Growth and Early Postimplantation Defects in Mice Deficient for the Bromodomain-Containing Protein Brd4{dagger}

In a gene trap screen we recovered a mouse mutant line in whichan insertion generated a null allele of the Brd4 gene. Brd4belongs to the Fsh/Brd family, a group of structurally relatedproteins characterized by the association of two bromodomainsand one extraterminal domain. Members of this family includeBrd2/Ring3/Fsrg1 in mammals, fs(1)h in Drosophila, and Bdf1in Saccharomyces cerevisiae. Brd4 heterozygotes display pre-and postnatal growth defects associated with a reduced proliferationrate. These mice also exhibit a variety of anatomical abnormalities:head malformations, absence of subcutaneous fat, cataracts,and abnormal liver cells. In primary cell cultures, heterozygouscells also display reduced proliferation rates and moderatesensitivity to methyl methanesulfonate. Embryos nullizygousfor Brd4 die shortly after implantation and are compromisedin their ability to maintain an inner cell mass in vitro, suggestinga role in fundamental cellular processes. Finally, sequencecomparisons suggest that Brd4 is likely to correspond to theBrd-like element of the mediator of transcriptional regulationisolated by Y. W. Jiang, P. Veschambre, H. Erdjument-Bromage,P. Tempst, J. W. Conaway, R. C. Conaway, and R. D. Kornberg(Proc. Natl. Acad. Sci. USA 95:8538-8543, 1998) and the Brd4mutant phenotype is discussed in light of this result. Together,our results provide the first genetic evidence for an in vivorole in mammals for a member of the Fsh/Brd family.

INTRODUCTION

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Introduction

The bromodomain is a conserved 110-amino acid motif which specificallyinteracts with acetyl-lysines, at least in the context of shorthistone H3 and H4 peptides (reference 9; see also reference39). Although bromodomains have now been found in more than40 different proteins (13, 17, 39), the function of this motifis poorly understood. The association of two N-terminal bromodomainswith a C-terminal extraterminal (ET) domain defines the Fsh/Brdsubgroup (23), which includes members in many species: Saccharomycescerevisiae (Bdf1 and Bdf2) (5, 23, 24), Caenorhabditis elegans(cefsh) (36), Drosophila [fs(1)h, previously called fsh (Flybasenomenclature)] (14), and hagfish (hffsh; GenBank accession no.AF191032). In vertebrates, four members of the Fsh/Brd subgrouphave now been identified: Brd2/RING3/fsrg1 (1, 32, 36), Brd3/ORFX/fsrg2(37), Brd4/HUNK1/MCAP (8), and Brd5/BRDT (19).

The yeast Bdf1 protein interacts with the general transcriptionfactor TFIID (24) as well as with histones H3 and H4 (28), andthe Bdf1 mutant phenotype shares features with those of mutantsaffected in general transcription (23). Bdf1 mutants displaya reduced rate of vegetative growth, failure to undergo oneor both meiotic divisions, and sensitivity to the DNA-damagingagent methyl methanesulfonate (MMS). Some alleles of Drosophilafs(1)h cause partial or complete loss of segments and homeotictransformations in progeny of mutant females (10, 14), but littleis known about the molecular properties of its product. In vertebrates,the human Brd2 protein has been identified as a mitogen-activatednuclear protein associated with a kinase activity (6, 30, 32)and a regulator of E2F-dependent cell cycle genes (7).

In a gene trap screen designed to identify genes involved inmouse development, we recovered an integration producing a nullallele of Brd4/MCAP, another member of the Brd/Fsh family (8).We showed that the Brd4 nullizygous condition results in earlyembryonic lethality. We also demonstrated that only one functionalallele of Brd4 is not able to sustain normal development. Heterozygosityfor Brd4 leads to pre- and postnatal growth defects that areassociated with reduced proliferation in vitro and in vivo.Together, our results provide the first genetic evidence ofan in vivo role for a Fsh/Brd family member in mammals.

MATERIALS AND METHODS

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Materials and Methods

Gene trap and rapid amplification of cDNA ends (RACE). Production of gene trap embryonic stem (ES) cells and the genetrap vector (a kind gift of W. Skarnes) was performed as describedpreviously (4). The vector contained a splice acceptor sequenceupstream of the CD4 transmembrane domain sequence, a lacZ-neomycin-phosphotransferasefusion (ß-geo), and a polyadenylation signal suchthat integrations into introns of transcriptionally active genesproduce a ß-galactosidase fusion protein that containsan endogenous sequence amino terminal to the site of insertion.The inclusion of a transmembrane domain-encoding region in thevector was designed to enrich for insertions downstream of secretorysignal sequences of type I transmembrane domains, because ß-galactosidaseactivity is only preserved when the enzyme is retained in thecytoplasm (34). Therefore, an unexpected aspect of our resultswhile using this vector was the isolation of a nuclear protein(34). In a systematic screen using a similar vector, J. Brennanand W. Skarnes isolated genes coding for nonsecreted proteinsin 5 out of 12 cases, 3 of these involving insertion of thevector into the part of the gene corresponding to the 5' untranslatedregion (UTR), as is the case for Brd4. In these cases, a functionalß-geo protein was presumably produced via internalinitiation of translation (reference 33 and J. Brennan, personalcommunication).

RACE was performed as described previously (4). The ST132 RACEfragment was used to screen an embryonic day 13 (E13) testiscDNA library (27) by standard procedures. The cDNA sequencematched precisely the MCAP sequence described in the study citedin reference 8. The 3' UTR of the 3.5-kb Brd4 clone was obtainedby 3' RACE using a Marathon-Ready cDNA kit (Clontech) and wascloned into a Topo II vector (Invitrogen). Reverse transcription-PCR(RT-PCR) was performed using an Advantage One-Step RT-PCR Kit(Clontech) (primer 1f, AAA TCA GCT CAC CAG GCTG T; primer 1r,TCT TGG GCT TGT TAG GGT TG; primer 2r, CAC CAC CAG GTT CAC TTCCT). Sequence comparison of the different members of the Brdfamily was performed with ClustalX software (17, 35). Sequencealignment of the Ring3-like peptides of the mediator with thedifferent members of the Brd family was performed with Lasergenesoftware (DNASTAR).

Mouse strains. CGR8 ES cells (129/Ola) were used to generate the gene trapcell lines (4, 34). Chimeras and their heterozygous progenywere backcrossed for five to eight generations onto a C57BL6/Jbackground. Heterozygous individuals were obtained by matinga Brd4^gt/Brd4⁺ male with a C57BL6/J female in order to avoidpossible maternal effects of the Brd4^gt allele. The day of detectionof the vaginal plug was defined as E0.

Radiation hybrid mapping. The mouse-hamster radiation hybrid (RH) panel was used accordingto the supplier's instructions (Research Genetics). Notch31f(AGG GAC CTG TGT GGA TGG CGT GAG C) was used as an upstreamprimer and Notch31r (CCA TTC TGA CAG GGT GCC TGG CTG C) wasused as a downstream primer to specifically amplify a 228-bpfragment from the mouse Notch3 locus. 18f (CAA TCT GAC TGA GCTGTT CAT ATA GGT C) was used as an upstream primer and 18r (GTTGTA GAC ATT TGG GAG GTT TCT AGT C) was used as a downstreamprimer to specifically amplify a 414-bp fragment from the mouseBrd4 locus. Maps and extensive information on mouse RH can befound in The Jackson Laboratory RH database site (http://lena.jax.org/resources/documents/cmdata/).

Histology, antibody staining, and in situ hybridization. The Brd4 common probe contains the first 1,820 nucleotides ofthe published cDNA (NCBI accession number NM 0205508). X-Galstaining was performed as described previously (16). For insitu hybridization on blastocyst explants, blastocysts weregrown on gelatinized Lab-Tek chamber slide (catalog no. 177445;Nunc) in ES cell culture medium without leukemia-inhibitingfactor (LIF) (15) for 2 to 5 days. The resulting culture wasrinsed twice in phosphate-buffered saline, fixed for 4 h infresh 4% paraformaldehyde in phosphate-buffered saline, washedtwice in phosphate-buffered saline, and hybridized as previouslydescribed (25). The anti-phospho-histone H3 antibody (catalogno. 06570; Upstate Biotechnology) was used according to thespecifications of the supplier (5 µg/ml on paraffin sectionswith antigen retrieval). For eosin-hematoxylin staining, organswere fixed in Bouin's fixative. After fixation, samples wereprocessed, embedded in paraffin, sectioned, and stained accordingto standard procedures. For skeleton preparations, skulls from2-month-old males were skinned, dehydrated in 95% ethanol for24 h, fat dissolved in acetone for 24 h, cleared in 2% KOH forabout 3 h, and stained for 3 days in 0.015% alcian blue-0.005%alizarin red-5% acetic acid in 60% ethanol. The skulls werethen cleared in 2% KOH.

Mouse embryonic fibroblast culture and proliferation. Mouse embryonic fibroblasts were isolated and cultured as describedin the study cited in reference 15 except that minced embryoswere only trypsinized once at 37°C for 10 min and spun downfor 5 min and the pellet was plated. For the proliferation test,1.00 x 10⁵ cells were harvested at day 0 and cells were countedat different time points by using a hemocytometer. The MMS wassupplied by Aldrich (catalog no. 12,992-5). UV irradiation wasdone in a Stratalinker apparatus using 254-nm bulbs.

RESULTS

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Results

Isolation and characterization of the Brd4 gene. Isolation of the Brd4 gene was performed as follows. ST132 wasan ES cell clone selected from a gene trap screen originallydesigned to identify integrations into genes expressed duringearly murine organogenesis (4, 34). After demonstration of theexpression of the lacZ reporter in midgestation chimeras, amouse strain carrying the ST132 integration was established(see Materials and Methods).

5' RACE was used to clone a 209-bp fragment of the trapped genefrom the ST132 cell line. This fragment showed no open readingframe or obvious homology to any sequence from the databases.A probe corresponding to the ST132 5' RACE product was usedto isolate a 1,890-bp clone from a mouse embryonic testis cDNAlibrary (27). The sequencing of the complete cDNA was achievedby a combination of expressed sequence tag alignments, RT-PCR,and 3' RACE (Fig. 1a). The junction between the 5' RACE fragmentand the cDNA sequence, as well as the junction between the 5'RACE fragment and the gene trap sequence, was confirmed by RT-PCR(Fig. 1b). While this work was in progress, the same cDNA wasreported under the name MCAP (mitotic chromosome-associatedprotein (8). However, we have designated it Brd4 for reasonsof consistency with the nomenclature used for the members ofthis family, as recommended by the Mouse Gene Informatics NomenclatureCommittee.

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FIG. 1. The gene trap strategy and Brd4 gene. (a) Structure of the Brd4 and Brd4^gt transcripts. A long Brd4 mRNA (6 kb, encoding a 1,400-amino acid protein) is transcribed from 20 exons (1 to 20), and a short Brd4 mRNA (3.5 kb, encoding a 722-amino acid protein) is formed by the use of an alternative 13th exon (13S). The fusion transcript containing the first two noncoding exons from Brd4 fused to the gene trap sequences is transcribed from the Brd4^gt allele. 1f, 1r, and 2r are the specific primers used for RT-PCR in Fig. 1b. Dotted boxes, UTR; plain boxes, coding exons; nis, nuclear localization signal; TM, transmembrane domain. (b) RT-PCR with primers 1f (5' RACE fragment specific), 1r (Brd4-specific coding sequence), and 2r (gene trap-specific primer). Primers 1f and 2r (1f-2r) amplified the expected 338-bp product in total RNA from Brd4^gt/Brd4⁺ (h) but not Brd4⁺/Brd4⁺(w) individuals. Primers 1f and 1r (1f-1r) amplified the expected 256-bp fragment in total RNA from both wild types and heterozygotes. (c and d) Brd4⁺/Brd4⁺ (c) and Brd4^gt/Brd4^gt (d) blastocyst explants cultured for 2 days and hybridized with a probe covering exons 1 to 9 and part of exon 10. No transcript could be detected in the homozygote.

The Brd4 gene is transcribed from 20 exons. The Brd4 gene encodes a putative 1,400-amino acid protein characterizedby the presence of two bromodomains in the N-terminal regionof the protein and an ET domain in the more C-terminal region.The presence of several nuclear localization sequences is inagreement with the reported nuclear localization of the protein(8). The presence of several PEST sequences suggests that Brd4may be unstable (31).

Sequence comparison of the murine Brd4 cDNA (NCBI accessionnumber NM 0205508) with murine genomic sequences (GenBank accessionno. AC074208, AC073765, and AC073779) revealed that Brd4 istranscribed from 20 exons spanning at least 110 kb (GenBankaccession no. AF461395) (Fig. 1a). As noted in reference 8,the Brd4 gene shares the highest sequence identity with theuncharacterized human HUNK1 cDNA (NCBI accession number NM 014299),which encodes a protein of 722 amino acids, which is much smallerthan Brd4. Using 3' RACE performed on the E11 cDNA library andRT-PCR procedures performed on adult tissues, we were able toreveal the existence of an alternative 13th exon (13S [short])that encodes three C-terminal amino acids as well as an alternative3' UTR. Use of the exon 13L (long) is predicted to lead to thesynthesis of a 6-kb transcript coding for a 1,400-amino acidprotein. Use of the exon 13S is predicted to lead to the synthesisof a 3.5-kb transcript coding for a 722-amino acid protein with96% identity with the HUNK1 protein (Fig. 1a) (GenBank accessionno. AF461396). Sequence comparison of the murine and human Brd4cDNAs to human genomic sequences (NT 011290) revealed that thehuman gene contains the same number of exons as the mouse geneand covers 43.6 kb. In both species, exon 13S is located betweenexons 12 and 13L and is highly conserved (85% identity over414 bp). However, although we were able to reveal the existenceof this short transcript by RT-PCR and 3' RACE, we were unableto detect it by Northern blotting or in situ hybridization,a result in agreement with the single-size protein detectedby Western blotting (8). Therefore, the biological significanceof this short transcript remains elusive. In conclusion, boththe high sequence identity and the similarities in genomic organizationstrongly suggest that Brd4 is the mouse orthologue of HUNK1.

Brd4 belongs to a group of structurally related proteins. Several proteins that are characterized by the association oftwo N-terminal bromodomains with a more C-terminal ET domainhave now been identified. We compared the motif organizationalstructures (Fig. 2a) and sequences (Fig. 2b) of the differentmembers of this family. Besides their bromodomains and ET domains,all the vertebrate members of the Fsh/Brd family as well asthe Drosophila fs(1)h protein contain identical short stretchesof 11 amino acids (GVKRKADTTTP) (Fig. 2a). This motif was notencountered in any other protein present in the databases, andits function is presently unknown.

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FIG. 2. Structure of the Brd4 gene and conservation of the Brd family. (a) Domain organizations of the different members of the Brd/fs(1)h family. A convenient nomenclature for the vertebrate members of this family based on the root fsrg (female sterile homeotic related) was proposed by Rhee et al. (32). However, here we use the nomenclature retained by the Mouse Gene Informatics Nomenclature Committee, who proposed the root Brd to designate the members of this bromodomain-containing family. Bromodomains are shown in blue, ET domains are shown in red, and the conserved 10-amino acid motif is shown in orange. Note that the hffsh sequence is only partial and terminates in the ET domain. (b) Similarity tree of the members of the Brd family (vertebrate members of the family are underlined). Note that Brd2/RING3 and Brd3/ORFX group together, in agreement with the important conservation of synteny observed between human chromosomes 6 and 9 (see reference 21 for review). Brd5/BRDT, the only member of the Brd family that exhibits a nonubiquitous pattern of expression (19), is found outside the mammalian group, suggesting a possible divergent function. Bdf1 and Bdf2 group together, suggesting a duplication after the separation between yeast and metazoans. Alignments of bromodomains also confirmed this result (data not shown). (c) Sequence comparison of the Ring3-like peptides (p66, p96a1, and p96a2) (isolated in the study cited in reference 18) with those of the four mammalian members of the Brd family. Arrows point at the sequences of the peptides and Brd4.

The Brd2/RING3 protein was originally described as a mitogen-activatednuclear kinase (6), although the existence of kinase activityof Brd2 remains controversial (7, 30, 32). We compared the sequencesof the kinase motifs described for Brd2 with the Brd4 sequence.Neither the Brd2 putative ATP binding motif (LGPSGFGPS) northe putative catalytic lysine (ATK) was conserved in Brd4. Therefore,if Brd2 has a kinase activity, it is unlikely to be conservedin Brd4. However, it remains possible that Brd4 is able to recruitanother protein with kinase activity, since such a functionhas been proposed for Brd2 (30).

One member of the Fsh/Brd family has been isolated in the mousecounterpart of the yeast mediator of transcriptional regulation,the role of which is to integrate the positive and negativeregulatory information originating from enhancer and silencerelements. It then transmits this information to the RNA polymeraseII, modulating its activity in promoter-dependent transcription(18). At the time, Jiang et al. could not identify the memberof the family that they had purified. When we compared the proteinsequences of the three Brd-like peptides that they isolatedwith those of the four members of the Fsh/Brd family, we foundthat they were 100% identical to parts of the bromodomain II(peptides p66 and p96a1) and ET domains (peptide p96a2) of Brd4(46 amino acids out of 46) (Fig. 2c). The other members of themammalian Brd family did not share 100% identity with the peptides(41-of-46 amino acid identity for Brd2; 40 of 46 for Brd3; 40of 46 for Brd5). These peptides are not predicted to be encodedby any other putative open reading frame presently identifiedin the human or mouse genome. Therefore, we conclude that Brd4is very likely to be the protein identified by Jiang et al.as a component of the mediator.

Brd4 is located on mouse chromosome 17 and reveals a conservation of synteny with human chromosome 19p13.1. BRD4/HUNK1 is located on human chromosome 19p13.1, 35.6 kb fromthe Notch3 gene (NT 011290). The mouse-hamster RH panel wasused to map both mouse Brd4 and Notch3 on the mouse genome.We localized Brd4 and Notch3 on mouse chromosome 17 betweenmarkers D17Mit181 and D17Mit191, a result confirmed by the presenceof both genes on the draft genome sequence (GenBank accessionno. AC074208). This localization reveals a short conservationof synteny between Brd4/Notch3 on chromosome 17 on the mousegenome and HUNK1/NOTCH3 on chromosome 19p13.1 on the human genome.This conservation of synteny gives further evidence for theorthology between Brd4 and HUNK1. Interestingly, the three othermembers of the Fsh/Brd family also map close to members of theNotch family, at least in humans. Brd2 maps next to Notch4 onhuman chromosome 6, Brd3 next to Notch1 on human chromosome9, and Brd5 next to Notch2 on human chromosome 1 (reviewed inreference 20).

Brd4^gt is a null allele of Brd4. The gene trap vector integrated within the second intron ofBrd4. A fusion mRNA containing the noncoding exons 1 and 2 fromBrd4 spliced to the gene trap sequences is predicted to be transcribedfrom the trapped locus (Fig. 1a and b). Therefore, the transcriptencoded by the gene trap allele (designated Brd4^gt) should notcontain any Brd4 coding sequences. In order to confirm thatwe had indeed generated a null allele of Brd4, we cultured blastocystsfrom Brd4^gt/Brd4⁺ x Brd4^gt/Brd4⁺ matings. Using in situ hybridizationwith a probe spanning Brd4 exons 1 to 9 and part of exon 10,we showed that blastocyst explants homozygous for Brd4^gt areclearly recognizable by the absence of Brd4 transcript, evenafter extended periods of staining (compare Fig. 1c and d).In order to ensure a homogeneous genetic background and to eliminateany independent mutation that may have occurred in the ES cellclone, we backcrossed the heterozygotes onto a C57BL6/J backgroundfor five to eight generations. In parallel, fluorescent in situhybridization analysis of the G-banded metaphase spread of ST132ES cells revealed that only a single gene trap vector insertionwas evident and that it coincided grossly with the positionof Brd4 (data not shown). We conclude from these results thatthe phenotype in Brd4^gt mice (see below) is attributable toa null allele of Brd4.

Expression pattern of Brd4. In Brd4^gt/Brd4⁺ embryos, we detected a faint expression of thelacZ reporter gene from the blastocyst stage onwards. We alsoshowed that the gene is strongly expressed in blastocyst explants(Fig. 1c and Fig. 6d and f). In E6 embryos, the expression ofthe reporter gene was ubiquitous but the intensity of the signalappeared to be stronger in the embryonic than in the extraembryonicpart of the conceptus (see Fig. 6a). The highest level of expressionwas observed in E10 and E11 embryos (data not shown). At laterstages of development and after birth, Brd4 remains expressedin all tissues tested, although the levels of expression differ(reference 8 and D. Houzelstein, unpublished data).

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FIG. 6. The homozygote phenotype. (a to c) E6 embryos. Black arrows demarcate the embryonic part of the conceptus, whereas the black arrowhead points at the extraembryonic part. Whole-mount ß-galactosidase staining of a Brd4^gt/Brd4⁺ embryo (a) and hematoxylin-eosin staining of sections of Brd4⁺/Brd4⁺ (b) and Brd4^gt/Brd4^gt (c) embryos are shown. Note that the embryonic part which normally expresses the gene strongly is missing in the homozygote. (d to f) Blastocyst explants stained with ß-galactosidase after 5 days in culture. The arrows point at the giant trophoblastic cells, and the arrowhead points at the inner cell mass. In Fig. 6e, the limit of trophoblast giant cell spread is indicated by a dotted line. In Fig. 6f, the inner cell mass of the homozygote has degenerated and is missing.

Postnatal death of Brd4^gt/Brd4⁺ individuals. Shortly after birth, the expected 1:1 ratio of heterozygousto wild-type individuals was observed from Brd4^gt/Brd4⁺ x Brd4⁺/Brd4⁺matings (Fig. 3a). However, 12 to 17 days after birth (P12 toP17), about 50% of the heterozygotes were missing, whereas afterP17 the proportions of heterozygous individuals remained relativelyconstant, indicating a critical period for survival during thefirst 2 weeks of postnatal life. No significant numbers of deathsoccurred in wild-type C57BL6/J littermates over this period.

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FIG. 3. In vivo phenotype of the heterozygotes. (a) Postnatal lethality: comparison between the average numbers of Brd4⁺/Brd4⁺ (plain line) and Brd4^gt/Brd4⁺ (dotted line) individuals per litter from matings of Brd4^gt/Brd4⁺ males with Brd4⁺/Brd4⁺ females, from birth to adulthood. The number indicated above each point represents the average number of individuals per litter. Note that the expected ratio of heterozygous to wild-type individuals was observed at birth. n, number of individuals counted. (b) Growth defect before birth: average weights of Brd4⁺/Brd4⁺ and Brd4^gt/Brd4⁺ individuals from E10 to E19. At least three littermate individuals from each genotype were counted at every point. Heterozygote versus wild-type weight percentages are indicated. Note that the difference in weight between heterozygous and wild-type individuals is most pronounced between E10 and E11. (c) Growth defect after birth. Six Brd4^gt/Brd4⁺ and five Brd4⁺/Brd4⁺ Brd4^gt/Brd4⁺ males were weighed over a period of 320 days. Wild-type individuals (plain line) were significantly heavier than their heterozygous (dotted line) littermates. Weights of both males and females from several litters gave results quantitatively and qualitatively similar. The average weight is indicated over each point, and heterozygote versus wild-type weight percentages are indicated in the squares. (d) Growth defect of individual organs. Individual organs from Brd4⁺/Brd4⁺ and Brd4^gt/Brd4⁺ 10-month-old females were weighed separately. Every organ was affected by the mutation. (e) Reduced numbers of mitotic cells in the Brd4^gt/Brd4⁺ individuals. An anti-phospho-histone H3 antibody was used to reveal mitotic cells from E15 liver and lung sections. An enlargement of part of such a section is shown. The average numbers of mitotic cells per section are indicated. There were significantly fewer mitotic cells in sections from heterozygous embryos than in those from wild-type embryos, although the cell density was unaffected.

Prenatal and postnatal growth defects of the Brd4^gt/Brd4⁺ individuals. While the cause of heterozygous death remained unclear, theseindividuals appeared consistently smaller than wild-type littermates.Therefore, heterozygous and wild-type individuals were systematicallyweighed before and after birth. At E10, the heterozygotes weighed,on average, 25% less than their wild-type littermates. Thisgrowth defect was maintained throughout the prenatal perioduntil birth (Fig. 3b). After birth, the defect was less pronounced,since heterozygotes were only 10% lighter than the wild types(Fig. 3c). The weights of several organs from heterozygous adultswere reduced in similar proportion compared to those of wildtypes (Fig. 3d). Since the growth defect was maintained throughoutlife, it is unlikely to result from an abnormal growth hormonepathway, which acts only after birth. A defect in nutrient supplyfrom the mother also seems unlikely, since the growth defectwas maintained after birth and weaning (Fig. 3a). To addressthe cause of the growth defect, we performed immunolabelingexperiments on lung and liver sections by using an antibodydirected against phospho-histone H3, a specific marker of mitoticcells (14) (Fig. 3e). We found a significantly reduced numberof mitotic cells in heterozygotes compared to that in wild-typetissue (Fig. 3c). Taken together, these results indicate thatthe general growth restriction observed in heterozygous tissuesis likely to be due to a reduction in the proliferation rate.

Anatomical defects of the Brd4^gt/Brd4⁺ individuals. A few weeks after birth, the heterozygotes were easily recognizablebecause of the abnormal shape of their heads. Skeleton preparationsfrom 8-week-old heterozygous and wild-type animals revealedthat this recognizable facies was due to a shorter incisivebone as well as a shorter and bent nasal bone (Fig. 4a and b).The mandible was also reduced in length. No discernible differencesbetween the axial and appendicular skeletons of the wild typesand heterozygotes could be found (data not shown).

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FIG. 4. Anatomical and histological defects in the heterozygotes. (a, a', b, and b') Skeletal preparations of two-month-old Brd4⁺/Brd4⁺ (a) and Brd4^gt/Brd4⁺ (b) male skulls, respectively; bone is stained in red and cartilage in blue. Note the shorter and bent nasal bone (arrow), the shorter incisor bone (arrowhead), and the shorter mandible of the heterozygote. f, frontal bone; i, incisive bone; ip, interparietal bone; lf, lacrimal foramen; m, mandible; mp, mastoid process; n, nasal bone; o, occipital bone; os, orbital surface of the frontal bone; p, parietal bone; t, temporal bone; tb, tympanic bulla; z, zygomatic bone. (c and d) Sections through the skin of two Brd4⁺/Brd4⁺ (c) and Brd4^gt/Brd4⁺ (d) 1-month-old individuals. In the heterozygote, the epidermis (e) was thickened and the amount of subcutaneous adipose tissue (s) was reduced and contained degenerating adipose cells surrounded by phagocytic cells (black arrow). d, dermis; m, muscle. (e and f) Sections through the liver of two Brd4⁺/Brd4⁺ (e) and Brd4^gt/Brd4⁺ (f) 2-month-old individuals. The black arrow in Fig. 4f points to degenerating hepatocytes, and the white arrowheads point at surrounding phagocytes in the heterozygote.

Various morphological abnormalities were seen in the skin, liver,testis (epididymal duct and vas deferens), and brain of heterozygotes.In the skin, the epidermis appeared abnormally thick, althoughthe layers were clearly recognizable. The subcutaneous adiposetissue was considerably reduced and contained degenerating cellssurrounded by infiltrating macrophages. The dermis also containedan increased number of neutrophils and mature fibrocytes (Fig.4c and d). In the heterozygous liver, many hepatocytes containedone large aberrant nucleus or even two nuclei. Degenerated andnecrotic hepatocytes surrounded by phagocytes (Küpffercells and neutrophils) were also detected (Fig. 4e and f). Inthe testis, several abnormal seminiferous tubules lacking spermatogeniccells that might indicate impaired spermatogenesis were alsonoted. In the epididymis and vas deferens, many cells of theepithelium lining the lumen of ducts had abnormal polyploidnuclei and were altered in size and shape. Some of the epithelialcells also showed features of degeneration. In the brain, afew necrotic neurons were observed but classical histologicalprocedures could not reveal any defect in myelination (datanot shown).

In a sample population of 34 heterozygotes between 6 weeks and6 months of age, four individuals had one or both eyes reducedin size and seven had a cataract. No eye defects were evidentin a control group of 48 wild-type animals (data not shown).

Heterozygous mouse embryonic fibroblasts (MEFs) have a reduced proliferation rate and increased sensitivity to MMS. To investigate further the cellular impact of the Brd4 heterozygouscondition, we isolated fibroblasts from E13 embryos. The doublingtime of heterozygous MEFs was significantly longer (4 days)than that of wild-type control MEFs (2 days) (Fig. 5a). We concludethat the growth restriction observed in heterozygous embryoswas maintained in cell culture. Therefore, MEFs constitute anin vitro system suitable for the further analysis of the Brd4growth phenotype. We showed that medium conditioned with wild-typeMEFs could not rescue the phenotype of heterozygous MEFs, suggestingthat the growth restriction phenotype is cell autonomous (datanot shown).

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FIG. 5. Effect of DNA damaging agents on survival of MEFs. (a) 1.00 x 10⁵ Brd4⁺/Brd4⁺ (plain line) or Brd4^gt/Brd4⁺ (dotted line) MEFs were harvested on individual plates at day 0 and counted at 4-day intervals. (b to d) MEFs were plated as for Fig. 5a. After they adhered to the plate, they were submitted to increasing doses of IR (b), MMS (c), or UV (d), cultured for 8 days, and counted.

Some aspects of the heterozygous growth restriction phenotypeare reminiscent of those observed in various mutants affectedin different DNA damage repair pathways (see reference 11 forreview). Thus, we tested the survival of heterozygous cellsafter exposure to ionizing radiation (IR). IR is known to inducelesions that are the substrate for the nonhomologous end joiningrepair pathway and the transcription-coupled repair pathway(21). For the doses tested, no difference could be seen betweenheterozygous and wild-type MEFs (Fig. 5b). When heterozygousthymocytes were tested, the relative insensitivity to IR wasalso observed and the absence of nonhomologous end joining deficiencyin these cells was corroborated by the normal generation ofT-cell receptor variable region diversity (data not shown).

We then measured the survival of heterozygous cells followingexposure to MMS. MMS induces lesions, such as N-methylpurines(29), which are substrates for the base excision repair pathway(see reference 22 for a recent review). We detected up to atwofold increase in sensitivity to MMS of heterozygous cellscompared to that detected in wild-type cells (Fig. 5c).

Finally, the survival of heterozygous cells following exposureto UV radiation was assayed. Lesions induced by UV radiation,such as pyrimidine dimers, are substrates for the nucleotideexcision repair pathway. No clear difference could be detectedbetween heterozygous and wild-type cells in these conditions(Fig. 5d).

Characterization of the homozygous phenotype in vivo and in vitro. To investigate the phenotype of individuals nullizygous forBrd4, heterozygous individuals were mated. No Brd4^gt/Brd4^gthomozygous progeny could be identified by in situ hybridizationperformed on midgestation litters (0 of 22). Out of 43 deciduarecovered between E7 and E9, wild-type (10 of 43) and heterozygousembryos (24 of 43) were observed in the expected proportions.The remaining 25% of decidua (9 of 43), likely to correspondto the missing homozygotes, were empty, suggesting postimplantationlethality. When heterozygous females were mated with wild-typemales, no empty site was recovered (0 of 23), indicating thatthe lethality observed in heterozygous matings was not due toa maternal defect. A number of E6 decidua were processed forhistological examination. In 7 out of 37 cases, the embryonicregion was absent (Fig. 6c; compare with a wild type in Fig.6b). The embryonic region was the part of the conceptus whereexpression of the reporter gene was predominantly detected (Fig.6a), whereas the extraembryonic region, which normally expressesBrd4 at lower levels, was less affected in the homozygous mutants(Fig. 6a to c).

To test whether homozygous embryos survive to the blastocyststage, embryos were isolated at E3, at which point they showedno overt developmental delay and were morphologically indistinguishablefrom wild-type blastocysts (data not shown). The growth potentialof wild-type and mutant blastocysts was monitored in vitro overa period of 2 to 5 days. During the first 2 days, both wild-typeand homozygous blastocysts hatched out of the zona pellucidaand attached to the slide. During this period, explants grownfrom homozygous embryos, as assayed by the absence of Brd4 transcript,appeared morphologically normal, albeit smaller than wild types(compare Fig. 1c and d). After 5 days in culture, two cell populationswere clearly recognizable morphologically in explants from wild-typeblastocysts: flat, spread-out, giant trophoblast cells (Fig.6d) and the smaller inner-cell-mass cells (Fig. 6d and 6e).In explants grown from homozygous embryos, trophoblast giantcells were clearly recognizable and did not appear necrotic,whereas cells from the inner cell mass completely degenerated(Fig. 6f). Therefore, at least one copy of Brd4 is requiredfor the maintenance of the inner cell mass.

DISCUSSION

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Discussion

We have examined the consequences of a gene trap insertion resultingin a null mutation in the Brd4 gene. We showed that Brd4 isexpressed ubiquitously from the blastocyst stage and that nullizygousmutant embryos implant in the uterine wall but arrest in theirdevelopment shortly thereafter, indicating that Brd4 exertscellular functions essential for early postimplantation development.We have also found that individuals with only one functionalallele of the Brd4 gene are growth restricted both before andafter birth. Growth restriction is also observed in primarycell cultures and is therefore not dependent on diffusible growthfactors, either in the medium or from wild-type cells. Thus,the defect appears to be cell autonomous, consistent with apredicted nuclear location for Brd4. We showed that, in vivo,deletion of only one Brd4 allele results in a reduction of thenumber of mitotic cells in at least two embryonic tissues. Thisresult is consistent with the in vitro observation that microinjectionof anti-Brd4 antibodies into HeLa cell nuclei completely inhibitsentry into mitosis (8). The reduced mitotic activity is likelyto underlie the growth restriction during the whole life ofBrd4^gt heterozygotes. This growth restriction is most pronouncedat E10 and E11 (i.e., the earliest stage tested), when celldivision is relatively rapid and the reduced proliferative abilityof the Brd4^gt cells is likely to be most detrimental.

The complex set of anatomical defects exhibited by the Brd4^gt/Brd4⁺mutants is reminiscent of some of the defects observed in tworelated human syndromes, Cockayne syndrome (CS) and trichothiodystrophy(TTD). They include partial lethality, impaired growth, peculiarfacies, lack of subcutaneous fat, and cataracts (see references2 and 3 for recent reviews). Patients suffering from these syndromesare affected in DNA repair pathways resulting, in particular,in severe sensitivity to UV radiation. However, we demonstratethat Brd4-deficient cells do not display sensitivity to ionizingand UV radiation, excluding a major role for Brd4 in these pathways.Heterozygous cells appeared significantly more sensitive toMMS than wild-type cells, suggesting a defect in DNA damagerepair. However, this could be a primary or secondary consequenceof the mutation. Interestingly, a similar phenotype (slow growth,sensitivity to MMS, and relative insensitivity to IR) was alsodescribed for the yeast Bdf1 mutant.

However, since TTD and CS are now known to be the consequenceof defects in both DNA repair and transcription (see references2 and 3 for recent reviews), impaired transcription in the Brd4^gtmutants remains a possibility to explain the phenotypic similaritiesbetween the Brd4^gt mutants and TTD-CS patients. Consistent withthis possibility is our demonstration that Brd4 is very likelyto be a component of the mediator of transcriptional regulationisolated in the study cited in reference 18. However, the transcriptionalcoactivator function of the murine mediator has yet to be demonstrated(18) and many other mammalian mediator-related complexes havebeen characterized extensively and do not appear to containany Brd family member as a constitutive unit (see references12 and 26 for reviews). Therefore, the question of whether Brd4or other members of the Fsh/Brd family act as a constitutiveunit of the mediator awaits detailed biochemical analyses. Nevertheless,it is noteworthy that the defects in Brd4 nullizygous embryosare very similar to those found when Srb7, which encodes theonly other protein isolated as part of the mediator whose functionhas been inactivated in mice, is deleted. Srb7 nullizygous embryosalso die shortly after implantation, and the inner cell massof blastocysts grown in culture degenerates (38).

Summary.

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Summary.

Our results suggest that a fundamental cellular process is impairedin Brd4^gt cells and that the complex pathological features observedin Brd4^gt/Brd4⁺ individuals might be explained by a global deficiencyin a basic mechanism, resulting in reduced proliferative ability.Indeed, the most remarkable defect of Brd4^gt/Brd4⁺ mice is theirpre- and postnatal growth defect. Given that this phenotypeis associated with haploinsufficiency in the mouse, heterozygousmutations in the Brd human homologues could also be associatedwith pre- and postnatal growth defects. Therefore, Brds maybe candidate genes for some human disorders characterized bya short stature. Thus, the Brd4^gt mouse mutant will be of greatvalue, not only in elucidating Brd protein function during pre-and postnatal life but also as a model for human pathology.

ACKNOWLEDGMENTS

We are especially grateful to Françoise Poirier for herinvaluable help during the preparation of the manuscript. Wealso thank Margaret Buckingham, Alain Sarasin, Thierry Magnaldo,and Jean-Pierre Rousset for comments on the manuscript. We thankDimitris Kioussis and Owen Williams for thymocyte cultures andJudy Fletcher for FISH.

This work was supported by the MRC and by a Marie Curie traininggrant from the European Commission to D.H.

D.H. and S.L.B. contributed equally to this work.

FOOTNOTES

* Corresponding author. Present address: Laboratoire de Génétique et Développement, Institut Jacques Monod, 2, place Jussieu, 75251 Paris Cedex 05, France. Phone: 33 1 44 27 40 35. Fax: 33 1 44 27 52 65. E-mail: houzelstein{at}ijm.jussieu.fr.

This article is dedicated to the memory of Rosa Beddington.

Present address: Developmental Genetics Laboratory, ImperialCancer Research Fund, London WC2A 3PX, United Kingdom.

Present address: Centre for Genome Research, University of Edinburgh,Edinburgh EH9 3JQ, United Kingdom.

REFERENCES

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