The authors have declared that no competing interests exist.
Conceived and designed the experiments: CMT KH. Performed the experiments: CMT AL. Analyzed the data: CMT AL KH. Contributed reagents/materials/analysis tools: CMT. Wrote the paper: CMT KH.
Embryonic development requires chromatin remodeling for dynamic regulation of gene expression patterns to ensure silencing of pluripotent transcription factors and activation of developmental regulators. Demethylation of H3K27me3 by the histone demethylases
Mouse embryonic stem cells (ESCs) are pluripotent cells derived from the inner cell mass of a blastocyst that can be propagated as a cell line in tissue culture. They have the capability of self-renewal and are able to differentiate into all three germ layers (ectoderm, mesoderm and endoderm) that will later differentiate into the distinct cell types present in adult mice. During this process changes in chromatin structure is accompanied by the de-repression of lineage-specific genes and repression of pluripotent transcription factors, such as Oct4 and Nanog
Several mechanisms are involved in the regulation of chromatin architecture in ESCs, including post-translational modifications of histone tails like acetylation, methylation, phosphorylation, and ubiquitylation
Lysine residues of histone tails can be mono-, di- or tri-methylated and the degree of methylation as well as the specific residue methylated influences which proteins can bind to chromatin and modify the chromatin state
The Polycomb repressive complex 2 (PRC2), and more specifically its catalytic subunit Ezh2, is responsible for di- and tri-methylation of H3K27. Whereas the PRC2 core components Ezh2, Suz12 and Eed are essential for early embryonic development, they are not required for ESC proliferation
Utx (Kdm6a) and Jmjd3 (Kdm6b) catalyze the demethylation of H3K27me3 and H3K27me2.
UTX is required for the activation of homeotic genes
Utx homologues in zebrafish (Utx-1 Utx-2) and
A demethylase independent role of UTX has indeed been described in
ESCs were grown on 0.2% gelatin (Sigma) coated tissue culture plates (Nunc) and cultured in 2i medium
Homogeneous cardiomyocytes were generated as previously described
All knockdown experiments were done using shRNAs. Viral transductions were performed using pLKO vectors from Sigma (NM_009484.1-809s1c1 (Uty-shRNA1), NM_009484.1-1113s1c1 (Uty-shRNA2), NM-009484.1-808s1c1 (Uty-shRNA3), NM_001017426.1-2839s1c1 (Jmjd3-shRNA1), NM_001017426.1-5159s1c1 (Jmjd3-shRNA2), NM_001017426.1-3013s1c1 (Jmjd3-shRNA3) and SHC201 (
We designed a conditional targeting vector that after deletion of exon 3 produce a frame shift and introduce a translational stop codon. The Utx conditional construct was generated using the methodology described by Zhang et al.
Briefly, using the mouse BAC RP23-214H5 that covers the mouse
Utx tagged BACs (wild type: +Wt, and catalytic inactive mutant: +Mut) were electroporated, using Amaxa - nucleofector II (Lonza) into
Utx polyclonal antibody was generated by immunizing rabbits with bacterially expressed affinity-purified GST–UTX (amino acids 453–753). The polyclonal antibody was affinity-purified using GST–UTX-coupled Sepharose
Total RNA was isolated using the RNAeasy Minikit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized using the TaqMan Reverse Transcription kit (Applied Biosystems). qPCR was performed using SYBR Green 2× PCR Master mix (Applied Biosystems) on an ABI Prism 7300 Real-Time PCR system (Applied Biosystems) or on a LightCycler 480 System (Roche Applied Science), using the LightCycler 480 SYBR Green I Master mix (Roche Applied Science) according to the manufacturers’ instructions. Error bars represent standard deviation of three PCR amplifications for each sample. Similar results were obtained in at least three independent experiments. Primer sequences are provided in
Expression primers | Primer Fw 5′–3′ | Primer Rv 5′–3′ |
Oct4 |
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Nanog |
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Sox2 |
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Msi1 |
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Sox1 |
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Otx2 |
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Pax6 |
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Tubb3 |
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Gfap |
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Nestin |
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T |
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Flk1 |
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Gata4 |
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Sall4 |
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FoxA2 |
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Pax3 |
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Utx |
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Jmjd3 |
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Uty |
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rpPO |
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Afp |
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Sox17 |
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Hoxb1 |
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Hoxb1 |
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T |
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Flk1 |
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Msi1 |
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Sox1 |
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FoxA1 |
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ChIP analyses were performed as described
Cells were fixed in 70% ethanol and stained with primary antibody for 1 h, followed by 1 h incubation with Alexa Fluor 488 or 647 anti-rabbit (Invitrogen). Cells were pulsed with 33 µM bromodeoxyuridine (BrdU) for 30 min. DNA was counterstained by 0.1 mg/ml propidium iodide supplemented with RNase for 1 h at 37°C. Analysis was performed on a FACS Calibur using CellQuest software (BD). Quantification and analysis of cell-cycle profiles were obtained using FlowJo (Tree Star, Inc).
3×105 ESCs were seeded in a 6 cm-dish during 48 h, and then ESCs were trypsinized and counted with a counting chamber. 3×105 cells were seeded again in another 6-cm dish. This process was repeated 3 more times. Cumulative population doublings at each sub-cultivation was calculated and added to the previous population doubling level, to yield the cumulative population doubling level.
Histological analysis was performed according to standard procedures. Briefly, ten days differentiated embryoid bodies were embedded in OCT (Sakura, 25608-930) and frozen for cryosectioning. Cuts of 8–10 µM sections were deposited over a super frost plus slide (VWR-Menzel, 631–9483) and stained with hematoxylin and eosin, dehydrated and mounted using VectaMount (Vector).
To study the function of Utx in ESC self-renewal and differentiation, we generated a Utx conditional construct that was used to target male 129/Sv mouse derived R1 ESCs, introducing two loxP sites surrounding exon 3 (
(A) Overview of the functional domains in Utx, the genomic locus of
For the analysis of Utx function we also generated cells expressing wild type Utx or catalytic inactive mutant in Utx KO ESCs by introducing BAC transgenes encoding a tagged version of the wild type and mutated
Utx is highly expressed in mouse ESCs (
(A) mRNA expression levels of Oct4, Nanog and Sox2. RT-qPCR normalized for Rplp0 and R1. (B–C) Immunoblots showing Oct4 and Nanog levels. Vinculin was used as loading control. (D) Morphology of Controls (R1, D02), KO (KO1, KO2) and complemented (+WT, +Mut) ESCs. (E) Cell proliferation analysis of the indicated ESCs measured at the indicated days. (F) DNA/BrdU flow cytometry analysis of WT (D02) and KO1 cells. Control represents cells without BrdU pulsing. PI is propidium iodine. (G) Cell cycle analysis by flow cytometry of WT and KO1.
Next, we assessed the involvement of Utx in ESC differentiation in vitro using two well-described differentiation strategies: 1) All-trans retinoic acid (RA) treatment to induce monolayer differentiation of ESCs and 2) Spontaneous embryoid body (EB) formation. In these assays we did not observe any difference in phenotypes between floxed D02, D05 and wild type R1 ESCs, and we therefore only included results from the floxed D02 or D05 as controls.
For the differentiation of ESCs in monolayer, the ESCs were treated with 1 µM RA for 24, 48 and 72 hours (
(A) Scheme of ESC differentiation. Samples were taken 24, 48 and 72 hours after RA treatment and analyzed by RT-qPCR. All RT-qPCRs were normalized to Rplp0 and the levels in D02 at T0 (B) ESC morphology 72 h after RA treatment. (C) The expression of Utx mRNA during the time course (D, E) Pluripotency marker Oct4 mRNA and protein expression levels during differentiation. Vinculin was used as loading control. (F–N) Expression analysis of the indicated genes during differentiation. Ectoderm markers: Msi1, Sox1, Otx2, Pax6, Nes, Gfap, Tubb3; mesoderm markers: Flk1, T. Error bars represent SD, n = 3 independent assays (***p<0.0005, two tailed Student’s test).
(A–F) Expression analysis of endoderm markers FoxA2, Sox17, Sall4, Gata4, Afp and homeobox gene Hoxb1. Error bars represent SD, n = 3 independent assays (***p<0.0005, two tailed Student’s test). (G–H) H3K27me3 protein levels during differentiation. ß-tubulin and H3 were used as loading controls.
Hoxb1 is a member of the homeobox transcription factor family that confers tissue identity along the anterior-posterior axis during mouse and human development. Interestingly, full activation of Hoxb1 also requires Utx, and although the levels are not completely rescued by the re-expression of catalytic inactive Utx, the expression is higher than in knockout cells (
EBs are the in vitro developmental equivalent of the mouse embryo at early developmental stages
(A) Morphology of embryoid bodies 9 days after formation using the indicated ESCs as a starting material. White arrowheads depict some internal cavitation. (B) H&E staining of EBs harvested at day 10 post differentiation. (C) Pluripotency markers Oct4 and Nanog expression after 6 and 9 days of differentiation. (D) Utx expression levels before and after 3, 6 and 9 days of EB differentiation. (E) Western blot for Utx, Ezh2 and H3K27me3 during EB formation of ESCs. Vinculin, H3 and ß-tubulin were used as loading controls. (F, G, I) Gene activation of ectodermal (Msi1, Sox1), mesodermal (Flk1) and endodermal (FoxA2, Pax3) markers after 9 days of EB differentiation. (H) Expression levels of mesodermal marker Brachyury after 6 days of differentiation. All RT-qPCRs were normalized to the expression in D02 at T0 and Rplp0. (J) R1, D02, KO2, +Wt and +Mut percentage of beating EBs after EB formation and cardiac lineage differentiation. Error bars represent SD, n = 3 independent assays (***p<0.0005, two tailed Student’s test)
Similar.to the monolayer differentiation assay, Utx is also required for the proper activation of ectodermal (
To further understand the role of Utx in mesodermal differentiation, we differentiated wild type, floxed, Utx knockout, +Wt and +Mut ESCs into cardiomyocytes (CM), which exhibit spontaneous contractile activity. In agreement with our previous results, Utx KO cells failed to differentiate into cardiomyocytes (mesoderm derived lineage) and did not produce contractile cells (
In summary, our results show that Utx is required for ectoderm and mesoderm differentiation in vitro, independently of its catalytic activity. However, a subset of genes, like Hoxb1, requires the catalytic activity of Utx for their normal activation during differentiation
To investigate the mechanism by which Utx regulates ESC differentiation, we tested if Utx binds to genes involved in ectoderm, mesoderm and endoderm differentiation. As a control we showed that Utx is recruited to the previously identified Utx target gene,
(A) ChIP assays of Utx and the indicated histone modifications on the
Deletion of
(A) The expression levels of the Kdm6 family in ESCs measured by RT-qPCR and normalized to Rplp0 and D02. (B) Expression levels of Jmjd3 in D02, KO1, and KO2 ESCs. Vinculin was used as loading control. (C–D) Western blots showing the H3K27me3 and H3K4me3 levels in the indicated cell lines. ß-tubulin and H3 were used as loading controls. (E) The efficiency of Jmjd3-shRNA1 and Uty-shRNA2 knockdown in the indicated cell lines as measured by RT-qPCR and normalized to Rplp0. (F) Cell proliferation analysis of the indicated cells lines. (G) Western blot showing H3K27m3 levels in the indicated cell lines before and after 72h of RA differentiation (H) mRNA expression levels of Utx target genes in Utx knockout (KO2) cells with and without knocking down Jmjd3 or Uty knockdown cells. All RT-qPCRs were normalized to Rplp0 and the expression levels in D02 Scr at T0. Error bars represent SD, n = 3 independent assays (**p<0.005; ***p<0.0005, two tailed Student’s test).
To address this, we knocked down (kd) the expression of Jmjd3 or Uty in floxed (DO2) and
In this study we demonstrate that Utx is dispensable for ESC proliferation and that its deletion does not lead to a global increase in H3K27me3 levels. Moreover, we show that Utx is required for the proper differentiation of ESCs into ectoderm and mesoderm, independently of its catalytic activity. Our results show that Utx is required for the expression of several genes involved in regulating differentiation, and the binding of Utx to the promoters of these suggest that it directly regulates their transcription. While the catalytic activity of Utx is not required for the regulation of several genes involved in ectoderm and mesoderm specification, the catalytic activity of Utx is required for the activation of a subset of genes, including
The expression of Utx is maintained during ESC differentiation, and in agreement with the notion that regulation of chromatin structure by posttranslational modifications is important for regulating gene expression during differentiation, we have shown that Utx is a critical regulator of this process. Interestingly, we found that while the expression level of pluripotency marker Oct4 is decreased in Utx knockout ESCs undergoing differentiation, several differentiation markers were not induced. This suggests that Utx is not essential for initiating the differentiation program in ESCs, but for the activation of genes contributing to differentiation.
Specifically, we have shown that genes involved in ectoderm formation such as
During vertebrate development, the formation of the central nervous system (CNS) and neural plate (future neural tube) from a region of the primitive ectoderm is a result of the activation of specific genes, which in turn promote the formation of the nervous system. Otx2 is a member of the bicoid sub-family of home domain-containing transcription factors. Otx2 protein plays an important role in brain and sensory organ development. It is activated in the entire ectoderm before gastrulation, and is one of the earliest genes expressed in the anterior neuroectoderm, demarcating rostral brain regions
We have also shown that Utx is enriched at the Flk1/Kdr/Vegfr2 and Brachyury (T) promoters during ESC differentiation, and that Utx is required for the activation of these two genes. Flk1 is a type III transmembrane kinase receptor that plays a critical role in vascular endothelial cell development. Flk1-deficient mice die in utero between E8.5 and E9.5 due to defective development of yolk sac blood islands, endothelial and hematopoietic cells
Brachyury (T) is a DNA binding protein that functions as a transcription factor and form part of the T-Box family of proteins. In mice, Brachyury affects the development of the posterior mesoderm during gastrulation in a dose dependent-manner. Homozygous mutant mice die around E10 dpc with a general failure in notochord morphogenesis
In summary, based on the observation that Utx is required for the activation of
Our analysis of gene expression during ESC differentiation also showed that Uty and Jmjd3 contribute to the regulation of tissue specific genes, and that they might partially compensate for the loss of Utx in a demethylase-independent and -dependent manner, respectively. Nevertheless, it has been shown that Jmjd3 also exhibits demethylase independent activity by recruiting chromatin remodeling complexes at the promoter of T-box genes
In summary we have shown that Utx is essential for normal ESC differentiation, that it is required for the activation of mesodermal and ectodermal genes independently of its catalytic activity. Our results suggest that Utx, Jmjd3 and Uty are dispensable for ESC proliferation and that they have partially overlapping functions during ESC differentiation. More in vivo experiments are required to completely to understand the function of this interesting family of proteins and its catalytic activity.
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We thank Gustavo Stadthagen, and members of the Helin laboratory for fruitful discussions.