19 research outputs found
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
A lateral protrusion latticework connects neuroepithelial cells and is regulated during neurogenesis
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 µm<sup>3</sup>. (<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