The SM50 gene encodes a minor matrix protein of the sea urchin embryo spicule. We carried out a detailed functional analysis of a cis-regulatory region of this gene, extending 440 bp upstream and 120 bp downstream of the transcription start site, that had been shown earlier to confer accurate skeletogenic expression of an injected expression vector. The distal portion of this fragment contains elements controlling amplitude of expression, while the region from −200 to +105 contains spatial control elements that position expression accurately in the skeletogenic lineages of the embryo. A systematic mutagenesis analysis of this region revealed four adjacent regulatory elements, viz two copies of a positively acting sequence (element D) that are positioned just upstream of the transcription start site; an indispensable spatial control element (element C) that is positioned downstream of the start site; and further downstream, a second positively acting sequence (element A). We then constructed a series of synthetic expression constructs. These contained oligonucleotides representing normal and mutated versions of elements D, C, and A, in various combinations. We also changed the promoter of the SM50 gene from a TATA-less to a canonical TATA box form, without any effect on function. Perfect spatial regulation was also produced by a final series of constructs that consisted entirely of heterologous enhancers from the CyIIIa gene, the SV40 early promoter, and synthetic D, C, and A elements. We demonstrate that element C exercises the primary spatial control function of the region we analyzed. We term this a ‘locator’ element. This differs from conventional ‘tissue-specific enhancers’ in that while it is essential for expression, it has no transcriptional activity on its own, and it requires other, separable, positive regulatory elements for activity. In the normal configuration these ancillary positive functions are mediated by elements A and D. Only positively acting control elements were observed in the SM50 regulatory domain throughout this analysis.

Reference

Anstrom
J. A.
,
Chin
J. E.
,
Leaf
D. S.
,
Parks
A. L.
,
Raff
R. A.
(
1987
)
Localization and expression of msp130, a primary mesenchyme lineage-specific cell surface protein of the sea urchin embryo.
Development
101
,
255
265
Benson
S. C.
,
Sucov
H. M.
,
Stephens
L.
,
Davidson
E. H.
,
Wilt
F.
(
1987
)
A lineage-specific gene encoding a major matrix protein of the sea urchin embryo spicule. I. Authentication of the cloned gene and its developmental expression.
Dev. Biol
120
,
499
506
Briggs
M. R.
,
Kadonaga
J. T.
,
Bell
S. P.
,
Tjian
R.
(
1986
)
Purification and biochemical characterization of the promoter-specific transcription factor Sp1.
Science
234
,
47
52
Calzone
F. J.
,
Theze
N.
,
Thiebaud
P.
,
Hill
R. L.
,
Britten
R. J.
,
Davidson
E. H.
(
1988
)
Developmental appearance of factors that bind specifically to cis-regulatory sequences of a gene expressed in the sea urchin embryo.
Genes Dev
2
,
1074
1088
Calzone
F. J.
,
Höög
C.
,
Teplow
D. B.
,
Cutting
A. E.
,
Zeller
R. W.
,
Britten
R. J.
,
Davidson
E. H.
(
1991
)
Gene regulatory factors of the sea urchin embryo. I. Purification by affinity chromatography and cloning of P3A2, a novel DNA-binding protein.
Development
112
,
335
350
Cameron
R. A.
,
Hough-Evans
B. R.
,
Britten
R. J.
,
Davidson
E. H.
(
1987
)
Lineage and fate of each blastomere of the eight-cell sea urchin embryo.
Genes Dev
1
,
75
85
Chen
J.-L.
,
Attardi
L. D.
,
Verrijzer
C. P.
,
Yokomori
K.
,
Tjian
R.
(
1994
)
Assembly of recombinant TFIID reveals differential coactivator requirements for distinct transcriptional activators.
Cell
79
,
93
105
Coffman
J. A.
,
Davidson
E. H.
(
1992
)
Expression of spatially regulated genes in the sea urchin embryo.
Curr. Opinion Genet. Dev
2
,
260
268
Davidson
E. H.
(
1989
)
Lineage-specific gene expression and the regulative capacities of the sea urchin embryo: a proposed mechanism.
Development
105
,
421
445
Davidson
E. H.
(
1990
)
How embryos work: A comparative view of diverse modes of cell fate specification.
Development
108
,
365
389
Drager
B. J.
,
Harkey
M. A.
,
Iwata
M.
,
Whiteley
A. H.
(
1989
)
The expression of embryonic primary mesenchyme genes of the sea urchin, Strongylocentrotus purpuratus, in the adult skeletogenic tissues of this and other species of echinoderms.
Dev. Biol
133
,
14
23
Ernst
S. G.
,
Hough-Evans
B. R.
,
Britten
R. J.
,
Davidson
E. H.
(
1980
)
Limited complexity of the RNA in micromeres of sixteen-cell sea urchin embryos.
Dev. Biol
79
,
119
127
Fischer
J. A.
,
Maniatis
T.
(
1988
)
Drosophila Adh: a promoter element expands the tissue specificity of an enhancer.
Cell
53
,
451
461
Flytzanis
C. N.
,
McMahon
A. P.
,
Hough-Evans
B. R.
,
Katula
K. S.
,
Britten
R. J.
,
Davidson
E. H.
(
1985
)
Persistence and integration of cloned DNA in postembryonic sea urchins.
Dev. Biol
108
,
431
442
Flytzanis
C. N.
,
Britten
R. J.
,
Davidson
E. H.
(
1987
)
Ontogenic activation of a fusion gene introduced into sea urchin eggs.
Proc. Natl. Acad. Sci. USA
84
,
151
155
Franks
R. R.
,
Hough-Evans
B. R.
,
Britten
R. J.
,
Davidson
E. H.
(
1988
)
Direct introduction of cloned DNA into the sea urchin zygote nucleus, and fate of injected DNA.
Development
102
,
287
299
Franks
R. R.
,
Anderson
R.
,
Moore
J. G.
,
Hough-Evans
B. R.
,
Britten
R. J.
,
Davidson
E. H.
(
1990
)
Competitive titration in living sea urchin embryos of regulatory factors required for expression of the CyIIIa actin gene.
Development
110
,
31
40
Gan
L.
,
Wessel
G. M.
,
Klein
W. H.
(
1990
)
Regulatory elements from the related Spec genes of Strongylocentrotus purpuratus yield different spatial patterns with a lacZ reporter gene.
Dev. Biol
142
,
346
359
George
N. C.
,
Killian
C. E.
,
Wilt
F. H.
(
1991
).
Characterization and expression of a gene encoding a 30.6 kDa Strongylocentrotus purpuratus spicule matrix protein.
Dev. Biol
147
,
334
342
Gorman
C.
,
Moffat
L. F.
,
Howard
B. H.
(
1982
)
Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells.
Mol. Cell. Biol
2
,
1044
1051
Hapgood
H.
,
Patterton
D.
(
1994
)
Purification of an oligo(dG)•oligo(dC)-binding sea urchin nuclear protein, suGF1: a family of G-string factors involved in gene regulation during development.
Mol. Cell Biol
14
,
1402
1409
Harkey
M. A.
,
Whiteley
H. R.
,
Whiteley
A. H.
(
1992
)
Differential expression of the msp130 gene among skeletal lineage cells in the sea urchin embryo: A three dimensional in situ hybridization analysis.
Mech. Dev
37
,
173
184
Henry
J. J.
,
Amemiya
S.
,
Wray
G. A.
,
Raff
R. A.
(
1989
)
Early inductive interactions are involved in restricting cell fates of mesomeres in sea urchin embryos.
Dev. Biol
136
,
140
153
Hörstadius
S.
(
1939
)
The mechanics of sea urchin development, studied by operative methods.
Biol. Rev. Cambr. Phil. Soc
14
,
132
179
Hough-Evans
B. R.
,
Britten
R. J.
,
Davidson
E. H.
(
1988
)
Mosaic incorporation of an exogenous fusion gene expressed exclusively in aboral ectoderm cells of the sea urchin embryo.
Dev. Biol
129
,
198
208
Hough-Evans
B. R.
,
Franks
R. R.
,
Zeller
R. W.
,
Britten
R. J.
,
Davidson
E. H.
(
1990
)
Negative spatial regulation of the lineage specific CyIIIa actin gene in the sea urchin embryo.
Development
108
,
41
50
Kabakoff
B.
,
Hwang
S.-P.L.
,
Lennarz
W. J.
(
1992
)
Characterization of post-translational modifications common to three primary mesenchyme cell-specific glycoproteins involved in sea urchin embryonic skeleton formation.
Dev. Biol
150
,
294
305
Katoh-Fukui
Y.
,
Noce
T.
,
Ueda
T.
,
Fujiwara
Y.
,
Hashimoto
N.
,
Higashinakagawa
T.
,
Killian
C. E.
,
Livingston
B. T.
,
Wilt
F. H.
,
Benson
S. C.
,
Sucov
H. M.
,
Davidson
E. H.
(
1991
)
The corrected structure of the SM50 spicule matrix protein of Strongylocentrotus purpuratus.
Dev. Biol
145
,
201
202
Kaufmann
J.
,
Smale
S. T.
(
1994
)
Direct recognition of initiator elementsby a component of the transcription factor IID complex.
Genes Dev
8
,
821
829
Khaner
O.
,
Wilt
F.
(
1990
)
The influence of cell interactions and tissue mass on differentiation of sea urchin mesomeres.
Development
109
,
625
634
Khaner
O.
,
Wilt
F.
(
1991
)
Interactions of different vegetal cells with mesomeres during early stages of sea urchin development.
Development
112
,
881
890
Khoury
G.
,
Gruss
P.
(
1983
)
Enhancer elements.
Cell
33
,
313
314
Killian
C. E.
,
Wilt
F. H.
(
1989
)
The accumulation and translation of a spicule matrix protein mRNA during sea urchin embryo development.
Dev. Biol
133
,
148
156
Leaf
D. S.
,
Anstrom
J. A.
,
Chin
J. E.
,
Harkey
M. A.
,
Showman
R. M.
,
Raff
R. A.
(
1987
)
Antibodies to a fusion protein identify a cDNA clone encoding msp130, a primary mesenchyme-specific cell surface protein of the sea urchin embryo.
Dev. Biol
121
,
29
40
Leahy
P. S.
(
1986
)
Laboratory culture of Strongylocentrotus purpuratus adults, embryos, and larvae.
Meth. Cell Biol
27
,
1
13
Livant
D.
,
Cutting
A.
,
Britten
R. J.
,
Davidson
E. H.
(
1988
)
An in vivo titration of regulatory factors required for expression of a fusion gene in transgenic sea urchin embryos.
Proc. Natl. Acad. Sci. USA
85
,
7607
7611
Livant
D. L.
,
Hough-Evans
B. R.
,
Moore
J. G.
,
Britten
R. J.
,
Davidson
E. H.
(
1991
)
Differential stability of expression of similarly specified endogenous and exogenous genes in the sea urchin embryo.
Development
113
,
385
398
Livingston
B. T.
,
Wilt
F. H.
(
1989
)
Lithium evokes expression of vegetal-specific molecules in the animal blastomeres of sea urchin embryos.
Proc. Natl. Acad. Sci. USA
86
,
3669
3673
McMahon
A. P.
,
Flytzanis
C. N.
,
Hough-Evans
B. R.
,
Katula
K. S.
,
Britten
R. J.
,
Davidson
E. H.
(
1985
)
Introduction of cloned DNA into sea urchin egg cytoplasm: Replication and persistence during embryogenesis.
Dev. Biol
198
,
420
430
Okazaki
K.
(
1975
)
Spicule formation by isolated micromeres of the sea urchin embryo.
Am. Zool
15
,
567
581
Ransick
A.
,
Ernst
S.
,
Britten
R. J.
,
Davidson
E. H.
(
1993
)
Whole mount in situ hybridization shows Endo16 to be a marker for the vegetal plate territory in sea urchin embryos.
Mech. Dev
42
,
117
124
Richardson
W.
,
Kitajima
T.
,
Wilt
F.
,
Benson
S.
(
1989
)
Expression of an embryonic spicule matrix gene in calcified tissues of adult sea urchins.
Dev. Biol
132
,
266
269
Ruffins
S. W.
,
Ettensohn
C. A.
(
1993
)
A clonal analysis of secondary mesenchyme cell fates in the sea urchin embryo.
Dev. Biol
160
,
285
288
Seed
B.
,
Sheen
J.-Y.
(
1988
)
A simple phase-extraction assay for chloramphenicol acetyltransferase activity.
Gene
67
,
271
277
Sucov
H. M.
,
Benson
S.
,
Robinson
J. J.
,
Britten
R. J.
,
Wilt
F.
,
Davidson
E. H.
(
1987
)
A lineage-specific gene encoding a major matrix protein of the sea urchin embryo spicule.
Dev. Biol
120
,
507
519
Sucov
H. M.
,
Hough-Evans
B. R.
,
Franks
R. R.
,
Britten
R. J.
,
Davidson
E. H.
(
1988
)
A regulatory domain that directs lineage-specific expression of a skeletal matrix protein gene in the sea urchin embryo.
Genes Dev
2
,
1238
1250
Theze
N.
,
Calzone
F. J.
,
Thiebaud
P.
,
Hill
R. L.
,
Britten
R. J.
,
Davidson
E. H.
(
1990
)
Sequences of the CyIIIa actin gene regulatory domain bound specifically by sea urchin embryo nuclear proteins.
Mol. Reprod. Dev
25
,
110
122
Wang
D. G.-W.
,
Kirchhamer
C. V.
,
Britten
R. J.
,
Davidson
E. H.
(
1995
)
SpZ12-1, a negative regulator required for spatial control of the territory-specific CyIIIa gene in the sea urchin embryo.
Development
121
,
1111
1122
Xiang
M.
,
Lu
S.-Y.
,
Musso
M.
,
Karsenty
G.
,
Klein
W. H.
(
1991
)
A G-string positive cis -regulatory element in the LpS1 promoter binds two distinct nuclear factors distributed non-uniformly in Lytechinus pictus embryos.
Development
113
,
1345
1355
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