- Email: [email protected]
Octamania: The POU factors in murine development
I]~EVIEWS
S t u d i e s of Drosophila embryogenesis suggest that a sequential activation of a hierarchy of regulatory genes occurs during the development of multicellular organisms 1. These genes regulate the transformation of genetic information into morphological structure by orchestrating a precise temporal and spatial expression of effector genes, which in turn govern cell type specificities and eventually organ identity. Numerous developmental control genes have been isolated from Drosophila and Caenorhabditis elegans by genetic means. A similar approach has not been possible in vertebrates since not epough developmental mutants are available and they are hard to generate. Instead, a variety of mouse genes have been identified from their sequence similarity to Drosophila regulatory genes. Many of these, such as the Hox and Pax genes, are probably involved in pattern formation during or after gastmlation 23. An alternative approach to isolating regulatory genes is to use promoter and enhancer elements to screen for trans-activating factors playing a role in developmental processes. By this method a novel family of transcription factors, the POU family, was discovered; certain members of this family are expressed at pregastrulation stages. The POU family was defined by the sequence homology of three mammalian transcription factors and one nematode regulatory protein (Pit-1/GHF-1, Oct-l, Oct-2 and Unc-86) 4. Subsequently, several other members of this family have been identified and characterized. These proteins have two con~rved domains: a homeodomain distantly related to the prototype Antennapedia homeoo domain 5 and, nearby on the amino-temdnal side, a domain designated the POUspecific domain. The regions outside these two domains are highly divergent and contain domains required for transcriptional activation. Oct-1
Octamer-binding proteins and the POU
family
The octamer motif (ATGCAAAT) is a DNA sequence required for both ubiquitous and tissue-specific expression of various genes 6. It was first identified in the promoters of the histone H2B and the light and heavy chain immunoglobulin genes. The apparent paradox that the same element is required both for ubiquitous and B-cell-specific gene expression was resolved when two different proteins interacting with this sequence were characterized and cloned. Oct-1 was present in all cell types tested, while Oct-2 was detected only in B lymphocytes. Subsequently,
Octamania: The POU factors in murine development HANS R. SCH()LER Much effort has been directed towards the investigation of regulatory processes in the earlg mouse embryo. Several multigenefamilies of developmental control genes have been identified The POUfamily is a group of related transcription factors containing a particular type of bipartite DNA-bindingdomaim Members of this farai~ show distinct expression patterns during embryonic development. Two members, Oct.4 and Oct-6 are expressed as early as in the preimplantation embryo and thus may regulate early events of murine development several additional proteins that bind to the octamer motif have been identified at various developmental stages and in a variety of organs and tissues of the mouse (Fig. 1). Those proteins characterized so far all belong to the POU family (Table 1). cDNAs corresponding to several other POU genes have been cloned 7 but the proteins encoded by them have not been tested for DNA binding.
Structure of the POU region o~
~
Oct-1
NOct-2 i NOct-3 Oct-6
~
Oct-6
Oct-7
~
Oct-4A Oct-4B Oct-5
Oct-8~ ~ Oct-9- I Oct-10"'" MiniOct-2
The conserved POU-specific and POU homeodomains are 81 and 60 amino acids, respectively, and are separated by 14-26 variable amino acids. In the POUspecific domain, clusters of acidic amino acids are located near both ends. The POU-specific domain contains 28 invariant residues clustered in two subdomains of particularly high sequence homology (Fig. 2A), Each subdomain has two features in common: a cluster of basic amino acids in the center and a predicted (x-helix at the carboxy-terminal end. The homeodomain is a DNA-binding domain that plays a central role in eukaryotic gene regulation 5. It contains three well-defined helices, with helices 2 and 3 forming a helix-turn-helix motif similar to that in a number of prokaryotic DNA-binding proteins 8. However, the homeodomain helices are longer, and binding to DNA differs in several respects9. In the prokaryotic proteinDNA complex the second helix of the helix-turn-helix motif, often referred to as the recognition helix, makes critical
HcO
Survey of murine octamer-binding proteins with the electrophoretic mobility shift assay. Extracts of newborn mouse cerebellum and embryonic stem cells (D3) were incubated with a DNA fragment containing the octamer motif and applied to polyacrylamide gel electrophoresis. The ge] was dried and exposed to an X-ray film. These ~-o sources represent all known octamer-binding proteins. All except Oct-4and Oct 5 appear to be expressed in brain. The proteins were numbered according to their mobility, which reflects the size, charge and shape of the protein-DNA complexes. TIG OCTOBER ~ {1991 I!lst~ iur Htiun~c Publi>her~ Lid I I "KI o t 6 8
9 t ~ 9 91 NI}2 !)(I
1991 vOL 7 No. 10 I
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f~EVIEWS
TAmE 1. Properties o f routine Oct factors Ad~tiomd
Oct
Oct-I
Oct-2
of prot,~.
~
Hllnlan: OTF-t; NF-A1; NF-III; OBP 100
743
Oct-1
Hum,aft:
Oct-2A: 451 463 Oct-2B: 583
OTF-2; NF-A2
N-Oct2~ N-Oct-2
Fa,l,no
Adult
~t~tts
Ubiquitous
Ubiquitous
ATATGATAATGA 6, 24 TAATGARAT CTCATGA (heptamer)
Neural tube; in entire brain except telencephalon
Lymphoid cells; nervous system; intestine; testis; kidney
CTCATGA (heptamer) 6,24,28,42
Nervous system
Nervous system (astrocytes, certain glioblastoma and neuroblastoma cell lines)
Nervous system
Nervous system; primary spermatids
(1) Oct-2
(7)
Human:
N4)ct2~;
MiniOct-2
232
Oct-2
(7)
N-Oct-3
352 324
(strong expression in developing nasal neuroepithelium)
kfs
24, 25, 28, 42
Nervous system
Nervous system (certain glioblastoma and neurobhstorna cell lines)
Totipotent and
Oocytes
TrAA.AATrc_&
Blastocyst; ES and EC cells: brain
Nervous system: testis
TAATGARAT 21,24-27 d TFAAAATrCA CTCATGA (heptamer)
24,25,42
17-21,23,24
Oct-4A Oct4B Oct-5
NF-A3;Oct-3
Oct-6
Rat: Tst-1; SCIP
Oct-7
Human: N-Oct4
Nervous system
Nervous system
24, 25, 42
Oc,-8 Oct-9 Oct-10
Human:
Nervous system
Nervous system (astroo/tes)
24, 25, 42
Oct4
(17)
pturipotent stem cells
of the pregastrulation embryo, embryonic ectoderm, primordial germ cells; testis, ovary 448 449
Oct45 (4)
N-Oct5a N4k't5b
aNumber of amino acids, determined from cDNA. ~ m m x ~ m e location in parentheses. cA. Stoykova and P. Gruss, pets. commun.
OH. RohdewoMd and P. Gruss, pers. commun.
contacts via residues near its amino-terminal end. In contrast, such residues in the homeodomain of the Drosophila engrailed protein are near the center of the recognition helix9. POU homeodomains contain 22 invariant residues, ten of which are identical to those in the 'classical' Antennapedia homeodomain (Fig. 2B). Clusters of basic amino acids are present at the amino- and carboxy-terminal boundaries. POU homeodomains probably also contain three ~-helices 9,1°. The recognition helix is the most conserved part of the POU homeodomain, containing 11 invariant residues. The RVWFCN motif therein harbors a cysteine residue, at position nine of the helix, that is specific for the POU family, whereas the other residues are extremely well conserved in all homeodomain proteins. A serine residue at the same position is typical of the pairedtype homeodomain, whereas the corresponding residue in most other homeodomains is glutamine. When
the cysteine residue in the recognition helix of Pit-1 is changed to a glutamine, specificity and affinity for the Pit-l-binding site are unaffected, indicating that other amino acid residues outside this region are critical for recognition n. Thus, for the POU family the term 'recognition helix' is not entirely valid for helix 3. Pairwise comparison of all POU homeodomain and POU subdomain sequences divides the POU family into five classes (Fig. 2C)7. This classification is supported by the similarity of the linker sequence between the POU-specific domain and POU homeodomain among members of classes II and III. The rat genes Brn-1 and Brn-2 probably encode proteins that bind to the octamer motif. Both are widely expressed in the developing and adult brain and are probably rat homologs of mouse Oct proteins. They are grouped in class III, with a recognition helix identical to that of Oct-6 and a POU-specific domain that differs at only one position 7.
WiG OCTOBER 1991 VOL. 7 NO. 10
]]REVIEWS
A
POU-SPECIFIC DOMAIN
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a ~- . . . . .
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0
B
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POU HOMEODOMAIN KRKK S RRKRRRT TIE
(
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-Kps
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LE
R
K
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.o,,x,
F
class III
Oct-6, Brn-1, Bin-2, Cfla, Ceh-6
c,ass,v
Brn-3, Unc-86
class V
Oct-4
FIG~ Consensus sequence of the POU region and highlighted structural properties. The POU region consists of two well-conserved domains: the POU-specific domain (A) and POU homeodomain (B), connected by a variable linker sequence of 14-26 amino acids. The POU-specffic domain contains subdomains A and B, two regions of particularly high homology. Each subdomain has a predicted (x-helix at the carboxy-terminal boundary. The three helices indicated in the POU homeodomain correspond to those established for Antennapedia and engrailed homeodomains; positions identical to the 'classical' Antennapedia homeodomain are underlined with thick bars. Large letters represent residues found in all known POU proteins; two small letters represent two possible amino acids at that position, the upper letter representing the one found more frequently; a single small letter represents the predominant amino acid when more than two are found at that position. Hyphens indicate variable positions. Clusters of acidic (+) and basic (-) amino acid residues are indicated. According to the homology between the POU domains and POU subdomains, POU proteins can be subdivided into five classes (C). The classification holds for the linker sequences of classes II and III, but not for class IV.
Function of the POU region Whereas an intact homeodomain is required for DNA binding of all POU proteins, the contribution of the POU-specific domain varies, depending both on the DNA-binding site and on the POU protein. The POU homeodomain of Pit-1 is sufficient for low-affinity binding to AT-rich DNA sequences, but addition of the POU-specific domain increases both the affinity and specificity of binding to DNA1L The recognition helix and the predicted helix A must be intact for efficient DNA binding, whereas (in contrast to the engrailed homeodomain9) the other helices play only a minor role. The contribution of the Oct-1 h o m e o d o m a i n and POU-specific domain to DNA binding has been studied in detail for the Ad2 octamer motif 12,13. The POU-specific domain and the homeodomain each make half of the base contacts with the octamer sequence, whereas the majority of the backbone contacts are made by the homeodomain. Since base contacts determine the sequence specificity of a DNA-binding protein, while the binding energy stems mainly from electrostatic interactions between the protein and the DNA backbone 14, the h o m e o d o m a i n of Oct-1 contributes most of the binding energy, whereas the POUspecific domain provides half of the specificity for the octamer motif. The Oct-1 homeodomain binds with significantly lower affinity to the octamer motif than does the complete POU region. However, with other Oct-lbinding sites (Table 1), addition of the POU-specific domain of Oct-1 has no effect or can decrease affinity 12. The POU-specific domain is also required for protein-protein interactions 11, which can confer additional regulatory specificity. An inhibitory effect of one POU factor on another has recently been described for two
Drosophila proteins, I-POU and Cfl-a (Ref. 15). Cfl-a is monomeric in solution but dimeric when it binds to the Cfl-a regulatory site in the dopa decarboxylase gene. I-POU, which is closely related to Unc-86, lacks two of the five basic residues that are present in the amino-terminal portion of other POU homeodomains. Since these residues are critical for binding to DNA, I-POU alone remains as a m o n o m e r in solution. Coexpression of both factors leads to stable heterodimers, resulting in inhibition of DNA binding and suppression of transcription. Both POU regions are presumably required for dimerization, although this has only been shown for the POU region of Cfl-a. The POU-specific domain does not contain a known DNA-binding structure, and attempts to show binding of an isolated POU-specific domain to certain DNA motifs have failed. However, this does not exclude the possibility that the POU-specific domain can bind by itself to some other DNA motif. In this respect, it may be helpful to compare POU proteins with members of the Pax family 16. The paired domain of these proteins is associated with a paired-type home0domain in certain Drosophila and mouse proteins, perhaps constituting a domain analogous to the POU region (Fig. 3). Interestingly, several Pax proteins lacking a homeodomain bind via their paired domain to specific DNA sequences that are distinct from homeodomain-binding sites (G. Chalepakis and P. Gruss, pers. commun.). Perhaps homeodomain-less POU proteins also exist, although none has so far been reported.
Expression of POU factors in mouse development Most if not all POU genes are differentially expressed throughout embryogenesis. However, unlike
"rig OCTOBER'1991 vot. 7 NO. 10
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~'~EVIEWS mutations prevent the differentiation of mother cells into daughter cells: HOMEODOMAIN [ SPECIFICDOMAIN one of the two daughter cells of variable 60 I prototypestructure a division fails to assume its normal fate and instead propagates the phenotype of the mother cell. Thus PAIREDHOMEODOMAIN PAIREDDOMAIN Unc-86 represents an important com128 I 60 ~--- Pax-3;Pax-6; ponent of a mechanism linking- cell variable Pax-7 identity to cell lineage30. PAIREDDOMAIN The expression patterns of some Pax-1; Pax-2; 128 POU genes are increased in Pax-8 complexity by differential splicing. Oct-2 splicing variants are found in POU-SPEC.DOMAIN POU HOMEODOMAIN distinct regions of the murine nerOct--factors Pit-1 vous systemzS. One of these transcripts encodes a protein consisting POU-SPEC. DOMAIN homeodornain-less almost entirely of the Oct-2 POU POU protein ? domain, hence its name MiniOct-2 (A. Stoykova and P. Gruss, pers, bTGIR commun.). During development, Comparison of two different families containing a homeodomain and a specific MiniOct-2 RNA expression is very high in the olfactory neuroepithelium. domain, In the POU and in the Pax families (U. Deutsch, pers. commun, and Ref. 16) the homeodomain is linked via a variable sequence to a domain specific for In adult brain it is found in the mitral each family. Certain members of the Pax familylack the homeodomain, whereas cell layer of the olfactory bulb. The homeodomain-less POU proteins have not been described. Examples for each olfactory system, including the mitral combination of homeodomain and specific domain are presented. cells, is the only region in the mammalian nervous system where growth the Hox and Pax gene families, no unifying regularity and differentiation of sensory neurons continue throughout adult life31. As a result, the olfactory of their expression patterns is obvious. Oct-4 (also called Oct-3 and NF-A3) and Oct-6 are elements are constantly reinervated and are kept in a expressed in early embryogenesis 17-25. Oct-4 is found proliferative state. The Pit-1/GHF-1 gene is transiently expressed in in the totipotent and pluripotent stem cells of the pregastrulation embryo and is downregulated during dif- the neural tube and later reappears in the pituitary. ferentiation of these cells, eventually becoming con- The development of this organ results in the temfined to the germ-line lineage. The expression pattern porally precise appearance of five distinct cell types of Oct-4 (detailed in the legend to Fig. 4) thus seems that are derived from a common lineage 32. In the to correlate with an undifferentiated cell phenotype, In mouse, Pit-I/GHF-1 transcripts are detected within embryonic stem (ES) and embryonal carcinoma (EC) 24 h of the first observable events in anterior pituitary cell lines, both Oct-4 and Oct-6 are downregulated differentiation (13.5 p.c.). Pit-1/GHF-1 protein is not when the cells are induced to differentiate by retinoic detected until about three days later and correlates with the onset of growth hormone and prolactin acid. In addition to its expression at early embryonic expression 29. Therefore, Pit-1/GHF-1 expression stages, Oct-6 is found in specific neurons of the devel- appears to be controlled at both the transcriptional oping and adult brain and also in testis. SCIP/tst-1, the and post-transcriptional levels. Snell dwarf (dw) and rat homolog of Oct-6, is expressed postnatally by devel- dwarf Jackson (dw.l) mice, which contain disrupted oping Schwann cells of the peripheral nervous system. Pit-1/GHF-1 genes, lack growth hormone, prolactin Because SCIP/tst-1/Oct-6 is only transiently expressed and thyroid-stimulating hormone, and show hypoplasia during the period of rapid cell division separating the of the target cell types of these hormones33. The premyelinating and myelinating phases of Schwann Pit-1/GHF-1 protein of Snell dwarf mice cannot cell differentiation, it might play a role in the progress- bind to its target sequence because the tryptophan ive determination of these cells2<27. Other POU genes residue in the recognition helix has been changed to are also expressed in a distinct temporal and spatial a cysteine. Rescue experiments using wild-type pattern in the developing rat or mouse nervous system. Pit-1/GHF-1 will show whether Pit-1/GHF-1 is a deterOct-l, Oct-2, Brn-1, Brn-2, Brn-3 and Pit-1/GHF-1 are minant of the distinct pituitary cell types. all expressed in the neural tube, and at later stages in Are Oct4 and Oct-6 'master genes'? distinct patterns in the n e r v o u s system 7,28,29. Whereas most murine developmental control genes The rat gene Brn-3 is highly homologous to unc-86 of C. elegans and is also expresmd in sensory ganglion that have been identified by sequence similarity to cells. Their similar expression patterns and protein Drosophila genes are expressed only at postimplansequences suggest that the proteins may have con- tation stages, Oct-4 and Oct-6 are expressed in the preserved roles in highly diverged organisms. The unc-86 implantation embryo. The functional analysis of Oct gene determines the development of neuronal pheno- factors in ES and EC cells may give some indications types involving sensory neurons, with loss-of function about the function of POU genes in early developmutations altering three neuroblast lineages30. These ment. Both activation and repression via the octamer
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4.5 d
Exc
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-Allantois
Exc Ee
=
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LPen ICM BC
3.5 d
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6.ha
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Z!
5.5d
TE __.....
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Expression of the Oct-4 gene in early murine development. Oct-4 mRNA is found in maturing and ovulated oocytes and not in sperm. Upon fertilization Oct-4 message decreases and is at background level at the four-cell stage. At the eight-cell stage, the mRNA level is similar to that in growing oocytes, indicating that the zygotic Oct-4 gene expression is activated between the four- and eight-cell stage. Between the 16-cell and the expanding blastocyst stage, the embryos contain abundant Oct-4 mRNA, evenly distributed throughout the embryo. In hatched blastocysts this RNA is predominantly expressed in the inner cell mass (ICM), while an intermediate level is found in the primitive endoderm and none in trophectodermal cells. After implantation, Oct-4 RNA is confined to the embryonic ectoderm and later to neuroectoderm until about day 8 p.c. At day 7 p.c., mesodermal cells have started to differentiate from the embryonic ectoderm. Very little Oct-4 mRNA is detectable in mesodermal cells or at day 8 p.c. in neuroectodermal cells, indicating that Oct-4 expression is downregulated in these cells. After 8.5 days p.c., expression cannot be detected in somatic cells but is restricted to primordial germ cells. These arise at the posterior end of the embryo where the allantois, the amniotic membrane, and the three germ layers of the embryo converge. Top: Expression is schematically shown for the oocyte (day 0), morula (day 2.5), expanding (day 3.5) and hatched (day 4.5) blastocyst, egg cylinder stage (day 5.5 and 6.5), early (day 7.5) and late (day 8.0) primitive streak stage. Orange represents intermediate or low Oct-4 expression, and red represents high expression. Green and blue coloring indicates extraembryonic tissues, green representing the proximal endoderm, blue the rest of the extraembryonic tissues. BC, blastocoel; Den, distal endoderm; Ed, endoderm; Ee, embryonic ectoderm; Exc, exocoelon; lCM, inner cell mass; M, mesoderm; Mo, morula; Mtr, mural trophectoderm; Oo, oocyte; Pen, proximal endoderm; Ptr, polar trophectoderm; TE, trophectoderm; Zp, zona pellucida, l~ttom: A sagittal section of a day 8.5 to day 9 embryo stained for alkaline phosphatase as a marker for primordial germ cells arising at the base of the allantois (left). A higher magnification shows coexpression of alkaline phosphatase (upper right) and Oct-4 (lower right), indicating that Oct-4 is expressed in primordial germ cells. Oct-4 mRNA is detected by in situ hybridization as dark grains. Bottom figure kindly provided by Gregory R. Dressier. motif have been demonstrated in EC cells 23,34. Whether the octamer confers activation or repression is determined by its context with other regulatory elements, and the amount of the Oct factor(s) present e3,24,35.
Some recent evidence suggests that there may be an interaction b e t w e e n Oct-4 and an (as yet unidentified) E1A-like factor in the early embryo. W h e n mouse embryos are infected with SV40 or polyoma
"FIGOCTOBER1991 VOL. 7 NO. 10 j,
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[]~EVIEWS virus, the early multipotent cell lineages - the same tissues that express Oct-4 - do not support viral gene expression39. The same block is observed in EC cells, where it seems to be mediated by an endogenous EIAlike factor (see Ref. 35), and it is tempting to suggest that Oct-4 and this factor might interact. Indeed, an interaction has been shown between EIA and Oct-4 cotransfected into HeLa cells: E1A acts as a bridging factor for transcriptional regulation by Oct-4 (Ref. 35). Several of the developmental control genes that are expressed after Oct-4 and Oct-6, such as H o x 1.3, harbor octamer motif(s) in their promoter regions and thus might be regulated by Oct-4 and/or Oct-6. H o x 1.3 expression is not detectable in undifferentiated EC cells but is induced during differentiation3637. Since H o x 1.3 and both Oct factors are inversely expressed, H o x 1.3 might be negatively regulated by one of the Oct factors. A possible candidate for gene activation by these Oct factors is the hst/k-FGF proto-oncogene. This gene is controlled by an enhancer within its 3' noncoding region that is active in undifferentiated but not in differentiated EC cells. Analysis of the smallest enhancer fragment conferring transcriptional activity revealed two sequences that are highly related to the octamer motif 38.
Possible roles of POUfactors in murine development These results taken together may indicate that Oct-4 acts at the beginning of a cascade of control events. Its importance for the very early embryonic stages has been demonstrated by injection of antisense Oct-4 oligonucleotides into fertilized oocytes 4°. Microinjection resulted in a loss of Oct-4 mRNA and as a consequence in an inhibition of DNA synthesis and arrest of the embryo at the one-cell stage. The block was rescued by mRNA synthesized in vitro, indicating that Oct-4 protein is required for the first embryonic cell division. A requirement for transcription at that stage seems unlikely, because the murine zygotic genome is expressed from the two-cell stage onwards. Instead, Oct-4 might regulate DNA replication at the one-cell stage. There is a precedent for POU transcription factors activating both transcription and replication41: both Oct-1 and Oct-2 can stimulate DNA replication in vitro and, as shown for Oct-l, an intact POU region suffices. In agreement with the notion that POU factors are involved in replication, some are highly expressed during periods of increased cellular proliferation. SCIP/tst-1/Oct-6 expression is transiently activated during a period of rapid cell division and thus might reflect coupling of POU protein expression with cellular proliferation. MiniOct-2 is continuously expressed in the olfactory neuroepithelium and thus might play a role in the proliferation processes of this region of the brain. The absence of a trans-activation domain in this Oct-2 variant might ensure that gene expression in these cells is not disturbed. Perhaps Unc-86 is involved in DNA replication in certain neuronal lineages, which cease proliferation when Unc-86 is missing (see above). Finally, in cell culture Oct-1 is highly expressed in rapidly dividing cells whereas expression is low when the cells are confluent (H. Sch61er, unpublished).
All the evidence available is consistent with a role for POU factors in cellular proliferation. With respect to their function during development, the question remains whether POU factors can also specify a certain cell lineage. The u n c - 8 6 mutations might give us a first hint to an answer because Unc-86 seems to represent an important component of a mechanism linking cell identity to cell lineage. One of the major tasks for the future will be to determine whether such a linkage can also be shown for mammalian POU proteins.
Acknowledgements I thank the members of the Gruss lab for many valuable discussions and comments, in particular Michael Gross, Rudi Bailing, Andreas Ptischel, Urban Deutsch and Ahmed Mansouri. Lastly, l am indebted in many respects to Peter Gruss. I also thank Anastasia Stoykova, Heidi Rohdewohld and Georges Chalepakis for the communication of unpublished results. I hope that my colleagues understand that due to the constraints of this article I regretfully was unable to cite all individual references.
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Raven Press 32 Ingraham, H.A. etal. (1990) Annu. Rev. Physiol. 52, 773-791 33 Li, S. et al. (1990) Nature 347, 528-533 34 Sch61er, H.R. et al. (1989) EMBO,L 8, 2551-2557 35 Sch61er, H.R., Ciesiolka, T. and Gruss, P. (1991) Ce1166, 291-304 36 Fibi, M. et al. (1988) Development 102, 349-359 37 Murphy, S.P. el al. (1988) Proc. Natl Acad. Sci. USA 85, 5587-5591 38 Curatola, A.M. and Basilico, C. (1990) Mol. Cell. BioL 10, 2475-2484
40 Rosner, M.H., De Santo, R.J., Arnheiter, H. and Staudt, L.M. (1991) Cel164, 1103-1110 41 Verrijzer, C.P., Kal, A.J. and van der VileR, P.C. (1990) ~ B O J . 9, 1883-1888 42 Schreiher, E. et al. (1990) Nucleic Acids Res. 18, 5495-5503
I t is now well established that genes containing a h o m e o b o x are regulatory genes coding for nuclear proteins that probably act as transcription factors 1. The h o m e o d o m a i n encoded by the h o m e o b o x is simply a protein domain capable of recognizing and binding to specific DNA sequences 2. Through the recognition properties of their homeodomain, homeoproteins are believed to regulate the expression of batteries of target genes. Among h o m e o b o x genes of vertebrates, those belonging to the Hox family show a number of interesting characteristics. First, they occur in clusters that have been confined in compact genomic regions since the remote evolutionary past. The first evidence for such clustering came from the study of Drosophila homeotic genes 3, located in two genomic regions called the Antennapedia (ANT-C) and Bithorax (BX-C) complexes. Subsequently, evidence has accumulated that these two complexes present in flies may have arisen from a single complex, called HOM-C, early in insect radiation s. The ancestral cluster corresponding to the HOM-C of insects has undergone duplications in the lineage leading to vertebrates ~',7. Higher vertebrates have four such homologous clusters, termed HOX loci in humans ~ and Hox loci in the mouse 9. Within these clusters, all Hox genes share the same transcriptional orientation 8. Hox genes were originally identified on the basis of the primary sequence of their homeodomains m. In fact, most Hox genes encode proteins containing an homeodomain closely related to the archetypal Antennapedia (Antp) homeodomain, often referred to as a class I homeodomain. However, since the resemblance is very slight for Hox genes at the ends of the four clusters (Fig. 1), it is more convenient to define Hox genes on the basis both of the primary sequence of the encoded products and of their physical location on chromosomes. Second, Hox genes appear to cooperate in providing positional information along the anteroposterior axis 9 of animals with bilateral symmetry, and perhaps even of all metazoans. This was originally recognized in flies, where homeotic genes specify the fate of segmental units within the metameric body plan3. Evidence is accumulating that they may provide positional information along other axes as well, for example in developing limb buds 11.
H.R. SCHOLER IS IN THE MAX-PLANCg INSTITUTE OFI BIOPHYSICAL CHEMISTRY,DEPARTMENTOF MOLECULARCELLI BIOLOGY, 1)-3400 GOTTINGEN, FRG; PRESENT ADDRESX" EMBL MEYERHOFSTRASSEI, D-6900 HEIDELBERG,FRG.
HOXgeneactivation by retin0ic acid EDOARDO BONCINELLI,ANTONIO S1MEONE, DARIO ACAMPORAAND FULVIOMAVIHO Vertebrate homeohox genes of the Hox family are, like Drosophila homeotic genes, organized in gene clusters and show a strict correspondence, or coUinearity, between the order of the genes (3' to 5') within the chromosomal cluster and that of their expression domains (anterior to posterior) in the embryo. Recent data obtained from embryonal carcinoma cells induced to differentiate by retinoic acid cast some light on the molecular mechanisms underlying the collinear expression of the Hox genes. Third, the genomic organization of Hox genes is reflected in their expression pattern. All the Hox genes are developmentally regulated in mouse 9, frog 12 and human 13 embryos and are expressed in partially overlapping domains. The anterior boundaries of the expression domains of the various genes of a given cluster uniformly follow a 5'-posterior/Y-anterior role along the embryonic anteroposterior body axis 1~,1s (reviewed in Ref. 4). This phenomenon was first observed for the homeotic genes in the BX-C of Drosophila and has been termed collinearity 3. A similar collinear expression pattern seems to apply to the genes of the four human HOX loci in differentiating embryonal carcinoma (EC) cells.
Human HOX gene organization So far, 38 h u m a n H O X g e n e s have b e e n identified 16. T h e y are distributed a m o n g four h o m o l o gous c h r o m o s o m a l loci, H O X I to HOX4, on chrom o s o m e s 7, 17, 12 a n d 2, respectively. Each cluster contains at least nine g e n e s organized in a h o m o l o gous linear arrangement. The 38 genes can be
Nomenclature: Hox: Vertebrate homeobox genes (species not defined). Hox: Mot,se homeobox genes. HOX: Human homeobox genes.
TIG OCTOBER]991 VOL. 7 xo. 10
S t u d i e s of Drosophila embryogenesis suggest that a sequential activation of a hierarchy of regulatory genes occurs during the development of multicellular organisms 1. These genes regulate the transformation of genetic information into morphological structure by orchestrating a precise temporal and spatial expression of effector genes, which in turn govern cell type specificities and eventually organ identity. Numerous developmental control genes have been isolated from Drosophila and Caenorhabditis elegans by genetic means. A similar approach has not been possible in vertebrates since not epough developmental mutants are available and they are hard to generate. Instead, a variety of mouse genes have been identified from their sequence similarity to Drosophila regulatory genes. Many of these, such as the Hox and Pax genes, are probably involved in pattern formation during or after gastmlation 23. An alternative approach to isolating regulatory genes is to use promoter and enhancer elements to screen for trans-activating factors playing a role in developmental processes. By this method a novel family of transcription factors, the POU family, was discovered; certain members of this family are expressed at pregastrulation stages. The POU family was defined by the sequence homology of three mammalian transcription factors and one nematode regulatory protein (Pit-1/GHF-1, Oct-l, Oct-2 and Unc-86) 4. Subsequently, several other members of this family have been identified and characterized. These proteins have two con~rved domains: a homeodomain distantly related to the prototype Antennapedia homeoo domain 5 and, nearby on the amino-temdnal side, a domain designated the POUspecific domain. The regions outside these two domains are highly divergent and contain domains required for transcriptional activation. Oct-1
Octamer-binding proteins and the POU
family
The octamer motif (ATGCAAAT) is a DNA sequence required for both ubiquitous and tissue-specific expression of various genes 6. It was first identified in the promoters of the histone H2B and the light and heavy chain immunoglobulin genes. The apparent paradox that the same element is required both for ubiquitous and B-cell-specific gene expression was resolved when two different proteins interacting with this sequence were characterized and cloned. Oct-1 was present in all cell types tested, while Oct-2 was detected only in B lymphocytes. Subsequently,
Octamania: The POU factors in murine development HANS R. SCH()LER Much effort has been directed towards the investigation of regulatory processes in the earlg mouse embryo. Several multigenefamilies of developmental control genes have been identified The POUfamily is a group of related transcription factors containing a particular type of bipartite DNA-bindingdomaim Members of this farai~ show distinct expression patterns during embryonic development. Two members, Oct.4 and Oct-6 are expressed as early as in the preimplantation embryo and thus may regulate early events of murine development several additional proteins that bind to the octamer motif have been identified at various developmental stages and in a variety of organs and tissues of the mouse (Fig. 1). Those proteins characterized so far all belong to the POU family (Table 1). cDNAs corresponding to several other POU genes have been cloned 7 but the proteins encoded by them have not been tested for DNA binding.
Structure of the POU region o~
~
Oct-1
NOct-2 i NOct-3 Oct-6
~
Oct-6
Oct-7
~
Oct-4A Oct-4B Oct-5
Oct-8~ ~ Oct-9- I Oct-10"'" MiniOct-2
The conserved POU-specific and POU homeodomains are 81 and 60 amino acids, respectively, and are separated by 14-26 variable amino acids. In the POUspecific domain, clusters of acidic amino acids are located near both ends. The POU-specific domain contains 28 invariant residues clustered in two subdomains of particularly high sequence homology (Fig. 2A), Each subdomain has two features in common: a cluster of basic amino acids in the center and a predicted (x-helix at the carboxy-terminal end. The homeodomain is a DNA-binding domain that plays a central role in eukaryotic gene regulation 5. It contains three well-defined helices, with helices 2 and 3 forming a helix-turn-helix motif similar to that in a number of prokaryotic DNA-binding proteins 8. However, the homeodomain helices are longer, and binding to DNA differs in several respects9. In the prokaryotic proteinDNA complex the second helix of the helix-turn-helix motif, often referred to as the recognition helix, makes critical
HcO
Survey of murine octamer-binding proteins with the electrophoretic mobility shift assay. Extracts of newborn mouse cerebellum and embryonic stem cells (D3) were incubated with a DNA fragment containing the octamer motif and applied to polyacrylamide gel electrophoresis. The ge] was dried and exposed to an X-ray film. These ~-o sources represent all known octamer-binding proteins. All except Oct-4and Oct 5 appear to be expressed in brain. The proteins were numbered according to their mobility, which reflects the size, charge and shape of the protein-DNA complexes. TIG OCTOBER ~ {1991 I!lst~ iur Htiun~c Publi>her~ Lid I I "KI o t 6 8
9 t ~ 9 91 NI}2 !)(I
1991 vOL 7 No. 10 I
I
f~EVIEWS
TAmE 1. Properties o f routine Oct factors Ad~tiomd
Oct
Oct-I
Oct-2
of prot,~.
~
Hllnlan: OTF-t; NF-A1; NF-III; OBP 100
743
Oct-1
Hum,aft:
Oct-2A: 451 463 Oct-2B: 583
OTF-2; NF-A2
N-Oct2~ N-Oct-2
Fa,l,no
Adult
~t~tts
Ubiquitous
Ubiquitous
ATATGATAATGA 6, 24 TAATGARAT CTCATGA (heptamer)
Neural tube; in entire brain except telencephalon
Lymphoid cells; nervous system; intestine; testis; kidney
CTCATGA (heptamer) 6,24,28,42
Nervous system
Nervous system (astrocytes, certain glioblastoma and neuroblastoma cell lines)
Nervous system
Nervous system; primary spermatids
(1) Oct-2
(7)
Human:
N4)ct2~;
MiniOct-2
232
Oct-2
(7)
N-Oct-3
352 324
(strong expression in developing nasal neuroepithelium)
kfs
24, 25, 28, 42
Nervous system
Nervous system (certain glioblastoma and neurobhstorna cell lines)
Totipotent and
Oocytes
TrAA.AATrc_&
Blastocyst; ES and EC cells: brain
Nervous system: testis
TAATGARAT 21,24-27 d TFAAAATrCA CTCATGA (heptamer)
24,25,42
17-21,23,24
Oct-4A Oct4B Oct-5
NF-A3;Oct-3
Oct-6
Rat: Tst-1; SCIP
Oct-7
Human: N-Oct4
Nervous system
Nervous system
24, 25, 42
Oc,-8 Oct-9 Oct-10
Human:
Nervous system
Nervous system (astroo/tes)
24, 25, 42
Oct4
(17)
pturipotent stem cells
of the pregastrulation embryo, embryonic ectoderm, primordial germ cells; testis, ovary 448 449
Oct45 (4)
N-Oct5a N4k't5b
aNumber of amino acids, determined from cDNA. ~ m m x ~ m e location in parentheses. cA. Stoykova and P. Gruss, pets. commun.
OH. RohdewoMd and P. Gruss, pers. commun.
contacts via residues near its amino-terminal end. In contrast, such residues in the homeodomain of the Drosophila engrailed protein are near the center of the recognition helix9. POU homeodomains contain 22 invariant residues, ten of which are identical to those in the 'classical' Antennapedia homeodomain (Fig. 2B). Clusters of basic amino acids are present at the amino- and carboxy-terminal boundaries. POU homeodomains probably also contain three ~-helices 9,1°. The recognition helix is the most conserved part of the POU homeodomain, containing 11 invariant residues. The RVWFCN motif therein harbors a cysteine residue, at position nine of the helix, that is specific for the POU family, whereas the other residues are extremely well conserved in all homeodomain proteins. A serine residue at the same position is typical of the pairedtype homeodomain, whereas the corresponding residue in most other homeodomains is glutamine. When
the cysteine residue in the recognition helix of Pit-1 is changed to a glutamine, specificity and affinity for the Pit-l-binding site are unaffected, indicating that other amino acid residues outside this region are critical for recognition n. Thus, for the POU family the term 'recognition helix' is not entirely valid for helix 3. Pairwise comparison of all POU homeodomain and POU subdomain sequences divides the POU family into five classes (Fig. 2C)7. This classification is supported by the similarity of the linker sequence between the POU-specific domain and POU homeodomain among members of classes II and III. The rat genes Brn-1 and Brn-2 probably encode proteins that bind to the octamer motif. Both are widely expressed in the developing and adult brain and are probably rat homologs of mouse Oct proteins. They are grouped in class III, with a recognition helix identical to that of Oct-6 and a POU-specific domain that differs at only one position 7.
WiG OCTOBER 1991 VOL. 7 NO. 10
]]REVIEWS
A
POU-SPECIFIC DOMAIN
I
~o I i
EE-S
SUBDOMAINA ] 5o SUBDOMAINB 8° ] 2O 30 40 60 70 ALG vL . . . . v~S Q srZl sCR F E * LQ LSFNNK AvAMCK E LEQFAK LF KvQ RKR I zKLG TQ*N° ~lt~..~ L TMa |K-L.~RI~I L SK"'LE - A D ¥ ¥t:TE vN
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C
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LE
R
K
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F
class III
Oct-6, Brn-1, Bin-2, Cfla, Ceh-6
c,ass,v
Brn-3, Unc-86
class V
Oct-4
FIG~ Consensus sequence of the POU region and highlighted structural properties. The POU region consists of two well-conserved domains: the POU-specific domain (A) and POU homeodomain (B), connected by a variable linker sequence of 14-26 amino acids. The POU-specffic domain contains subdomains A and B, two regions of particularly high homology. Each subdomain has a predicted (x-helix at the carboxy-terminal boundary. The three helices indicated in the POU homeodomain correspond to those established for Antennapedia and engrailed homeodomains; positions identical to the 'classical' Antennapedia homeodomain are underlined with thick bars. Large letters represent residues found in all known POU proteins; two small letters represent two possible amino acids at that position, the upper letter representing the one found more frequently; a single small letter represents the predominant amino acid when more than two are found at that position. Hyphens indicate variable positions. Clusters of acidic (+) and basic (-) amino acid residues are indicated. According to the homology between the POU domains and POU subdomains, POU proteins can be subdivided into five classes (C). The classification holds for the linker sequences of classes II and III, but not for class IV.
Function of the POU region Whereas an intact homeodomain is required for DNA binding of all POU proteins, the contribution of the POU-specific domain varies, depending both on the DNA-binding site and on the POU protein. The POU homeodomain of Pit-1 is sufficient for low-affinity binding to AT-rich DNA sequences, but addition of the POU-specific domain increases both the affinity and specificity of binding to DNA1L The recognition helix and the predicted helix A must be intact for efficient DNA binding, whereas (in contrast to the engrailed homeodomain9) the other helices play only a minor role. The contribution of the Oct-1 h o m e o d o m a i n and POU-specific domain to DNA binding has been studied in detail for the Ad2 octamer motif 12,13. The POU-specific domain and the homeodomain each make half of the base contacts with the octamer sequence, whereas the majority of the backbone contacts are made by the homeodomain. Since base contacts determine the sequence specificity of a DNA-binding protein, while the binding energy stems mainly from electrostatic interactions between the protein and the DNA backbone 14, the h o m e o d o m a i n of Oct-1 contributes most of the binding energy, whereas the POUspecific domain provides half of the specificity for the octamer motif. The Oct-1 homeodomain binds with significantly lower affinity to the octamer motif than does the complete POU region. However, with other Oct-lbinding sites (Table 1), addition of the POU-specific domain of Oct-1 has no effect or can decrease affinity 12. The POU-specific domain is also required for protein-protein interactions 11, which can confer additional regulatory specificity. An inhibitory effect of one POU factor on another has recently been described for two
Drosophila proteins, I-POU and Cfl-a (Ref. 15). Cfl-a is monomeric in solution but dimeric when it binds to the Cfl-a regulatory site in the dopa decarboxylase gene. I-POU, which is closely related to Unc-86, lacks two of the five basic residues that are present in the amino-terminal portion of other POU homeodomains. Since these residues are critical for binding to DNA, I-POU alone remains as a m o n o m e r in solution. Coexpression of both factors leads to stable heterodimers, resulting in inhibition of DNA binding and suppression of transcription. Both POU regions are presumably required for dimerization, although this has only been shown for the POU region of Cfl-a. The POU-specific domain does not contain a known DNA-binding structure, and attempts to show binding of an isolated POU-specific domain to certain DNA motifs have failed. However, this does not exclude the possibility that the POU-specific domain can bind by itself to some other DNA motif. In this respect, it may be helpful to compare POU proteins with members of the Pax family 16. The paired domain of these proteins is associated with a paired-type home0domain in certain Drosophila and mouse proteins, perhaps constituting a domain analogous to the POU region (Fig. 3). Interestingly, several Pax proteins lacking a homeodomain bind via their paired domain to specific DNA sequences that are distinct from homeodomain-binding sites (G. Chalepakis and P. Gruss, pers. commun.). Perhaps homeodomain-less POU proteins also exist, although none has so far been reported.
Expression of POU factors in mouse development Most if not all POU genes are differentially expressed throughout embryogenesis. However, unlike
"rig OCTOBER'1991 vot. 7 NO. 10
m
~'~EVIEWS mutations prevent the differentiation of mother cells into daughter cells: HOMEODOMAIN [ SPECIFICDOMAIN one of the two daughter cells of variable 60 I prototypestructure a division fails to assume its normal fate and instead propagates the phenotype of the mother cell. Thus PAIREDHOMEODOMAIN PAIREDDOMAIN Unc-86 represents an important com128 I 60 ~--- Pax-3;Pax-6; ponent of a mechanism linking- cell variable Pax-7 identity to cell lineage30. PAIREDDOMAIN The expression patterns of some Pax-1; Pax-2; 128 POU genes are increased in Pax-8 complexity by differential splicing. Oct-2 splicing variants are found in POU-SPEC.DOMAIN POU HOMEODOMAIN distinct regions of the murine nerOct--factors Pit-1 vous systemzS. One of these transcripts encodes a protein consisting POU-SPEC. DOMAIN homeodornain-less almost entirely of the Oct-2 POU POU protein ? domain, hence its name MiniOct-2 (A. Stoykova and P. Gruss, pers, bTGIR commun.). During development, Comparison of two different families containing a homeodomain and a specific MiniOct-2 RNA expression is very high in the olfactory neuroepithelium. domain, In the POU and in the Pax families (U. Deutsch, pers. commun, and Ref. 16) the homeodomain is linked via a variable sequence to a domain specific for In adult brain it is found in the mitral each family. Certain members of the Pax familylack the homeodomain, whereas cell layer of the olfactory bulb. The homeodomain-less POU proteins have not been described. Examples for each olfactory system, including the mitral combination of homeodomain and specific domain are presented. cells, is the only region in the mammalian nervous system where growth the Hox and Pax gene families, no unifying regularity and differentiation of sensory neurons continue throughout adult life31. As a result, the olfactory of their expression patterns is obvious. Oct-4 (also called Oct-3 and NF-A3) and Oct-6 are elements are constantly reinervated and are kept in a expressed in early embryogenesis 17-25. Oct-4 is found proliferative state. The Pit-1/GHF-1 gene is transiently expressed in in the totipotent and pluripotent stem cells of the pregastrulation embryo and is downregulated during dif- the neural tube and later reappears in the pituitary. ferentiation of these cells, eventually becoming con- The development of this organ results in the temfined to the germ-line lineage. The expression pattern porally precise appearance of five distinct cell types of Oct-4 (detailed in the legend to Fig. 4) thus seems that are derived from a common lineage 32. In the to correlate with an undifferentiated cell phenotype, In mouse, Pit-I/GHF-1 transcripts are detected within embryonic stem (ES) and embryonal carcinoma (EC) 24 h of the first observable events in anterior pituitary cell lines, both Oct-4 and Oct-6 are downregulated differentiation (13.5 p.c.). Pit-1/GHF-1 protein is not when the cells are induced to differentiate by retinoic detected until about three days later and correlates with the onset of growth hormone and prolactin acid. In addition to its expression at early embryonic expression 29. Therefore, Pit-1/GHF-1 expression stages, Oct-6 is found in specific neurons of the devel- appears to be controlled at both the transcriptional oping and adult brain and also in testis. SCIP/tst-1, the and post-transcriptional levels. Snell dwarf (dw) and rat homolog of Oct-6, is expressed postnatally by devel- dwarf Jackson (dw.l) mice, which contain disrupted oping Schwann cells of the peripheral nervous system. Pit-1/GHF-1 genes, lack growth hormone, prolactin Because SCIP/tst-1/Oct-6 is only transiently expressed and thyroid-stimulating hormone, and show hypoplasia during the period of rapid cell division separating the of the target cell types of these hormones33. The premyelinating and myelinating phases of Schwann Pit-1/GHF-1 protein of Snell dwarf mice cannot cell differentiation, it might play a role in the progress- bind to its target sequence because the tryptophan ive determination of these cells2<27. Other POU genes residue in the recognition helix has been changed to are also expressed in a distinct temporal and spatial a cysteine. Rescue experiments using wild-type pattern in the developing rat or mouse nervous system. Pit-1/GHF-1 will show whether Pit-1/GHF-1 is a deterOct-l, Oct-2, Brn-1, Brn-2, Brn-3 and Pit-1/GHF-1 are minant of the distinct pituitary cell types. all expressed in the neural tube, and at later stages in Are Oct4 and Oct-6 'master genes'? distinct patterns in the n e r v o u s system 7,28,29. Whereas most murine developmental control genes The rat gene Brn-3 is highly homologous to unc-86 of C. elegans and is also expresmd in sensory ganglion that have been identified by sequence similarity to cells. Their similar expression patterns and protein Drosophila genes are expressed only at postimplansequences suggest that the proteins may have con- tation stages, Oct-4 and Oct-6 are expressed in the preserved roles in highly diverged organisms. The unc-86 implantation embryo. The functional analysis of Oct gene determines the development of neuronal pheno- factors in ES and EC cells may give some indications types involving sensory neurons, with loss-of function about the function of POU genes in early developmutations altering three neuroblast lineages30. These ment. Both activation and repression via the octamer
--{
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Exc Ee
=
M
LPen ICM BC
3.5 d
I
6.ha
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Z!
5.5d
TE __.....
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FIG~I
Expression of the Oct-4 gene in early murine development. Oct-4 mRNA is found in maturing and ovulated oocytes and not in sperm. Upon fertilization Oct-4 message decreases and is at background level at the four-cell stage. At the eight-cell stage, the mRNA level is similar to that in growing oocytes, indicating that the zygotic Oct-4 gene expression is activated between the four- and eight-cell stage. Between the 16-cell and the expanding blastocyst stage, the embryos contain abundant Oct-4 mRNA, evenly distributed throughout the embryo. In hatched blastocysts this RNA is predominantly expressed in the inner cell mass (ICM), while an intermediate level is found in the primitive endoderm and none in trophectodermal cells. After implantation, Oct-4 RNA is confined to the embryonic ectoderm and later to neuroectoderm until about day 8 p.c. At day 7 p.c., mesodermal cells have started to differentiate from the embryonic ectoderm. Very little Oct-4 mRNA is detectable in mesodermal cells or at day 8 p.c. in neuroectodermal cells, indicating that Oct-4 expression is downregulated in these cells. After 8.5 days p.c., expression cannot be detected in somatic cells but is restricted to primordial germ cells. These arise at the posterior end of the embryo where the allantois, the amniotic membrane, and the three germ layers of the embryo converge. Top: Expression is schematically shown for the oocyte (day 0), morula (day 2.5), expanding (day 3.5) and hatched (day 4.5) blastocyst, egg cylinder stage (day 5.5 and 6.5), early (day 7.5) and late (day 8.0) primitive streak stage. Orange represents intermediate or low Oct-4 expression, and red represents high expression. Green and blue coloring indicates extraembryonic tissues, green representing the proximal endoderm, blue the rest of the extraembryonic tissues. BC, blastocoel; Den, distal endoderm; Ed, endoderm; Ee, embryonic ectoderm; Exc, exocoelon; lCM, inner cell mass; M, mesoderm; Mo, morula; Mtr, mural trophectoderm; Oo, oocyte; Pen, proximal endoderm; Ptr, polar trophectoderm; TE, trophectoderm; Zp, zona pellucida, l~ttom: A sagittal section of a day 8.5 to day 9 embryo stained for alkaline phosphatase as a marker for primordial germ cells arising at the base of the allantois (left). A higher magnification shows coexpression of alkaline phosphatase (upper right) and Oct-4 (lower right), indicating that Oct-4 is expressed in primordial germ cells. Oct-4 mRNA is detected by in situ hybridization as dark grains. Bottom figure kindly provided by Gregory R. Dressier. motif have been demonstrated in EC cells 23,34. Whether the octamer confers activation or repression is determined by its context with other regulatory elements, and the amount of the Oct factor(s) present e3,24,35.
Some recent evidence suggests that there may be an interaction b e t w e e n Oct-4 and an (as yet unidentified) E1A-like factor in the early embryo. W h e n mouse embryos are infected with SV40 or polyoma
"FIGOCTOBER1991 VOL. 7 NO. 10 j,
)=-
[]~EVIEWS virus, the early multipotent cell lineages - the same tissues that express Oct-4 - do not support viral gene expression39. The same block is observed in EC cells, where it seems to be mediated by an endogenous EIAlike factor (see Ref. 35), and it is tempting to suggest that Oct-4 and this factor might interact. Indeed, an interaction has been shown between EIA and Oct-4 cotransfected into HeLa cells: E1A acts as a bridging factor for transcriptional regulation by Oct-4 (Ref. 35). Several of the developmental control genes that are expressed after Oct-4 and Oct-6, such as H o x 1.3, harbor octamer motif(s) in their promoter regions and thus might be regulated by Oct-4 and/or Oct-6. H o x 1.3 expression is not detectable in undifferentiated EC cells but is induced during differentiation3637. Since H o x 1.3 and both Oct factors are inversely expressed, H o x 1.3 might be negatively regulated by one of the Oct factors. A possible candidate for gene activation by these Oct factors is the hst/k-FGF proto-oncogene. This gene is controlled by an enhancer within its 3' noncoding region that is active in undifferentiated but not in differentiated EC cells. Analysis of the smallest enhancer fragment conferring transcriptional activity revealed two sequences that are highly related to the octamer motif 38.
Possible roles of POUfactors in murine development These results taken together may indicate that Oct-4 acts at the beginning of a cascade of control events. Its importance for the very early embryonic stages has been demonstrated by injection of antisense Oct-4 oligonucleotides into fertilized oocytes 4°. Microinjection resulted in a loss of Oct-4 mRNA and as a consequence in an inhibition of DNA synthesis and arrest of the embryo at the one-cell stage. The block was rescued by mRNA synthesized in vitro, indicating that Oct-4 protein is required for the first embryonic cell division. A requirement for transcription at that stage seems unlikely, because the murine zygotic genome is expressed from the two-cell stage onwards. Instead, Oct-4 might regulate DNA replication at the one-cell stage. There is a precedent for POU transcription factors activating both transcription and replication41: both Oct-1 and Oct-2 can stimulate DNA replication in vitro and, as shown for Oct-l, an intact POU region suffices. In agreement with the notion that POU factors are involved in replication, some are highly expressed during periods of increased cellular proliferation. SCIP/tst-1/Oct-6 expression is transiently activated during a period of rapid cell division and thus might reflect coupling of POU protein expression with cellular proliferation. MiniOct-2 is continuously expressed in the olfactory neuroepithelium and thus might play a role in the proliferation processes of this region of the brain. The absence of a trans-activation domain in this Oct-2 variant might ensure that gene expression in these cells is not disturbed. Perhaps Unc-86 is involved in DNA replication in certain neuronal lineages, which cease proliferation when Unc-86 is missing (see above). Finally, in cell culture Oct-1 is highly expressed in rapidly dividing cells whereas expression is low when the cells are confluent (H. Sch61er, unpublished).
All the evidence available is consistent with a role for POU factors in cellular proliferation. With respect to their function during development, the question remains whether POU factors can also specify a certain cell lineage. The u n c - 8 6 mutations might give us a first hint to an answer because Unc-86 seems to represent an important component of a mechanism linking cell identity to cell lineage. One of the major tasks for the future will be to determine whether such a linkage can also be shown for mammalian POU proteins.
Acknowledgements I thank the members of the Gruss lab for many valuable discussions and comments, in particular Michael Gross, Rudi Bailing, Andreas Ptischel, Urban Deutsch and Ahmed Mansouri. Lastly, l am indebted in many respects to Peter Gruss. I also thank Anastasia Stoykova, Heidi Rohdewohld and Georges Chalepakis for the communication of unpublished results. I hope that my colleagues understand that due to the constraints of this article I regretfully was unable to cite all individual references.
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I t is now well established that genes containing a h o m e o b o x are regulatory genes coding for nuclear proteins that probably act as transcription factors 1. The h o m e o d o m a i n encoded by the h o m e o b o x is simply a protein domain capable of recognizing and binding to specific DNA sequences 2. Through the recognition properties of their homeodomain, homeoproteins are believed to regulate the expression of batteries of target genes. Among h o m e o b o x genes of vertebrates, those belonging to the Hox family show a number of interesting characteristics. First, they occur in clusters that have been confined in compact genomic regions since the remote evolutionary past. The first evidence for such clustering came from the study of Drosophila homeotic genes 3, located in two genomic regions called the Antennapedia (ANT-C) and Bithorax (BX-C) complexes. Subsequently, evidence has accumulated that these two complexes present in flies may have arisen from a single complex, called HOM-C, early in insect radiation s. The ancestral cluster corresponding to the HOM-C of insects has undergone duplications in the lineage leading to vertebrates ~',7. Higher vertebrates have four such homologous clusters, termed HOX loci in humans ~ and Hox loci in the mouse 9. Within these clusters, all Hox genes share the same transcriptional orientation 8. Hox genes were originally identified on the basis of the primary sequence of their homeodomains m. In fact, most Hox genes encode proteins containing an homeodomain closely related to the archetypal Antennapedia (Antp) homeodomain, often referred to as a class I homeodomain. However, since the resemblance is very slight for Hox genes at the ends of the four clusters (Fig. 1), it is more convenient to define Hox genes on the basis both of the primary sequence of the encoded products and of their physical location on chromosomes. Second, Hox genes appear to cooperate in providing positional information along the anteroposterior axis 9 of animals with bilateral symmetry, and perhaps even of all metazoans. This was originally recognized in flies, where homeotic genes specify the fate of segmental units within the metameric body plan3. Evidence is accumulating that they may provide positional information along other axes as well, for example in developing limb buds 11.
H.R. SCHOLER IS IN THE MAX-PLANCg INSTITUTE OFI BIOPHYSICAL CHEMISTRY,DEPARTMENTOF MOLECULARCELLI BIOLOGY, 1)-3400 GOTTINGEN, FRG; PRESENT ADDRESX" EMBL MEYERHOFSTRASSEI, D-6900 HEIDELBERG,FRG.
HOXgeneactivation by retin0ic acid EDOARDO BONCINELLI,ANTONIO S1MEONE, DARIO ACAMPORAAND FULVIOMAVIHO Vertebrate homeohox genes of the Hox family are, like Drosophila homeotic genes, organized in gene clusters and show a strict correspondence, or coUinearity, between the order of the genes (3' to 5') within the chromosomal cluster and that of their expression domains (anterior to posterior) in the embryo. Recent data obtained from embryonal carcinoma cells induced to differentiate by retinoic acid cast some light on the molecular mechanisms underlying the collinear expression of the Hox genes. Third, the genomic organization of Hox genes is reflected in their expression pattern. All the Hox genes are developmentally regulated in mouse 9, frog 12 and human 13 embryos and are expressed in partially overlapping domains. The anterior boundaries of the expression domains of the various genes of a given cluster uniformly follow a 5'-posterior/Y-anterior role along the embryonic anteroposterior body axis 1~,1s (reviewed in Ref. 4). This phenomenon was first observed for the homeotic genes in the BX-C of Drosophila and has been termed collinearity 3. A similar collinear expression pattern seems to apply to the genes of the four human HOX loci in differentiating embryonal carcinoma (EC) cells.
Human HOX gene organization So far, 38 h u m a n H O X g e n e s have b e e n identified 16. T h e y are distributed a m o n g four h o m o l o gous c h r o m o s o m a l loci, H O X I to HOX4, on chrom o s o m e s 7, 17, 12 a n d 2, respectively. Each cluster contains at least nine g e n e s organized in a h o m o l o gous linear arrangement. The 38 genes can be
Nomenclature: Hox: Vertebrate homeobox genes (species not defined). Hox: Mot,se homeobox genes. HOX: Human homeobox genes.
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