Analysis of the 5′ flanking sequences from the human protein kinase p58 (PITSLREβ1)-encoding gene

Gene, 145(1994)279%282 0 1994 Elsevier Science B.V. All rights reserved.

279

0378-1119/94/$07.00

GENE 07948

Analysis of the 5’ flanking sequences from the human protein kinase ~58 (PITSLREPl) -encoding gene (Promoter;

cell cycle; gene regulation;

transcription

start point; apoptosis)

John Eastona,b and Vincent J. Kidd” ‘Department of Tumor Cell Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA; and bDepartment ofCell Biology, University of Alabama at Birmingham, Birmingham, AL35294, USA Received by J.A. Engler: 6 December

1993; Accepted:

31 January

1994; Received at publishers:

10 March

1994

SUMtiARY

The ~58 (PITSLREbl) protein kinase (PK) is a member of a large supergene family related to the master mitotic protein kinase, ~34”~“‘. This PK is also a member of a sub-family itself, with at least six additional related PITSLRE PK isoforms expressed by alternative splicing and promoter utilization from three duplicated genes. Minimal overproduction of the PITSLREbl PK in Chinese hamster ovary cells results in a late mitotic delay, suggesting that this PK’s function may be related to the cell cycle [Bunnell et al., Proc. Natl. Acad. Sci. USA 87 (1990) 7467-74711. Further studies using structural and functional mutants have shown that PITSLRE PKs are involved in signaling apoptosis. The gene encoding the PITSLREPl PK has previously been isolated and structurally characterized [Eipers et al., Genomics 13 (1992) 613-6211. Here we characterize the minimal essential promoter for this gene. Analysis of a 1.18-kb stretch of DNA located upstream from the PITSLREpl start codon demonstrates that significant cat gene expression can be driven by a construct containing this sequence. Deletion studies of this DNA fragment have defined a minimal promoter that extends 144 bp 5’ of the previously mapped transcription start point (tsp), and 521 bp 5’ of the start codon. This region of PITSLREjl DNA does not contain canonical TATA-box sequences or G+C-rich sequences associated with many promoters, yet it has approximately 20% of the promoting activity when compared to the SV40 early promoter. This suggests that this DNA sequence is a relatively strong basal promoter of a previously uncharacterized type.

INTRODUCTION

The ~58 PK gene encodes a member of the p34cdc2related supergene family, whose minimal overexpression leads to a late mitotic delay due to an apparent failure Correspondence to: Dr. V.J. Kidd, Department St. Jude Children’s Research TN 38105, USA. Tel. (l-901)

Hospital, 522-0469;

of Tumor

Cell Biology,

332 N. Lauderdale, Memphis, Fax (l-901) 531-2381.

Abbreviations: aa, amino acid(s); AP, alkaline phosphatase; bp, base pairs(s); CAT, Cm acetyltransferase; cat, gene encoding CAT; cdc2, cell division control gene product; Cm, chloramphenicol; ds, double strand(ed); kb, kilobase or 1000 bp; MCS, multiple cloning site(s); nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; PCR, polymerase chain reaction; PK, protein kinase(s); PK, gene encoding PK; SV40, simian virus 40; tsp, transcription start point. SSDI 0378-1119(94)00159-P

of cytokinesis 1992). Further cells expressing active p58 PK,

(Bunnell et al., 1990; Meyerson et characterization of these cells, as well structurally altered and enzymatically has demonstrated that this PK is part

al., as inof

an apoptotic signaling pathway (Lahti et al., 1994). The ability to signal apoptosis was shown to be independent of cell-cycle delay or mitotic catastrophes in these studies. The ~58 PK is also a member of a smaller sub-family of PKs, with at least six additional mRNAs and proteins generated by alternative splicing from three duplicated and linked genes (Xiang et al., 1994). In keeping with a convention established by Meyerson et al. (1992), this family of PKs has been renamed PITSLREcl, p, or y l-3 based on the single aa code of the conserved PSTAIREbox region of these protein kinases and the gene

280 -1180

ggatcccagtggccgactcccaacagagttcccggctcacacacctgcttgggtgggacgctgggaacgcaaacctgcacaccagccccggcacagaccactccacgccgctgggcctcg -1060

gcctgtgggcaggccgcctgctactgcaagggagtggcaaagcccagggccaggctgacctctggcttctagaggtgctgaggggaaatccaacctccaatagctgctcaggtgaggacg -940

ggaacccaggtgcagtcgcagctctcggcagccagcccctgccccacttcccctgcctttgtggggtgaggggaccccacccacctgttttcttgtcttttgctcgtagaccactcgata -820

ggtgccctcctcgatcctgttcaggcactggaactcctcgacgctccggc~gccctaaaaaaaaaacctatatgaggtctcagtggcc~tgcc~gctggagggagggcggcgtccgc~gg -700 cacggcacaccggcacgggcaggtgcaggcgagagccttggactgggccaggggtggagccgggagcagctcagttctttcaaagtctctttccttgcaaaaccatctgac~ctttatt~ -580 tgaaacaaaaccagtgtgaacaaaaggccatcccagccaaatacacaGa~ctcaaqcctgt~attccagcactttgggagggcaaggcaggaggattgcttgagcccaggagttcaagac -460 cagcctggccaacatagcaacactgttttctttttttctttttQagatggclgtctcgctctgtcaccc~ggct~aataca~taataaa~tctcggctcactgcaacctccacctcctcctgg -340 gttcaggcgattctcctgcctcccaatcctagtagctgagtatcaggtgagtcgcagccccaacgcacgcccggcataatttttttatttttagtcgagacgggtttcaccacgttggcc -220 aggctggtctcgaactcctgacctcaggtg~tccacccgccttcggctccc~aagcactgggaattac~ggcgtg~gccaccgcgcccggc~ccatatccattcttgggacacttgttg +1 -100 tgcttagctgaacggegcccgcatgctgtggcagcactcgccccggtgctggtctgagcag~cgcctcctttctcttgcagaagaagtaagtgaggaagaaatg

Fig. 1. Nucleotide

sequence

of the 5’ untranslated

of a 1.18-kb BumHI-XhrrI

fragment

previously

rsp (indicated

untranslated

mapped

major

that extends

region and a small portion

region and upstream

regulatory

from near the start codon

by the downward of the promoter

arrowhead).

sequences

for the human

of the PITSLREBl

protein

The bold and italicized

region. A region of dyad symmetry

sequence

is indicated

PITSLRE

13gene. Shown is the sequence

to a region located identifies

647 bp upstream

an .4/u repeat

by the horizontal

arrows

The location of oligos used for deletion analysis of this promoter are underlined below the sequence. Locations of pertinent deletion analysis of this promoter, as well as pertinent sequence numbers, are shown above the sequence. Both the sequence and that previously as previously under

reported

described

accession

(from nt - 1056 to + 1) (Eipers et al., 1992) are shown.

(Bunnell

et al.. 1990: Eipers et al., 1992). The sequence

DNA sequence

will appear

analysis

in the 5’

above the sequence.

restriction determined

was performed

in the GenBank;EMBL

from the

element

sites used for in this study

on ds DNA templates

Nucleotide

Sequence

Database

No. U03874

(PITSLRE A, B, or C) from which the mRNA is transcribed (i.e., a PITSLRE A gene transcript is designated PITSLREd ). Previously, our laboratory cloned and characterized the p58 (PITSLRE B) gene, including a portion of the putative promoter (Eipers et al., 1991; 1992). This study identified the major tsp as a region 422 nt upstream from the start codon, which includes the portion of an intact Alu-type remajor peat oriented in a 3’ to 5’ direction in the 5’ untranslated region of PZTSLREjIl (Bunnell et al., 1990; Eipers et al., 1992). The identification of multiple PITSLRE-related PK isoforms generated from three highly conserved and physically linked genes suggests that regulation of PITSLRE gene expression is most likely complex. To further understand the nature of the PZTSLRE B gene promoter and to extend our knowledge of this gene family, we undertook an analysis of this gene’s upstream region to identify the minimal essential promoter. In this study we extend the known 5’ flanking sequence of the PITSLRE B gene to a BamHI site located 1.18-kb upstream from the start codon, demonstrate promoter activity from the 5’ flanking region using a cut reporter system, and localize the minimal promoter element within the 5’ flanking sequence. Furthermore, it appears that deletion of a region between -343 and - 124 5’ of the tsp of this gene results in a twofold increase in promoter activity, suggesting that this portion of the promoter may contain sequences which normally repress transcription of the gene.

EXPERIMENTAL

AND DISCUSSION

(a) Sequence analysis of the element The region a portion

of the PITSLRE

of the 5’ upstream

BamH -1180

3

-1180

1

I

B gene corresponding region

has previously

-422

to been

ATG

+’

I

-974

t-1

-765

t-1

PITSLREblpromoter

= 200nt

1-t

-546

1-1

-377

~58-5

-377

~58-24

-377

~58-14

-377

~58-12

Fig. 2. Schematic representation of the deletion constructs used in the CAT production assays for the PITSLRE B gene promoter. Shown are schematic representations of progressively deleted PITSLRE B promoter sequence (nt - 1180 to - 377, ~58-5) cloned into the promoterless cat vector. A series of staggered 5’ to 3’ oligos located at nt -974 (~58-24) - 765 (p58-l4), and - 546 (~58-12) and a fixed oligo corresponding to the 3’ to 5’ orientation at nt -377 (p58R) were used to generate the PCR fragments used in this study. The position of the oligos depicted in this figure correspond to the underlined sequences in Fig. 1 (i.e., -974 is the corresponding nt for the first oligo sequence used to generate the corresponding construct). Also shown is the position of the first rsp(bent arrow at nt -422) relative to the translational start. All constructs were sequenced in their entirety as detailed in Fig. I.

281 reported (Eipers et al., 1992). We extended this sequence upstream from the start codon to a BamHI site at nt - 1180 to include possible transcriptional control elements (Fig. 1). This region did not contain any significant ORFs, but did contain a perfect copy of an Alu repeat element oriented in a 3’ to 5’ direction in the 5’ untranslated region of the PITSLRE B gene. A region of dyad symmetry was noted near the tsp (Fig. l), but the possible importance of this sequence feature is not clear. The PlTSLREfll promoter sequence was analyzed for the presence of any known eukaryotic transcriptional control sequences, but we could not find evidence of either TATA box or SPl-binding sequences. The absence of TATA or G + C-rich sequences from this DNA region suggests that this promoter may belong to a unique class.

activity in human HeLa cells (Gorman et al., 1982). Transfection of these constructs and analysis of their CAT production indicated that this PZTSLRE I3 gene fragment functioned as a promoter in this system (Fig. 3). To define the minimal essential promoter element, similar constructs were made in the same vectors using progressively less of the BarnHI-BglII restriction fragment, with deletions originating at the 5’ end of the fragment (Fig. 2). These constructs were generated by PCR, progressively moving the 5’ primer towards the tsp, while anchoring the 3’ primer in one location (see Figs. 1 and 2). All of

(b) Promoter activity The upstream BarnHI-BglII fragment was cloned into enhancer + cat expression vectors to determine promoter of the BamHI-BglII PITSLRE B gene deletions of this fragment as compared

Fig. 3. Promoter activity fragment and progressive

with the SV40 early promoter.

(Panel

A) CAT production

from HeLa cells transfected with the indicated constructs. of the various non-acetylated and acetylated Cm also

shown.

with

the indicated

B) AP

(Panel

activity

~58 constructs

measured

after

or controls.

co-transfection

2.0

-

BumHI-BglII

fragment

(~58-5)

was

directionally

cloned

Progressive deletions of the original BamHI-BglII fragment were made by PCR with the following oligos: hp58-5, 5’-GGGGAAGCTTAAAGGGCCTGTGT;

hp58-24,5’-GGGGAAGCTTTGGAAGAA-

hp58-14,

1.5 .Y_ 1.0 cz EE jg

5’-GGGGAAGCTTGGTGCACAGGAC-

0.5

Hind111 restriction site was introduced on the 5’ end of the hp58-(5,12,14,24), and a synthetic XbaI restriction site was introduced to 40% confluency

for cloning

purposes.

HeLa

mid, using the Ca.phosphate precipitation technique (Wigler et al., 1977). The cells were then harvested after 48 h. Cell lysate was obtained by repeated concentration

freeze-thaw cycles (Gorman et al., 1982) and the protein was determined using the Bio-Rad protein assay kit. AP

activity was measured as described by others (Berger et al., 1988; Langer et al., 1992). CAT production was assayed by measuring the conversion of [14C] Cm to its acetylated forms by using 10 pg of protein and analyzing the products by thin-layer chromatography as described by others (Gorman et al., 1982). The thin-layer plate was developed with a Molecular Dynamics 400A Phosphorimager. ImageQuant software was used to allow accurate quantitation of the Cm acetylated forms. The results of these CAT assays were then normalized to the AP results to accurately reflect transfection efficiency between different constructs. This is reported as percent acetylation.

-I

~

0

with 10 pg

of ~58-5, ~58-24, ~58-14, ~58-12, pCATcontro1, or pCATenh and 10 pg of pSEAP (Langer et al., 1992): the alkaline phosphatase control plas-

-,s=:::

q

0.0

cells were grown

(lOO-mm dish) and were then transfected

:i::l::-:-::: 95

i

4

TCAGGC; hp58-12,5’-GGAGTGCAATGGTGAGATTCTAGAGGG; hp58R, 5’-GGGTCTAGAATCTCACCATTGCACTCC. A synthetic

on the 5’ end of hp58R

chloramphenicol

+

origin

+crncmk

into

a MCS immediately upstream contains the SV40 early pro-

moter cloned into the MCS. The negative control (none) is the pCATENH vector without a promoter sequence cloned into the MCS.

TCCAACCTCCAGTAG;

+

-12 +14 -24 x+5 as"40 -Oh

bar 3, ~58-14; bar 4, ~58-24; bar 5, ~58-5; bar 6, pSV40; bar 7, enhancer. the vector pCATenh which contains from the cat gene. pCATcontrol(SV40)

mono-acetylated

B

C) Relative

CAT enzyme activity after normalization for transfection efficiency based on the results shown in panel B. (Bar 1, mock; bar 2, ~58-12; The

di-acetylated

+

measured

The locations products are

(Panel

+

,

i 10

I

w ,

20

,

30

c ,

/

40

-.

/

,

60

60

Time (min)

C

CAT As**“*

70

282 these constructs

were sequenced

that

the complete

from

the original

DNA

in their entirety

sequence

sequence

isolated

clone. We found that a construct sequences

of efficiently

activity

was approximately

BarnHI-BglII

containing

cut expression,

promoter,

an

fragment

pSEAP

Center

for information

family members. grant

GM

regarding

This research

44088-04

other

was supported

to V.J.K.,

by a Cancer

Core grant from the NIH, and by assistance

the tsp) was

the

and that its

(ALSAC).

American

Lebanese

Syrian

Associated

from

Charities

driven

and the enzyme

(Fig. 3, panel

due to CAT enzyme

was then normalized

A and

et al.. 1992)

ity assayed (Berger et al., 1988). The resulting

construct

by NIH

nt

efficiency,

gene

(Langer

with our constructs

of Cm acetylation

upstream

and J. Xiang

PITSLRE

the genomic

(Fig. 3, panels

transfection

AP-encoding

from these cells is shown

J. Lahti

to insure unaltered

twofold higher than the intact

for transient

containing

co-transfected

driving

restriction

C). To control B-actin

from

from - 546 to - 377 bp (including

capable

vector

remained

a was

activ-

AP activity

B). The percent activity

REFERENCES

by a

for each

(Fig. 3, panel C) using the

results of the AP assay (panel B). This experiment demonstrates that the minimal essential promoter responsible for producing the PZTSLREPl mRNA from the PITSLRE B gene is encoded by sequences extending from -546 to -377 bp upstream from the start codon (- 124 bp from the tsp), and that the region between - 1180 and - 546 bp upstream from the start codon (-552 to - 124 bp relative to the tsp) contains DNA sequences that may repress normal promoter function. A search of this DNA sequence against known transcriptional data bases did not reveal a match with any of the known major promoter sequences. However. a region of dyad symmetry is contained within this limited sequence (Fig. 1). These data suggest that this sequence may define a new class of basal promoter element.

Berger. .I.. Hauber. placental

J., Hauber,

alkaline

tor of gene expression Bunnell,

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P.G.,

Barnoski.

B.L.. Han,

.I., Carroll,

A.J. and

Kidd,

V.J.:

Localization of the expressed human p58 protein kinase chromosomal gene to chromosome 1~36 and a highly related sequence to chromosome 15. Genomics 11 (199 I) 621-629. Eipers. P.G.. Lahti, J.M. and Kidd, V.J.: Structure the human

p%clk-I

protein

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G.,

M.. Xiang, J.. Jones,

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A.T.. and

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V.J.: Alterations

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Langer, S.J., Bortner, D.M., Roussel, M.F., Sherr, C.J. and Ostrowski. M.C.: Mitogenic signaling by colony-stimulating factor 1 and ~(1,sis suppressed

by the rts-2 DNA-binding

domain

and restored

overexpression. Mol. Cell. Biol. 12 ( 1992) 5355. 5362. Meyerson, M., Enders, G.H., Wu, C.-L.. Su, L.-K., Gorka,

Wigler, M., Silverstein,

S.. Lee, L.S.. Pellicer, A., Cheng,

R.: Transfer of purified herpes virus thymidine tured mouse cells. Cell 11 (1977) 2233232. Xiang, J.. Lahti, J.M., Grenet,

The authors appreciate the help provided by Dr. M. Roussel in establishing CAT production systems, and Dr.

Kidd.

Increased expression of a 58-kDa protein kinase leads to changes in the CHO cell cycle. Proc. Natl. Acad. Sci. USA 87 (1990)

C.. Harlow, E. and Tsai. L.-H.: A family of human protein kinases. EMBO J. I I ( 1992) 2909-2917.

ACKNOWLEDGEMENTS

indical-10.

J., Easton.

by r?tyc’

C., Nelson. cdc2-related

Y.C. and Axe].

kinase

gene to cul-

J. and Kidd, V.J.: Molecular

cloning and expression of alternatively spliced kinase isoforms. J. Biol. Chem. ( 1994) in press.

PITSLRE

protein