In vivo effect of NCoR

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TRANSGENIC TARGETING OF A DOMINANT NEGATIVE COREPRESSOR TO

LIVER BLOCKS BASAL REPRESSION BY THYROID HORMONE RECEPTOR AND

INCREASES CELL PROLIFERATION

Xu Feng, Yuan Jiang*, Paul Meltzer* and Paul M. Yen

Mol. Regulation and Neuroendocrinology Section, Clinical Endocrinology Branch

NIDDK, and *Cancer Genetics Branch, NHGRI, National Institutes of Health,

Bethesda, MD 20892

Key Words: co-repressor, thyroid hormone receptor, liver, gene regulation,

microarray

Running Title: In vivo effects of NCoR

Corresponding Author: Paul M. Yen, M.D. Molecular Regulation and

Neuroendocrinology Section, Clinical Endocrinology Branch, NIDDK, NIH,

Bethesda, MD 20892

Tel: 301-594-6797 FAX: 301-402-4136

email: pauly@bdg10.niddk.nih.gov

JBC Papers in Press. Published on January 29, 2001 as Manuscript M011027200 by guest on D

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Abstract

Unliganded thyroid hormone receptors (TRs) interact with

corepressors and repress basal transcription of target genes in co-transfection

and in vitro studies. Currently, little is known about the function of

corepressors in vivo . We thus used a mouse albumin promoter to generate

several transgenic mouse lines that overexpressed a dominant negative

mutant corepressor, NCoRi, in liver. The transgenic mice had normal liver

weight, appearance, and minimal changes in enzyme activity. To study the

effects of NCoRi on transcription of hepatic target genes, we examined T3-

regulated gene expression of hypo- and hyperthyroid transgenic mice. In

hypothyroid mice, hepatic expression of Spot 14, bcl-3, glucose 6-phosphatase,

and 5’ deiodinase mRNA was higher in transgenic mice than littermate

controls whereas these genes were induced to similar levels in T3-treated

mice. Derepression was not observed for malic enzyme mRNA expression i n

hypothyroid mice. Thus, NCoRi selectively blocked basal transcription of

several thyroid hormone-responsive genes, but had no effect on ligand-

mediated transcription. Additionally, compensatory increases in endogenous

SMRT and NCoR mRNA were observed in hypothyroid transgenic mice.

Interestingly, hepatocyte proliferation as detected by BrdU incorporation was

increased in transgenic mice. The gene profile in transgenic mouse livers was

studied by cDNA microarray, and several genes related to cell proliferation

were induced. In summary, our studies show that NCoR plays important

roles in mediating basal repression by TRs and may prevent cellular

proliferation in vivo.

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Introduction

Thyroid hormone receptors (TRs) and retinoic acid receptors (RARs)

can repress basal transcription in the absence of ligand, and activate

transcription upon ligand-binding, in positively-regulated target genes. TRs

and RARs mediate basal repression through interactions with corepressors,

such as NCoR ( n uclear receptor cor epressor) and SMRT ( s ilencing m ediator

for r etinoid and t hyroid hormone receptors) (1,2). NCoR and SMRT are both

270 kDa in size and their overall amino acid identity is 43% (3,4). These co-

repressors have two nuclear hormone receptor interaction domains in the

carboxy-terminus and three transferable repression domains in the amino-

terminus (5,6). NCoR binds to sin3, which in turn, recruits histone

deacetylases (7-11). The formation of the NCoR/sin3/HDAC complex by T R

leads to hypoacetylation of local histones resulting in conformational changes

in the nucleosome structure and decreased access of enhancers and

components of the basal transcriptional machinery to the promoter region

and transcriptional start site (7-10). In contrast, transcriptional activation,

occurs via ligand-dependent recruitment of p160 co-activators by TR, which

then forms complexes that contain histone acetyltransferase activity (12).

These complexes may then exchange with other complexes that share

components with the RNA pol II transcriptional initiation complex (13,14).

The NCoR/Sin3/HDAC complex not only mediates transcriptional

repression by TR and RAR, but also by other transcription factors such as Rev-

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Erb, COUP-TF, MyoD as well as Mad/Max and Mad/Mxi dimers, (7-10,15-17).

Additionally, co-repressors can interact with PML-RARα and the PLZF-RARα

and LAZ3/BCL6 which are involved in acute promyelocytic leukemia and

non-Hodgkin lymphomas, respectively (18-21). Recent evidence also suggests

that NCoR and Sin3 may interact directly with the key components of the

basal transcriptional machinery to inhibit basal transcription in an alternative

repression pathway (22).

Almost all studies of NCoR function to date have been performed in i n

vitro transcription and co-transfection systems. Currently, little is known

about the physiological and developmental roles played by NCoR in v i v o .

Hollenberg et al. have shown that a variant form of NCoR, NCoRi, lacks the

repression domains in the amino-terminus but retains the nuclear receptor

interaction domains. NCoRi also had dominant negative activity on TR-

mediated basal repression in co-transfection assays (23). The liver has long

been known to be a major target organ for TH, with more than 50 target genes

identified recently by cDNA microarray (24). Thus, to examine the roles of

NCoR in vivo , we created several transgenic mouse lines in which the

dominant negative NCoRi was targeted to liver via an albumin promoter.

Our studies showed that NCoRi selectively blocked basal repression of several

TH-regulated target genes but had no effect on transcriptional activation.

Additionally, hepatocellular proliferation was increased in the transgenic

mice. Our study demonstrates that NCoR plays an important role i n

mediating basal repression by TRs in vivo, and also may be involved i n

cellular proliferation in the liver.

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Materials and Methods

Construction of liver-specific expression vectors for NCoRi

To construct a vector containing the albumin promoter and NCoRi

sequence, a 3.1 kb NCoRi fragment was obtained by EcoRI digestion from

PKCR2-NCoRi (gift of A. Hollenberg, Beth Israel Hospital, Boston, MA) (23)

and inserted into multiple cloning sites in the albumin promoter cassette

PGEMLB-SVPA (gift from Dr. Jack Liang, NIDDK, NIH), resulting i n

PGEMLB-SVPA-NCoRi vector (25). A Flag (2x) sequence also was inserted

into N-terminus of NCoRi cDNA. All sequences were verified by sequencing.

Creation of transgenic mice

Alb-NCoRi (6.8 kb) fragment containing the mouse albumin promoter,

NCoRi cDNA, and SV40 intron/polyA sequence (Figure 1A) was liberated

from the plasmid vector PGEMLB-SVPA-NCoRi by digestion with AatII and

MluI, separated by electrophoresis on 1% agarose gel, and purified through

Qiagen gel extraction kit (Qiagen), following by Elutip column (Schleicher and

Schuell). Microinjection was carried out by the NIDDK Transgenic Facility,

NIH.

Identification of transgenic mice by PCR genotyping

Transgenic mice carrying Alb-NCoRi was identified by PCR genotyping

of tail DNA using primers specific to albumin promoter and NCoRi cDNA.

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PCR amplification was carried out for 30 cycles using 200-400 ng of mouse tail

DNA, with each cycle set at 94oC for 30 sec, 60oC for 30 sec, 72oC for 3 min.

Southern blotting analysis of genomic DNA from transgenic mice

5ug genomic DNA from mouse tails was digested with EcoRV

restriction enzyme. Digestion products were separated by agarose gel

electrophoresis and transferred to nylon transfer membrane (Schleicher and

Schuell). cDNA probe preparation, hybridization and washes were carried out

as described for Northern blotting (see below).

Regulation of thyroid hormone status in mice

All animals used in these experiments were 8-12 weeks old.

Hypothyroid mice were fed a low iodine (loI) diet supplemented with 0.15%

propylthiouracil (PTU) purchased from Harlan Teklad Co. (Madison, WI) for

four weeks (26). Serum TSH measurements (kindly measured by Dr. Samuel

Refetoff, University of Chicago, Chicago, IL) showed that mice littermate

control and transgenic mice treated with PTU were profoundly hypothyroid

(TSH 27.0 -/+ 7.2 mU/ml and 37.1 -/+ 8.0 mU/ml, respectively n=4; nl 0.005-

0.034 mU/ml). After four weeks on this diet, hypothyroid mice were injected

intraperitoneally with 100µg L-T3 (Sigma) per 100g mouse body weight i n

phosphate buffered saline for six hours before sacrifice and liver harvest.

Control mice were injected with the same volume of phosphate buffered

saline alone for six hours before sacrifice and liver harvest. Euthyroid mice

were fed a normal diet. Control and transgenic mice were bled from their

tails, and sera were analyzed by the Clinical Chemistry Laboratory, NIH.

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RNA preparation and labeling for microarray

Total RNA was isolated from individual mouse livers by RNeasy kit

(Qiagen) and further purified by TRIZOL reagent (Gibco). 100ug total RNA

was converted to cDNA by using SuperScript II RNA reverse transcriptase

(Gibco) as previously described (27). RNA isolated from a littermate control

mouse liver was used to prepare cDNA probes labeled with Cy3-deoxyuridine

triphosphate (dUTP) dUTP (Amersham Life Science), and RNA isolated from

a trangenic mouse liver was used to prepare cDNA labeled with Cy5-dUTP

(Amersham Life Science). Labeled cDNA was purified using MicroCon 30

filters(Amicon). Hybridization and cDNA microarray analyses were

performed as described previously (24).

RNA extraction, probe preparation, and Northern blotting hybridization

Total RNA was prepared from mouse livers using TRIZOL reagent

(Life Technologies, Inc.) according to the manufacturer’s instructions. Poly

(A+) was isolated using PolyA tract mRNA isolation kit (Promega). Poly (A+)

RNA (2ug) was separated on 1% agarose-formaldehyde gel, then transferred

to a nylon transfer membrane (Schleicher and Schuell). The blots were

probed with gel purified α-[32P] dCTP-labeled fragments, probes were purified

using NICK column (Pharmacia) according to manufacturer’s instruction.

mRNA signals were quantified using ImageQuant software (Molecular

Dynamics), and normalized with corresponding thyroid hormone-insensitive

36B4 signals. Fold-induction was determined from transgenic mice signal

values divided by control mice signal values within the same experiment.

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Histological examination and measurement of cell proliferation

Mouse liver sections were taken from mouse and fixed in 10% neutral

buffered formalin solution (Sigma). The specimens then were embedded i n

paraffin, sectioned at 5um, and stained with hemotoxylin-eosin by American

Histolab (Rockville, MD). To detect proliferating hepatocytes, mice were

injected intraperitoneally with 5-bromo-2’-deoxyuridine (BrdU, 50 mg per kg

of body weight) (Boeringer-Mannhein). After 1 hr, the mice were killed

immediately and liver biopsies were fixed in 10% neutral buffered formalin

solution (Sigma). Immunostaining of BrdU and TUNEL analysis was carried

out by Molecular Histology Laboratories (Rockville, MD).

Results

In order to study basal repression in vivo, we constructed an expression

vector containing the NCoRi cDNA and the mouse albumin promoter to

target expression of NCoRi to the liver (25) (Fig. 1A). NCoRi is derived from

a 3.1 kb cDNA that was originally isolated from a human placental library.

NCoRi protein contains the TR interaction domains and surrounding amino

acids (AA 1539-2453), but lacks the repressor domains (AA 1-1120) present i n

full-length murine NCoR (23). It also has been shown that NCoRi has

dominant negative activity on endogenous NCoR as it blocked basal

repression by TR (23).

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DNA fragments containing the Alb-NCoRi transgene then were

isolated and microinjected into fertilized mouse eggs. Newborn mice were

screened for the presence of Alb-NCoRi transgene by PCR genotyping using

primers specific to mouse albumin promoter and NCoRi cDNA. Thirteen

out of a total 96 pups were identified as Alb-NCoRi transgene carriers and did

not show developmental abnormalities. Southern blot analysis of founder

mouse tail DNA showed that the founders carried 2-80 copies of the Alb-

NCoRi transgene (data not shown).

We outbred five of these transgenic founders and examined their F1-F4

heterozygous progeny in more detail. To determine whether these mice

expressed NCoRi mRNA, poly(A+)RNA was extracted from the mouse livers

and analyzed by Northern blotting. As shown in Fig. 1B, a 4 kb mRNA

corresponding to NCoRi mRNA was detected from the transgenic mice but

not their littermate controls. NCoRi mRNA was expressed 17 to 146 times

higher than endogenous NCoR mRNA in these mouse lines in the euthyroid

state. Moreover, endogenous NCoR mRNA did not increase in the

transgenic lines (Figs. 1B and 2B). To determine whether the transgene was

specifically expressed in liver, we examined the tissue distribution of NCoRi

mRNA by Northern blot analysis and found NCoRi mRNA was detected

only in the liver in trasgenic line 210 (Figure 1C).

Mice from all the transgenic lines had normal appearance, size, and

weight when compared to littermate controls (data not shown). Livers were

normal in size and appearance. Small differences in serum alkaline

phosphatase (transgenic 60 +/- s.d. 10 vs. control 34.5 +/- s.d.10, n=4) and

triglycerides (transgenic 41.25 +/- s.d. 0.9 vs. control 78.5 +/- s.d. 24.9, n=4)

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were observed between euthyroid transgenic and littermate control mice. No

differences in serum glucose, cholesterol, triglycerides, alkline phosphatase,

SGOT, SGPT, total bilibrubin were found between hypothyroid transgenic and

littermate control mice.

SMRT is another nuclear corepressor expressed in various tissues

(2,28). Like NCoR, SMRT interacts with the unliganded thyroid hormone

and retinoic acid receptors via conserved nuclear receptor interaction

domains and strongly represses basal transcription in co-transfection studies.

To determine whether expression of NCoRi changed the expression of

endogenous SMRT and NCoR mRNA, we examined their expression i n

transgenic mice from line F210, which expressed the highest amount of

NCoRi mRNA, and their littermate controls. Hepatic SMRT mRNA was

slightly increased in the euthyroid transgenic mice (Figure 2A). Since basal

repression is mediated by unliganded TR in the hypothyroid state, we

examined whether hepatic SMRT expression was increased in transgenic

mice rendered hypothyroid after 4 weeks of treatment with propylthiouracil

(PTU). SMRT mRNA was expressed at three-fold higher levels i n

hypothyroid control mice than in euthyroid control mice suggesting that

SMRT gene expression is negatively-regulated by T3 in liver. Hepatic SMRT

mRNA increased seven-fold in transgenic mice suggesting a compensatory

increase in hypothyroid transgenic mice. Similarly, endogenous NCoR

mRNA was increased in hypothyroid transgenic mice (Figure 2B).

To study whether overexpression of NCoRi affected basal repression of

target genes, we examined mRNA expression levels of six different thyroid

hormone-responsive genes, Spot 14, 5’ deiodinase, bcl3, glucose 6

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phosphatase, malic enzyme, and sialyltransferase in hypothyroid and

hyperthyroid mice. With the exception of sialyltransferase which is

negatively-regulated, all of these target genes are positively-regulated (24).

For these studies, transgenic mice from line F183 (which had a medium level

of NCoRi mRNA expression) and their control littermates were used. W e

first examined hepatic spot 14 (S14) gene expression in hypothyroid transgenic

mice, and found they exhibited 4-fold higher expression than their littermate

controls (Fig. 3A), suggesting that NCoRi was able to de-repress basal

repression in the absence of ligand. In contrast, hepatic S14 mRNA

expression was similar in hyperthyroid transgenic and littermate control

mice, consistent with the notions that NCoR only interacts with TR in the

absence of ligand and co-activator complexes are involved in ligand-

dependent transcriptional activation. Similar results were found in several

other thyroid hormone-responsive genes: bcl3, glucose 6-phosphatase, and 5’

deiodinase (Fig. 3B-3D). Of note, not all of the thyroid hormone responsive

genes studied were affected by overexpression of NCoRi. No difference i n

malic enzyme mRNA expression was observed between transgenic and

control mice in the absence or presence of T3 (Fig. 3E). These findings suggest

that NCoRi selectively blocked basal reperssion of several positively-regulated

thyroid-hormone responsive genes, but had no effect on their ligand-

dependent transcriptional activation.

We also studied NCoR’s role on the gene expression of a negatively-

regulated target gene, sialyltransferase. We previously used cDNA microarray

to identify this gene as negatively-regulated by T3 in liver (24). In some

negatively-regulated target genes, there is ligand-independent activation by

TR (29). Northern blotting analysis showed that ligand-independent

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activation in the hypothyroid state was decreased in the transgenic mice

compared to littermate controls (Figure 4). These findings result in decreased

negative-regulation of sialyltransferase mRNA in the transgenic mice as the

difference between mRNA expression in the hypothyroid state and the

hyperthyroid state is less in the transgenic mice than wild-type control mice.

To investigate other effects of NCoRi expression on hepatic function,

we injected control and transgenic mice intraperitoneally with 5-bromo-2’-

deoxyuridine (BrdU). Proliferating cells that incorporated BrdU into their

genomic DNA were visualized by staining liver sections with an antibody

specific for BrdU (Fig. 5A). As shown in Fig. 5B, proliferating cells were

increased by 120% in the highest expressing line (F210), and 50% in the

medium expressing line (F183) when compared to control littermates.

Additionally, TUNEL analyses was performed and did not show any

significant apoptosis in the transgenic or littermate control mice (data not

shown).

cDNA microarray analyses was undertaken to study the patterns of

gene expression in the transgenic mouse livers. cDNAs obtained from hepatic

RNA from two different transgenic lines were labeled with the fluorescent

dye, cy5, whereas cDNA obtained from control mice RNA were labeled with a

second fluorescent dye, cy3. The labeled cDNAs were hybridized on an array

containing a total 4500 mouse gene elements and their expression pattern

determined. We observed 28 genes that were up-regulated by more than two-

fold and 10 genes that were down-regulated by more than 60% in two

different transgenic lines. The full results of this microarray study will be

reported elsewhere. Interstingly, we found up-regulation of a number of

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genes that were either markers of proliferation or correlated with hepatocyte

proliferation (Table 1). Additionally, we observed a strong down-regulation

of retinoblastoma (Rb) mRNA which is a key regulator of G1 to S phase

transition. Northern blotting performed on RNA samples from each of the

transgenic lines confirmed the up-regulation of these genes (Fig. 6).

Discussion

Unliganded thyroid and retinoic acid receptors can repress

transcription in co-transfection and in vitro systems by interacting with co-

repressors such as NCoR and SMRT. Previously, it was not known whether

basal repression of target gene expression by TRs occurs in physiological

conditions, and what its mechanism might be if it did occur. Our Alb-NCoRi

transgenic mouse model provides in v ivo evidence that nuclear hormone

corepressors are involved in the basal repression by TRs in absence of ligand.

Earlier in vitro studies showed that overexpression of NCoRi and

several regions of NCoR involved in nuclear receptor interaction blocked

RAR- and TR-mediated basal repression (23,30). The mechanism for this

derepression was dominant negative activity by the mutant NCoRs on wild-

type NCoR activity. Our in v ivo data are consistent with these in vitro

findings, as overexpression of NCoRi in transgenic livers resulted i n

derepression of several positively-regulated thyroid hormone-responsive

genes, but had no effect on ligand-dependent transcriptional activation.

Additionally, NCoRi did not affect basal transcription of at least one thyroid

hormone-responsive gene, malic enzyme, suggesting that specific

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corepressors may mediate basal repression of target genes. We also found

that overexpression of NCoRi partially blocked the basal transcription of a

negatively-regulated target gene, sialyltransferase, in the absence of hormone.

These results are consistent with the findings of Tagami et al. who showed

that NCoR can augment basal transcription of negatively-regulated target

genes in co-transfection studies (29).

The importance of basal repression in development is exemplified by

recent studies with double knockout mice which lack both TRα and

TRβ (31,32) These mice are viable, reproduce, and have a milder phenotype

than mice with congenital or neonatal hypothyroidism. Thus the absence of

TR produces a milder phenotype than the absence of thyroid hormone,

suggesting basal repression of target genes may have deleterious effects. Since

our studies on basal transcription were performed in hypothyroid mice, it is

possible that basal repression of target genes in the hypothyroid state may

contribute to the clinical phenotype observed in congenital hypothyroidism.

Additionally, basal repression of target genes may have a normal

physiological function as it is possible that transient hypothyroidism may

occur in the fetus before its thyroid gland is competent (33). Basal repression

also may occur in specific tissues which have decreased transport or

deiodination of T4. While this manuscript was in preparation, Rosenfeld and

colleagues reported a NCoR gene-deleted mouse which died in utero with

defects in erythrocyte, thymocyte, and CNS development (11). In coinjection

studies of mouse embryo fibroblasts, they observed reversal of basal

repression by retinoic acid receptor using an artificial direct repeat 5 reporter.

Our results demonstrate that blockade of NCoR function can reverse basal

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repression of endogenous target genes in live animals, and support a

physiological role for co-repressors.

We also observed that SMRT mRNA was negatively-regulated by T3.

This suggests that SMRT expression increases in the hypothyroid state when

it is involved in basal repression of target genes. Moreover, there was a

compensatory increase in endogenous SMRT and NCoR mRNA expression

in hypothyroid transgenic mice. It currently is not known whether NCoRi

directly affects the transcription of these genes or whether derepression of

other regulatory genes may alter their expression. Interestingly, a similar

compensatory mechanism was observed for co-activators as TIF2 mRNA was

increased in the SRC-1 knockout mouse (34).

Our data also showed that there was increased DNA synthesis in the

hepatocytes from the transgenic mice as measured by BrdU incorporation.

These findings are consistent with increased hepatocyte proliferation (35,36);

furthermore, we did not detect any change in apoptosis. In parallel with the

increase in proliferation, we detected increased gene expression of several

markers of cell proliferation in the livers of transgenic mice using cDNA

microarray: alpha fetoprotein, MAP kinase phosphatase-1 (MKP-1), and cyclin

A2, and decreased gene expression of a cell cycle repressor, Rb (Table 1). Alpha

fetoprotein (AFP) is a critical serum transport protein produced by the liver

during embryogenesis (37) and is used clinically as prenatal marker for a

number of fetal abnormalities including spina bifida and Down’s syndrome.

It also is expressed in hepatocellular carcinoma, and is associated with

dedifferentiation and proliferation of hepatocytes. MKP-1 is an immediate

early gene that can be rapidly induced by mitogens, heat shock, or oxidative

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stress, and thus is induced in parallel with MAP kinases which also are

stimulated by mitogens (38). Interestingly, a recent study showed that MAP

kinase stimulation phosphorylates SMRT, and decreases its interaction with

nuclear hormone receptors (39). These findings suggest that stimulation of

MKP-1 mRNA could potentially enhance SMRT activity. Cyclin A2 mRNA

is upregulated at the beginning of S phase and has been used as a marker of

hepatocyte proliferation (40,41). Rb blocks the progression fom G1 to S phase,

and decreased expression or mutations of Rb correlate with cell proliferation

and hepatocellular carcinoma (42,43). While these markers may not have a

primary role in inducing proliferation, they nonetheless reflect the

proliferative state of the transgenic livers.

Since this proliferation occurred in euthyroid mice, it is possible that

this proliferation may be mediated by pathways that do not require thyroid

hormone. Recently, it was shown that Mad/Max/Sin3/NCoR/HDAC

complexes can repress transcription and potentially inhibit growth (7-10). Mxi

also can recruit msin3 (44). Thus Mad/Max and Mxi1/Max dimers can act as

antagonists of Myc binding to Myc enhancer sites, and repress transcription by

recruiting co-repressors (44,45). Taken together, these findings suggest that

overexpression of NCoRi, could disrupt the repression of myc-mediated

proliferation as well as other pathways repressed by NCoR.

Several novel target genes that are involved in cell proliferation were

identified by(Table 1). Among them was cAMP response element modulator

(CREM), which regulates cAMP-induced transcription. Recent studies have

shown that CREM is increased during liver regeneration following partial

hepatectomy (46). Moreover, CREM knockout mouse showed decreased cell

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proliferation and delayed liver regeneration following partial hepatectomy,

confirming the important role of CREM in liver regeneration (47). Similarly,

tumor necrosis factor receptor (TNFR) mRNA was induced in transgenic

mice. Tumor necrosis factor has been shown to be important for liver

regeneration and growth (48). Furthermore, recent studies on TNFR

knockout mice showed they have markedly decreased DNA synthesis after

partial hepatectomy (49). The mechanism for TNF-mediated regeneration

likely involves STAT3-mediated transcription. Insulin-like growth factor

binding protein-1 (IGFBP-1) mRNA also was increased in the transgenic mice.

Insulin-like growth factors (IGFs) are potent mitogens that are important for

growth and development. IGF binding proteins (IGFBPs) are serum proteins

that bind IGFs with high affinity. Recently, IGFBP-1 was identified as one of

the most highly expressed immediate-early genes in liver regeneration

(50,51). It also is highly expressed in fetal rat liver suggesting that it may play a

role in hepatic growth and development (50). Although IGFBPs can have

growth inhibitory properties by competitively binding IGFs, and prevent IGF

interaction with IGF receptors, sequestration of IGF can enhance IGF-1 effects

by allowing a slow release of IGF-1, and thereby protect IGF receptors from

down-regulation (52). It also is possible that IGFBPs can enhance IGF-1

activity independently of IGF-1 receptors (50). Although the precise

mechanism by which NCoR represses the expression of these proliferative

genes is not known, our findings demonstrate that NCoR has either direct or

indirect effects on several key signaling pathways that affect hepatocellular

proliferation.

Another potential contributor to hepatocyte proliferation is bcl3 (53),

which had increased mRNA expression in the transgenic mice. Bcl3, an IκB

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protein, interacts directly with (NF)κB homodimers (54). Bcl3 also interacts

with transcription integrators such as SRC-1 and CBP/p300 (53), and can

function as a co-activator of retinoid X receptor and AP1-mediated

transcription (53,55,56). Preliminary analysis of the bcl3 promoter showed

that it contains potential myc and thyroid hormone response elements (57)

suggesting that bcl3 could be a target gene of Myc/Max/Mad and TR. W e

recently showed that bcl3 is a target gene of thyroid hormone (24). Thus, the

interaction of NCoR with members of both the nuclear hormone receptor

and the Myc superfamilies raise the possibility that there may be co-

operativity between the two pathways.

In conclusion, we have generated a transgenic mouse model in which

a dominant negative corepressor, NCoRi is targeted to liver. NCoRi

selectively blocked basal repression of target genes by TRs suggesting that

NCoR may mediate basal repression in certain physiological conditions.

Moreover, NCoRi also stimulated hepatocyte proliferation in euthyroid mice

and induced several genes that may be involved in hepatocyte proliferation

in transgenic mice. These findings suggest that NCoR may modulate

transcription from TR-mediated and other signaling pathways. Recently,

steroid hormone receptors bound to antagonists have been shown to recruit

corepressors (58-60). RARs also can bind corepressors and mediate basal

repression in the absence of ligand (1,2). Thus, this system should be useful

in characterizing the in v ivo functions of NCoR for such antagonist-bound

nuclear receptors as well as other nuclear hormone receptors such as RARs

which bind corepressors in the absence of ligand. It also may help define

novel pathways for corepressor action.

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Acknowledgements

We would like to thank Dr. Samuel Refetoff (University of Chicago,

Chicago, IL) for measuring TSH levels in the hypothyroid mice, Dr. Anthony

Hollenberg (Beth Israel Hospital, Boston, MA) for providing PKCR2-NCoRi

vector, and Dr. Jake Liang (NIDDK, Bethesda, MD) for providing the albumin

promoter in PGEMLB-SVPA.

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Figure Legends

Fig 1. Characterization of transgenic mice carrying Alb-NCoRi.

A) Structure of liver specific expression construct, Alb-NCoRi, used i n

preparation of transgenic mice. The size of each DNA fragment is shown

below the corresponding structure. Restriction sites are also indicated below

the construct.

B) Northern blotting analysis of poly (A+) RNA (2ug) from control and

transgenic mouse lines to determine relative NCoRi mRNA expression.

mRNA signals corresponding to endogenous NCoR or NCoRi transgene are

indicated. mRNA ratio of NCoRi transgene and endogenous NCoR are

shown below the blot. Control probe (36B4) was used as a RNA loading

control. Position of endogenous NCoR mRNA was confirmed using a 5’

probe specific for full-length NCoR in a separate Northern blot.

C) Northern blotting analysis of poly (A+) RNA to demonstrate liver-specific

expression of NcoRi in transgenic mouse line F210. Upper panel,

autoradiography of Northern blot containing 2ug poly (A+) RNA from liver,

intestine, stomach, kidney, spleen. heart, and lung. Lower panel,

autoradiography of control probe 36B4 used as a RNA loading control.

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Fig 2. Endogenous SMRT and NCoR gene expression in livers of transgenic

mice. Northern blotting performed as described in Materials and Methods.

All signals were normalized to control signal 36B4. Each bar represents the

mean of four samples. Star indicates statistical difference between transgenic

and littermate control mice, P < 0.05.

A) Bar graph showing hepatic SMRT mRNA levels from hypothyroid and

euthyroid mice from line F210 and littermate controls.

B) Bar graph showing endogenous hepatic NCoR mRNA levels from

hypothyroid and euthyroid mice from line F210 and littermate controls. A

probe against the 5’ region of NCoR was used to better visualize

endogenous NCoR.

Fig 3. Hepatic target gene expression in transgenic and control mice under

hypothyroid and hyperthyroid conditions. In the hypothyroid state,

derepression by NCoRi is examined. In the hyperthyroid state, NCoRi effects

on ligand-dependent transcriptional activation are studied. Poly(A+) RNA

was prepared from livers of hypothyroid control and transgenic mice (line

F183), and mice treated six hours with T3 , and Northern blotting performed,

as described in Materials and Methods. Shown are the means and s.d. of 4-6

samples from control and transgenic mice under hypothyroid and

hyperthyroid conditions. All signals were normalized to control signal 36B4

mRNA expression. Star indicates statistical difference between transgenic and

littermate control mice, P < 0.05.

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A) S14 mRNA

61. Bcl3 mRNA

62. Glucose 6-phosphatase

63. 5’ deiodinase mRNA (Note different scales for hypo- and hyperthyroid

conditions)

64. malic enzyme mRNA

Fig. 4. Northern Blot analysis of a negatively-regulated target gene,

sialyltransferase. In the hypothyroid state, NCoRi effects on ligand-

independent activation are examined. In the hyperthyroid state, NCoRi

effects on ligand-dependent negative regulation are studied. NCoRiPoly (A+)

RNA preparation from line F183, and Northern blotting were performed

similar to the previous Figure. Shown are the means and s.d. of at least four

samples from control and transgenic mice under hypo- and hyperthyroid

conditions. All signals were normalized to control signal 36B4 mRNA

expression.

Fig. 5. Effect of NCoRi on hepatic cell proliferation.

Control and transgenic mice from lines F210 and F183 were injected withBrdU,

and hepatic slices analyzed as described in Materials and Methods.

A. Representative micrographs showing BrdU-labeled hepatocytes (red) i n

liver sections from adult transgenic mice (transgenic) and their littermate

controls (control).

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In vivo effect of NCoR

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B. Bar graph showing relative scores of BrdU labeled hepatocytes in 10,000

cells (n=3-4).

Fig. 6. Northern Blot analysis of hepatic genes induced in transgenic mice

and associated with cell proliferation. Poly (A+) RNA from livers of euthyroid

transgenic mice from line F210 and their littermate controls were prepared

and analyzed by Northern blot as in Figure 3. Shown are the means and s.d. of

at least four samples from transgenic and littermate control mice.

Table1. cDNA microarray analysis of genes related to cell

proliferation in transgenic mouse livers . Hepatic RNA from individual

transgenic mice (lines F210 (high-copy number) and F220 (medium-copy

number)) and their littermate controls was prepared, labeled, and analyzed by

cDNA microarray analysis as described in Materials and Methods. Similar

results were obtained in a repeat analysis with different mice from the same

lines.

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A.

B.

0

1

2

3

4

5

6

7

8

9

euthyroid hypothyroid

SM

RT

mR

NA

leve

l

control

transgenic

0

1

2

3

4

5

6

euthyroid hypothyroid

NC

oR m

RN

A le

vel

control

transgenic

Fig2

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A B C

D E

0

2

4

6

8

10

12

14

-T3 +T3

Sp

ot

14 m

RN

A le

vel control

transgenic

0

2

4

6

8

10

12

14

-T3 +T3

Bcl

3 m

RN

A le

vel

control

transgenic

0

2

4

6

8

10

12

14

-T3 +T3

G6P

mR

NA

leve

l

control

transgenic

0

1

2

3

4

5

6

Control Transgenic

5' D

I mR

NA

leve

l

0

30

60

90

120

150

180

control Transgenic

5'D

I mR

NA

leve

l

+T3

0

2

4

6

8

10

12

14

-T3 +T3

Mal

ic e

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mR

NA

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transgenic

-T3

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Fig4

0

0.2

0.4

0.6

0.8

1

1.2

-T3 +T3

sial

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transgeni

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Control Transgenic

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

1 4 0

C TR L F 210 F 183

BrdU

-lab

eled

cel

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0,00

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Fig5

A

B

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Fig6

0

1

2

3

4

5

6

CREM MKP-1 IGFBP-1 TNFR Bcl3

mR

NA

exp

ress

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leve

l

control

transgenic

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Fold-Induction

Clone ID Gene F210 F220

387802 alpha fetoprotein 3.36 2.48597868 bcl3 5.05 2.84515968 cAMP response element modulator 3.57 2.97552363 tumor necrosis factor receptor 5.26 4.44582081 MAP kinase phosphatase-1 4.69 5.56483145 insulin-like growth binding protein 3.69 8.4779163 cyclin A2 3.68 4.06463944 zinc finger protein 90 5.56 3.05619049 retinoblastoma-binding protein 0.166 0.157

table1

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Xu Feng, Yuan Jiang, Paul Meltzer and Paul M. Yenrepression by the thyroid hormone receptor and increases cell proliferation

Transgenic targeting of a dominant negative corepressor to liver blocks basal

published online January 29, 2001J. Biol. Chem. 

  10.1074/jbc.M011027200Access the most updated version of this article at doi:

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