111

Mechanisms o f Development, 39 (1992) 111-123

© 1992 Elsevier Scientific Publishers Ireland, Ltd. 0925-4773/92/$05.00 MOD 00123

The mouse Enhancer trap locus 1 ( Etl-1): a novel mammalian gene related to Drosophila and yeast transcriptional regulator genes Raija Soininen a, Michael Schoor a, Ulf Henseling a,1, Claudia Tepe Brigitte Kisters-Woike b, Janet Rossant c and Achim Gossler a

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a Max-Delbriick-Laboratorium in der Max-Planck-Gesellschaft, KEln, FRG, t, Institut fiir Genetik der Universitiit zu KEln, Weyertal, KEln, FRG and c Samuel LunenfeM Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada

(Received 10 July 1992; accepted 13 August 1992)

A novel mouse gene, Enhancer trap locus 1 (Etl-1), was identified in close proximity to a iacZ enhancer trap integration in the mouse genome showing a specific fl-galactosidase staining pattern during development. In situ analysis revealed a widespread but not ubiquitous expression of Etl-1 throughout development with particularly high levels in the central nervous system and epithelial cells. The amino acid sequence of the Etl-1 protein deduced from the cDNA shows strong similarity, over a stretch of 500 amino acids, to the Drosophila brahma protein involved in the regulation of homeotic genes and to the yeast transcriptional activator protein SNF2 / SWI2 as well as to the RAD54 protein and the recently described helicase-related yeast proteins STH1 and MOT1. Etl-1 is the first mammalian member of this group of proteins that are implicated in gene regulation a n d / o r influencing chromatin structure. The homology to the regulatory proteins SNF2/SWI2 and brahma and the expression pattern during embryogenesis suggest that Etl-1 protein might be involved in gene regulating pathways during mouse development. Enhancer trap; Mouse; Development; Brahma-like protein; SNF2-1ike protein

Introduction One objective of developmental genetic studies is the identification and isolation of genes that are involved in the control of differentiation processes during development. In mouse various approaches have been pursued towards this aim. Developmentally important genes have been isolated based on analysis of mutants (e.g. Woychik et al., 1985; Herrmann et al., 1990), based on differential cDNA screening protocols (e.g. Poirier et al., 1991) and, most successfully, based on sequence homologies to known developmental control genes in other species (for reviews see Kessel and Gruss, 1990; McGinnis and Krumlauf, 1992). More

Correspondence to: A. Gossler, Max-Delbriick-Laboratorium in der

Max-Planck-Gesellschaft, Carl-von-Linn~ Weg 10, 5000 K61n 30, FRG. 1 Present address: Boehringer Mannheim, Nonnenwald, 8122 Penzberg, FRG.

recent strategies use various types of lacZ reporter gene constructs to detect and isolate genes which are expressed in spatially and temporally regulated patterns during embryogenesis (Allen et al., 1988; Kothary et al., 1988; Gossler et al., 1989; Skarnes, 1990; Friedrich and Soriano, 1991; Skarnes et al., 1992; Von Melchner et al., 1992). We have focused on the use of enhancer trap constructs to detect and isolate genes differentially expressed during early mouse embryogenesis. Enhancer trap constructs carry the lacZ gene under the control of a weak promoter. Upon integration into the genome the promoter can be activated by endogenous cis-acting elements close to or at the site of integration leading to expression of the reporter gene. This strategy is based on the assumption that these elements regulate endogenous genes whose expression is visualized by the reporter gene. Enhancer traps to detect genes expressed in patterns during development have first been developed in Drosophila (O'Kane and Gehring, 1987). By P-element mediated transposition thousands of Drosophila strains

112 carrying enhancer trap integrations in their genome have been generated and analysed for/3-galactosidase staining patterns. About 65% of the integrations were transcriptionally activated in patterns during development (Bellen et al., 1989; Bier et al., 1989). A high proportion of the /3-galactosidase staining patterns reflected expression patterns of endogenous genes whose control elements directed lacZ expression in a manner indistinguishable from or very similar to that of the endogenous gene (Wilson et al., 1989), although exceptions have been reported (Bier et al., 1990). In mouse, lacZ enhancer trap vector expression was first analysed in transgenic mice produced by DNA microinjection (Allen et al., 1988; Kothary et al., 1988). About 20-30% of such integrations gave rise to specific /3-galactosidase expression patterns during embryogenesis. These experiments demonstrated that lacZ reporter gene constructs can be used to detect transcriptional activation patterns in mouse embryos and suggested that it might be feasible to detect and isolate genes differentially expressed during early mouse development with the help of enhancer trap integrations. However, thus far cloning of an endogenous gene from transgenic mouse lines showing lacZ staining patterns has not been reported. We have devised a strategy employing embryonic stem (ES) cell chimaeras to analyze enhancer trap integrations for staining patterns. This strategy involves the introduction of enhancer trap vectors in ES cells by electroporation, which results in many cases in single-copy-integrations of the vector. Clonal ES cell lines representing independent integrations are then used to produce chimeric embryos which after subsequent development in foster females are stained for /3-galactosidase activity (Gossler et al., 1989). Cell lines carrying enhancer trap vectors that either expressed or did not express detectable levels of /3-galactosidase in undifferentiated stem cells have been analyzed in chimaeras. With both types of cell lines staining patterns were observed at a high frequency (Korn et al., 1992). In this report we describe the analysis at the molecular level of an enhancer trap integration (cell line 6028) that expressed the lacZ gene in undifferentiated stem cells and that gave rise to a spatially and temporally regulated staining pattern in embryos starting at around day 8 of development (see Fig. 5 in the accompanying paper of Korn et al. (1992) and Fig. 2 in Gossler et al., 1989). In close proximity to the enhancer trap integration site in cell line 6028 we have identified a novel mouse gene which we call Etl-1 ( E n h a n c e r trap locus 1) gene. Etl-1 provides the first example of a mouse gene isolated from a lacZ enhancer trap integration and supports the validity of this approach in mouse. The deduced protein sequence encoded by the Etl-1 gene shows strong similarities to several proteins that are implicated in gene regulation or chro-

matin structure modulation such as the transcriptional activator S N F 2 / S W I 2 of Saccharomyces cereuisiae (Laurent et al., 1991), the regulator of homeotic genes, brahma, in Drosophila. (Tamkun et al., 1992), the RAD54 protein (Emery et al., 1991) and the helicaserelated yeast proteins STH1 and M O T I (Davis et al., 1992; Laurent et al., 1992). Etl-1 is the first mammalian member of this group of proteins. The Etl-1 gene is expressed in many cell types throughout development and in adult tissues at highly variable levels suggesting functions in multiple processes during embryogenesis.

Results

Isolation of flanking sequences, wild type locus and cDNA clones The 6028 cell line was generated by electroporating the enhancer trap construct 6LSN into D3 embryonic stem (ES) cells (Gossler et al., 1989). Southern blot analyses using enhancer trap construct fragments as probes showed that the cells carry in their genome one copy of the construct (data not shown). To clone the integration site, the 3' end of the 6LSN construct and about 700 bp genomic DNA flanking the integration site were amplified by ligation mediated PCR and cloned as described in the Experimental Procedures. A 500-bp genomic fragment of the PCR clone, called A H / H 500, was used as a probe to analyze DNA from 6028 cells and untransfected ES cells. RFLPs were detected with several restriction digestions indicating that the cloned DNA fragment was derived from the integration site (Fig. IA). A genomic clone containing the pre-integration site was isolated from a C 5 7 B L / 6 mouse genomic library using A H / H 5 0 0 as a probe. The restriction map of the pre-integration site is consistent with Southern and RFLP data obtained with genomic DNA from 6028 and untransfected ES cells (Fig. 1B). No rearrangements were detected at this level of analysis. The integration site was localized to an internal EcoR1 fragment that was partially sequenced, and at one end of this fragment the sequence was identical to the sequence of the PCR amplified fragment (except some single base changes probably due to the different genetic background of the two DNA sources). Several unique probes were generated from different regions of the pre-integration site and analyzed further. Two of them, 1.1 H / S and A H / H 5 0 0 (Fig. 1B), hybridized to DNA from zebrafish, Xenopus, chicken, rabbit and man (not shown) and were therefore used to screen a mouse day 12.5 embryo cDNA library (provided by Dr. A. Joyner, Toronto). The screen with the probe 1.1H/S yielded two clones that

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were used to rescreen the day 12.5 and a day 8.5 cDNA library (gift of Dr. B. Hogan, Nashville) and probes from the resulting overlapping clones were used to extensively screen these and a random primed cDNA

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a Fig. 2. Northern blot with poly(A) + RNA from embryonal carcinoma F9 cells hybridized with a probe covering 2.4 kb of the coding sequence (bp 220 to bp 2636) (a) and a probe 3' to the first polyadenylation signal (bp 4805 to bp 5095) showing that the two transcripts are derived from the Etl-1 gene due to differential usage of two polyadenylation signals in the 3' region of the mRNA (b). Sizes of the RNA markers in 1000 nucleotides are shown at the right. 4.8 is the level of 28S rRNA.

library from mouse Sendl cells (provided by Drs. Vestweber and Weller, Freiburg). The resulting clones comprised 5.1 kb of cDNA sequence that contained the 3' end of the m R N A including a poly(A) tail and an open reading frame that started at the 5' end of the cDNA sequence but no consensus sequence for a start of translation. Thus far, cDNA clones extending the sequence further 5' have not been obtained. Probes from the coding region of the Etl-1 cDNA detected two transcripts of about 5.0 and 5.4 kb in Northern blot analysis with RNA from F9 embryonal carcinoma cells whereas a 300-bp probe from the very 3' end of the cDNA detected only the larger transcript (Fig. 2) indicating that the shorter transcript arises from the use of the first of two polyadenylation signals which are present 447 nucleotides apart in the untranslated region of the mRNA. Since the isolated cDNA contains the 3' end of the larger of the two Etl-1 transcripts and covers 5.1 kb, we estimate that about 300 bases are missing from the 5' end of the complete mRNA. To obtain additional information about the 5' end of the Etl-1 gene, a 1.8-kb genomic D N A fragment hybridizing to the most 5' cDNA fragment was sequenced. An exon of 212 bp, an intron of 244 bp and an exon containing the 5' most cDNA sequence were present in the genomic sequence which extended the open reading frame of the cDNA (Fig. 1C) but the exact site of the start of transcription has not been unambiguously characterized so far. The Etl-1 sequence derived from the overlapping cDNA clones comprises 5105 bp and is shown in Fig. 3. The sequence begins with a long open reading frame of 3408 bp which can encode a protein of 1136 amino acids. Even if most of the uncloned 5' sequences were

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Fig. 3. Sequence of the Etl-1 cDNA and the deduced amino acid sequence of the EtL1 protein. The termination codon is indicated by an asterisk. The two polyadenylation signals are underlined. Locations of introns at the 5' end of the gene are indicated by arrowheads. coding, the protein can be expected to extend only about 5 0 - 8 0 a m i n o acids further towards the Nterminus. Thus, most o f the c o d i n g s e q u e n c e is present in the c D N A c l o n e s that have b e e n o b t a i n e d thus far.

Expression of Etl-1 during det~elopment Expression of the Etl-1 g e n e during e m b r y o g e n e s i s was analyzed by R N A in situ hybridization on frozen

115 sections of embryos from days 10.5, 12.5, 15.5 and 18 of development. Etl-1 expression was widespread and Etl-1 mRNA was detected in many cell types and tissues throughout the embryo at all analyzed stages. However, the expression levels varied considerably between different cell types and tissues and in some tissues levels changed significantly during development. In the central nervous system Etl-1 transcripts were abundant throughout embryogenesis. At day 10 and 12.5 Etl-1 was expressed uniformly throughout all parts of the brain and spinal cord at comparable levels (Fig. 4) whereas beginning at day 15.5 levels were highest in the retina and in various parts of the brain (for details see Figs. 5, 6; Table 1). Etl-1 mRNA was also abundant in various epithelia of ectodermal, mesodermal and endodermal origin. Expression in the epidermis was first detected at day 12.5 with particularly high levels in cells on the snout and distal on fore- and hind-limbs. This expression was maintained during later stages of development where the basal layer of the epidermis was found to be the main Etl-1 expressing cell type in the skin. Also the oral and nasal epithelia expressed Etl-1 at clearly detectable levels from day 12.5 onwards. In these epithelia Etl-1 expression remained very similar throughout development, whereas the epithelium of the gut showed the highest level of expression on day 15.5 and lower levels at earlier and later stages (Fig. 5h). Between day 10 and 12.5 Etl-1 transcripts were also abundant in mesenchymal cells of the branchial bars and limb buds (Fig. 4), whereas at later stages mesenchymal tissue showed no or only low levels of Etl-1 expression. High levels of Etl-1 expression on days 15.5 and 18.5 were seen also in thymus, teeth and salivary gland, whereas heart, skeletal muscle and connective and bone tissue showed no or barely detectable levels of expression (Figs. 5, 6; Table 1) throughout development. Additionally, Etl-1 transcripts were detected in trophectodermal tissue (see Fig. 4) and uterine epithilium ceils (not shown). The results of the in situ hybridizations are compiled and compared to the/3-galactosidase staining in Table 1. Many aspects of the complex expression pattern of Etl-1 during development were reflected by the /3galactosidase staining pattern of the enhancer trap integration in 6028 ceils whereas some were not. Particularly at early stages of development cells expressing Etl-1 at high levels did not always show high levels of /3-galactosidase activity, e.g., liver and forebrain. At later stages the staining pattern resembled the distribution of Etl-1 transcripts more closely (see Table 1). For example, there were high levels of/3-galactosidase and Etl-1 in the basal layer of the epidermis, the salivary gland, eye and gut although there were still tissues such as thymus, forebrain and developing teeth in which Etl-1 transcripts were abundant but /3-galactosidase activity was low or undetectable. No tissues

have been found thus far that expressed the lacZ gene but not Etl-1. Similarities between Etl-1 and other proteins The Etl-1 cDNA begins with a long open reading frame which could encode a protein of 1136 amino acids. This makes up at least 90% of the Etl-1 protein assuming that up to 100 amino acids are missing from the N-terminus of the protein. No motives such as transmembrane regions, zinc fingers, leucine zippers or homeodomains were found in the sequence. However, using the TFASTA program (Pearson and Lipman, 1988) we have found five proteins in data base searches that are related to the protein encoded by Etl-1. Strong homology was found to the SNF2 protein of Saccharomyces cerevisiae (Laurent et al., 1991) and to the brahma protein of Drosophila. (Tamkun et al., 1992) which both code for transcriptional regulators. Additional proteins with strong homology to the Ed-1 protein are the helicase-related yeast proteins STH1 (Laurent et al., 1992) and MOT1 (Davis et al., 1992) as well as the less similar RAD54 protein (Emery et al., 1989). The strong homology between Etl-1 and these five proteins is confined to the 540 amino acids in the C-terminal half of the Etl-1 protein where the overall similarity ranges from 63 to 53% (Fig. 7). When compared individually, between 40 and 32% of the amino acid residues in the C-terminus of Etl-1 are identical to amino acid residues in the corresponding domains of the other proteins (Fig. 7). Within this region several stretches of striking homology and identity are found, where up to 11 out of 20 amino acids are identical in all six proteins (LNGILADEMGLGKTIQxI 'motif' in Fig. 7, region 1 in Fig. 8). The proteins SNF2, STH1 and brahma are most similar to Etl-1, 17 out of 20 amino acids being identical in the most conserved region I (Fig. 8). Parts of the homologous regions resemble the seven motifs found in known and putative helicases as indicated by superscript roman numbers in Fig. 8, and include a motif very similar to a sequence found in ATP and GTP binding proteins. The spacing between some of the motifs however, differs between the members of this group of proteins. In particular, the distance between region IV and V is larger than in (putative) helicases. Etl-1 contains about 150 additional residues and brahma, SNF2, STH1, MOT1 and RAD54 about 100 additional residues in that region as compared to about 30 residues in known or putative helicases used to generate a consensus sequence by Gorbalenya et al. (1989). SNF2 and brahma exhibit homology in two additional smaller stretches in the amino terminal part of the proteins (Tamkun et al., 1992). The corresponding regions are also similar in Etl-1 and show a better homology to brahma than to SNF2. However, Etl-1 is

116 400-500 amino acids shorter than SNF2 and brahma, and the region corresponding to a fourth domain of homology between SNF2 and brahma in their C-terminal region ('bromodomain', T a m k u n et al., 1992) is absent from Etl-1.

Discussion LacZ enhancer trap constructs have successfully been employed in Drosophila to detect and isolate genes expressed in patterns during fly development (Bellen et al., 1989; Bier et al., 1989, 1990; Wilson et al., 1989). Previous experiments with enhancer trap constructs in transgenic mice (Allen et al., 1988) and ES cell chimaeras (Gossler et al., 1989) suggested that the isolation of novel genes whose expression is spatially and temporally regulated during development might also be feasible in m o u s e - - h o w e v e r , an analysis of enhancer trap integrations at the molecular levels has yet not been reported. H e r e we described the isolation of the first mouse gene from the integration site of a lacZ enhancer trap insertion that gave rise to a specific staining pattern during embryogenesis and demonstrate that novel mouse genes can be identified and isolated by this approach. Transcribed sequences were detected about 2.5 kb upstream of the reporter gene integration, and this integration does not interfere with Etl-1 gene expression (M. Schoor and A. Gossler, unpublished). In accordance with this, mice homozygous for the enhancer trap integration display no phenotypic abnormalities (Korn et al., 1992). This suggests that the reporter gene integrated outside the coding region of Etl-1 potentially close to or even into the regulatory region of the gene. The lacZ staining pattern does not completely reflect Etl-1 expression. This may be due to the reporter gene responding to only part of the regulatory elements of the Etl-1 gene, or to enhancers from other genes further 5' or 3' to the integration site influencing expression of the reporter gene. Several lines of evidence argue in favour of the first possibility. First, lacZ expression is observed only in tissues which express Etl-1. At no stage did we find tissues that expressed the lacZ gene but did not express Etl-1. If regulatory elements of genes other than Etl-1 directed lacZ expression one might expect to find tissues that ex-

pressed the lacZ but not the Etl-1 gene. Second, while Etl-1 expression was clearly more widespread than the staining at earlier stages of development, the correlation became better at later stages and in some tissues the staining pattern and the distribution of Etl-1 transcripts are superimposable. The response to some but not all regulatory elements of Etl-1 by the reporter gene could be due to the opposite direction of transcription of Etl-1 and the lacZ gene. In addition, some distinct regulatory elements directing Etl-1 expression in different tissues or at different stages during embryogenesis may exert their influence on the reporter gene while others do not, the result being a staining pattern that reveals many hut not all aspects of the Etl-1 expression pattern during development. The protein encoded by the the Etl-1 gene shows in its C-terminal half strong homology to the brahma protein of Drosophila (Tamkun et al., 1992) and to the yeast proteins SNF2 (Laurent et al., 1991), STH1 (Laurent et al., 1992), MOT1 (Davis et al., 1992) and R A D 5 4 (Emery et al., 1991). Brahma and SNF2 encode transcriptional activators (Laurent et al., 1991; T a m k u n et al., 1992), M O T I acts, formally, as a negative regulator of transcription (Davis et al., 1992) and R A D 5 4 is involved in D N A repair and mitotic recombination ( G a m e and Mortimer, 1974). The function of STH1 is not known so far. The regions of homology between these proteins contain all seven protein signatures assigned to known or putative helieases but are not restricted to these signatures. Thus, these proteins might comprise the first members of a novel group of (nuclear) proteins related to but distinct from the two suggested helicase superfamilies (Gorbalenya et al., 1989). In many cases structural similarities point to similar biological functions. Data available thus far for SNF2 and brahma suggest that also in this case these proteins may have similar biological functions. SNF2 is a nuclear protein required for transcription of many differently regulated genes in S. cerer,isiae and is required for healthy growth and sporulation of diploid cells but is not essential for cell viability (Winston et al., 1987; Happel et al., 1991; Laurent et al., 1991). The Drosophila b r a h m a protein acts as an activator of multiple homeotic genes of the A n t e n n a p e d i a and Bithorax complex and is involved in the maintenance of the spatially restricted expression patterns of these genes

Fig. 4. In situ hybridization of cross-sections of a day 10 embryo with its deciduum (a-e) and sagittal sections of a day 12.5 embryo hybridized with an antisense (a, d, g, i) and sense probe (c) (a, c, d, g, i, darkfield photographs; b, e, h, j, brightfield photographs). The planes of sections in b and e are indicated in f; i and j show details of g and h. Strong Etl-1 expression is found throughout the developing central nervous system at both stages, additionally, high levels of Etl-I transcripts are present at day 10 in the mesenchyme of the maxillary process (mp), mandibular arches (ma) and the limb buds (lb), on day 12.5 in ectodermal cells around the snout and limb buds, in liver and spinal ganglia. Etl-1 mRNA was detected in the placenta outside the embryo, fl, forelimb bud; h, heart; hi, hind limb bud; lb, limb bud; li, liver; mp, maxillary process; ma, mandibular arch; me, mesencephalon; pl, placenta; re, rhombencephalon; sc, spinal cord; sg, spinal ganglion; t. tail; ve, vertebra, bar: a-e 250 /~m; g,h 1 mm; i,j, 150 p.m.

117

!

:,

.

118 ( T a m k u n et al., 1992). brm is an essential gene and flies h o m o z y g o u s for strong alleles die as u n h a t c h e d larvae w i t h o u t obvious s e g m e n t a t i o n defects or h o m e o t i c transformations. T h e lack of segmentation defects was suggested to be due to the large maternal contribution of the brm gene p r o d u c t ( T a m k u n et al., 1992). S N F 2 acts in concert with o t h e r proteins, notably SNF5 and SNF6, and it has b e e n p r o p o s e d that these and additional proteins may be parts of a multiprotein complex necessary for transcriptional activation (Laurent et al., 1991). S N F 2 is identical to SWI2, a gene required for expression of the H O gene and is thus

essential for mating type switching in S. cerevisiae ( T a m k u n et al., 1992). S N F 2 / S W I 2 antagonizes the negative regulator of H O expression SIN1 (Kruger and Herskowitz, 1991) which e n c o d e s a non-specific D N A binding protein related to H M G 1 , a non-histone chromosomal protein. SIN1 is t h o u g h t to repress H O expression by influencing chromatin structure (Kruger and Herskowitz, 1991). Similarly, Polycomb, a negative regulator of homeotic genes in Drosophila, may also repress gene activity by influencing chromatin structure. It has been suggested that Pc is involved in establishing heterochromatin-like complexes in certain regions of c h r o m o s o m e s and thus maintains repressed

ii

1

!

Fig. 5 In situ hybridization of sagittal sections of a day 15.5 embryo hybridized with an antisense Etl-1 probe (a, d, h, i, j, darkfield photographs; b, c, d, e, f, brightfield photographs), (c-j) show details of (a) and (b), the areas that are enlarged are framed in (b). Etl-1 transcripts are detected in many tissues throughout the embryo. Particularly high levels of transcripts in addition to the central nervous system were detected in epithelial cells of the nasal chambers (c,d), of the gut (e,h), and in the epidermis (gJ) as well as in thymus and salivary gland (f,i). gu, gut; he, heart; li, liver; sg, salivary gland; th, thymus, bar, a,b 1 mm; c-j, 200/xm.

119 states of genes within these regions (Paro, 1990; Gaunt and Singh, 1990). Brahma has been isolated in a genetic screen as a suppressor of polycomb mutations

and like the trithorax gene (Capdevila and Garcia-Bellido, 1981; Kennison and Tamkun, 1988), brm counteracts the repressing activity of polycomb (Tamkun et

Fig. 6. In situ hybridization of sagittal and cross-sections of a day 18.5 embryo hybridized with an antisense Etl-1 probe (a and c, darkfield photographs; b and d, brightfield photographs). The line in b indicates the approximate plane of section in c and d. Etl-1 transcripts are most abundant in all parts of the central nervous system and spinal ganglia, thymus, salivary gland and epithelial cells, bl, bladder; he, heart; ki, Kidney; li, liver; lu, lung; sc, spinal cord; sg, salivary gland; th, thymus, bar, 1 mm.

120

al., 1992) as does SNF2/SWI2 for SIN1. Together, the

homeotic gene expression during development after these patterns have initially been set up by the segmentation genes.

polycomb and trithorax group genes maintain but do not establish the spatially

restricted patterns of

TABLE I Summary of Etl-1 expression between day 10 and 18.5 of development as assessed by RNA in situ hybridization and/3-galactosidase staining in embryos heterozygous for integration 6028 obtained after germ line transmission Tissue

Etl-1 Expression in embryos on day 10

Telencephalon + Olfactory bulb / Hippocampus / Cerebrum / Mesencephalon + Rhombencephalon + Pons / Cerebellum / Spinal cord Ependymal layer + Mantel layer + Spinal ganglia + Eye + Retina / Inner nuclear layer / Outer nuclear layer / Pigmented layer / Lens epithelium + Lacrimal gland / Inner ear Sensory epithelia / Lung / Bronchi epithelium / Connective tissue / Gut + Epithelium / Mesenchyme / Kidney / Glomeruli / Mesenchyme / Liver / Spleen / Integument Stratum germinativum / Hair follicles (ext. root sheet) / Dermis / Thymus / Submandibular gland / Teeth / Dental papilla / Ameloblasts / Odontoblasts / Bones/cartilage / Periosteum / Vertebrae / Long bones / Skull / Muscles Trunk muscles / Limb muscles / Heart muscle + Diaphragm /

12.5 +

+ +

+ +

+

15.5

lacZ Expression in embryos on day 18.5

+ + / / / + + + + / /

+ + na na na + + + + na na

+ + + + + + / / / / + + /

+ + + + +

+ + + + + +

+ + na na na + + na

+ + + + +

/ + / /

na + na na

+ + /

+ + + +

+ + + + +

+ + + +

+ + + + + +

+ + + ±

10

12.5

15.5

+ / / / + + ++ / /

+

+

/

na

+

/

na

+ +

/

fla

+

+ +++

+ ++

+

/

na

+ +

/

na

+++

+ + + + / / / / + /

+ + + + / / / / + /

+ + +

++ + +

++ ++ ++ ++ + ++

++ ++ ++ + ++

/ / / / _+

/

na

/

na

++ + +

++

++

+

+

/

+ + +

+ +

/

/

+

±

/

+ + ± + na

+ + _+ + +

+ + + + _+ + + + + + + na na na

+ + + + +

_+ / /

+ + _+ + _+

+ + _+ + +_

/ / / / / / / / / / / / / / / / / / / /

na na _+ /

na na + -

+ _+ -

/ / /

/ + / / + + / + / + + / / / / / / / _+

+ + + + + +

+_ + + + + + +

18.5

na

/ / +

±

i

/ + / +++

na

na

++ +++

+++ +++

/

+++

+++

/

+

/ / /

na na na

/ /

++ ++

/ /

/

+ + + , high; + + , moderate; + , low; +_, barely detectable; - , no Etl-1 expression or/3-galactosidase staining; / , tissue not yet discernable; ha, not analysed in detail.

121 I LNGILADEMGLGKTIQ

[

Etl-1

brm

[ 33%

SNF2/SWI2

I 24%I/41%S

23% I / 38% S

[

STH1

40% I / 62% S I

39% I / 63% S

MOT1

[

-! 36% I / 59% S

RAD54

[

-1

II 32% I / 53% S

Fig. 7. Schematic comparison showing the regions of homology between the Etl-1 and brahma, SNF2, STH1, MOT1 and RAD54 proteins. Stippled parts indicate regions homologousto Etl-1. The percentage of identical (%1) amino acids and the overall similarity(%S) between the various proteins and Etl-1 in the stippled area is given below each box. Comparisons were done using the GCG package (Devereuxet al., 1984). The hatched areas in brm, SNF2 and STH1 indicate a gap present in all three proteins as compared to Etl-1. The broken line at the N-terminus of Etl-1 indicates the additional residues missing from the sequence. The most conserved sequence is indicated above Etl-1 and proteins have been aligned around this sequence. The sequence homology of the Etl-1 protein to the SNF2 and brahma proteins suggests that Etl-1 may serve functions similar although not neccessarily identical to (brm) and SNF2. Thus, Etl-1 could be the first mammalian member of a group of transcriptional activators that antagonizes gene repression caused by modulation of chromatin structure. The identification of Etl-1 and the recent isolation of mouse genes with homology to the Drosophila polycomb genes Pc (Pearce et al., 1992) and Psc (van Lohuizen et al., 1991) lend support to the idea that, in mouse as well as in Drosophila, determined states may be achieved and inherited in daughter cells by the combined action of proteins maintaining either repressed (polycomb-like proteins) or activated (brm-like proteins) states of gene expression once specific expression patterns have been set up during development. It is an intriguing possibility that Etl-1, like brm, might be involved in activating homeobox genes or in maintaining their active state during embryogenesis. The widespread but not ubiquitous expression of Etl-1 would be consistent with a role in transcriptional activation of a number of genes which are otherwise differentially regulated by distinct transcriptional regulators. If functionally similar to SNF2 and brm, the Etl-1 protein might act also in a complex with other proteins. We are currently raising antibodies against different parts of the Etl-1 protein to address this question. The predicted Etl-1 protein is considerably shorter than SNF2 and brahma and contains only three out of four regions of homology shared by these two proteins suggesting that Etl-1 may share

some but not all functional properties with SNF2 and brm. Generating mice mutated in the Etl-1 gene will address these and other questions related to Etl-1 function in gene regulation during development.

Experimental Procedures Cloning of the flanking genomic sequences The D N A from 6028 ceils was digested into completion with EcoRI, then ligated overnight. 100 ng of the ligated D N A was used as a template in the PCR reaction. Primers were from the neo region of the construct, 'neo 5' out', G G A G A A C C T G C G T G C A A T C C , and 'neo 3' out', C T C A T G C T G G A G T T C I T C G C , and amplification conditions were 2 min at 94°C, 2 min at 60°C, 10 min at 72°C, 40 cycles. The amplified fragment of 1.3 kb was digested with EcoRI and XbaI which cut in the construct sequence so that only a 250-bp SV40 polyadenylation sequence remained joined to the genomic flank. The generated 0.8-kb fragment was cloned into pUC vector. Digestion of this clone with HinclI generated a unique 500-bp fragment, A H / H 5 0 0 .

Isolation and characterization of the genomic and cDNA clones The genomic C 5 7 B L / 6 mouse library in E M B L 3 was screened with the A H / H 5 0 0 probe under stan-

122 dard conditions (Sambrook et al., 1989). D N A from clones hybridizing to A H / H 5 0 0 was isolated and characterized by restriction mapping. Genomic fragments z l H / H 5 0 0 and H / S 1.1 were used to screen a mouse day 12.5 embryonic c D N A library under standard conditions, and inserts from isolated clones were used to further screen day 8.5 and Send-1 c D N A libraries. Fragments of the genomic clone and inserts from the isolated c D N A clones were subcloned into the Bluescript vector and sequenced using Sequenase Kit (United States Biochemicals) and vector derived universal and reverse primers, or primers derived from within the c D N A or genomic sequence.

in a 1% agarose-2.2 M formaldehyde gel (Sambrook et al., 1989). The gel was stained with ethidium bromide to visualize the markers and the R N A was transferred to a Hybond membrane (Amersham) and hybridized in 50% formamide, 2 × SSC, 0.1% SDS, salmon sperm D N A 50 p~g/ml, 1 × Denhardt's solution at 42°C. Filters were washed to the stringency of 0.5 × SSC, 0.1% SDS.

In situ hybridization Embryos were washed once in PBS, embedded in Tissue Tec and frozen at - 2 0 ° C . 0.5-10-/xm sections were made at - 15-18°C, mounted on pretreated slides (soaked in 2% Hellmanex o / n , washed 30 min in running water, dipped in g e l a t i n e / c h r o m a l u n solution and baked for 2 h at 250°C) and air-dryed at 55°C. The probe covered bp 220 to bp 3096 of the c D N A and was prepared as described in Wilkinson and Green (1990). In situ hybridization was carried out under stringent conditions essentially as described in Wilkinson and Green (1990). After dehydration the slides were processed for autoradiography using Kodak NTB2 emulsion diluted 1:1 in water and exposed between 2-3 weeks at 4°C. Slides were developed for 3 rain in

RNA isolation and Northern blot analysis Poly(A) enriched R N A was isolated directly from mouse F9 cells according to the instructions of the Fast Track m R N A Isolation Kit (Invitrogen). Cell lysis buffer was 0.01 M Tris (pH 7.5), 4 mM E D T A , 0.5% SDS, Prot. K 100 /xg/ml; binding buffer 0.01 M Tris, (pH 7.5), 4 mM E D T A , 0.5% SDS, 0.5 M LiCI, and elution buffer 0.01 M Tris, 4 mM E D T A . For Northern analysis, 10/xg of poly(A) R N A and 4 /xg R N A size marker (Promega) was electrophoresed

I ETLt BRM SNF2 STHI MOTt

~'''E'OF : ~?SN. IDYL'A'AFNTSSEOPL'''EKAL'':: C1" :..... OM''P'KA'PFKLPIA. E TGGWNR'0 ....... ::KAT : L': 'O'T''N'R'

Con=

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ETLI ~N LC v . OEE KQIRFNIHNKYEDY E~RM . . . . . =ATKI. SNF2 . . . . . . . . . NE ~ . . . . . CG STHI MOT1 B ~ I , , I F .... I.l,,l~,r RPQLSOADI, ~ADS4 N T L T P L A V D G K K S S M G G I N T T V S Q A I H A W A Q A Q G R N I V I Con=

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EL .

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C .

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N G .

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DE

.

.

.

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DL . .

.

RVSGKFELLOR . . . . . .

.

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LPKLKA . . . .

.

SQHRVLLF .

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ETLI BRM SNF2 STHI MOT1 RAD~

Conl

ER _

sig

ELLR -

FNAPDSD

FCFLLSTRAGGLGLNLQTAOTVIIFD

-

DWNPHQDLOAQDRAHRIGOKNEVRVLRLITTGSVEE

K

o

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RA .

KL .

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ID .

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.

.

FD

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.

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L

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o

Fig. 8. Sequence alignment of the Etl-1 and brahma, SNF2, STH1, MOT1 and RAD54 proteins in the C-terminal region of Etl-l. The alignment

starts at position 571 in Etl-1. The consensus sequence calculated for the six proteins is given below the alignment as well as the signatures for the helicase superfamily members as deduced by Gorbalenya et al. (1989). The seven regions containing the motifs found in the helicase supeffamilies are indicated by bars and roman numbers above the sequence. When more than one amino acid or motif sequence was present at a position in the helicase signature the residue or motif identical or most similar to the consensus sequence between the six proteins is shown. * indicates hydrophobic(I, L, V, M, F, Y, W), o, charged or polar residues (S, T, D, E, N, Q, K, R).

123 Kodak D19 developer, washed for 1 min in 1% acidic acid and fixed for 3 min in 30% thiosulfate. Sections were stained with Giemsa and embedded with Eukitt.

Acknowledgements We are grateful to Dr. Alexandra Joyner for providing the day 12.5, Dr. Brigid Hogan for the day 8.5 embryo cDNA library, to Drs. Dietmar Vestweber and Andreas Weller for the Send-1 cDNA library and to Dr. Kenji Imai for providing the genomic C57BL/6 library. R.S was supported by an EMBO long term fellowship. This work was supported by the Ministry of Research and Technology, FRG (A.G.) and the National Institutes of Health Grant 5R01HD2533403 (J.R.).

References Allen, N.D., Cran, D.G., Barton, S.C., Hettle, S., Reik, W. and Surani, M.A. (1988) Nature 333, 852-855. Bellen, H.J., O'Kane, C.J., Wilson, C., Grossniklaus, U., Kurth Pearson, R. and Gehring, W.J. (1989) Genes Dev. 3, 1288-1300. Bier, E., Vaessin, H., Shepherd, S., Lee, K., McCall, K., Barbel, S., Ackerman, L., Carretto, R., Uemura, T., Grell, E., Jan, L.Y. and Jan Y.N. (1989) Genes Dev. 3, 1273-1287. Bier, E., Jan, L.Y. and Jan Y.N. (1990) Genes Dev. 4, 190-203. Capdevila, M.P. and Garcia-Bellido, A. (1981) Roux's Arch. Dev. Biol. 190, 339-350. Davis, J.L., Kunisawa, R. and Thorner, J. (1992) Mol. Cell. Biol. 12, 1879-1892. Devereux, J., Haeberli, P. and Smithies, O. (1984) Nucleic Acids Res. 12, 387-394. Emery, H.S, Schild, D., Kellogg, D.E. and Mortimer, R.K. (1991) Gene 104, 103-106. Friedrich, G., and Soriano, P. (1991) Genes Dev. 5, 1513-1523. Game, J.C. and Mortimer, R.K. (1974) Mutat. Res. 24, 281-291. Gaunt J.S. and Singh, P.B. (1990) Trends Genet. 6, 208-212. Gorbalenya, A.E., Koonin, E.V., Donchenko, A.P. and Blinov, V.M. (1989) Nucleic Acids Res. 17, 4713-4730. Gossler, A., Joyner, A.L., Rossant, J. and Skarnes, W.C. (1989) Science 244, 463-465.

Happel, A.M., Swanson, M.S. and Winston, F. (1991) Genetics 128, 69-77. Herrmann, B.G., Labeit, S., Poustka, A., King, T.R. and Lehrach, H. (1990) Nature 343, 617-622. Hope, I.A. (1991) Development 113, 399-408. Kennison, J.A. and Tamkun, J.W. (1988) Proc. Natl. Acad. Sci. USA 85, 8136-8140. Kessel, M. and Gruss, P. (1990) Science 249, 374-379. Korn, R., Schoor, M., Neuhaus, H., Henseling, U., Soininen, R., Zachgo, J. and Gossler, A. (1992) Mech. Dev. 39, 95-109. Kothary, R., Clapoff, S., Brown, A., Campbell, R., Peterson, A. and Rossant, R. (1988) Nature 335, 435-437. Kruger, W. and Herskowitz, I. (1991) Mol. Cell. Biol. 11, 4135-4146. Laurent B.C., Treitel, M.A. and Carlson, M. (1991) Proc. Natl. Acad. Sci. USA 88, 2687-2691. Laurent B.C., Yang, X. and Carlson, M. (1992) Mol. Cell. Biol. 12, 1893-1902. McGinnis, W. and Krumlauf, R. (1992) Cell 68, 283-302. O'Kane, C.J. and Gehring, W.J. (1987) Proc.Natl.Acad.Sci. USA 84, 9123-9127. Paro, R. (1990) Trends Genet. 6, 416-421. Pearce, J.J.H., Singh, P.B. and Gaunt, S.J. (1992) Development 114, 921-929. Pearson, W.R. and Lipman, D.J. (1988) Proc. Natl. Acad. Sci. USA, 85, 2444-2448. Poirier, F., Chan, C.-T.J., Timmons, P., Robertson, E.J. and Rigby, P.W.J. (1991) Development 113, 1105-1114. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY. Skarnes, W.C. (1990) Bio/Technol. 8, 827-831. Skarnes, W.C., Auerbach, A. and Joyner, A.L. (1992) Genes Dev. 6, 903-918. Tamkun, J.W., Deuring, R., Scott, M.P., Kissinger, M., Pattatucci, A.M., Kaufman, T.C. and Kennison, J.A. (1992) Cell 68, 561-572. Van Lohuizen, M., Frasch, M. Wientjens, E. and Berns, A. (1991) Nature 353, 353-355. Von Melchner, H., Degregori, J.V., Rayburn, H., Reddy, S., Friedel, C. and Ruley, H.E. (1992) Genes Dev. 6, 919-927. Wilkinson, D., G. and Green J. (1990) In: Postimplantation mammalian embryos (A.J. Copp and D.L. Cockroft, Eds.) IRL Press, Oxford. Wilson, C., Kurth Pearson, R., Bellen, H.J., O'Kane, C.J., Grossniklaus, U. and Gehring, W.J. (1989) Genes Dev. 3, 1301-1313. Winston, F., Dollard, C., Malone, E.A., Clare, J., Kapakos, J.G., Farabaugh, P. and Minehart, P.L. (1987) Genetics 115, 649-656. Woychik, R.P., Stewart, T.A., Davis, L.G., D'Eustachio, P. and Leder, P. (1985) Nature 318, 36-40.