The EIF4EBP3 translational repressor is a marker of CDC73 tumor suppressor haploinsufficiency in a parathyroid cancer syndrome

Abstract

Germline mutation of the tumor suppressor gene CDC73 confers susceptibility to the hyperparathyroidism-jaw tumor syndrome associated with a high risk of parathyroid malignancy. Inactivating CDC73 mutations have also been implicated in sporadic parathyroid cancer, but are rare in sporadic benign parathyroid tumors. The molecular pathways that distinguish malignant from benign parathyroid transformation remain elusive. We previously showed that a hypomorphic allele of hyrax (hyx), the Drosophila homolog of CDC73, rescues the loss-of-ventral-eye phenotype of lobe, encoding the fly homolog of Akt1s1/ PRAS40. We report now an interaction between hyx and Tor, a central regulator of cell growth and autophagy, and show that eukaryotic translation initiation factor 4E-binding protein (EIF4EBP), a translational repressor and effector of mammalian target of rapamycin (mTOR), is a conserved target of hyx/CDC73. Flies heterozygous for Tor and hyx, but not Mnn1, the homolog of the multiple endocrine neoplasia type 1 (MEN1) tumor suppressor associated with benign parathyroid tumors, are starvation resistant with reduced basal levels of Thor/4E-BP. Human peripheral blood cell levels of EIF4EBP3 were reduced in patients with CDC73, but not MEN1, heterozygosity. Chromatin immunoprecipitation demonstrated occupancy of EIF4EBP3 by endogenous parafibromin. These results show that EIF4EBP3 is a peripheral marker of CDC73 function distinct from MEN1-regulated pathways, and suggest a model whereby starvation resistance and/or translational de-repression contributes to parathyroid malignant transformation.

Citation: Cell Death and Disease (2012) 3, e266; doi:10.1038/cddis.2012.6

& 2012 Macmillan Publishers Limited All rights reserved 2041-4889/12 http://www.nature.com/cddis

Web End =www.nature.com/cddis

The EIF4EBP3 translational repressor is a marker of CDC73 tumor suppressor haploinsufciency in a parathyroid cancer syndrome

J-H Zhang1, EM Seigneur1, M Pandey1, A Loshakov1, PK Dagur2, PS Connelly3, L Koo4, LM Panicker1,5 and WF Simonds*,1

Germline mutation of the tumor suppressor gene CDC73 confers susceptibility to the hyperparathyroidism-jaw tumor syndrome associated with a high risk of parathyroid malignancy. Inactivating CDC73 mutations have also been implicated in sporadic parathyroid cancer, but are rare in sporadic benign parathyroid tumors. The molecular pathways that distinguish malignant from benign parathyroid transformation remain elusive. We previously showed that a hypomorphic allele of hyrax (hyx), the Drosophila homolog of CDC73, rescues the loss-of-ventral-eye phenotype of lobe, encoding the y homolog of Akt1s1/ PRAS40. We report now an interaction between hyx and Tor, a central regulator of cell growth and autophagy, and show that eukaryotic translation initiation factor 4E-binding protein (EIF4EBP), a translational repressor and effector of mammalian target of rapamycin (mTOR), is a conserved target of hyx/CDC73. Flies heterozygous for Tor and hyx, but not Mnn1, the homolog of the multiple endocrine neoplasia type 1 (MEN1) tumor suppressor associated with benign parathyroid tumors, are starvation resistant with reduced basal levels of Thor/4E-BP. Human peripheral blood cell levels of EIF4EBP3 were reduced in patients with CDC73, but not MEN1, heterozygosity. Chromatin immunoprecipitation demonstrated occupancy of EIF4EBP3 by endogenous parabromin. These results show that EIF4EBP3 is a peripheral marker of CDC73 function distinct from MEN1-regulated pathways, and suggest a model whereby starvation resistance and/or translational de-repression contributes to parathyroid malignant transformation.

Cell Death and Disease (2012) 3, 266; doi:http://dx.doi.org/10.1038/cddis.2012.6

Web End =10.1038/cddis.2012.6 ; published online 2 February 2012

Subject Category: Cancer

Germline loss-of-function mutation of the tumor suppressor gene CDC73 (also known as HRPT2) confers susceptibility to the hyperparathyroidism-jaw tumor syndrome (HPT-JT), an autosomal dominant familial cancer syndrome associated with a high risk of parathyroid malignancy.16 Analysis of multiple kindreds with HPTJT by Carpten et al.7 led to the identication of CDC73/HRPT2 by positional candidate cloning. Inactivating somatic and/or germline CDC73/HRPT2 mutations have also been implicated in sporadic parathyroid cancer.8,9 CDC73 encodes parabromin,7 a 531-amino acid putative tumor suppressor protein with sequence homology to yeast Cdc73p.10 As was originally shown in yeast for Cdc73p,11 evidence in humans suggests that parabromin interacts with RNA polymerase II as a part of the PAF1 complex.1214 The components of the PAF1 complex are highly conserved in Drosophila as well, including hyrax (hyx) a homolog of CDC73.15

Despite its strong association with parathyroid cancer, inactivation of CDC73 is quite rare in sporadic, benign parathyroid tumors.16,17 In contrast, inactivation of the multiple endocrine neoplasia type 1 (MEN1) tumor suppressor gene is nearly always associated with benign parathyroid tumors in the context of familial or sporadic MEN1,18 and

somatic inactivation of MEN1 in sporadic benign parathyroid tumors is common.19,20 Despite the identication of these key parathyroid tumor suppressor genes, the mechanisms and pathways that distinguish benign from malignant transformation of the parathyroid remain elusive. Furthermore, the development of a Cdc73/Hrpt2 knockout mouse model21

and other experimental approaches15,22,23 have so far yielded

little insight into the pathway(s) whereby CDC73 loss-of-function might promote tumorigenesis.

Using Drosophila as a model system, we recently showed that hyx mutant ies were resistant to starvation and that hyx

1Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA; 2Flow Cytometry Core Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA;

3Electron Microscopy Core Facility, National Heart, Lung and Blood Institute, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA and 4Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA *Corresponding author: WF Simonds, Metabolic Diseases Branch, National Institutes of Health, Building 10, Room 8C-101, 10 Center Dr. MSC 1752, Bethesda, MD 20892-1752, USA. Tel: [phone]; Fax: [phone]; E-mail: mailto:[email protected]

Web End [email protected]

5Present address: Department of Microbiology and Immunology, University of Maryland School of Medicine, 660 West Redwood Street, Howard Hall, Room 319C, Baltimore, MD 21201, USA.

Keywords: PAF1 complex; HRPT2; apoptosis; eIF4E; 4E-BP; s6kAbbreviations: HPT-JT, hyperparathyroidism-jaw tumor syndrome; Hyx, hyrax; EIF4EBP, eukaryotic translation initiation factor 4E-binding protein; MEN1, multiple endocrine neoplasia type 1; PRAS40, proline-rich AKT substrate of 40 kDa; AKT1S1, Akt1 substrate 1; mTOR, mammalian target of rapamycin; FBS, fetal bovine serum; APF, after pupal formation; TUNEL, terminal deoxynucleotidyl transferase mediated dUTP nick end labeling; ChIP, chromatin immunoprecipitation; WBC, cultured human peripheral blood mononuclear cells

Received 18.7.11; revised 19.12.11; accepted 09.1.12; Edited by A Stephanou

EIF4EBP3 is a marker of the CDC73 tumor suppressor J-H Zhang et al

2

rescued the loss-of-ventral-eye phenotype of lobe, the y homolog of Akt1 substrate 1 (AKT1S1).24 Akts1, also called 40 kDa proline-rich AKT substrate (PRAS40), is a negative regulator of mammalian target of rapamycin (mTOR) complex 1.25 The protein kinase mTOR functions as a cellular sensor of nutrient and energy availability, and regulates cell growth and autophagy.26

One well-characterized effector of mTOR is the eukaryotic translation initiation factor 4E-binding protein (eIF4EBP or 4E-BP) that competes with eIF-4G for binding to eIF-4E to inhibit mRNA cap-dependent translational initiation.27,28

Phosphorylation of eIF4EBP by mTOR reduces its afnity for eIF-4E and promotes the initiation of protein synthesis from a pool of transcripts enriched in mRNAs encoding proteins involved in angiogenesis, growth, and survival.29 Enhanced function of eIF-4E, often a result of inactivation of eIF4EBP by phosphorylation, is linked to poor prognosis, tumor progression, and metastasis in squamous cell,30 breast,31,32 ovarian, 33 and other cancers.

We demonstrate now that EIF4EBP is an evolutionarily conserved target of hyx/CDC73, and show that basal and serum starvation-stimulated levels of EIF4EBP3 transcript are reduced in peripheral mononuclear cells from patients heterozygous for CDC73, but not MEN1. We show that the expression of the EIF4EBP3 translational repressor represents a peripheral marker of CDC73 function and propose a model whereby starvation resistance, mediated at least in part by de-repression of protein translation, may contribute to parathyroid malignant transformation.

Results

We previously showed that in Drosophila a hypomorphic allele of hyx rescues the eye phenotype of lobe and that hyx heterozygous ies were resistant to starvation.24 Thus, Lsi/ ; hyxEY6898/ double heterozygotes have normal eyes,

even though heterozygous Lsi/ ies show a loss-of-ventral

eye phenotype (Figure 1e, cf. 1ac), and provide a sensitive genetic background to identify additional genes that interact with L, hyx, or both by eye phenotype screening.24

On the basis of the rescue of lobe by hyx and the enhanced resistance to starvation seen in hyx heterozygous ies,24 we

used the eye phenotype screening assay to look for an interaction of hyx with Tor, a key regulator of cellular nutritional homeostasis26 and target of lobe/ Akt1S1/ PRAS40 inhibition.25 To this end we crossed doubly heterozygous (Lsi/ ; hyxEY6898/ ) ies with a TorMB07988 mutant strain

(Figures 1hq, s, and t; Table 1). Approximately, 40% of the triple heterozygote (Lsi/TorMB07988 hyxEY6898/ ) ies had NOG

eye phenotypes (notch and dysplastic overgrowth), including half eyes with dysplastic overgrowths24 (Figures 1hk; Table 1, cross 8). The overgrowths typically appeared in or near the missing ventral eye region (Figures 1iq). The ommatidia and the sensory bristles were deformed and in disarray (Figures 1s and t, cf. 1r). Testing of another Tor mutant allele, TorK17004,

gave similar results (Table 1, cross 10). In contrast, 90% or more of the doubly heterozygous mutant ies carrying the Lsi

allele (Lsi/TorMB07988L si/TorK17004 Lsi/ ;hyxEY6898/ ) had

normal eyes (Figures 1e and f; Table 1, crosses 3, 7, and11). As the other possible doubly heterozygous mutant

combinations from the same matings produced ies with normal eyes (Figure 1g; Table 1, crosses 9 and 12), the observed strong eye phenotype must result from the specic combination of hyx and Tor loss-of-function.

We previously showed that the eye discs of Lsi/ ;

hyxEY6898/orb2BG02373 triple heterozygotes with a propensity

to later develop the NOG phenotype were characterized by an abnormal pattern of apoptosis at the larval stage.24 In

contrast, the larval eye discs of Lsi/TorMB07988 hyxEY6898/

triple heterozygotes showed no gross abnormalities in the pattern or extent of apoptosis compared with w1118 controls

(data not shown).

We, therefore, compared the pupal eye discs of wild-type and Lsi/TorMB07988 hyxEY6898/ triple heterozygotes to look

for developmental abnormalities that might account for the subsequent development of the NOG phenotype in the triple mutants. Normal eye development in Drosophila involves a wave of cell apoptosis between 35 and 50 h after pupation that eliminates a large fraction of undifferentiated interommatidial cells.34 The pupal eye discs of wild-type and Lsi/TorMB07988 hyxEY6898/ triple heterozygote ies were, therefore, exam

ined at 45 h after pupation using the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay to identify the nuclei of cells undergoing apoptosis (Figure 2). Eye discs from Lsi/TorMB07988 hyxEY6898/ triple heterozygote

pupae showed a disordered pattern of apoptosis as well as a signicant decrease in TUNEL-positive apoptotic nuclei compared with eyes from w1118 controls (Figures 2a and b). Thus, the eye discs of Lsi/TorMB07988 hyxEY6898/ triple

heterozygotes with a propensity to later develop the NOG phenotype were characterized by a substantial reduction in the usual burst of apoptosis between 35 and 50 h after pupation.

As current models suggest that Tor acts as a nutrient sensor, and regulator of cell growth and autophagy,26 we

studied starvation resistance in Tor and hyx mutant ies. Consistent with our previous results,24 hyx heterozygous ies were resistant to starvation compared with w1118 controls

(Figure 3a). Resistance to starvation was also found to be higher in Tor (TorMB07988/ ) heterozygous ies (Figure 3b).

Double hyx/ Tor heterozygotes (TorMB07988/ ; hyxEY6898/ )

were also resistant to starvation without any obvious additivity of the effect (Figure 3c).

A separate set of experiments used a different mutant allele of Tor (TorK17004) and showed that TorK17004/ heterozy

gotes, like TorMB07988/ heterozygous ies, exhibited resis

tance to starvation (Figure 3d). A double hyx/ Tor heterozygote strain demonstrated comparable resistance to starvation as seen with the single heterozygotes (Figure 3e, cf. 3a and d). We tested for rescue of the phenotype exploiting the fact that hyx can be overexpressed in strains carrying the GAL4-sensitive hyxEY6898 allele by mating to GAL4-over-

expressing strains.15,24 As shown in Figure 3f, overexpression

of hyx in ies carrying the ubiquitously expressed act5C-Gal4 driver and doubly heterozygous for hyx/ Tor mutation restored starvation sensitivity to near control levels, whereas control ies with 5C-actin promoter-driven GAL4 expression only had slightly enhanced starvation resistance (Figure 3f). In contrast to the hyx mutants, ies heterozygous for Mnn1 (Mnn1DG30701/ ) were sensitive to starvation like the w1118

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w1118 hyxEY6898/+

TorMB07988/+;hyxEY6898/+ Lsi/+/TorMB07988/+;

hyxEY6898/+

Lsi/+

TorMB07988/ MB07988

Lsi/+;hyxEY6898/+

Lsi/TorMB07988

Lsi/+/TorMB07988/+; hyxEY6898/+

Figure 1 Genetic interaction among lobe/Akt1s1, hyx/CDC73, and Tor evident from Drosophila eye phenotypes. Genetic interactions were recognized by the formation of novel NOG structures at the ventral part of the adult eye after crosses between ies with different genotypes. Shown are representative eye phenotypes captured by scanning electron microscopy for w1118 control (a), heterozygous Lsi mutant of lobe/AKT1S1 (b), heterozygous hyxEY6898 mutant of hyx/CDC73 (c), homozygous TorMB07988 mutant of Tor (d); double heterozygous mutants of lobe and hyx/CDC73 (e), lobe and Tor (f), and hyx/CDC73 and Tor (g); and triple heterozygous hybrid mutants of lobe, hyx/CDC73 and Tor strains (h). Images of additional eyes from triple heterozygous hybrid mutants of lobe, hyx/CDC73 and Tor strains are shown in (j) and (k) (image (i) is a duplicate of (h)). Higher magnication images of dysplastic overgrowth regions of (ik) are shown in (ln) and (oq). Higher magnication images of the ommatidial and sensory bristle phenotype of the triple heterozygous lobe, hyx/CDC73 and Tor mutant are shown (s and t), with corresponding image of wild-type (r)

controls (Figure 3g). Thus, heterozygosity of hyx and Tor, but not Mnn1, confers starvation resistance. Furthermore, hyx gene overexpression, at least at time points o100 h in a model system using the GAL4-sensitive hyxEY6898 allele and

the act5C-Gal4 driver, can rescue the starvation-resistant phenotype of Tor.

To better understand the interaction between hyx and Tor, quantitative RT-PCR was used to measure transcript levels in hyx or Tor mutant y strains. Levels of hyx transcript were partially reduced in TorMB07988 heterozygotes and by some 30% in TorMB07988 homozygotes compared to with control (Figure 4a). In contrast, adult ies heterozygous for hyxEY6898

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Table 1 Genetic interactions of L, hyrax, and Tor

CrossNo. Parent 1 Parent 2 F1 genotype

% ies with NOGa eye phenotype

% NOG in both eyes

Molecular function

1 w1118 w1118 w1118 0 0 02 Lsi/si w1118 Lsi/+ 0 0 100 L (lobe) is the y homolog of mammalian AKT1S1 (also called PRAS40)

3 hyxEY6898/+ Lsi/+;hyx EY6898/+ 6 0 11 Hyx (hyrax) is the y homolog of CDC73/ HRPT2

4 hyxEY6898/+ w1118 hyxEY6898/+ 0 0 0

5 TorMB07988/

MB07988 TorMB07988/MB07988 TorMB07988/MB07988 0 0 0 Tor is a key cellular kinase

involved in the regulation of cell growth and nutritional homeostasis

6 w1118 TorMB07988/+ 0 0 07 Lsi/+ Lsi/TorMB07988 6 0 28 Lsi/+;hyx EY6898/+ Lsi/TorMB07988; hyx EY6898/+

% notch w/o OG

40*** 7 3

9 hyxEY6898/+ TorMB07988/+; hyxEY6898/+ 0 0 010 TorK17004/+ Lsi/+; hyx EY6898/+ Lsi/TorK17004; hyxEY6898/+ 45*** 8 711 Lsi/TorK17004 10 o1 9 12 hyxEY6898/+ TorK17004/+; hyxEY6898/+ 0 0 0

aNOG: notch and overgrowth (OG), with notches and variably sized dysplastic overgrowths in the ventral eye elds. ***Po0.0001 compared with w1118n, and all double and single heterozygote mutants

had normal levels of Tor transcript (Figure 4b), even though Tor message levels were successively reduced in heterozygous and homozygous TorMB07988 mutant ies (Figure 4b).

To test the effect of Mtor inactivation on Cdc73 expression in a mammalian model, the expression of parabromin in several tissues harvested from wild-type and Mtor heterozygous mice was compared by quantitative immunoblotting. In several tissues examined, including pancreas, liver, and kidney, the expression of parabromin was reduced in Mtor heterozygous mice relative to b-actin (Supplementary Figure S1).

The translational repressor 4E-BP (eIF4EBP), which competes with eIF-4G for binding to eIF-4E to regulate translational initiation,35 is a downstream target of the Tor kinase in mammals28 and in ies.36 As our results indicate an interaction between hyx and Tor at the transcriptional level, we looked for a possible interaction between hyx and Thor, the y ortholog of EIF4EBP. Transcript levels of Thor/EIF4EBP are reduced by B50% in hyx heterozygotes (hyxEY6898/ )

(Figure 4c). This reduction in Thor/EIF4EBP expression was reversed by the GAL4-induced overexpression of hyx (Figure 4c). The level of Thor/EIF4EBP transcript in ies expressing the act5C-Gal4 driver alone was comparable to the controls (Figure 4d). The level of Thor/EIF4EBP transcript was reduced by B30% in Tor heterozygotes and additionally reduced to B50% in double hyx/Tor heterozygotes, a reduction rescued by overexpression of hyx through the hyxEY6898 allele (Figure 4e). Heterozygosity of hyx had no effect on the expression of S6k, the y ortholog of RPS6KB1 encoding the ribosomal protein S6 kinase, another well-characterized downstream target of the Tor kinase in mammals37 and in ies36 (Figure 4f).

Next the effect of starvation on relevant transcript levels in control and mutant ies was determined (Figures 5ae). Although starvation of ies had no effect on Tor transcript levels (Figure 5a), hyx expression was increased (Figure 5b). The induction of hyx by starvation persisted in Tor

heterozygotes, suggesting nutritional cues may regulate hyx transcription independently of Tor activity (Figure 5c). Starvation also increased Thor/EIF4EBP transcript expression compared with fed controls; however, the level of Thor/ EIF4EBP transcript in starved hyx heterozygous ies was reduced by B80% compared with starved w1118 controls

(Figure 5d). There was no difference in the basal Thor/ EIF4EBP transcript level between w1118 control and Mnn1 heterozygous ies, although the level of starvation-induced Thor/EIF4EBP was somewhat lower in the Mnn1 mutants (Figure 5e). These results suggest that the starvation resistance of both hyx and Tor heterozygous ies results, at least in part, from impaired upregulation of Thor/EIF4EBP in response to caloric deprivation.

To look for these interactions in human cellular models, the effect of serum starvation on the expression of CDC73 and the three mammalian EIF4EBP isoforms was determined in HEK293 human embryonic kidney cells and cultured human peripheral blood mononuclear cells (WBC). Serum starvation for 48 h signicantly increased CDC73, EIF4EBP1, EIF4EBP2, and EIF4EBP3 transcript expression in HEK293 cells (Figures 5fi). In control WBC, as in HEK293 cells, serum starvation signicantly increased CDC73 transcript expression (Supplementary Figure S2).

The expression of EIF4EBP isoforms was compared in WBC collected from normal healthy volunteer controls and from patients with the HPT-JT (CDC73 heterozygotes) or MEN1 (MEN1 heterozygotes) (Supplementary Table S1). Compared with control WBC, basal levels of all three EIF4EBP isoforms were reduced in CDC73 heterozygotes with the most striking reduction in EIF4EBP3 (Figure 6). In WBC from MEN1 heterozygous patients, the basal level of EIF4EBP1 transcript was reduced compared with control; however, there was no difference in the basal EIF4EBP2 level, and basal EIF4EBP3 was increased (Figure 6). Serum starvation-induced levels of EIF4EBP1 and EIF4EBP2

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w1118

L/Tor; hyx/+

300

Apoptotic cells per

pupal eye disc, total

200

100

***

0 w1118 L / Tor; hyx/+

Figure 2 Loss of normal apoptosis during pupal development correlates with the NOG eye phenotype observed in the L/Tor;hyx triple heterozygous mutant ies. Normal eye development in Drosophila involves a wave of cellular apoptosis between 35 and 50 h after pupal formation (APF) that eliminates a large fraction of undifferentiated interommatidial cells. The eye discs of Lsi/TorMB07988 hyxEY6898/

triple heterozygotes with a propensity to later develop the NOG phenotype were characterized by a substantial reduction in this usual burst of apoptosis APF compared with w1118 controls. (a) 40,6-diamidino-2-phenylindole nuclear staining (left), TUNEL analysis (middle), and merged images (right) of pupal eye discs dissected B45 h APF reveal a loss and mis-localization of apoptotic-positive cell nuclei in Lsi/TorMB07988 hyxEY6898/ triple heterozygote ies compared with

w1118 controls. The difference in size between w1118 control and Lsi/TorMB07988 hyxEY6898/ triple heterozygote eye discs shown represents a random variation

and was not reective of a consistent difference between the two. (b) Quantication of apoptotic cells per pupal eye disc shows signicant (B60%) reduction in total apoptotic-positive cell nuclei in Lsi/TorMB07988 hyxEY6898/ triple heterozygotes

compared with w1118 controls (***Po0.0001, unpaired Students t-test; w1118 control, n 16; Lsi/TorMB07988 hyxEY6898/ , n 18)

transcript did not discriminate between CDC73 and MEN1 heterozygous patients, and both groups of patients had lower induced transcript levels than the controls (Figure 6). In contrast, the serum starvation-induced level of EIF4EBP3 transcript from CDC73 heterozygous patients was markedly lower than that from both control and MEN1 heterozygous patient WBCs (Figure 6). Thus, reduced basal and serum starvation-induced EIF4EBP3 transcript in peripheral WBC represents a phenotype that correlates strongly with CDC73 heterozygosity, but not with that of MEN1.

Chromatin immunoprecipitation (ChIP) was used in HEK293 cells to determine if the effect of CDC73 allele dosage on EIF4EBP3 transcript levels was consistent with regulation at the transcriptional level. Three pairs of primers were used to interrogate anti-parabromin immunoprecipitates in a ChIP assay by quantitative PCR, with one primer set (P1) targeting the upstream EIF4EBP3 promoter region, one set (P2) targeting the internal gene coding sequence at

exon 2, and one set (P3) directed at a anking region downstream of the putative poly A signal (Figure 7a). Antibodies to other components of the PAF1 complex, Leo1, and Paf1 were also used. As shown in Figure 7b, primer pairs P1 and P2 produced signicantly higher signals from the anti-parabromin, anti-Leo1, and anti-Paf1 immunoprecipitates than from those using control IgG. The specic anti-parabromin ChIP signal was much stronger in the P1 and P2 regions of EIF4EBP3 than in the P3 region, and no signal at all was detectable in the P3 region with anti-Leo1 or anti-Paf1 ChIP (Figure 7b). Taken together, these data suggest that the PAF1 complex might be involved in both EIF4EBP3 transcript initiation and elongation, consistent with PAF1 complex function at other gene loci.1214,38

Discussion

Gene expression proling studies of parathyroid tumors by Haven et al.39 strongly suggest that sporadic parathyroid malignancies and tumors associated with CDC73 inactivation follow a pathway distinct from benign sporadic and MEN1-related parathyroid tumors. This dichotomy is reinforced by the clinical observations that inactivation of CDC73 is frequent in parathyroid cancer,8,9 but rare in sporadic benign parathyroid tumors,16,17 whereas mutation of MEN1 is nearly always associated with benign parathyroid adenomas in both sporadic and MEN1-associated cases.

We show here that EIF4EBP3 is distinctly regulated by CDC73 and MEN1, that parabromin and other PAF1 complex components occupy the EIF4EBP3 promoter and coding regions, and that loss of EIF4EBP3 expression represents a marker of CDC73 haploinsufciency and heterozygosity, but not MEN1 loss-of-function, in a human WBC model system. As current PCR-based CDC73 germline mutation testing methods may miss some carriers,7 our results indicate it may be possible to independently assess CDC73 allele dosage by the determination of EIF4EBP3 expression in a clinically practical manner.

The strong evolutionary conservation of the link between EIF4EBP and CDC73 is striking. Taken together with our previous nding that hyx opposes lobe/Akt1S1 in the eye phenotype assay,24 the present work strengthens the notion that TOR and CDC73 interact with an overlapping set of regulators and effector targets. Functioning as a sensor of nutrient availability, the protein kinase mTOR regulates cell growth and autophagy.26 Under physiological conditions, growth signals acting through mTOR promote the phosphorylation of eIF4EBP and reduce its afnity for eIF-4E; thus initiating protein synthesis from a pool of transcripts critical for cell growth and survival. Under pathological conditions, the enhanced function of eIF-4E, frequently a result of inactivation of eIF4EBP by mTor phosphorylation, is linked to poor prognosis and metastasis in multiple malignancies.3033

Unlike the inactivation of eIF4EBP by phosphorylation, via the canonical mTOR pathway, loss of CDC73 exerts its negative effect on EIF4EBP3 through loss of gene expression. Our ndings suggest a model in which de-repression of cap-dependent protein translation and heightened survival resulting from CDC73 loss-of-function may enhance carcino-genesis in parathyroid tumors, conferring selective advantage

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* __________

***

_____

* ___________

***

100

100

80

________

**

80

60

60

%Survival

%Survival

40

40

20

w1118

w1118

20

w1118

Tork17004/+

___

*

hyxEY6898/+

*

0

0 0 24 48 72 96 120

0 50 100

Hours of Starvation Hours of Starvation

* ___

** ________________

***

*** **

100

100

__________

* * *

80

80

60

%Survival

60

%Survival

40

40

20

20

w1118

TorMB07988/+

Tork17004; hyxEY6898

0 0 50 100

Hours of Starvation

0 0 24 48 72 96 120

Hours of Starvation

0 0 24 48 72 96 120

Hours of Starvation

Hours of Starvation

100

100

______

80

80

%Survival

______

*

60

%Survival

60

40

40

*

w1118

20

act-GAL4/+ w1118

act-GAL4/ Tork17004;hyxEY6898

20 TorMB07988/+;hyxEY6898/+

0 0 50 100

100

80

60

%Survival

40

20

w1118

Mnn1+/-

0 0 50 100

Hours of Starvation

Figure 3 Enhanced starvation resistance in hyx/CDC73 and Tor mutant ies. Survival upon starvation of the indicated single or double hyx/CDC73 and Tor heterozygous mutant y strains is shown. Flies ag were supplied only with water to test starvation resistance. The number of surviving ies was recorded every 12 h. For each experiment, 20 or more vials were used and two or more independent experiments were conducted for each y line. Vials contained 10 ies each. Each data point represents the pooled mean survival from 20 to 30 vials of the indicated genotype (*Po0.05; **Po0.001; ***Po0.0001; versus wt for the indicated time points, twotailed P-values for unpaired

Students t-test). The experiment shown in (f) tested the rescue of the starvation resistance of the TorK17004/ ; hyxEY6898/ double mutant (cf. e) by overexpression of hyx

from the hyxEY6898 allele upon mating with a driver strain expressing GAL4 from the 5C-actin promoter (act-GAL4), with the driver-only control shown (act-GAL4/ ; dashed

gray line). In panel f, the P values for the unpaired Students t-test comparing the % survival of w1118 control strain with the act-Gal4/TorK17004/ ; hyxEY6898/ strain were:

84 h, P 0.32; 96 h, P 0.14; 108 h, P 0.036

to hypermetabolic neoplastic cells for clonal expansion in the face of nutritional stress.

Materials and MethodsHuman subjects. All patients participated in the protocols approved by the Investigational Review Boards of the National Institute of Diabetes and Digestive and Kidney Diseases and each gave written consent. Normal volunteers had no

personal or family history of hyperparathyroidism, HPT-JT, or MEN1. Patients with HPT-JT or MEN1 had documented heterozygous germline mutation of CDC73/ HRPT2 or MEN1, respectively. Patient characteristics are summarized in Supplementary Table 1.

Fly stocks. The enhancer trapped y lines from the Japanese NP Consortium

Gal4 Enhancer Trap Insertion Database were obtained from the Drosophila Genetic

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0.015

0.020

Expression of hyx

Expression of Tor

0.010

0.015

** ***

***

0.010

***

0.005

***

0.005

0.000

0.000

w1118 Tor +/- Tor +/- Tor +/-

w1118 w1118 w1118;

act5C-GAL4

hyx +/-

w1118 Tor +/- Tor -/- hyx +/-

0.20

0.25

0.20

0.15

Expression of

Thor/ 4E-BP

***

Expression of

Thor/ 4E-BP

0.15

0.10

0.10

0.05

0.05

0.00

0.00

hyx +/-;

act5C-GAL4

0.06

0.3

0.04

Expression of

Thor/ 4E-BP

Expression of

S6k/ RPS6KB1

0.2

**

***

0.1

0.02

0.0

0.0

w1118 Tor +/- Tor +/-;

hyx+/-

Tor +/-;

hyx +/-;

act5C-GAL4

w1118 hyx +/-

Figure 4 Transcriptional regulation of Tor, hyx, and Thor in y. Heterozygous Tor/ (TorMB07988/ ) and homozygous Tor / (TorMB07988/MB07988) mutant ies have a

dose-dependent reduction in both Tor and hyx transcript compared with w1118 controls (a and b). Heterozygous hyx (hyxEY6898/ ) mutant ies show a reduction in hyx, but not

in Tor transcript expression (a and b). Thor/4E-BP transcript expression is signicantly reduced in hyx/CDC73/ ies compared with w1118 controls, and is rescued back to control levels following act5C-GAL4-driven overexpression of hyx/CDC73 from the hyxEY6898 allele (c). The act5C-GAL4/ driver alone has no effect on Thor/4E-BP

transcript levels (d). Tor/ heterozygosity and Tor/ ;hyx/ double heterozygosity results in a decrease in Thor/4E-BP transcript levels, and is similarly rescued following act5C-GAL4-driven overexpression of hyx/CDC73 from the hyxEY6898 allele in the Tor/ ;hyx/ double heterozygous mutant background (e). Heterozygosity of hyx/ CDC73/ has no effect on transcript levels of s6k/RPS6KB1, which is, like 4E-BP, a major effector of Tor (f). For a, b, c, and e **Po0.005; ***Po0.0005 compared with w1118 control, using unpaired Students t-test

Resource Center, Kyoto Institute of Technology (Kyoto, Japan). The y line bearing the hypomorphic allele, hyxEY6898, which contains a P-element (P[EPgy2]) insertion located 36 bp upstream of the hyx translational start site in the 50 untranslated transcript region (Stock No. 16768), and all the other y lines used, including Mnn1DG30701 (Stock No. 21335), TorK17004/CyO (Stock No. 11218), TorMB07988 (Stock No. 25363), and Act5C-GAL4/CyO (Stock no. 4414), were from the Bloomington Drosophila Stock Center at Indiana University (Bloomington, IN, USA).

Scanning electron microscopy. Flies were xed in 4% paraformaldehyde (Electron Microscopy Sciences, Hateld, PA, USA). Before examination, the samples were dehydrated through 100% ethanol, and then critically point dried (Tousimis model Samdri-795, Rockville, MD, USA). A 10-nm gold coating was deposited in a sputter coater (Electron Microscopy Sciences model 575 ), and the

detailed structure of the y eyes were studied at 5 kV on a scanning electron microscope (Hitachi S-3400N, Pleasanton, CA, USA).

Pupal eye disc apoptosis analysis. The ApopTag Red In situ Apoptosis Detection Kit (Millipore, Billerica, MA, USA) was used for TUNEL analysis. Eye discs collected B45 h following pupation were dissected in PBS and analyzed for apoptosis following the same method as previously described for the eye discs of third instar y larvae.24

Fly starvation resistance. For starvation stress tests, ies with desired genotypes that eclosed within 24 h were collected. After ageing for 8 days at 251C,

mated female ies were placed into vials containing paper discs (d 2.3 cm)

soaked in 400 ml of H2O (Whatman, Piscataway, NJ, USA). H2O was replenished throughout the experiment as needed. A total of 2030 vials (10 ies per vial) were used for each test, and at least two independent experiments were performed. Dead ies were recorded every 12 h. For data analysis, each vial was treated as a data point.

mRNA quantication. Gene expression levels were estimated on the basis of the transcript abundance as measured by quantitative RT-PCR. Quantitative RT-PCR was performed with one-step quantitative RT-PCR master mix (Agilent Technologies, Santa Clara, CA, USA) using a Stratagene MX 3005P real time PCR machine (Agilent Technologies) and analyzed using the accompanying software. Each reaction was conducted in triplicate and three to nine biological samples prepared independently were used in data analysis. The Prism software version5.0c (GraphPad Software, Inc., La Jolla, CA, USA) was used for graphing the analyzed data set. Total RNA was prepared from both mutant and control samples using QIAGEN RNeasy kit (Valencia, CA, USA). Primer sequences were developed using PrimerQuest software (Integrated DNA Technologies, Coralville, IA, USA), and were as follows: Drosophila Tor forward (50-TGG CAA AGC AAC TGG GCA

AGA A-30) and reverse (50-TCG GGA TCC ACA ATG CGA TGT T-30); hyx forward (50-TTT GCG TGC CAT CCT GGA CTA T-30) and reverse (50-TCT TGC CCG TGC TTT GCA GAA T-30); Mnn1 forward (50-CAA ATG GAT AGA CGG ACT GCT CGT-30) and reverse (50GTC AAC AGT TCG TAA CAA GGA TTT GC-30); Thor forward

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J-H Zhang et al

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***

0.015

***

*** ***

0.015

Starv. - +

0.015

0.010

Expression of Tor

Expression of

hyx/ CDC73

0.010

0.010

0.005

Expression of

hyx/ CDC73

0.005

0.005

0.000

0.000

0.000

w1118 w1118

Starv. - +

w1118 w1118

Starv. - + - +

w1118 w1118 Tor +/- Tor +/-

***

***

***

***

*** ***

Expression of

EIF4EBP3

1.5

0.5

0.4

Expression of

Thor/ 4E-BP

Expression of

Thor/ 4E-BP

1.0

0.3

***

0.2

0.5

0.1

0.0

0.0

w1118 w1118 hyx +/- hyx +/-

Starv. - + - +

w1118 w1118 Mnn1+/- Mnn1+/-

Starv. - + - +

0.08

0.020 *

0.08

1.0

**

***

***

0.06

0.8

0.015

0.06

Expression of

CDC73/ HRPT2

0.04

Expression of

EIF4EBP1

0.6

0.010

Expression of

EIF4EBP2

0.04

0.4

0.02

0.005

0.02

0.2

0.00

0.000

0.00

0.0

Serum starv. - + Serum starv. - + Serum starv. - + Serum starv. - +

Figure 5 Transcriptional regulation of hyx/CDC73 and Thor/4E-BP in response to caloric or serum starvation. Upregulation in w1118 control or mutant ies of hyx/CDC73 and Thor/4E-BP, but not of Tor transcript, compared with fed controls following 48 h of starvation (ad). The basal reduction of hyx transcript observed in Tor/ (TorMB0798/ )

heterozygous ies was lost following 48 h of starvation, suggesting that nutritional cues may regulate hyx transcription independently of Tor activity (c). Conversely, the basal reduction in Thor/4E-BP transcript observed in hyx/ heterozygous ies persists following 48 h of starvation, suggesting that hyx is necessary for the starvation-induced increase in Thor/4E-BP transcript (d). Basal expression of Thor/4E-BP transcript was unchanged in Mnn1/ (Mnn1DG30701/ ) heterozygous ies compared with w1118

controls; however, the level of Thor transcript was somewhat reduced following 48 h of starvation (e). Expression of human CDC73, EIF4EBP1, EIF4EBP2, and EIF4EBP3 transcript was signicantly increased in HEK293 cells following 48 h of serum starvation (fi). For bi *Po0.05, **Po0.005; ***Po0.0005 compared with control, using unpaired Students t-test

(50-CCA TGA TCA CCA GGA AGG TTG TCA-30) and reverse (50-TCT TCA TGA AAG CCC GCT CGT AGA-30); s6k forward (50-ATG CGG CGG CTG TTC AAA TAC A-30) and reverse (50-TGG CAC TTT CGC TTA GCG TTG T-30). Human CDC73 forward (50-AGA TGC AAC CAG GGG GCA CTG-30) and reverse (50-GCA GGA CCC TGC ACA AAA ACG G-30); EIF4EBP1 forward (50-TGG ACA AGA ACG AAC CCT TCC T-30) and reverse (50-AGG GAG CTT TCC CAA GCA CAT-30); EIF4EBP2 forward (50-TTT GCA TTC ACC CTC CTT CCC A-30) and reverse (50-AGG GCA CCA AAT CCA ACC AGA A-30); EIF4EBP3 forward (50-AAG TTC CTG CTG GAG TGC AAG A-30) and reverse (50-TCT CCT GCT CCT TCA GCT CCT C-30).

Immunoblotting and infrared imaging. Tissues from 6-week old female wild-type C57BL/6 and Mtor heterozygous mice (Jackson Laboratory, Bar Harbor, ME, USA, strain B6.129S5-MtorMtorGt(OST92090)Lex/J, Stock No. 013190) were stored at 801 and then thawed on ice. For every 50 mg of mouse tissue, 1 ml of

1 RIPA cell lysis buffer (Cell Signaling, Beverly, MA, USA, Cat. No. 9806)

including 1 protease inhibitors (Calbiochem, EMD Serono, Inc., Rockland, MA,

USA, Cat. No. 539134) was added followed by homogenization on ice in a 1.5-ml microcentrifuge tube using a tissue homogenizer (Omni International, Kennesaw, GA, USA, Model TH-115) at high speed for 30 s. Homogenates were combined with an equal volume of Laemmlis 2 gel loading buffer, vortexed, and heated at 951C

for 10 min before gel loading. Electrophoresis by SDS-PAGE was followed by the

transfer of the proteins on to 0.45-micron nitrocellulose membrane. Membranes were blocked with TBS or PBS (pH 7.4) containing 0.1% Tween 20 and 5% nonfat dry milk (blocking buffer), and incubated overnight with primary antibodies in the same buffer. Primary antibodies used were rabbit anti-parabromin polyclonal (GRAPE antibody40) and mouse monoclonal anti-b- actin (Sigma-Aldrich, St. Louis,

MO, USA, Cat. No. A5316). Membranes were then washed, and IR-labeled secondary antibodies (dilution 1 : 20 000) were used for detecting the protein signals in conjunction with the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). Quantication of parabromin immunoreactivity used the b-actin signal in the same lane for normalization. IR secondary antibodies (anti-rabbit IR 800 and anti-mouse Green) were from LI-COR Biosciences.

Mammalian cell culture. Human embryonic kidney HEK293 cells (American Type Culture Collection, Manassas, VA, USA, Cat. No. CRL-1573) were grown in 75-cm2 asks in DMEM supplemented with 10% fetal bovine serum (FBS), 4 mML-glutamine and penicillin/streptomycin at 371C and 5% CO2. For serum-starvation experiments, HEK293 cells newly seeded the night before were either fed with medium supplemented with 10% FBS (control) or not (starved) for 48 h. Cells were then detached using TrypsinEDTA, pelleted in microcentrifuge tubes, and either used immediately or stored at 801C.

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EIF4EBP3 is a marker of the CDC73 tumor suppressor J-H Zhang et al

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***

***

***

***

***

***

***

***

0.020

0.010

Expression ofEIF4EBP1

*

Expression ofEIF4EBP1

0.008

0.015

*

0.006

***

0.010

***

0.004

0.005

0.002

0.000

CDC73+/-

MEN1+/-

- MEN1 Con CDC73+/-

+/-

CDC73+/-

MEN1+/-

- MEN1 Con CDC73+/-

+/-

0.000

Serum starv.

Con

-

-

+

+

+

Serum starv.

Con

-

-

+

+

+

***

***

*

*

0.06

0.020

Expression ofEIF4EBP2

*

*

*

Expression ofEIF4EBP2

*

0.015

0.04

0.010

0.02

0.005

MEN1+/-

- MEN1 Con CDC73+/-

+/-

MEN1+/-

- MEN1 Con CDC73+/-

+/-

0.00

0.000

CDC73+/-

CDC73+/-

Serum starv.

Con

-

-

+

+

+

Serum starv.

Con

-

-

+

+

+

**

***

**

***

***

***

2.5

8

***

***

2.0

Expression ofEIF4EBP3

4

Expression ofEIF4EBP3

*

6

*

1.5

**

**

1.0

2

0.5

CDC73+/-

MEN1+/-

- MEN1 Con CDC73+/-

+/-

CDC73+/-

MEN1+/-

- MEN1 Con CDC73+/-

+/-

0.0

Serum starv.

0 Con

- Serum starv.

Con

-

+

+

+

-

-

+

+

+

Figure 6 Heterozygosity of CDC73 results in a reduction of EIF4EBP transcript levels in human WBCs. WBCs were collected from healthy volunteers or from patients with HPT-JT (CDC73 heterozygotes) or MEN1 (MEN1 heterozygotes). The expression of transcripts for the indicated isoforms of EIF4EBP was determined by quantitative RT-PCR following 72 h of control culture conditions or serum starvation, as indicated. The same data are presented as scatter dot plots on the left (a, c and e) and histograms on the right (b, d and f). Control, n 4362 transcript measurements from eight cell harvests from eight normal volunteers. CDC73/ , n 2234 transcript measurements from

seven cell harvests from ve patients. MEN1/ , n 3747 transcript measurements from 11 cell harvests from 9 patients. For af *Po0.05, **Po0.005; ***Po0.0005 for

indicated comparisons, using unpaired Students t-test

A mononuclear cell fraction was isolated from a morning whole blood sample drawn from non-fasting normal volunteers, CDC73 or MEN1 heterozygotes (collected at room temperature in heparinized tubes) processed using Lymphocyte Separation Medium (Lonza, Walkersville, MD, USA, Cat. No. 17829E). The buffy layer, enriched in lymphocytes and mononuclear cells, was collected, washed once in PBS after which the cells were resuspended in complete RPMI medium 1640 (GIBCO, Life Technologies, Grand Island, NY, USA) containing 10% FBS, plated on to 12-well cell culture plates and incubated at 371C with 5% CO2. After 24 h of incubation, non-adherent cells were gently removed and the adherent cell population, enriched in

mononuclear cells, was divided and further incubated with either fresh complete RPMI medium (control cells) or the RPMI medium without serum (serum-starved cells) for an additional 72 h. The cells were then detached by trypsinEDTA treatment, pelleted in microcentrifuge tubes, and stored at 801C until use.

ChIP assay. ChIP assay kit from Millipore (Cat. No. 17295) was used in the analysis of HEK293 cells following the manufacturers instructions except that the QIAquick PCR purication kit (Qiagen, Cat. No. 28104) was used for DNA purication. Puried DNA was used as a template for the amplication of selected

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Conict of Interest

The authors declare no conict of interest.

Acknowledgements. We are grateful to Sunita Agarwal and Stephen Marx for encouragement and helpful discussions. This research was supported by the Intramural Research Programs of the National Institute of Diabetes and Digestive and Kidney Diseases, the National Heart, Lung and Blood Institute, and the National Institute of Allergy and Infectious Diseases.

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2. Mallette LE, Malini S, Rappaport MP, Kirkland JL. Familial cystic parathyroid adenomatosis. Ann Intern Med 1987; 107: 5460.

3. Teh BT, Farnebo F, Kristoffersson U, Sundelin B, Cardinal J, Axelson R et al. Autosomal dominant primary hyperparathyroidism and jaw tumor syndrome associated with renal hamartomas and cystic kidney disease: linkage to 1q21-q32 and loss of the wild type allele in renal hamartomas. J Clin Endocrinol Metab 1996; 81: 42044211.

4. Teh BT, Farnebo F, Twigg S, Hg A, Kytla S, Korpi-Hyvalti E et al. Familial isolated hyperparathyroidism maps to the hyperparathyroidism-jaw tumor locus in 1q21-q32 in a subset of families. J Clin Endocrinol Metab 1998; 83: 21142120.

5. Simonds WF, James-Newton LA, Agarwal SK, Yang B, Skarulis MC, Hendy GN et al. Familial isolated hyperparathyroidism: Clinical and genetic characteristics of thirty-six kindreds. Medicine (Baltimore) 2002; 81: 126.

6. Simonds WF, Robbins CM, Agarwal SK, Hendy GN, Carpten JD, Marx SJ. Familial isolated hyperparathyroidism is rarely caused by germline mutation in HRPT2, the gene for the hyperparathyroidism-jaw tumor syndrome. J Clin Endocrinol Metab 2004; 89: 96102.

7. Carpten JD, Robbins CM, Villablanca A, Forsberg L, Presciuttini S, Bailey-Wilson J et al. HRPT2, encoding parabromin, is mutated in hyperparathyroidism-jaw tumor syndrome. Nat Genet 2002; 32: 676680.

8. Shattuck TM, Valimaki S, Obara T, Gaz RD, Clark OH, Shoback D et al. Somatic and germ-line mutations of the HRPT2 gene in sporadic parathyroid carcinoma. N Engl J Med 2003; 349: 17221729.

9. Cetani F, Pardi E, Borsari S, Viacava P, Dipollina G, Cianferotti L et al. Genetic analyses of the HRPT2 gene in primary hyperparathyroidism: germline and somatic mutations in familial and sporadic parathyroid tumors. J Clin Endocrinol Metab 2004; 89: 55835591.

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1

Human EIF4EBP3

1911 2513

P2 P3

-600

P1

-368

Exon 1

Exon 2

Exon 3

1206

Poly A signal

-180

1412 1948

2238

1.5

1.0

% Input

0.5

0.0

P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 Control IgG

anti-Paf1

anti-Pfb

anti-Leo1

Primer set 1 (P1)

Primer set 2 (P2)

Primer set 3 (P3)

500-300-200-100-

M. DNA Markers1. anti-Pfb ChIP2. anti-Leo1 ChIP3. anti-Paf1 ChIP4. control IgG ChIP5. no template

500-

300-200-

100-

400-

M

1

2

3

4

5

M

1

2

3

4

5

M

1

2

3

4

5

Figure 7 ChIP demonstrates occupancy at EIF4EBP3 by the PAF1 complex. The physical association of endogenous parabromin and other components of the PAF1 complex (including the Paf1 and Leo1 proteins) with human EIF4EBP3 was examined by ChIP in HEK293 cells. (a) Schematic diagram showing the relative location of the three PCR primer sets used in the ChIP assay (P1, P2, and P3) along the human EIF4EBP3 gene (HGNC ID: 3290) and anking regions (not to scale). The numbering shown is relative to the rst base of exon 1 as 1 as indicated.

(b) ChIP analysis using primer sets targeting upstream (P1), exon 2 (P2) and downstream (P3) sequence of EIF4EBP3 using antibodies against parabromin, Leo1 or Paf11 or control rabbit IgG as shown. Results shown are mean percentage of input signalS.E.M. (c) Qualitative analysis of ChIP PCR-derived DNA products by agarose gel electrophoresis and ethidium bromide staining. All experiments are representative of three or more independent biological repeats

regions of EIF4EBP3 and its anking sequences using the following primer pairs (Figure 7a): P1 (upstream promoter region, PCR product 191 bp, forward 50-TCC

CTT CCT GCA AGA TGT GTG ACT-30, reverse 50-TGG CCC AGC GCT GGC CAA CCT GCC-30); P2 (exon 2 region, PCR product 206 bp, forward 50-TCC TGA CTC TTA CCT CAG TCC CAA-30, reverse 50-TAC CGG GTA TCT CTT CCT CTG TCT-30); P3 (downstream region following putative polyA signal, PCR product 288 bp, forward 50-TCT CAT CTC AGC CAC ACA GCT GAA-30, reverse 50-ATA GGC TGG GAC AGC

ACC TCT T-30). Antibodies used for the ChIP assay were anti-parabromin (GRAPE antibody40), anti-Leo1 (Bethyl Laboratories, Montgomery, TX, USA, Cat. No. A300-175A) and anti-Paf1 (Upstate (Millipore), Cat. No. 07653), and control rabbit IgG (Santa Cruz Biotechnologies, Santa Cruz, CA, USA, Cat. No. sc-2027). Real-time quantitative PCR was performed using 2 Brilliant II SYBR green QPCR master mix

(Agilent Technologies, Cat. No. 600828-51) using the Stratagene Mx3005P QPCR system. PCR parameters used were 951C 10 min, 40 cycles of 941C 30 s, 601C 1 min, and 721C 1 min, followed by 1 cycle of dissociation curve analysis. The size and quality of the PCR-amplied DNA was assessed by agarose gel electrophoresis and ethidium bromide staining in comparison to DNA markers (GeneRuler DNA Ladder Mix, Fermentas, Glen Burnie, MD, USA, Cat. No. SM0333). Data analysis and graphing were performed using Prism software version 5.0c (GraphPad Software, Inc.) with results expressed as percentage input signal after normalizing Ct values from each primer set.

Statistical analysis. Data were analyzed by Prism software version 5.0c. (GraphPad Software, Inc.). Unpaired Students t-test was used to evaluate for statistical signicance.

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