combinatorial action of transcription factors orchestrates cell cycle-dependent expression of the...

Combinatorial action of transcription factors orchestratescell cycle-dependent expression of the ribosomal proteingenes and ribosome biogenesisNagisa Nosrati, Neetu R. Kapoor and Vijay Kumar

Virology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India

Keywords

cell cycle; ChIP; histone acetyl transferase;

RPS27a; transcriptional regulation

Correspondence

V. Kumar, Group Leader and Staff Scientist,

Virology Group, International Centre for

Genetic Engineering and Biotechnology

(ICGEB), Aruna Asaf Ali Marg, New

Delhi 110067, India

Fax: +91 11 2674 2316

Tel: +91 11 2674 2360

E-mail: [email protected]

(Received 15 September 2013, revised 1

March 2014, accepted 17 March 2014)

doi:10.1111/febs.12786

Nucleolar assembly begins at the early G1 phase of the cell cycle and is a

hub of ribosomal DNA transcription and rRNA biosynthesis. The newly-

formed rRNAs together with ribosomal proteins (RPs) constitute the build-

ing block of the ribosomal machinery. Although RPs play a major role in

protein biosynthesis, their own regulation and expression is rather poorly

understood. In the present study, we investigated the regulation of RP

genes RPS27a, RPS24, RPS6, RPL9 and RPL4 in synchronized mamma-

lian cell culture. Quantitative RT-PCR analysis indicated their expression

during the mid to late G1 phase, whereas the rRNA genes were expressed

during the early G1 phase of the cell cycle. The promoter reporter analysis

of the RPS27a gene revealed that it could be synergistically stimulated by

the transcription factors specificity protein 1 (Sp1) and cAMP response ele-

ment-binding protein (CREB). However, E2F transcription factor 1 (E2F1)

appeared to negatively regulate gene expression. Chromatin immunoprecip-

itation studies confirmed the promoter occupancy of Sp1, CREB and

E2F1. Although Sp1 and CREB binding enhanced the promoter occupancy

of histone acetyltransferases PCAF, p300 and CREB binding protein,

E2F1 facilitated the recruitment of histone deacetylases. Both acetylation

(histone H4 pan-acetyl, histone H3 acetyl Lys 14) and methylation (histone

H3 trimethyl Lys 9) marks were observed in the RPS27a promoter region,

suggesting their important regulatory role in gene expression. Because the

promoter regions of most RP genes are well conserved, we propose that

their orchestrated regulation and synthesis during the cell cycle facilitates

ribosome biogenesis.

Introduction

Ribosomes are cell organelles that are responsible for

protein biosynthesis (translation) in the cell. Ribo-

somes are typically composed of two subunits: a ribo-

somal protein (RP) large subunit (RPL) and a

ribosomal protein small subunit (RPS), each composed

of specific ribonucleoprotein complexes made up of

four types of rRNAs and approximately 80 RPs [1,2].

Although rRNAs are transcribed in the nucleolar com-

partment of the eukaryotic cells, RPs are synthesized

in the cytoplasm and transported to the nucleolus for

the assembly of ribosomal subunits [3–5]. Ribosome

biogenesis is closely associated with growth and prolif-

Abbreviations

CBP, CREB binding protein; ChIP, chromatin immunoprecipitation; CREB, cAMP response element-binding protein; E2F1, E2F transcription

factor 1; FACS, fluorescence-activated cell sorting; H3K14ac, histone H3 acetyl Lys 14; H3K9me3, histone H3 trimethyl Lys 9; H4ac, histone

H4 pan-acetyl; HATs, histone acetyltransferases; HDAC1, histone deacetylase 1; IHH, immortalized human hepatocyte; NPM,

nucleophosmin; Rb, retinoblastoma; RPL, ribosomal protein large subunit; RP, ribosomal protein; RPS, ribosomal protein small subunit;

shRNA, small hairpin RNA; Sp1, specificity protein 1; UBCEP80 gene, ubiquitin carboxyl extension protein 80 gene.

FEBS Journal 281 (2014) 2339–2352 ª 2014 FEBS 2339

eration of cells and thus is tightly regulated at tran-

scriptional, translational and post-translational levels

to prevent aberrant cell proliferation [2,3]. Many cell

cycle regulators and tumor suppressors participate in

ribosome biogenesis and therefore also regulate cell

proliferation [6,7]. Some nucleolar and RPs also exhi-

bit a regulatory role as a stress sensor and activate

p53-mediated cell cycle arrest, apoptosis and prema-

ture senescence [8]. For example, RPs such as RPL5,

RPL11 and RPL23 exhibit anti-cancer properties by

inducing p53 expression, leading to cell cycle arrest

and cell death. On the other hand, RPS13 and RPL13

promote cancer and therefore could serve as cancer

biomarkers [9]. The level of RPS3a was found to be

up-regulated in hepatitis B virus-related hepatocellular

carcinoma and to be associated with increased nuclear

factor-jB signaling [10]. Similarly, RPS27a is also

over-expressed in tissues of renal, breast and colon

carcinomas [11–13]. More recently, we reported the

over-expression of the RPS27a gene in an oncomouse

model of hepatocellular carcinoma [14]. The RPS27a

levels decreased sharply following a chemopreventive

intervention [15]. Despite the emerging significance of

RP genes in cancer development, cell cycle and cellular

senescence, little is known about their regulatory

control mechanisms.

RPS27a is a fusion protein encoded by ubiquitin

carboxyl extension protein 80 gene (UBCEP80 gene).

The N-terminal domain of UBCEP80 gene codes for

monoubiquitin, whereas the C-terminal region codes

for a carboxyl extension protein, which eventually

becomes a part of the 40S ribosomal subunit [16,17].

Although a cell cycle-dependent transcription of RP

genes has been suggested [18,19], their transcriptional

regulation is not well understood. In the present

study, we aimed to assess cell cycle dependence of the

expression of RP genes and to delineate the regulatory

‘cis’ elements involved in their transcriptional regula-

tion. Because the promoter regions of RP genes are

highly conserved across species [20,21], we studied the

regulation of five genes, RPS27a, RPS24 and RPL9

[with specificity protein 1 (Sp1), cAMP response ele-

ment-binding protein (CREB) and E2F transcription

factor 1 (E2F1) binding sites] and RPS6 and RPL4

(without Sp1, CREB and E2F1 binding sites), to

broaden the scope of the present study. We show that

the RP genes are expressed in a cell cycle-dependent

manner and are under the regulatory control of

specific transcription factors, along with some fine

interplay of histone modifiers. Thus, we propose that

a coordinated and synchronized synthesis of ribosomal

genes during the cell cycle could be essential for ribo-

some biogenesis.

Results

RP genes are expressed during the G1 phase of

the cell cycle

To understand the mechanism of RP-dependent cell

cycle regulation, we investigated the expression of five

RP genes, RPS27a, RPS24, RPL9, RPS6 and RPL4,

in a synchronized population of immortalized human

hepatocyte (IHH) cells. Fluorescence-activated cell

sorting (FACS) analysis revealed approximately 80%

arrest of cells in the G0/G1 phase (Fig. S1). Cells were

harvested at different time points post serum stimula-

tion and the RPS27a transcript levels were measured

by quantitative RT-PCR. There was a marked increase

in the expression of the RPS27a gene at 12 h post-

serum stimulation corresponding to the mid to late G1

phase of the cell cycle (Fig. 1A). Interestingly, a simi-

lar expression pattern was also observed for other RP

genes, albeit at higher (RPS24 and RPL9) or moderate

levels (RPS6 and RPL4), suggesting a synchronized

expression of RP genes during the cell cycle. Further-

more, the rRNA genes (5S, 5.8S, 18S and pre-rRNA)

were also synchronously synthesized during the early

G1 phase (Fig. 1B).

Sp1 and CREB are key activators of the RPS27a

promoter

Because the RP genes exhibited a synchronized expres-

sion during the cell cycle, we next investigated the

mechanism of transcriptional regulation of RP genes.

The 2-kb promoter regions of RP genes were subjected

to bioinformatic analyses using TFSEARCH to character-

ize the ‘cis’ elements and trans-activator proteins

involved in their regulation. The putative binding sites

of common transcriptional regulators such as Sp1,

CREB and E2F1 in the promoter regions of RP genes

are shown schematically in Fig. S2. The DNA

sequence of 1.6-kb full-length human RPS27a gene

(FL-RPS27a), along with its regulatory elements, is

also shown in Fig. S3. The transcriptional regulation

of FL-RPS27a and its three deletion mutants, D1-, D2-and D3-RPS27a (Fig. 2A), was investigated using the

luciferase reporter assay after transiently transfecting

cells with some common transcription factors and cell

cycle regulators such as Sp1, c-ETS1, E2F1, cyclin D1,

nucleophosmin (NPM), CREB, c-Fos, c-Jun and c-

Myc. As shown in Fig. 2B, there was a significant

(eight- to ten-fold) increase in the FL-RPS27a reporter

gene activity in the presence of Sp1 and CREB. NPM,

c-Fos and c-Jun also showed a marked (four- to five-

fold) increase in the luciferase activity, whereas there

2340 FEBS Journal 281 (2014) 2339–2352 ª 2014 FEBS

Regulation of ribosomal protein genes N. Nosrati et al.

was only a moderate to no increase (approximately

two-fold) in the presence of c-ETS1, E2F1, cyclin D1

and c-Myc. In addition, an up-regulation in the

RPS27a transcript levels was also observed in the

presence of Sp1 and CREB, confirming their role in

the activation of the RPS27a gene (Fig. 2C). The

expression level of RPS27a was down-regulated by

RNA interference against Sp1, confirming the essential

role played by Sp1 in RPS27a gene stimulation

(Fig. 2D). Interestingly, the RPS24 and RPL9 genes

(with Sp1, CREB and E2F1 binding sites in their

promoters) were more responsive to Sp1 and

CREB-mediated transcriptional up-regulation com-

pared to the RPS6 and RPL4 genes (Fig. 2E).

Because Sp1 and CREB appeared to be key regula-

tors of the RPS27a promoter, these transcription

factors were used for further studies. The involvement

of Sp1 was investigated using the RPS27a promoter

deletion mutants D1-, D2- and D3-RPS27a. Although

there was no change in the D2-RPS27a reporter gene

activity in the presence of Sp1, D1-RPS27a exhibited

higher (1.5-fold) and D3-RPS27a exhibited subdued

luciferase activity compared to FL-RPS27a as a result

of the deletion of Sp1-binding sites (Fig. 3A). Next,

we analyzed the promoter occupancy of Sp1 and

CREB in IHH cells by chromatin immunoprecipitation

(ChIP)-quantitative PCR. There was a marked increase

in the recruitment of Sp1 (approximately five-fold) and

CREB (approximately 16-fold) compared to respective

mock samples (Fig. 3B). Furthermore, in agreement

with the transcriptional activation of the RPS27a gene

in the mid G1 phase (Fig. 1A), the peak promoter

occupancy of Sp1 and CREB was observed during the

mid to late G1 phase (Fig. 3C,D). As expected, the

RPL9 gene with both Sp1 and CREB binding sites in

its promoter also showed increased promoter occu-

pancy, whereas the RPS6 gene promoter, lacking these

binding sites, showed little or no promoter binding of

these factors (Fig. 3E). Thus, these results strongly

indicate that Sp1 and CREB are the key transcrip-

tional regulators of the RPS27a gene.

E2F1 acts as a negative regulator of Sp1 and

CREB-mediated transcriptional activation of

RPS27a

Sp1 and CREB are well known to act synergistically

and stimulate genes such as Chromogranin A and

Human SOX9 [22–24]. E2F1, on the other hand, can

both stimulate and suppress Sp1-regulated genes

[25,26]. Because the RPS27a gene appeared to be

responsive to Sp1 and CREB, we next investigated its

regulation by E2F1 using the FL-RPS27a reporter

gene. There was a significant inhibition in the Sp1-

and CREB-stimulated reporter gene activity in the

presence of E2F1 (Fig. 4A,B). Moreover, we observed

a synergy between Sp1 and CREB (P < 0.001) in the

stimulation of RPS27a promoter activity (Fig. 4C).

Interestingly, the E2F1-mediated inhibition was

reversed in the presence of the DE2F1 mutant that

lacks a transactivation domain. Note that E2F1 over-

expression alone did not inhibit the basal promoter

activity, suggesting context-dependent suppression of

the RPS27a gene. Quantitative RT-PCR analysis of

the endogenous RPS27a transcripts corroborated the

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Pre-rRNA

Fig. 1. Synchronized expression of the RP genes and ribosomal

RNAs during the cell cycle. The synchronized IHH cells were

stimulated with 10% serum and harvested at the indicated time

points. Total RNA was isolated from the cells and the transcript

levels of RPS27a, RPS24, RPS6, RPL9 and RPL4 genes (A) or pre-

rRNA, 18S, 5.8S and 5S rRNA genes (B) were measured by

quantitative RT-PCR. Results are represented as the mean � SD of

three independent experiments.


N. Nosrati et al. Regulation of ribosomal protein genes

reporter assay results, confirming E2F1 to be a nega-

tive regulator of the RPS27a gene, which could be

relieved in the presence of DE2F1 (Fig. 4D). Taken

together, these results strongly suggest that Sp1 and

CREB are positive transcriptional regulators, whereas

E2F1 is a negative regulator of RPS27a gene tran-

scription.

Functional interaction between Sp1 and CREB in

RPS27a promoter activation

Because the Sp1 and CREB binding sites are localized

in close proximity in the human RPS27a promoter

(Figs 2A and S3), we were interested in determining

whether the two trans-activator proteins also inter-

acted with each other for promoter activation. There-

fore, after establishing a functional interaction between

Sp1 and CREB (Fig. 4C), we probed their interaction

in co-immunoprecipitation experiments. As shown in

Fig. S4A, Sp1 and CREB apparently interacted with

each other in a hepatic microenvironment. However, it

is not clear whether there was any direct interaction

between the two transactivators or whether this was

mediated by cofactors. Nonetheless, the co-operativity

between Sp1 and CREB was substantiated by ChIP-

quantitative PCR experiments where the presence of

Luciferase

Luciferase FL-RPS27a

+1

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Δ1-RPS27aLuciferase

+1

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Fig. 2. Transcriptional regulation of the RP genes. (A) Schematic representation of the RPS27a luciferase reporter constructs: the full-length

RPS27a promoter reporter (FL-RPS27a) is shown at the top along with its three luciferase reporter deletion mutant constructs D1, D2 and

D3. The binding sites for transcription factors are shown relative to each other. +1 indicates the position of the transcriptional start site. (B)

HEK-293 cells were transfected with FL-RPS27a along with either vector control or expression plasmids for Sp1, CREB, E2F1, c-ETS1, c-

Fos, c-Jun and c-Myc as indicated. Cells were harvested after 48 h and total cell lysates were used for the luciferase assay. The relative

reporter activity was normalized with IRES2-EGFP control. (C, D) HEK-293 cells were transiently transfected either with control, Sp1 or

CREB expression vectors (C) or with Sp1, Sp1-shRNA or both Sp1 and Sp1-shRNA expression vectors (D) and total RNA was used to

measure the levels of RPS27a transcripts by quantitative RT-PCR after normalization with the GAPDH mRNA level. (E) IHH cells were

transfected with the indicated expression plasmids and total RNA was used to measure the levels of RPS24, RPS6, RPL9 and RPL4 gene

transcripts by quantitative RT-PCR. The results are represented as pooled data of three independent observations (mean � SD). *P < 0.001

versus control/mock.



one trans-activator facilitated the binding of other fac-

tor (Fig. S4B,C). Thus, these results strongly suggest a

synergism between Sp1 and CREB, both for promoter

occupancy and transactivation of the RPS27a gene.

E2F1-dependent repression involves dissociation

of transactivator proteins from the RPS27a gene

promoter

To clarify the molecular mechanism of E2F1-mediated

inhibition of the RPS27a promoter, we titrated the

Sp1-dependent promoter activation using increasing

amounts of the E2F1 expression vector. As expected,

we observed dose-dependent inhibition in RPS27a

luciferase activity in the presence of E2F1 (Fig. 5A).

Intriguingly, there was a 16-fold increase in the occu-

pancy of Sp1 on the promoter in presence of E2F1

(Fig. 5B). Notably the endogenous E2F1 showed an

approximately ten-fold enrichment on the promoter

compared to mock samples (Fig. S5). Because the ini-

tial binding of Sp1 to a gene promoter is essential for

the E2F1-mediated recruitment of co-repressors [27],

we next monitored the recruitment of transcriptional

co-repressors such as retinoblastoma (Rb) and histone

deacetylase 1 (HDAC1) on the RPS27a promoter in

the presence of E2F1. Overexpression of E2F1 led to a

significant five-fold increase in Rb enrichment and an

approximately two-fold increase in HDAC1 binding to

the RPS27a promoter, and these were reversed in the

presence of DE2F1 mutant (Fig. 5C,D). Furthermore,

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Fig. 3. Promoter occupancy and

localization of binding sites for

transcription factors on the RP gene

promoters. (A) HEK-293 cells were

transiently transfected with FL-RPS27a or

its deletion mutants D1, D2 and D3

RPS27a, along with either control or Sp1

expression vector. Cells were harvested

after 48 h and total cell lysates were used

for the luciferase assay. The relative

reporter activity was determined after

normalizing with IRES2-EGFP control. (B)

The promoter occupancy of Sp1 and

CREB was analyzed in asynchronously

growing IHH cells by ChIP-quantitative

PCR. CDK2 promoter served as a positive

control. (C, D) Synchronized IHH cells

were analyzed for the occupancy of Sp1

and CREB on RPS27a promoter at the

indicated time points by ChIP-quantitative

PCR after serum stimulation. (E) The

promoter occupancy of Sp1, CREB and

E2F1 was analyzed in asynchronously

growing IHH cells. The results represent

the pooled data from three independent

experiments (mean � SD). *P < 0.001

versus control/mock.



a prior promoter occupancy of E2F1 was essential for

Sp1 binding because there was a marked reduction in

Sp1 enrichment, as shown by the ChIP assay, even

though the E2F1 occupancy largely remained unaf-

fected (Fig. 5E). By contrast, Sp1 knockdown by

RNA interference had no bearing on the promoter

recruitment of E2F1 (Fig. 5F). Apparently, E2F1

appeared to act like a repressor without affecting the

promoter occupancy of activators.

Recruitment of histone acetyltransferases (HATs)

and chromatin remodeling near the RPS27a

promoter

Histone acetylation is associated with euchromatin and

is linked to transcriptional activation. Therefore, the

recruitment of HATs should facilitate the binding of

transcription factors to the RPS27a promoter and,

eventually, chromatin remodeling. Therefore, we aimed

to decode the histone modification marks that are

known to play an exclusive role in gene regulation.

Our ChIP studies showed a significant enrichment of

HATs on the RPS27a promoter. Although P300 and

CREB binding protein (CBP), respectively, showed

moderate 1.2- and 1.5-fold increases in promoter occu-

pancy, PCAF exhibited a significant four-fold increase

in promoter binding (Fig. 6A). Interestingly, the acety-

lation patterns of histone H4 pan-acetyl (H4ac) and

histone H3 acetyl Lys 14 (H3K14ac) near the RPS27a

promoter appeared to coincide with gene transcription

during the G1 phase of the cell cycle (Fig. 6B). Unex-

pectedly, the methylation pattern of histone H3

trimethyl Lys 9 (H3K9me3) also peaked around the

same time. Taken together, these results strengthen

our argument that the RPS27a gene is specifically

transcribed during the mid to late G1 phase of the cell

cycle.

Discussion

The ribosome is a complex cellular machine that serves

as a primary site of protein biosynthesis. In eukary-

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Fig. 4. Cooperation between transcription factors in the regulation of RPS27a promoter. (A, B) HEK-293 cells were transfected with FL-

RPS27a luciferase reporter along with Sp1, CREB, E2F1 or DE2F1 either alone or in indicated combinations (in a 1 : 1 ratio) and luciferase

activity was measured. The reporter activity was normalized with vector control. (C) The co-operation between Sp1 and CREB in RPS27a

promoter regulation was investigated by transiently transfecting HEK-293 cells with FL-RPS27a luciferase reporter construct along with

control or Sp1 expression plasmid (500 ng) alone or with an increasing concentration of CREB expression plasmid (0.5, 1, 1.5 and 2 lg) and

luciferase activity was measured. (D) Total RNA was isolated from HEK-293 cells transfected with the indicated transcription factors and the

levels of RPS27a transcripts were measured by quantitative RT-PCR. The results represent the pooled data from three independent

experiments (mean � SD). *P < 0.001 and **P < 0.05 versus control.



otes, ribosome biogenesis begins with the synthesis of

large primary RNA transcripts or pre rRNA by RNA

polymerase I in the nucleolus and RP synthesis in the

cytoplasm, followed by their processing and assembly

into mature ribosomal subunits. Although ribosome

biogenesis is highly dependent on the timely synthesis

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Fig. 5. Regulation of the Sp1-dependent stimulation of the RPS27a gene by E2F1. (A) HEK-293 cells were transiently transfected with FL-

RPS27a luciferase reporter construct along with control or Sp1 expression plasmid (500 ng) alone or with an increasing concentration of

E2F1 expression plasmid (0.5, 1, 1.5 and 2 lg) and luciferase activity was measured. (B) IHH cells were transiently transfected with E2F1

expression vector (5 lg) and the RPS27a promoter occupancy of Sp1 was measured in asynchronously growing cells by ChIP-quantitative

PCR. (C, D) IHH cells were transiently transfected either with E2F1 or DE2F1 expression vector (5 lg each) and the promoter occupancy of

Rb and HDAC1 was measured. (E) IHH cells were transfected with DE2F1 (5 lg) and the promoter occupancy of Sp1 and E2F1 was

measured in asynchronously growing cells. (F) IHH cells were transfected with vector or Sp1-shRNA (5 lg), synchronized and analyzed 20 h

after serum stimulation for the promoter occupancy of Sp1 or E2F1 by ChIP-quantitative PCR. Data represent the mean � SD performed in

triplicate. *P < 0.001; **P < 0.05 versus control mock.



of RPs, there are no reports to suggest a synchronized

synthesis of RPs and linking that to ribosome biogene-

sis [28–31]. Studies on RP genes have suggested the

role of basal transcriptional machinery in their regula-

tion [20,21,32]. The present study focused on the tran-

scriptional regulation of RPS27a gene during the cell

cycle as a prototype RP gene. The small RP, RPS27a,

is an important component of the small ribosomal

subunit that, along with other RPs and rRNAs, consti-

tutes the scaffold of the ribosome [33]. It is reported to

play an essential role in the activation of cellular

checkpoints such as p53 expression under stress condi-

tions linking ribosome biogenesis to cell cycle progres-

sion [34,35].

The present data suggested that the distal regulatory

region of the RPS27a gene promoter (�940 to

�640 bp), which harbors binding sites for some key

transcription factors, played an important role in the

gene regulation. Therefore, we were interested in deter-

mining whether a similar regulatory mechanism was

operational in other RP gene promoters. Sequence

homology analysis of the human RPS27a gene pro-

moter (1640 bp) with 74 other RP gene promoters

(approximately 2000 bp) using ENSEMBL GENOME BROW-

SER displayed a striking homology. Furthermore, the

TFSEARCH analyses of the transcription factors binding

sites on selected 31 RP gene promoters indicated a

strong conservation of Sp1, CREB and E2F1 binding

sites (Fig. S2). Accordingly, we investigated the regula-

tion of RPS24, RPL9, RPS6 and RPL4 genes to test

our hypothesis of a cell cycle-dependent and coordi-

nated expression of the RPs. We observed that the

expression of RP genes was specifically induced during

the mid to late G1 phase of the cell cycle (Fig. 1A),

most likely to ensure the availability of RPs during

ribosome biogenesis. Interestingly, the transcription of

rRNA genes preceded the expression of RP genes

during the G1 phase of cell cycle (Fig. 1). These data

strongly suggest that the regulation of ribosomal com-

ponents in a synchronized and cell cycle-dependent

manner.

To identify the transcription factors involved RP

gene regulation, we studied the regulation of RPS27a

gene in detail. Bioinformatics analysis of the promoter

sequence of human RPS27a gene predicted binding

sites for some key transcription factors such as Sp1,

CREB and E2F1 (Figs 2A and S3). Our transcrip-

tional and reporter gene studies corroborated a signifi-

cant contribution of Sp1 and CREB in the positive

regulation of RP genes. The deletion of their binding

sites led to a decrease in reporter gene activity

(Fig. 3A). The stimulatory role of Sp1 and CREB was

also evident from an increased promoter occupancy of

these factors in ChIP assays (Fig. 3B) and a specific

reduction in RPS27a gene transcription by RNA inter-

ference (Fig. 2D). Interestingly, the RP promoter not

harboring SP1 and CREB regulatory sites in the pro-

moter responded poorly to these factors and also

showed no enrichment of SP1 or CREB (Fig. 3E).

Furthermore, the binding kinetics of Sp1 and CREB

on the RPS27a promoter showed an overlap with the

gene expression during the late G1 phase of the cell

cycle (Fig. 3C,D). Note that Sp1 regulates the expres-

sion of many cell cycle-specific genes and their levels

fluctuate with Sp1 expression during the G1 phase

[36]. Furthermore, Sp1 is well known to interact with

A B

*

0

1

2

3

4

5

6

p300 CBP pCAF

Fold

enr

ichm

ent

Mock

HAT

**

*

0

4

8

12

16

20

24

Fold

enr

ichm

ent

Binding post serum stimulation (h)

H4acH3K14acH3K9 me3

G0 G1 S

0 30

Fig. 6. Epigenetic modifications on the RPS27a gene promoter. (A) The RPS27a promoter occupancy of histone acetyl transferases was

analyzed in asynchronously growing IHH cells by ChIP-quantitative PCR. The cell lysates were immunoprecipitated either with control serum

(Mock) or with anti-P300, anti-CBP or anti-PCAF sera. (B) Synchronized IHH cells were also analyzed for H4ac, H3K14ac and H3K9me3

occupancy on the RPS27a promoter at the indicated time points after serum stimulation. The result represents pooled data from three

independent experiments (mean � SD). *P < 0.001; **P < 0.05 versus control/mock.



many co-activators, co-repressors and transcription

factors, which may account for its divergent functions.

Therefore, an increase in the recruitment of Sp1 on the

RPS27a promoter may facilitate the recruitment of

HATs, leading to enhanced gene expression [37].

As reported previously [23,24], we also observed a

synergy between Sp1 and CREB in the activation of

the RPS27a gene (Fig. 4) and promoter occupancy of

the two key factors also exhibited interdependence;

however, this point needs to be investigated further.

Nevertheless, our co-immunoprecipitation studies also

indicated an interaction between Sp1 and CREB,

which may be responsible for their co-operative action

in target gene activation (Fig. S4). Similarly, the inter-

action between Sp1 and E2F1 could also play an

important role in the regulation of the RPS27a gene

[27,38,39]. Accordingly, there was an inhibition of

Sp1-dependent RPS27a promoter activation in the

presence of E2F1, which could be alleviated after the

co-expression of a dominant negative DE2F1 mutant

that lacks a transactivation domain (Figs 4A,C and

5A). The expression of E2F1 alone in the cell does not

exhibit similar regulation, indicating that its suppres-

sive nature is unraveled only in case of Sp1 and CREB

overexpression; thus, E2F1 could be an important

checkpoint regulator for controlling the synthesis of

RP genes. Apparently, this observation was qualita-

tively different from an earlier study in which super

activation of Sp1 by E2F1 was observed in the case of

dhfr gene expression [40]. Therefore, such differences

may represent a promoter-specific phenomenon.

Although E2F1 negatively regulated the Sp1-medi-

ated transcriptional activation of the RPS27a gene, it

did not interfere (and rather enhanced) Sp1 binding

to the RPS27a promoter (Fig. 5B). Nevertheless, the

transcriptional activity of Sp1 was hampered as a

result of the increased recruitment of negative regula-

tors such as Rb and HDACs. The enrichment of Rb

and HDAC1 on the RPS27a promoter was inhibited

in the presence of DE2F1 mutant (Fig. 5C,D), indi-

cating a specific requirement of E2F1 in the recruit-

ment of co-repressors. Interestingly, the enrichment

of Sp1 on the RPS27a promoter was abrogated in

the presence of the DE2F1 mutant, suggesting the

important role played by E2F1 in regulating Sp1

binding on the promoter (Fig. 5E). Notably, the

expression of DE2F1 drastically increased the occu-

pancy of E2F1 on the RPS27a promoter because this

still carries the DNA binding domain. Although

E2F1 enhanced the recruitment of Sp1 on the

RPS27a promoter (Fig. 5B), Sp1 knockdown had no

effect on E2F1 recruitment (Fig. 5F). These results

are in agreement with earlier studies indicating the

importance of E2F1 with respect to keeping a check

on the transcription activity of some promoters [27].

As in the case of Sp1, E2F1 co-expression led to a

down-regulation in CREB-mediated transcriptional

activation, which was reversed in the presence of

DE2F1 (Fig. 4B). Thus, these findings are in agree-

ment with the findings of a previous study [27] sug-

gesting that E2F1 can recruit transcriptional

suppressors such as Rb and HDAC1 to promoter

regions and inhibit gene transcription.

The positive regulation of the RPS27a gene expres-

sion apparently coincided with the promoter recruit-

ment of HATs such as PCAF, P300 and CBP, with

PCAF as the major player (Fig. 6A). Our epigenetic

studies indicated both acetylation (H4ac, H3K14ac)

and methylation (H3K9me3) marks on the RPS27a

promoter (Fig. 6B). Expectantly, the histone tails were

maximally acetylated at the G1 phase, correlating with

maximal RPS27a expression. However, a concomitant

histone methylation, which normally coincides with

transcription repression, was a rather unexpected

observation. However, the H3K9me3 signature may

not necessarily be an exclusive mark of heterochroma-

tin because this has been reported for some actively

transcribed mammalian genes, such as MHC class II,

b-major, Band 3 and AHSP [41,42]. Therefore, it will

be interesting to determine the role of histone methyla-

tion in RPS27a gene expression.

The data reported in the present study strongly sug-

gest that the regulatory mechanism established here

for the human RPS27a gene could be extrapolated to

other RP gene promoters. Our bioinformatic analyses

support the hypothesis that a common regulatory

network may be in place, which would allow the

synchronized expression of RP genes and the assembly

of ribosomes to prepare cells for cell division (Fig. 7).

Synchronized synthesis of rRNA in the same phase of

the cell cycle further strengthens the argument that all

of the raw material for ribosome biogenesis is

expressed in a coordinated fashion.

Materials and methods

Chemicals and antibodies

All of the restriction enzymes used were obtained from Fer-

mentas (Burlington, Canada). Protein-A or A/G sepharose

and protease inhibitor cocktail were obtained from Amer-

sham Biotech (Little Chalfont, UK). The luciferase assay

system was obtained from Promega (Madison, WI, USA).

DMEM was obtained from Gibco (Gaithersburg, MD,

USA). Thymidine and SYBR Green were obtained from

Sigma-Aldrich (St Louis, MO, USA).



Antibodies for Sp1, CREB, E2F1, Rb, P300, CBP and

PCAF were purchased from Santa Cruz Biotechnologies

(Santa Cruz, CA, USA). Antibodies for HDAC1, H4ac,

H3K14ac and H3K9me3 were obtained from Upstate Bio-

technology (Lake Placid, NY, USA).

Cloning, expression vectors and reporter

constructs

The development of expression vectors for the human

RPS27a and c-Myc genes has been described previously

[14,43]. IRES-EGFP and pEGFP-N3 were procured from

Clontech, (Palo Alto, CA, USA), pSilencer 1.0-U6 was

from Ambion (Austin, TX, USA). The expression vectors

for wild-type E2F1 (pCMV-E2F1) and its transactivation

defective mutant pCMV-E2F1-DC (1–374) were kind gifts

from X. Lu [44]. The expression vectors for c-ETS1, c-Jun

and c-Fos, nucleophosmin 1, cyclin D1, CREB and pFlag-

Sp1(1-778)-HA were kindly provided by H. Sugimoto [45],

A. Weisz (University of Napoli, Naples, Italy), M. Komad-

a [46], M. E. Ewen [47], C. Vinson [48] and M. Spengler

[49], respectively.

The full-length RPS27a reporter construct (FL-RPS27a)

was developed by PCR amplifying a 1.6-kb promoter

region using the human genomic DNA as template and

primers 1F and 1R (Table S1). The DNA fragment was

cloned into the pGL3 basic luciferase vector between SmaI

and HindIII sites (Promega). Three deletion mutants of

RPS27a promoter were derived from the FL-RPS27a

reporter construct using a combination of restriction

enzymes (D1, NheI/NdeI; D2 NheI/SpeI and D3, PstI). All

of the RPS27a reporter constructs were verified by restric-

tion digestion and DNA sequencing.

Cell culture and transfection

The IHH cell line was kindly provided by F. Danniel [50].

The human embryonic kidney HEK-293 cells (ATCC

E2F1

A

B

Me

H3K9me3 e.g., H3K14ac,H4ac

RP gene promoters

Histone tailN

P300 CBP

PCAF

RPs

Small subunit

Large subunit

RPs

Sp1

S

G1

M

G2

Sp1

CREBCREB

Me Ac

Ac

Ac

S

G1

M

G2

RP gene promoters

E2F1

Histone tailN

P300 CBP

PCAFSmall subunitLarge

subunit

Sp1 Sp1

CREBCREB

Rb Rb

Fig. 7. Model depicting the regulation of

RP genes during the cell cycle. (A) During

late G1 phase, the RP gene promoters are

activated by binding of specific

transcription factors such as Sp1, CREB

and E2F1 to their respective binding

elements. This facilitates the recruitment

of HATs such as P300, CBP and PCAF,

leading to the opening up of nucleosomal

DNA by epigenetic modifications of

histones such as H4ac, H3K14ac and

H3K9me3 and activation of RP gene

transcription. Post-translationally, the RPs

are translocated into the nucleolus where

the assembly of both subunits of

ribosomes takes place along with their

constituent ribosomal RNAs. (B) After a

threshold level of RPs has been achieved,

a negative feedback mechanism activates

E2F1 that facilitates the recruitment of

HDAC1 and co-repressors such as Rb on

the promoter. This results in the

dissociation of HATs from chromatin and

the removal of the epigenetic marks

causing the inhibition of RP gene

expression.



CRL-1573) and IHH cells were maintained in complete med-

ium comprising DMEM with antibiotics (penicillin/strepto-

mycin) and 10% fetal bovine serum (HyClone, Logan, UT,

USA) at 37 °C in a humidified incubator with 5% CO2. Cells

were transiently transfected with Lipofectamine (Invitrogen,

Carlsbad, CA, USA) in accordance with the manufacturer’s

instructions. In general, cells were transfected at a density of

0.6 9 106 or 1.5 9 106 (approximately 60%) in 60- or 100-

mm culture dishes with 2 or 5 lg, respectively, of the indi-

cated plasmid DNA or small hairpin RNAs (shRNAs).

IRES-EGFP, EGFP-N3 and pSilencer 1.0-U6 were used as a

vector and transfection controls in parallel set of experi-

ments. Typically, a 60–70% cell transfection level was

accomplished in these experiments. Because HEK293 cells

gave better transfection efficiency compared to IHH, all of

the luciferase and quantitative RT-PCR experiments were

performed using HEK293 cells.

Cell synchronization and cell cycle analysis

IHH cells were synchronized by serum starvation. Cells

were first serum starved for 72 h followed by incubation

with complete medium for the indicated time points and

then harvested for further analysis.

Cell cycle analysis was performed as described previously

[51]. Briefly, cells were washed with NaCl/Pi (91), fixed in

70% ethanol and stained with propidium iodide

(50 mg�mL–1). The cell suspension was analyzed by FACS

analysis using a FACSCalibur flow cytometer (BD Biosci-

ence, San Jose, CA, USA). Percentages of cell cycle distri-

bution were determined manually.

ChIP and quantitative RT-PCR

The ChIP assay was carried out as described previously

[51]. Briefly, IHH cells were cross-linked with formalde-

hyde (1%), lysed and sonicated over ice (seven pulses at

30% amplitude) and centrifuged at 15 000 g. for 10 min

to obtain the supernatant. Samples were pre-cleared for

1–2 h with protein A-Sepharose beads and incubated

overnight with specific antibodies. The immune complexes

were pulled down using protein A-Sepharose beads. After

a series of washing steps, the beads were extracted in

500 lL of elution buffer (0.1 M NaHCO3, 1% SDS) and

analyzed by SYBR green quantitative RT-PCR with

RPS27a ChIP primers 3F and 3R (Table S1). CDK2 pro-

moter was used as control in these experiments using

ChIP primers 4F and 4R (Table S1). The ChIP primers

for studying promoter occupancy of RPL4, RPL9, RPS6

and RPS24 genes are also given in Table S1. The PCR

amplified products for RPS27a and CDK2 promoters

were 450 and 208 bp, respectively. Data were normalized

with input DNA and expressed as fold enrichment over

mock. The results were analyzed by using the comparative

DDCT method [52].

Immunoprecipitation and western blot analysis

Cell lysates were prepared in cell lysis buffer (Promega).

The protein concentration was determined in the cleared ly-

sates using Bradford reagent and equal amounts of protein

were incubated with the indicated antibodies overnight at

4 °C. Protein A sepharose beads were added and further

incubation was carried out at 4 °C. Subsequently, the beads

bound to protein complexes were washed with lysis buffer

and then re-suspended in 29 SDS dye, boiled and eluates

were resolved on 10–15% SDS/PAGE followed by western

blotting [53]. The protein bands were visualized using elect-

rochemiluminescence or Fluorchem (Protein Simple, Santa

Clara, CA, USA).

Luciferase assay

Luciferase assay was performed in accordance with the

manufacturer’s instructions (Promega). Relative luciferase

activity was measured after normalizing each sample with

the protein amount and transfection efficiency.

RNA isolation and quantitative RT-PCR assay

Total RNA was isolated from cells using TRIzol reagent in

accordance with the manufacturer’s instructions (Invitro-

gen). RT-PCR was performed with M-MuLV reverse trans-

criptase (Fermentas) in accordance with the manufacturer’s

instructions. Quantitative RT-PCR was carried out using

specific primers for the transcripts of RPS27a (2F and 2R),

RPL4, RPL9, RPS6 and RPS24 genes (Table S1). The

sequence of oligonucleotide primers for r-RNA 5S, 5.8S,

18S and pre-rRNA transcripts is also provided in Table S1.

The quantitative RT-PCR results were analyzed using the

comparative DDCT method [52].

Bioinformatic analysis

TFSEARCH software relating to the TRANSFAC database

was used to predict transcription factor binding sites on the

RPS27a promoter sequence (http://www.cbrc.jp/research/

db/TFSEARCH.html). ENSEMBL GENOME BROWSER was used

for comparisons of human RPS27a promoter and other

human RPs promoter sequences (http://www.ensembl.org).

Statistical analysis

Data are expressed as the mean � SE. Statistical signifi-

cance was calculated using Student’s t-test. P < 0.05 was

considered statistically significant.

Acknowledgements

This work was supported in part by a J .C. Bose

National Fellowship (Grant Number SR/S2/JCB-80/



http://www.cbrc.jp/research/db/TFSEARCH.html

http://www.cbrc.jp/research/db/TFSEARCH.html

http://www.ensembl.org

2012) from the Department of Science and Technol-

ogy, Government of India, New Delhi (to V.K.). We

thank our colleagues, Vijaya Pandey, Kaveri Sidhu

and Richa Ahuja, for helpful discussions. We are

grateful to the following scientists for kindly providing

us the expression vectors for key regulatory proteins:

Dr Xin Lu (Ludwig Institute for Cancer Research,

Cambridge, UK) for wild-type E2F1 (pCMV-E2F1)

and its transactivation defective mutant pCMV-E2F1-

DC (1–374); Dr Hiroyuki Sugimoto (Dokkyo Medical

University School of Medicine, Mibu, Japan) for c-

ETS1; Dr A. Weisz (University of Napoli, Naples,

Italy) for c-Jun and c-Fos; Dr Masayuki Komada

(Tokyo Institute of Technology, Yokohama, Japan)

for NPM1; Dr M. E. Ewen (Dana-Farber Cancer

Institute, Boston, MA, USA) for cyclin D1; Dr

Charles Vinson (National Institute of Health, Beth-

esda, MD, USA) for CREB and Dr M. Spengler (Ros-

well Park Cancer Institute, Buffalo, NY, USA) for

Sp1. We thank Pallavi Rajput (ICGEB, New Delhi)

for Sp1-shRNA. The IHH cell line was kindly pro-

vided by F. Danniel (Institut National de la Sant�e et

de la Recherche M�edicale Unite 481, Universite Paris

7, Paris, France). Technical assistance by R. Kumar

and T. Choedon is gratefully acknowledged. Nagisa

Nosrati received a pre-doctoral fellowship from the

International Centre for Genetic Engineering and Bio-

technology (ICGEB) and Neetu Rohit Kapoor

received a fast track fellowship for young investigators

from the Department of Science and Technology

(DST), Government of India.

Author contributions

Nagisa Nosrati performed the experiments and pre-

pared the manuscript. Neetu Kapoor supervised and

contributed to the experiments. Vijay Kumar designed

the experiments, arranged funds and finalized the

manuscripts.

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Supporting information

Additional supporting information may be found in

the online version of this article at the publisher’s web

site:Fig. S1. Cell cycle analysis of IHH cells.

Fig. S2. Schematic representation of the promoter

region of 32 different RP genes.

Fig. S3. DNA sequence of the human RPS27a gene

promoter.

Fig. S4. Interaction between Sp1 and CREB and their

occupancy on the RPS27a promoter.

Fig. S5. Occupancy of E2F1 on the RPS27a gene pro-

moter.

Table S1. Oligonucleotide primers.



combinatorial action of transcription factors orchestrates cell cycle-dependent expression of the...

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