Jmjd5, an H3K36me2 histone demethylase,
modulates embryonic cell proliferation throughthe regulation of Cdkn1a expression.
メタデータ

言語: English
出版者:
公開日: 2017-10-05
キーワード (Ja):
キーワード (En):作成者: Ishimura, Akihiko, Minehata, Ken-ichi,
Terashima, Minoru, Kondoh, Gen, Hara, Takahiko,
Suzuki, Takeshi
メールアドレス:所属:

URL

http://hdl.handle.net/2297/30098

Title: Jmjd5, an H3K36me2 histone demethylase, modulates embryonic cell proliferation

through the regulation of Cdkn1a expression.

Akihiko Ishimura1, Ken-ichi Minehata2, Minoru Terashima1, Gen Kondoh3, Takahiko Hara2 andTakeshi Suzuki1,*

1

Division of Functional Genomics, Cancer Research Institute, Kanazawa University,

Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan; 2Stem Cell Project Group, Tokyo

Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506,
Japan; 3Laboratory of Animal Experiments for Regeneration, Institute for Frontier Medical

Science, Kyoto University, Shogoin-kawahara-cho 53, Sakyo-ku, Kyoto 606-8507, Japan

Short title: Role of Jmjd5 in mouse embryos*

Author for correspondence (suzuki-t@staff.kanazawa-u.ac.jp)

Takeshi Suzuki

Division of Functional Genomics, Cancer Research Institute, Kanazawa

University, Kakuma-machi, Kanazawa 920-1192, Ishikawa, Japan

Phone: +81-76-264-6741; Fax: +81-76-234-4502

2

SUMMARY

Covalent modifications of histones play an important role in chromatin architecture anddynamics. In particular, histone lysine methylation is important for transcriptional control

during diverse biological processes. A nuclear protein, Jmjd5 (also called Kdm8) is a histonelysine demethylase that contains a JmjC domain at the C-terminal region. In this study, we havegenerated Jmjd5-deficient mice (Jmjd5) to investigate the in vivo function of Jmjd5. Jmjd5
embryos showed severe growth retardation, resulting in embryonic lethality at the midgestation

stage. Mouse embryonic fibroblasts (MEFs) derived from Jmjd5 hypomorphic embryos(Jmjd5neo/neo) also showed growth defect. Quantitative PCR analysis for various cell cycle

regulators indicated that only Cdkn1a expression was up-regulated in Jmjd5neo/neo MEFs andJmjd5 embryos. Knock-down assay with Cdkn1a-specific small interfering RNAs revealed
that the growth defect of Jmjd5neo/neo MEFs was significantly rescued. In addition, a genetic
study using Jmjd5; Cdkn1adouble knock-outmice showed that the growth retardation of
Jmjd5 embryos was partially rescued by Cdkn1a deficiency. Chromatin immunoprecipitation
analysis showed that increased di-methylated lysine 36 of histone H3 (H3K36me2) and reduced

recruitment of endogenous Jmjd5 were detected in the transcribed regions of Cdkn1a in

3Jmjd5neo/neo MEFs. Taken together, these results suggest that Jmjd5 physiologically moderates

embryonic cell proliferation through the epigenetic control of Cdkn1a expression.

KEY WORDS: Jmjd5 (Kdm8), Cdkn1a, cell proliferation

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INTRODUCTION

In the eukaryotic nuclei, genomic DNA is efficiently packed into chromatin which isconstructed by numerous nucleosomes. In each nucleosome, approximately 146 base pairs of

DNA are wrapped around a histone octamer containing two copies of four core histones: H2A,

H2B, H3 and H4. Histone H1, the linker histone, helps further compact the nucleosomal DNA

into higher-order structures. In addition, the flexible amino-termini extending from core

histones (termed as histone tails) post-translationally receive various covalent modifications

such as methylation, acetylation, phosphorylation, ubiquitylation and SUMOylation. These

histone modifications can alter the structure of the nucleosome and may also change theassembly between chromatin and the nuclear proteins that recognize specific histone

modifications (Mosammaparast and Shi, 2011).

Among these modifications, methylation of specific lysine residues on histone H3(e.g., H3K4, H3K9, H3K27 and H3K36) is closely related to gene expression (Martin andZhang, 2005). During transcriptional processes, methylation at H3K4 and H3K36 correlateswith transcriptionally active genes, whereas methylation at H3K9 and H3K27 is linked totranscriptional repression. To add more complexity, each of the lysine residues has four distinct

5methylation states that are un-, mono- (me1), di- (me2) or tri- (me3) methylated. Importantly,

methylation is a reversible modification regulated by histone lysine methyltransferases (KMTs)

or histone lysine demethylases (KDMs). The methylation status of histone H3 is dynamicallyregulated by KMTs and KDMs, and is implicated in diverse biological processes including

cellular proliferation, differentiation, DNA repair and recombination.

Two classes of KDMs have been identified (Klose et al., 2006). KDM1 (also called

LSD1) encodes a flavin adenine dinucleotide-dependent amine oxidase, and is known to remove

mono- and di-methylated lysine modifications. The Jumonji C (JmjC)-domain family of KDMs
contains proteins that are Fe (II) and -ketoglutarate-dependent enzymes. Unlike KDM1, JmjC
proteins can remove all three methylation states. Recent studies suggest that the deregulation of

JmjC members is associated with human diseases such as neurological disorders and cancers(Pedersen and Helin, 2010). For instance, mutations in JARID1C/KDM5C were found inpatients with X-linked mental retardation (XLMR, Jensen et al., 2005), and PHF8 mutations

were also observed in patients with XLMR and cleft lip/palate (Abidi et al., 2007; Koivisto etal., 2007; Laumonnier et al., 2005). Gene amplification of JMJD2C/KDM4A was detected inesophageal squamous carcinomas, medulloblastoma and breast cancer (Ehrbrecht et al., 2006;

6

Liu et al., 2009; Northcott et al., 2009; Yang et al., 2000), and elevated expression of

PLU1/KDM5B was reported in breast, prostate and lung cancers (Hayami et al., 2010; Lu et al.,

1999; Xiang et al., 2007). Furthermore, inactivating somatic mutations of UTX/KDM6A havebeen reported in multiple tumor types (van Haaften et al., 2009).

Genetically modified mice for several JmjC members have previously been

generated suggesting the involvement of JmjC proteins in various pathological/developmental

processes. For example, deletion of Jmjd1a/Kdm3a showed an adult-onset obesity phenotype

and down-regulation of several metabolic genes (Inagaki et al., 2009; Tateishi et al., 2009).

Deletion of Fbxl10/Kdm2b showed an exencephalic phenotype and caused increased cellproliferation and cell death in neural progenitor cells, indicating an essential role for this geneduring neural development (Fukuda et al., 2011).

Recently, Jmjd5, a member of the JmjC family, was reported as a demethylase fordi-methylated lysine 36 of histone H3 (H3K36me2), regulating the expression of the CCNA1

gene in MCF7 breast cancer cells (Hsia et al., 2010). In Arabidopsis thaliana, Jmjd5 wasco-regulated with evening-phased clock components and was positively associated with clock

genes expressed at dawn (Jones et al., 2010). They also showed that knock-down of Jmjd5 in

7

U2OS osteosarcoma cells led to fast-running circadian oscillations, suggesting its

interchangeable role in both plant and human circadian systems. However, the physiologicalrole of Jmjd5 in the context of a whole mammalian organism remains unknown.

In order to understand the function of Jmjd5 in cellular and developmental processes,

we have successfully generated Jmjd5 mutant mice. We found that the deletion of Jmjd5 allelecaused severe growth retardation, resulting in embryonic lethality at the midgestation stage.

MEFs derived from Jmjd5 hypomorphic embryos also showed the defect in cell proliferation.

We found the increased expression of Cdkn1a as one of the mechanisms leading to cell growth

retardation. ChIP analysis indicated that Jmjd5 was directly recruited on the Cdkn1a locus,

resulting in the alteration of H3K36me2 status of the Cdkn1a gene. These results suggest that

Jmjd5 histone demethylase is involved in embryonic cell proliferation by fine-tuning theexpression of Cdkn1a.

8

MATERIALS AND METHODS

Generation of Jmjd5-deficient mice

The targeting vector for Jmjd5-deficient mice was constructed by a recombineering system (Liuet al., 2003). The vector was first linearized by Pme I digestion and used for electroporation into

the V6.5 clone derived from C57BL/6 and 129Sv ES cells (Eggan et al., 2001). The cells were
subjected to G418 selection (150 g/ml), and independent colonies were screened by Southernblotting. Genomic DNAs isolated from ES clones were digested with Cla I or Eco RV andhybridized with a 5’-probe (nucleotides 4641 to 5187 in NCBI Reference Sequence:

NC_000073.5) and a 3’-probe (nucleotides 13709 to 14028), respectively. These probes weregenerated by polymerase chain reaction (PCR) using the following primers: for 5’-probe,

5’-TCCCTTACACGGCTATGGTC-3’

and5’-AGCTGAGAGAATTCAGGGCA-3’;

for

3’-probe, 5’-GGGTGAGTCAGAATAGTGTA-3’ and 5’-TCATTGGCCAAGGATTCAGA-3’.

One derivative of each ES cell line containing the floxed Jmjd5 allele was injected

into the blastocoel cavity of 3.5-day C57BL/6 blastocysts. Chimeric mice were generated at

UNITECH Co., Inc. (Japan) and mated with C57BL/6 females. The offspring (Jmjd5+/neo) was

screened for germline transmission by PCR. F1 mice were backcrossed with C57BL/6 mice.

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CAG-Flp transgenic mice (Ando et al., 2000), Pgk2-Cre transgenic mice (Kanki et al., 2006),

and Cdkn1a-deficient mice (Deng et al., 1995) were used for the generation of Jmjd5flox/flox mice,Jmjd5 mice and Jmjd5; Cdkn1a mice, respectively. All animal experiments wereapproved by the Animal Care and Use Committee of Kanazawa University.

Genotyping for Jmjd5-deficient mice

For the genotyping of adult mice, the tails were lysed in lysis buffer (20 mM Tris-HCl pH 8.0, 5

mM EDTA, 400 mM NaCl, 0.3% SDS and 0.2 mg/ml Proteinase K) for DNA extraction.

Genomic DNA was used for PCR using Ex-Taq DNA polymerase (Takara) or Southern blotting.

For the genotyping of embryos, a part of the yolk sac was lysed and the lysate was used for

direct PCR using MightyAmp DNA polymerase (Takara). The primers used for genotyping arelisted in Table S1. For Southern blotting, genomic DNA was digested with Stu I or Kpn I andhybridized with a Jmjd5-specific probe (nucleotides 10833 to 11233 in NCBI Reference

Sequence: NC_000073.5). Signals were visualized with the Gene Image Random-Prime

Labeling and Detection System (Amersham). The probe was amplified using the primers:

5’-TTCTGACCTCCACATATGACACAG-3’ and 5’-CCAACCTGACTCAACCTACTCC-3’.

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Preparation of Jmjd5 hypomorphic MEFs

MEFs were established from wild-type and Jmjd5neo/neo embryos using standard protocols (Nagyet al., 2003) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal

bovine serum, 2 mM glutamine and penicillin/streptomycin at 37˚C in 5% CO2. For cell growth
assay, 3 x 104 cells were plated on 12-well plates, and the cell number was counted every 24
hours. For retroviral infection, 1.5 x 105 cells were plated on 6-well plates, and the cell number

was counted at 48 hours after infection.

Preparation of plasmids and anti-Jmjd5 antibody

For whole mount in situ hybridization, a fragment of Jmjd5 cDNA (nucleotides 73 to 1502 in

NCBI Reference Sequence: NM_029842.5) or Cdkn1a cDNA (nucleotides 104 to 587 in NCBI

Reference Sequence: NM_007669.4) was amplified and cloned into pSTBlue-1 vector(Novagen). For luciferase assay, pTAL-Luc (Clontech) was used as a control vector (tk-luc). To

preparep53-responsive

luciferase

vector5’-GAGAACATGTCCCAACATGTTG-3’(p53BS),and11

complementaryoligonucleotides,

5’-TCCAACATGTTGGGACATGTTC-3’,

corresponding to the p53-binding site from human Cdkn1a locus (El-Deiry et al., 1993), weresynthesized. Six annealed oligonucleotides were tandemly ligated and inserted into pTAL-Luc.

Mouse Jmjd5 cDNA was tagged with FLAG-His6-tag and cloned into pDON-5 Neo
plasmid (Takara) to produce retroviruses. A Jmjd5 H319A mutant, which corresponds to thecatalytically inactive JMJD5 mutant H321A in humans (Hsia et al., 2010), was generated byusing a PCR-based Mutagenesis kit (Takara). The shRNA-expressing retroviruses wereconstructed and produced as described previously (Yoshida et al., 2011). Oligonucleotides forshRNA production are listed in Table S2.

For the preparation of anti-Jmjd5 rabbit polyclonal antibody, histidine-tagged

C-terminal Jmjd5 (D175 to Y396) proteins purified from genetically engineered Escherichia

coli were used.

Quantitative reverse transcription-PCR (qRT-PCR)

Total RNA was extracted with Trizol (Invitrogen) and synthesized to cDNA using SuperScript

VILO cDNA synthesis kit (Invitrogen). qRT-PCR analysis was performed with FastStart

Universal SYBR Green Master (Roche) using 7900HT Fast Real-Time PCR System (Applied

12

Biosystems). PCR data were normalized with the expression of Gapdh. Primers designed for theqPCR are listed in Table S1.

Chromatin immunoprecipitation (ChIP) analysis.

ChIP experiments were performed as described previously (Kimura et al., 2008). The complexes

that were immunopreciptated with anti-K36me2 mouse monoclonal antibody or anti-Jmjd5

antibody were collected by Dynabeads Protein A or G (Invitrogen). The precipitates were used

for the detection of Cdkn1a genomic regions by qPCR. Primers designed for ChIP are listed inTable S1.

Knock-down assay.

Each Stealth RNAi for Cdkn1a (Invitrogen) was introduced into the cells using Lipofectamine

RNAiMAX (Invitrogen) by a reverse transfection procedure. The cells were collected for RNA

extraction after 48 hours, or the cell number was counted after 72 hours. Stealth RNAi Negative

Control Kit (Invitrogen) was used for control. The siRNAs used for this study are listed in Table

S2.

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Immunoblotting.

MEFs were lysed in RIPA buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1% NP-40, 0.1%

SDS, 1 mM EDTA supplemented with protease inhibitors). The lysates were separated on

SuperSep Ace 5-20% running gel (Wako). Anti-p21 (SX118, BD Biosciences), anti-p53 (FL393,

Santa Cruz) and anti-GAPDH (6C5, Millipore) antibodies were used. As a positive control forthe detection of endogenous p21 and p53, wild-type MEFs were treated with 0.03 g/ml
adriamycin for 24 hours.

Senescence-associated -galactosidase (SA--gal) staining and Immunofluorescence.

SA--gal staining was performed according to the protocol described in the reference (Kitajima
et al., 2010). As a positive control for the detection of senescent cells, MEFs (1 x 104 cells) weresparsely seeded on 6-well plates and cultured for 15 days. For immunofluorescence, MEFs were

fixed with 4% paraformaldehyde in phosphate-buffered saline. The specimens were incubatedwith anti-single stranded DNA (ssDNA) antibody (Dako) and treated with Alexa546-conjugatedanti-rabbit

IgG

antibody(Invitrogen).

14

Nucleiwerevisualizedwith

4’,6-diamidino-2-phenylindole (DAPI). As a positive control for the detection of apoptotic cells,
wild-type MEFs were treated with 0.06 g/ml adriamycin for 24 hours.

Whole mount in situ hybridization (WISH) analysis.

WISH of mouse embryos was performed as described previously (Yamaguchi et al., 1993) withthe exception that hybridization was performed at 70°C. Digoxygenin (DIG)-labeled RNAprobes were synthesized with DIG RNA Labeling Mix (Roche).

Luciferase assay.

Reporter vector was introduced into wild-type MEFs or Jmjd5neo/neo MEFs by Fugene HD

Transfection Reagent (Roche). The cells were collected after 48 hours, and the luciferase

activities were determined with Dual-Luciferase Reporter Assay System (Promega).

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RESULTS

Jmjd5-deficient mice are embryonic lethal at the midgestation stage.

In order to investigate the in vivo function of Jmjd5, we generated a conditional Jmjd5

knock-out mice (Jmjd5flox/flox) by the targeted disruption of exon IV, which is where the JmjC

domain starts to get encoded (Fig. 1A). The recombination of the Jmjd5flox allele by Cre

recombinase causes a frame-shift mutation, resulting in the lack of production for Jmjd5 proteinwith JmjC domain. We mated Jmjd5flox/flox female mice with spermatocytes specificCre-expressing transgenic male mice (Pgk2-Cre, Ando et al., 2000), and mutant mice with a
Jmjd5 deletion allele (Jmjd5) were established. Jmjd5 heterozygous mice were viable andfertile, and no morphological changes were observed compared with wild-type mice. To
generate Jmjd5 null mice (Jmjd5), Jmjd5 mice were intercrossed, and the genotype for eachmouse was determined by Southern blot analysis. For Southern blot, genomic DNAs isolated
from wild-type or Jmjd5 mice tails were digested with Stu I or Kpn I, and were hybridized
with the Jmjd5-specific probe. As shown in Fig. 1B, this probe was able to recognize the 2.3 kb

wild-type and the 1.4 kb mutant fragments from the Stu I digested genomic DNA as well as the

1.6 kb wild-type and the 0.7 kb mutant fragments from the Kpn I digested genomic DNA.

16

However, we could not find Jmjd5 homozygous mice in the offspring (Table S3), suggestingthat Jmjd5 mice were embryonic lethal. Thus we next performed further genotyping analyses
at various embryonic stages. The genotype for each embryo was determined by polymerase

chain reaction (PCR) analysis, which could detect a 1.5 kb band for the wild-type allele and a
0.5 kb band for the mutant allele, respectively (Fig. 1C). Jmjd5 embryos could be detectedfrom embryonic day (E) 6.5 to E 11.5 (Fig. 1C and Table S3), but were almost absorbed at E

11.5 (5 of 5), suggesting that they died around E 11.0.

At the same time, we analyzed the temporal and spatial expression pattern ofendogenous Jmjd5 during early embryogenesis. Quantitative reverse transcription (qRT)-PCR

analysis showed that Jmjd5 was expressed at all embryonic stages examined (Fig. 1D), and theexpression of Jmjd5 was maintained at and just beyond the lethal stage (E11.0) of embryonicdevelopment. Whole mount in situ hybridization (WISH) analysis using the anti-sense RNAprobe for Jmjd5 indicated that endogenous Jmjd5 was ubiquitously expressed in the whole

embryo at E 8.5 (5 of 5, Fig. 1E left panel), while the complementary sense RNA probe detected

no signal (2 of 2, Fig. 1E right panel). Thus these results suggested an important role of Jmjd5

during embryogenesis at least after the post-implantation stages.

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Next, we mated Jmjd5 mice and dissected the pregnant mice to observe thephenotype of Jmjd5 embryos and the littermates from E 6.5 to E 10.5 (Fig. 2). Although theresults showed that there was no phenotypic difference between Jmjd5 embryos and thecontrol littermates at E 6.5 (8 of 8, Fig. 2A,A’), the growth of Jmjd5 embryos at E 9.0 was
obviously delayed compared with that of control embryos (8 of 8, Fig. 2B,B’). The Jmjd5
embryos at E 9.0 looked similar with the wild-type embryo at E 8.5. At this stage, we were notable to observe other morphological defects in the appearance of Jmjd5 embryos. At E 10.5,
Jmjd5 embryos showed the severe growth retardation including the incomplete embryonicturning. We also observed typical angiogenesis defects in Jmjd5 embryos that contained
avascular yolk sacs (8 of 8, Fig. 2C’) and the clumps of red blood cells (7 of 8, Fig. 2D’). Thissuggested the possibility that the abnormal vascular development might lead to the insufficient
circulatory system in Jmjd5 embryos, resulting in the observed embryonic growth retardation.
However, a slight growth retardation of Jmjd5 embryos has already been confirmed at E 7.5
when endothelial precursors (angioblasts) start developing in the mouse embryo (data notshown). Thus we concluded that Jmjd5 may be a critical factor for embryonic survival,

especially for embryonic cell proliferation after the post-implantation stages.

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Jmjd5 regulates the cell proliferation of mouse embryonic fibroblasts (MEFs).

To examine whether Jmjd5 could intrinsically regulate the embryonic cell proliferation, we

attempted to prepare Jmjd5-deficient MEFs to see the effect of Jmjd5 on the cell growth.
However, it was difficult to establish Jmjd5 MEFs, because Jmjd5embryos did not survive
to E 14.5, one of the most suitable stages for the preparation of MEFs. Interestingly, we foundthe Jmjd5neo/neo homozygous mice were also embryonic lethal without any excision of Jmjd5

allele (See Fig. 1 and Table S4). At E 10.5, whereas the majority of Jmjd5neo/neo embryos (25 of37) showed severe growth retardation as observed for Jmjd5 embryos (Fig. S1, panel B), the
12 remaining Jmjd5neo/neo embryos seemed to be normal except for the smaller embryonic body(Fig. S1, panel C) and survived until E 14.5. This result indicated that Jmjd5neo/neo embryos

displayed a hypomorphic phenotype. Furthermore, RT-PCR analysis indicated that theendogenous Jmjd5 expression was significantly reduced in Jmjd5neo/neo embryos (Fig. S2). Since

Jmjd5flox/flox mice, in which Pgk-Neo cassette has been already removed by mating with

CAG-Flp mice, were viable and looked normal (data not shown), it was suggested thatexogenous Pgk-Neo cassette from our targeting vector could be interfering with the expression

19

of endogenous Jmjd5, resulting in the hypomorphic phenotype of Jmjd5neo/neo mice. Thus we

used these Jmjd5neo/neo embryos to establish Jmjd5 hypomorphic MEFs for further analyses.

Pregnant mice were dissected at 14.5 days post coitum (p.c.), and MEFs wereprepared from each embryo. Genotypes were determined by PCR analysis, and a single band of

0.5 kb corresponding to Jmjd5neo allele was detected for Jmjd5neo/neo MEF (Fig. 3A).

Approximately a 75% reduction of endogenous Jmjd5 expression was observed by qRT-PCR

analysis in the prepared MEFs (Fig. 3B), and immunoblotting with anti-Jmjd5 antibody also

showed that the expression of Jmjd5 was significantly lower than wild-type (Fig. 3C). Next, we

compared the cell growth between wild-type and Jmjd5neo/neo MEFs (Fig. 3D). The wild-type

MEFs proliferated exponentially and became confluent after 72 hours culture (n = 3). However,

the growth of Jmjd5neo/neo MEFs was significantly reduced compared with wild-type within 48

hours after passage (n = 3, p < 0.01), and the cell number was decreased to approximately 50%

of control cells at 72 hours after passage (Fig. 3D). This result suggested that Jmjd5 played an

important role in embryonic cell proliferation.

To examine whether the decreased cell number of Jmjd5neo/neo MEFs might be due tothe induced premature senescence, we carried out senescence-associated β-galactosidase20

staining. The β-galactosidase positive cells were not detected in both Jmjd5neo/neo MEFs andwild-type MEFs (Fig. 3E, middle and upper panels), although β-galactosidase positive cells

could clearly be detected in the control sparsely seeded MEFs (Fig. 3E, lower panel). Next, we

performed immunofluorescence assay with anti-single stranded DNA (ssDNA) antibody to

examine whether the cells undergo apoptosis at a higher rate. Although many cells were stainedwith anti-ssDNA antibody in the positive controls (adriamycin-treated MEFs, Fig. 3F, lower

panels), we did not observe the elevated rate of the ssDNA positive cells in Jmjd5neo/neo MEFs

compared with wild-type MEFs (Fig. 3F, middle and upper panels). These results indicated that

down-regulation of Jmjd5 did not increase premature senescence and apoptosis, but rather

decreased embryonic cell proliferation.

Next we examined whether the growth retardation of Jmjd5neo/neo MEFs wasdependent on Jmjd5 demethylase activity. The Jmjd5neo/neo MEFs were infected with control

retrovirus, the retrovirus expressing wild-type Jmjd5 or the catalytically inactive mutant H319A.

The expression of Jmjd5 was increased at the same level when MEFs were infected withwild-type or inactive Jmjd5 expressing virus (Fig. S3). The cell number of the infected MEFs

was counted at 48 hours after infection, and the cell growth defect of Jmjd5neo/neo MEFs was21significantly rescued by re-expressing wild-type Jmjd5 (Fig. 3G). No significant change wasobserved with the expression of inactive H319A mutant compared to the control. These results

suggested that the demethylase activity of Jmjd5 was required for normal embryonic cellproliferation.

The expression of Cdkn1a/p21 is up-regulated in Jmjd5neo/neo MEFs and Jmjd5 embryos.
A recent report showed that Jmjd5 activated the expression of the CCNA1 gene in MCF7 breast

cancer cells by demethylating H3K36me2 on the CCNA1 locus, resulting in cell cycle

progression (Hsia et al., 2010). Thus we first examined whether loss of Jmjd5 caused thedown-regulation of Ccna1 in mouse embryonic cells. We extracted total RNA from Jmjd5neo/neo
MEFs or Jmjd5 embryos to synthesize cDNA, and performed qRT-PCR analysis using
Ccna1-specific primers. Unexpectedly, there was no change in the expression of Ccna1 in
Jmjd5neo/neo MEFs or Jmjd5 embryos compared with wild-type (Fig. 4A,D). This indicatedthat there might be a different regulatory function of Jmjd5 in mouse embryonic cells andhuman cancer cell lines.

Various genetic studies using knock-out mice have previously shown that a number22

of cell cycle regulators are involved in normal mouse development (Ciemerych and Sicinski,2005). Therefore, we tried to examine whether the loss of Jmjd5 affected the expression ofrepresentative cell cycle regulators, including eleven Cyclins, ten CDKs, eight CDK inhibitors

and Rb family, for mouse embryogenesis. Comparing the expression of cell cycle regulators

between wild-type MEFs and Jmjd5neo/neo MEFs, the expressions of Ccnd2, Cdk6 and Cdk7

were 1.57-fold, 1.44-fold and 1.43-fold higher respectively in Jmjd5neo/neo MEFs (Fig. 4A,B). In

addition, the expressions of Cdkn1a and Cdkn2a/p16Ink4a were 1.93-fold and 1.69-fold higher

than those of wild-type, respectively (Fig. 4C). On the other hand, the expressions of other

Cyclins, CDKs, CDK inhibitors and Rb family were not significantly changed in Jmjd5neo/neo

MEFs (Fig. 4A-C, S4). Next, we examined the expression of Ccnd2, Cdk6, Cdk7, Cdkn1a andCdkn2a/p16Ink4a in wild-type embryos at the early somite stage (E 8.25, n = 3) and in thestage-matched Jmjd5embryos (n = 7) by qRT-PCR analysis. In agreement with our data from
Jmjd5neo/neo MEFs, we detected a significant increase of Cdkn1a transcripts (4.17-fold, p < 0.01)
in Jmjd5embryos (Fig. 4D). However, unlike the up-regulation in Jmjd5neo/neo MEFs, theexpression of other candidates was not altered in Jmjd5embryos, and the expression of
Ccnd2 showed a 1.52-fold reduction (p < 0.01). Since Cdkn1a expression was commonly23

elevated in both Jmjd5neo/neo MEFs and Jmjd5 embryos, we picked up Cdkn1a as one of themost probable transcriptional targets for Jmjd5.
To evaluate the spatial alteration of Cdkn1a expression in Jmjd5embryos, we also
performed WISH analysis (Fig. 4E). The elevation of Cdkn1a was detected in the posterior halfof the Jmjd5embryo including the primitive streak where endogenous Cdkn1a was highly
expressed in wild-type embryo. The ectopic expression of Cdkn1a was also observed in theanterior half of the Jmjd5embryo (7 of 10). A complementary sense probe for Cdkn1a was
not able to detect any signals (4 of 4). These results suggested that endogenous Jmjd5 could be

responsible for the spatial expression pattern of Cdkn1a in whole embryos.

Next we examined whether the knock-down of Jmjd5 expression could induce theup-regulation of Cdkn1a. We used the retroviruses expressing two different small hairpin RNAs(shRNAs) for Jmjd5 (Jmjd5 sh#1 and sh#2). MEFs were infected with the control retrovirus or

the retrovirus expressing each Jmjd5 shRNA. qRT-PCR indicated that Jmjd5 transcripts weresignificantly reduced with the infection of both Jmjd5 shRNA expressing retroviruses, but notwith the control retrovirus (Fig. 4F, left). We then examined the effect of Jmjd5 knock-down in

the expression of Cdkn1a. Both Jmjd5-specific shRNAs led to the significant increase of24

Cdkn1a transcripts (1.59-fold and 1.65-fold) (Fig. 4F, right). These observations suggested that

Jmjd5 might be directly involved in the regulation of Cdkn1a expression in MEFs.

Jmjd5 regulates the expression of Cdkn1a intrinsically without the stimulation of p53-p21

pathway.

According to the results shown in Fig. 3 and 4, we speculated that Cdkn1a up-regulation causedby the reduction in Jmjd5 levels might be a major reason for the observed growth retardation.

To test our hypothesis, we performed a knock-down assay using Cdkn1a-specific siRNAs andinvestigated whether the reduction of Cdkn1a could rescue the inhibition of cell proliferation in

Jmjd5neo/neo MEFs. Two Cdkn1a-specific siRNAs (siRNA#1 and #2) were designed, and theknock-down efficiency of each siRNA was shown to be approximately 80 % (Fig. 5A). Cdkn1a

siRNA#1, #2 or control unrelated siRNA was transfected into Jmjd5neo/neo MEFs, and the growthof the transfected MEFs was compared. Knock-down of Cdkn1a in Jmjd5neo/neo MEFs caused asignificant increase of cell proliferation, compared with the proliferation of Jmjd5neo/neo MEFs

treated with the control siRNA as well as untreated Jmjd5neo/neo MEFs (n = 3, p < 0.01, Fig. 5B).

The cell number of Jmjd5neo/neo MEFs treated with the Cdkn1a-specific siRNAs was still less25

than that of wild-type MEFs, indicating a partial rescue of cell growth by Cdkn1a knock-down.

These results suggested that the regulation of Cdkn1a expression by Jmjd5 played an importantbut not exclusive role in embryonic proliferation, although the incomplete knock-down of

Cdkn1a may be responsible for the partial rescue phenotype.

Since it is known that Cdkn1a is a major direct target for p53 pathway (Wood andShilatifard, 2006), we examined whether the elevation of Cdkn1a was dependent on the

induction of endogenous p53 transcripts (Trp53) in Jmjd5-deficient cells. qRT-PCR showed that
there was no difference in the expression of Trp53 in Jmjd5neo/neo MEFs and Jmjd5 embryos
compared with control cells (Fig. 5C). Immunoblotting also indicated that there was noalteration in the level of p53 protein between wild-type and Jmjd5neo/neo MEFs, despite theenrichment of Cdkn1a/p21 protein in Jmjd5neo/neo MEFs (Fig. 5D). We next measured thetranscriptional activity of p53 in wild-type and Jmjd5neo/neo MEFs by luciferase assay. We

constructed a p53BS-tk-luciferase reporter (p53BS) containing p53 response elements derived

from human CDKN1A (El-Deiry et al, 1993), and transfected the reporter plasmid or control

plasmid (tk-luc) with a Renilla luciferase reference plasmid into wild-type or Jmjd5neo/neo MEFs.

The result showed that there was no significant difference in the luciferase activity (n = 4, Fig.

265E), indicating that the alteration of p53 transcriptional activity did not occur in Jmjd5neo/neo

MEFs.

We investigated whether the growth retardation caused by the loss of Jmjd5 wasclosely associated with the deregulation of Cdkn1a in the embryos. Jmjd5-deficient mice wereintercrossed with Cdkn1a-deficient mice (Cdkn1a), which were morphologically normal at E
10.5 (5 of 5, Fig 6B; Deng et al., 1995), to generate Jmjd5 and Cdkn1a double knock-out mice
(Jmjd5; Cdkn1a). The phenotypes were compared between the double knock-out mice andJmjd5-deficient mice. The Jmjd5; Cdkn1amice were embryonic lethal as well as Jmjd5
mice (data not shown), and showed the severe growth retardation (4 of 4, Fig. 6D) comparedwith wild-type embryos (Fig. 6A). However, the whole sizes of Jmjd5; Cdkn1aembryos
were slightly bigger than Jmjd5 littermates (Fig. 6C). Other abnormal phenotypes such as theincomplete turning and the angiogenesis defect seemed not to be rescued in the double

knock-out embryos. These results suggested that the deficiency of Cdkn1a could partially rescue
the phenotype of Jmjd5 embryos, similarly to our result from the knock-down experiment(Fig. 5B). We demonstrated that a genetic interaction between Jmjd5 and Cdkn1a was at least

important for embryonic cell proliferation but also proposed that other factors regulated by27

Jmjd5 would contribute to normal embryonic cell growth.

Jmjd5 is involved in the maintenance of H3K36me2 in Cdkn1a locus.

Previous studies suggested that Jmjd5 was an H3K36me2 histone demethylase (Hsia et al.,2010), and that methylated K36 in histone H3 was a transcriptional active mark (Li et al., 2007).

Thus we assumed that the loss of Jmjd5 caused an elevation of H3K36me2 in Cdkn1a locus,

resulting in the activation of Cdkn1a in Jmjd5-deficient cells. To test our hypothesis, we

designed primers to amplify several genomic regions of Cdkn1a for chromatin

immunoprecipitation (ChIP) analysis (Fig. 7A). The crosslinked chromatin from wild-type or

Jmjd5neo/neo MEFs was immunopreciptated with anti-K36me2 or anti-Jmjd5 antibody. The

precipitates were used for the detection of Cdkn1a genomic regions by qPCR. The enrichmentof H3K36me2 was observed in intron II (regions a and b) and exon III (region c containing theopen reading frames of Cdkn1a) but not intron III (region d) of Cdkn1a (n > 9, p < 0.01) in

Jmjd5neo/neo MEFs (Fig. 7B). On the other hand, the recruitment of Jmjd5 into the Cdkn1a locus

was significantly reduced in regions a, b and c, while no significant change was observed inregion d (n > 9, p < 0.01, Fig. 7C). These results suggested that reduced recruitment of Jmjd5

28

into several regions of the Cdkn1a locus induced the increased H3K36me2 in the corresponding

regions of Cdkn1a. Based on the previous report showing the correlation between enriched

H3K36me2 and transcriptional active phase (Li et al., 2007), the increased H3K36me2 wasthought to induce Cdkn1a expression in Jmjd5neo/neo MEFs. We also performed ChIP analysis

with anti-H3K4me3 antibody to investigate whether another epigenetic mark, H3K4me3, wasaltered in regions a, b and c of Cdkn1a. The result showed that the enrichment of H3K4me3 wasobserved only in region a, and that significant changes were not detected in the other regions(Fig. S5). Since H3K4me3 is a transcriptional active mark (Martin and Zhang, 2005), thisalteration of H3K4me3 in Jmjd5-independent manner might reflect the enhanced transcriptional

activation of Cdkn1a in Jmjd5neo/neo MEFs. Taken together, our data demonstrates that Cdkn1a is

one of the downstream targets for Jmjd5 histone demethylase, and the maintenance of

H3K36me2 in Cdkn1a locus regulated by Jmjd5 may be required for normal embryonicproliferation.

29

Discussion

In this study, we generated Jmjd5-deficient mice and observed severe growth retardation in
Jmjd5embryos, leading to embryonic lethality around E 11.0. In Jmjd5embryos,
significant up-regulation of Cdkn1a was detected. Similarly, growth retardation as well as anelevated expression of Cdkn1a was observed in Jmjd5neo/neo MEFs. ChIP analysis indicated thatthe detection of H3K36me2 was significantly increased in several regions of the Cdkn1a locus

derived from Jmjd5neo/neo MEFs, where the recruitment of endogenous Jmjd5 was reduced. Thus

we demonstrate for the first time that Jmjd5 moderates the expression of Cdkn1a during

embryogenesis through the histone demethylase activity.

Currently, there are more than 30 known mammalian JmjC domain-encoding genes,

and some have been reported to be involved in cell proliferation by regulating the expression ofcell cycle inhibitors. Our recent study demonstrated that Utx/KDM6A directly demethylated

H3K27me3 in Rb and Rbl2 increasing their expression levels, resulting in the growth repressionof MEFs (Terashima et al., 2010). Jmjd3/KDM6B, an H3K27 demethylase, activated the

INK4A-ARF locus in response to oncogenic stress, and the overexpression of Jmjd3 led to theinhibition of cell proliferation (Agger et al., 2009; Barradas et al., 2009). Furthermore,

30

Fbxl10/KDM2b, an H3K36 demethylase, regulated cell proliferation through the modulation of

Cdkn2b (He et al., 2008). These results strongly suggest an essential role played by the JmjC

family in cell growth signaling. Our analysis with Jmjd5-deficient mice indicated a novelassociation of Jmjd5 and Cdkn1a expression, which is critical for cell proliferation during

normal mouse development.

Genome-wide profiling of methylated histone H3 from yeast to humans showed that

H3K36 methylation tended to be enriched across the transcribed regions of active genes rather

than promoter regions (Li et al., 2007). In Saccharomyces cerevisiae, methylation of histone H3

by Set2, an H3K36 KMT, is associated with transcriptional elongation by RNA polymerase II(Krogan et al., 2003; Schaft et al., 2003). However, there is still not much evidence for thephysiological function of H3K36me2 in mammals. Recently, knock-down of Fbxl10 in MEFs

was shown to induce the activation of Cdkn2b by the increased H3K36me2 in the Cdkn2b locus(He et al., 2008). Our study demonstrated that the disruption of Jmjd5 in mouse cells induced

the activation of Cdkn1a transcripts and the elevation of H3K36me2 at Cdkn1a transcribedregions, supporting the current model between transcriptional activation and methylated

H3K36.

31

We observed the induced expression of Cdkn1a in Jmjd5embryos and Jmjd5neo/neo
MEFs. Although Cdkn1a is a p53 target gene, we did not detect any significant changes of p53

expression at both the transcriptional and the translational levels. Reporter assay showed thatthe transcriptional activity of p53 was not changed in Jmjd5neo/neo MEFs, andimmunofluorescence and immunoprecipitation studies indicated no alteration of the subcellular

localization of p53 and no direct interaction between Jmjd5 and p53 (data not shown). These

results suggested that the aberrant induction of Cdkn1a seemed to occur without p53 induction.

However, we cannot exclude the possibility that p53 signaling is altered, since p53 protein

receives various post-translational modifications such as phosphorylation, methylation,acetylation and ubiquitylation, which are related to its stability, the specificity of target genes

and the affinity to cofactors (Carter and Vousden, 2009). Therefore, more detailed analyses

would be necessary to prove whether there is a potential connection between Jmjd5 and p53 ornot.
In all of the Jmjd5embryos examined, severe growth retardation was observed,
and the expression of Cdkn1a was more than four-fold higher compared to wild-type embryos.

However, growth retardation is sometimes detected indirectly by abnormal placental

32

development in genetically modified mice (Han and Carter, 2001). In fact, endogenous Jmjd5

expression was detected in ectoplacental cone, a precursor of placenta, at E 8.5. The allantois

need to fuse with the chorion for normal placental development, but this was not observed insome Jmjd5 embryos at E 10.5 (3 of 8) (data not shown). Thus we could not exclude thepossibility that abnormal placental development might be a reason for the growth defect in
Jmjd5embryos. However, it was notable that high expression of endogenous Jmjd5 wasobserved in not only the ectoplacental cone but also the entire embryonic fraction. We also

demonstrated that although embryonic cells purified from Jmjd5neo/neo embryos are not underplacental control, these cells still showed the defect in proliferation and the elevated expressionof Cdkn1a. Furthermore, knock-down of endogenous Cdkn1a was able to rescue the growth
delay significantly in Jmjd5neo/neo MEFs. The growth retardation in Jmjd5embryos was also
partially rescued under the Cdkn1agenetic background. These results indicated that theregulation of Cdkn1a by Jmjd5 played an important role in cell proliferation and embryonicdevelopment. Since the rescue effect was partial, it was also suggested that additional factors

regulated by Jmjd5 would contribute to embryonic development. We are now trying to identify

other target genes regulated by Jmjd5 by the experiments including microarray and

33

ChIP-sequence, and hope to demonstrate the detailed mechanism of Jmjd5 function inembryonic cell proliferation in the near future.
Similarly to the phenotype observed in Jmjd5embryos, the up-regulation ofendogenous Cdkn1a has been reported in several mutant mice, resulting in the severe growth

retardation (Ruland et al., 2001; Rantakari et al., 2010; Suzuki et al., 1997). Notably, theelevation of Cdkn1a in Brca1- or Mdm4-deficient mice has been observed (Hakem et al., 1996;

Migliorini et al., 2002), and importantly, intercrossing with Cdkn1a-deficient mice partiallyrescued the phenotype, suggesting that the aberrant expression of Cdkn1a is a major, but not

exclusive, reason for the embryonic death (Hakem et al., 1997; Steinman et al., 2004).

Interestingly, the phenotype of Histone deacetylase 1 (Hdac1)–deficient mice was similar,
although more severe, with Jmjd5mice. Disruption of Hdac1 resulted in embryonic lethality
before E 10.5 due to the severe growth retardation, and the expression of Cdkn1a and Cdkn1b

was up-regulated (Laggeret al., 2002). This result suggested that several epigenetic controls,

including histone methylation and acetylation, were closely associated with normal expressionof Cdkn1a during embryogenesis. Therefore, the clarification of the relationship between Jmjd5

and other histone modifying enzymes would be helpful for understanding the detailed

34

mechanism of Cdkn1a regulation by Jmjd5.

35

Acknowledgment

We thank Dr. N. Nozaki (Hokkaido Univ., Japan) for providing anti-di-methylated H3K36

antibody. We thank Drs. J. Takeda (Osaka Univ., Japan), N. A. Jenkins and N. G. Copeland(IMCB, Singapore) for providing gene targeting vectors. We thank Drs. Y. Nakanishi and K.

Yoshioka (Kanazawa Univ., Japan) for providing Pgk2-Cre transgenic mice, Dr. S. Itohara(RIKEN, Japan) for CAG-Flp transgenic mice, and Dr. A. Hirao (Kanazawa Univ., Japan) for

Cdkn1a-deficient mice. This study was supported by Grant-in-Aid for Young Scientist (B) and

Scientific Research (C) from the Ministry of Education, Culture, Sports, Science andTechnology of Japan.

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Figure legends

Fig. 1.

Generation of Jmjd5-deficient mice and the expression of Jmjd5 during early

embryogenesis. (A) Exon-intron structure of Jmjd5 allele (Jmjd5+) and targeting strategy to

generate Jmjd5-deficient mice. Jmjd5+ allele contains eight exons (black boxes), and exon IV

contains the 5’ region of the JmjC domain (blue boxes). The targeting vector contains PGK-Neo

cassette (white box), a pair of loxP sequences (white arrowheads) and FRT sequences (orange

arrowheads). In Jmjd5neo allele, the targeting vector is inserted in the Jmjd5+ allele. The

PGK-Neo cassette is removed by mating with CAG-Flp mice to generate the Jmjd5flox allele.

Jmjd5flox/flox mice are mated with Pgk2-Cre mice to establish mutant mice with Jmjd5 deletion(Jmjd5). (B) Southern blotting of the tail DNA to distinguish wild-type and Jmjd5+ mice. (C)Genotyping PCR using genomic DNA from E 10.5 littermates of Jmjd5+ mice intercrosses.
(D) qRT-PCR analysis of endogenous Jmjd5 expression during embryogenesis using

Jmjd5-specific primers. (E) WISH analysis of E 8.5 embryo with the sense (right) or thecomplementary anti-sense (left) probe for Jmjd5. Scale bars: 400 m.

Fig. 2.

The phenotypic observation of Jmjd5 embryos. (A-D) Phenotypes of control

47

embryos from E 6.5 to E 10.5. (A’-D’) Phenotypes of Jmjd5 embryos from E 6.5 to E 10.5.
Scale bars: 400 m.

Fig. 3.

Jmjd5neo/neo hypomorphic MEFs proliferate more slowly than wild-type MEFs. (A)Genotyping PCR using genomic DNA from Jmjd5+/+ or Jmjd5neo/neo MEFs. (B) qRT-PCR

analysis of Jmjd5 expression in Jmjd5+/+(grey bar) or Jmjd5neo/neo MEFs (black bar).

(C)

Immunoblotting with anti-Jmjd5 antibody showed significant reduction of endogenous Jmjd5

protein in Jmjd5neo/neo MEFs. *non-specific band. (D) Growth curve for Jmjd5+/+ (grey) or

Jmjd5neo/neo MEFs (black). The mean ± SD from at least three experiments are shown, and thestatistical difference is determined by Student’s t-test. **p < 0.01. (E) Senescence-associatedβ-galactosidase assays for Jmjd5+/+ (upper panel) or Jmjd5neo/neo MEFs (middle panel). As apositive control for the detection of senescent cells, sparsely seeded MEFs were stained (lower
panel). Scale bars: 200 m. (F) Immunofluorescence assay with anti-ssDNA antibody for
Jmjd5+/+ (upper panels) or Jmjd5neo/neo MEFs (middle panels). The cells were treated with

anti-ssDNA antibody (left panels) and counterstained with DAPI (right panels). As a positive
control, MEFs treated with adriamycin were stained (lower panels). Scale bars: 50 m. (G) The48

growth defect of Jmjd5neo/neo MEFs was rescued by the re-expression of wild-type Jmjd5. The

Jmjd5neo/neo or Jmjd5+/+ MEFs were infected with the retrovirus expressing either without insert(pDON-5) or with wild-type Jmjd5 or H319A mutant. The cell number was counted at 48 hours

after infection. **p < 0.01.

Fig. 4.

The expression of Cdkn1a is up-regulated in Jmjd5-deficient cells. (A-C) The

expression of Cyclins (A), CDKs (B) and CDK inhibitors (C) in Jmjd5+/+ MEFs (grey bar) andJmjd5neo/neo MEFs (black bar) detected by qRT-PCR. (D) The expression of Ccna1, Ccnd2, Cdk6,

Cdk7, Cdkn1a and Cdkn2a/p16 in Jmjd5+/+ embryos at E 8.25 (grey bar) and stage-matched
Jmjd5 embryos (red bar). The mean ± SD from at least three experiments are shown. **p <0.01. (E) WISH analysis of Jmjd5+/+ and Jmjd5 embryo with the sense or the anti-sense probe
for Cdkn1a. Arrowheads show the primitive streak, and the arrow shows the ectopic expressionof Cdkn1a in the anterior half. Scale bars: 400 m. (F) qRT-PCR analysis of Jmjd5 (left panel)
and Cdkn1a (right panel) transcripts in MEFs after knock-down of Jmjd5. (-) shows uninfected

MEFs.

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Fig. 5.

Jmjd5 intrinsically regulates the expression of Cdkn1a without the transcriptional

activation of p53. (A) qRT-PCR analysis was used for the validation of Cdkn1a-specificsiRNAs. (B) Comparative growth assay between control and Cdkn1a knock-down Jmjd5neo/neo

MEFs. Each cell number for Jmjd5+/+ (grey bar), Jmjd5neo/neo (black bar), control siRNA-treated(green bar) or Cdkn1a specific siRNAs-treated Jmjd5neo/neo MEFs (blue bar) was counted at 72

hours after passage. The mean ± SD from at least three experiments are shown. **p < 0.01. (C)qRT-PCR analysis for the detection of Trp53 in MEFs (left) and E 8.25 embryos (right). (D)Immunoblotting with anti-p53 antibody (left panel) or anti-Cdkn1a/p21 (right panel). The lysateof adriamycin-treated wild-type MEFs was used as a positive control for the induction of p53 (aquarter amount of the samples) or p21 protein (the same amount). Anti-GAPDH antibody wasused for loading controls. (E) Luciferase assay with tk promoter luciferase vector (tk-luc) or

p53-responsive luciferase vector (p53BS) showed no significant change in wild-type (grey bar)and Jmjd5neo/neo MEFs (black bar).

Fig. 6.

Phenotypic analysis of Jmjd5; Cdkn1adouble knock-out embryos. Jmjd5+;Cdkn1a+double heterozygous mice were intercrossed. (A) Wild-type embryos, (B)50

Cdkn1a-deficientembryos, (C) Jmjd5-deficient embryos and (D) Jmjd5; Cdkn1a embryos
were observed at E 10.5. Scale bars: 500 m.

Fig. 7.

Elevated level of H3K36me2 and decreased recruitment of Jmjd5 in Cdkn1a locus

from Jmjd5neo/neo MEFs. (A) Schematic structure of the Cdkn1a locus and the distribution of

the amplified regions (a to d) for ChIP analysis. The coding region is indicated by black boxes.

(B) ChIP analysis with anti-H3K36me2 antibody to determine the sites of H3K36me2

enrichment in four regions at the Cdkn1 locus in Jmjd5neo/neo MEFs (black bar) compared withwild-type MEFs (grey bar). (C) ChIP analysis with anti-Jmjd5 antibody to confirm therecruitment of endogenous Jmjd5. The mean ± SD from at least nine experiments are shown.

**p < 0.01.

51