doi:10.1182/blood-2006-03-012013Prepublished online September 26, 2006;   

 Vladimir Lazar, Philippe Dessen, Roberto Mantovani, Luc Aguilar and Jean-Philippe GirardCorinne Cayrol, Chrystelle LaCroix, Catherine Mathe, Vincent Ecochard, Michele Ceribelli, Emilie Loreau, through modulation of pRB/E2F cell cycle target genesThe THAP-zinc finger protein THAP1 regulates endothelial cell proliferation

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The THAP-zinc finger protein THAP1 regulates endothelial cell

proliferation through modulation of pRB/E2F cell cycle target genes

Corinne CAYROL*, Chrystelle LACROIX*, Catherine MATHE*, Vincent ECOCHARD*,

Michele CERIBELLI§, Emilie LOREAU+, Vladimir LAZAR#, Philippe DESSEN#, Roberto

MANTOVANI§, Luc AGUILAR+ and Jean-Philippe GIRARD*§

*Laboratoire de Biologie Vasculaire, Equipe labellisée ‘La Ligue 2006’, Institut de

Pharmacologie et de Biologie Structurale, CNRS UMR 5089, 205 route de Narbonne, 31077

Toulouse, France ; +ENDOCUBE, Prologue Biotech, BP700, rue Pierre et Marie Curie, 31319

Labège cedex, France ; #Unité de Génomique Fonctionnelle et Bioinformatique, Institut Gustave

Roussy, Pavillon de Recherche 2, 39 rue Camille Desmoulins, 94805 Villejuif, France ;

Dipartimento di Scienze Biomolecolari e Biotecnologie, Università di Milano, Via Celoria 26,

20133 Milano, Italy.

Running head: THAP zinc finger protein in EC proliferation

Scientific category: Hemostasis, Thrombosis and Vascular Biology

Word count : text, 4977 words ; abstract, 200 words

This work was supported by grants from Ligue Nationale contre le Cancer (Equipe labellisée),

ANR-programme blanc “Cuboïdale” and Ministère de la Recherche Action Concerté Incitative-

Cancéropôle Grand Sud-Ouest 2004 (Network ‘Angiogenesis and Invasion’).

§ Correspondence should be addressed to Dr. Jean-Philippe Girard, IPBS-CNRS UMR 5089,

205 route de Narbonne, 31077 Toulouse, France; Phone: 33-5-61-17-59-67, Fax: 33-5-61-17-

59-94, e-mail: Jean-Philippe.Girard@ipbs.fr

Blood First Edition Paper, prepublished online September 26, 2006; DOI 10.1182/blood-2006-03-012013

Copyright © 2006 American Society of Hematology

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Author Contribution Statement

Corinne Cayrol, performed research and analyzed data (cell proliferation/cell cycle

analyses, apoptosis assays, antibody generation and validation, western blot

and immunofluorescence analyses, qPCR experiments, retroviral transduction, RNA

interference, ChIP-qPCR assays); Chrystelle Lacroix, performed research (ChIP assays);

Catherine Mathe, performed research (microarray data mining, bioinformatics); Vincent

Ecochard, performed research (in vitro DNA-binding assays); Michele Ceribelli, performed

research (ChIP assays); Emilie Loreau, performed research (qPCR experiments); Vladimir

Lazar, performed research (microarray experiments at the IGR Microarray Platform); Philippe

Dessen, performed research (bioinformatics analyses at the IGR Microarray Platform); Roberto

Mantovani, analyzed data (supervised ChIP assays); Luc Aguilar, analyzed data; Jean-Philippe

Girard, performed research (Northern blot), designed research, analyzed data and wrote the

manuscript.

Abstract

We recently cloned a novel human nuclear factor (designated THAP1) from post-capillary

venule endothelial cells (ECs), that contains a DNA-binding THAP domain, shared with

zebrafish E2F6 and several Caenorhabditis elegans proteins interacting genetically with pRB.

Here, we show that THAP1 is a physiological regulator of EC proliferation and cell cycle

progression, two essential processes for angiogenesis. Retroviral mediated-gene transfer of

THAP1 into primary human ECs inhibited proliferation, and large scale expression profiling

with microarrays revealed that THAP1-mediated growth inhibition is due to coordinated

repression of pRB/E2F cell cycle target genes. Silencing of endogenous THAP1 through RNA

interference similarly inhibited EC proliferation and G1/S cell cycle progression, and resulted in

downregulation of several pRB/E2F cell cycle target genes, including RRM1, a gene required for

S-phase DNA synthesis. Chromatin immunoprecipitation assays in proliferating ECs showed

that endogenous THAP1 associates in vivo with a consensus THAP1-binding site found in the

RRM1 promoter, indicating that RRM1 is a direct transcriptional target of THAP1. The similar

phenotypes observed after THAP1 overexpression and silencing suggest that an optimal range of

THAP1 expression is essential for EC proliferation. Together, these data provide the first links

in mammals between THAP proteins, cell proliferation and pRB/E2F cell cycle pathways.

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Introduction

In the adult, endothelial cells (ECs) normally are quiescent and play an important role in

maintaining the integrity of blood vessels1. However in several physiological conditions (e.g.

ovarian and uterine vascular beds) and diverse pathological conditions such as cancer,

rheumatoid arthritis, diabetic retinopathy and atherosclerosis, ECs have the ability to leave their

normal quiescent state, reenter the cell cycle, proliferate and form neo-vessels in a process called

neovascularization or angiogenesis2-4. A better understanding of the molecular mechanisms and

factors controlling EC proliferation and cell cycle progression may therefore lead to novel

therapeutic approaches for the control of angiogenesis-dependent diseases, including cancer and

chronic inflammatory diseases.

Whereas many secreted angiogenic growth factors (including VEGF and FGF family

members), angiogenesis inhibitors (angiostatin, endostatin, …) and their corresponding

receptors, have been described which modulate EC growth and survival during angiogenesis2-4,

comparatively little is known about the nuclear transcription factors involved in the control of

EC proliferation and cell cycle progression5. We recently identified a novel nuclear factor in a

cDNA library from post-capillary high endothelial venule ECs (HEVEC), isolated from human

tonsils6. This factor, designated THAP1, is the prototype of a previously uncharacterized family

of cellular factors, the THAP proteins (> 100 distinct members in the animal kingdom), defined

by the presence at their amino-terminus of an evolutionarily conserved ~ 90-residues protein

motif, the THAP domain7. We demonstrated that the THAP domain of THAP1 is an atypical

zinc-dependent sequence-specific DNA-binding domain belonging to the zinc finger

superfamily8. We also showed that the THAP domain shares its metal-coordinating C2CH

signature (CX2-4C-X35-53-CX2H) with the site-specific DNA-binding domain of Drosophila P

element transposase, indicating that the THAP domain constitutes a novel example of a DNA-

binding domain shared between cellular proteins and transposases from mobile genomic

parasites7,8.

Although orthologous relationships with the human THAP proteins were not obvious,

analysis of THAP proteins in model animal organisms gave interesting clues to the functions of

these proteins in vivo8. First, in zebrafish and two other unrelated fish species, the orthologue of

cell cycle transcription factor E2F6 was found to contain a THAP domain at its amino-terminus

which exhibits significant homologies with the THAP domain of human THAP18. Although

E2F6 does not bind pRB family members, it functions as a repressor of E2F-dependent

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transcription during S phase that is critical to distinguish G1/S and G2/M transcription during

the cell cycle9. The identification of the THAP-E2F6 fusion gene in fish species provided the

first link in vertebrates between the THAP proteins and the pRB/E2F pathway, and

complemented nicely previous findings in Caenorhabditis elegans that have revealed the

existence of genetic interactions between LIN-35/Rb, the sole C. elegans retinoblastoma

homolog, and four distinct C. elegans THAP proteins (LIN-36, LIN-15B, LIN-15A and HIM-

17), initially characterized for their role in the specification of vulval cell fates (synthetic

Multivulva genes, synMuv) or meiotic chromosome segregation10-13. Among these, LIN-36 and

LIN-15B, have been found to function as inhibitors of the G1/S cell cycle transition12. LIN-36

behaved most similar to LIN-35/Rb and Efl-1/E2F12,14, the orthologue of mammalian cell cycle

transcription factors E2F4/515, and was therefore proposed to act in a pathway or complex with

LIN-35/Rb and Efl-1/E2F, to repress G1/S control genes12 16. Finally, a third C. elegans THAP

protein, GON-14, was recently shown to play a critical role in cell proliferation and development

since gon-14 null mutants were found to exhibit larval growth arrest associated with cell

division defects in intestine, gonad and vulva 17. Together, these observations in zebrafish and

C. elegans suggested important roles for THAP proteins in cell proliferation and cell cycle

control.

Although several protein partners of THAP1, THAP0/DAP4 and THAP7 have been

identified6,18-20, the biological roles of human THAP proteins remain largely unknown and

potential functions in cell proliferation or cell cycle regulation have not yet been described. In

the present manuscript, we show that THAP1 is an endogenous physiological regulator of EC

proliferation and G1/S cell cycle progression, which modulates expression of several pRb/E2F

cell cycle target genes. In addition, we identify RRM1, a G1/S-regulated gene required for S-

phase DNA synthesis, as a direct transcriptional target of endogenous THAP1.

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Materials and Methods

Retroviral vector construction, virus packaging, and transduction of primary

human ECs. A THAP1 retroviral expression vector (pMLV-THAP1) was generated using a

pseudotyped vesicular stomatitis virus G protein (VSV-G)-Moloney murine leukaemia virus

(MuLV)-derived retrovirus vector which allows very efficient transduction (> 90 %) of

proliferating HUVECs. The multiple-cloning site of retroviral vector pBullet21 was modified by

incorporation of synthetic oligonucleotides containing NaeI and MfeI restriction sites, between

the NcoI and BamHI sites of the polylinker. The modified vector was called pMLV-MCS. The

full length coding region of human THAP16 was amplified by PCR with primers: THAP1-5’ (5’-

ATGGTGCAGTCCTGCTCCGC-3’) and THAP1-MfeI-3’ (5’-

GCCAATTGTTATGCTGGTACTTCAACTATTT-3’), and cloned into the pMLV-MCS vector

cleaved with NaeI and MfeI restriction enzymes, to generate the pMLV-THAP1 retroviral

expression vector. Retroviral vectors were produced by transient triple transfection of the

packaging plasmid (pVPack-GP, Stratagene), the envelope plasmid (pVPack-VSV-G,

Stratagene) and the transducing vectors pMLV-MCS or pMLV-THAP1, in 293T cells (ATCC

No. CRL11268, ATCC, Rockville, MD), using a DNA-calcium phosphate procedure. Cell

supernatants containing viral particles were harvested 48 hours after the transfection, clarified of

cell debris using low-speed centrifugation and filtered on 0.45 µm filters. A total of 106 primary

HUVECs were transduced in a 75 cm2 plate with viral supernatant in the presence of 8 µg/ml of

polybren (Sigma) as previously described22. After 4 hours, the supernatant was replaced by fresh

endothelial cell medium consisting of MCDB131 (Invitrogen) supplemented with 20% heat-

inactivated serum, endothelial cell growth factor (ECGS, Sigma Chemical Co.) and 5 U/ml

sodium heparin. When applicable, a second transduction was performed using the same protocol

a day after the first transduction. Forty-eight hours after infection, cells were trypsinized and

pelleted for RNA preparation. Total RNA was isolated from 106 cells with the Absolutely RNA

miniprep kit according to manufacturer’s instructions (Stratagene, La Jolla, CA, USA).

Antibody production, Western blotting and immunofluorescence microscopy.

Affinity-purified rabbit polyclonal antibodies were raised against the peptide

FQKEKDDVSERGYVI, corresponding to amino acids 186-200 of the human THAP1 sequence

(Eurogentec). For western blotting, lysates from retrovirally-transduced HUVECs or siRNA-

treated HUVECs (each corresponding to ~105 cells) were fractionated by sodium dodecyl

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sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10%). Detection was performed with

rabbit antiserum to THAP1 (1/500) or mouse antibody to α-tubulin (1/20000; Sigma), followed

by HRP-conjugated goat anti-rabbit or anti-mouse Ig (1/10000; Promega), and finally an

enhanced chemiluminescence kit (Amersham Bioscience). Indirect immunofluorescence staining

was performed as previously described6, with rabbit polyclonal anti-THAP1 (1/500) or mouse

monoclonal anti-Ki-67 (1/250; clone B56; BD Pharmingen) antibodies.

Analysis of DNA synthesis by BrdU labeling, FACS and TUNEL assays. DNA

synthesis in retrovirally transduced- or siRNA-treated HUVECs was analyzed by measuring

BrdU incorporation as described previously23. For cell cycle analysis, 1 × 106 cells were

collected, washed in PBS, fixed with 70% cold ethanol for at least 30 min, washed with PBS,

treated for 30 min at 37 °C with RNase A (0.1 mg/ml), and stained with propidium iodide

(69 µM) (Sigma) in 38 mM sodium citrate. Cell cycle analysis was carried out with a

fluorescence-activated cell sorter (FACScan; Becton Dickinson). Analyses were performed three

times on 40,000 cells with similar results. TUNEL assays were performed as described

previously6.

Colony assay. Human U2OS osteosarcoma cells were transfected with pcDNA3,

pcDNA3-THAP1 or pcDNA3-p21 expression vectors, using a calcium-phosphate procedure.

Transfected cells were selected in neomycin (750 ug/ml) and the ability to form colonies was

assayed after 2 weeks by counting crystal violet stained cells.

Microarray analysis. Total RNA quality control was performed by running 100 ng on

an RNA 6000 Nano assay (Agilent) using Bio-analyser 2100. Probe synthesis and labelling with

Cy3- or Cy5- fluorescent CTP (Perkin-Elmer) were carried out with 500 ng total RNAs using

low RNA input fluorescent linear amplification kit (Agilent) according to manufacturer's

instruction. One microgram of fragmented Cy3 and Cy5 labeled cRNAs were hybridized to

Agilent human genome 1A oligonucleotide array (Agilent G4110A, 22 000 unique 60-nt

oligonucleotide probes representing > 17 000 human genes) for 17 hours at 60 °C, then washed

with 0.6X and 0.01X SSC buffers containing triton, and dried with a nitrogen gun. Microarrays

were scanned using the Agilent DNA microarray scanner. Data analysis was performed with the

Feature extraction software (Agilent) and Resolver software (Rosetta). Dye swap experiments

were performed to eliminate the effect of dye bias. The final ratio (i.e., fold change) is the

average of the two individual ratios from dye swap experiments (combined experiments).

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Microarray data have been submitted to ArrayExpress (http://www.ebi.ac.uk/arrayexpress/ ; Acc

# E-TABM-24).

Quantitative real-time PCR (qPCR). qPCR was performed using the ABI7700 Prism

SDS Real-Time PCR Detection System (Applied Biosystems, Foster City, CA, USA) with a

SYBR Green PCR Master Mix kit (Applied Biosystems) and a standard temperature protocol as

previously described24. GAPDH and NF-KB2 p100 were used as control genes for normalization.

Primer sequences are available upon request.

DNase I footprinting and EMSA. DNase I footprinting assays were performed by

incubating a 130-bp 32P end-labelled RRM1 promoter DNA fragment (bp –190 to -61) with

recombinant THAP domain of human THAP18, at 28°C for 20 min in 20µl of binding buffer (20

mM Tris, 100 mM NaCl, 0.1 % NP40, 5 % glycerol, 100 µg/ml BSA). After 1 min of cleavage

by DNase I (50 ng/ml) in the presence of CaCl2 (1 mM), the reaction was stopped with 5 µl of

stop solution (1,5 M NaOAc, 250 mM EDTA), DNA was purified and analysed on 10% Urea-

polyacrylamide gel and autoradiographed. Sequence references were obtained by sequencing the

identical PCR fragments with the Maxam and Gilbert sequencing method. EMSA experiments

were performed as described previously8, using the upstream THAP1-binding sequence

(THABS) of the RRM1 promoter as probe

(5’CGAACTCGGCTTGCCCACACAAAACATGGTA-3’). A 50-fold molar excess of wild-type

THABS or mutated THABS (mutTHABS) unlabeled oligonucleotides8 were used for

competition experiments.

Chromatin immunoprecipitations (ChIP). ChIP assays were performed as previously

described25,26, using 5 µg of control anti-Flag antibody (Sigma) or 5 µg of antibodies specific for

NF-YB25 or THAP1. Semi-quantitative PCRs were performed with the following primers: NF-YA

5’-GGAGGCCCGATTCCCCTTTG-3’, 5’GGTCAGCGAGACCCGCCAAT-3’; RRM1 5’-

CAGCGGGCTCTAGGTGCTAC-3’, 5’-CGGGGTAGGCTTCACAGACT-3’. For ChIP-qPCR

assays, ChIP was performed as described27 and quantified in triplicate by qPCR using the % of

input method28. In brief, the amount of genomic DNA co-precipitated with anti-THAP1

antibodies was calculated as a % of total input the following way: ∆CT=CT(input)-CT(THAP1-

IP), %input=2∆CTx0.25 (0.25% of total input was used). The following primers were used for the

ChIP-qPCR assays: qRRM1 5'-CCCATGGGAGAGGCGTAGT-3', 5'-

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CAGACTGACAGGCGACGTGTA-3'; qNF-YA 5'-CCGGTACTGGAGCCAATCA-3', 5'-

GGATATTGGCTCCTCACACTCAC-3' .

RNA interference. Control GL2 luciferase and THAP1 SMARTpool and individual

siRNA duplexes were purchased from Dharmacon (Lafayette, CO). Two successive transfections

of HUVECs were performed at 24h hour-interval by incubating cells for 6 hours with siRNA

duplexes at 20 nM final concentration in Oligofectamine and serum-free Opti-MEM-1

(Invitrogen). Samples for qPCR, western blot, BrdU labeling, determination of cell numbers,

FACS and TUNEL assays were taken at the indicated time points after the second siRNA

transfection.

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Results

Retroviral-mediated gene transfer of THAP1 in primary human ECs inhibits proliferation

and G1/S cell cycle progression

To better understand the function of THAP1 as a nuclear factor in the vasculature, we

sought to ectopically express THAP1 in primary human vascular ECs using retroviral gene

transfer. Primary HUVECs were transduced with pMLV-THAP1 or pMLV-MCS (as the

negative infection control) retroviral expression vectors, and ectopic expression of THAP1 in

transduced endothelial cells was verified by Western blotting and indirect immunofluorescence

staining (Figure 1A-B). Overexpression of THAP1 was found to result in an inhibition of

endothelial cell proliferation (Figure 1C). This effect was specific for the THAP1 gene since it

was not observed after retroviral-mediated transfer of other genes, including the unrelated

nuclear factor NF-HEV, the chemokines CCL21 and CXCL9 or the Green Fluorescent Protein

(data not shown). The loss of cell proliferation was not due to non-specific toxic effects of

THAP1 expression since the presence of floaters or non-trypan blue excluding cells in THAP1-

expressing cultures was not observed and TUNEL assays failed to reveal enhanced apoptosis

(Figure 1D). In addition, the levels of apoptosis were also similar when the cells were incubated

in low serum media for 24h (Figure 1E). Thus, the antiproliferative effects of THAP1 on

primary human endothelial cells were not dependent on induction of apoptosis.

To further characterize the antiproliferative properties of THAP1, we analyzed cell cycle

progression in THAP1-expressing cells by staining cells for bromodeoxyuridine (BrdU)

incorporation and nuclear proliferation marker Ki-67. We found that retroviral-mediated ectopic

expression of THAP1 into HUVECs greatly inhibited DNA synthesis compared with uninfected

cells or HUVECs transduced with pMLV-MCS control vector (Figure 2A). The percentage of

cells in S-phase incorporating BrdU falled from ~ 30 % in control HUVECs to 5 % in cells

expressing THAP1 (Figure 2B). Nuclear proliferation marker Ki-67 which stains cells in mid to

late G1 and S-G2/M cell cycle phases was also down-regulated (Figure 2C). Cell cycle analysis

of HUVEC-THAP1 cells by flow cytometry revealed a significant reduction in both the S- and

G2/M-phase cell populations and a corresponding increase in the number of cells in G1 (Figure

2D-E), indicating that ectopic expression of THAP1 into primary HUVECs inhibits cell cycle

progression at the G1/S transition. Another difference between THAP1-expressing HUVECs and

control HUVECs was seen in mitotic cells several days after the retroviral transduction; while

the majority of mitotic figures in HUVECs transduced with pMLV-MCS control vector

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appeared morphologically normal, many of the mitotic figures in HUVECs transduced with

THAP1 retroviral expression vector showed abnormalities (data not shown).

To determine whether the cell cycle inhibitory properties of THAP1 are specific for

primary ECs, we analysed the effects of THAP1 ectopic expression in human epithelial cell

lines, which express endogenous THAP1 at levels similar to those found in ECs (supplementary

Figure 1). Transient transfection of the THAP1 plasmid into human U2OS osteosarcoma cancer

cells (Figure 2F) or human 293T embryonic kidney cell line (data not shown) resulted in

significant reduction of BrdU incorporation. In addition, ectopic expression of THAP1

significantly reduced the colony-forming ability of U2OS cells (Figure 2G), indicating that

THAP1 overexpression inhibits proliferation of epithelial cancer cells.

Overexpression of THAP1 in primary human ECs downregulates mRNA levels of critical

cell-cycle regulators and pRB-E2F target genes

To gain further insight into the effects of THAP1 on EC growth regulatory pathways,

DNA microarray experiments were performed after one (#1xTHAP1) or two (#2xTHAP1)

consecutive transductions of HUVECs with pMLV-MCS or pMLV-THAP1 retroviral expression

vectors. Although fold increase or decrease for each gene probe were significantly lower after the

single retroviral transduction because of lower transduction efficiency (only 251 genes showing

differential expression with p value < 0.01 in the #1xTHAP1 experiment), this stringent analysis

identified gene probes reproducibly affected by THAP1 expression in fully independent

experiments (independent HUVEC primary cell cultures, independent transductions with

independent retroviral supernatants, independent microarray analyses). Using a threshold of 1.5-

fold increase or decrease, we identified 80 gene probes with decreased expression and 16 gene

probes with increased expression in response to retroviral-mediated gene transfer of THAP1 into

primary HUVECs (Table 1).

Strikingly, most of the genes encoding proteins with known functions that are

downregulated after THAP1 overexpression (54 distinct genes out of 80) corresponded to genes

encoding proteins associated with cell cycle/cell proliferation (Table 1), indicating a highly

selective nature of THAP1-mediated inhibition of gene expression. The list of down-regulated

genes included genes with a well-established role in cell cycle control, genes involved in DNA

replication and DNA repair, as well as genes encoding proteins that function in central mitotic

processes, such as chromosome condensation and segregation, mitotic spindle checkpoint,

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mitotic spindle assembly and cytokinesis (Table 1). Interestingly, many of these genes have

previously been found to be regulated by the pRB-E2F pathway (38 genes out of 54, see Table

1), exhibiting down-regulation upon expression of pRB and up-regulation upon overexpression

of E2F29-36.

In addition to the cell cycle-specific genes, a series of genes with diverse biological roles

were also identified as target genes down-regulated or up-regulated by THAP1 but these could

not be grouped into any functional category (Table 1, Miscellaneous), and the effect of THAP1

on the regulation of these genes was not further studied.

To validate the microarray data, RNA samples from HUVECs transduced with pMLV-

MCS or pMLV-THAP1 retroviral expression vectors were analyzed by qPCR, using specific

primers for 15 distinct genes selected from Table 1. As shown in figure 3, the qPCR data were in

good agreement with the microarray results and showed that expression of all these genes was

decreased more that 2-fold after retroviral-mediated gene transfer of THAP1 into primary

HUVECs. Together, the microarray and qPCR data indicated that overexpression of THAP1 into

primary HUVECs inhibits cell proliferation through coordinated repression of critical cell cycle

regulators and pRb/E2F target genes.

Endogenous THAP1 is required for EC proliferation, S-phase DNA synthesis and G1/S

cell cycle progression

To determine whether endogenous THAP1 regulates EC proliferation and cell cycle

progression, we performed a knockdown of THAP1 in HUVECs using RNA interference with

siRNAs. For an efficient depletion of THAP1, we used a THAP1 siRNA SMARTpool

(siTHAP1) at a final concentration of 20nM which is known to avoid off-target effects of the

siRNAs37. Analysis of THAP1 expression by qPCR and western blot at 24 h and 48 h after

THAP1 siRNA transfection revealed up to 80% reduction of THAP1 mRNA and protein levels

when compared with samples treated with a control luciferase siRNA (Figure 4A-B). We

measured BrdU incorporation in cells treated with siTHAP1 or siLuc control siRNA and found

that THAP1 knockdown resulted in inhibition of S-phase DNA synthesis (Figure 4C).

Specificity of the effect was demonstrated by the use of four individual siRNAs targeted at

different areas of the THAP1 coding region, that led to similar (siTHAP1-2, siTHAP1-4) or even

higher (siTHAP1-1, siTHAP1-3) reductions of BrdU incorporation when compared to EC

treated with siLuc control siRNA (Figure 4C). Inspection of transfected cultures indicated that

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silencing of THAP1 resulted in less proliferation (Figure 4D). Quantification of the proliferation

rate of ECs treated with the four individual THAP1 siRNAs revealed a significant inhibition of

cell proliferation compared to untransfected ECs or ECs transfected with the siLuc control

siRNA (Figure 4E). Cell cycle analysis of ECs transfected with THAP1 siRNAs revealed a

reduction in both the S- and G2/M-phase cell populations and a corresponding increase in the

number of cells in G1 (Figure 4F-G), indicating that endogenous THAP1 is required for G1/S

cell cycle progression. TUNEL assays showed that knockdown of THAP1 did not enhance

apoptosis levels (Figure 4H). Together, these data indicate that endogenous THAP1 is essential

for EC proliferation and cell cycle progression at the G1/S transition.

To determine whether THAP1 is expressed in proliferating ECs in vivo, we performed in

situ hybridization and immunohistochemistry experiments, but, we failed to detect expression,

probably because endogenous THAP1 expression levels are below the detection limits of these

techniques (data not shown). However, we succeeded in showing expression of endogenous

THAP1 mRNA in ECs freshly purified from different human tissues (Supplementary Figure 1),

including rheumatoid arthritis synovium, a tissue associated with high levels of angiogenesis

and EC proliferation. Therefore, THAP1 is likely to be expressed in proliferating ECs in vivo.

Actually, the results obtained with proliferating and quiescent HUVECs indicated that THAP1 is

expressed at slightly higher levels in proliferating ECs than in quiescent growth arrested ECs

(Supplementary Figure 2).

Silencing of endogenous THAP1 inhibits expression of pRB/E2F cell cycle target genes

To address the physiological role of THAP1 in the regulation of pRB/E2F cell cycle

target genes, relative mRNA levels of these genes were quantified by qPCR in ECs treated with

siTHAP1 or siLuc control siRNAs. Knockdown of THAP1 using either siTHAP1-1 or siTHAP1-

3 siRNAs led to a significant decrease in the expression of the eight tested pRB/E2F cell cycle

target genes (RRM1, Mad2, survivin, HMMR, RRM2, CDC2, cyclin B1 and DLG7), when

compared to ECs treated with control siRNA (Figure 5A-B). There was no significant change in

the expression level of actin between ECs treated with siTHAP1 and siLuc control siRNAs,

indicating that THAP1 knockdown led to a specific reduction of mRNA levels of the pRB/E2F

cell cycle target genes RRM1, Mad2, survivin, HMMR, RRM2, CDC2, cyclin B1 and DLG7.

Endogenous THAP1 associates in vivo with the promoter of RRM1, a pRb/E2F cell cycle

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target gene required for S-phase DNA synthesis

The presence of the sequence-specific DNA-binding THAP domain in THAP18

suggested that THAP1 may bind directly to the promoters of some of the pRb-E2F cell cycle-

specific genes identified. In agreement with this possibility, we found candidate THAP1-binding

sites (THABS)8 in the promoters of several genes: RRM1, RRM2, BIRC5/Survivin, cyclin B1,

USP16. We selected the RRM1 gene for further in vitro and in vivo characterization of potential

THAP1/promoter interactions. RRM1, a gene activated at the G1/S cell cycle transition, encodes

the ribonucleotide reductase M1 subunit, which is essential for S-phase DNA synthesis38,39. The

RRM1 promoter exhibited two consensus THABS ~ 100 nt upstream the 5’ end of the mRNA

(Figure 6A). The upstream THABS sequence was found to be protected in DNase I footprinting

assays (Figure 6B), suggesting direct binding of the THAP domain of THAP1 to these sites. In

vitro EMSA protein/DNA-binding assays confirmed interaction of THAP1 with the RRM1

THABS motif and demonstrated the specificity of binding (Figure 6C). To examine possible

association of endogenous THAP1 with the RRM1 promoter in vivo, we performed chromatin

immunoprecipitation (ChIP) assays with cross-linked extracts from proliferating HUVECs,

using anti-THAP1, anti-NF-YB and anti-Flag antibodies (Figure 6D). As expected, the NF-YB

transcription factor, used as a positive control25, bound to the NF-YA and RRM1 promoters

which contain CCAAT boxes (NF-Y binding sites). THAP1 bound to the endogenous RRM1

promoter, but not to the NF-YA promoter which doesn’t exhibit THABS motifs. Consistent with

the specificity of our ChIP assay, the control anti-Flag antibody did not immunoprecipitate

significant levels of the different promoters analyzed. ChIP-qPCR assays were performed to

confirm and quantify the ChIP results. This revealed significant binding of endogenous THAP1

to the RRM1 promoter (1.4 % of total input DNA precipitated) while binding of THAP1 to the

NF-YA promoter was similar to background levels, obtained with anti-Flag antibodies on both

the RRM1 and NF-YA promoters (Figure 6E). Quantification indicated a ~ 50-fold enrichment of

THAP1 on the RRM1 promoter compared to the NF-YA promoter (Figure 6F). Taken together

with the in vitro DNA-binding assays (Figure 6B-C), and the downregulation of RRM1 mRNA

observed after knockdown of endogenous THAP1 (Figure 5), these in vivo ChIP assays indicate

that RRM1 is a direct transcriptional target of endogenous THAP1.

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Discussion

In this manuscript, we demonstrate that the human THAP-zinc finger protein THAP1 is

an endogenous physiological regulator of EC proliferation. Silencing of THAP1 by RNA

interference in human primary ECs resulted in inhibition of G1/S cell cycle progression and

down-modulation of several pRb/E2F cell cycle target genes, including RRM1, a gene activated

at the G1/S transition, essential for S-phase DNA synthesis. ChIP assays in proliferating ECs

revealed that endogenous THAP1 associates in vivo with a consensus THAP1-binding site found

in the RRM1 promoter, indicating that RRM1 is a direct target gene of THAP1, and providing

important mechanistic insights about the role of endogenous THAP1 in the regulation of EC

proliferation. Together, our results suggest that THAP1 may constitute a potential target for

inhibition of EC proliferation in angiogenesis-dependent diseases, such as cancer and chronic

inflammatory diseases.

THAP-zinc finger proteins represent a previously uncharacterized family of cellular

factors with ~ 100 distinct members in model animal organisms7,8, including zebrafish

orthologue of cell cycle regulator E2F6, and five C. elegans proteins, LIN-36, LIN-15A, LIN-

15B, HIM-17 and GON-14, interacting genetically with LIN-35/Rb, the C. elegans orthologue

of the tumor suppressor pRB10-13,17. While several of these THAP proteins (LIN-36, LIN-15B)

have been shown to function as inhibitors of G1/S cell cycle transition and cell

proliferation12,14,16, our data indicate that human THAP1 may rather play a positive role in cell

proliferation and G1/S cell cycle progression. Interestingly, cell division defects were recently

observed in the intestine, gonad and vulva of gon-14 null mutants, suggesting that C. elegans

THAP family member GON-14 may also function as a positive regulator of cell proliferation17.

Therefore, both in humans and model animal organisms, THAP-zinc finger proteins appear to be

critical regulators of cell proliferation and cell cycle progression.

The similar phenotypes observed after THAP1 overexpression through retroviral-

mediated gene transfer or silencing through RNA interference suggest that THAP1

overexpression inhibit the function of endogenous THAP1 in cell proliferation and cell cycle

progression. Similar observations have been made in ECs for components of the Notch pathway;

silencing or overexpression of the Notch ligand DLL4 in ECs led to a significant inhibition of

proliferation40,41, and overexpression or knock-out of Notch4 produced similar vascular

phenotypes in mice42. This indicated that there may be a window of appropriate Notch

expression levels for EC proliferation. Our data suggest that an optimal range of THAP1

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expression also appears to be critical for EC proliferation and cell cycle progression. One could

imagine that overexpression of THAP1 may inhibit the function of endogenous THAP1 protein

complexes by disrupting the normal stoichiometry of these complexes.

Since large scale gene expression profiling showed a selective modulation of pRB/E2F

cell cycle target genes in ECs overexpressing THAP1 (Table 1), we asked whether a similar

modulation of pRb-E2F cell cycle target genes could be observed in ECs transfected with

THAP1 siRNAs. We found that silencing of endogenous THAP1 by RNA interference resulted

in down-modulation of the eight pRB/E2F cell cycle target genes analyzed, RRM1, Mad2,

survivin, HMMR, RRM2, CDC2, cyclin B1 and DLG7. Therefore, THAP1 may play a role in the

activation or the maintenance of the activated state of cell-cycle specific genes in proliferating

cells. Interestingly, we identified potential THAP1-binding sites in the promoters of several

genes (RRM1, RRM2, BIRC5/Survivin, cyclin B1) down-modulated after THAP1 silencing. We

selected RRM1, a critical pRb/E2F target gene activated at the G1/S transition29-32,34-36, for

further characterization, and we could demonstrate direct binding of the THAP domain of

human THAP1 to the RRM1 promoter in vitro using EMSA and footprinting assays and

association of endogenous THAP1 with the RRM1 promoter in vivo using quantitative ChIP

assays in proliferating ECs. Together, these data indicate that RRM1 is a direct transcriptional

target of THAP1, and suggest that THAP1 may associate with the promoters of other pRb/E2F

cell-cycle target genes and modulate their activity. This model is supported by observations

made in model animal organisms. In zebrafish and other fish species, the THAP-E2F6 fusion

proteins8 are likely to associate with cell-cycle specific promoters since this has clearly been

shown for mouse and human E2F6 orthologues using ChIP assays9. In C. elegans, the THAP

protein LIN-36 has been proposed to act in a transcriptional repressor complex with LIN-35/Rb

and Efl-1/E2F4-5, to repress G1/S control genes12,16.

Genetic data obtained in C. elegans suggest that THAP1 may act at the level of

chromatin regulation. Indeed, several members of the C. elegans THAP family (LIN-36, LIN-

15B, LIN-15A, HIM-17, GON-14) have been found to interact genetically with known

components of chromatin-modifying and/or chromatin-remodeling complexes, including

members of the Rb and Nucleosome Remodeling Deacetylase (NuRD) complexes and

components of the Tip60/NuA4 histone acetyltransferase complex10-13,17,43,44. Interestingly, the

human THAP7 protein has also been found to interact with chromatin-modifying enzymes19,20.

Together, these observations suggest that THAP1 may function at the level of chromatin

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regulation, by recruiting chromatin-modifying and/or chromatin remodeling complexes to

specific DNA sites in target genes (i.e. RRM1 promoter and potentially other pRb/E2F cell cycle

target genes), resulting in transcriptional activation of these target genes during cell

proliferation.

In summary, the results presented in this manuscript are important because they provide

the first link in humans between the THAP proteins, the cell cycle and the pRB/E2F pathway. In

addition, they suggest, that THAP proteins may play important roles in the control of cell

proliferation and cell cycle progression in humans, similarly to what has been found in C.

elegans. Future studies should aim at determining whether other human THAP proteins play a

role in cell proliferation, defining the composition of the endogenous THAP1 protein complexes

and characterizing the potential links between THAP1 and E2F family members in the

regulation of cell-cycle specific promoters.

Acknowledgments

We are grateful to Dr RA Willemsen (Daniel den Hoed Cancer Center, Erasmus MC,

Rotterdam) for the gift of the pBULLET retroviral expression vector and to the Laboratoire de

Biothérapies (Inserm IFR31-Ligue Régionale contre le Cancer, Toulouse) and Dr P Bouille and

R Gayon (Endocube, Labège) for help with the retroviral transduction experiments. We thank F

Viala for iconography, and N Ortega and S Roga (IPBS-CNRS, Toulouse) for help with HUVEC

culture and qPCR experiments, respectively. Special thanks go to Pr F Amalric (IPBS-CNRS,

Toulouse) for stimulating discussions.

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Table 1. Genes regulated after ectopic expression of THAP1 in endothelial cells. The Unigene ID, gene symbol, gene name and description are indicated for each gene as well as the fold changes obtained after two retroviral transductions of HUVECs with pMLV-MCS or pMLV-THAP1 expression vectors (#2xTHAP1 experiment). The table only includes genes reproducibly affected by THAP1 expression (p value < 0.01) in the two fully independent microarray experiments (#1xTHAP1 and #2xTHAP1). Genes previously identified as targets of the pRB/E2F pathway are indicated in bold together with the corresponding references.

Unigene ID

Gene Symbol

Gene Name and Description Fold Change

E2F target

Rb target

Genes down-regulated after ectopic expression of THAP1 in primary EC Cell cycle control

Hs.374378 CKS1B CDC28 protein kinase regulatory subunit 1B (CKS1) -2.19 35 31

Hs.520506 FBXO5 F-box protein 5 (APCcdh1 inhibitor Emi1) -1.83 32 32

Hs.334562 CDC2 cell division cycle 2, G1 to S and G2 to M -1.81 33-36 29-31,36

Hs.194698 CCNB2 cyclin B2 -1.66 34,36 29-31,36

Hs.23960 CCNB1 cyclin B1 -1.61 34,36 29-31,36

DNA replication / DNA repair Hs.234896 GMNN geminin, DNA replication inhibitor -2.02 29

Hs.198363 MCM10 MCM10 minichromosome maintenance deficient 10 (S. cerevisiae) -2.01

Hs.409065 FEN1 flap structure-specific endonuclease 1 -2 32-36 29,31,32,36

Hs.202672 DNMT1 DNA (cytosine-5-)-methyltransferase 1 -2 33 29,31

Hs.5199 HSPC150 similar to ubiquitin-conjugating enzyme Ubc13p (S. cerevisiae) -1.92 35,36 36

Hs.383396 RRM1 ribonucleotide reductase M1 polypeptide -1.79 32,34-36 29-32,36

Hs.438720 MCM7 MCM7 minichromosome maintenance deficient 7 (S. cerevisiae) -1.75 32-34 29-32

Hs.489037 ASK activator of S phase kinase -1.74 35 31

Hs.433180 PSF2 DNA replication complex GINS protein PSF2 -1.73

Hs.226390 RRM2 ribonucleotide reductase M2 polypeptide -1.58 32,34 29-32

Chromosome condensation and segregation Hs.99819 USP16 ubiquitin specific protease 16 (associated with mitotic chromosomes) -3.37

Hs.497741 CENPF centromere protein F, 350/400ka (mitosin) -2.8 36 31,36

Hs.75573 CENPE centromere protein E, 312kDa -2.15 35,36 31,36

Hs.434953 HMGB2 high-mobility group box 2 (associated with mitotic chromosomes) -2.09 32-34 29-32

Hs.42650 ZWINT kinetochore protein ZW10 interactor -1.78 31

Hs.14559 C10orf3 chromosome 10 open reading frame 3 (SMC-domain protein) -1.72

Hs.5719 CNAP1 chromosome condensation-related SMC-associated protein 1 -1.65

Hs.350966 PTTG1 securin (pituitary tumor-transforming 1) -1.63 34-36 29,31,36

Hs.521097 PTTG3 pituitary tumor-transforming 3 -1.62

Hs.511755 PTTG2 pituitary tumor-transforming 2 -1.52

Mitotic spindle checkpoint Hs.421956 SPC25 kinetochore protein Spc25 homolog (S. cerevisiae) -2.04 36 29,31,36

Hs.234545 CDCA1 kinetochore protein Nuf2 (cell division cycle associated 1) -1.91

Hs.533185 MAD2L1 mitotic spindle assembly checkpoint protein MAD2 (MAD2-like 1) -1.91 35,36 31,36

Hs.36708 BUB1B mitotic checkpoint serine/threonine-protein kinase BUBR1 -1.85 33,34,36 29,31,36

Hs.414407 KNTC2 kinetochore protein Hec1 (kinetochore associated 2) -1.79 35,36 31,36

Hs.469649 BUB1 mitotic checkpoint serine/threonine-protein kinase BUB1 -1.76 29-31

Hs.524947 CDC20 CDC20 cell division cycle 20 homolog (S. cerevisiae) -1.6 34 29-31

Mitotic spindle assembly Hs.121028 ASPM asp (abnormal spindle)-like, microcephaly associated (Drosophila) -2.05

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Hs.514033 SPAG5 mitotic spindle coiled-coil related protein SPAG5 (MAP126) -1.91

Hs.77695 DLG7 HURP/KIAA0008, discs large homolog 7 (Drosophila) -1.9 29,31

Hs.104019 TACC3 transforming, acidic coiled-coil containing protein 3 -1.8

Hs.119324 KIF22 kinesin family member 22 (KNSL4, kinesin-like 4) -1.76 35,36 29,31,36

Hs.90073 CSE1L CSE1 chromosome segregation 1-like (yeast) -1.63 31

Hs.514527 BIRC5 survivin (baculoviral IAP repeat-containing 5) -1.62 36 31,36

Hs.72550 HMMR hyaluronan-mediated motility receptor (RHAMM) -1.6 35 29,31

Hs.73625 KIF20A kinesin family member 20A (RAB6KIFL) -1.59 36 29,30,36

Hs.532793 KPNB1 karyopherin (importin) beta 1 -1.58 36 36

Hs.69360 KIF2C kinesin family member 2C (KNSL6, kinesin-like 6) -1.53 36 31,36

Cytokinesis

Hs.126774 RAMP RA-regulated nuclear matrix-associated protein -1.69

Hs.459362 PRC1 protein regulator of cytokinesis 1 -1.66 35,36 29,36

Hs.62180 ANLN anillin, actin binding protein (scraps homolog, Drosophila) -1.61 35,36 29,36

Cell cycle/ cell proliferation miscellaneous Hs.470654 CDCA7 cell division cycle associated 7 (c-myc target gene JPO1) -2.55

Hs.104741 PBK PDZ binding kinase (mitotic kinase TOPK) -1.89

Hs.434886 CDCA5 cell division cycle associated 5 -1.88

Hs.436187 TRIP13 thyroid hormone receptor interactor 13 -1.78 30,31

Hs.239 FOXM1 forkhead box M1 -1.65 31

Hs.89497 LMNB1 lamin B1 -1.65 33 29-31

Hs.484813 DEK DEK oncogene (DNA binding) -1.61 32 32

Hs.208912 C22orf18 Proliferation associated nuclear element (PANE1) -1.56

Miscellaneous Hs.421907 GLTSCR2 glioma tumor suppressor candidate region gene 2 -4.41

Hs.528306 U1SNRNPBP U11/U12 snRNP 35K -3.77

Hs.497636 LAMB3 laminin, beta 3 -2.91

Hs.160411 TSHR thyroid stimulating hormone receptor -2.55

Hs.7432 THAP1 THAP domain containing, apoptosis associated protein 1 -2.13

Hs.248941 TAF9 TAF9 RNA polymerase II -2.08

Hs.472054 C20orf42 chromosome 20 open reading frame 42 (kindlerin KIND1) -1.86

Hs.71827 RRS1 RRS1 ribosome biogenesis regulator homolog (S. cerevisiae) -1.84

Hs.296310 GJA4 gap junction protein, alpha 4, 37kDa (connexin 37) -1.68

Hs.472119 MKKS McKusick-Kaufman syndrome -1.62

Hs.166539 ITGB3BP integrin beta 3 binding protein (beta3-endonexin, NRIF3) -1.55

Predicted proteins with unknown functions Hs.327252 LZIC leucine zipper and CTNNBIP1 domain containing -2.18

Hs.388087 ZCSL2 zinc finger, CSL domain containing 2 -2.07

Hs.525764 ARHGAP11A Rho GTPase activating protein 11A -2.03

Hs.529778 FKSG14 leucine zipper protein FKSG14 -2.01 32 32

Hs.508292 FLJ10514 hypothetical protein FLJ10514 -1.89

Hs.404323 FLJ10156 hypothetical protein FLJ10156 -1.81

Hs.121536 FAM54A family with sequence similarity 54, member A -1.79

Hs.162717 MGC15668 hypothetical protein MGC15668 -1.7

Hs.330663 FLJ20641 hypothetical protein FLJ20641 -1.66

Hs.127767 LOC130502 similar to CG14894-PA -1.63

Hs.426696 FLJ20516 timeless-interacting protein -1.62

Hs.209715 FLJ22624 FLJ22624 protein -1.6

Hs.283532 BM039 uncharacterized bone marrow protein BM039 -1.58

Hs.550564 RPL7L1 ribosomal protein L7-like 1 -1.53

Hs.30696 TCFL5 transcription factor-like 5 (basic helix-loop-helix) -1.5

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Genes up-regulated after ectopic expression of THAP1 in primary EC Miscellaneous

Hs.531081 LGALS3 lectin, galactoside-binding, soluble, 3 (galectin 3) 1.52

Hs.198308 WRB tryptophan rich basic protein 1.58

Hs.387794 CHGN chondroitin beta1,4 N-acetylgalactosaminyltransferase 1.6

Hs.45140 TMEM35 transmembrane protein 35 1.65

Hs.505141 FLJ14337 similar to Peptidylprolyl isomerase A, isoform 1 1.67

Hs.549129 RP11-529I10.4 deleted in a mouse model of primary ciliary dyskinesia 1.69

Hs.508835 TEP1 telomerase-associated protein 1 1.7

Hs.518731 UCHL1 ubiquitin carboxyl-terminal esterase L1 (ubiquitin thiolesterase) 1.71

Hs.106511 PCDH17 protocadherin 17 1.71

Hs.492261 TP53INP1 tumor protein p53 inducible nuclear protein 1 1.89

Hs.126598 TMEM45A transmembrane protein 45A 1.96

Hs.391561 FABP4 fatty acid binding protein 4, adipocyte 1.98

Hs.386470 NMB neuromedin B 2.01

Hs.517581 HMOX1 heme oxygenase (decycling) 1 3.06

Hs.289292 FOXL2 forkhead box L2 4.21

Hs.546296 SECTM1 secreted and transmembrane 1 5.51

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Figure Legends

Figure 1. Retroviral-mediated gene transfer of THAP1 inhibits EC proliferation. (A)

Indirect immunofluorescence analysis of HUVECs transduced with pMLV-MCS (HUVEC-

MCS) or pMLV-THAP1 (HUVEC-THAP1) retroviral expression vectors, with anti-THAP1

polyclonal antibodies. Nuclei were counterstained with DAPI. (B) Western blot analysis of

HUVEC-MCS and HUVEC-THAP1 with anti-THAP1 polyclonal antibodies or anti-α−Tubulin

(loading control) mouse monoclonal antibody. (C) Analysis of cell proliferation in HUVEC-

MCS and HUVEC-THAP1. Expression of THAP1 results in inhibition of EC proliferation. (D)

TUNEL labelling of apoptotic nuclei in HUVECs transduced with pMLV-MCS (HUVEC-MCS)

or pMLV-THAP1 (HUVEC-THAP1) retroviral expression vectors, and incubated in low-serum

media for 24 h. Nuclei were counterstained with DAPI. (E) Apoptosis levels were quantified in

HUVEC-MCS and HUVEC-THAP1 incubated in high-serum (20% FCS) or low-serum (0.5%

FCS) media for 24 h. TUNEL labelling of apoptotic nuclei was performed at day 4 post-

infection and at least 500 cells were counted for each condition. Results are mean of two

independent retroviral transduction experiments.

Figure 2. Ectopic expression of THAP1 into primary human EC inhibits cell cycle

progression and blocks S-phase DNA synthesis. (A) Inhibition of DNA synthesis in EC

expressing THAP1. HUVECs transduced with pMLV-MCS (HUVEC-MCS) or pMLV-THAP1

(HUVEC-THAP1) retroviral expression vectors were pulse-labeled with BrdU for 90 min. Cy2-

conjugated anti-BrdU antibody was used to identify cells incorporating BrdU in the S phase of

the cell cycle. Cells were visualized for BrdU incorporation (upper panels) and THAP1

expression (lower panels). (B) The graph shows the percentage of BrdU+ cells in HUVEC,

HUVEC-MCS and HUVEC-THAP1 cell populations. At least 500 cells were counted for each

condition. Results are mean of two independent retroviral transduction experiments. (C) Down-

regulation of nuclear proliferation marker Ki-67 in EC expressing THAP1. The graph shows the

percentage of Ki-67+ cells in HUVEC-MCS and HUVEC-THAP1 cell populations. Ki-67 is a

marker of cycling cells in late G1 and S-G2/M cell cycle phases; therefore, reduction in the

number of Ki-67+ cells in HUVEC-THAP1 indicates an inhibition of cell cycle progression in

EC expressing THAP1. (D) Flow cytometry analysis of cell cycle distribution in HUVEC-MCS

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and HUVEC-THAP1 cell populations. (E) Ectopic expression of THAP1 into HUVECs inhibits

cell cycle progression at the G1/S transition. The graph shows the percentage of cells in G1, S

and G2/M phases of the cell cycle in HUVEC-MCS and HUVEC-THAP1 cell populations.

Results are mean of two independent retroviral transduction experiments. (F) Ectopic expression

of THAP1 into human U2OS osteosarcoma cancer cells inhibits S-phase DNA-synthesis. The

graph shows the percentage of BrdU+ cells in control untransfected cells or in U2OS cell

populations expressing THAP1 (% BrdU+ cells in the THAP1+ population) or the unrelated

nuclear factor PAPSS1 (% BrdU+ cells in the PAPSS1+ population). Analysis was performed

48h after transfection. (G) Ectopic expression of THAP1 impairs growth of U2OS cells. Cells

transfected with indicated pcDNA3 expression vectors, were selected in neomycin for 14 days

prior to crystal violet staining.

Figure 3. Quantitative real-time RT-PCR analysis of gene expression repressed in

response to retroviral-mediated gene transfer of THAP1 into primary human EC. RNA

samples from HUVECs transduced with pMLV-MCS (HUVEC-MCS) or pMLV-THAP1

(HUVEC-THAP1) retroviral expression vectors were analysed by qPCR. The fold change in the

mRNA levels for 15 selected genes (identified in the DNA microarray experiments; Table 1),

between HUVEC-MCS and HUVEC-THAP1, was calculated. The mean and standard error for

two independent data sets are shown (Q, black bars). For comparison, the fold changes obtained

in the DNA microarray experiments (p value < 0.01) are indicated (M, white bars). The levels of

down-regulation observed in THAP1 expressing ECs were generally higher in the qPCR

experiments than in the microarrays experiments.

Figure 4. Silencing of endogenous THAP1 inhibits EC proliferation, S-phase DNA

synthesis and G1/S cell cycle progression. (A) siRNA-mediated knock-down of THAP1

mRNA in primary human ECs. Levels of THAP1 mRNA were analysed by qPCR at different

time points after transfection of siTHAP1 or siLuc control siRNAs (20 nM final concentration).

(B) siRNA-mediated silencing of THAP1 protein in primary human ECs. Levels of THAP1 and

α−Tubulin proteins were analysed by western blot at different time points after transfection of

siTHAP1 or siLuc control siRNAs (20 nM final concentration). (C) Knockdown of THAP1 in

primary human ECs inhibits S-phase DNA synthesis. The graph shows the percentage of BrdU+

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cells in HUVEC transfected with siLuc, siTHAP1 (pool), siTHAP1-1, siTHAP1-2, siTHAP1-3

or siTHAP1-4 siRNAs. At least 500 cells were counted for each condition. Results are mean of

two independent experiments performed 48h after siRNA transfection. (D,E) siRNA-mediated

knockdown of THAP1 inhibits proliferation of primary human EC. Representative photos (D) of

HUVEC treated with siLuc or the four individual THAP1 siRNAs are shown. Cell count assay

(E) showing inhibition of proliferation in ECs treated with the four individual THAP1 siRNAs

compared to untreated cells or ECs treated with siLuc control siRNA. Results are mean of three

independent experiments. (F-G) Knockdown of THAP1 in primary human ECs inhibits G1/S

cell cycle progression. Flow cytometry analysis of cell cycle distribution (F) in HUVECs

transfected with siLuc, siTHAP1-1 or siTHAP1-3 siRNAs. The graph shows the percentage of

cells in G1, S and G2/M phases of the cell cycle (G) in HUVECs transfected with siLuc,

siTHAP1-1 or siTHAP1-3 siRNAs. Results are mean of three independent experiments

performed 48h after siRNA transfection. (H) Apoptosis levels were quantified in HUVECs

transfected with siLuc, siTHAP1-1 or siTHAP1-3 siRNAs. TUNEL labelling of apoptotic nuclei

was performed 48h after siRNA transfection, and at least 500 cells were counted for each

condition. Results are mean of three independent experiments.

Figure 5: Knock down of endogenous THAP1 in human primary EC inhibits expression of

pRB/E2F cell cycle target genes RRM1, Mad2, survivin, HMMR, RRM2, CDC2, cyclin B1

and DLG7. (A,B) Expression level analysis by qPCR following siRNA-mediated knock-down of

THAP1 in primary human ECs. RNA was isolated from ECs transfected with siTHAP1-1 (A),

siTHAP1-3 (B) or siLuc siRNAs, 48h after siRNA transfection, and used for qPCR analysis

with the indicated human gene primers (control gene: actin; pRB/E2F cell cycle target genes:

RRM1, Mad2, survivin, HMMR, RRM2, CDC2, cyclin B1 and DLG7). NF-KB2 p100 was used

as a control gene for normalization. The mean and standard error for two independent data sets

are shown.

Figure 6. THAP1 binds to the RRM1 promoter in vitro and in vivo. (A) Sequence of the

RRM1 promoter indicating the positions of THAP1 binding sites (THABS), E2F cell cycle

regulatory elements, and NF-Y/CCAAT box. mRNA region is indicated in uppercase letters. The

initiating methionine is shown in bold. (B) Dnase I footprinting analysis of RRM1 promoter

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region. Lane 0, partial DnaseI cleavage of DNA fragment without incubation with protein. Lanes

0.1 and 1, partial DnaseI cleavage after incubation with 0.1 or 1 µM THAP domain of human

THAP1, respectively. Lanes C+T and A+G, Maxam-Gilbert chemical sequencing references

(cleavage after pyrimidine C+T and purine A+G). (C) In vitro EMSA using the first THABS

motif of RRM1 promoter and 0.1 µM THAP domain of human THAP1. A 50-fold molar excess

of wild type (THABS) and mutant (mutTHABS) cold competitor oligonucleotides were used to

show specificity of binding. (D) Identification of endogenous THAP1 on the RRM1 promoter in

vivo using ChIP assays. Cross-linked chromatin from proliferating HUVECs was subjected to

immunoprecipitation with antibodies against THAP1, NF-YB (positive control) and Flag

epitope (negative control). The NF-YA promoter was used as a positive control for the NF-YB

transcription factor and a negative control promoter for THAP1. (E) ChIP-qPCR assays were

used to quantify the amount of RRM1 or NF-YA promoter DNA precipitated by anti-THAP1 or

anti-Flag antibodies. Immunoprecipitated DNA was quantified in triplicate by qPCR using the

% of input method (see “Materials and methods”). A representative experiment out of three is

shown. (F) Fold enrichment of THAP1 on the RRM1 promoter was calculated by dividing the

amount of RRM1 promoter DNA precipitated by anti-THAP1 antibodies to the amount of DNA

precipitated from the NF-YA negative control promoter. No enrichment was observed with anti-

Flag negative control antibodies.

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