oxygen-sensing consensus sequences and tissue-specific transcriptional regulatory elements

Gene, 137(1993)2033210 0 1993 Elsevier Science Publishers

GENE

B.V. All rights reserved.

203

0378-l 119/93/$06.00

07534

The 3’ flanking region of the human erythropoietin-encoding gene contains nitrogen-regulatory/oxygen-sensing consensus sequences and tissue-specific transcriptional regulatory elements (DNA sequence;

enhancer

Sylvia Lee-Huanga, Paul Lee Huangc

elements;

growth hormone;

Jih-Jing Lin b, Hsiang-fu

erythropoiesis)

Kungb, Philip Lin Huangc, Leo Leeb and

“Department of‘ Biochemistry, New York Uniuersify School of’Medicine,New York, NY 10016, USA; bLaboratory ofBiochemical Physiology, Biological Response Modifiers Program, National Cancer InstituteeFrederick Cancer Research and Development Center, Frederick, MD 21701, USA. Tel. ( l-301 ) 846-5703; and ‘Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA. Tel. ( l-61 7 ) 726-2000 Received by M. Bagdasarian:

11 June 1993; Accepted:

25 June 1993; Received at publishers:

23 August

1993

SUMMARY

We have reported the identification of a classical canonical CAAT box, TATA boxes and other transcriptional regulatory elements in the 5’ flanking region of the human erythropoietin (hEp)-encoding gene [ Lee-Huang et al., Gene 128 (1993) 227-2361. These elements were not found in the hEp genomic clones reported by others. Our genomic clone extends in both directions beyond any reported clones, by 3.9 kb on the 5’ side and by 1.8 kb on the 3’ side. Many important regulatory elements are found in these extended flanking regions. We report here the genomic structure of the extended 3’ flanking region of hEp. This region contains the following regulatory elements: nitrogenregulatory/oxygen-sensing consensus sequences, 5’-TTTTGCA and 5’-CCCTGCA; tissue-specific regulatory elements, including binding sites for A-activator, 5’-GTGGTGCAA; for DBP, 5’-TGATTTTGT; for HNF, 5’-T(A/G)TTTGT; and for C/EBP, 5’-T(T/G) (T/G)TGCAAT; a lymphokine-responsive element, T-GTGAAACCCC (Rev), as well as binding sites for AP and Spl. In addition, the nucleotide (nt) sequence in this region is rich in inverted repeats (palindromes) that allow the formation of hairpin loops. A total of 14 potential stem loops with a maximum loop size of 20 nt are found. The identification of these regulatory elements in hEp should provide further insight into the tissuespecific and inducible expression of hEp. Such knowledge should be useful in the clinical modulation of erythropoiesis under physiologic and pathologic conditions.

INTRODUCTION

Erythropoietin (Ep), a glycoprotein hormone, is the prime regulator of red blood cell production. It regulates blood oxygen tension by modulating the number of circuCorrespondence to: Dr. S. Lee-Huang, Department of Biochemistry, New York, University School of Medicine, New York, NY 10016, USA. Tel. (l-212) 263-5135; Fax (l-212) 263-8166. Abbreviations: AABS, A-activator-binding site; AP, activator protein; bp, base pair(s); C/EBP, CCAAT/enhancer-binding protein; CHO, Chinese hamster ovary; DBP, D-site binding of albumin pro-

lating erythrocytes. Ep production is under feedback control by oxygen sensing (Throling et al., 1968); it is also tissue-specific, developmentally regulated and inducible. Ep is produced by the liver in the fetus (Zanjani et al., 1981), and by the kidney after birth (Jacobson et al., moter; Ep, erythropoietin; GRE, glucocorticoid-responsive element(s); hEp, human Ep; hEp, gene (DNA) encoding hEp; HNF, hepatic nuclear factor; IL, interleukin; IRE, IL-responsive element(s); kb, kilobase or 1000 bp; LFB, liver factor binding; MRE, metallo-responsive element(s); NF, nuclear factor; n$ nitrogen fixation gene(s); N/O, nitrogenregulatory/oxygen-sensing; ntr, nitrogen assimilation gene(s); nt, nucleotide(s); TF, transcription factor.

204 1975). Under

normal

duced constitutively of anemic

physiological

or hypoxic

stress, production

Ep is pro-

of this hormone expression.

It contains

a total of 4.5 kb at the 5’ flanking

oxygen

either

due to de-

2.6 kb at the 3’ flanking

of tissue

A common

blood oxygen-carrying

tension, capacity

reduced ambient oxygen concentration likely signal transduction mechanism.

in anemia,

or to

in hypoxia, is the However, little is

known about the regulatory pathway of oxygen sensing that controls the Ep gene expression. Enhancer-like activities for cell-type-specific and hypof Ep have been described.

expression

have been reported mouse

at the 3’ flanking genes

(Beth

These

region

of

been

9.3-kb nt sequence region, as compared

of our clone. region and to 0.6 kb at

the 5’ side and 0.8 kb or 0.6 kb at 3’ side reported by others (Jacobs et al., 1985; Lin et al., 1985). The coding region introns.

consists

of about

2.2 kb with five exons and four

The size of the exons and introns,

as well as the

exon/intron boundaries, are identical for the fetal liver gene. The genomic

with those reported structure of the ex-

tended

and its relationship

3’ flanking

with the remainder

region of hEpSLH

of hEp are shown in Fig. 1.

et al., 1991;

Imagawa et al., 1991; Pugh et al., 1991; Semenza et al., 1991a) as well as in the highly conserved 5’ flanking regions, exon I, intron I and exon V of these genes (Tmagawa et al., 1991). Recently, it was reported that the previously described cell-type-specific/hypoxia-induced Ep expression appears to be in error and that oxygenregulated gene expression identical or similar to that of Ep operates commonly in nature regardless of cell type (Maxwell et al., 1993). It was thus suggested that tissue specificity and hypoxia inducibility in Ep expression are regulated by distinct mechanisms. However, no defined oxygen-sensing consensus sequences or liver-specific regulatory elements have been found in the limited 3’ flanking region of the reported Ep clones (Jacobs et al., 1985; Lin et al., 1985). We report here that the 3’ flanking region of hEp contains nitrogen-regulatory/oxygen-sensing consensus sequences, as well as tissue-specific regulatory sequences. These findings provide a molecular basis for many of the observed features of cell-type-independent, hypoxia-induced Ep expression.

RESULTS

has

et al., 1993). We have

gene

Reduction

and

(Lee-Huang

and its relative

by others

stress-induced

this

the human

previously

of hEpSLH

reported

the complete

in

both

those

determined

involved

oxia-induced

described

organization

with

seems to be

increased.

elements

The genomic relationship

conditions

signal

is significantly

creased

conditions,

at low levels, but under

AND DISCUSSION

(a) Genomic structure of the 3’ flanking region of the hEpSLH clone ( 1) Characterization and organizution The genomic structure of the extended 3’ flanking region of the hEp gene was studied in a 9.3-kb genomic clone, hEpSLH. This clone was identified from a human leukocyte genomic library in hgtll, using 32P-labeled hEp cDNA as a probe (Lee-Huang, 1984). The 9.3-kb BumHI insert of hEpSLH was subcloned into pUC19 for large scale preparation. The clone was analyzed by restriction mapping and Southern blotting (Southern, 1975). The sequence of the genomic DNA was determined by the dideoxy method (Sanger et al., 1977) using M13mp18 and mp19 subclones.

(2 ) Nucleotide sequence Fig. 2 shows the nt sequence of the extended 3’ flanking region of hEpSLH. It extends from the last PstI site of the previously reported clones to the 3’ BumHI site. This newly described region consists of 1777 bp. A computeraided homology search of the entire sequence against the nt data in the GenBank did not reveal significant homology with any published nt sequence. Thus, the sequence reported here is new and unique. ( 3 ) Regulatory elements Table I presents a selected list of the regulatory elements and their nt positions at the extended 3’ flanking region of the hEp gene. There are nitrogen-regulatory/ oxygen-sensing consensus sequences, tissue-specific regulatory elements and cytokine responsive elements, and many other potential transcriptional regulatory elements. A schematic map of these regulatory elements is shown in Fig. 3a. The identification of these regulatory elements should provide useful information in the analysis of transcriptional regulation of Ep expression. (4 ) Stem-loops The extended 3’ flanking region of hEp contains many inverted repeats that allow the formation of stem-loop structures. A total of 14 possible stem-loops with maximum loop size of 20 nt were identified. Fig. 3b shows a

hEpSLH

clone 9.3 kb hiidlll

EmHI

,+--

3.9 kb -

3 6 kb Previously

reported

p+-sequence

1 777 bp Extended

+

3’ region

(see Figs 2 8 3)

Fig. 1. Genomic organization and structure of hEpSLH gene encoding human erythropoietin. A schematic representation of the genomic structure of the 9.3-kb hEpSLH and its comparison with the 3.6-kb genomic clone reported by others (Jacobs et al., 1985; Lin et al., 1985).

205

Fig. 2. Nucleotide sequence of the extended 3’ flanking region of hEpSLH. The nt sequence of the extended 3’ flanking region, from the last PsfI to BamHI sites of hEpSLH. This region consists of 1777 nt that are not present in the 3.6-kb hEp genomic clone reported by others (Jacobs et al., 1985; Lin et al., 1985). The oxygen-sensing and tissue-specific regulatory elements are underlined. The nt sequence before the last PstI site is provided to show how the new sequence

schematic map of DNA-looping is may be involved many genes. The

is extended

from the previously

reported

these stem-loops and their nt positions. an important regulatory feature that in the regulation of transcription of effects of enhancers are often mediated

a. Regulatory

3.6-kb sequence.

by loop formation. Stem-loop formation facilitates the interaction between DNA-binding proteins and their cisacting elements, and may also increase the stability of mRNA (Matthews, 1992).

elements BarnHI

Pstl

/I DBP

II CiEBP

AABS

GRE

CiEBP

I

I

I

1

500

1777 bp

b. Stem loop structures

1:59-100 5: 382-417 10: 682-718 2:130-157 6:387-430 3:130-164 7~396436 4~249-289 8:401-436 9:459-496

11:1092-1124 12:1390-142213:1579-1617 14~1584-1617

Fig. 3. Identification of the oxygen-sensing and tissue-specific consensus sequences and stem-loop structures in the 3’ extended flanking region of hEpSLH. The nt sequence of hEpSLH was analyzed using the CCC sequence analysis software package, (Genetic Computer Group, Madison, WI, USA). Sequence comparisons were carried out using BESTFIT and GAPSHOW programs. Localization of sequences corresponding to transcription factor binding sites was done using the FINDPATTERN program. (a) Schematic representation of locations of the nitrogen-regulatory/oxygen-sensing (N/O), tissue-specific and other selected transcriptional regulatory elements in the 3’ flanking region of hEpSLH. Table I shows the exact nt positions of these elements. (b) Schematic map of the locations of 14 potential stem-loop structures. The nt locations of these loops are shown underneath the map.

206 TABLE

1

Selected list of regulatory Regulatory” element

elements

and their positions Locationb (nt)

AABS

409

DBP-RS HNF

345 339 351

HNF/Rev

355 359 1140

NF-GM

region

of hEpSLH

Sequence (5’ to 3’)

Kaling

GTGGTGCAA TGATTTGTT

et al. (1991)

Muller et al. (1990) Frain et aI. ( 1989)

TATTTGT TGTTTGT TGTTTGT TGTTTGT ACAAACA ACAAACA

Frain et al. (1989)

410 1278

TGGTGCAAT

Xanthopoulos

985

1144 C/EBP

in the 3’ flanking

et al. (1989)

TTTTGCAAT

N/O (nif’H) CS N/O (nif) CS

1246 1278

GTGAAACCC CCCTGCA TTTTGCA

J. Biol. Chem. 266 (1991) 252-257 Ow et al. (1983)

Lymphokine/Rev GH API-CSl

985 524 486

GTGAAACCC TAAATTA GTGACTAA

Heinrich et al. (1990) J. Biol. Chem. 263 (1988) 7821 -7829

AP2-CS4 APZ/Rev

503 707

TCGCCATGCC GGGGGTGGGGG

AP2-CS5

708 26

GGGTGGGGGA GGGAGGCC

880 971

GGGAGGCC

AP2-CS5JRev GREfRev CR-uteroglobin

884 1419 1421

GRIPR-MMTV MREJRev

1421 402 845

NF-KB/Rev TFIID Spl (GCbox)

SPl

Cell 55 (1988) 395-397 Cell 51 (1987) 251-260 Cell 51 (1987) 251-260 Cell 50 (1987) 847-861

GCCTGGCC GGCCAGGC TCTGTTCT TGTTCT TGTTCT

Cell 50 (1987) 847-861 CABlOS 1 (1985) 95-104 Nucleic Acids Res. 15 ( 1987) 4535-4552 Nature 313 (1985) 706-709 Cell 48 (1987) 261-270

GAGTGCA GTGTGCA GAGTGCA GTGAAACCCCCC TATAAA

J. Biol. Chem. 266 (1991) 252-257 Proc. Natl. Acad. Sci. 84 (1987) 2203~-2207

TTTATA GGGCGG

Nature

312 (1984) 409-413

840 1354 61

GGGCGG GGGCGG GGGGCTGGT

Nature

316 (1985) 7744778

926 1062 1092

GAGGCTGAG GAGGCTGAG GAGGCGGAG

1585 985 287 333 253

“The locations of some of the transcription regulatory elements listed are shown in Fig. 3a. Regulatory elements are listed either as DNA sequence (italics) or as protein factors that recognize the regulatory elements (Roman). CS, consensus sequence; N/O, nitrogen-regulatory/oxygen-sensing; Rev, reverse sequence; RS, regulator sequence. bThe nt positions, measured from the first nt at the extended 3’ end of hEpSLH as shown in Fig. 2. “References are listed either by authors and year (those cited also in the text) or as journal, volume, year and pages (those not listed in References).

(b) The nitrogen-regulatory/oxygen-sensing consensus sequence In response to anemia or hypoxia, the level of Ep mRNA is increased several lOO-fold in mouse liver and kidney (Bondurant et al., 1986; Schuster et al., 1989). Analyses of Ep expression in transgenic mice as well as in human hepatoma cell lines HepG2 and Hep3B reveal that oxygen-sensing enhancer elements are located in the 3’ flanking region of the Ep gene (Goldberg et al., 1987; Beck et al., 1991; Imagawa et al., 1991; Pugh et al., 1991;

Semenza et al., 1991a; 1991b; Madan and Curtin, 1993; Maxwell et al., 1993; Wang and Semenza, 1993). A DNA fragment located at 120 nt 3’ to the polyadenylation site of the mouse Ep gene has been found to confer hypoxiaregulated expression of the Ep gene. About 70 bp of this fragment were reported to be necessary and sufficient for the enhancer activity. This enhancer element was originally reported to be cell-type-specific and active only in hepatoma cells but not in CHO and MEL cells (Pugh et al., 1991). Recently, it was reported that this observa-

207 tion

appears

density

to be in error

(Maxwell

due to the use of low cell

et al., 1993).

gene revealed that an oxygen-sensing system similar to that of Ep is commonly present in a variety of mammalian et al., 1993). This enhancer

is active not

only in hepatoma

cell lines but also in other

including

fetal

human

lung

fibroblast

cell types,

(MRCS),

fibroblast (lBR), monocytes/macrophage monkey renal fibroblast (COS-7), pig renal

skin

(U973), epithelium

(LLC-PKl ), Chinese hamster ovary (CHO, K 1) and mouse renal adenocarcinoma (RAG) cells. These results suggest

that

oxygen-regulated

an

identical gene

or

The most

common

and

system is found in nitrogen

Transient transfection of an a,-globin reporter gene coupled to the hypoxia-enhancer region of the mouse Ep

cells (Maxwell

genes.

similar

expression

may

mechanism operate

for com-

monly in biological systems. Parallel results in hEp suggest that a hypoxia-inducible nuclear factor HIF-1, which binds to an enhancer element in hEp, is present in cells that do not produce Ep, suggesting that similar responses to hypoxia may be present in many cell types. The nature of the hypoxia-enhancer element in hEp has not been well defined. It was reported that the highly conserved 5’ flanking region, exon I, intron I and exon V stimulated about two- to threefold expression of a cat reporter gene in Hep3B cells in response to hypoxic stress. A hypoxic-enhancer-like activity was also observed in a 255-bp fragment at the 3’ flanking region (Imagawa et al., 1991). However, this fragment shares no homology with the hypoxia-regulatory element of the mouse gene and demonstrated no enhancer-like activity when linked to an a,-globin reporter gene (Pugh et al., 1991), casting doubt as to its biologic relevance. Transient transfection of Hep3B cells with hEp mini-gene constructs has identified an enhancer in a 150-bp ApaI-PstI fragment 120 bp downstream from the polyadenylation site of hEp (Beck et al., 1991). This element shares about 80% homology with the mouse hypoxia-enhancer region, although it was reported to be cell-type-specific and active only in the hepatoma cell lines. Another hypoxia responsive enhancer element identified in the 3’ flanking region of hEp falls within an ApaIITuqI 38-bp fragment, of which a 24-bp sequence appears necessary for activity (Madan et al., 1993). This enhancer works with an SV40 promoter as well as with the hEp promoter, and results in an eightfold increase in transcription upon hypoxia. Despite the growing evidence for oxygen-sensing regulatory mechanisms that are common to a variety of biological systems, no generally operative enhancer elements have been identified thus far. Furthermore, the elements previously reported in hEp do not account for the lOO-fold increase in expression that is seen in vivo. It is well documented that oxygen is the primary physiological factor controlling the expression of many

The inhibitory

ancient

oxygen-sensing

fixation

bacteria

effect of atmospheric

oxygen

(Fay, 1992). on nitrogen

fixation was observed in free-living N,-fixing organisms as well as in symbiotic systems. The expression of the nitrogen fixation genes, the nif genes, is under oxygen-sensing control. These genes are transcribed

tight only

when the oxygen tension

level

(1% oxygen

is reduced

The regulation

of the @“genes in Klebsiellu pneumoniue

has been studied extensively for nitrogen/oxygen sensing ganism, adjacent

to microaerobic

and 99% nitrogen).

17 nif genes operons.

and used as a model system (Ausubel, 1984). In this or-

are transcribed

Transcription

pressed by oxygen. The regulation

in seven

or eight

of the nif genes is reis thought

to be medi-

ated at two levels. The first level is nzyspecific and mediated by the two regulatory genes of the nifLA operon, n{fA and nifL. The products of these genes act as the positive and negative controls of nif structural genes respectively. The nifA gene product is required for the transcription of the nifstructural genes whereas the nifL gene product acts as a negative control, preventing the expression of other nif operons in the presence of oxygen. The second level is regulated by the centralized nitrogen assimilation system, the ntrC and ntrA gene products. In response to NH: starvation, the gene ntrC and ntrA products activate the n{fLA operon and other operons involved in nitrogen assimilation, such as histidine utilization (hut) and proline utlization (put). The transcriptional controls on the sensing and signaling of cellular nitrogen and oxygen tension are closely coordinated. The nitrogen-regulatory/oxygen-sensing consensus sequences have been identified, and consist of the heptameric sequence 5’-NNYTGCA with the underlined core sequence TGCA (Beynon et al., 1983; Ow et al., 1983). The sequence 5’-TTTTGCA (with at most one mismatch) is most commonly found on the nifstructural genes, which are responsive to both nifA and ntrC activation. In contrast, the sequence 5’-CCCTGCA (with no mismatch) is unique; it is found exclusively in the nifH (nitrogenase) gene of K. pneumoniue, and it is nifA specific and cannot be activated by ntrC. Nitrogenase, the nitrogen fixation enzyme, fixes atmospheric dinitrogen to ammonia reductively: N, + 6H+ + 6e- +2NH,. This reaction is under stringent hypoxia regulation and inhibited by oxygen. Oxygen also represses the expression of the nitrogenase gene via the nifL oxygen-sensing regulation (Fay, 1992). The ntr and nif genes are evolutionarily related and they are conserved between species (Fay, 1992). We have found that the extended 3’ flanking region of our hEpSLH contains two copies of nitrogenregulatory/oxygen-sensing consensus sequences, namely 5’-TTTTGCA and 5’-CCCTGCA at nt positions 1278

208 and 1246 respectively. quence 5’-TTTTGCA

As described previously, the seand its homologues are common

nitrogen-re~ulatory/oxygen-sensing genes

but

nifr-, and

(nitrogen

assimilation)

fixation).

On the other

responsive

hand,

5’-CCCTGCA

site of the n$H

only to the n$4 gene product

important.

5’-TTTTGCA,

The

S-CCCTGCA,

core sequences

transcription

factors such as DBP (Mueller

(nitrogen

NF-IL6

has been

1988) also bind to the same or a very similar sequence. LFB/HNFl was originally identified as a liver-specific

(nitrogenase)

(Ow et al., 1983).

multiple copies of nitrogenconsensus sequences in hEp is

fact that

is responsive

tion and nitrogen

the enhancer

the ntrC

K-. p~e~f~o~iae, and it is

bacterium

The identification of regulatory/oxygen-sensing very

to both

and n&4 gene products

found only at the regulatory gene of the enterie

sites on all of the nif

are responsive

assimilation

is responsive

one

of these

elements,

to both of the nitrogen regulators, only to nitrogen

lung tissue (Xanthopoulos et al., 1989). It recognizes a wide array of sequences, including the CCAAT boxes and

fixa-

and the other, fixation reg-

ulation has important regulatory implications. These results suggest that nitrogen-regulatory/oxygen-sensing sequences may be evolutionarily conserved between the nitrogen fixation genes and hEp, and underscore that they may be a widespread regulatory mechanism common in different cell types. (c) Tissue-specific regulatory elements Many copies of tissue-specific regulatory elements are found in the extended 3’ flanking region of hEp. These include the binding sites for A-activator (AABS), 5’-GTGGTGCAA at nt position 409; for CCAAT/ enhancer binding protein (C/EBP), 5’-TGGTGCAAT and 5’-TTTTGCAAT at nt positions 410 and 1278, respectively; for D-site of albumin promoter (DBP), 5’-TGATTTTGT-3’ at position 345; for heptocyte nuclear factor (HNF), 5’-TATTTTGT at position 339, 5’-TGTTTGT at positions 351, 355, and 359 as well as its reverse sequence 5’-ACAAACA at positions 1, 140 and 1144. These &-acting nt sequences have been reported to be involved in liver-specific gene expression (De Simone et al., 1988). They are present in eukaryotic genes from Xenopus laevis to humans, including those encoding albumin (Kugler et al., 1988), aldolase (Tsutsumi et al., 1989), a,-antitrypsin (Grayson et al., 19X8), ol-fetoprotein (Kugler et al., 1988), B-fibrinogen (Courtois et al., 1987), carbamylphosphate synthetase I (Howell et al., 1989), transthyretin (Costa et al., 1988) and pyruvate kinase (Vaulont et al., 1989). AABS is a liver-specific transcriptional regulatory element with the consensus sequence of 5’-GTGNNGYAA. It is found in the A2 vitellogenin gene of X. laevis as well as in liver-specific, IL-6 responsive, acute-phase genes of humans including the hemopexin, haptoglobin, and c-reactive genes (Kaling et al., 1991). Both AABS and IL&RE interact with at least three distinctive transcriptional factors, C/EBP and LFB/HNFl. C/EBP is found in fully differentiated liver cells, fat and

kidney

et al., 1987). Other et al., 1990),

(Akira et al., 1990) and LAP (Descombes

transcriptional of liver-specific quently kidney.

(Johnson

et al.,

factor which recognizes the HP1 element genes (Frain et al., 1989), but it was subse-

found to be also present in the intestine and The presence of HNF in both the liver and the is intriguing

and raises the possibility

play a role in the regulation

that it may

of renal and hepatic expres-

sion of hEp. It is clear that tissue-specific

gene expression

involves

the coordination of many distinct regulatory units, not just a single &-acting element in a given cell type. The C/EBP binding site, 5’-TTTTGCAAT at nt position 1278, overlaps with the nitrogen-regulatory~oxygen-sensing consensus sequence, 5’-TTTTGCA. The physiological significance in the quantitative and distinctive usage of these regulatory elements in tissue-specific and hypoxiainduced expression of hEp will be important to investigate. The identification of an array of these tissue-specific and oxygen-sensing regulatory elements in hEp provides abundant resources for studying the control and regulation of expression of this hormone.

(d) Conclusions (I) The extended 3’ flanking sequence of hEp gene reported here contains many potential transcriptional regulatory elements, such as nitrogen-regulatory/oxygensensing consensus sequence, tissue-specific regulatory elements and lymphokine responsive elements. These elements have not been found in the Ep genomic clones thus far described (Jacobs et al., 1985; Lin et al., 1985). (2) The nt sequence of the extended 3’ flanking region from the Pstl site to BamHI site of hEpSLlfrl is a new and unreported sequence. A computer-aided homology search of the entire 1777 bp against all published nt sequences in the GenBank fails to reveal any significant homology. (3) The newly described 3’ Aanking region of the hEp gene contains many inverted repeats which enable the formation of stem-loops. A total of 14 stem-loops with loop size of 20 nt were identified. These stem-loops may participate in transcriptional regulation of tissue-specific and inducible expression of hEp. (4) The identification of distinct oxygen-sensing consensus sequence and liver-specific regulatory elements provides a molecular basis for many observed features of tissue-specific and inducible expression of hEp.

209 Jacobs,

ACKNOWLEDGEMENTS

K., Shoemaker,

Mufson,

We thank

David

Liu for assistance

of the 3’ flanking

region

Lee for excellent

technical

of hEpSLH

Kawakita,

in the sequencing and Cheng

assistance.

S.L.-H.

ization

Hsuan

acknowl-

C., Rudersdrof,

A., Seehra,

R., Neill, S.D., Kaufmann,

J.. Jones,

S.S., Hewick,

M., Shimizu, T. and Miyake, of genomic

and

cDNA

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gratitude

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