RE-1 silencing transcription factor (REST): Báo cáo khoa học về điều hòa phiên mã gen synaptophysin ở người thông qua trình tự DNA đặc hiệu

RE-1 silencing transcription factor (REST) regulates human

synaptophysin gene transcription through an intronic

sequence-specific DNA-binding site

Michael Lietz, Mathias Hohl and Gerald Thiel

Department of Medical Biochemistry and Molecular Biology, University of Saarland Medical Center, Homburg, Germany

Synaptophysin, one of the major proteins on synaptic vesi-

cles, is ubiquitously expressed throughout the brain. Syn-

aptophysin and synapsin I, another synaptic vesicle protein,

are also expressed by retinoic acid-induced neuronally dif-

ferentiated P19 teratocarcinoma cells. Here, we show that

inhibition of histone deacetylase activity in P19 cells is suf-

ﬁcient to activate transcription of the synaptophysin and

synapsin I genes, indicating that neuronal diﬀerentiation

and impairment of histone deacetylases results in a similar

gene expression pattern. The transcription factor REST, a

repressor of neuronal genes in non-neuronal tissues, has

been shown to function via recruitment of histone deacety-

lases to the transcription unit, indicating that modulation of

the chromatin structure via histone deacetylation is of major

importance for REST function and neuron-speciﬁc gene

transcription. Furthermore, REST has been shown to be the

major regulator of neuronal expression of synapsin I. Here,

we have identiﬁed a functional binding site for REST in the

ﬁrst intron of the human synaptophysin gene indicating that

REST blocks human synaptophysin gene transcription

through an intronic neuron-speciﬁc silencer element. The

synaptophysin promoter is, however, devoid of neuron-

speciﬁc genetic elements and directs transcription in both

neuronal and non-neuronal cells. Using a dominant-negat-

ive approach we have identiﬁed the transcription factor Sp1

as one of the regulators responsible for constitutive tran-

scription of the human synaptophysin gene.

Keywords:neuronalgenes;REST;Sp1;synapsin I;synapto-

physin.

Synaptophysin is a major integral membrane protein of

small synaptic vesicles [1]. Synaptophysin forms a hetero-

multimeric complex in the vesicle membrane, consisting of

at least four synaptophysin molecules and the synaptic

vesicle protein synaptobrevin [2]. Many functions have been

attributed to synaptophysin in the past: synaptophysin has

been proposed to form a channel/fusion pore [3], to function

as a Ca

sensor in the synapse [4], and to be essential for

neurotransmitter release [5]. Gene targeting experiments in

transgenic mice revealed, however, that synaptophysin is

not essential for neurotransmitter release. No difference

between wild-type and mutant mice in synaptic transmission

and short-term and long-term plasticity was observed [6]

suggesting that other synaptic vesicle proteins may com-

pensate for the lack of synaptophysin. Double knockout

mice lacking synaptophysin and the structurally related

synaptic vesicle protein synaptogyrin I showed a severe

reduction in short-term and long-term plasticity, indicating

that synaptophysin and synaptogyrin I perform essential,

redundant functions in synaptic plasticity [7]. Recently, a

further role of synaptophysin in regulating activity-depend-

ent synapse formation has been proposed [8].

Synaptophysin is ubiquitously expressed in neurons

throughout the brain and also in neuroendocrine cells [9].

During neuronal development, synaptophysin expression is

correlated with synaptogenesis [10] and synaptophysin has

been widely used as marker for neurons and nerve terminal

differentiation, due to its abundance and pan-neuronal

expression.

Here, we have analyzed the regulation of the human

synaptophysin gene, in comparison with the regulation of

the synapsin I gene. We show that expression of both genes

is sensitive to histone deacetylase inhibition, indicating that

chromatin structure governs synaptophysin and synapsin I

gene transcription. Furthermore, two transcription factors,

Sp1 and the RE-1 silencing transcription factor (REST)

were identiﬁed that are responsible for either constitutive or

neuron-speciﬁc transcription of the synaptophysin gene.

Experimental procedures

Reporter constructs

The reporter constructs are derivatives of pGL3-Basic

and pGL3-Promoter (Promega). To construct plasmid

pSyI

-2309/+47

luc we cut plasmid pSyCAT10 [11] with SalI

and ligated the fragment into the XhoIsiteofpGL3-Basic.

The genomic clone p10C-6 containing the promoter, exons I–

III and introns I and II of the human synaptophysin gene

Correspondence to G. Thiel, Department of Medical Biochemistry

and Molecular Biology, Building 44, University of Saarland

Medical Center, D-66421 Homburg, Germany.

Fax: + 49 6841 1626500, Tel.: + 49 6841 1622606,

E-mail: bcgthi@uniklinik-saarland.de

Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase;

GST, glutathione S-transferase; NRSE, neural-restrictive silencer

element; REST, RE-1 silencing transcription factor; Syp,

synaptophysin; TSA, trichostatin A.

(Received 8 August 2002, revised 6 November 2002,

accepted 11 November 2002)

Eur. J. Biochem. 270, 2–9 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03360.x

[12], was a kind gift of T. Su

¨dhof, HHMI, Dallas, TX, USA.

To construct a synaptophysin promoter/luciferase reporter

gene, we ﬁrst subcloned a KpnI fragment of the genomic

clone p10C-6 into pGEM7 (Promega). This plasmid was

modiﬁed by inserting the annealed oligonucleotides

5¢-CTAGCCGAGCCTCCCGCCCCCTGCATTGCTGG

TCGACGCATG-3¢and 5¢-CGTCGACCAGCAATGCA

GGGGGCGGGAGGCTCGG-3¢into the NheIandSphI

sites. This plasmid was subsequently cut with XbaI, ﬁlled in

with the Klenow fragment of Escherichia coli DNA poly-

merase I, and recut with SalI. The fragment encompassing the

5¢-ﬂanking region of the human synaptophysin gene (nucle-

otides 29255–31636, accession no. U93305) was isolated and

inserted into plasmid pGL3-Basic, generating plasmid pSyp

2356/+27

luc. Plasmid pSyp

Intron

luc, containing nucleotides 3–

427 of the ﬁrst intron of the human synaptophysin gene

(nucleotides 28788–29216, accession no. U93305) upstream

of the SV40 promoter, was constructed by inserting an RsaI

fragment derived from plasmid p10C-6 into Ecl136II-cut

pGL3-Promoter. Plasmids pSypNRSE

SV40luc and pSyI-

NRSE

SV40luc, containing two copies of the intronic NRSE

(neural-restrictive silencer element) derived from the synapt-

ophysin gene or two copies of the NRSE derived from the

human synapsin I promoter 5¢of the SV40 promoter, were

generated by subcloning of the synthetic oligonucleotides

5¢-TCGAGTCCAGCACCGTGGACAGAG CCG-3¢and

5¢-TCGACGGCTCTGTCCACGGTGCTG GAC-3¢(syn-

aptophysin gene) or 5¢-TCGAGCTTCAG CACCGCGG

ACAGTGCCTTG-3¢and 5¢-TCGACAA GGCACTGTC

CGCGGTGCTGAAGC-3¢(synapsin I gene) into the XhoI

and SalI sites of plasmid pHIVTATA CAT [13]. The

sequences were subsequently multimerized as described

previously [13], excised with XhoIandSalI and cloned into

the XhoI site of plasmid pGL3-Promoter.

Expression constructs

The REST expression vector pCMVFLAG-REST is iden-

tical to the previously described plasmid pCMVmycREST

[14], except that the myc epitope has been exchanged

for a triple FLAG epitope (sequence: MDYKDHDG

DYKDHDLDYKDDDDK). The expression vector enco-

ding a positive-dominant mutant of REST, FLAG-DP-

REST, is identical to the previously described plasmid

pCMVDP-REST [15,16], except that the hemagglutinin

epitope has been exchanged for a triple FLAG epitope. The

expression vector encoding myc-tagged REST4 [16] and

mammalian glutathione S-transferase (GST) encoding

expression vectors pEBGN and pEBGN-Sp1 [17,18] have

been described previously. Plasmids pRSVband pSV40lacZ

encodes for b-galactosidase of E. coli [19,20].

Cell culture and transfections

The murine neuroblastoma cell line NS20Y, the immortal-

ized septal cell line SN56, the murine teratocarcinoma cell

line P19 and human 293T cells were maintained as described

previously [11,19,21,22]. NS20Y cells were transfected using

the calcium phosphate coprecipitation method with 0.5–2 lg

of luciferase reporter plasmid, 100 ng of expression vector

encoding FLAG-REST, FLAG-DP-REST or myc-REST4,

and 0.5–1 lgofpRSVbinternal standard plasmid. 293T cells

were transfected as described previously [22] with 1 lgof

luciferase reporter plasmid, 1–5 lg of GST expression vector

and 0.2 lg of the internal standard plasmid pSV40lacZ. The

titration experiments with NS20Y cells were performed with

1lg of luciferase reporter plasmid, 1–5 lg of GST-expres-

sion vector and 0.2 lg of the internal standard plasmid

pSV40lacZ. P19 cells were grown as described previously

[23]. For neuronal differentiation, cells were aggregated and

treated with 0.5 l

all-trans-retinoid acid for 4 days. Cell

aggregates were then lightly trypsinized, plated onto tissue

culture plates and cultured for 5 days in the absence of

retinoid acid, but in the presence of 5 lgÆmL

of cytosine

-arabinofuranoside (Sigma C6645). Trichostatin A

(TSA) was purchased from Wako Chemicals GmbH (Neuss,

Germany) and used at a concentration of 100 ngÆmL

dissolved in dimethylsulfoxide.

RNase protection mapping

Cytoplasmic RNA of undifferentiated, differentiated,

dimethylsulfoxide and TSA-treated P19 cells was prepared

as described previously [23]. For RNase protection mapping,

20 lg RNA was used for the detection of synapsin I and

synaptophysin mRNA, and 2.5 lg RNA for the detection of

glyceraldehyde 3-phosphate dehydrogenase (GAPDH)

and b-actin mRNA. The template for mouse synapsin I

cRNA synthesis (plasmid pSP6-mSyI-2) has been described

previously [23]. To synthesize a synaptophysin-speciﬁc

riboprobe, a cDNA fragment encompassing nucleotides

1730–2011 of the mouse synaptophysin gene (accession no.

X95818) was ampliﬁed by PCR using the primers 5¢-

GGTTCTGGTCAGGATTGC-3¢and 5¢-TCAGTAAGG

GACATTTCG-3¢and cloned into the SmaIsiteofpBlue-

script to generate plasmid pT3mp38. Hybridization with

synapsin I and synaptophysin mRNA protected fragments

of 194 and 282 nucleotides, respectively, from RNase

digestion. Plasmids SP6-b-actin and pTRI-GAPDH-Rat,

used to synthesize b-actin and GAPDH-speciﬁc riboprobes,

were purchased from Ambion. Hybridization with b-actin

and GAPDH mRNA protected fragments of 250 and 316

nucleotides, respectively, from RNase digestion.

Reporter gene assays

Cell lysates of transfected cells were prepared 48 h post-

transfection using the cell culture lysis buffer (Promega), and

b-galactosidase and luciferase activities were determined as

described [20,21]. Each experiment included four separate

transfections for each experimental setting, and the experi-

ments were repeated at least twice giving consistent results.

Expression and purification of recombinant

GST fusion proteins

293T cells were transfected with plasmids pEBGN or

pEBGN-Sp1, encoding a nuclear targeted GST or a GST-

Sp1 fusion protein. Forty-eight hours post-transfection, cells

were harvested, washed with NaCl/P

and lyzed in RIPA

buffer (50 m

Tris/HCl, pH 7.5, 150 m

NaCl, 1 m

EDTA, 10% NP-40, 0.5% deoxycholate, 0.1% SDS) for

FEBS 2003 Regulation of synaptophysin gene transcription (Eur. J. Biochem. 270)3

30 min at 4 C and centrifuged for 10 min. The supernatant

was incubated with 50 lL glutathione-agaraose beads

(Pharmacia Biotech) for 30 min. The glutathione-agarose-

GST protein complexes were isolated by centrifugation,

washed twice with RIPA buffer and dissolved in 100 l

SDS-stop solution (125 m

Tris/HCl, pH 6.8, 3 m

EDTA, 20% glycerol, 9% SDS, 0.05% bromophenol blue).

Results

Induction of neuronal gene transcription in P19

teratocarcinoma cells by retinoid acid or TSA

The P19 teratocarcinoma cell line is frequently used as an

in vitro model system for neuronal differentiation, neurite

outgrowth and neuronal gene expression. We differentiated

P19 cells on bacterial plates in the presence of retinoid acid.

Four days later, the cell aggregates were lightly trypsinized

and plated onto tissue culture plates for 5 days in the

presence of cytosine b-

-arabinofuranoside to suppress

growth of non-neuronal cells. Cytoplasmic RNA was

prepared and analyzed by RNase protection mapping using

speciﬁc riboprobes for the detection of synapsin I, synapto-

physin, b-actin and GAPDH mRNA, respectively. Synap-

sin I and synaptophysin are synaptic vesicle proteins and

serve as marker proteins for neuronal differentiation. b-Actin

and GAPDH are constitutively expressed in P19 cells.

Figure 1A shows that neuronal differentiation of P19 cells

induced the expression of the synapsin I and synaptophysin

genes, conﬁrming previous results [23,24]. Neuronal expres-

sion of the synapsin I gene has been shown to be controlled

by REST [25], a transcriptional repressor of neuronal genes

in non-neuronal cells. REST contains two activetranscrip-

tional repression domains on its N- and C-termini. The

N-terminal repression domain of REST recruits histone

deacetylases to the target genes of REST [26,27]. Likewise,

repression mediated by this domain was shown to be sensitive

to inhibitors of histone deacetylases such as TSA. Moreover,

there are indications that the C-terminal repression domain

also functions via recruitment of histone deacetylases [28].

Histone deacetylation generates a compact chromatin struc-

ture that is not as accessible to the transcriptional machinery.

Thus, alterations of the chromatin structure are essential for

transcriptional repression via REST. Therefore, we tested

whether an inhibition of histone deacetylases in P19 cells is

sufﬁcient to induce neuronal gene transcription. P19 cells

weretreatedfor24hwithTSAandcytoplasmicRNAwas

prepared and analyzed by RNase protection mapping.

Figure 1B shows that inhibition of histone deacetylases by

TSA was sufﬁcient to induce synapsin I gene transcription.

Moreover, TSA treatment also induced transcription of the

synaptophysin gene, indicating that expression of both genes

is controlled by alterations of the chromatin structure.

The human synaptophysin promoter is devoid

of neuron-specific genetic elements

The synapsin I promoter contains a neuron-speciﬁc control

element, the REST binding site termed neuron-restrictive

silencer element (NRSE). Transfection of a human synap-

sin I promoter/luciferase reporter gene depicted in Fig. 2A

in neuronal and non-neuronal cells revealed that the

promoter mainly directs luciferase expression in the neur-

onal cell lines NS20Y and SN56, but not in the human

embryonic kidney cell line 293T (Fig. 2B, upper panel).

Both NS20Y and SN56 cells have been shown by RNase

protection mapping to express synapsin I and synaptophy-

sin (data not shown). A human synaptophysin promoter/

luciferase reporter gene, however, showed constitutive

transcriptional activity in NS20Y, SN56 and 293T cells,

indicating that neuron-speciﬁc expression of synaptophysin

is not regulated by genetic elements located in the

5¢-ﬂanking region of the synaptophysin gene.

Identification of a REST consensus binding site within

the first intron of the human synaptophysin gene

A data base search revealed the presence of an NRSE in the

ﬁrst intron of the synaptophysin gene of the rat [29]. We

analyzed the database of the human genome and found an

NRSE in the ﬁrst intron of the synaptophysin gene at an

identical position as in synaptophysin gene of the rat

(Fig. 3A). A comparison of the sequence revealed that the

intronic NRSE of the synaptophysin gene is 100%

conserved between the rat and human gene and has only

three mismatches in comparison to the NRSE found in the

human synapsin I promoter (Fig. 3B).

The intronic REST binding site confers REST regulation

to reporter genes

REST has been shown to repress transcription despite the

location or orientation of its binding site within a gene [14].

Fig. 1. Activation of synapsin I and synaptophysin gene transcription in

P19 teratocarcinoma cells. (A) P19 teratocarcinoma cells were diﬀer-

entiated via aggregation and treatment with retinoic acid for 4 days,

than plated and cultured for a further 5 days in the presence of cytosine

-arabinofuranoside. (B) P19 cells were treated for 24 h with the

histone deactylase inhibitor TSA or with the vehicle dimethylsulfoxide.

Cytoplasmic RNA from undiﬀerentiated (A, denoted –), neuronally

diﬀerentiated (A, denoted +), dimethylsulfoxide-treated (B, denoted

–) and TSA-treated (B, denoted +) P19 cells were isolated and

analyzed by RNase protection mapping using cRNAs speciﬁc for

synapsin I, synaptophysin, b-actin and GAPDH, respectively.

4 M. Lietz et al. (Eur. J. Biochem. 270)FEBS 2003

Thus, a functional NRSE of the synaptophysin gene should

operate from any position within the gene. We therefore

generated the reporter plasmid, pSyp

Intron

luc, consisting of

the luciferase open reading frame and the SV40 promoter.

To the 5¢of the SV40 promoter, we inserted 424 nucleotides

from the ﬁrst intron of the human synaptophysin gene

including the NRSE. As a control, we used plasmid pGL3-

Promoter, containing the luciferase gene under control of

the SV40 promoter (Fig. 4A). As expression vectors, we

transfected plasmids encoding either a FLAG-tagged

REST, a FLAG-tagged DP-REST or a myc-tagged

REST4. The modular structure of FLAG-DP-REST and

myc-REST4, in comparison to REST, is depicted in

Fig. 4B. DP-REST contains the DNA-binding domain of

REST fused to the activation domain of the herpes simplex

virus protein VP16. Following binding to the REST cognate

site, DP-REST strongly activates transcription, due to the

presence of a transcriptional activation domain [15,16].

REST4 contains the N-terminal repression domain of

REST and ﬁve of the eight zinc ﬁngers that constitutes the

DNA-binding domain [16]. REST4 binds only weakly to

the NRSE, due to the lack of zinc ﬁnger 7 that is important

for DNA binding [30]. REST4 was used as a negative

control in the experiment. Transient tranfections were

performed with NS20Y neuroblastoma cells. One of the

reporter plasmids, pSyp

Intron

luc or pGL3-Promoter, was

transfected into NS20Y cells together with plasmid pRSVb,

encoding b-galactosidase under the control of the Rous

sarcoma virus long-terminal repeat, to correct for variations

in transfection efﬁciencies. In addition, the emptyexpres-

sion vector pCMV5 (control) or expression vectors enco-

ding FLAG-REST, FLAG-DP-REST or myc-REST4 were

transfected. Forty-eight hours post-transfection, cells were

harvested, cell extracts prepared and the relative luciferase

activities determined. The results show that FLAG-REST

repressed transcription of the transcription unit containing

part of the ﬁrst intron of the human synaptophysin gene

(plasmid pSyp

Intron

luc). Likewise, expression of FLAG-DP-

REST increased reporter gene expression over the level

already obtained by the strong SV40 promoter (Fig. 4C, left

panel). In contrast, myc-REST4 did not show any tran-

scriptional activity as expected from previous experiments

[16]. Naturally, the SV40 promoter was not regulated by

either FLAG-REST, FLAG-DP-REST or myc-REST4

(Fig. 4C, right panel). These data indicate that the NRSE

derived from the human synaptophysin gene is biological

active and functions as a REST-regulated silencer.

The REST binding sites derived from the human

synaptophysin and synapsin I gene are

functionally indistinguisable

To compare the biological activity of the NRSEs derived

from the synapsin I promoter and the ﬁrst intron of the

human synaptophysin gene, we used model promoters

containing the luciferase gene as reporter, the SV40

promoter, and two copies of the NRSE derived from the

synapsin I and synaptophysin gene, respectively (reporter

Fig. 2. Activity of the human synapsin I and synaptophysin promoter in

neuronal and non-neuronal cells. (A) Schematic representation of the

synapsin I promoter/luciferase and synaptophysin promoter/luciferase

reporter genes pSyI

-2309/+47

luc and pSyp

-2356/+27

luc. (B) One of the

reporter plasmids pSyI

-2309/+47

luc or pSyp

-2356/+27

luc, was transfected

into NS20Y, SN56 (1 lg per plate) or 293T cells (0.5 lg per plate)

together with the reference plasmid pRSVb(NS20Y, SN56 cells:

0.5 lg per plate; 293T cells: 0.25 lg per plate) that encoded b-galac-

tosidase under control of the Rous sarcoma virus long-terminal repeat.

Forty-eight hours post-transfection cell extracts were prepared and the

b-galactosidase and luciferase activities of these extracts determined.

The data are presented as the ratio of luciferase activity (light units) to

b-galactosidase units (Aunits) measured in the cell extracts. At least

two experiments in quadruplicate were performed and the mean

±SEM is depicted.

Fig. 3. Localization of an NRSE in the human synaptophysin gene. (A)

Schematic representation of part of the human synaptophysin gene

containing the promoter region, exons I to III and introns I and II. The

location of the NRSE within the ﬁrst intron of the gene is indicated.

The accession number for the human synaptophysin gene is U93305.

The NRSE encompassed nucleotides 29190–29210. (B) Sequence of

the NRSEs derived from the human synapsin I gene and the human

and rat synaptophysin genes.

FEBS 2003 Regulation of synaptophysin gene transcription (Eur. J. Biochem. 270)5

plasmids pSyINRSE

SV40luc and pSypNRSE

SV40luc,

Fig. 5A). One of the reporter plasmids, the internal

reference plasmid pRSVband expression vectors encoding

FLAG-REST, FLAG-DP-REST or myc-REST4 were

transfected into NS20Y neuroblastoma cells. Forty-eight

hours post-transfection, cells were harvested, cell extracts

prepared and the relative luciferase activities determined.

The results show that the presence of a REST binding site,

either from the synapsin I or synaptophysin gene, together

with expression of FLAG-REST, caused a striking decrease

in transcription (Fig. 5B, upper panels). Likewise, expres-

sion of FLAG-DP-REST increased reporter gene transcrip-

tion signiﬁcantly (Fig. 5B, middle panels). The splice variant

of REST, REST4, however, did not activate or repress

transcription of NRSE-containing reporter genes (Fig. 5B,

lower panels), conﬁrming previous results [16]. Taken

together, no major differences were detected between the

NRSE derived from the synapsin I or synaptophysin gene,

indicating that the NRSE functions in both genes as a

neuron-restrictive silencer element.

The zinc finger transcription factor Sp1 is responsible

for constitutive transcription via the human

synaptophysin promoter

The presence of a functional NRSE in the ﬁrst intron of the

human synaptophysin gene indicates that REST blocks

human synaptophysin gene transcription through this

intronic neuron-speciﬁc silencer element. In contrast, no

neuron-speciﬁc genetic elements were found in the synapto-

physin 5¢-ﬂanking region. Rather, this part of the transcrip-

tion unit contained constitutive transcriptional elements

that are active in neuronal as well as in non-neuronal cells.

The 5¢-ﬂanking region of the human synaptophysin gene is

GC-rich, and potential binding sites for the transcription

factor Sp1 have been proposed [31]. To test whether Sp1 is

responsible for the constitutive transcriptional activity of the

human synaptophysin promoter, we used a dominant-

negative Sp1 mutant [17], consisting of the GST fused to the

DNA-binding domain of Sp1. Both domains were separ-

ated by a nuclear localization sequence, to ensure nuclear

targeting. As a control, an expression vector encoding a

nuclear-targeted GST was used (Fig. 6A). The expression

vectors were ﬁrst transiently transfected into 293T cells.

The recombinant proteins were puriﬁed by glutathione

afﬁnity chromatography and separated by SDS/PAGE.

Both proteins migrated on SDS/PAGE as expected.

Next, 293T and NS20Y cells were transfected with the

human synaptophysin promoter/luciferase reporter plasmid

pSyp

-2356/+27

luc, the GST-N encoding expression vector as

control, or increasing amounts of the expression vector

encoding GST-Sp1. The results show that the dominant-

negative Sp1 mutant decreased the constitutive transcrip-

tional activity of the synaptophysin promoter (Fig. 6C),

indicating that Sp1 is, at least in part, responsible for the

constitutive, tissue-unspeciﬁc activity of the 5¢-ﬂanking

region of the human synaptophysin gene.

Fig. 4. REST regulates the transcription activity of a strong viral pro-

moter via the intronic NRSE derived from the human synaptophysin gene.

(A) Reporter plasmid pSyp

Intron

luc and pGL3-Promoter containing the

luciferase reporter gene and the SV40 promoter. Plasmid pSyp

Intron

luc

contains a fragment of the ﬁrst intron of the human synaptophysin

gene, including the NRSE, 5¢of the SV40 promoter. (B) Modular

structure of FLAG-REST, FLAG-DP-REST, and myc-REST4. REST

contains a cluster of eight zinc ﬁngers that function as DNA-binding

domain, and two repressor domains on the N- and C-termini of the

molecule. FLAG-DP-REST, a positive-dominant mutant of REST,

retains the DNA-binding domain, but lacks the repression domains and

has instead a transcriptional activation domain derived from the VP16

protein of herpex simplex virus. REST4 is a neuron-speciﬁc splice

variant of REST that contains the N-terminal repression domain and

ﬁve of the eight zinc ﬁnger motifs of the DNA-binding domain. In

addition, FLAG-REST, FLAG-DP-REST, and myc-REST4 contain

recognition sequences (triple FLAG tag or myc-tag) on the N-termini.

Intron

luc or pGL3-Promoter (1 lg

per plate), 0.5 lg per plate of the pRSVbinternal standard plasmid and

either 100 ng per plate of the emptyexpression vector pCMV5 or one

of the expression vectors encoding FLAG-REST, FLAG-DP-REST,

or myc-REST4 were introduced into NS20Y cells. Transcription was

analyzed by determination of the b-galactosidase and luciferase activ-

ities of the cell extracts.

6 M. Lietz et al. (Eur. J. Biochem. 270)FEBS 2003

Báo cáo khoa học: RE-1 silencing transcription factor (REST) regulates human synaptophysin gene transcription through an intronic sequence-speciﬁc DNA-binding site Michael Lietz, Mathias Hohl and Gerald Thiel Department of Medical Biochemistry and Molecular

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