Structure of gene flanking regions and functional analysis of sequences upstream of the rat\u003c...

$: Structure of gene flanking regions and functional analysis of sequences upstream of the rat\u003c i\u003e hsp70. 1\u003c/i\u003e stress gene$
Structure of gene flanking regions and functional analysis of sequences

upstream of the rat hsp70.1 stress gene$

Anna Fiszer-Kierzkowska, Aleksandra Wysocka, Micha» Jarzab,Katarzyna Lisowska*, Zdzis»aw Krawczyk

Department of Tumor Biology, Center of Oncology, Maria Sk -lodowska Curie Memorial Institute, Wybrzeze Armii Krajowej 15, 44-100 Gliwice, Poland

Received 8 January 2002; received in revised form 4 September 2002; accepted 6 November 2002

Abstract

We present structural and comparative analysis of the flanking regions of the rat hsp70.1 stress gene. Several repetitive sequences,

microsatellites and short interspersed repetitive elements (SINEs) were found, as well as a significant gap in the 3V UTR, as compared to the

orthologous mouse gene. We also show that the complex microsatellite region composed of partially overlapping inverted repeat and long

homopurine–homopyrimidine sequence, which is localized 1.8 kbp upstream of the transcription start site, is capable to adopt non-B DNA

structures (an H-DNA and a cruciform structure) in vitro. Functional analysis performed with the use of various fragments of the 5Vendflanking regions ligated to the chloramphenicol acetyltransferase (CAT) reporter gene revealed a crucial role of cooperation between heat

shock element (HSE) regulatory sequences, while none of the three HSEs alone is able to drive efficient heat induced transcription of the

reporter gene. We also found that the microsatellite region does not influence transcription by itself, however, it abolishes the effect of the

adjacent putative silencing element. To our knowledge, this is a first extensive structural and functional analysis of the promoter region of the

mammalian heat inducible hsp70i gene localized distally to the hsp70-related spermatid-specific gene in the major histocompatibility

complex III.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Heat shock gene; hsp70 family; Transcription; Promoter sequence; MHC III; Non-B DNA structure

1. Introduction

Mammalian hsp70 family contains two to four hsp70i

(inducible) genes [1]. In human, rat and mouse two dupli-

cated, major histocompatibility complex (MHC) III-linked

hsp70i genes co-localize with the spermatid-specific hsp70-

related gene (see Fig. 1). Two additional hsp70i genes called

hsp70B and hsp70BVwere found in human genome, and

they also originated by ancestral duplication.

The nucleotide sequences of both MHC III-linked hsp70i

genes are highly similar and they code for virtually identical

chaperone proteins. A telomeric gene called hsp70A/

hsp70.1 in human [2] (hsp70.3 in mouse [3] and hsp70.2

in rat [4]) is positioned in head-to-head orientation with the

spermatid-specific hsc70t gene [4–6]. The transcription

initiation sites of these two types of genes are only about

600 bp apart. A centromeric MHC III-linked hsp70i gene,

called hsp70.2 in human [6], hsp70.1 in mice [7] and

hsp70.1 in rat [4,8–10], is separated from the telomeric

hsp70i gene by approximately 10 kb and is transcribed in

the same direction (Fig. 1). The functional significance of

the duplicated hsp70i locus coding for identical protein was

partially elucidated in the very elegant study employing

knock-out mice model, which showed differential expres-

sion of both MHC III-linked genes during ontogenesis [11].

These observations together with the results of other studies

0167-4781/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0167 -4781 (02 )00592 -4

Abbreviations: CAT, chloramphenicol acetyltransferase; DMEM, Dul-

becco’s modified Eagle’s medium; HAM, nutrient mixture; HSE, heat

shock element; HSF, heat shock factor; HSP, heat shock protein; hsp70,

gene encoding 70-kDa HSP; hsp70i, inducible hsp70; MHC, major

histocompatibility complex; SINE, short interspersed repetitive element;

tsp, transcription start point; UTR, untranslated region$ Nucleotide sequence of the rat hsp70.1 gene and its flanking regions

has been deposited in the EMBL Data Bank, Ac X74271.

* Corresponding author. Tel.: +48-32-278-9669; fax: +48-32-231-35-

12.

E-mail address: [email protected] (K. Lisowska).

www.bba-direct.com

Biochimica et Biophysica Acta 1625 (2003) 77–87

showing differential expression of both genes in response to

different stressors [12–15] suggest that these two genes

posses distinct regulatory mechanisms and each may

respond to different stimuli.

The hsp70i genes show a highly differentiated expression

pattern, from being strictly inducible in certain tissues to

being constitutively active in various tumors (reviewed in

Refs. [16,17]). Interestingly, an altered expression of the

hsp70i genes seems to be involved in the changes of cell

phenotype and in the etiology and progression of some

diseases, e.g. autoimmune, neurodegenerative or cancer.

The general inducing mechanism of hsp70i gene expression,

the interaction of heat shock factor(s) (HSF) with the heat

shock element (HSE) sequences, is relatively well recog-

nized (reviewed in Ref. [18]). Much less is known about

other regulatory interactions, e.g. with c-myc [19], B-myb

[20], c-myb [21], p53 [22,23], E1A [24,25] in trans or MAR

[26,27] in cis. This stresses the need for more extensive

functional and comparative investigation of DNA regions

flanking the hsp70i genes.

Of the mammalian hsp70i genes, the human hsp70.1

(telomeric, MHC III-linked) has been most extensively

studied since its cloning in 1985 [2,28–30]. Despite that,

it appeared recently that its structure is more complex than

suspected. An intron was detected at the 5V end of the gene

and it was found that transcription can be initiated from two

different sites and both promoter regions have distinct

regulatory elements [31]. Other human and rodent hsp70i

genes were less extensively studied. So far, only limited

functional studies of the rat hsp70.2 gene (telomeric, MHC

III-linked, [32]) and the orthologous mouse hsp70.3 gene

[3] have been performed. Interestingly, some silencing

activities were found in the region between telomeric hsp70i

gene and the adjacent spermatid-specific gene [32,33].

In contrast, neither the mouse nor the human promoter of

the centromeric MHC III-linked hsp70i gene has been a

subject of extensive functional analysis. Also, very limited

information is available about the regulation of expression

of the orthologous rat hsp70.1 gene. In our earlier work we

determined the most important promoter region for heat

inducibility of the gene [8], while Konishi et al. [34]

reported that only the second, more distal HSE element is

engaged in DNA–protein interactions under heat shock

conditions. We also found a complex microsatellite

sequence localized 1.8 kbp upstream of the transcription

start site that is potentially able to adopt non-B DNA

structure(s) in vitro [35]. Similar sequences, with a potential

to adopt cruciform or H-DNA structure, occur frequently in

the 5V flanking regions and UTRs of genes, and in some

cases were shown to influence transcription efficiency [36–

39]. Their effect on transcription may probably depend on

such factors as length of the sequence, perfection of its

symmetry, distance from the gene and possible interactions

with specific proteins recognizing either primary or secon-

dary DNA structure.

Here we present the results of structural and functional

studies of the 5Vend flanking region of the rat hsp70.1 gene.

Special attention has been paid to elucidate whether a

complex microsatellite sequence found upstream of the

transcription start site can adopt non-B DNA structure and

Fig. 1. Structure of the hsp70.1 gene locus. (A) Localization of three hsp70 genes within the MHC III complex of rat, mouse and human. Arrows indicate

direction of transcription. ‘MS’ indicates microsatellite region; ‘C2’ and ‘Bf’, genes of the complement complex; ‘TNF’, TNF genes. The telomeric hsp70i

gene is called hsp70.1 in rat [8,4], hsp70.1 in mouse [7] and hsp70.2 in human [6]. The centromeric hsp70i gene is called hsp70.2 in rat [4], hsp70.3 [3] in

mouse and hsp70.1 in human [2]. (B) Structure of the region containing the rat hsp70.1 gene and its flanking sequences. Boxes marked ID and B2 indicate

localization of these SINE sequences found around the gene (the 5V-end ID sequence represents class 4 ID elements and the 3V-end one class 3 [60]). The

arrowhead indicates the site of the suspected deletion in the 3VUTR (see text for details).

A. Fiszer-Kierzkowska et al. / Biochimica et Biophysica Acta 1625 (2003) 77–8778

affect expression of the gene. In addition, we present

comparative analysis of the gene flanking sequences of

the hsp70.1 and homologous, MHC III-linked hsp70i genes

from mouse and human.

2. Materials and methods

2.1. Plasmid constructs

Plasmids p68/1.0, p68/700, p68/600, p68/450 and p68/

300 contain different fragments of the rat hsp70.1 gene

upstream sequence [from � 1924 to � 869 region, relative

to the transcription start point (tsp)], cloned into pUC19

vector (see Fig. 3A). Plasmids used for transient trans-

fections contain different DNA fragments from between

� 1924 and + 85, cloned into pBLCAT6 vector bearing

the bacterial chloramphenicol acetyltransferase (CAT)

reporter gene [40]. For further details of plasmid content

and construction, see Table 1 and Fig. 6.

2.2. S1 nuclease cleavage and mapping of the cleavage site

Plasmid DNA was incubated in S1 buffer (final concen-

tration: 30 mM sodium acetate, pH 4.5, 50 mM zinc

chloride, and 5% glycerol) for 30 min on ice. S1 nuclease

(Pharmacia) was diluted in buffer containing 10 mM sodium

acetate, pH 5.0, 1 mM dithiothreitol, 0.1 mM zinc chloride

and 0.001% Triton X-100). Different concentrations of the

nuclease were added to the DNA samples and reactions

were carried out on ice for 10 min. Reactions were stopped

by the addition of Tris base and EDTA to the final concen-

trations of 90 and 45 mM, respectively. For mapping of the

cleavage site the S1-treated DNA was phenol-extracted and

ethanol-precipitated, then digested with appropriate restric-

tion endonucleases.

2.3. Construction of deletion plasmids

Plasmid p68/300 was digested with S1 nuclease and

cleavage products were separated electophoretically. DNA

band corresponding to the linear form of plasmid, contain-

ing products of complete (specific) digestion by S1 nucle-

ase, was isolated from agarose gel, blunted with Klenow

enzyme and ligated. Twenty-one clones were successfully

sequenced and their sequence was compared to that of p68/

300 in order to determine size and localization of the

deletion introduced by S1.

2.4. Cell culture and transient transfection

Rat hepatoma FTO cells were maintained in DMEM/

HAM medium supplemented with 10% foetal bovine serum

(ICN). For transient transfections cells were seeded into

100-mm petri dishes (6� 105 cells per dish) and grown in

10 ml of medium to 50–60% confluency. Transfections

were performed by the DEAE-dextran method using a

modification of a published procedure (Maniatis). Briefly,

cells were overlaid with 0.65 ml of mixture containing 2 Agof DNA and 0.5 mg/ml of DEAE-dextran in TBS*(P) buffer

(25 mM Tris–Cl, 137 mM NaCl, 5 mM KCl, 0.7 mM

CaCl2, 0.5 mM MgCl2 and 0.3 mM Na2HPO4) and incu-

bated at room temperature for 30 min. Then, cells were

washed with TBS*(P) and overlaid with 10% solution of

DMSO in DMEM/HAM medium for 2 min. After washing

and addition of medium with serum, cells were grown for

48 h at 37 jC (controls) or for 43 h, then heat shocked at

42.5 jC for 45 min and allowed to recover for 4 h.

Table 1

Structure of the plasmids

Plasmid name Coordinates of the hsp70.1 sequence Features

p68/1.0 � 1924 to � 869 MS, ID, and 600 bp nonrepetitive sequence

p68/300 � 1924 to � 1611 MS

p68/600 � 1924 to � 1322 MS, ID

p68/700 � 1611 to � 869 ID and 600 bp nonrepetitive sequence

p68/450 � 1322 to � 869 450 bp nonrepetitive sequence

p150/CAT6 � 64 to + 85 Sp1, TATA, 85 bp 5VUTRp250/CAT6 � 167 to + 85 HSE1, Sp1, TATA, 85 bp 5VUTRp350/CAT6 � 269 to + 85 HSE2, HSE1, Sp1, TATAS, 85 bp 5VUTRp350(DHSE1)/CAT6 � 269 to � 167 and � 64 to + 85 as p350/CAT6 except DHSE1

p550/CAT6 � 478 to + 85 HSE3, HSE2, HSE1, Sp1, TATA, 85 bp 5VUTRp550(DHSE1)/CAT6 � 478 to 167 and � 64 to + 85 as p550/CAT6 except DHSE1

p950/CAT6 � 869 to + 85 400 bp nonrepetitive sequence, HSE3, HSE2, HSE1, Sp1,

TATA, 85 bp 5VUTRp950(DHSE1+ 2)/CAT6 � 869 to � 280 and � 64 to + 85 as p950/CAT6 except DHSE1 and 2

p150–950/CAT6 � 1024 to + 85 as p950/CAT6+ 150 bp nonrepetitive sequence

p300–950/CAT6 � 1180 to + 85 as p950 + 300 bp nonrepetitive sequence

p450–950/CAT6 � 1322 to + 85 as p950 + 450 bp nonrepetitive sequence

p700–950/CAT6 � 1611 to + 85 as p950/CAT5+ 450 bp nonrepetitive sequence + ID

p1.0–960/CAT6 � 1924 to + 85 as p950/CAT6+ 450 bp nonrepetitive sequence + ID +MS

Coordinates shown in the second column concern the hsp70.1 sequence cloned in each plasmid; they are indicated relative to the transcription start point of the

gene. Third column contains information concerning specific features of the cloned DNA fragment, e.g. cis regulatory element or repetitive sequences.

A. Fiszer-Kierzkowska et al. / Biochimica et Biophysica Acta 1625 (2003) 77–87 79

Fig. 2. Nucleotide sequence of the regions flanking the rat hsp70.1 gene coding unit. The sequence of the coding region of the gene was published earlier [8]

and is not shown here. Inverted repeat and the homopurine/homopyrimidine sequence are boxed. TG repeats are underlined (dashed line). AT-rich sequences

are shown in italics, ID sequences are double underlined, and the B2 sequence is double overlined. Direct repeats flanking the ID sequence are shown in bold.

‘A’ indicates heat shock elements (HSE); ‘B’, CAAT-box; ‘C’, Sp1 sequences; ‘D’, inverted CAAT-box; ‘E’, TATA-box; ‘F’, polyadenylation signal;

arrowhead, site of putative deletion. The sequence shown here is deposited in EMBL Data Bank (acc. no. X74271).


2.5. CAT assay

Cell culture extracts were prepared using Reporter Lysis

Buffer (Promega) according to the manufacturer’s instruc-

tions. Protein concentration in extracts was determined by

Bradford method [41]. CAT assay was performed as a

modification of procedure described by others (Maniatis).

Briefly, 100 Al of cell extract containing 25 Ag of protein

was added to 100 Al of reaction mixture containing 0.25

Tris–HCl (pH 7.8), 1 mM EDTA, 4.5 Al of 100 mM acetyl-

CoA (Sigma) and 2.5 Al of [14C] chloramphenicol (2.5 ACi/ml; Amersham). Samples were incubated in 37jC for 4 h.

The reaction products were separated by thin layer chroma-

tography. To quantitate CAT activity, radioactive spots

representing acetylated and nonacetylated forms of chlor-

amphenicol were cut out from the plate and their activity

was measured in a scintillation counter. Counts were con-

verted to the percentage of acetylated chloramphenicol as

described by others [42]. Data for each experimental point

were gathered in quadruplicate.

3. Results

3.1. Structure of the hsp70.1 gene flanking regions

Fig. 2 shows DNA sequence of the regions flanking the

coding unit of rat hsp70.1 gene with distinguished repetitive

sequences. The most complex of these sequences is the

microsatellite sequence localized upstream of the tsp of the

gene. The core region of this sequence exhibits some unique

structural properties, being composed of a long inverted

repeat and a homopurine/homopyrimidine (Pu/Py) mirror

repeat. In close proximity to the microsatellite sequence we

detected three separate DNA regions highly enriched in AT

base pairs and poly(A) tracts (Fig. 2). One of the AT-rich

regions constitutes a part of the ID sequence (Fig. 2), a

rodent-specific short interspersed repetitive element (SINE)

[44]. We also found a second ID element located 128 bp

downstream of the polyadenylation signal. Another SINE

sequence, the B2 repeat, was identified 608 bp downstream

of the poly(A) signal on the complementary strand. We also

Fig. 3. S1 endonuclease sensitivity of plasmids containing various fragments of the DNA region located upstream from the rat hsp70.1 gene. (A) Structure of

plasmids. The EcoRI–PstI fragment located between positions � 1924 and � 869 (relative to tsp) and its shorter subfragments were cloned into pUC19.

Drawing of the 5Vgene flanking region shows restriction sites used for cloning. Dark grey box represents microsatellite sequence, light grey box indicates ID

repeat. (B) S1 nuclease assay. Plasmids described in (A) and control pUC19 plasmid were treated with S1 nuclease. Lanes marked ‘C’ contain native plasmid

(control); lanes ‘L’, plasmid cleaved with the restriction enzyme, which serve as a marker for lanes ‘S1’, indicating position of linear plasmid; lanes ‘S1’,

plasmid treated with S1 nuclease. Supercoiled plasmids with non-B DNA structures consisting partially of single-stranded fragments are cleaved by S1

nuclease to the OC form and subsequently to the linear form (depending on enzyme concentration and duration of reaction). Plasmids devoid of non-B DNA

structures remain intact.


found a triple tandem repeat of 33 nt within the 5VUTR and

a 239-bp gap within the 3VUTR as compared to the

orthologous mouse region (Figs. 1B and 2).

3.2. Structure of the Pu/Py region

The microsatellite region found about 1.8 kbp upstream

of the tsp of the gene is composed of a (GA)6CAG(TC)6inverted repeat and a partially overlapping (TC)24T mirror

repeat. This core sequence is flanked by imperfect (TG)9and (TG)11 repeats. Theoretically, both the long inverted

repeat and the Pu/Py mirror repeat are able to form non-B

DNA structures such as a cruciform or a triple-stranded H-

DNA, respectively. Our preliminary results indicated that

the microsatellite sequence cloned into the pUC19 vector

(plasmid p68/600) was susceptible to S1 nuclease which

digests single-stranded DNA regions that accompany differ-

ent non-B DNA structures [35]. We also observed a DNA

polymerase blockage at the beginning of the (TC)24 tract in

the sequencing reaction with the lower DNA strand as

template (not shown). Here we map more precisely the

region of S1 nuclease cleavage and attempt to define the

nature of the non-B DNA structure. The series of plasmids

was constructed (see Fig. 3A and Materials and methods)

that contained either all repetitive sequences present in

analyzed region, or some of them, or only the adjacent

nonrepetitive sequences. These plasmids were treated with

S1 nuclease and specific cleavage was observed in p68/1.0,

p68/600 and p68/300, each containing the microsatellite

region (Fig. 3B). Plasmid p68/450, devoid of any repetitive

sequences, was not susceptible to S1 digestion. Plasmid

p68/700 was very slightly cleaved by S1, probably due to

the lower melting temperature within the long poly(A) tract

present in its insert. In order to map the cleavage site, S1-

treated p68/300 plasmid was further digested with different

restriction enzymes (Fig. 4A). Two main cleavage sites

could be deduced from the restriction pattern, the first

within the CAG sequence (the core sequence of the inverted

repeat (GA)6CAG(TC)6) and the second in the center of the

homopurine–homopyrimidine mirror repeat (TC)24T (Fig.

4B and C). This suggested that under our experimental

conditions, part of the plasmid molecules adopted a cruci-

form structure while other part formed an H-DNA. To test

this hypothesis we re-ligated the S1-treated p68/300 plasmid

and sequenced 21 of the clones obtained. In four clones we

observed huge deletions encompassing the whole micro-

satellite region and some neighboring sequences which can

probably be ascribed to the S1 nuclease overdigestion and

unspecific activity on double-stranded DNA (Fig. 5, dele-

tions marked ‘‘A’’). Fifteen clones had deletions suggesting

the formation of an H-DNA structure (marked ‘‘C’’). Four-

teen of them had deletions ranging from 8 to 31 bp localized

within the Pu/Py sequence. In one clone the deletion started

in the middle of the Pu/Py sequence and proceeded far into

the neighboring nonrepetitive sequence which can be

explained similarly to the ‘‘A’’-type deletion. Only two

clones had deletions that can be interpreted as the result

of cruciform cleavage by S1 (marked ‘‘B’’). This result,

together with the results of restriction mapping of the S1

cleaved plasmids, shows that the equilibrium between two

possible non-B DNA structures may vary depending prob-

ably on slight differences in the experimental conditions

(e.g. buffer concentration and temperature). We also tested

Fig. 4. Low resolution mapping of the S1 nuclease sensitive sites in p68/

300 plasmid. (A) Samples of supercoiled p68/300 plasmid were treated

with S1 nuclease, then cleaved by restriction enzymes (lanes 1–4). Large

restriction fragments containing vector sequences were cut out off the

picture. Sizes of reference marker (lanes ‘M’) are indicated on the right.

Sizes of the restriction fragments were defined using ONE-D Scan software

(Scananalytics); they correspond well to the theoretical cleavage products

that may be obtained when one fraction of plasmid particles adopts H-DNA

structure, while the other adopts cruciform structure. Diffuse shape of the

bands may reflect differential activity of S1 nuclease (different plasmid

particles are digested more or less intensively). (B) Theoretical sizes of the

cleavage products. ‘S1A’ indicates most probable S1 cleavage site in the

case of cruciform structure formation; ‘S1B’, site of cleavage in the case of

H-DNA. Gray box at the top drawing represents insert in p68/300; line

represents vector sequences. (C) Localization of S1A and S1B sites within

the nucleotide sequence of the MS region.


the susceptibility of the deletion clones to the subsequent S1

nuclease cleavage. As expected, the clones with minor

deletions (TC)4 to (TC)8 were still cleaved by S1 while

clones with larger deletions were much more resistant (not

shown). Surprisingly, clones with huge deletions encom-

passing the whole microsatellite region were also cleaved by

S1, although very slightly. We think that this was due to the

high AT content (over 80%) in the remaining part of the

insert.

3.3. Functional analysis of the hsp70.1 gene 5V flanking

sequences

We made several constructs containing various fragments

of the 5V sequence of the hsp70.1 gene fused with a CAT

reporter gene (see Fig. 6). The first series of constructs was

made to study the basic heat shock regulatory elements of

the gene, the HSEs. Plasmids contained each of three HSEs

a lone (p250/CAT6, p350(DHSE1) /CAT6, p950

(DHSE1 + 2)/CAT6), combinations of two HSEs (p350/

CAT6, p550(DHSE1)/CAT6), all three HSEs (p550/CAT6)

or only proximal promoter sequences without any HSE

(p150/CAT6). These plasmids were transiently transfected

to FTO cells and CAT activity was analyzed in extracts of

control and heat shocked cells (Fig. 6B). No CAT activity

was observed in case of the plasmid p150/CAT6 containing

only proximal promoter sequences encompassing TATA-

box and Sp1 sequence, but without any HSE. Significant

constitutive as well as heat inducible CAT activities (12%

and 30% of acetylated chloramphenicol, respectively) were

achieved with the construct p350/CAT6 containing both

proximal HSEs. Addition of the third HSE (construct

p550/CAT6) caused only a slight, insignificant increase in

both types of CAT activity. Interestingly, we found that none

of HSEs alone is able to drive inducible transcription of the

reporter gene.

A further question was whether the more upstream

sequences, with special emphasis on the repetitive motifs,

have any impact on transcription efficiency. Two constructs

were made containing, in addition to the region with three

HSEs, a further upstream nonrepetitive sequences of 391 bp

(p950/CAT6) and 844 bp (p450–950/CAT6; compare with

plasmid p68/450, Fig. 3A). Next plasmid p700–950/CAT6

contained additionally the poly(A) tract of the ID sequence

(compare with p68/700), while p1.0–950/CAT6 contained

both the microsatellite sequence and the poly(A) tract.

Surprisingly, plasmid p950/CAT6, which differs from

p550/CAT6 by addition of 391 bp of upstream region devoid

of any evident regulatory elements, exhibited much higher

CAT activity (75% versus 35%). Plasmids p950/CAT6 and

p1.0–950/CAT6 showed comparable activity of the reporter

gene, although it seemed that the presence of the micro-

satellite sequence in the latter construct caused a decrease of

transcription in control cells. This is closer to the behavior of

the strictly inducible promoter of the rat hsp70.1 gene in

vivo. Surprisingly, in the case of the intermediate constructs

(p450–950/CAT6 and p700–950/CAT6) a significant

decrease in the CAT activity was observed, both in control

and heat shocked cells. This suggests the possible presence

of a silencing element in this DNA region, whose negative

influence on transcription can be reversed by the presence of

more distal regulatory elements (plasmid p1.0–950/CAT6).

Searching for a negative regulatory element we shortened

the p450–950/CAT6 plasmid (the shorter of two constructs

with diminished transcriptional efficiency) by 142 and 298

bp from the 5Vend. Both thus constructed plasmids (p300–

950/CAT6 and p150–950/CAT6) showed diminished heat

inducibility, which suggests that the silencing activity is

localized within the 155 bp of the HincII–PstI fragment (see

Fig. 6B).

3.4. Interspecies comparison of noncoding and flanking

DNA sequences of the hsp70.1 gene

In search for evolutionary conserved repetitive motifs we

compared the gene-flanking sequences of three orthologous

Fig. 5. Size and localization of deletions introduced in p68/300 plasmid by S1 nuclease. A sample of p68/300 plasmid was treated with the S1 nuclease.

Cleavage products were separated by agarose gel electrophoresis; linearized plasmid DNAwas isolated from gel, filled with Klenow enzyme to blunt possible

overhanging ends and ligated. Twenty-one of the obtained clones were sequenced. Upper line shows the DNA sequence within the microsatellite region

(positions from � 1792 to � 1691 relative to the tsp). Lines below indicate fragments of the sequence that were deleted by S1 digestion. Four types of deletion

were repeated more than once (indicated by the number on the left side of the line). Deletions marked by dashed lines are located within (TC)24 sequence and

due to the repetitive nature of that sequence, their precise localization cannot be established. For description of the deletions marked as A, B or C, see text.


(centromeric) MHC III-linked hsp70i genes—the human

hsp70.2 gene [6] (acc. no. AF134726), the mouse hsp70.1

gene [10] (acc. no. AF109906) and the rat hsp70.1 gene [7]

(acc. no. X74271). We observed similar distribution of

SINEs in the 5V flanking regions of all three genes. As in

the case of the rat hsp70.1 gene three SINE sequences were

found upstream of the mouse hsp70.1 gene (PB1D7,

PB1D10, MMB1F, beginning 1.07, 1.56 and 1.76 kbp

upstream of the ATG codon, respectively). Also, in the

region preceding the human hsp70.2 gene, three Alu

sequences were found at distances of 1.4, 1.65 and 1.76

kbp upstream of the ATG codon. As all these are species-

specific sequences, it seems that SINE sequences evolved in

this region of the mammalian genome independently, after

diversification to the distinct species.

Searching for conserved microsatellite sequences in the

mouse hsp70.1 gene, we found a long tract (CC(TC)9CTCCC(TC)11TT(TC)9(GT)19) composed of Pu/Py sequen-

ces devoid of perfect mirror symmetry and a (GT)19 repeat.

This sequence, with partial similarity to the microsatellite

sequence found in rat is, however, localized on the opposite

DNA strand, what probably excludes a common evolu-

tionary origin. In human there are no simple repeats neither

in the region corresponding to the position of microsatellite

sequence in the rat hsp70.1 gene nor in the whole 3.0-kbp

region upstream of the ATG codon.

Fig. 6. In vitro assay of the promoter activity. Left panel: Schematic representation of the plasmids used for transfections. Different fragments of the promoter

and further upstream region of the rat hsp70.1 gene were cloned in pBLCAT6 [40] vector. Drawing of the structure of analyzed DNA region (upper row) shows

restriction sites used for subcloning. Right panel: Data from transfection experiments. Plasmids were transiently transfected to the rat FTO cells and CAT

activity was determined in control (white bars) and heat shocked (gray bars) cells. CAT activity is shown as percentage of acetylated chloramphenicol. Each

value is expressed as a mean (with the standard deviation) of four transfections. (A) Experiments performed to evaluate function of the proximal promoter

region. (B) Functional analysis of the far upstream DNA region and repetitive sequences therein.


We also compared the nucleotide sequence of the

untranslated regions (UTRs) of the rat, mouse and human

genes. The 5VUTRs showed about 80% identity between

mouse and rat hsp70.1 genes, and about 50% identity

between rat hsp70.1 and human hsp70.2 genes (see also

Ref. [4]). We found a 33-nt triple tandem repeat (TTR) in

the 5VUTR of the rat hsp70.1 gene (Fig. 2), which is very

similar to the TTR described previously in the mouse

hsp70.1 gene [7]. A database search revealed that such a

TTR is also present in the 5V UTR of the second (telomeric)

MHC III-linked hsp70i gene of the mouse (hsp70.3 gene

[3]) and in the orthologous rat gene (hsp70.2 gene [4]) but is

absent from any other hsp70 gene. The functional meaning

of this TTR in hsp70 genes is unknown.

The nucleotide sequences of the 3V UTR of the rat

hsp70.1 and human hsp70.2 genes show 70% identity.

Interestingly, the nucleotide sequence of the rat gene is

evidently interrupted by a gap of 239 bp (Fig. 2) and the

identity between the DNA regions which flank the gap is

84%. The presence of the microsatellite sequence (AT)17flanking the 5Vend of this 239-bp sequence in the mouse

gene suggests a possible deletion/insertion event, and

according to the mammalian phylogenetic tree, it seems

that this diversity could have been caused by insertion rather

than by deletion.

4. Discussion

Functional analysis performed in this study revealed

several interesting features concerning differential effect of

various fragments of the promoter region on the level of

heat inducible expression of the rat hsp70.1 gene. Promoter

region of the gene contains 3 HSE elements, all localized

within the approximately 450 bp DNA fragment adjacent to

the transcription unit of the hsp70.1 gene. We found that

functional cooperation between these HSE sequences is

required for efficient heat inducible transcription. None of

HSEs alone, even the HSE1 with almost perfect match with

the consensus sequence, is able to activate transcription of

the reporter gene.

Our data show that although HSE elements are indis-

pensable for heat shock activation of the hsp70.1 promoter,

there are DNA sequences localized more upstream, which

can significantly affect transcription efficiency. This

upstream fragment, of approximately 1.5 kbp, was found

to contain regions which can exert either stimulatory or

inhibitory effect.

We observed a very significant enhancement of heat

inducible transcription when the promoter region containing

three HSE sequences was extended to approximately 900 bp

(up to � 869 PstI restriction site); see Fig. 6B. In contrast,

DNA sequences localized between positions � 869 and

� 1024 (relative to the tsp) significantly reduced the indu-

cibility of the rat hsp70.1 gene promoter. This silencing

effect could be, however, abolished when upstream sequen-

ces containing the microsatellite region were present in the

construct. This suggests possible cis– trans interactions at

various sites found within the 1.5-kbp distal promoter

region containing three HSEs. These interactions can prob-

ably affect transcription machinery and/or stabilize/destabi-

lize other crucial interactions. As a relatively large DNA

fragment has to be analyzed in search for cis regulatory

elements involved in modulation of HSE-HSF dependent

activation of the hsp70i genes, this would need a separate

study. It seems, however, that even the present preliminary

data may be useful, e.g. for construction of the vectors

containing inducible promoters.

It has to be noted that the differential effect exerted by

various fragments of the promoter region was already

observed by others in the case of telomeric hsp70i genes.

The 224-bp DNA fragment of the rat hsp70.2 gene, adjacent

to the transcription initiation site, was sufficient to govern

heat inducible expression of the CAT reporter gene; how-

ever, the induction level was rather low (approximately

threefold) and some activity was observed at physiological

temperature. Quite unexpectedly, a longer fragment of the

promoter region (of 387 bp) activated the reporter gene in a

constitutive way and the expression level was independent

of heat shock [32]. Similar studies of the mouse hsp70.3

gene revealed that a 2.3-kb fragment of the 5Vend sequences

was at least three times more effective as a heat inducible

promoter than a 292-bp fragment when assayed in HeLa

cells. However, the same DNA fragments showed compa-

rable promoter activities in mouse L cells [3].

Taken together, our data and those of others, it seems

evident that cis elements other than HSEs could be involved

in the regulation of hsp70i gene expression. It may well be

that such elements are responsible for frequently observed

tissue-specific differences in the induction level of the

hsp70i genes. It is also possible that such elements can play

a role in differential expression of two of a MHC III-linked

hsp70i genes. Differential expression in response to various

conditions such as hypertonicity [13], ischemia/reperfusion

[14], exposition to H2O2 and NaCl [12] or during differ-

entiation of hematopoietic cells [15] was already commu-

nicated. It remains to be established whether DNA

sequences localized upstream the HSEs have any functional

meaning in vivo.

The aim of our study was also to find out whether a

complex microsatellite region identified far upstream from

the beginning of the hsp70.1 gene transcription unit can

affect expression of the gene. Potential cruciform and H-

DNA forming sequences are relatively frequent in genomes

of higher eucaryotes. They are localized predominantly in

the 5V flanking regions and UTRs of genes but are almost

excluded from coding sequences [43,44]. Both types of

structures are suspected to play some functional roles, e.g. in

transcription, recombination or DNA replication. Cruciform

forming sequences may affect transcription by differential

DNA–protein interactions depending on the DNA confor-

mation (dsDNA versus cruciform) [45]. The effect of H-


DNA forming sequences on transcription efficiency is rather

unpredictable without performing functional studies; in fact,

positive [38,39,46–49], negative [50,51] or neutral [52–54]

or even variable [36,55] influence of Pu/Py sequences has

been found. Other observations suggest that in some cases

H-DNA may play the role of transcriptional insulator

separating the gene from the influence of its chromosomal

environment [56]. Looking at the results of the said studies,

it seems that the way such sequences can affect transcription

of a particular gene probably depends on such factors as the

length of the tract, the perfection of its symmetry, its

distance from the gene and even surrounding sequences

context. Although there is still a controversy concerning the

existence of H-DNA structures in vivo, crucial may be the

differential interaction with cellular proteins showing affin-

ity to either dsDNA or ssDNA or triplex structure formed

within the same sequence [47,57–59]. It has been even

proposed that a transcriptional mechanism functioning

through interconversion between ds- and ssDNA (being

part of H-DNA structure) is common, especially for

growth-related genes [39].

In our study we found that hsp70i-linked Py-Pu-contain-

ing region had the potential to reverse the effect of putative

silencing sequences. It remains to be found out what kind (if

any) of non-B DNA structure may be formed in vivo by the

microsatellite region identified by us and whether any

DNA–protein interaction(s) influencing the transcription

efficiency occur in this region.

Acknowledgements

We thank Mrs. K. Klyszcz for technical assistance and

B. Sarecka, M. Sci. for cooperation during preliminary

transfection assays. We are grateful to Dr. A. Sochanik for

critical reading of the manuscript. This work has been

supported by a grant from the State Committee for

Scientific Research (grant 6 P04A 036 15) to K.L., and

by a grant from the Foundation for Polish Science (grant

BIMOL 76/9) to Z.K.

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