- Email: [email protected]
Sox genes find their feet
338
Sax genes find their feet Larysa H Pevny* and Robin Lovell-Badge’ The identification
of the mammalian
SRY, led to the description transcription display
factors,
properties
architectural diverse
patterns
mutations factors
the SOX
gene family. SOX
of both classical
components
of SOX
genes
factor, encoding
proteins factors
The dynamic
mice and Drosophila
play key roles in decisions
developmental
transcription
of chromatin.
of expression
in humans,
testis-determining
of a new class of genes
and
and
and analysis
suggest
of cell fate during
of
diverse
Addresses Laboratory of Developmental Genetics, MRC National lnstltute for Medical Research, The RIdgeway, Mill HIII, London NW7 1AA, UK [email protected] [email protected]
Current
Opinion in Genetics
0 Current
Biology
& Development
1997, 7:338-344
Ltd ISSN 0959-437X
Abbreviations CD CNS Fgf-4 HMG sox TCF
campomelic
the strucwre,
expression
and fun&on
that SOX
processes.
‘e-mall: ‘e-mail:
made to characterize
of the members of the Sex gene family These primary analyes suggest that-as with the founding member of the family S’RY-S’ox genes are involved in governing cell face decisions in a number of diverse de\~elopmcncal processes.
dysplasia
central nervous system fibroblast growth factor-4 high mobility group SRY box T-cell factor
Introduction Throughout developmenr. transcriptional regulation of eukaryoric gene expression requires both the recruitment of tissue-specific transcription factors to a promoter and the proper establishment of local chromarin structure. In this review, we describe the SOX family, a group of proteins which appear to goTern cell fate decisions during embryogenesis by functioning both as classical transcription factors and architectural components of chromatin. The SOX factors comprise a novel group of proteins characterized by the presence of an SRY box (hence ‘SOX’), a 79 amino acid motif that encodes an HhlG-type DNA-binding domain. The genes encoding these factors, highly conserved across evolurion, were originally identified through homology as they contain an HhlG box closely related to that of the mammalian testis-determining gene SRY [1,2]. The SOX family falls into a subclass of HhlG box proteins, the members of which show highly restricted tissue distribution and bind to specific sequences at high affinities (reviewed in [3]). hlost strikingly, on binding, SOX proreins cause DNA to bend at an acute angle [4,5]. Compelling evidence for the developmental importance for individual members of the Sax gene family comes from mutational analyses in human [&lo], mouse [ll”] and Drosophih [12”,13”]. In this review. we concentrate on recent advances thar have been
High mobility group boxes and the SOX gene family To date, HRIG box proteins have been classified into two major subgroups according to sequence specificity of the DNA-binding and rhe number of HhlG DNA-binding domains within a single protein [14]. The first subgroup consists of HhlG-box proteins which concain more than one DNA-binding domain, are usually expressed ubiquitously and bind preferentially to bent DNA. hlembers of this group include the HhIG non-hisrone chromatin-associated proteins HhlGl and HMG2 [15.16]. In contrast. members of the second subgroup of proteins contain only a single HhlG box, show highly restricted expression patterns, and may bind prestructured DNA with either little or no sequence specificity hlembers of this class. however, can also bind to linear DNA at high affinities in a sequence-specific manner. Examples of this group include the T-cell factor (TCF) [17-191, \lATA [ZO-221 and the SOX ([1.2,6]: Table 1) families. HhlG-box proteins or genes can be further categorized into families on the basis of groups which share a high degree of homology within the HhlG box. The SOX family of proteins is defined by an HhlG box which has at least 50% sequence identity with the founding member of this group, mouse SRY. At present. this group includes at least 20 members and can be further divided into subfamilies on rhe basis of degree of homology both within and outside the HhlG box ([2,23-251; Table 1). Categorizing rhe Sax genes only on known sequence many of tvhich are incomplete, however, is at present ambiguous and may need recent cloning
co be refined and functional
on the basis studies.
of results
from
Individual SOX family members also show a high degree of conservation across species both lvithin and outside their HhlG domain. It is less easy, however, to idenrif! orthologues of a specific SOS gene in invertebrates bur in some cases the similarities are intriguingly high. An esample is human SOX2 [26] which shares 98% overall similarity with the mouse protein [27’]. 95% with the chicken homologue cSOX2 [28] and 88% with the putative Dr-osophih homologue Dir/eate (also called .5’(J?I7~~~1or /is/Moofi [ 12”,13**]). SRY is a major exception because very little sequence homology can be detected outside the HhlG domain even between closcl~ related hpecieh [29-30,31’].
Sox genes find
their
feet
Pevny and Love&Badge
339
Table 1 The SOX family. Orthologue
Gene Group
References
Expression
Mapping
A
SRY Group
Human,
rodent,
marsupial
Human
Y
Genital
b31
ridge and testis
B
sox7
Human,
mouse,
chicken
Human
8
sox2
Human,
mouse,
chicken
Human
3
Soxl: Sox2:
embryonic
CNS,
ICM, primitive
lens
ectoderm,
[2,27’,60,61] [26,27’,28,42”,55’,61
I
CNS, PNS, embryonic gut, endoderm; cSox2: embryonic
sox3 Soxl4 ZfSoxl9 sox15 Dichaete (Sox7OD, fish-hook) Group
mouse,
Human
chicken
X
Mouse Zebrafish
Sox3
CNS and PNS and cSox3: embryonic
CNS
[2,7,27’,28] [26]
Embryonic
CNS
[62,63]
Mouse
Drosophila
70Dl-2
1641 [12”, 13**1
Entire trunk of syncitial blastoderm;
seven
irregular
stripes
in cellular blastoderm; ventral cephalic neuroectoderm
and
C
sox4
Human,
SOXll
sox72 sox20 Group
Human,
Human,
mouse
mouse,
chicken
Human, mouse Human
Human
6
Embryonic heart and spinal cord, adult pre-B and pre-T cells
Human
2
cSox7 7: embryonic CNS, post-mitotic neurons
[2 311 * 111 35 41 57.91 l
[25,28,65] 1261
Human
(671
17
D
sox5 SOX6
Human, Human,
Human
mouse mouse
6
Sox5: Sox6:
adult testis
embryonic
CNS,
[23,32,36]
[23,681
adult testis Group E SOX8 sox9
SOXlO Group F sox-7 sox- 17 sox- 7 8
WV251
Human, mouse Human, mouse, chicken Human,
Human 17, Mouse 2 -
mouse
genital ridge and CNS, notochord
[23 25 33*‘] [9,48:‘,49,50”] [23,25]
1231
Mouse Mouse Mouse
ICM, inner cell mass; PNS, peripheral
Chondrocyte, adult testis,
Testis Mouse
nervous
2
Heart, lung, spleen, skeletal muscle, liver and brain of adult
[34’,69] [40,69,70]
system.
In addition to sequence similarities, all Sox genes studied to date with the exception of Sox5 [32], Sor9 [8,33*-l and Soxl7 [34*] display a monoexonic structure, at least with respect to their open reading frame. Gene-mapping studies show the Sex genes to be distributed randomly throughout the genome with no evidence of gene clustering (see Table 1). This genomic organization indicates a possible early divergence of subfamilies of HMG proteins during evolution. The ancestral sequence-specific HMG box could have contained an intron, which still occurs in the Ef genes and in some Sox genes but this intron may have been lost in other SOS genes. Apart from some expressed pseudogenes, PolII transcribed intron-less genes are fairly rare in the eukaryotic genome. Therefore, it is conceivable that the intron-less genes arose by reverse transcription and retroposition [35].
The biochemical properties of SOX proteins SOX proteins share several characteristics with classical transcription factors. Results from several in vitro studies involving SOX proteins demonstrated that the HMG domain of SOX proteins binds to a specific DNA
sequence motif (A/T A/T CAA A/T G) with high-affinity [5,27*,36+0] and several SOX proteins including mouse SOX4, SOX17, SOX18 [34*,40,41], chicken cSOX2 [42**], and human SOX9 [43*] have been shown to activate the transcription of reporter constructs. In several instances-such as SOX4, SOX9 and SOX18 - the putative transcriptional activation domains have been mapped but there appears to be no consensus sequence within this domain [40,41,43*]. In addition to acting as classical transcription factors, SOX proteins also have a number of properties which suggest that they function by organizing local chromatin structure. Unlike most transcription factors, binding of SOX proteins occurs in the minor groove and results in the induction of a dramatic bend within the DNA [4,5,38,44**,45”]. This was first demonstrated for the HMG box protein LEFl which induces a dramatic bend of 130”. The ability to alter DNA structure is likely to be a common property of all the sequence-specific class of HMG box containing proteins as two members of the SOX family, SRY and SOX.5, have also been shown to induce strong DNA bending (85” for SRY,
340
Genetics of disease
73-90” for SOXS [5,39,46]). The architectural role for SOX proteins is further supported by studies on SRY mutations in human XY females (see below). \Vhy bend at specific facilitate adjacent
DNA? By altering local chromatin structure binding sites. SOX proteins may act to
the interaction between other factors sites or allow the interaction of distant
nucleoprotein complexes machinery. For example, HMG box protein, LEFl,
bound at enhancer
with the basal transcription the bending induced by the may allow the interaction of
DNA-binding factors located on opposite sides of the LEFl-binding site [S]. Alternatively, the binding of SOX proteins and the local changes in chromatin structure may lead to the recruitment of higher-order architectural factors, such as the polycomb or the trithorax group of proteins which, in turn, could serve to act over long distances. Conversely, the severe distortion brought about by bending the DNA helix could act in a negative fashion by simply preventing the binding of factors to adjacent sites in the major groove.
Expression of Sox genes Sox genes show diverse and dynamic
patterns of expression throughout embryogenesis and in a variety of adult tissue types. As shown in Table 1, there is a member of the Sax gene family expressed in almost every tissue of the developing embryo. Can the comparison of the expression patterns of individual Sox genes or subfamilies be used to make any predictions of SO,Kgene function?
the expression
patterns
implicate
the
genes
as playing
a
role in establishing cell fates but the specific cell type will depend on the tissue in question, implying that there must be alternative cooperating factors and different sets of target
genes
Functional
in the various
tissue
lineages.
analysis of SOX genes
The first demonstration of a functional role genes in a genetic disease and a developmental
for SOX process
came from studies on human sex reversal. Genetic and molecular analysis of XY sex reversal (61, arising from mutations in SRI: and studies involving autosomal sex reversal in campomelic dysplasia (CD) patients, resulting from mutations in SOXY [%-lo] suggest that SOX genes may act to initiate or bias early cell fate decisions. This hypothesis is supported by the finding that misexpression of S’ry in the mouse diverts cells destined to become ovaries to differentiate as testes [6,51]. Expression of J’I;)~ alone, hovvever, may be insufficient for cell fate conversion as, in a number of naturally occurring and experimental conditions, the sex reversal shows varying degrees of penetrance (reviewed in [SZ]). This data is consistent with the variable sex reversal phenotypes displayed by campomelic dysplasia patients carrying identical SOXY mutations and the variable phenotype displayed in null alleles of the Drosophila Sox gene Dichnete. Collectively. these studies suggest that SOX genes may act in a dose-dependent manner. An idea that is supported by the studies showing that the dominant phenotype campomelic dysplasia patients must be caused largely by haploinsufficiency rather than a dominant negative
S~J is expressed in the undifferentiated male gonad and is quickly downregulated once the decision is made to
effect
initiate male development [6,31’,33”,46]. Similarly, the expression patterns of many of the other Sox family
Detailed molecular analyses carried out in these studies provide in vivo evidence that SOX proteins can function as both classical transcription factors as well as architectural proteins. For example, studies on mutations found within SRY in human XY females demonstrate that binding affinity and bending function as assayed in vitro are distinct. hloreover, one case of sex reversal associated with an SRY mutation appears to derive from the reduced DNA bending activity of the mutant protein [44”,53]. Finlike
members throughout development also appear to correlate with early cell fate decisions. For example, during the early phases of neural induction in both mouse and chick, the neuroepithelium shows a dramatic upregulation of 3’0x1 and Sod expression and a high level of SoxL expression [27’,28,47*]; but then, coincident with the differentiation of neural precursors, expression of all three genes is rapidly downregulated. The three genes therefore seem to respond both to signals involved in neural induction as well as signals which trigger the loss of potential and, thus, their expression largely defines the uncommitted neuroblast population. The expression of a particular Sox gene is not necessarily restricted to a particular cell type or lineage. For example, Sox9 other than being expressed in the differentiating male Sertoli cells is also expressed in mesenchymal condensations of the limb and somite prior to cartilage formation, in the CNS, and in a number of other organ systems [9,33”,48”,49,50”]. Similarly, Sox4 is expressed in the immune system, in pre-B and pre-T cells prior to terminal differentiation, but also in the developing heart, CNS, as well as numerous other tissues [41]. In each case,
[S].
SRY, vvhere nearly all missense mutations in sex-reversed XY females occurs in the HRIG box, mutations in J’OX9 resulting in campomelic dysplasia are distributed throughout the coding region. Some SOX9 mutations lead to the truncation of a transactvation domain, suggesting that the phenotypes in these cases result, at least in part, from loss of transcriptional transactivation of downstream genes
[43’].
Specificity/redundancy
of Sox gene function
The in vivo analysis of Sox genes together with in vitro assays provide evidence for potential redundancy within the So,v gene family, This is supported by studies which demonstrate that all SOX proteins, including SRY’. can bind to the same DNA sequence (4,461. Therefore. the specificity may be caused in part by the temporal
Sox genes find their feet Pevny and Lovell-Badge
and gene
spatial family;
expression this
of
individual
may explain
the
members
phenotype
mutant embryos [ 1 l”]. Animals homozygous disruption of So.~l display severe cardiac
of
the
of So_&-
for a targeted malformations
of Fgf4
in teratocarcinoma
consistent with peri-implantation the
crystallin
proposal
is
the expression of all three genes mouse embryos. Similar to studies
in on
promoter,
cells
both
[55*]. This
341
SOX2
and OCT3/4
appear
and lack B lymphocytes. These tissues appear to only express SO.Y-4 and no other known SO,Y family member.
to bind necessary
Sox-4, however, is also expressed in other regions of the embryo-such as the developing CNS-which are unaffected in null mutants [41]. hlany other Sox family members, most notably Soxl-3, are co-expressed with
co-workers [56] have shown that the HMG box of HMGl interacts with the homeodomain of specific HOX proteins. It is therefore conceivable that SOXZ and OCT3/4 also interact through their DNA-binding domains.
Sox-4 in the CNS [27*]. Similarly, mental retardation is associated with deletions of the region of the human X chromosome which encompasses SOX.? but the phenotype appears to be mild compared with the extent of its expression throughout the developing CNS [7.27-l, and it seems highly likely that SOXl and SOXZ could largely compensate for the lack of SOX3 as they are very similar proteins and show considerable overlaps in tissue distribution.
Target genes and protein-protein
interactions
There is good evidence for several SOX protein-target gene interactions from detailed itz vitro studies. For example, SOXZ and (probably) SOXl most likely participate in the regulation of 6- and y-crystallin which are expressed in the lenses of chick and mouse, respectively [42”]. These studies -which focused on the characterization of an enhancer required for tissue-specific expression of the &crystallin gene-led to the identification of binding sites for three factors within the enhancer core: a repressor, 6EF1, and two activators, 6EF2 and 6EF3. 6EF2 appears to be a complex of several proteins with similar DNA-binding properties, one of which upon the cloning of its encoding gene was shown to be SOXZ. In transient transfection assays, it was demonstrated that activation of the enhancer was dependent on the presence of both cSOX2 and 6EF3; neither factor alone was sufficient for activity. Critically, increasing amounts of cSOX2 led to a level of activation which rapidly plateaued, presumably because of limiting amounts of 6EF3. The plateau may be caused by the binding of cSOX2 to the minor groove which, in turn, would prevent the binding of the repressor 6EFl. Perhaps through direct protein-protein interactions, cSOX2 and 6EF3 then cooperate to give activation of the crystallin gene. SOXl can substitute for SOX2 in these assays consistent with proposals for possible redundancy amongst SOX subfamily members. The data is also consistent with the expression of the Soxl-3, subfamily: Soxl’ and Sox3 are expressed at high levels in the lens placode during early development whereas Soxl is expressed after lens invagination coincident with upregulation of &crystallin or y-crystallin [42”,54]. It will be interesting to determine if members of other subfamilies can work in this system and, if not, which parts of the protein are critical. SOX2 is also likely domain transcription
to participate, factor OCT3/4,
along with the POU in the transactivation
to adjacent sites in the DNA and may be for target gene expression. Recently, Bianchi and
In a variety of other studies, different SOX proteins have been implicated in transcriptional control in a number of discrete tissue types. For example, human SOX4 was shown to trans-activate the CD2 enhancer in T cells [57”] and SOX9 has been strongly implicated in the regulation of type II collagen gene expression [58,59]. In Drosophila, Dictiaete is probably involved in the regulation of pair-rule gene expression, perhaps cooperating with the products of gap and primary pair-rule genes, although there is no direct evidence for this at present [12”,13”]. It is too early to know if these in vitro results reflect in ZWO situations, however, general trends are emerging. In all the stated cases, the SOX protein interacts with enhancers located distal to the basal promoters. The significance of this, however, is not clear. It is conceivable that the bending of DNA induced by SOX binding may facilitate the interaction of the enhancer elements with the core promoter region. Alternatively, the SOX proteins may act locally to organize chromatin structure within the enhancer region. It is also likely that specificity of action of a SOX protein is context-dependent, relying on both the direct interaction of the SOX protein with its DNA-binding site as well as protein contacts with other transcription factors. As suggested by studies with SOX2, the interacting partners need not be the same in different cell types (e.g. Ort3/4 is not expressed in lens tissue). If there is a reliance on specific interacting factors, then it is likely that ectopic expression of a Sor gene may not necessarily have widespread consequences which may explain the relatively mild phenotype of the dominant Dicfiaete mutations, wing are affected.
in which
only
specific
regions
of the
Conclusions It is still too early to get a clear picture of how the SOX family functions. There is increasing evidence, however, that SOX proteins are most likely involved in many aspects of transcriptional regulation. The action of SOX proteins appears to be very context dependent, perhaps relying on protein-protein interaction as much as the interaction with DNA. It is therefore critical to determine the types of proteins SOX factors interact with and establish if there are any common rules. hlembers of the LEF/TCF family have recently been shown to interact with B catenin, placing them within many important pathways involving Wnt signaling. Similarly, SOX proteins could also be involved in
342
Genetics of disease
the immediate as implicated
response by aspects
to signal transduction pathways. of their expression patterns.
SOX proteins
have several
properties
which
allow them
12. ..
to
bring a high degree of specificity to the regulation of gene expression. It is therefore easy to imagine them occupying pivotal
roles.
For
this
reason,
it will
not
be a surprise
Russell SRH, Sanchez-Soriano N, Wright CR, Ashburner M: The Dichaefe gene of Drosophila melanogasfer encodes a SOX-domain protein required for embryonic segmentation. Development 1996, 122:3669-3676. The authors describe the cloning and charactenzation of the first Drosophila Sox gene - Sox7OD. They provide evidence that Sox7OD corresponds to the dominant wing mutation Dichaete. (simultaneously cloned by Nambu and Nambu [13”]). The authors argue that the variable phenotypes observed in Dichaefe mutants supports the role of Sax genes as architectural proteins.
involved in cell fate decisions, as exemplified by the founding father, 5~. The SOX genes are beginning to find their feet in the world of developmentally important
Nambu PA, Nambu JR: The Drosophila fishhook gene encodes a HMG domain protein essential for segmentation and CNS development. Development 1996, 122:3467-3475. The authors describe the isolation and analysis of the first Drosophila Sox gene (fish-hook; simultaneously cloned by Russell et al. [l 2”]). Mutational analyses indicate an essential role for hsh-hook in anterior/posterior pattern formation and nervous system development and suggest a potential function in modulating the activities of gap and pair rule proteins.
genes.
14.
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1 7.
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if results of mutation analyses-which are underway in many laboratories-confirm that Sax genes are indeed
References
and recommended
reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
. .. 1.
2.
of special interest of outstanding interest Sinclair AH, Berta P, Palmer MS, Hawkins JR, Griffiths BL, Smith MJ, Foster JW, Frischauf AM, Lovell-Badge R, Goodfellow PN: A gene from the human sex determining region encodes a protein with homology to a conserved DNA binding motif. Nature 1990, 346:240-244. Gubbay J, Collignon J, Koopman P, Cape1 B, Economou A, Munsterberg A, Vivian N, Goodfellow P, Lovell-Badge R: A gene mapping to the sex determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes. fv’afure 1990, 346:245-250.
3.
Ner SS: HMGs everywhere.
Curr Viol 1992, 2:206-210.
4.
Ferrari S, Harley VR, Pontiggia A, Goodfellow PN, Lovell-Badge R, Bianchi M: SRY, like HMGl. recognizes sharp angles in DNA. EM60 I 1992, 11:4497-4506.
5.
Giese K, Cox J, Grosschedl R: The HMG domain of lymphoid enhancer factor 1 bends DNA and facilitates assembly of functional nucleoprotein structures. Cell 1992, 69:165-l 95.
6.
Goodfellow PN, Love&Badge R: SRY and sex determination mammals. Annu Rev Genet 1993, 27:71-92.
7.
Stevanovic M, Lovell-Badge R, Collignon J, Goodfellow P: SOX3 is an X-linked gene related to SRY. Hum MO/ Gener 1993, 2:2013-2016.
6.
Foster JW, Dominguez-Steglick MA, Guioli S, Kwok G, Weller PA, Stevanovic M, Weissenbach J, Mansour S, Young ID, Goodfellow PN et a/.: Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY related gene. Nature 1994, 372:525-530.
9.
10.
in
Wagner T, Wirth J, Meyer J, Zabel B, Held M, Zimmer J, Pasantes J, Bricarelli FD, Keutel J, Hustert E, Wolf V et al: Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Cell 1994, 79:lll l-l 120. Kwok C, Weller PA, Guioli S, Foster JW, Mansour S, Zuffardl 0, Punnett HH, Dominguez-Steglich MA, Brook JD, Young ID et al.: Mutations in SOXS, the gene responsible for campomelic dysplasia and autosomal sex reversal. Am J Hum Genet 1995, 57:1026-l 036.
Schillam MW, Oostenvegel MA, Moerer P, Ya J, De Boer PAJ, Van de Wetering M, Verbeek S, Lamers WH. Kruisbeek AM, Cumano A, Clevers H: Defects in cardiac outilow tract formation and pro-B-lymphocyte expansion in mice lacking Sox-4. Nature 1996, 380:71 l-71 4. This paper reports the first functional analysts of a Sow gene in mice. It describes the targeted disruption of Sox4 and its effect on both development of endocardial ridges and B cell differentiation. The observed range of septation defects caused by the lack of Sox4 in mice is compared to ‘common arterial trunk’ condition in man. In addition, the authors demonstrate that B-cell development is blocked at the pro-B cell stage in Sox4 mutant mice.
11. ..
13. ..
region.
Collignon J, Sockanathan S, Hacker A, Cohen-Tannoudji M, Norris D, Rastan S, Stevanovic M, Goodfellow PN, LovellBadge R: A comparison of the properties of Sox-3 with Sty and two related genes, Sox-f and Sox-2. Development 1996, 122:509-520. The authors describe the cloning and sequencing of Soxl, Sow.2, and Sox3 from the mouse and show that Sow3 is most closely related to Sry. The discussion focuses on the evolutionary link between the genes and supports the model that Sry has evolved from Sox3. 2 7. .
26.
Uwanogho D, Rex M, Cartwright EJ, Pearl G, Healy C, Scatting PJ, Sharpe PT: Embryonic expression of the chicken Soxf, Sox3 and Soxf 7 genes suggests an interactive role in neuronal development Mech Dev 1995,49:23-36.
29.
Whitfield LS. Lovell-Badge R, Goodfellow PN: Rapid sequence evolution of the mammalian sex determining gene SRV. Nature 1993, 364:713-715.
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Tucker PK, Lundrigan BL: Rapid evolution of the sex determining locus in Old World mice and rats. Nature 1993, 364:715-717.
Hacker A, Cape1 B, Goodfellow P, Lovell-Badge R: Expression of Sry, the mouse sex determining gene. Development 1995, 121:1603-1614. Using RNAse protection assays, the authors characterize the genital ridge specific transcript of the mouse Sry gene. The authors demonstrate that the Sry genital ridge transcript is derived from a single exon and is a linear mRNA. This is in contrast to the Sry transcript expressed in adult mouse testis which exists as a circular RNA. The linear transcript is shown to start in a unique region of the Sry locus whereas the circular transcript starts with the 5’ repeat arm suggesting that the two transcripts are generated by the use of different promoters. This study also defines the critical period during gonadal development when Sry acts to initiate testes differentiation (10.5-l 1.5 dpc). 31. .
32.
Wunderle VM, Critcher R, Ashworth A, Goodfellow PN: Cloning and characterization of SOX5, a new member of the human SOX gene family. Genomics 1996, 36:354-358.
33. ..
Wright E, Hargrave MR, Christiansen J, Cooper L, Kun J, Evans T, Gangadharan U, Greenfield A, Koopman P: The Sly-related gene Sox9 is expressed during chondrogenesis in mouse embryos. Nat Genet 1995, 9:15-20. The authors of this work report the isolation and characterization of the mouse orthologue Sox9 of the human SOX9 gene, mutations of which are associated with skeletal dysmorphology (CD) and sex reversal. By wholemount in situ hybridization, the authors demonstrate that Sow9 is expressed in mesenchymal cells during mouse embryogenesis in a pattern suggestive of a role in skeletal formation. The authors go on to map Sox9 to mouse chromosome 11 in the region of the Tail short mutation, which has a strikingly similar phenotype to human CD syndrome. 34. .
Kanai Y, Kanai-Azuma M, Note T, Saido TC, Shiroishi T, Hayashi Y, Yazaki K: Identification of two Soxl7 messenger RNA isoforms, with and without the high mobility group box region, and their differential expression in mouse spermatogenesis. 1 Cell Biol 1996, 133:1-l 5. The authors report the isolation of two different mRNA isoforms, with and without an HMG box, of the mouse Sox77 gene. One form (Sox77) is expressed in spermatogonia, binds DNA and can activate transcription of a reporter construct. The second form (f-Soxl7) is accumulated in round spermatids and shows no apparent DNA-brnding actrvity. The authors propose the Sox77 may function as a transcriptional activator in premerotic germ cells and that a splicing switch Into t-Sox77 may lead to the loss of its function in the postmeiotic germ cells. 35.
Schillam MW, Van Eyk M, Van de Wetering M, Clevers HC: The murine Sox-4 protein is encoded on a single exon. Nucleic Acids Res 1993, 21:2009.
36.
Denny P, Swift S, Connor F, Ashworth A: An SRY related gene expressed during spermatogenesis in the mouse encodes a sequence specific DNA binding protein. EM80 J 1992, 11:3705-3712.
37.
Harley VR, Lovell-Badge R, Goodfellow PN: Definition of a consensus DNA binding site for SRY. Nucleic Acids Res 1994, 22:1500-l 501.
38.
Van de Wetering M, Clevers H: Sequence-specific interaction of the HMG box proteins TCF-1 and SRY occurs within the minor groove of a Watson-Crick double helix. EMBO J 1992, 11:3039-3044.
39.
Connor F, Cary PD, Read CM, Preston NS, Driscoll PC, Denny P, Crane-Robinson C, Ashworth A: DNA binding and bending properties of the post-meiotically expressed Sry related protein Sox-5. Nucleic Acids Res 1994, 22:3339-3346.
40.
Hosking BM, Muscat GEO, Koopman P, Dowhan DH, Dunn TL: Trans.-activation and DNA-binding properties of the transcription factor, Sox-18. Nucleic Acids Res 1995, 23:2626-2628.
41.
Van de Wetering M, Oosterwegel M, Van Norren K, Clevers H: Sox-4, an Sly like HMG box protein, is a transcriptional activator in lymphocytes. EMBO J 1993, 12:3847-3854.
43. .
Sudbeck P, Lienhard-Schmitz M, Baeuerle PA, Scherer G: Sexreversal by loss of the C-terminal transactivation domain of human SOX9. Nat Genet 1996, 13:230-232. The authors show that SOX9 can bind to the consensus motif AACAAAG, can transactivate transcription of a reporter construct, and map a putative transcriptional activation domain in the carboxyl terminus of the protein. The authors further describe campomelic dysplasia patients in which this carboxy-terminal domain of SOX9 has been truncated. The authors argue that the possibility remains that the phenotype of campomelic dysplasia and autosomal sex reversal in some cases can be caused by a dominant-negative effect rather than haploinsufficiency. 44. ..
Werner MH, Huth JR, Gronenborn AM, Clore M: Molecular basis of human 46 XY sex reversal revealed from the threedimensional solution structure of the human SRY-DNA complex. Cell 1995, 81:705-714. The three-dimensional solution structure of the human SRY HMG box (hSryHMG) bound to a DNA octamer, comprising a specific target sequence in the MIS promoter was determined using multidimensional nuclear magnetic resonance. The authors propose that, from a structural view, mutations resulting in sex reversal either affect the packing of residues with the HMG box or residues which come in direct contact with the DNA. 45. ..
Love JJ, Li X, Case DA, Giese K, Grosshedl R, Wright PE: Structural basis for DNA binding by architectural transcription factor LEF-1. Nature 1995, 376:791-795. Multidimensional nuclear magnetic resonance was used to determine the three-dimensional structure of the HMG domain of LEF-1 complexed to a 15 bp oligonucleotide containing the optimal binding site from the TCRa gene enhancer. The DNA duplex binds to the concave surface of the LEF-1 domain and is bent toward the major groove. The structure of the LEF-1 HG domain complexed with DNA provides new insights into the molecular basis for manor groove binding and protein-induced bending. LEF-1 and proteins such as TBP and SRY induce large angle bends in DNA by a common mechanism involving the opening of the minor groove and partial insertion of one or more hydrophobic side chains into the base stack from the minor groove side. 46.
Koopman P, Munsterberg A, Cape1 B, Vivian N, Lovell-Badge R: Expression of a candidate sex-determining gene during mouse testis differentiation. Nafure 1990, 348:450-452.
47. .
Streit A, Sockanathan S, Perez L, Rex M, Scatting PJ, Sharpe PT, Lovell-Badge R, Stern CD: Preventing the loss of competence for neural induction HGF/SF, L5 and Sox-2. Development 1997, 124:1191-l 202. The authors compare the expression patterns of L5 and SOX2 during early chick embryogenesrs. They show that early during embryogenesis (stg3) expression of L5 is included in a broader SOX2 domain in the area opaca but both become restricted to identical domains defining the neural plate. In a series of grafting experiments, the authors demonstrate that the embryonic region competent to respond to neural induction is confined to the LS-expressing portion. 48. ..
Morais-da-Silva S, Hacker A, Harley V, Goodfellow P, Swam A, Lovell-Badge R: Sox9 expression during gonadal development implies a conserved role for the gene in testis differentiation in mammals and birds. Nat Genet 1996, 14:62-68. The results of these expression studies imply that Sox9 plays an essential role in sex determination, possibly immediately downstream of Sry in mammals, and that it functions as a critical Sertoli cell differentiation factor. These studies suggest that the role of SOX9 in sex determination may be conserved in vertebrates, as illustrated by the identical expression patterns of Sox9 in both mouse and chicken in the developing gonad. 49.
Healy C, Uwanogho D, Sharpe PT: Expression of the chicken Sox9 gene marks the onset of cartilage differentiation. Ann NY Acad Sci 1996, 785:261-262.
50. ..
Kent J, Theatley SC, Andrews JE, Sinclair AH, Koopman P: A male specific role for SOX9 in vertebrate sex determination. Development 1996, 122:2813-2822. This paper describes the expression of Sox9 during sex-determination in the mouse and chick. In both species, Sox9 is expressed in male but not female genital rrdges. This conserved expression of Sox9 between mouse and chick, which have divergent mechanisms of sex determination - the former being dependent on Sry - provrdes compelling evidence for the critical role of Sow9 in vertebrate testis determmation. 51.
Koopman P, Gubbay J, Vivian N, Goodfellow P, Love&Badge R: Male development of chromosomally female mice transgenic for Sry. Nature 1991,351:117-121.
52.
Cape1 B: New bedfellows in the mammalian affair. fiends Genet 1995, 11 :161-l 63.
53.
Pontiggra A, Rimini R, Harley VR, Goodfellow PN, Love+Badge Bianchi ME: Sex reversing mutations affect the architecture SRY-DNA complexes. EMBO J 1994, 13:6115-6124.
42. ..
Kamachi Y, Sockanathan S, Liu Q, Brerman M, Lovell-Badge R, Kondoh H: Involvement of SOX proteins in lens-specific activation of crystallin genes. EM50 I 1995 14:351 O-351 9. This paper reports that the nuclear factor 6EF2a, which binds the crystallin enhancer, is encoded by the cSox2 gene. It is also demonstrated that cSOX2 actrvates the 61 crystalltn enhancer core fragment in the lens. This work is one of the first examples of the discovery of direct regulatory targets of SOX proteins.
343
sex-determination R, of
344
Genetics of disease
54.
Collignon J: Study of a new family of genes related Mammalian Testis Determining gene [PhD Thesis]. Council for National Academic Awards; 1992.
to the London:
55. .
Yuan H, Corbi N, Basilic0 C, Dailey L: Developmental specific activity of the FGF-4 enhancer requires the synergistic action of Sox2 and Ott-3. Genes Dev 1995, 9:2635-2645. The authors present in vitro evidence suggesting that Fgf-4 is a target gene of SoxP and O&3/4. When Sox-2 and Ocf3/4 were cotransfected toaether. an 1 1 -fold induction of chloramohenicol acetvltransferase (CAT) acti& was observed but each one alone iailed to activate CAT expression. These in vitro results suggest that Sox2 and Oct3/4 may activate Fgf-4 expression in viva during earii embryogenesis, a time when -all three genes are known to be expressed. I
56.
61.
Prior HM, Walter MA: Characterization of the human SOX2 genes. Am J Hum Genet 1996, 57:774.
62.
Vriz S, Lovell-Badge R: The zebrafish Zf-Sox 19 protein: a novel member of the Sox family which reveals highly conserved motifs outside of the DNA-binding domain. Gene 1995, 1531275-276.
63.
Vriz S, Joly C, Boulebache H, Condamine, H: Zygotic expression of the zebrafish Sox-19, an HMG box-containing gene, suggests an involvement in central nervous system development. MO/ Brain Res 1996, 40:221-228.
64.
Van de Wetering M, Clevers H: Sox-75, a novel member of the murine Sox family of HMG box transcription factors. Nucleic Acids Res 1993, 21 :1669.
65.
Jay P, Goze C, Marsollier C, Taviaux S, Hardelin JP, Koopman P, Berta P: The human SOXI 1 gene: cloning, chromosomal assignment and tissue expression. Genom& 1995, 29:541-545.
66.
Komatsu N, Hiraoka Y, Shiozawa M, Ogawa M, Aiso S: Cloning and expression of Xenopus laevfs xSoxl2 cDNk Biochim Biophys Acta 1996, 1305:117-l 19.
67.
Meyer J, Wirth J, Held M, Schempp W, Scherer G: SOX20, a new member of the SOX gene family, is located on chromosome 17~13. Cytogenet Cell Genet 1996, 72:246-249.
68.
Connor F, Wright E, Denny P, Koopman P, Ashworth A: The Sryrelated HMG box-containing gene Sox6 is exDressed in the adult testis and developing-n&ous system of the mouse. Nucleic Acids Res 1995, 23:3365-3372.
69.
Dunn TL, Mynett-Johnson L, Hosking BM, Koopman PA, Muscat GEO: Sequence and expression of Sox-18, a new HMG-box transcription factor. Gene 1995, 161:223-225.
70.
Greenfield A, Dunn T, Muscate G, Koopman P: The SRY related gene SOX18 maps to the distal mouse chromosome 2. Genomics 1996, 36:558-559.
I
Zappavigna V, Falciola L, Citterich MH, Mavilio F, Bianchi ME: HMGI interacts with HOX proteins and enhances their DNA-binding and transcriptional activation. EM60 J 1996, 15:4961-4991.
57. ..
Wotton D, Lake RA, Farr CJ, Owen MJ: The high-mobility group transcription factor, SOX4, transactivates the human CD2 enhancer. J Biof Chem 1995, 270:7515-7522. This paper reports that one of the transcription factors to bind to the CD2 enhancer is an HMG box protein, SOX4. It is demonstrated that SOX4 is also able to transactivate the CD2 gene. The sequence which SOX4 binds in the CD2 enhancer (AACAATA), however, differs from the normal HMG site (WWCAAAG), supporting the evidence that SOX proteins preferentially recognize a DNA sequence different from the TCF/LEF HMG proteins. 56.
Ng U, Wheatley S, Muscat GEO, Conway-Campbell J, Bowles J, Wright E, Bell DM, Tam PLP, Cheah KSE, Koopman P: SOX9 binds-DNA, activates transcription, and coexpiesses with type II collagen during chondrogenesis in the mouse. Dev Biol 1997,183:108-121.
59.
Lefebre V, Huang WD, Harley VR, Goodfellow PN, Decrombruggle B: SOXg is a potent activator of the chondrocyte-specific enhancer of the pro-alpha 1 (II) collagen gene. MO/ Cell Biol 1997, 17:2336-2346.
60.
Malas S, Sartor M, Duthie S, Hadjantonakis K, Lovell-Badge R, Episkopou V: Genetic and physical mapping of the murine Soxf gene. Mamm Genome 1996, 7:620-621.
SOXI
and
Sax genes find their feet Larysa H Pevny* and Robin Lovell-Badge’ The identification
of the mammalian
SRY, led to the description transcription display
factors,
properties
architectural diverse
patterns
mutations factors
the SOX
gene family. SOX
of both classical
components
of SOX
genes
factor, encoding
proteins factors
The dynamic
mice and Drosophila
play key roles in decisions
developmental
transcription
of chromatin.
of expression
in humans,
testis-determining
of a new class of genes
and
and
and analysis
suggest
of cell fate during
of
diverse
Addresses Laboratory of Developmental Genetics, MRC National lnstltute for Medical Research, The RIdgeway, Mill HIII, London NW7 1AA, UK [email protected] [email protected]
Current
Opinion in Genetics
0 Current
Biology
& Development
1997, 7:338-344
Ltd ISSN 0959-437X
Abbreviations CD CNS Fgf-4 HMG sox TCF
campomelic
the strucwre,
expression
and fun&on
that SOX
processes.
‘e-mall: ‘e-mail:
made to characterize
of the members of the Sex gene family These primary analyes suggest that-as with the founding member of the family S’RY-S’ox genes are involved in governing cell face decisions in a number of diverse de\~elopmcncal processes.
dysplasia
central nervous system fibroblast growth factor-4 high mobility group SRY box T-cell factor
Introduction Throughout developmenr. transcriptional regulation of eukaryoric gene expression requires both the recruitment of tissue-specific transcription factors to a promoter and the proper establishment of local chromarin structure. In this review, we describe the SOX family, a group of proteins which appear to goTern cell fate decisions during embryogenesis by functioning both as classical transcription factors and architectural components of chromatin. The SOX factors comprise a novel group of proteins characterized by the presence of an SRY box (hence ‘SOX’), a 79 amino acid motif that encodes an HhlG-type DNA-binding domain. The genes encoding these factors, highly conserved across evolurion, were originally identified through homology as they contain an HhlG box closely related to that of the mammalian testis-determining gene SRY [1,2]. The SOX family falls into a subclass of HhlG box proteins, the members of which show highly restricted tissue distribution and bind to specific sequences at high affinities (reviewed in [3]). hlost strikingly, on binding, SOX proreins cause DNA to bend at an acute angle [4,5]. Compelling evidence for the developmental importance for individual members of the Sax gene family comes from mutational analyses in human [&lo], mouse [ll”] and Drosophih [12”,13”]. In this review. we concentrate on recent advances thar have been
High mobility group boxes and the SOX gene family To date, HRIG box proteins have been classified into two major subgroups according to sequence specificity of the DNA-binding and rhe number of HhlG DNA-binding domains within a single protein [14]. The first subgroup consists of HhlG-box proteins which concain more than one DNA-binding domain, are usually expressed ubiquitously and bind preferentially to bent DNA. hlembers of this group include the HhIG non-hisrone chromatin-associated proteins HhlGl and HMG2 [15.16]. In contrast. members of the second subgroup of proteins contain only a single HhlG box, show highly restricted expression patterns, and may bind prestructured DNA with either little or no sequence specificity hlembers of this class. however, can also bind to linear DNA at high affinities in a sequence-specific manner. Examples of this group include the T-cell factor (TCF) [17-191, \lATA [ZO-221 and the SOX ([1.2,6]: Table 1) families. HhlG-box proteins or genes can be further categorized into families on the basis of groups which share a high degree of homology within the HhlG box. The SOX family of proteins is defined by an HhlG box which has at least 50% sequence identity with the founding member of this group, mouse SRY. At present. this group includes at least 20 members and can be further divided into subfamilies on rhe basis of degree of homology both within and outside the HhlG box ([2,23-251; Table 1). Categorizing rhe Sax genes only on known sequence many of tvhich are incomplete, however, is at present ambiguous and may need recent cloning
co be refined and functional
on the basis studies.
of results
from
Individual SOX family members also show a high degree of conservation across species both lvithin and outside their HhlG domain. It is less easy, however, to idenrif! orthologues of a specific SOS gene in invertebrates bur in some cases the similarities are intriguingly high. An esample is human SOX2 [26] which shares 98% overall similarity with the mouse protein [27’]. 95% with the chicken homologue cSOX2 [28] and 88% with the putative Dr-osophih homologue Dir/eate (also called .5’(J?I7~~~1or /is/Moofi [ 12”,13**]). SRY is a major exception because very little sequence homology can be detected outside the HhlG domain even between closcl~ related hpecieh [29-30,31’].
Sox genes find
their
feet
Pevny and Love&Badge
339
Table 1 The SOX family. Orthologue
Gene Group
References
Expression
Mapping
A
SRY Group
Human,
rodent,
marsupial
Human
Y
Genital
b31
ridge and testis
B
sox7
Human,
mouse,
chicken
Human
8
sox2
Human,
mouse,
chicken
Human
3
Soxl: Sox2:
embryonic
CNS,
ICM, primitive
lens
ectoderm,
[2,27’,60,61] [26,27’,28,42”,55’,61
I
CNS, PNS, embryonic gut, endoderm; cSox2: embryonic
sox3 Soxl4 ZfSoxl9 sox15 Dichaete (Sox7OD, fish-hook) Group
mouse,
Human
chicken
X
Mouse Zebrafish
Sox3
CNS and PNS and cSox3: embryonic
CNS
[2,7,27’,28] [26]
Embryonic
CNS
[62,63]
Mouse
Drosophila
70Dl-2
1641 [12”, 13**1
Entire trunk of syncitial blastoderm;
seven
irregular
stripes
in cellular blastoderm; ventral cephalic neuroectoderm
and
C
sox4
Human,
SOXll
sox72 sox20 Group
Human,
Human,
mouse
mouse,
chicken
Human, mouse Human
Human
6
Embryonic heart and spinal cord, adult pre-B and pre-T cells
Human
2
cSox7 7: embryonic CNS, post-mitotic neurons
[2 311 * 111 35 41 57.91 l
[25,28,65] 1261
Human
(671
17
D
sox5 SOX6
Human, Human,
Human
mouse mouse
6
Sox5: Sox6:
adult testis
embryonic
CNS,
[23,32,36]
[23,681
adult testis Group E SOX8 sox9
SOXlO Group F sox-7 sox- 17 sox- 7 8
WV251
Human, mouse Human, mouse, chicken Human,
Human 17, Mouse 2 -
mouse
genital ridge and CNS, notochord
[23 25 33*‘] [9,48:‘,49,50”] [23,25]
1231
Mouse Mouse Mouse
ICM, inner cell mass; PNS, peripheral
Chondrocyte, adult testis,
Testis Mouse
nervous
2
Heart, lung, spleen, skeletal muscle, liver and brain of adult
[34’,69] [40,69,70]
system.
In addition to sequence similarities, all Sox genes studied to date with the exception of Sox5 [32], Sor9 [8,33*-l and Soxl7 [34*] display a monoexonic structure, at least with respect to their open reading frame. Gene-mapping studies show the Sex genes to be distributed randomly throughout the genome with no evidence of gene clustering (see Table 1). This genomic organization indicates a possible early divergence of subfamilies of HMG proteins during evolution. The ancestral sequence-specific HMG box could have contained an intron, which still occurs in the Ef genes and in some Sox genes but this intron may have been lost in other SOS genes. Apart from some expressed pseudogenes, PolII transcribed intron-less genes are fairly rare in the eukaryotic genome. Therefore, it is conceivable that the intron-less genes arose by reverse transcription and retroposition [35].
The biochemical properties of SOX proteins SOX proteins share several characteristics with classical transcription factors. Results from several in vitro studies involving SOX proteins demonstrated that the HMG domain of SOX proteins binds to a specific DNA
sequence motif (A/T A/T CAA A/T G) with high-affinity [5,27*,36+0] and several SOX proteins including mouse SOX4, SOX17, SOX18 [34*,40,41], chicken cSOX2 [42**], and human SOX9 [43*] have been shown to activate the transcription of reporter constructs. In several instances-such as SOX4, SOX9 and SOX18 - the putative transcriptional activation domains have been mapped but there appears to be no consensus sequence within this domain [40,41,43*]. In addition to acting as classical transcription factors, SOX proteins also have a number of properties which suggest that they function by organizing local chromatin structure. Unlike most transcription factors, binding of SOX proteins occurs in the minor groove and results in the induction of a dramatic bend within the DNA [4,5,38,44**,45”]. This was first demonstrated for the HMG box protein LEFl which induces a dramatic bend of 130”. The ability to alter DNA structure is likely to be a common property of all the sequence-specific class of HMG box containing proteins as two members of the SOX family, SRY and SOX.5, have also been shown to induce strong DNA bending (85” for SRY,
340
Genetics of disease
73-90” for SOXS [5,39,46]). The architectural role for SOX proteins is further supported by studies on SRY mutations in human XY females (see below). \Vhy bend at specific facilitate adjacent
DNA? By altering local chromatin structure binding sites. SOX proteins may act to
the interaction between other factors sites or allow the interaction of distant
nucleoprotein complexes machinery. For example, HMG box protein, LEFl,
bound at enhancer
with the basal transcription the bending induced by the may allow the interaction of
DNA-binding factors located on opposite sides of the LEFl-binding site [S]. Alternatively, the binding of SOX proteins and the local changes in chromatin structure may lead to the recruitment of higher-order architectural factors, such as the polycomb or the trithorax group of proteins which, in turn, could serve to act over long distances. Conversely, the severe distortion brought about by bending the DNA helix could act in a negative fashion by simply preventing the binding of factors to adjacent sites in the major groove.
Expression of Sox genes Sox genes show diverse and dynamic
patterns of expression throughout embryogenesis and in a variety of adult tissue types. As shown in Table 1, there is a member of the Sax gene family expressed in almost every tissue of the developing embryo. Can the comparison of the expression patterns of individual Sox genes or subfamilies be used to make any predictions of SO,Kgene function?
the expression
patterns
implicate
the
genes
as playing
a
role in establishing cell fates but the specific cell type will depend on the tissue in question, implying that there must be alternative cooperating factors and different sets of target
genes
Functional
in the various
tissue
lineages.
analysis of SOX genes
The first demonstration of a functional role genes in a genetic disease and a developmental
for SOX process
came from studies on human sex reversal. Genetic and molecular analysis of XY sex reversal (61, arising from mutations in SRI: and studies involving autosomal sex reversal in campomelic dysplasia (CD) patients, resulting from mutations in SOXY [%-lo] suggest that SOX genes may act to initiate or bias early cell fate decisions. This hypothesis is supported by the finding that misexpression of S’ry in the mouse diverts cells destined to become ovaries to differentiate as testes [6,51]. Expression of J’I;)~ alone, hovvever, may be insufficient for cell fate conversion as, in a number of naturally occurring and experimental conditions, the sex reversal shows varying degrees of penetrance (reviewed in [SZ]). This data is consistent with the variable sex reversal phenotypes displayed by campomelic dysplasia patients carrying identical SOXY mutations and the variable phenotype displayed in null alleles of the Drosophila Sox gene Dichnete. Collectively. these studies suggest that SOX genes may act in a dose-dependent manner. An idea that is supported by the studies showing that the dominant phenotype campomelic dysplasia patients must be caused largely by haploinsufficiency rather than a dominant negative
S~J is expressed in the undifferentiated male gonad and is quickly downregulated once the decision is made to
effect
initiate male development [6,31’,33”,46]. Similarly, the expression patterns of many of the other Sox family
Detailed molecular analyses carried out in these studies provide in vivo evidence that SOX proteins can function as both classical transcription factors as well as architectural proteins. For example, studies on mutations found within SRY in human XY females demonstrate that binding affinity and bending function as assayed in vitro are distinct. hloreover, one case of sex reversal associated with an SRY mutation appears to derive from the reduced DNA bending activity of the mutant protein [44”,53]. Finlike
members throughout development also appear to correlate with early cell fate decisions. For example, during the early phases of neural induction in both mouse and chick, the neuroepithelium shows a dramatic upregulation of 3’0x1 and Sod expression and a high level of SoxL expression [27’,28,47*]; but then, coincident with the differentiation of neural precursors, expression of all three genes is rapidly downregulated. The three genes therefore seem to respond both to signals involved in neural induction as well as signals which trigger the loss of potential and, thus, their expression largely defines the uncommitted neuroblast population. The expression of a particular Sox gene is not necessarily restricted to a particular cell type or lineage. For example, Sox9 other than being expressed in the differentiating male Sertoli cells is also expressed in mesenchymal condensations of the limb and somite prior to cartilage formation, in the CNS, and in a number of other organ systems [9,33”,48”,49,50”]. Similarly, Sox4 is expressed in the immune system, in pre-B and pre-T cells prior to terminal differentiation, but also in the developing heart, CNS, as well as numerous other tissues [41]. In each case,
[S].
SRY, vvhere nearly all missense mutations in sex-reversed XY females occurs in the HRIG box, mutations in J’OX9 resulting in campomelic dysplasia are distributed throughout the coding region. Some SOX9 mutations lead to the truncation of a transactvation domain, suggesting that the phenotypes in these cases result, at least in part, from loss of transcriptional transactivation of downstream genes
[43’].
Specificity/redundancy
of Sox gene function
The in vivo analysis of Sox genes together with in vitro assays provide evidence for potential redundancy within the So,v gene family, This is supported by studies which demonstrate that all SOX proteins, including SRY’. can bind to the same DNA sequence (4,461. Therefore. the specificity may be caused in part by the temporal
Sox genes find their feet Pevny and Lovell-Badge
and gene
spatial family;
expression this
of
individual
may explain
the
members
phenotype
mutant embryos [ 1 l”]. Animals homozygous disruption of So.~l display severe cardiac
of
the
of So_&-
for a targeted malformations
of Fgf4
in teratocarcinoma
consistent with peri-implantation the
crystallin
proposal
is
the expression of all three genes mouse embryos. Similar to studies
in on
promoter,
cells
both
[55*]. This
341
SOX2
and OCT3/4
appear
and lack B lymphocytes. These tissues appear to only express SO.Y-4 and no other known SO,Y family member.
to bind necessary
Sox-4, however, is also expressed in other regions of the embryo-such as the developing CNS-which are unaffected in null mutants [41]. hlany other Sox family members, most notably Soxl-3, are co-expressed with
co-workers [56] have shown that the HMG box of HMGl interacts with the homeodomain of specific HOX proteins. It is therefore conceivable that SOXZ and OCT3/4 also interact through their DNA-binding domains.
Sox-4 in the CNS [27*]. Similarly, mental retardation is associated with deletions of the region of the human X chromosome which encompasses SOX.? but the phenotype appears to be mild compared with the extent of its expression throughout the developing CNS [7.27-l, and it seems highly likely that SOXl and SOXZ could largely compensate for the lack of SOX3 as they are very similar proteins and show considerable overlaps in tissue distribution.
Target genes and protein-protein
interactions
There is good evidence for several SOX protein-target gene interactions from detailed itz vitro studies. For example, SOXZ and (probably) SOXl most likely participate in the regulation of 6- and y-crystallin which are expressed in the lenses of chick and mouse, respectively [42”]. These studies -which focused on the characterization of an enhancer required for tissue-specific expression of the &crystallin gene-led to the identification of binding sites for three factors within the enhancer core: a repressor, 6EF1, and two activators, 6EF2 and 6EF3. 6EF2 appears to be a complex of several proteins with similar DNA-binding properties, one of which upon the cloning of its encoding gene was shown to be SOXZ. In transient transfection assays, it was demonstrated that activation of the enhancer was dependent on the presence of both cSOX2 and 6EF3; neither factor alone was sufficient for activity. Critically, increasing amounts of cSOX2 led to a level of activation which rapidly plateaued, presumably because of limiting amounts of 6EF3. The plateau may be caused by the binding of cSOX2 to the minor groove which, in turn, would prevent the binding of the repressor 6EFl. Perhaps through direct protein-protein interactions, cSOX2 and 6EF3 then cooperate to give activation of the crystallin gene. SOXl can substitute for SOX2 in these assays consistent with proposals for possible redundancy amongst SOX subfamily members. The data is also consistent with the expression of the Soxl-3, subfamily: Soxl’ and Sox3 are expressed at high levels in the lens placode during early development whereas Soxl is expressed after lens invagination coincident with upregulation of &crystallin or y-crystallin [42”,54]. It will be interesting to determine if members of other subfamilies can work in this system and, if not, which parts of the protein are critical. SOX2 is also likely domain transcription
to participate, factor OCT3/4,
along with the POU in the transactivation
to adjacent sites in the DNA and may be for target gene expression. Recently, Bianchi and
In a variety of other studies, different SOX proteins have been implicated in transcriptional control in a number of discrete tissue types. For example, human SOX4 was shown to trans-activate the CD2 enhancer in T cells [57”] and SOX9 has been strongly implicated in the regulation of type II collagen gene expression [58,59]. In Drosophila, Dictiaete is probably involved in the regulation of pair-rule gene expression, perhaps cooperating with the products of gap and primary pair-rule genes, although there is no direct evidence for this at present [12”,13”]. It is too early to know if these in vitro results reflect in ZWO situations, however, general trends are emerging. In all the stated cases, the SOX protein interacts with enhancers located distal to the basal promoters. The significance of this, however, is not clear. It is conceivable that the bending of DNA induced by SOX binding may facilitate the interaction of the enhancer elements with the core promoter region. Alternatively, the SOX proteins may act locally to organize chromatin structure within the enhancer region. It is also likely that specificity of action of a SOX protein is context-dependent, relying on both the direct interaction of the SOX protein with its DNA-binding site as well as protein contacts with other transcription factors. As suggested by studies with SOX2, the interacting partners need not be the same in different cell types (e.g. Ort3/4 is not expressed in lens tissue). If there is a reliance on specific interacting factors, then it is likely that ectopic expression of a Sor gene may not necessarily have widespread consequences which may explain the relatively mild phenotype of the dominant Dicfiaete mutations, wing are affected.
in which
only
specific
regions
of the
Conclusions It is still too early to get a clear picture of how the SOX family functions. There is increasing evidence, however, that SOX proteins are most likely involved in many aspects of transcriptional regulation. The action of SOX proteins appears to be very context dependent, perhaps relying on protein-protein interaction as much as the interaction with DNA. It is therefore critical to determine the types of proteins SOX factors interact with and establish if there are any common rules. hlembers of the LEF/TCF family have recently been shown to interact with B catenin, placing them within many important pathways involving Wnt signaling. Similarly, SOX proteins could also be involved in
342
Genetics of disease
the immediate as implicated
response by aspects
to signal transduction pathways. of their expression patterns.
SOX proteins
have several
properties
which
allow them
12. ..
to
bring a high degree of specificity to the regulation of gene expression. It is therefore easy to imagine them occupying pivotal
roles.
For
this
reason,
it will
not
be a surprise
Russell SRH, Sanchez-Soriano N, Wright CR, Ashburner M: The Dichaefe gene of Drosophila melanogasfer encodes a SOX-domain protein required for embryonic segmentation. Development 1996, 122:3669-3676. The authors describe the cloning and charactenzation of the first Drosophila Sox gene - Sox7OD. They provide evidence that Sox7OD corresponds to the dominant wing mutation Dichaete. (simultaneously cloned by Nambu and Nambu [13”]). The authors argue that the variable phenotypes observed in Dichaefe mutants supports the role of Sax genes as architectural proteins.
involved in cell fate decisions, as exemplified by the founding father, 5~. The SOX genes are beginning to find their feet in the world of developmentally important
Nambu PA, Nambu JR: The Drosophila fishhook gene encodes a HMG domain protein essential for segmentation and CNS development. Development 1996, 122:3467-3475. The authors describe the isolation and analysis of the first Drosophila Sox gene (fish-hook; simultaneously cloned by Russell et al. [l 2”]). Mutational analyses indicate an essential role for hsh-hook in anterior/posterior pattern formation and nervous system development and suggest a potential function in modulating the activities of gap and pair rule proteins.
genes.
14.
Laudet V, Stehelin D, Clevers H: Ancestry and diversity of the HMG box superfamily. Nucleic Acids Res 1993, 21:2493-2501.
15.
Jantzen H-M, Admon A, Bell SP, Tjian R: Nucleolar transcription factor hUBF contains a DNA-binding motif with homology to HMG proteins. Nature 1990, 344:830-636.
16.
Grosshedl R, Giese K, Pagel J: HMG domain proteins: architectural elements in the assembly of nucleoprotein structures. Trends Genet 1994, 10:94-99.
1 7.
Waterman M, Jones K: Purification of TCF-1 n, a T-cell-specific transcription factor that activates the T-cell receptor Ca gene enhancer in a context-dependent manner. New Biol 1990, 2:621-636.
18.
Travis A, Amsterdam A, Belanger C, Grosschedl R: Lef-7, a gene encoding a lymphoid- specific protein with an HMG domain, regulates T-cell receptor a enhancer function. Genes Dev 1991) 5:660-694.
19.
Van de Wetering M, Oosterwegel M, Dooijes D, Clevers H: Identification and cloning of TCF-1. a T lymphocyte-specific transcription factor containing a sequence specific HMG box. EMBO J 1991, IO:1 23-l 32.
20.
Kellv M. Burke J. Smith M. Klar A. Beach D: Four mating-hrpe genes control iexual differentiation in the fission yeastEM60 J 1966, 7:1537-l 547.
21.
Staben C, Yanofsky C: Neurospora crassa a mating-type Proc Nat/ Acad Sci USA 1990, 67:4917-4921.
22.
Sugimoto A, lino Y, Maeda T, Watanabe Y, Yamamoto M: Schizosaccharomyces pombe stell+ encodes a transcription factor with an HMG motif that is a critical regulator of sexual development Genes Dev 1990, 5:1990-l 999.
23.
Denny P, Swift S, Brand N, Dabhade N, Barton P, Ashworth A: A conserved family of genes related to the testis determining gene, SRV. Nucleic Acids Res 1992, 20:2667.
24.
Coriat AM, Muller U, Harry JL, Uwanogho D, Sharpe PT: PCR amplification of Sry-related sequences reveals evolutionary conservation of the SRY-box motif. PCR Methods Appl 1993, 2:216-222.
25.
Wright EM, Snopek B, Koopman P: Seven new members of the Sox gene family expressed during mouse development Nucleic Acids Res 1992, 21:744.
26.
Stevanovic M, Zuffardi 0, Collignon J, Lovell-Badge R, Goodfellow PN: The cDNA sequence and chromosomal location of the human SOX2 gene. Mamm Genome 1994, 5:640-642.
if results of mutation analyses-which are underway in many laboratories-confirm that Sax genes are indeed
References
and recommended
reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
. .. 1.
2.
of special interest of outstanding interest Sinclair AH, Berta P, Palmer MS, Hawkins JR, Griffiths BL, Smith MJ, Foster JW, Frischauf AM, Lovell-Badge R, Goodfellow PN: A gene from the human sex determining region encodes a protein with homology to a conserved DNA binding motif. Nature 1990, 346:240-244. Gubbay J, Collignon J, Koopman P, Cape1 B, Economou A, Munsterberg A, Vivian N, Goodfellow P, Lovell-Badge R: A gene mapping to the sex determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes. fv’afure 1990, 346:245-250.
3.
Ner SS: HMGs everywhere.
Curr Viol 1992, 2:206-210.
4.
Ferrari S, Harley VR, Pontiggia A, Goodfellow PN, Lovell-Badge R, Bianchi M: SRY, like HMGl. recognizes sharp angles in DNA. EM60 I 1992, 11:4497-4506.
5.
Giese K, Cox J, Grosschedl R: The HMG domain of lymphoid enhancer factor 1 bends DNA and facilitates assembly of functional nucleoprotein structures. Cell 1992, 69:165-l 95.
6.
Goodfellow PN, Love&Badge R: SRY and sex determination mammals. Annu Rev Genet 1993, 27:71-92.
7.
Stevanovic M, Lovell-Badge R, Collignon J, Goodfellow P: SOX3 is an X-linked gene related to SRY. Hum MO/ Gener 1993, 2:2013-2016.
6.
Foster JW, Dominguez-Steglick MA, Guioli S, Kwok G, Weller PA, Stevanovic M, Weissenbach J, Mansour S, Young ID, Goodfellow PN et a/.: Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY related gene. Nature 1994, 372:525-530.
9.
10.
in
Wagner T, Wirth J, Meyer J, Zabel B, Held M, Zimmer J, Pasantes J, Bricarelli FD, Keutel J, Hustert E, Wolf V et al: Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Cell 1994, 79:lll l-l 120. Kwok C, Weller PA, Guioli S, Foster JW, Mansour S, Zuffardl 0, Punnett HH, Dominguez-Steglich MA, Brook JD, Young ID et al.: Mutations in SOXS, the gene responsible for campomelic dysplasia and autosomal sex reversal. Am J Hum Genet 1995, 57:1026-l 036.
Schillam MW, Oostenvegel MA, Moerer P, Ya J, De Boer PAJ, Van de Wetering M, Verbeek S, Lamers WH. Kruisbeek AM, Cumano A, Clevers H: Defects in cardiac outilow tract formation and pro-B-lymphocyte expansion in mice lacking Sox-4. Nature 1996, 380:71 l-71 4. This paper reports the first functional analysts of a Sow gene in mice. It describes the targeted disruption of Sox4 and its effect on both development of endocardial ridges and B cell differentiation. The observed range of septation defects caused by the lack of Sox4 in mice is compared to ‘common arterial trunk’ condition in man. In addition, the authors demonstrate that B-cell development is blocked at the pro-B cell stage in Sox4 mutant mice.
11. ..
13. ..
region.
Collignon J, Sockanathan S, Hacker A, Cohen-Tannoudji M, Norris D, Rastan S, Stevanovic M, Goodfellow PN, LovellBadge R: A comparison of the properties of Sox-3 with Sty and two related genes, Sox-f and Sox-2. Development 1996, 122:509-520. The authors describe the cloning and sequencing of Soxl, Sow.2, and Sox3 from the mouse and show that Sow3 is most closely related to Sry. The discussion focuses on the evolutionary link between the genes and supports the model that Sry has evolved from Sox3. 2 7. .
26.
Uwanogho D, Rex M, Cartwright EJ, Pearl G, Healy C, Scatting PJ, Sharpe PT: Embryonic expression of the chicken Soxf, Sox3 and Soxf 7 genes suggests an interactive role in neuronal development Mech Dev 1995,49:23-36.
29.
Whitfield LS. Lovell-Badge R, Goodfellow PN: Rapid sequence evolution of the mammalian sex determining gene SRV. Nature 1993, 364:713-715.
Sox genes find their feet Pevny and Lovell-Badge
30.
Tucker PK, Lundrigan BL: Rapid evolution of the sex determining locus in Old World mice and rats. Nature 1993, 364:715-717.
Hacker A, Cape1 B, Goodfellow P, Lovell-Badge R: Expression of Sry, the mouse sex determining gene. Development 1995, 121:1603-1614. Using RNAse protection assays, the authors characterize the genital ridge specific transcript of the mouse Sry gene. The authors demonstrate that the Sry genital ridge transcript is derived from a single exon and is a linear mRNA. This is in contrast to the Sry transcript expressed in adult mouse testis which exists as a circular RNA. The linear transcript is shown to start in a unique region of the Sry locus whereas the circular transcript starts with the 5’ repeat arm suggesting that the two transcripts are generated by the use of different promoters. This study also defines the critical period during gonadal development when Sry acts to initiate testes differentiation (10.5-l 1.5 dpc). 31. .
32.
Wunderle VM, Critcher R, Ashworth A, Goodfellow PN: Cloning and characterization of SOX5, a new member of the human SOX gene family. Genomics 1996, 36:354-358.
33. ..
Wright E, Hargrave MR, Christiansen J, Cooper L, Kun J, Evans T, Gangadharan U, Greenfield A, Koopman P: The Sly-related gene Sox9 is expressed during chondrogenesis in mouse embryos. Nat Genet 1995, 9:15-20. The authors of this work report the isolation and characterization of the mouse orthologue Sox9 of the human SOX9 gene, mutations of which are associated with skeletal dysmorphology (CD) and sex reversal. By wholemount in situ hybridization, the authors demonstrate that Sow9 is expressed in mesenchymal cells during mouse embryogenesis in a pattern suggestive of a role in skeletal formation. The authors go on to map Sox9 to mouse chromosome 11 in the region of the Tail short mutation, which has a strikingly similar phenotype to human CD syndrome. 34. .
Kanai Y, Kanai-Azuma M, Note T, Saido TC, Shiroishi T, Hayashi Y, Yazaki K: Identification of two Soxl7 messenger RNA isoforms, with and without the high mobility group box region, and their differential expression in mouse spermatogenesis. 1 Cell Biol 1996, 133:1-l 5. The authors report the isolation of two different mRNA isoforms, with and without an HMG box, of the mouse Sox77 gene. One form (Sox77) is expressed in spermatogonia, binds DNA and can activate transcription of a reporter construct. The second form (f-Soxl7) is accumulated in round spermatids and shows no apparent DNA-brnding actrvity. The authors propose the Sox77 may function as a transcriptional activator in premerotic germ cells and that a splicing switch Into t-Sox77 may lead to the loss of its function in the postmeiotic germ cells. 35.
Schillam MW, Van Eyk M, Van de Wetering M, Clevers HC: The murine Sox-4 protein is encoded on a single exon. Nucleic Acids Res 1993, 21:2009.
36.
Denny P, Swift S, Connor F, Ashworth A: An SRY related gene expressed during spermatogenesis in the mouse encodes a sequence specific DNA binding protein. EM80 J 1992, 11:3705-3712.
37.
Harley VR, Lovell-Badge R, Goodfellow PN: Definition of a consensus DNA binding site for SRY. Nucleic Acids Res 1994, 22:1500-l 501.
38.
Van de Wetering M, Clevers H: Sequence-specific interaction of the HMG box proteins TCF-1 and SRY occurs within the minor groove of a Watson-Crick double helix. EMBO J 1992, 11:3039-3044.
39.
Connor F, Cary PD, Read CM, Preston NS, Driscoll PC, Denny P, Crane-Robinson C, Ashworth A: DNA binding and bending properties of the post-meiotically expressed Sry related protein Sox-5. Nucleic Acids Res 1994, 22:3339-3346.
40.
Hosking BM, Muscat GEO, Koopman P, Dowhan DH, Dunn TL: Trans.-activation and DNA-binding properties of the transcription factor, Sox-18. Nucleic Acids Res 1995, 23:2626-2628.
41.
Van de Wetering M, Oosterwegel M, Van Norren K, Clevers H: Sox-4, an Sly like HMG box protein, is a transcriptional activator in lymphocytes. EMBO J 1993, 12:3847-3854.
43. .
Sudbeck P, Lienhard-Schmitz M, Baeuerle PA, Scherer G: Sexreversal by loss of the C-terminal transactivation domain of human SOX9. Nat Genet 1996, 13:230-232. The authors show that SOX9 can bind to the consensus motif AACAAAG, can transactivate transcription of a reporter construct, and map a putative transcriptional activation domain in the carboxyl terminus of the protein. The authors further describe campomelic dysplasia patients in which this carboxy-terminal domain of SOX9 has been truncated. The authors argue that the possibility remains that the phenotype of campomelic dysplasia and autosomal sex reversal in some cases can be caused by a dominant-negative effect rather than haploinsufficiency. 44. ..
Werner MH, Huth JR, Gronenborn AM, Clore M: Molecular basis of human 46 XY sex reversal revealed from the threedimensional solution structure of the human SRY-DNA complex. Cell 1995, 81:705-714. The three-dimensional solution structure of the human SRY HMG box (hSryHMG) bound to a DNA octamer, comprising a specific target sequence in the MIS promoter was determined using multidimensional nuclear magnetic resonance. The authors propose that, from a structural view, mutations resulting in sex reversal either affect the packing of residues with the HMG box or residues which come in direct contact with the DNA. 45. ..
Love JJ, Li X, Case DA, Giese K, Grosshedl R, Wright PE: Structural basis for DNA binding by architectural transcription factor LEF-1. Nature 1995, 376:791-795. Multidimensional nuclear magnetic resonance was used to determine the three-dimensional structure of the HMG domain of LEF-1 complexed to a 15 bp oligonucleotide containing the optimal binding site from the TCRa gene enhancer. The DNA duplex binds to the concave surface of the LEF-1 domain and is bent toward the major groove. The structure of the LEF-1 HG domain complexed with DNA provides new insights into the molecular basis for manor groove binding and protein-induced bending. LEF-1 and proteins such as TBP and SRY induce large angle bends in DNA by a common mechanism involving the opening of the minor groove and partial insertion of one or more hydrophobic side chains into the base stack from the minor groove side. 46.
Koopman P, Munsterberg A, Cape1 B, Vivian N, Lovell-Badge R: Expression of a candidate sex-determining gene during mouse testis differentiation. Nafure 1990, 348:450-452.
47. .
Streit A, Sockanathan S, Perez L, Rex M, Scatting PJ, Sharpe PT, Lovell-Badge R, Stern CD: Preventing the loss of competence for neural induction HGF/SF, L5 and Sox-2. Development 1997, 124:1191-l 202. The authors compare the expression patterns of L5 and SOX2 during early chick embryogenesrs. They show that early during embryogenesis (stg3) expression of L5 is included in a broader SOX2 domain in the area opaca but both become restricted to identical domains defining the neural plate. In a series of grafting experiments, the authors demonstrate that the embryonic region competent to respond to neural induction is confined to the LS-expressing portion. 48. ..
Morais-da-Silva S, Hacker A, Harley V, Goodfellow P, Swam A, Lovell-Badge R: Sox9 expression during gonadal development implies a conserved role for the gene in testis differentiation in mammals and birds. Nat Genet 1996, 14:62-68. The results of these expression studies imply that Sox9 plays an essential role in sex determination, possibly immediately downstream of Sry in mammals, and that it functions as a critical Sertoli cell differentiation factor. These studies suggest that the role of SOX9 in sex determination may be conserved in vertebrates, as illustrated by the identical expression patterns of Sox9 in both mouse and chicken in the developing gonad. 49.
Healy C, Uwanogho D, Sharpe PT: Expression of the chicken Sox9 gene marks the onset of cartilage differentiation. Ann NY Acad Sci 1996, 785:261-262.
50. ..
Kent J, Theatley SC, Andrews JE, Sinclair AH, Koopman P: A male specific role for SOX9 in vertebrate sex determination. Development 1996, 122:2813-2822. This paper describes the expression of Sox9 during sex-determination in the mouse and chick. In both species, Sox9 is expressed in male but not female genital rrdges. This conserved expression of Sox9 between mouse and chick, which have divergent mechanisms of sex determination - the former being dependent on Sry - provrdes compelling evidence for the critical role of Sow9 in vertebrate testis determmation. 51.
Koopman P, Gubbay J, Vivian N, Goodfellow P, Love&Badge R: Male development of chromosomally female mice transgenic for Sry. Nature 1991,351:117-121.
52.
Cape1 B: New bedfellows in the mammalian affair. fiends Genet 1995, 11 :161-l 63.
53.
Pontiggra A, Rimini R, Harley VR, Goodfellow PN, Love+Badge Bianchi ME: Sex reversing mutations affect the architecture SRY-DNA complexes. EMBO J 1994, 13:6115-6124.
42. ..
Kamachi Y, Sockanathan S, Liu Q, Brerman M, Lovell-Badge R, Kondoh H: Involvement of SOX proteins in lens-specific activation of crystallin genes. EM50 I 1995 14:351 O-351 9. This paper reports that the nuclear factor 6EF2a, which binds the crystallin enhancer, is encoded by the cSox2 gene. It is also demonstrated that cSOX2 actrvates the 61 crystalltn enhancer core fragment in the lens. This work is one of the first examples of the discovery of direct regulatory targets of SOX proteins.
343
sex-determination R, of
344
Genetics of disease
54.
Collignon J: Study of a new family of genes related Mammalian Testis Determining gene [PhD Thesis]. Council for National Academic Awards; 1992.
to the London:
55. .
Yuan H, Corbi N, Basilic0 C, Dailey L: Developmental specific activity of the FGF-4 enhancer requires the synergistic action of Sox2 and Ott-3. Genes Dev 1995, 9:2635-2645. The authors present in vitro evidence suggesting that Fgf-4 is a target gene of SoxP and O&3/4. When Sox-2 and Ocf3/4 were cotransfected toaether. an 1 1 -fold induction of chloramohenicol acetvltransferase (CAT) acti& was observed but each one alone iailed to activate CAT expression. These in vitro results suggest that Sox2 and Oct3/4 may activate Fgf-4 expression in viva during earii embryogenesis, a time when -all three genes are known to be expressed. I
56.
61.
Prior HM, Walter MA: Characterization of the human SOX2 genes. Am J Hum Genet 1996, 57:774.
62.
Vriz S, Lovell-Badge R: The zebrafish Zf-Sox 19 protein: a novel member of the Sox family which reveals highly conserved motifs outside of the DNA-binding domain. Gene 1995, 1531275-276.
63.
Vriz S, Joly C, Boulebache H, Condamine, H: Zygotic expression of the zebrafish Sox-19, an HMG box-containing gene, suggests an involvement in central nervous system development. MO/ Brain Res 1996, 40:221-228.
64.
Van de Wetering M, Clevers H: Sox-75, a novel member of the murine Sox family of HMG box transcription factors. Nucleic Acids Res 1993, 21 :1669.
65.
Jay P, Goze C, Marsollier C, Taviaux S, Hardelin JP, Koopman P, Berta P: The human SOXI 1 gene: cloning, chromosomal assignment and tissue expression. Genom& 1995, 29:541-545.
66.
Komatsu N, Hiraoka Y, Shiozawa M, Ogawa M, Aiso S: Cloning and expression of Xenopus laevfs xSoxl2 cDNk Biochim Biophys Acta 1996, 1305:117-l 19.
67.
Meyer J, Wirth J, Held M, Schempp W, Scherer G: SOX20, a new member of the SOX gene family, is located on chromosome 17~13. Cytogenet Cell Genet 1996, 72:246-249.
68.
Connor F, Wright E, Denny P, Koopman P, Ashworth A: The Sryrelated HMG box-containing gene Sox6 is exDressed in the adult testis and developing-n&ous system of the mouse. Nucleic Acids Res 1995, 23:3365-3372.
69.
Dunn TL, Mynett-Johnson L, Hosking BM, Koopman PA, Muscat GEO: Sequence and expression of Sox-18, a new HMG-box transcription factor. Gene 1995, 161:223-225.
70.
Greenfield A, Dunn T, Muscate G, Koopman P: The SRY related gene SOX18 maps to the distal mouse chromosome 2. Genomics 1996, 36:558-559.
I
Zappavigna V, Falciola L, Citterich MH, Mavilio F, Bianchi ME: HMGI interacts with HOX proteins and enhances their DNA-binding and transcriptional activation. EM60 J 1996, 15:4961-4991.
57. ..
Wotton D, Lake RA, Farr CJ, Owen MJ: The high-mobility group transcription factor, SOX4, transactivates the human CD2 enhancer. J Biof Chem 1995, 270:7515-7522. This paper reports that one of the transcription factors to bind to the CD2 enhancer is an HMG box protein, SOX4. It is demonstrated that SOX4 is also able to transactivate the CD2 gene. The sequence which SOX4 binds in the CD2 enhancer (AACAATA), however, differs from the normal HMG site (WWCAAAG), supporting the evidence that SOX proteins preferentially recognize a DNA sequence different from the TCF/LEF HMG proteins. 56.
Ng U, Wheatley S, Muscat GEO, Conway-Campbell J, Bowles J, Wright E, Bell DM, Tam PLP, Cheah KSE, Koopman P: SOX9 binds-DNA, activates transcription, and coexpiesses with type II collagen during chondrogenesis in the mouse. Dev Biol 1997,183:108-121.
59.
Lefebre V, Huang WD, Harley VR, Goodfellow PN, Decrombruggle B: SOXg is a potent activator of the chondrocyte-specific enhancer of the pro-alpha 1 (II) collagen gene. MO/ Cell Biol 1997, 17:2336-2346.
60.
Malas S, Sartor M, Duthie S, Hadjantonakis K, Lovell-Badge R, Episkopou V: Genetic and physical mapping of the murine Soxf gene. Mamm Genome 1996, 7:620-621.
SOXI
and