当前位置：文档库 › p53 is regulated by the lysine demet Source Nature SO 2007 Sep 6 449 7158 105 8[PMIDM17805299]

p53 is regulated by the lysine demet Source Nature SO 2007 Sep 6 449 7158 105 8[PMIDM17805299]

LETTERS

p53is regulated by the lysine demethylase LSD1

Jing Huang 1,Roopsha Sengupta 2,Alexsandra B.Espejo 3,Min Gyu Lee 1,Jean A.Dorsey 1,Mario Richter 2,Susanne Opravil 2,Ramin Shiekhattar 1,Mark T.Bedford 3,Thomas Jenuwein 2&Shelley L.Berger 1

p53,the tumour suppressor and transcriptional activator,is regu-lated by numerous post-translational modifications,including lysine methylation 1,2.Histone lysine methylation has recently been shown to be reversible;however,it is not known whether non-histone proteins are substrates for demethylation.Here we show that,in human cells,the histone lysine-specific demethylase LSD1(refs 3,4)interacts with p53to repress p53-mediated transcriptional activation and to inhibit the role of p53in promoting apoptosis.We find that,in vitro ,LSD1removes both monomethylation (K370me1)and dimethylation (K370me2)at K370,a previously identified Smyd2-dependent monomethylation site 2.However,in vivo ,LSD1shows a strong preference to reverse K370me2,which is performed by a distinct,but unknown,methyltransferase.Our results indicate that K370me2has a different role in regulating p53from that of K370me1:K370me1represses p53function,whereas K370me2promotes association with the coactivator 53BP1(p53-binding protein 1)through tandem Tudor domains in 53BP1.Further,LSD1represses p53function through the inhibition of interaction of p53with 53BP1.These observations show that p53is dynamically regulated by lysine methylation and demethylation and that the methylation status at a single lysine residue confers distinct regula-tory output.Lysine methylation therefore provides similar regula-tory complexity for non-histone proteins and for histones.

Previous observations of p53methylation led us to test whether LSD1,also known as BHC110(ref.3),has a role in p53signalling.We used co-immunoprecipitation to examine whether LSD1binds to p53.Ectopically expressed tagged LSD1and p53associated in HEK-293cells (Fig.1a).HDAC2,which is present in a stable complex with LSD1(ref.5),also co-immunoprecipitated with p53(Fig.1a).Using nuclear extract,we detected interaction between endogenous LSD1and p53in MCF7cells with or without treatment by adriamy-cin,a DNA-damaging reagent that activates p53(Fig.1b).Similar results were obtained in HEK-293cells (Supplementary Fig.1).We studied whether LSD1interacts directly with p53.Purified recombinant Flag–LSD1from insect cells (Supplementary Fig.3a)and glutathione S -transferase (GST)-tagged p53from bacteria were used in GST pull-down assays.We observed that GST–p53,but not GST alone,associates with Flag–LSD1,suggesting a direct interaction between p53and LSD1(Fig.1c).

LSD1can act either as a transcriptional coactivator or as a co-repressor 4,6.We investigated the role of LSD1in p53-mediated tran-scriptional regulation in either mock-treated or adriamycin-treated U2OS cells.Ablation of LSD1with short interfering RNA (siRNA)results in increased expression of p21and mdm2(Fig.2a),two well-characterized p53target genes.The increased expression of p21and mdm2was not caused by an increased steady-state level of p53after treatment with LSD1siRNA (Supplementary Fig.2a).In fact,we consistently observed a slight decrease in the steady-state level of p53without adriamycin treatment,which probably resulted from feedback proteolysis of p53caused by an elevated level of mdm2

when that of LSD1was decreased.Similar results were observed in MCF7cells (Supplementary Fig.2b,c).To examine whether the effect of LSD1knockdown on the expression of p21and mdm2is p53-dependent,we used the cell lines BJ and BJ-DNp53(refs 2,7).BJ-DNp53cells contain a stably incorporated dominant-negative p53(DNp53)that prevents endogenous p53from binding to DNA 7,8.We observed that a decrease in LSD1level increased the expression of p21in BJ cells,but not in BJ-DNp53cells (Fig.2b and Supplementary Fig.2d).A similar dependence of LSD1-mediated repression on p53was observed in another pair of cell lines,HCT116(p531/1)and HCT116(p532/2)(Supplementary Fig.2e).Taken together,these results show that LSD1represses the transcriptional activity of p53.

Because LSD1does not regulate the activity of p53in BJ-DNp53cells in which the DNA-binding ability of p53is impaired,we rea-soned that LSD1might affect the binding of DNA by p53,thus

1The Wistar Institute,3601Spruce Street,Philadelphia,Pennsylvania 19104,USA.2Research Institute of Molecular Pathology (IMP),The Vienna Biocenter,1030Vienna,Austria.3

M.D.Anderson Cancer Center,Department of Carcinogenesis,University of Texas,Smithville,Texas 78957,

USA.

LSD1

Colloidal

Western

G S T

G S T –p 53

I n p u t

Adriamycin (8 h)

Adriamycin (8 h)p53 blot

p53 IP

LSD1 IP

LSD1 blot

Anti-LSD1IgG p53 blot Anti-p53

Input

IgG 1

123456

Input

HA–p53F-LSD1+++

–HA–p53

HA–p53F-LSD1IP Input Input IP HDAC2LSD1+

–+

–

–+–+–+

–

F-p53a

F-p53

Figure 1|LSD1interacts with p53.a ,b ,Reciprocal co-immunoprecipitation assays were used to detect interaction between ectopic (a )and endogenous (b )LSD1and p53in HEK-293cells (a )and MCF7cells (b ).c ,GST pull-down assay.F,Flag;HA,haemagglutinin;IP,immunoprecipitation.

Vol 449|6September 2007|doi:10.1038/nature06092

105

altering the equilibrium between DNA-bound and free p53.To test this hypothesis,we used chromatin immunoprecipitation (ChIP)and real-time polymerase chain reaction (PCR).We found signifi-cantly higher levels of p53at the p21promoter in U2OS cells than in control cells when the level of LSD1was decreased by siRNA treat-ment (Fig.2c).These results indicate that LSD1represses the activity of p53in part through decreasing the binding of p53to DNA.

After DNA damage,p53activates the expression of genes that are involved in either cell cycle arrest or apoptosis.Because LSD1represses the transcriptional activation activity of p53,we proposed that LSD1inhibits p53-mediated apoptosis and/or cell cycle arrest.Without adriamycin treatment,decreasing LSD1levels with lenti-virus-based short hairpin RNA (shRNA)did not affect the percentage of apoptotic cells (sub-G1population)in U2OS cells (Fig.2d,upper panels,and Supplementary Fig.2f).However,the apoptotic cell population increased (to 19%)in response to adriamycin treatment,and increased further (to 33.3%)when LSD1levels were decreased with shRNA (Fig.2d,lower panels).These results indicate that LSD1represses p53-mediated apoptosis.

LSD1was the first lysine demethylase to be characterized;it demethylates lysine 4or lysine 9on histone H3(refs 4–6).Because LSD1binds directly to p53(Fig.1c),we examined whether LSD1demethylates p53.To assay the two known p53methylation sites in vitro ,we developed a methylation–demethylation assay (Supple-mentary Fig.3a–c and Methods).We found that LSD1specifically removes Smyd2-mediated methylation but not methylation cata-lysed by Set9(Fig.3a).Because K370is the only amino acid residue modified by Smyd2in vitro (Supplementary Fig.3d),we conclude that LSD1demethylates p53at K370in vitro .

Smyd2is a monomethyl transferase 2,and LSD1is able to remove both monomethylation and dimethylation from histone substrates 4.To determine whether LSD1can remove dimethylation at p53K370,we used peptides representing monomethylated (K370me1),dime-thylated (K370me2)and trimethylated (K370me3)substrate,as well as monomethylated (K372me1)p53K372(Fig.3b).Reaction pro-ducts were subjected to dot-blot analysis to assess the different methylation sites and levels of methylation by using specific antibod-ies 1,2.We found that LSD1decreases the level of K370me1and K370me2but not that of K370me3(Fig.3b),supporting previous findings that LSD1demethylates only monomethylated and dimethylated lysine residues on histone substrates 4.Consistent with the observation on methylated GST–p53(Fig.3a)was our obser-vation that LSD1was not able to demethylate K372me1peptide (Fig.3b).These results show that LSD1specifically removes both K370me1and K370me2in vitro .

We next investigated whether LSD1affects K370or K372methyla-tion in vivo .The level of LSD1was decreased in U2OS cells by using siRNA,and then K370me1,K370me2and K372me1signals were determined by western blot analysis after immunoprecipitation with p53antibody.On adriamycin treatment,a decrease in LSD1level significantly increased that of K370me2,whereas that of

K370me1

Control shRNA LSD1shRNA

Adr. (48 h)

Mock

Sub-G1 (P < 0.05)G1 (P > 0.1)S (P > 0.1)

G2/M (P < 0.05)

3.1 ± 0.6 2.4 ± 0.755.3 ± 0.75

4.6 ± 2.319.3 ± 0.120.9 ± 1.322.7 ± 0.122.6 ± 1.718.9 ±

5.633.3 ± 2.519.6 ± 3.61

6.1 ± 3.323.6 ± 1.921.3 ± 0.338.4 ± 3.9

29.8 ± 1.1

Sub-G1 (P > 0.1)G1 (P > 0.1)S (P > 0.1)G2/M (P > 0.1)

P e r c e n t a g e o f i n p u t (%)c

Adr. (h)

p21p21mdm2R e l a t i v e m R N A l e v e l

R e l a t i v e m R N A l e v e l

Adriamycin

Figure 2|LSD1represses the activity of p53.a ,Quantitative real-time PCR to assess the relative mRNA levels of p21and mdm2in U2OS cells

transfected with control (grey columns)or LSD1(hatched columns)siRNA followed by 24h of mock treatment or treatment with adriamycin.

b ,Quantitative real-time PCR was used to measure the relative mRNA levels of p21in BJ and BJ-DNp53cells transfected with control (grey columns)or LSD1(hatched columns)siRNA followed by treatment with adriamycin (Adr.)for 0,2,4or 8h.

c ,ChIP assay to measure p53boun

d to its respons

e element (53RE)and to a control site (non-RE)within the p21promoter in U2OS cells transfected with control (grey columns)or LSD1(hatched columns)siRNA followed by treatment with adriamycin for 8h.d ,Flow cytometry analysis to assess the effect o

f LSD1on the percentage of apoptotic (that is,sub-G1)U2OS cells.Results are shown as means 6

s.d.BSA +–+–Set9Smyd2K 370m e 1

K 370m e 2

K 370m e 3

K 372m e 1

BSA LSD1

Mock b

R e l a t i v e l u c i f e r a s e a c t i v i t y (%)

LSD1 vector (ng)

C o n t r o l L S

D 1

C o n t r o l

L S D 1

Mock Adriamycin p53K370me2p53

p53K370me1

2.9

5.5

LSD1

LSD1(K661A)

R e l a t i v e l u c i f e r a s e a c t i v i t y (f o l d c h a n g e )

––+–––

–

Figure 3|LSD1demethylates p53at K370.a ,Top:fluorography,showing demethylation of p53by LSD1.Bottom:colloidal staining,showing

loadings.b ,Dot-blot analysis,showing demethylation of p53peptides by LSD1.c ,Western blot analysis of DO1immunoprecipitation in U2OS cells transfected with control or LSD1siRNA followed by treatment with

adriamycin for 8h.Numbers below the western blots show the ratio of the signal with LSD1knockdown to control knockdown,then to p53levels.d ,e ,Dual luciferase assay in H1299cells with a reporter bearing p21promoter to show that LSD1-mediated repression is dependent on its

demethylation activity (d )and K370in p53(e ).In e :grey columns,wild-type p53;stippled columns,p53(K370R).f ,ChIP assay to detect p53K370me2and p53K382ac recruitment to p21promoter in U2OS cells transfected with control (grey columns)or LSD1(hatched columns)siRNA followed by treatment with adriamycin (Adr.)for 8h.Top:absolute percentage of input.Bottom:ratio of modification ChIP to total p53ChIP.Results in d –f are shown as means 6s.d.

106

was affected only slightly(Fig.3c).Therefore,although LSD1is able to remove both K370me1and K370me2in vitro,it seems to demethylate K370me2preferentially in vivo.Other histone demethylases also exhibit a preference for specific methylation levels9,10.The underlying mechanism for these preferences is unclear but may involve other post-translational modifications on the sub-strate or demethylase-associated proteins.Under these assay condi-tions,endogenous K370me3and K372me1signals are not detected, even after long exposures in western blot analysis(data not shown). We note that Smyd2is a monomethyl transferase2;our results there-fore indicate that K370me2is performed by a distinct,currently unidentified methyltransferase.Taken together,our results reveal that p53K370me2is demethylated by LSD1.

To assess whether the demethylation activity of LSD1is required for its repression of the transcriptional activity of p53,we ectopically expressed LSD1and LSD1(K661A),a demethylation-defective mutant5,together with p53in H1299cells.A luciferase reporter bearing the p21promoter was used to assess transcriptional activa-tion driven by p53(Fig.3d).We observed that LSD1but not LSD1(K661A)decreases the activity of p53(Fig.3d).LSD1and LSD1(K661A)interact with p53comparably(Supplementary Fig. 3e),suggesting that LSD1represses the activity of p53in a demethy-lation-dependent manner.To test whether LSD1represses the activity of p53in a K370-dependent manner,we co-transfected H1299cells with vectors expressing LSD1along with either p53or p53(K370R).We found that LSD1decreased luciferase activity mediated by wild-type p53but not by p53(K370R)(Fig.3e),dem-onstrating that LSD1represses the activity of p53through K370. We determined whether p53dimethylated at K370is present at the p21promoter in U2OS cells by ChIP assay with DO1(unmodi-fied p53),p53K370me2and p53K382acetylation(K382ac)anti-bodies(Fig.3f).The K382ac antibody serves as a positive control because it detects a well-characterized activating modification of p53during DNA damage11,12.Treatment with adriamycin results in increased K370me2and K382ac ChIP signals(Fig.3f,grey bars), suggesting that K370me2is an activating modification for p53. Decreasing the level of LSD1by siRNA results in a strongly increased K370me2signal,whereas the K382ac signal is enhanced only slightly (Fig.3f,upper panel).The K370me2level increases even after nor-malization to the total p53level(Fig.3f,lower panel),showing that higher K370me2is due not only to elevated p53levels at the promoter caused by treatment with LSD1siRNA(Fig.2c).Thus,p53K370me2 increases at the p21promoter after DNA damage and this recruit-ment is regulated by LSD1.

These results suggest that K370me2may have a distinct role in regulating p53in comparison with repression-linked Smyd2-mediated K370me1.Recent studies show that specific lysine methy-lation states in histones serve as recognition sites for specific binding proteins13–17.We investigated binding proteins that recognize specific methylation states at K370by screening a GST protein domain microarray18(Fig.4a and Supplementary Fig.4).We found that the tandem Tudor domains of53BP1preferentially recognize K370me2peptide,compared with K370me0,K370me1or K370me3 (Fig.4a).We used peptide pull-down to confirm that53BP1binds to the K370me2peptide more strongly than to K370me1or K370me3 peptide,and that there is no binding to the K370me0peptide (Supplementary Fig.5a).These results are similar to recent observa-tions in vitro that the tandem Tudor domains of53BP1preferentially bind to histone dimethylated at H3K79and H4K20,and bind more weakly to monomethylated H3K79and H4K20(refs18–20).

We next examined whether53BP1associates with dimethylated p53K370more strongly than with monomethylated p53K370in vivo. We performed a Flag immunoprecipitation assay to pull down Flag–53BP1-associated proteins followed by western blot analysis to detect p53K370me1and K370me2(Fig.4b).Because the p53K370me1sig-nal is weaker than that of K370me2,Smyd2was coexpressed with p53 to provide equivalent K370me1input.Under the same loading conditions of input signals,we detected more K370me2signal than K370me1in the Flag–53BP1immunoprecipitation eluates(Fig.4b). These results in vivo support the hypothesis that53BP1binds to p53K370me2more strongly than to p53K370me1.

Physical interaction between p53and53BP1was discovered in a yeast two-hybrid analysis21,and in vivo interaction between p53and53BP1has recently been reported22,23.We are able repro-ducibly to detect interaction between endogenous p53and53BP1 in vivo(Supplementary Fig.5b).The specificity of this

interaction GST proteins K370me0 K370me1 K370me2 K370me3 a

p53

β-actin

LSD1

p53

F-53BP1

Flag IP

Input

F-53BP1

LSD1 shRNA

d 293

F-p53

HA–53BP1

F-p53

HA–53BP1

F-p53

F-p53(K370A)

F-p53(K370R)

HA–53BP1

+ + + +

– – + +

– + – +

– – – +

– – + –

– + – –

Flag IP

Input

c H1299

LSD1

53BP1

p53

mdm2

p21

β-actin

LSD1 siRNA

–

1 2 3 4 5 6 7 8 9 10 11 12

Adr. (8 h)

Control

shRNA

53BP1

shRNA no. 1

53BP1

shRNA no. 2

e MCF7

PUMA

Mono-

methylation

Di-

methylation

Activating

b 293

2% input

+ +

– –

+ +

– –

+ +

– –

Flag IP

F-53BP1

K370me1

K370me2

10% input

0.5% input

Flag IP

p53

53BP1

Repressing

Figure4|LSD1represses the activity of p53through53BP1.a,GST protein domain array to identify binding proteins for p53K370me0,

p53K370me1,p53K370me2and p53K370me3.The K370me2-dependent interaction with tandem Tudor domains of53BP1is circled.Part of the protein array(C3of Tudor domain section)is shown.For the entire array, see Supplementary Fig.4.b,Flag immunoprecipitation(IP)to assay the relative binding of53BP1to K370me1and K370me2in vivo.c,Flag IP to determine the requirement of K370for the interaction between53BP1and p53.d,Flag IP to study the effect of LSD1knockdown on the53BP1–p53 interaction.e,Western blot analysis to test whether LSD1-mediated repression is53BP1-dependent.Adr.,adriamycin.f,Schematic model for the dynamic regulation of K370methylation.

NATURE|Vol449|6September2007LETTERS

107

was analysed in several approaches.First,we generated53BP1bear-ing substitution mutations in conserved residues in the Tudor domains previously shown to be required for binding to histone H3K79me2and H4K20me2(Y1502Q or D1521R)19,20.These substi-tutions in53BP1strongly decreased co-immunoprecipitation with p53(Supplementary Fig.5c).Second,p53substituted at K370 (K370A or K370R)showed greatly decreased interaction with 53BP1(Fig.4c).Last,decreasing the level of LSD1with shRNA increased the interaction between p53and53BP1(Fig.4d),indi-cating that LSD1represses the activity of p53through decreasing the interaction with53BP1.

53BP1has been functionally linked to p53as a potential coactiva-tor21,24,25.We directly assayed whether53BP1is required for trans-criptional activation by p53.53BP1levels in MCF7cells were stably reduced by lentivirus-based shRNAs(Fig.4e).Two different shRNAs targeting53BP1for knockdown resulted in decreased protein levels of p21,mdm2and PUMA(p53upregulated modulator of apoptosis) (Fig.4e,compare lane2with lane6or10)and messenger RNA (Supplementary Fig.5d).As described above(Fig.2and Supplemen-tary Fig.2),treatment with LSD1siRNA increased the expression of p21,mdm2and PUMA in MCF7cells(Fig.4e,compare lanes2and4). In contrast,gene expression did not increase in the LSD1knockdown in cells also carrying53BP1shRNAs(Fig.4e,compare lanes2and4 with lanes6and8or10and12).Together,these results strongly indicate that LSD1regulates the activity of p53through its coactiva-tor53BP1.

Our results demonstrate that LSD1demethylates p53at K370and displays a strong preference in vivo for demethylating p53K370me2. In addition,although we did not detect K372me1as a substrate, previous observation that K372me1is transient during p53activa-tion1indicates that other demethylases might target methylated p53. It is well established in histones that various methylation levels elicit specific biological outcomes,through recognition by different binding proteins13–15.In this study we found that K370me2,in con-trast to K370me1,has an activating role in p53regulation through providing an interaction surface for the binding of53BP1.These results add another layer of complexity to the methylation-mediated regulation of p53.Methylation,similarly to other post-translational modifications of p53,seems to fine-tune p53function26.

We propose a dynamic model for the regulation of p53through lysine methylation(Fig.4f).During gene activation,p53,by means of K370me2,binds to53BP1.In contrast,during gene repression,LSD1 prevents the accumulation of K370me2by demethylating the site, thereby disallowing the binding of53BP1to p53.The demethylation activity of LSD1thus maintains p53in an inactive state,to prevent binding to DNA.53BP1and p53synergize to inhibit tumorigenesis27; our results suggest a mechanism contributing to their cooperative suppression of cancer.

METHODS SUMMARY

Co-immunoprecipitation and GST pull-down assays were used to detect pro-tein–protein interaction.Transient transfection of siRNA and lentivirus trans-duction of shRNA were used to decrease the levels of specific proteins.Flow cytometry was used to assess the apoptosis and cell cycle arrest caused by p53. ChIP assay was performed to detect the recruitment of p53and modified p53to DNA.We used real-time PCR to measure the mRNA level and immuno-precipitated DNA in a ChIP assay.An in vitro demethylation assay was used to determine whether LSD1demethylates p53.A protein domain microarray was used to identify proteins that bind to p53K370me1,p53K370me2or p53K370me3.

Full Methods and any associated references are available in the online version of the paper at https://www.wendangku.net/doc/7f1598654.html,/nature.

Received5April;accepted9July2007.

1.Chuikov,S.et al.Regulation of p53activity through lysine methylation.Nature432,

353–360(2004).2.Huang,J.et al.Repression of p53activity by Smyd2-mediated methylation.Nature

444,629–632(2006).

3.Hakimi,M.A.,Dong,Y.,Lane,W.S.,Speicher,D.W.&Shiekhattar,R.A candidate

X-linked mental retardation gene is a component of a new family of histone

deacetylase-containing complexes.J.Biol.Chem.278,7234–7239(2003).

4.Shi,Y.et al.Histone demethylation mediated by the nuclear amine oxidase

homolog LSD1.Cell119,941–953(2004).

5.Lee,M.G.,Wynder,C.,Cooch,N.&Shiekhattar,R.An essential role for CoREST in

nucleosomal histone3lysine4demethylation.Nature437,432–435(2005). 6.Metzger,E.et al.LSD1demethylates repressive histone marks to promote

androgen-receptor-dependent transcription.Nature437,436–439(2005).

7.Vaziri,H.&Benchimol,S.Reconstitution of telomerase activity in normal human

cells leads to elongation of telomeres and extended replicative life span.Curr.Biol.

8,279–282(1998).

8.Shaulian,E.,Zauberman,A.,Ginsberg,D.&Oren,M.Identification of a minimal

transforming domain of p53:negative dominance through abrogation of

sequence-specific DNA binding.Mol.Cell.Biol.12,5581–5592(1992).

9.Tsukada,Y.et al.Histone demethylation by a family of JmjC domain-containing

proteins.Nature439,811–816(2006).

10.Yamane,K.et al.JHDM2A,a JmjC-containing H3K9demethylase,facilitates

transcription activation by androgen receptor.Cell125,483–495(2006).

11.Barlev,N.A.et al.Acetylation of p53activates transcription through recruitment

of coactivators/histone acetyltransferases.Mol.Cell8,1243–1254(2001). 12.Gu,W.&Roeder,R.G.Activation of p53sequence-specific DNA binding by

acetylation of the p53C-terminal domain.Cell90,595–606(1997).

13.Wysocka,J.et al.WDR5associates with histone H3methylated at K4and is

essential for H3K4methylation and vertebrate development.Cell121,859–872 (2005).

14.Wysocka,J.et al.A PHD finger of NURF couples histone H3lysine4

trimethylation with chromatin remodelling.Nature442,86–90(2006).

15.Shi,X.et al.ING2PHD domain links histone H3lysine4methylation to active gene

repression.Nature442,96–99(2006).

16.Pena,P.V.et al.Molecular mechanism of histone H3K4me3recognition by plant

homeodomain of ING2.Nature442,100–103(2006).

17.Li,H.et al.Molecular basis for site-specific read-out of histone H3K4me3by the

BPTF PHD finger of NURF.Nature442,91–95(2006).

18.Kim,J.et al.Tudor,MBT and chromo domains gauge the degree of lysine

methylation.EMBO Rep.7,397–403(2006).

19.Huyen,Y.et al.Methylated lysine79of histone H3targets53BP1to DNA double-

strand breaks.Nature432,406–411(2004).

20.Botuyan,M.V.et al.Structural basis for the methylation state-specific recognition

of histone H4–K20by53BP1and Crb2in DNA repair.Cell127,1361–1373(2006).

21.Iwabuchi,K.,Bartel,P.L.,Li,B.,Marraccino,R.&Fields,S.Two cellular proteins

that bind to wild-type but not mutant p53.Proc.Natl https://www.wendangku.net/doc/7f1598654.html,A91,

6098–6102(1994).

22.Sengupta,S.et al.Functional interaction between BLM helicase and53BP1in a

Chk1-mediated pathway during S-phase arrest.J.Cell Biol.166,801–813(2004).

23.Ward,I.et al.The tandem BRCT domain of53BP1is not required for its repair

function.J.Biol.Chem.281,38472–38477(2006).

24.Iwabuchi,K.et al.Stimulation of p53-mediated transcriptional activation by the

p53-binding proteins,53BP1and53BP2.J.Biol.Chem.273,26061–26068(1998).

25.Brummelkamp,T.R.et al.An shRNA barcode screen provides insight into cancer

cell vulnerability to MDM2inhibitors.Nature Chem.Biol.2,202–206(2006). 26.Toledo,F.&Wahl,G.M.Regulating the p53pathway:in vitro hypotheses,in vivo

veritas.Nature Rev.Cancer6,909–923(2006).

27.Morales,J.C.et al.53BP1and p53synergize to suppress genomic instability and

lymphomagenesis.Proc.Natl https://www.wendangku.net/doc/7f1598654.html,A103,3310–3315(2006). Supplementary Information is linked to the online version of the paper at https://www.wendangku.net/doc/7f1598654.html,/nature.

Acknowledgements We thank N.Barlev for the Set9expression vector;D.Reinberg for the p53K372me1antibody;S.Benchimol for the BJ and BJ-DNp53cell lines; T.Halazonetis for53BP1plasmid;R.Schule for p21luciferase vector;and members of the T.J.and https://www.wendangku.net/doc/7f1598654.html,boratories for discussions.This project is funded,in part,by a AACR-Pennsylvania Department of Health Fellows grant and Leukemia and Lymphoma Society Special Fellow grant(J.H.).M.T.B.is supported by a Welch Foundation grant.Research in the laboratory of T.J.is supported by the IMP through Boehringer Ingelheim and by grants from the European Union and the Austrian GEN-AU initiative,which is financed by the Austrian Ministry of Education,Science and Culture.Research support to S.L.B.was provided by a grant from the National Cancer Institute at NIH and the Commonwealth Universal Research Enhancement Program of the Pennsylvania Department of Health. Author Contributions J.H.,R.S.,A.B.E.,M.G.L.,J.A.D.,M.R.and S.O.performed the experimental work;R.S.,M.T.B.,T.J.and S.L.B.were responsible for project planning and data analysis.

Author Information Reprints and permissions information is available at

https://www.wendangku.net/doc/7f1598654.html,/reprints.The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to S.L.B. (berger@https://www.wendangku.net/doc/7f1598654.html,).

LETTERS NATURE|Vol449|6September2007 108

METHODS

Antibodies.Anti-Smyd2(amino terminus)and anti-p53K370me1,me2and me3antibodies were generated and described previously2.Anti-p53K370me2 antibody was antigen-purified from crude serum.Other antibodies were as follows:anti-p53K372me1(gift from D.Reinberg),anti-LSD1(Bethyl),anti-p53(DO1;Santa Cruz),anti-p53(FL393;Santa Cruz),anti-haemagglutinin (Roche),anti-GST(Upstate),anti-53BP1(monoclonal;Upstate),anti-53BP1 (polyclonal;Bethyl),anti-b-actin(Sigma),anti-Puma N terminus(Sigma), anti-Flag(Sigma)and mouse and rabbit IgG(Santa Cruz).

Co-immunoprecipitation assay.In the standard co-immunoprecipitation assay,cells were lysed in NET0.1%buffer(50mM Tris-HCl pH7.4,150mM NaCl,5mM EDTA,0.1%Nonidet P40,freshly added1mM phenylmethyl-sulphonyl fluoride,protease inhibitors)and sonicated with a Bioruptor (Diagenode)for5min,with30-s‘on’and1-min‘off’cycles.After removal of cell debris by centrifugation,2m g of antibody was added to1mg of clarified whole cell extract(WCE)and incubated overnight at4u C.The next day,40m l of Protein A-agarose beads(Upstate)was added and incubated for a further2h. Beads then were subjected to three washes with NET0.l%buffer and boiled with 13SDS loading buffer.A modified co-immunoprecipitation assay was used to detect interaction between p53and53BP1,in which nuclei were prepared in accordance with the protocol from a Nuclear Extraction Kit(catalogue no. AY2002;Panomics).Nuclear extract(NE)was prepared by lysing the nuclei with NET0.1%supplemented with2mM sodium orthovanadate,50mM NaF and 50mM b-glycerolphosphate as described above for WCE.Typically,1mg of NE (3–5m g m l21)was used for each immunoprecipitation with2m g of specific anti-body.For Flag immunoprecipitation,to assess the binding of Flag–53BP1to K370me1and K370me2in vivo,Smyd2and p53were coexpressed with or with-out Flag–53BP1.The same amounts of input and Flag immunoprecipitation eluates were loaded in western blot analysis for the detection of K370me1and K370me2.

GST pull-down assay.Bacterial lysate for GST–p53or GST was pre-bound to 10m l of GST beads(Pharmacia)at25u C.Beads were then preblocked with in vitro pull-down buffer(20mM HEPES pH7.9,150mM KCl,1mM EDTA, 0.1%Nonidet P40,10%glycerol,1m g m l21BSA and freshly added1mM dithiothreitol and1mM phenylmethylsulphonyl fluoride)at4u C for1h. Recombinant Flag–LSD1(2m g)purified from baculovirus-infected Spodoptera frugiperda(Sf9)cells was added and incubated for a further3h.Beads were washed three times with pull-down buffer,then boiled with20m l of13SDS loading buffer.Anti-Flag antibody was used to detect Flag–LSD1.

RNA interference.siRNAs were purchased from Dharmacon,and100nM siRNA was transfected into cells with DharmaFECT1.siRNA sequences for luciferase control were as described previously2.LSD1siRNA sequences were: 59-UGAAUUAGCUGAAACACAAUU-39(sense sequence)and59-pUUGUG-UUUCAGCUAAUUCAUU-39(antisense sequence).Lentivirus-based shRNA vectors for luciferase control and LSD1were generated with the BLOCK-it U6 RNAi Expression System(Invitrogen).shRNA vectors for53BP1#1and53BP1#2 were purchased from Sigma.The sequences of the short hairpins were:luciferase control,59-CACCTAAGGCTATGAAGAGATACCGAAGTATCTCTTCATAG-CCTTA-39(top strand)and59-AAAATAAGGCTATGAAGAGATACTTCGG-TATCTCTTCATAGCCTTA-39(bottom strand);LSD1,59-CACCGCACCTTA-TAACAGTGATACTCGAAAGTATCACTGTTATAAGGTGC-39(top strand) and59-AAAAGCACCTTATAACAGTGATACTTTCGAGTATCACTGTTAT-AAGGTGC-39(bottom strand);53BP1#1,59-CCGGGATACTTGGTCTTA-CTGGTTTCTCGAGAAACCAGTAAGACCAAGTATCTTTTT-39(top strand)and59-AAAAAGATACTTGGTCTTACTGGTTTCTCGAGAAACCAGTAAGA-CCAAGTATCCCGG-39(bottom strand);53BP1#2,59-CCGGCCAGTGTGA-TTAGTATTGATTCTCGAGAATCAATACTAATCACACTGGTTTTT-39(top strand)and59-AAAAACCAGTGTGATTAGTATTGATTCTCGAGAATCAAT-ACTAATCACACTGGCCGG-39(bottom strand).

We found that lentivirus-based knockdown of LSD1by shRNA was not stable and the level of LSD1recovered two weeks after transduction(data not shown).To overcome this technical difficulty,we repeated the cell transduction two weeks after the first transduction.By this‘double transduction’procedure, we were able to maintain a low level of LSD1in U2OS,BJ and BJ-DNp53cells for a month.All the experiments were performed within a month of the first transduction.

Chromatin immunoprecipitation assay.In brief,cells were fixed with1%form-aldehyde and lysed with lysis buffer(50mM HEPES-KOH pH7.5,140mM NaCl,1mM EDTA,0.1%Triton X-100,0.1%sodium deoxycholate,with prote-ase inhibitors).The cell lysate was sonicated with a Bioruptor machine for a total of20min with30-s‘on’and1-min‘off’cycles,to shear the DNA to a final size of 200–500base pairs.After preclearing with Protein A or G beads(Upstate), antibody was added and incubated overnight at4u C.The next day,Protein A-agarose or Protein G-agarose beads were added and incubated for a further 1–2h.The complex was washed twice with lysis buffer,once with high salt buffer (50mM HEPES-KOH pH7.5,500mM NaCl,1mM EDTA,0.1%Triton X-100, 0.1%sodium deoxycholate),twice with LiCl buffer(10mM Tris-HCl pH8.0, 0.25M LiCl,0.5%Nonidet P40,0.5%sodium deoxycholate,1mM EDTA)and once with TE buffer,followed by elution in TE buffer containing1%SDS.The crosslinks were reversed,the DNA was purified with a QIAquick8PCR puri-fication kit(Qiagen)and subjected to analysis by quantitative real-time PCR. In vitro demethylation assay.Recombinant Set9,GST–p53and LSD1were purified as described previously1,5,11.Recombinant Smyd2was purified from insect(Sf9)cells infected with baculovirus.Methylation assays were performed as described previously2.GST–p53(10m g)and Set9(5m g)or Smyd2(5m g)were used in a50-m l methylation reaction.After passage of the methylation reaction products through a Microspin column30(Bio-Rad),1m l of flowthrough was subjected to scintillation counting to measure the level of methylated GST–p53. Comparable scintillation counts within the flowthroughs were used in the demethylation assays with3–5m g of LSD1in demethylation buffer(50mM HEPES/NaOH pH8.0,25%glycerol)4,5,28.For peptide demethylation,100pmol of peptide and5m g of LSD1purified from bacteria were used in a50m l total volume.After reaction,products were subjected to dot-blot analysis with specific antibodies.

Dual luciferase assay.Dual luciferase assays were performed in accordance with the manufacturer’s protocol(Promega).In brief,H1299cells were seeded in a 24-well plate.At16h after transfection,cells were lysed with passive lysis buffer and lysates were subjected to dual luciferase analysis with a Wallac1420multi-label counter(PerkinElmer).A Renilla luciferase vector(pRL-CMV)was used as an internal control.

Protein domain microarray.Chromo,PhD,Tudor,SANT,MBT and PWWP domains from about100chromatin-associated proteins were fused to GST and spotted on a slide18.Biotin-labelled p53K370me0(unmethylated),K370me1, K370me2and K370me3peptides were individually hybridized to the protein domain array;binding was then detected with Cy5-Streptavidin18.

28.Forneris,F.,Binda,C.,Vanoni,M.A.,Battaglioli,E.&Mattevi,A.Human histone

demethylase LSD1reads the histone code.J.Biol.Chem.280,41360–41365(2005).

doi:10.1038/nature06092

英语阅读系列·有趣的字母

A biscuit can be a bear. 一块饼干可以是小熊。 A biscuit can be a butterfly. 一块饼干可以是蝴蝶。 But…If birds are nearby…但是…..如果小鸟恰好在旁边的话…… The biscuits can only be birds.饼干就只是小鸟啦。 Lesson 3 Cool Cat 酷猫卡里 Cary is a cool cat. 卡里是只酷酷的小猫。 Cary can cut carrots like this. 卡里能像这样切胡萝卜。 Cary can climb a coconut tree like this. 卡里能像这样爬树。 Cary can clean a crocodile like this. 卡里能像这样清理鳄鱼。 Cary can catch flies like this. 卡里能像这样抓苍蝇。 Can Cary color like this? 卡里能像这样涂色吗？ Yes! Cary is a cool cat. 是的！卡里是只酷酷的小猫。 Lesson 4 Dancing Dad 爸爸爱跳舞

一年级攀登英语一级A教学计划

一年级攀登英语一级A教学计划一年级攀登英语一级A教学计划 1、激发学生学习英语的兴趣,培养他们学习英语的积极态度,使他们初步建立学习英语的自信心。 2、培养学生具有一定的语感和良好的语音、语调及书写基础,以及良好的学习习惯。 3、培养学生的观察、记忆、思维、想象和创造能力。一年级攀登英语一级A教学计划 (20**学年度第一学期) 本学期我担任一年级4个班的攀登英语教学工作,现我对本学期教学工作做如下计划: 一、学生情况分析一年级的孩子的年龄多为6周岁,具有好奇、好活动、爱表现、善模仿等特点。他们的学习兴趣很浓,接受能力、模仿能力很强,学习习惯初步养成,因此在本学期应注重培养学生良好的学习习惯,训练学生的听说能力,调动他们的自主能动性、积极性,营造互帮互助,共同学习英语的语境。二、教学目标 1、激发学生学习英语的兴趣,培养他们学习英语的积极态度,使他们初步建立学习英语的自信心。 2、培养学生具有一定的语感和良好的语音、语调及书写基础,以及良好的学习习惯。

3、培养学生的观察、记忆、思维、想象和创造能力能在图片、手势、情境等非语言提示的帮助下,听懂清晰的话语和录音。三、教材分析本书共由五部分构成:本册教材学习内容包括:日常英语(Everyday English)、歌曲童谣(Songs and Chants).动画英语(Cartoon English)等 1、日常英语:与学生日常生活密切相关的情景对话,共12个话题,每个话题包括2-4句情景对话。 2、动画英语:以《迪士尼神奇英语》(上)VCD为基本学习材料。共6张VCD,12课,每课4个小节,由学生熟悉和喜欢的迪士尼动画片片段组合而成。 3、歌曲童谣:包括歌曲和童谣两大块,其中有精心制作的传统英文经典歌曲,也有攀登英语独创的童谣日常英语替换内容:以日常英语重点句型为主线,结合歌曲童谣和动画英语中高频出现的词汇进行的开放性交替。 5、节奏英语:将日常英语替换内容编成12段朗朗上口的韵文,并配以节奏明快的背景音乐形成的独特学习内容。四、教学措施 1、考虑到小学生好动爱玩的特点,以活动为课堂教学的主要形式,设计丰富多彩的教学活动,让学生在乐中学、学中用,从而保证学生英语学习的可持续性发展。 2、通过听、说、读、写、唱、游、演、画、做等形式,进行大量的语言操练和练习。

英语阅读系列·有趣的字母

攀登英语阅读系列·有趣的字母北京师范大学“认知神经科学与学习”国家重点实验室攀登英语项目组编著 Lesson1Frank the Rat 大老鼠弗兰克 Frank the rat is in a bag. 大老鼠弗兰克在袋子里呢。 Frank is in a hat. 他在圆顶帽子里呢。 Frank is in a pan. 他在平底锅里呢。 Frank is on an apple. 他在苹果上呢。 Frank is on a bat. 他在球棒上呢。 “Oh, no!” Frank is on a cat. “噢，糟了！”弗兰克落在了猫身上！ Lesson 2 The Biscuits 我们来做饼干 “I’m hungry!”“我饿了！” A biscuit…一块饼干可以是...... A biscuit can be a bus.一块饼干可以是公共汽车。 A biscuit canbe a bike. 和一块饼干可以是自行车。 A biscuit can be a boat. 一块饼干可以是小船。 A biscuit can be a banana. 一块饼干可以是香蕉。 A biscuit can be a bear. 一块饼干可以是小熊。 A biscuit can be a butterfly. 一块饼干可以是蝴蝶。 But…If birds are nearby…但是…..如果小鸟恰好在旁边的话…… The biscuits can only be birds.饼干就只是小鸟啦。 Lesson 3 Cool Cat 酷猫卡里 Cary is a cool cat. 卡里是只酷酷的小猫。 Cary can cut carrots like this. 卡里能像这样切胡萝卜。 Cary can climb a coconut tree like this. 卡里能像这样爬树。 Cary can clean a crocodile like this. 卡里能像这样清理鳄鱼。 Cary can catch flies like this.卡里能像这样抓苍蝇。 Can Cary color like this? 卡里能像这样涂色吗？ Yes! Cary is a cool cat. 是的！卡里是只酷酷的小猫。 Lesson 4 Dancing Dad 爸爸爱跳舞 Dad loves dancing! 爸爸爱跳舞！ Dad is dancing with the desk. 他和桌子一起跳。 Dad is dancing with the duck. 他和鸭子一起跳。 Dad is dancing with the door. 他和门一起跳。 Dad is dancing with the dog. 他和小狗一起跳。 Dad is dancing with the deer. 他和小鹿一起跳。 Where is Dad? 爸爸在哪儿呀？ Wow！哇！ What a dancing Dad! 真是个爱跳舞的爸爸！ Lesson 5 Red Ben 小本的红色世界 Ben likes red. 小本喜欢红色。 Ben paints the eggs red. 他把鸡蛋涂成红色。 Ben paints the eggplants red. 他把茄子涂成红色。 Ben paints the lemons red. 他把柠檬涂成红色。

(完整版)攀登英语-有趣的字母(中英文_纯文字_26篇全)

攀登英语——有趣的字母 A: Frank the rat 老鼠弗兰克 Frank the rat is in a bag. 大老鼠弗兰克在袋子里。 Frank is in a hat. 弗兰克在圆顶帽子里。 Frank is in a pan. 弗兰克在平底锅里。 Frank is on an apple. 弗兰克在苹果里。 Frank is on a bat. 弗兰克在球棒上。 “Oh/no!”F r ank is on a cat. “哦?不！”弗兰克落在了猫身上。 B：The Biscuits 饼干 “I’m h ungry!”“我饿了!” A biscuit, —块饼干 A biscuit can be a bus. —块饼干可以是公共汽车。 A biscuit can be a bike. 可以是自行车。 A biscuit can be a boat. 可以是船。 A biscuit can be a banana. 可以是香蕉。 A biscuit can be a bear.可以是小熊。 A biscuit can be a butterfly.可以是蝴蝶。 But，if birds are nearby,但是，如果鸟儿靠近 The biscuits can only be birds.饼干只能是小鸟啦。 C： Cool Cat酷猫卡里 Cary is a cool cat.卡里是只酷酷的小猫。 Cary can cut carrots like this.卡里能像这样切胡萝卜。 Cary can climb a coconut tree like this. 卡里能像这样爬树。Cary can clean a crocodile like this.卡里能像这样淸洁鳄鱼。Cary can catch flies like this.卡里能像这样抓苍蝇。 Can Cary color like this? 卡里能像这样涂色吗？ Yes!是的 Cary is a cool cat.卡里是只酷酷的小猫。 D: Dancing Dad爸爸爱跳舞 Dad loves dancing!爸爸爱跳舞！ Dad is dancing with the desk.他和桌子一起跳。

攀登英语-有趣的字母(中英文_纯文字_26篇全)教案资料

攀登英语-有趣的字母(中英文_纯文字_26 篇全)

英语阅读系列·有趣的字母

英语阅读系列·有趣的字母攀登英语阅读系列?有趣的字母攀登英语阅读系列·有趣的字母北京师范大学“认知神经科学与学习”国家重点实验室攀登英语项目组编著 Lesson1 Frank the Rat 大老鼠弗兰克 Frank the rat is in a bag. 大老鼠弗兰克在袋子里呢。

Frank is in a hat. 他在圆顶帽子里呢。Frank is in a pan. 他在平底锅里呢。Frank is on an apple. 他在苹果上呢。Frank is on a bat. 他在球棒上呢。“Oh, no!” Frank is on a cat. “噢，糟了！”弗兰克落在了猫身上！ Lesson 2 The Biscuits 我们来做饼干 “I'm hungry!”“我饿了！” A biscuit…一块饼干可以是...... A biscuit can be a bus. 一块饼干可以是公共汽车。 A biscuit can be a bike. 和一块饼干可以是自行车。 A biscuit can be a boat. 一块饼干可以是小船。 A biscuit can be a banana. 一块饼干可以是香蕉。．攀登英语阅读系列?有趣的字母 A biscuit can be a bear. 一块饼干可以是小熊。 A biscuit can be a butterfly. 一

块饼干可以是蝴蝶。 But…If birds are nearby…但是…..如果小鸟恰好在旁边的话…… The biscuits can only be birds.饼干就只是小鸟啦。 Lesson 3 Cool Cat 酷猫卡里 Cary is a cool cat. 卡里是只酷酷的小猫。 Cary can cut carrots like this. 卡里能像这样切胡萝卜。 Cary can climb a coconut tree like this. 卡里能像这样爬树。 Cary can clean a crocodile like this. 卡里能像这样清理鳄鱼。 Cary can catch flies like this. 卡里能像这样抓苍蝇。 Can Cary color like this? 卡里能像这样涂色吗？ Yes! Cary is a cool cat. 是的！卡里是只酷酷的小猫。爸爸爱跳舞Dancing Dad Lesson 4 攀登英语阅读系列?有趣的字母

攀登英语-有趣的字母(中英文_纯文字_26篇全)

攀登英语——有趣的字母 A:Franktherat老鼠弗兰克 Franktheratisinabag.大老鼠弗兰克在袋子里。Frankisinahat.弗兰克在圆顶帽子里。 Frankisinapan.弗兰克在平底锅里。 Frankisonanapple.弗兰克在苹果里。 Frankisonabat.弗兰克在球棒上。 “Oh/no!”F rankisonacat.“哦?不！”弗兰克落在了猫身上。 B：TheBiscuits饼干 “I’mhungry!”“我饿了!” Abiscuit,—块饼干 Abiscuitcanbeabus.—块饼干可以是公共汽车。Abiscuitcanbeabike.可以是自行车。 Abiscuitcanbeaboat.可以是船。 Abiscuitcanbeabanana.可以是香蕉。 Abiscuitcanbeabear.可以是小熊。Abiscuitcanbeabutterfly.可以是蝴蝶。 But，ifbirdsarenearby,但是，如果鸟儿靠近Thebiscuitscanonlybebirds.饼干只能是小鸟啦。 C：CoolCat酷猫卡里 Caryisacoolcat.卡里是只酷酷的小猫。Carycancutcarrotslikethis.卡里能像这样切胡萝卜。Carycanclimbacoconuttreelikethis.卡里能像这样爬树。Carycancleanacrocodilelikethis.卡里能像这样淸洁鳄鱼。Carycancatchflieslikethis.卡里能像这样抓苍蝇。CanCarycolorlikethis?卡里能像这样涂色吗？ Yes!是的 Caryisacoolcat.卡里是只酷酷的小猫。 D:DancingDad爸爸爱跳舞 Dadlovesdancing!爸爸爱跳舞！Dadisdancingwiththedesk.他和桌子一起跳。