cdkn1a - journal of cell science › content › joces › early › 2014 › 09 › 01 ›...

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RESEARCH ARTICLE

CRL4B interacts with and coordinates the SIN3A-HDAC complexto repress CDKN1A and drive cell cycle progression

Qinghong Ji, Huili Hu, Fan Yang, Jupeng Yuan, Yang Yang, Liangqian Jiang, Yanyan Qian, Baichun Jiang,Yongxin Zou, Yan Wang, Changshun Shao and Yaoqin Gong*

ABSTRACT

CUL4B, a scaffold protein that assembles the CRL4B ubiquitin

ligase complex, participates in the regulation of a broad spectrum of

biological processes. Here, we demonstrate a crucial role of CUL4B

in driving cell cycle progression. We show that loss of CUL4B

results in a significant reduction in cell proliferation and causes G1

cell cycle arrest, accompanied by the upregulation of the cyclin-

dependent kinase (CDK) inhibitors (CKIs) p21 and p57 (encoded by

CDKN1A and CDKN1C, respectively). Strikingly, CUL4B was found

to negatively regulate the function of p21 through transcriptional

repression, but not through proteolysis. Furthermore, we

demonstrate that CRL4B and SIN3A-HDAC complexes interact

with each other and co-occupy the CDKN1A and CDKN1C

promoters. Lack of CUL4B led to a decreased retention of SIN3A-

HDAC components and increased levels of acetylated H3 and H4.

Interestingly, the ubiquitylation function of CRL4B is not required for

the stable retention of SIN3A-HDAC on the promoters of target

genes. Thus, in addition to directly contributing to epigenetic

silencing by catalyzing H2AK119 monoubiquitylation, CRL4B also

facilitates the deacetylation function of SIN3A-HDAC. Our findings

reveal a coordinated action between CRL4B and SIN3A-HDAC

complexes in transcriptional repression.

KEY WORDS: CRL4B, CDKN1A, SIN3A-HDAC, Cell cycle

progression

INTRODUCTIONHistones are subject to a variety of post-translational

modifications that affect chromatin configuration, transcriptionand the DNA damage response (Berger, 2007; Iizuka and Smith,

2003; van Attikum and Gasser, 2009). These modifications

include phosphorylation, methylation, acetylation, ubiquitylationand sumoylation (Peterson and Laniel, 2004; Tan et al., 2011).

Acetylation of histones, occurring mostly at lysine residues on the

N-terminal tails of histones H3 and H4, is linked to the opening ofchromatin and activation of gene expression, either through

altering the affinity of histones for DNA or by creating binding

sites for the proteins that regulate chromatin accessibility. Theantagonistic activities of two types of enzymes, histone

acetyltransferases (HATs) and histone deacetylases (HDACs),

control the reversible acetylation state (Kuo and Allis, 1998;

Shahbazian and Grunstein, 2007). HATs catalyze the acetylation

of histones and other proteins, whereas HDACs catalyze the

removal of the acetyl moieties from acetylated proteins. To date,

18 mammalian HDAC isoforms have been characterized and are

classified into class I, class II, class III and class IV (de Ruijter

et al., 2003). Among them, HDAC1 and HDAC2, members of

class I, represent two of the best-characterized HDACs to date.

They function in a number of deacetylase complexes – including

SIN3A-HDAC, NuRD-HDAC, the BCH10-containing complex

and the CoREST-HDAC complex – and they are generally

associated with transcriptional repression (Hayakawa and

Nakayama, 2011; Laherty et al., 1997; Wang et al., 2009b).

Cells lacking both HDAC1 and HDAC2 show G1 cell cycle arrest

accompanied by upregulation of the cyclin-dependent kinase

(CDK) inhibitors (CKIs) p21 and p57 (encoded by CDKN1A and

CDKN1C, respectively) (Gui et al., 2004; Wilting et al., 2010;

Yamaguchi et al., 2010; Zupkovitz et al., 2010).

Cullin 4 (CUL4) acts as the scaffold of the E3 ligase complex

CRL4. By interaction (at its C-terminal end) with a small RING

finger protein [either ROC1 (also known as RBX1) or ROC2

(also known as RBX2)], CUL4 recruits the E2 ubiquitin-

conjugating enzyme (E2) charged with a ubiquitin ready for

transfer to the substrate (Jackson and Xiong, 2009). The N-

terminal domain of CUL4 binds to the substrate adaptor DNA

damage binding protein 1 (DDB1), which recruits various

substrate-recognition proteins (DCAF proteins), resulting in a

large family of distinct CRL4 E3 ubiquitin ligase complexes

(Higa et al., 2006; Lee and Zhou, 2007; Scott et al., 2006). CRL4

targets different substrates for proteasomal degradation or for

protein modification, and thus regulates a broad variety of

physiologically and developmentally controlled processes (Higa

and Zhang, 2007). Although earlier studies focused on the

redundant function and common substrates of members of the

CUL4 family, CUL4B has been recently reported to function

distinctly from CUL4A in transcriptional repression, neuronal

gene regulation, response to reactive oxygen species (ROS) and

microRNA regulation (Hu et al., 2012; Li et al., 2011; Nakagawa

and Xiong, 2011; Zou et al., 2013). Although CUL4A has been

proved to regulate the cell cycle by targeting CDT1 and CKIs

such as p21 and p27 (encoded by CDKN1B) for proteolysis in

cultured cells, it seems to be dispensable for embryonic

development (Abbas et al., 2008; Hu et al., 2004; Li et al.,

2006). However, studies from three independent groups have

demonstrated that Cul4b-null embryos are impaired in

development (Chen et al., 2012; Jiang et al., 2012; Liu et al.,

2012), yet the underlying mechanisms still need to be elucidated.

Moreover, recent studies have established CRL4, especially

CRL4B, as important epigenetic regulators. CUL4B ablation

could block the degradation of WDR5, a core subunit of the

Key Laboratory of Experimental Teratology, Ministry of Education, Institute ofMolecular Medicine and Genetics, Shandong University School of Medicine,Jinan, 250012, China.

*Author for correspondence ([email protected])

Received 1 April 2014; Accepted 16 August 2014

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 4679–4691 doi:10.1242/jcs.154245

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histone H3 lysine 4 (H3K4) methyltransferase complex, and thusincrease H3K4 trimethylation (H3K4me3) on some neuronal gene

promoters, leading to their upregulation (Nakagawa and Xiong,2011). Recently, we have shown that CRL4B functions as atranscriptional co-repressor of tumor suppressors bymonoubiquitylating H2AK119 and coordinating with either the

PRC2 complex or with DNA methyltransferase, HP1 andSUV39H1 to regulate histone methylation or DNA methylation(Hu et al., 2012; Yang et al., 2013). Here, we show that CUL4B

depletion can inhibit cell proliferation owing to the upregulationof Cdkn1a and Cdkn1c. Importantly, we demonstrate that theCRL4B complex interacts and coordinates with SIN3A-HDAC to

exert its repressive effect.

RESULTSLack of CUL4B inhibits cell proliferation in MEFsOur previous results have shown that reduced cell proliferationand increased apoptosis can lead to developmental arrest inCul4b-null embryos (Jiang et al., 2012). We also noted that

CUL4B-deficient cells are severely selected against in vivo inhuman and mouse heterozygotes (Jiang et al., 2012; Zou et al.,2007). To further understand the mechanism by which CUL4B

regulates cell survival during embryonic development, wegenerated viable Cul4b-null mice by crossing Cul4b-floxedmice to Sox2-cre transgenic mice, and we then prepared wild-

type and Cul4b-null murine embryonic fibroblasts (MEFs), whichare designated as Cul4bf/Y and Cul4b-/Y, respectively. As shown inFig. 1A, no CUL4B protein was detected in Cul4b-/Y MEFs,

whereas CUL4A was detected at comparable levels betweenCul4bf/Y and Cul4b-/Y MEFs. To determine whether the two typesof MEFs have differential proliferation, we seeded equal numbersof Cul4bf/Y and Cul4b-/Y MEFs onto replicate plates and

monitored the fractions of CUL4B-positive cells at differentpassages by immunofluorescent staining of CUL4B. We observedthat the percentages of CUL4B-positive cells were significantly

increased with increased passage number (Fig. 1B), indicatingthat Cul4b-/Y MEFs were selected against. The proliferation ofCul4bf/Y and Cul4b-/Y MEFs was further evaluated by MTT and

EdU cell proliferation assays. The proliferation of Cul4b-/Y MEFswas significantly reduced compared with that of the MEFs fromwild-type littermates (Fig. 1C–E). Similar results were obtainedwith Cul4b-null MEFs generated with 4-hydroxytamoxifen

(OHT)-inducible Cre transgenic mice (Fig. 1C–E). Also, asshown by the results of flow cytometry, Cul4b-/Y MEFs showedan increased accumulation of cells in G1 phase of the cell cycle,

with concomitant decreases in the proportion of cells in G2 and Sphases compared with wild-type MEFs (Fig. 1F). Taken together,these results indicate that CUL4B positively regulates cell cycle

progression.

CUL4B depletion results in upregulation of Cdkn1a in a p53-independent mannerA recent report has shown that silencing of CUL4B expression inan extra-embryonic cell line results in the accumulation of p21protein (Liu et al., 2012). Thus, we next examined the effect of

CUL4B depletion on the expression of this CKI in MEFs.Consistent with the previous report, immunoblotting showed thatloss of CUL4B resulted in a significant accumulation of p21

(Fig. 2A, upper panel). Similar results were obtained withCUL4B-knockdown HEK293 and HeLa cells (Fig. 2A, middleand lower panels). Importantly, a quantitative RT-PCR (qRT-

PCR) assay indicated that the transcript level of Cdkn1a, which

encodes p21, was 2.3-fold higher in Cul4b-null MEFs than inwild-type MEFs (Fig. 2B, upper panel), and the CDKN1A mRNA

levels were 2.0-fold and 1.8-fold higher in CUL4B-knockdownHEK293 and HeLa cells (Fig. 2B, middle and lower panels),respectively, suggesting that CUL4B might repress CDKN1Aexpression at the transcriptional level. Given that CDKN1A is a

major transcriptional target of p53, we next examined whether theincreased transcription of CDKN1A was due to increasedactivation of p53 in CUL4B-deficient cells. Western blotting

and qRT-PCR assays showed that there was no difference in thelevel of p53 in CUL4B-deficient cells as compared with that ofcontrols (Fig. 2C), suggesting that the basal (stress-free)

upregulation of p21 is independent of p53. We alsodemonstrated that the negative effect of CUL4B on the p21level was not due to CUL4B-mediated protein degradation,

because treatment with the proteasome inhibitor MG132 did notnarrow the difference between CUL4B-overexpressing andcontrol cells (Fig. 2D). Furthermore, examination of p21 decayrates, performed by adding cycloheximide to the culture medium

to inhibit new protein synthesis, did not reveal a significantdifference between CUL4B-knockdown and control cells(Fig. 2E). Taken together, these results demonstrate that

CUL4B might function to repress the basal transcription ofCDKN1A.

To determine whether the compromised proliferation observed

in CUL4B-deficient cells was mediated by the upregulation ofp21, we performed rescue experiments in which we transfectedCul4b-/Y MEFs with siRNA targeting Cdkn1a or with control

siRNA. As shown in Fig. 2F and supplementary material Fig. S1,knockdown of Cdkn1a could partially rescue the proliferationdefects caused by CUL4B depletion, supporting the idea thatCUL4B promotes cell proliferation at least partially through

repressing the transcription of Cdkn1a.

The CRL4B complex is physically associated with the SIN3A-HDAC complexThe CDKN1A gene has been shown to be targeted by the SIN3A-HDAC complex (Cowley et al., 2005; Dannenberg et al., 2005;

Doyon et al., 2006; Yamaguchi et al., 2010). To test whether theSIN3A-HDAC complex is required for CUL4B-mediatedtranscriptional regulation of CDKN1A, we first determinedwhether CRL4B and SIN3A-HDAC complexes physically

interact. To this end, total protein lysates were extracted fromHEK293 cells and were subjected to co-immunoprecipitationexperiments. Immunoprecipitation with antibodies against CUL4B

or DDB1 was followed by immunoblotting with antibodies againstkey components of the SIN3A-HDAC complex, includingHDAC1, HDAC2, SIN3A, SAP180 (also known as ARID4B),

SAP130, SAP45 (also known as SDS3), SAP30 and RBP1 (alsoknown as ARID4A), with RING1B as a negative control.The results showed that CUL4B and DDB1 could be co-

immunoprecipitated with the components of the SIN3A-HDACcomplex (Fig. 3A). Reciprocally, immunoprecipitation withantibodies against HDAC1, HDAC2, SIN3A, SAP180, SAP130,SAP45, SAP30 or RBP1 followed by immunoblotting with

antibodies against CUL4B or DDB1 also revealed that theproteins in the two complexes could be co-immunoprecipitated(Fig. 3B). These results suggest that the CRL4B complex is

physically associated with the SIN3A-HDAC complex.To further investigate the molecular basis for the interaction

between the CRL4B and SIN3A-HDAC complexes, glutathione

S-transferase (GST) pull-down experiments were performed

RESEARCH ARTICLE Journal of Cell Science (2014) 127, 4679–4691 doi:10.1242/jcs.154245

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using GST-fused CUL4B or GST-fused DDB1 proteins and in

vitro transcribed and translated individual components of theSIN3A-HDAC complex, including SIN3A, SAP45, SAP180,SAP130, HDAC1 and HDAC2. With RbAp46 and RbAp48 (also

known as RBBP7 and RBBP4, respectively, both of which areknown to physically interact with SIN3A) serving as the positivecontrols, these experiments revealed that both CUL4B and DDB1could directly interact with SIN3A, but not with the other SIN3A-

HDAC components tested (Fig. 3C).SIN3A interacts with a large number of transcriptional factors and

co-regulators through six conserved domains, including four paired

amphipathic helices (PAH1–4), one histone deacetylase interactiondomain (HID) and one highly conserved region (HCR), among

which PAH1 and PAH2 are reserved for interactions with various

transcription factors, whereas PAH3, PAH4 and HID serve as ascaffold for other subunits of the co-repressor complex, such asHDACs (Silverstein and Ekwall, 2005; Wang et al., 1990). To

determine which region in the SIN3A protein can directly interactwith CUL4B and DDB1, we generated three GST-fused SIN3Aconstructs that contained different domains, GST–SIN3A S1 (aminoacids 1–400), S2 (amino acids 401–657) and S3 (amino acids 658–

1269). GST pull-down experiments demonstrated that the S2fragment, which contained the PAH3 and HID domains of SIN3A,could directly bind to CUL4B and DDB1 (Fig. 3D). Collectively,

these results demonstrate a physical association between CRL4Band SIN3A-HDAC, with SIN3A serving as a linker.

Fig. 1. Reduced cell proliferation inCul4b-null MEFs. (A) Theexpression of Cul4a and Cul4b inwild-type (Cul4bf/Y) and Cul4b-null(Cul4b-/Y) MEFs was determined byusing a qRT-PCR assay (upperpanel) and western blotting analysis(lower panel). (B) Competitive growthassay of Cul4bf/Y and Cul4b-/Y MEFs.Equal numbers of Cul4bf/Y andCul4b-/Y MEFs were mixed andseeded onto replicate plates. Cellsfrom different passages (P1–P3)were stained with the CUL4B-specificantibody and counterstained withDAPI. The percentages of positivelystained cells are shown (left) andwere counted in three random fields(right). (C–E) Knockout of Cul4binhibits cell proliferation. (C) Wild-type(Cul4bf/Y and 4OHT2) and Cul4b-null(Cul4b-/Y and 4OHT+) MEFs weresubjected to the MTT assay. (D,E) AnEdU incorporation assay wasperformed in wild-type and Cul4b-nullMEFs. Representativephotomicrographs are shown (D).The percentages of EdU-positivecells were scored in three randomfields (E). (F) The cell cycledistribution of Cul4b-null MEFs asdetermined by flow cytometricanalysis is shown. The x and y axesshow DNA content and cell number,respectively. The data shown arerepresentative of three independentexperiments. Quantitative data in A–C,E show the mean6s.d. (threeindependent experiments); *P,0.05;**P,0.01; ***P,0.001; N.S., nosignificant difference.


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The CUL4B-containing complex is associated with histonedeacetylase activityThe physical association between CRL4B and SIN3A-HDACprompted us to determine the role of CRL4B in the histone

deacetylase activity. To this end, FLAG–CUL4B was expressedin HEK293 cells, and cellular extracts were prepared by affinitypurification using M2 affinity gel. As expected, the components

of both CRL4B and SIN3A-HDAC complexes were detected inthe affinity-purified fractions (Fig. 4A, left). We then determinedthe histone deacetylase activities of the affinity-purified CUL4B-

associated complex in vitro. Increasing amounts of the purifiedCUL4B chromatographic fractions were incubated with calf

thymus bulk histone in the buffer for histone deacetylationmeasurement (HDM). The reaction products were then detectedby western blotting with antibodies against acetylated histones. Asshown in Fig. 4A (right), the amounts of acetylated H3 (AcH3) and

H4 (AcH4), AcH4K8 and AcH4K16 were gradually decreasedwith increasing amounts of purified CUL4B fractions, supportingthe notion that the CUL4B-associated complex possesses histone

deacetylase activity. To further confirm the authenticity of theCUL4B-associated activity, we added trichostatin A (TSA, aninhibitor of class I and II HDACs) to the reaction and found that

treatment with TSA significantly reduced the deacetylase activityof the purified CUL4B-associated complex, indicating that the

Fig. 2. CUL4B depletion results inupregulation of Cdkn1a expressionin a p53-independent manner.(A,B) Western blotting (A) and qRT-PCR analysis (B) showed thatdepletion of CUL4B resulted in theupregulation of p21 in MEFs, HEK293and HeLa cells at both the mRNA andprotein levels. shCUL4B, small-hairpin (sh)RNA against CUL4B;shNC, control shRNA. (C) CUL4Bdepletion in MEFs and HEK293 cellsdid not change the basal level of p53as determined by qRT-PCR (upperpanels) and western blot analysis(lower panels). (D) CUL4B-overexpressing HEK293 cells weretreated with MG132 or DMSO(MG132-) and proteins wereextracted and analyzed by westernblotting. (E) CUL4B-knockdown andcontrol HEK293 cells were treatedwith 50 mg/ml cycloheximide (CHX)and harvested at the indicated time-points. Equal amounts of proteinswere analyzed by western blotting(left) and quantified by densitometricanalysis. Expression is representedas the percentage remaining relativeto t50 (right). (F) Knockdown ofCdkn1a in Cul4b-null MEFs couldpartially rescue the proliferationdefects of Cul4b-null MEFs. Cul4b-null MEFs were transfected withCdkn1a-specific siRNA (siCdkn1a) orcontrol siRNA (siNC). At 48 hoursafter transfection, cells were fixed andimmunostained. Representativephotos are shown on the left and thepercentages of EdU-positive cellswere counted in three random fields(right). Quantitative data representthe mean6s.d. (three replicates);*P,0.05; **P,0.01; ***P,0.001;N.S., no significant difference.


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deacetylase activity of CUL4B precipitates is attributed to TSA-

sensitive histone deacetylases bound to the CUL4B-containingcomplex. We next examined the effect of CUL4B deletion on

histone acetylation in MEFs. Compared with wild-type MEFs, the

acetylation levels of bulk H3 and H4 were increased in Cul4b-nullMEFs (Fig. 4B). To determine whether there is a bias towards

Fig. 3. The CRL4B complex is physically associated with the SIN3A-HDAC complex. (A,B) Co-immunoprecipitation analysis of the association between theCRL4B and SIN3A-HDAC complexes. Whole-cell lysates were immunoprecipitated (IP) with the antibodies against the indicated proteins, with IgG as thenegative control. Immunocomplexes were then western blotted (WB) using the indicated antibodies. (C) Molecular interaction between CUL4B–DDB1 andSIN3A-HDAC subunits. GST pulldown experiments were performed with bacterially expressed GST–CUL4B and GST–DDB1 and the in vitro transcribed andtranslated SIN3A-HDAC subunit proteins, as indicated. GST–RbAp46 and GST–RbAp48 were used as positive controls. Coomassie Blue staining of CUL4B,DDB1, RbAp46, RbAp48 and GST control purified in GST pulldown experiments is shown on the left. Black arrows indicate specific bands. M, molecularmass marker. (D) Identification of the essential domains required for SIN3A to directly interact with CUL4B–DDB1. GST pulldown experiments were performedwith bacterially expressed GST–S1, GST–S2 and GST–S3 of SIN3A and in vitro transcribed and translated proteins of CUL4B or DDB1. Coomassie Bluestaining of GST–S1, GST–S2 and GST–S3 purified in GST pulldown experiments is shown on the left.


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deacetylation of a particular histone residue for the CRL4B

complex, we also examined the acetylation levels of individualresidues and observed significantly increased levels of acetylationat H3K27, H4K8 and H4K16 in Cul4b-null MEFs compared with

that of wild-type MEFs (Fig. 4B). Similar results were alsoobtained with CUL4B-knockdown HEK293 and HeLa cells(Fig. 4C). Taken together, these observations suggest thatCRL4B might function as a transcriptional repressor by

supporting the function of histone deacetylases.

HDAC1 and HDAC2 are involved in CUL4B-mediatedtranscriptional repressionThe data shown above indicate that the CRL4B complex isphysically associated with the SIN3A-HDAC complex, and that

CUL4B depletion increases histone acetylation. We thenexamined whether HDAC1 and HDAC2 are involved inCUL4B-mediated transcriptional regulation. For this purpose,

we transfected Gal4–CUL4B into HeLa cells with stableexpression of a luciferase reporter driven by 56Gal4-bindingdomain (Gal4-UAS cells), and treated cells with or withoutTSA. Although the expression of Gal4–CUL4B led to a

remarkable decrease in the expression of the reporter gene in a

dose-dependent manner as observed previously, TSA treatment

could efficiently attenuate the reduction in reporter geneexpression caused by the expression of Gal4–CUL4B (Fig. 5A),suggesting that TSA-sensitive HDACs are involved in CUL4B-

mediated transcriptional regulation. Furthermore, knockdown ofHDAC1 and HDAC2 in combination (HDAC1/2) also led to asignificant reduction in the transcriptional repressive activity ofCUL4B (Fig. 5B), supporting the notion that HDAC1 and

HDAC2 are involved in CUL4B-mediated transcriptionalrepression. To further confirm this notion, we performed aquantitative chromatin immunoprecipitation (qChIP) assay in

Gal4-UAS cells to investigate the recruitment of DDB1, HDAC1,HDAC2 and SIN3A to the Gal4 promoter after transfection withGal4–CUL4B or control Gal4–DBD vectors. Compared with

control (Gal4–DBD), transfection of Gal4–CUL4B resulted in anincrease in the recruitment of DDB1, HDAC1, HDAC2 andSIN3A (Fig. 5C). Consequently, the level of H2AK119ub1 was

significantly increased, whereas that of acetylated histones H3and H4 was decreased when Gal4–CUL4B was expressed(Fig. 5C). Treatment with TSA blocked the effect on histonedeacetylation caused by Gal4–CUL4B, although the binding of

SIN3A-HDAC to the Gal4 promoter was not affected (Fig. 5C).

Fig. 4. The CRL4B complex isassociated with histonedeacetylase activity. (A) TheCUL4B-associated complexpossesses histone deacetylaseactivity. Cellular extracts wereobtained from HEK293 cells stablyexpressing FLAG–CUL4B and wereimmunoprecipitated with the anti-FLAG antibody. Western blottinganalysis of the CUL4B-purifiedfractions using antibodies against theindicated proteins (left). The reactionmixtures were analyzed by westernblotting using antibodies against theindicated histones or modifiedhistones (right). CON, control.(B,C) Western blotting analysisshowed increased levels ofacetylated histones in Cul4b-nullMEFs (B) and CUL4B-knockdownHEK293 and HeLa cells(C) compared with control cells.shCUL4B, shRNA against CUL4B;shNC, control shRNA.


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However, compared with Gal4–CUL4B, the mutant construct(Gal4–DCullin, in which the cullin domain is deleted) lost theability to catalyze H2AK119 monoubiquitylation, but retained theability to recruit SIN3A-HDAC to the promoter, suggesting thatthe E3 ligase activity of CRL4B is dispensable for its ability to

recruit the SIN3A-HDAC complex to target promoters. To furtherdetermine the role of the E3 ligase activity of CRL4B and its

associated deacetylase activity in transcriptional repression, weoverexpressed CUL4B or CUL4B-DCullin in HEK293 cells andtreated the cells with the HDAC inhibitor TSA. As expected,overexpression of CUL4B resulted in a several-fold reduction ofCDKN1A expression at both RNA and protein levels, and TSA

treatment could partially block the reduction (Fig. 5D,E).However, CDKN1A expression was reduced by ,50% when

Fig. 5. See next page for legend.


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CUL4B-DCullin was expressed, although TSA could further relievethe repression by CUL4B-DCullin (Fig. 5D,E). Consistent with theseobservations, knockdown of SIN3A could also derepress CDKN1A(Fig. 5F). Importantly, simultaneous knockdown of SIN3A and

CUL4B resulted in further derepression than individual knockdown(Fig. 5F). Taken together, these results suggest that CRL4B mightrepress its target genes by promoting H2AK119 ubiquitylation and by

coordinating with the SIN3A-HDAC complex.

CUL4B promotes stable retention of the SIN3A-HDACcomplex at the CDKN1A promoterIn order to define the functional interplay between the CRL4Band SIN3A-HDAC complexes in the transcriptional repression ofCDKN1A, we next performed a qChIP assay to profile the binding

pattern of CUL4B, DDB1, HDAC1/2 and SIN3A on a 13-kbregion that surrounds the transcription start site (TSS) ofCDKN1A, using a panel of 13 pairs of oligonucleotide primers.

The qChIP assay revealed that the occupancy sites of theseproteins overlap and that the binding of CRL4B and SIN3A-HDAC both peaked in the region around 2800 bp to 2300 bp(Fig. 6A), suggesting that CRL4B and SIN3A-HDAC complexesmight co-occupy the CDKN1A promoter in the same complex.We also examined the binding profile of p53, which is known to

have binding sites on the CDKN1A promoter. As shown inFig. 6A, p53 binds primarily on the more upstream regions.To further test this proposition, ChIP-Re-ChIP assayswere performed. In these experiments, chromatin was first

immunoprecipitated with antibodies against CUL4B, DDB1,HDAC1/2 or SIN3A. The immunoprecipitates were subsequentlyre-immunoprecipitated with appropriate antibodies. The results

revealed that the CDKN1A promoter could be re-immunoprecipitated with antibodies against DDB1, HDAC1/2or SIN3A from CUL4B immunoprecipitates (Fig. 6B). Similar

results were obtained when the initial ChIP was performed withantibodies against DDB1, HDAC1/2 or SIN3A (Fig. 6B).

We next examined the effect of CUL4B depletion on the

recruitment of the SIN3A-HDAC complex to the CDKN1A

promoter. As shown in Fig. 6C, knockdown of CUL4B did notalter the expression of SIN3A, HDAC1 or HDAC2 and vice

versa. Importantly, knockdown of CUL4B resulted in a markedreduction in the levels of HDAC1, HDAC2 and SIN3A bound tothe CDKN1A promoter (Fig. 6D). Consistent with theseobservations, the level of H2AK119ub1 was greatly reduced

and those of acetylated histones, including AcH3, AcH4,AcH4K8 and AcH4K16, were increased upon CUL4Bknockdown (Fig. 6D). However, knockdown of SIN3A or

HDAC1/2 did not affect the recruitment of CUL4B and DDB1to the CDKN1A promoter (Fig. 6E). Knockdown of RbAp46/48did not alter the recruitment of CUL4B, SIN3A and HDAC1/2 to

the CDKN1A promoter (Fig. 6F, left), despite the fact that itderepressed the expression of p21(Fig. 6F, right). Collectively,these data support the argument that CRL4B contributes to

histone deacetylation, probably by facilitating the retention ofSIN3A-HDAC at the CDKN1A promoter.

CRL4B participates in the regulation of a subset of HDAC1/2target genesTo further understand the functional association between CRL4Band SIN3A-HDAC complexes, we used Cul4b-null MEFs to

examine the expression of ten cell cycle regulators that werepreviously identified as HDAC1/2 target genes in primaryfibroblasts. Among ten genes examined, five were upregulated

in Cul4b-null MEFs compared with wild-type MEFs (Fig. 7A,B),suggesting that the CRL4B complex is involved in regulating asubset of HDAC1/2 target genes. Interestingly, Cdkn1c, another

member of the Cip/Kip CKI family, was also significantlyupregulated in Cul4b-null MEFs compared with wild-type cells.The negative effect of CUL4B on the expression of CDKN1C wasconfirmed in CUL4B-knockdown and CUL4B-overexpressing

HEK293 cells (Fig. 7C,D). A ChIP-Re-ChIP assay showed thatthe CRL4B and SIN3A-HDAC complexes co-occupied theCDKN1C promoter as well (Fig. 7E,F).

DISCUSSIONTranscriptional repression of p21 by CUL4BOur previous results showed that reduced cell proliferation andincreased apoptosis could lead to the developmental arrest ofCul4b-null embryos (Jiang et al., 2012). Here, we demonstrate thecrucial function of CUL4B in cell cycle progression. We show

that loss of CUL4B in MEFs results in significantly reduced cellproliferation and G1 cell cycle arrest, accompanied by anupregulation of the CKI p21. Because knockdown of p21 in

Cul4b-null MEFs could rescue their proliferation defects, weconclude that CUL4B might promote cell cycle progression atleast partially through the negative regulation of p21.

By assembling the CRL4B ubiquitin ligase complex withDDB1 and ROC1, CUL4B participates in the regulation of abroad spectrum of biological processes by targeting different

substrates for proteasomal degradation or for protein modification(Higa and Zhang, 2007; Jackson and Xiong, 2009). Ourobservation that the loss of CUL4B in MEFs could cause theaccumulation of p21 is consistent with a recent report of

increased accumulation of p21 in Cul4b-deficient extra-embryonic cells (Liu et al., 2012). However, our quantitativeRT-PCR assay indicates that the transcription of the gene

encoding p21, Cdkn1a, was significantly upregulated in Cul4b-null MEFs compared with that of control MEFs. Meanwhile,CUL4B was not found to influence the degradation of p21, as

shown by treatment with the proteasome inhibitor MG132 and

Fig. 5. HDAC1 and HDAC2 are involved in CUL4B-mediatedtranscriptional regulation. (A) TSA treatment decreases the intrinsictranscriptional repressive activity of CUL4B. Construct encoding Gal4–CUL4B or control Gal4–DBD was transfected into HeLa cells expressing theGal4-UAS reporter, and then the cells were treated with or without TSA for24 h. Gal4 luciferase reporter activity was measured with six repeats.(B) HDAC knockdown impairs the potential transcriptional repressive activityof CUL4B. Gal4-UAS-reporter cells were transfected with construct encodingGal4–CUL4B and siRNA against HDAC1 and HDAC2 (siHDACs) or negativecontrol siRNA (siNC). Luciferase activity was measured (left) and theknockdown efficiency of HDAC1 and HDAC2 was analyzed by westernblotting (right). (C) CUL4B confers repressive activity through HDACs andsubsequent histone deacetylation. qChIP experiments using antibodiesagainst the indicated proteins were performed in cells that were transientlytransfected with constructs encoding Gal4–DBD, Gal4–CUL4B or Gal4–DCullin and treated with TSA. Enrichment of the indicated proteins at theGal4 promoter was analyzed. (D,E) CRL4B represses the transcription ofCDKN1A through its ubiquitylation function and its associated deacetylaseactivity. HEK293 cells were transfected with CUL4B, CUL4B-DCullin orcontrol construct and treated with TSA. CDNK1A expression was determinedby qRT-PCR (D) and western blotting (E). CON, control. (F) Simultaneousknockdown of SIN3A and CUL4B resulted in more severe effect thanindividual knockdown. siSIN3A or siNC were transfected into CUL4B-knockdown HEK293 cells (shCUL4B), then the RNA and protein levels ofp21 were analyzed. shNC, control shRNA. Quantitative data show themean6s.d. (three independent experiments); *P,0.05; **P,0.01;***P,0.001; N.S., no significant difference.


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Fig. 6. CRL4B and SIN3A-HDAC complexes co-occupy the CDKN1A promoter. (A) qChIP analyses were conducted to determine the binding region ofCUL4B, DDB1, HDAC1/2, SIN3A and p53 on the CDKN1A gene locus. Rabbit normal IgG served as a negative control. The results of ChIP enrichments arepresented as the percentage of bound/input signal. (B) ChIP and Re-ChIP experiments were performed in HEK293 cells with the indicated antibodies.(C) Knockdown efficiency was analyzed by western blotting in cells transfected with shRNA targeting CUL4B (shCUL4B) or siRNA targeting SIN3A (siSIN3A) orHDAC1 and HDAC2 (siHDAC1/2). (D) qChIP analysis of the enrichment of the indicated proteins at the CDKN1A promoter in HEK293 cells after transfectionwith control shRNA (shNC) or shRNA targeting CUL4B. (E) qChIP analysis of the recruitment of the CUL4B and DDB1 proteins to the CDKN1A promoter inHEK293 cells after transfection with control siRNA (siNC) or siRNAs targeting HDAC1/2 or SIN3A. (F) Left, the recruitment of CUL4B, HDAC and SIN3A to theCDKN1A promoter in HEK293 cells after transfection with control siRNA or siRNAs targeting RbAp46 and RbAp48 (siRbAp46/48) by qChIP analysis. Right,knockdown efficiency of RbAp46/48 and p21 level were analyzed by western blotting. For D–F, data represent the mean6s.d. (three independent experiments);*P,0.05; **P,0.01; ***P,0.001; N.S., no significant difference.


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half-life analysis. These results suggest that CUL4B negativelyregulates the function of p21 by transcriptional repression, but notby proteolysis.

As a major regulator of cell cycle progression in mammaliancells, p21 is primarily regulated at the transcriptional level (Xionget al., 1993). A variety of transcription factors as well as

Fig. 7. CRL4B transcriptionally represses a subset of HDAC1/2 target genes. (A,B) qPCR analysis of the expression of HDAC1/2 target genes in Cul4b-/Y

MEFs (A) or 4-OHT-inducible knockout MEFs (B) compared with that of control cells. (C,D) CDKN1C expression in CUL4B-knockdown (shCUL4B) andCUL4B-overexpressing cells as well as control (shNC, CON) HEK293 cells was determined by qRT-PCR (C) and western blot analysis (D). For A–C, datarepresent the mean6s.d. (three independent experiments); *P,0.05; **P,0.01, ***P,0.001; N.S., no significant difference. (E,F) The CRL4B and SIN3A-HDACcomplexes co-target the CDKN1C promoter. ChIP (E) and Re-ChIP (F) analysis of the recruitment of CUL4B, DDB1, HDAC1/2 and SIN3A to the CDKN1C genein HEK293 cells is shown. (G) A model depicting the association between the CRL4B and SIN3A-HDAC complexes in transcriptional repression.


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co-regulators (co-activators and co-repressors) have beenreported to be involved in the regulation of p21 expression

(Gartel and Radhakrishnan, 2005). Although p21 can be inducedby a p53-dependent mechanism in response to DNA damage toensure cell cycle arrest and DNA repair, many factors, such asHDAC inhibitors, can upregulate p21 independently of p53

(Huang et al., 2000; Yamaguchi et al., 2010). In this study, wefound that in the absence of extrinsic stress p53 activation is notinvolved in the regulation of p21 expression by CUL4B. Instead,

HDACs play a crucial role in this process.

Involvement of HDAC1 and HDAC2 in CUL4B-mediatedtranscriptional repression of CDKN1AHDAC1 and HDAC2 are well-documented co-repressors of p21regulation. In this study, we presented several lines of evidence

indicating that HDAC1 and HDAC2 are also involved in CUL4B-mediated transcriptional repression of CDKN1A. First, CUL4Band DDB1, the key components of the CRL4B complex, arephysically associated with HDAC1 and HDAC2. Second, an

affinity-purified CUL4B-associated complex was found topossess a deacetylase activity that could be blocked by theHDAC inhibitor TSA. Third, significantly increased acetylation

of histones 3 and 4 was detected in Cul4b-null MEFs comparedwith that of wild-type cells. Fourth, TSA treatment could partiallyblock the reduction of CDKN1A expression and reporter

expression caused by CUL4B overexpression. Lastly,knockdown of HDAC1/2 also led to a significant reduction inthe transcriptional repressive activity of the CUL4B-containing

complex. Taken together, these results support the idea thatHDAC1 and HDAC2 are involved in CUL4B-mediatedtranscriptional repression. More studies are needed to evaluatewhether other HDACs are involved in CUL4B-mediated

transcriptional repression.

Physical and functional association of CRL4Bwith the SIN3A-HDAC complexHDAC1 and HDAC2 are present in a variety of repressorcomplexes such as SIN3A, NuRD and REST, which acquire their

regional activities in part by interacting with sequence-specifictranscription factors (Yang and Seto, 2008). Because the SIN3Acomplex has been shown to target the CDKN1A gene, weexamined the interplay between the CRL4B and SIN3A-HDAC

complexes. A co-immunoprecipitation assay showed that bothCUL4B and DDB1 are physically associated with the componentsof the SIN3A-HDAC complex, including SIN3A, SAP30,

SAP130, SAP18, SAP45, HDAC1 and HDAC2. We furtherdemonstrated that the direct interaction between the CRL4B andSIN3A-HDAC complexes is bridged by the fragment containing

the PAH3 and HID domains in SIN3A, which is also responsiblefor the association of SIN3A with SAP30 and HDACs. TheCRL4B and SIN3A-HDAC complexes thus form a higher-order

complex in catalyzing H2AK119 monoubiquitylation and histonedeacetylation at target genes such as CDKN1A and CDKN1C.Accordingly, a Gal4–CUL4B fusion protein could recruitHDAC1/2 and SIN3A to the artificial reporter and led to

decreased levels of AcH3 and AcH4. qChIP assays revealed aperfect overlap of the occupancy sites of the CRL4B and SIN3A-HDAC complexes on the promoter of CDKN1A. Importantly,

knockdown of CUL4B resulted in a marked reduction in therecruitment of HDAC1, HDAC2 and SIN3A to the CDKN1Apromoter, increased the levels of histone acetylation and

decreased those of H2AK119ub1. However, knockdown of

SIN3A or HDAC1/2 did not affect the recruitment of CUL4Band DDB1, suggesting that CRL4B promotes histone

deacetylation by influencing the recruitment and/or retention ofHDAC1 and HDAC2 at target gene promoters. Furthermore, theinteraction between CRL4B and SIN3A-HDAC did not dependon the ubiquitylation function of CRL4B. Thus, in addition to

directly contributing to epigenetic silencing by catalyzingH2AK119 monoubiquitylation, CRL4B also facilitates thedeacetylation function of SIN3A-HDAC. Taken together, we

propose that, under physiological conditions, CRL4B and SIN3A-HDAC form a large complex that occupies the promoters ofgenes encoding CKIs such as p21 and p57 and inhibits their

expression by catalyzing H2AK119 monoubiquitylation andhistone deacetylation. When CUL4B is depleted, CKIs becomederepressed and accumulate, leading to impaired cell cycle

progression (Fig. 7G). Notably, neither CRL4B nor SIN3A-HDAC subunits contain DNA-binding domains that couldcontribute to their recruitment to promoters. Clearly, futurestudies are needed to determine the identity and function of

additional elements involved in CRL4B–SIN3A-HDACfunctionality. In conclusion, our study reveals that CRL4Binteracts with SIN3A and promotes the recruitment and stable

retention of SIN3A-HDAC to the CDKN1A and CDKN1Cpromoters, leading to increased H2AK119 monoubiquitylationand decreased histone acetylation and, consequently, to

epigenetic silencing of CKIs, thus driving cell cycle progression.

MATERIALS AND METHODSGeneration of Cul4b-null MEFsThe generation of Cul4b-floxed mice and genotyping were described

previously (Jiang et al., 2012). Mice carrying Cul4bflox/flox were

intercrossed with Sox2-Cre+/2 male mice and primary MEFs were

obtained from embryonic day (E)13.5–14.5 embryos according to

standard protocols. Inducible conditional knockouts were created by

crossing Cul4bflox/flox female mice to CAG-Cre/Ers1 male mice.

Cell culture and transfectionMEFs were cultured in Dulbecco’s modified Eagle’s medium (Gibco,

Grand Island, NY; Invitrogen, Carlsbad, CA) with 10% FBS, 100 units/

ml penicillin and 100 mg/ml streptomycin. The inducible-knockout cellswere treated with 200 nM 4-OHT (Sigma, St Louis, MO) for 48 h to

activate Cre recombinase. HeLa and HEK293 cells with CUL4B

knockdown or overexpression were as described previously (Zou et al.,

2009). Transfection of plasmids or siRNA was performed according to

standard protocols using calcium phosphate precipitation (Polysciences

Warrington, PA) or Lipofectamine 2000 (Invitrogen), respectively.

Real-time quantitative RT-PCRCells were lysed with Trizol reagent, and total RNA was isolated

following the manufacturer’s instructions (Invitrogen). cDNA was

prepared with the MMLV Reverse Transcriptase (Thermo Scientific,

Rockford, IL). Relative quantification of target genes was performed and

analyzed by using a Roche 480 machine. The expression of GAPDH was

determined as an internal control. All primers are listed in supplementary

material Table S2.

Plasmid constructionFLAG–CUL4B, FLAG–DDB1, Gal4–CUL4B and Gal4–DCullin were asdescribed previously (Hu et al., 2012). FLAG–SIN3A, FLAG–SAP180,

FLAG–SAP130, FLAG–SAP45, FLAG–SAP30, FLAG–HDAC1 and

FLAG–HDAC2 were generated by inserting full-length SIN3A, SAP180,

SAP130, SAP45, SAP30, HDAC1 or HDAC2 into pCMV-Tag2B. GST–

CUL4B and GST–DDB1, GST–RbAp46 and GST–RbAp48 were created

by inserting full-length CUL4B, DDB1, RbAp46 or RbAp48 into the

pGEX-4T-3 expression vector. Using the FLAG–SIN3A construct as a


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template, we generated three GST constructs that express GST-fusion

proteins corresponding to different domains of SIN3A, i.e. GST–S1

(amino acids 1–400), GST–S2 (amino acids 401–657) and GST–S3

(amino acids 658–1269).

Antibodies and reagentsThe primary antibodies used in this study were mouse anti-FLAG (Sigma),

rabbit anti-CUL4B (Sigma), rabbit anti-DDB1 (Santa Cruz Biotechnology,

Dallas, TX), rabbit anti-p21 (Cell Signaling Technology, Beverly,

MA), mouse anti-b-actin (Anbo), rabbit anti-SIN3A (Santa CruzBiotechnology), rabbit anti-HDAC1 (Abcam, Hong Kong, China), rabbit

anti-HDAC2 (Abcam), rabbit anti-RbAp46/48 (Sigma), rabbit anti-p53

(Santa Cruz Biotechnology), rabbit anti-RING1B (Santa Cruz

Biotechnology) and rabbit antibodies against SAP180, SAP130, SAP45,

SAP30 and RBP1 (Bethyl, Montgomery, Texas). Protein-A/G–Sepharose

CL-4B and glutathione–Sepharose beads were purchased from Amersham

Biosciences. Protease inhibitor cocktail was from Roche Applied Science.

siRNA against murine p21 and siRNA against human HDAC1/2 and

RbAp46/48 were synthesized by Sigma (supplementary material Table

S1). TSA, MG132 and cycloheximide (CHX) were purchased from Sigma.

Proliferation assays3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT)

substrate (Sigma) and 5-ethynyl-29-deoxyuridine (EdU) proliferationassays (Ribobio) were performed according to the manufacturer’s

instructions to measure cell proliferation. Briefly, cells were seeded at

26103 per well in 96-well plates with eight repeats for each group. Then,at 24 h, 48 h, 72 h and 96 h, the MTT reagent was added at a

concentration of 5 mg/ml to the cell medium and the cells were incubated

at 37 C̊ for an additional 4 h. The reaction was stopped by adding 100 mlof DMSO per well and the cells were then lysed for 20 min, followed by

measurement of the absorbance values. For the EdU assay, the cells were

incubated with EdU for 4 h before being fixed with 4% formaldehyde for

30 min and incubated with 2 mg/ml glycine for 5 min. Then, 0.5% Triton

X-100 was used for permeabilization and 16reaction cocktail was added.Finally, 4,6-diamidino-2-phenylindole (DAPI) was used to stain the

nuclei.

ImmunofluorescenceEqual numbers of Cul4b-null and wild-type MEFs were mixed and plated

onto coverslips for culture. After being washed with PBS and fixed in 4%

paraformaldehyde, cells were permeabilized with 0.2% Triton X-100,

blocked with 5% normal goat serum (Sigma) and incubated with CUL4B-

specific antibody overnight, followed by staining with FITC-conjugated

secondary antibody for 1 h at room temperature on the second day. Cells

were washed four times and nuclei were stained with DAPI at a final

concentration of 0.1 mg/ml.

Flow cytometryCul4b-null and wild-type MEFs at passage 3 were harvested by

trypsinization and fixed with 70% ethanol overnight. Then cells were

washed with cold PBS and incubated in RNase A and propidium iodide

(Sigma) staining solution. A single-cell suspension was analyzed

by using a flow cytometer equipped with CellQuest software. The

experiment was repeated three times.

Luciferase reporter assaysHeLa cells stably expressing the Gal4-UAS reporter (Gal4-UAS) were

cultured in 24-well plates and transfected with 50–800 ng of Gal4-CUL4B

or Gal4-DBD fusion assay vectors with 40 ng pRLTK (Renilla) as the

control. Total cell lysates were prepared after 48 h, and luciferase activities

were determined using the Dual-Luciferase Reporter Assay System

(Promega, Madison, Wisconsin). All samples were analyzed in triplicate.

In vitro histone deacetylation assayFLAG–CUL4B was transfected into HEK 293 cells, then the CUL4B-

associated complex was immunoprecipitated with the anti-FLAG antibody.

The immunoprecipitates were incubated with bulk histones in the buffer for

histone deacetylation measurement (HDM; 50 mM Tris, 50 mM KCl, 5 mM

MgCl2, 5% glycerin, 1 mM DTT, 1mM PMSF, adjust pH to 8.5 with HCl),

with or without TSA for 12 h at 37 C̊, and the levels of acetylated histones

in the reactions were then analyzed by western blotting with the indicated

antibodies.

Immunoprecipitation and GST pulldownImmunoprecipitation assays were performed as described previously (Hu

et al., 2012; Yang et al., 2013). Briefly, 16107 cells were collected, washedwith cold PBS and then lysed with lysis buffer at 4 C̊ for 30 min. Primary

antibodies or normal rabbit or mouse immunoglobulin G (IgG) was added

to cellular extracts and incubated at 4 C̊ overnight. Subsequently, Protein-

A/G–Sepharose CL-4B beads were prepared and added to bind with the

antibody for 2 h at 4 C̊. Beads were washed four to five times with lysis

buffer and the immune complexes were subjected to western blot assay.

GST fusion constructs were expressed in BL21 Escherichia coli cells, and

crude bacterial lysates were prepared by sonication in cold PBS in the

presence of the protease inhibitor mixture. The in vitro transcription and

translation experiments were accomplished with rabbit reticulocyte lysate

(TNT Systems; Promega). In the GST pulldown assay, ,10 mg of theappropriate GST fusion proteins were incubated with 30 ml of glutathione–Sepharose beads at 4 C̊ for 1 h, followed by extensive washing with cold

PBS. Then, GST fusion proteins immobilized on glutathione–Sepharose

beads were preincubated in binding buffer (0.8% BSA in PBS in the

presence of the protease inhibitor mixture) at 4 C̊ for 15 min, followed by

incubating with transcribed and translated products at 4 C̊ for 2 h. The

beads were collected, washed five times with washing buffer and

resuspended in 30 ml of 26 SDS-PAGE loading buffer. Protein bandswere detected with specific antibodies by western blotting.

ChIP and ChIP-Re-ChIPChIP and ChIP-Re-ChIP assays were performed as described previously

(Wang et al., 2009a; Zhang et al., 2004). Briefly, 56107 cells werecrosslinked with 1% formaldehyde, sonicated, pre-cleared and incubated

with 5–10 mg of antibody per reaction at 4 C̊ overnight. Subsequently,Protein-A/G–Sepharose CL-4B beads were prepared and added to bind

with the antibody for 2 h at 4 C̊. Then, complexes were washed with low

and high salt buffers, and the DNA was extracted and precipitated. For

Re-ChIP assays, immune complexes were eluted from the beads with

20 mM dithiothreitol. The eluates were then diluted 30-fold with ChIP

dilution buffer and subjected to a second immunoprecipitation reaction.

The final elution step was performed using elution buffer (pH 8.0). The

enrichment of the DNA template was analyzed by conventional PCR

using primers specific for each target gene promoter. All primers are

listed in supplementary material Table S3.

Statistical analysisData represent the mean6s.d.; statistical significance was evaluated byusing a two-tailed unpaired Student’s t-test by using GraphPad Prism

(GraphPad Software, San Diego, CA).

Competing interestsThe authors declare no competing interests.

Author contributionsQ.J., H.H., Y.W. and Y.G. conceived of the project. Q.J. performed the majority ofthe experiments and data analysis. H.H., F.Y., J.Y., Y.Y., L.J., Y.Q., B.J., Y.Z. andC.S. provided technical assistance and discussion; Q.J., H.H., C.S., and Y.G.wrote the manuscript.

FundingThis work was supported by grants from the National Basic Research Program ofChina (973 Program) [grant numbers 2011CB966200 and 2013CB910900]; andthe National Natural Science Foundation of China [grant numbers 8133005 and81321061].

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.154245/-/DC1


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