos (<b>F</b>), asymmetric with more on the right side (<b>G</b>) or symmetric (<b>H</b>). (<b>I</b>) A table showing the percentage of embryos with normal, reversed or bilateral parapineal organs, using different staining methods. Scale bars = 25 µm. See also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s009" target="_blank">Figure S9</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s010" target="_blank">S10</a></b> and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s015" target="_blank">Movie S4</a>.</b></p

Sox2 and Notch have synergistic effect on neurogenesis and complementary effects on cell-fate determination.

<p>(<b>A–D</b>) The number of pineal neurons (Isl1-positive cells) is increased in <i>sox2</i> morphants (<b>B</b>) and in DAPT-treated embryos (<b>C</b>), when compared to DMSO-treated controls (<b>A</b>). A synergistic effect is observed when both <i>sox2</i> and Notch are knocked down (<b>D</b>). (<b>E–H</b>) <i>Tg(aanat2:GFP)</i> drives GFP expression in the pineal PhRs in DMSO-treated controls (<b>E</b>). The number of PhRs is increased in <i>sox2</i> morphants (<b>F</b>), but remains unaffected in DAPT-treated embryos (<b>G</b>). The knockdown of both <i>sox2</i> and Notch results in an increased number of GFP-positive cells (<b>H</b>), comparable to the one observed in <i>sox2</i> morphants. (<b>I–L</b>) <i>Tg(elavl3:GFP)</i> expresses GFP in PNs. There is no difference in the number of GFP-positive in <i>sox2</i> morphants (<b>I</b>) when compared to DMSO-treated controls (<b>I</b>). The knockdown of Notch alone (<b>K</b>) or simultaneously with <i>sox2</i> (<b>L</b>) results in similar increase in GFP expression. (<b>M–O</b>) Average number of Isl1-positive cells (<b>M</b>), PhRs (<b>N</b>) and PNs (<b>O</b>) in untreated controls, DMSO-treated controls, untreated <i>sox2</i> morphants, DMSO-treated <i>sox2</i> morphants, DAPT-treated embryos and DAPT-treated <i>sox2</i> morphants. Confocal maximum projections of 28 hpf embryos, scale bars = 25 µm, error bars represent ± standard error, * = p-value <0.05 (MWU test), ** = p-value <0.001 (MWU test). See also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s005" target="_blank">Figure S5</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s007" target="_blank">S7</a></b> and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s012" target="_blank">Movie S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s013" target="_blank">S2</a></b>.</p