Vivian J. Bardwell

ETO, a target of t(8;21) in acute leukemia, interacts with the N-CoR and mSin3 corepressors

ABSTRACT t(8;21) is one of the most frequent translocations associated with acute myeloid leukemia. It produces a chimeric protein, acute myeloid leukemia-1 (AML-1)–eight-twenty-one (ETO), that contains the amino-terminal DNA binding domain of the AML-1 transcriptional regulator fused to nearly all of ETO. Here we demonstrate that ETO interacts with the nuclear receptor corepressor N-CoR, the mSin3 corepressors, and histone deacetylases. Endogenous ETO also cosediments on sucrose gradients with mSin3A, N-CoR, and histone deacetylases, suggesting that it is a component of one or more corepressor complexes. Deletion mutagenesis indicates that ETO interacts with mSin3A independently of its association with N-CoR. Single amino acid mutations that impair the ability of ETO to interact with the central portion of N-CoR affect the ability of the t(8;21) fusion protein to repress transcription. Finally, AML-1/ETO associates with histone deacetylase activity and a histone deacetylase inhibitor impairs the ability of the fusion protein to repress transcription. Thus, t(8;21) fuses a component of a corepressor complex to AML-1 to repress transcription.

DMRT1 prevents female reprogramming in the postnatal mammalian testis

Sex in mammals is determined in the fetal gonad by the presence or absence of the Y chromosome gene Sry, which controls whether bipotential precursor cells differentiate into testicular Sertoli cells or ovarian granulosa cells. This pivotal decision in a single gonadal cell type ultimately controls sexual differentiation throughout the body. Sex determination can be viewed as a battle for primacy in the fetal gonad between a male regulatory gene network in which Sry activates Sox9 and a female network involving WNT/β-catenin signalling. In females the primary sex-determining decision is not final: loss of the FOXL2 transcription factor in adult granulosa cells can reprogram granulosa cells into Sertoli cells. Here we show that sexual fate is also surprisingly labile in the testis: loss of the DMRT1 transcription factor in mouse Sertoli cells, even in adults, activates Foxl2 and reprograms Sertoli cells into granulosa cells. In this environment, theca cells form, oestrogen is produced and germ cells appear feminized. Thus Dmrt1 is essential to maintain mammalian testis determination, and competing regulatory networks maintain gonadal sex long after the fetal choice between male and female. Dmrt1 and Foxl2 are conserved throughout vertebrates and Dmrt1-related sexual regulators are conserved throughout metazoans. Antagonism between Dmrt1 and Foxl2 for control of gonadal sex may therefore extend beyond mammals. Reprogramming due to loss of Dmrt1 also may help explain the aetiology of human syndromes linked to DMRT1, including disorders of sexual differentiation and testicular cancer.Sex in mammals is determined in the fetal gonad by the presence or absence of the Y chromosome gene Sry, which controls whether bipotential precursor cells differentiate into testicular Sertoli cells or ovarian granulosa cells. This pivotal decision in a single gonadal cell type ultimately controls sexual differentiation throughout the body. Sex determination can be viewed as a battle for primacy in the fetal gonad between a male regulatory gene network in which Sry activates Sox9 and a female network involving WNT/β-catenin signalling. In females the primary sex-determining decision is not final: loss of the FOXL2 transcription factor in adult granulosa cells can reprogram granulosa cells into Sertoli cells. Here we show that sexual fate is also surprisingly labile in the testis: loss of the DMRT1 transcription factor in mouse Sertoli cells, even in adults, activates Foxl2 and reprograms Sertoli cells into granulosa cells. In this environment, theca cells form, oestrogen is produced and germ cells appear feminized. Thus Dmrt1 is essential to maintain mammalian testis determination, and competing regulatory networks maintain gonadal sex long after the fetal choice between male and female. Dmrt1 and Foxl2 are conserved throughout vertebrates and Dmrt1-related sexual regulators are conserved throughout metazoans. Antagonism between Dmrt1 and Foxl2 for control of gonadal sex may therefore extend beyond mammals. Reprogramming due to loss of Dmrt1 also may help explain the aetiology of human syndromes linked to DMRT1, including disorders of sexual differentiation and testicular cancer.

Polycomb group and SCF ubiquitin ligases are found in a novel BCOR complex that is recruited to BCL6 targets.

ABSTRACT The corepressor BCOR potentiates transcriptional repression by the proto-oncoprotein BCL6 and suppresses the transcriptional activity of a common mixed-lineage leukemia fusion partner, AF9. Mutations in human BCOR cause male lethal, X-linked oculofaciocardiodental syndrome. We identified a BCOR complex containing Polycomb group (PcG) and Skp-Cullin-F-box subcomplexes. The PcG proteins include RING1, RYBP, NSPC1, a Posterior Sex Combs homolog, and RNF2, an E3 ligase for the mono-ubiquitylation of H2A. BCOR complex components and mono-ubiquitylated H2A localize to BCL6 targets, indicating that the BCOR complex employs PcG proteins to expand the repertoire of enzymatic activities that can be recruited by BCL6. This also suggests that BCL6 can target PcG proteins to DNA. In addition, the BCOR complex contains components of a second ubiquitin E3 ligase, namely, SKP1 and FBXL10 (JHDM1B). We show that BCOR coimmunoprecipitates isoforms of FBXL10 which contain a JmjC domain that recently has been determined to have histone H3K36 demethylase activity. The recruitment of two distinct classes of E3 ubiquitin ligases and a histone demethylase by BCOR suggests that BCOR uses a unique combination of epigenetic modifications to direct gene silencing.

The BCL-6 POZ domain and other POZ domains interact with the co-repressors N-CoR and SMRT

Virtually all diffuse large cell lymphomas and a significant fraction of follicular lymphomas contain translocations and/or point mutations in the 5′ non-coding region of the putative oncogene BCL-6, that are presumed to deregulate its expression. BCL-6 encodes a Cys2-His2 zinc finger transcriptional repressor with a POZ domain at its amino-terminus. The POZ (or BTB) domain, a 120-amino-acid motif, mediates homomeric and, in some proteins, heteromeric POZ-POZ interactions. In addition, the POZ domain is required for transcriptional repression of several proteins, including BCL-6. Using a yeast two-hybrid screen, we identified N-CoR and SMRT as BCL-6 interacting proteins. Both N-CoR and SMRT, which were originally identified as co-repressors for the unliganded nuclear thyroid hormone and retinoic acid receptors, are components of large complexes containing histone deacetylases. We show that the interaction between BCL-6 and these co-repressors is also detected in the more physiologically relevant mammalian two-hybrid assay. The POZ domain is necessary and sufficient for interaction with these co-repressors. BCL-6 and N-CoR co-localize to punctate regions of the nucleus. Furthermore, when BCL-6 is bound to its consensus recognition sequence in vivo, it can interact with N-CoR and SMRT. We find, in vitro, that POZ domains from a variety of other POZ domain-containing proteins, including the transcriptional repressor PLZF, as well as ZID, GAGA and a vaccinia virus protein, SalF17R, also interact with varying affinities with N-CoR and SMRT. We find that BCL-6 POZ domain mutations that disrupt the interaction with N-CoR and SMRT no longer repress transcription. In addition, these mutations no longer self associate suggesting that self interaction is required for interaction with the co-repressors and for repression. More recently N-CoR has also been implicated in transcriptional repression by the Mad/Mxi proteins. Our demonstration that N-CoR and SMRT interact with the POZ domain containing proteins indicates that these co-repressors are likely involved in the mediation of repression by multiple classes of repressors and may explain, in part, how POZ domain containing repressors mediate transcriptional repression.

Oculofaciocardiodental and Lenz microphthalmia syndromes result from distinct classes of mutations in BCOR.

Lenz microphthalmia is inherited in an X-linked recessive pattern and comprises microphthalmia, mental retardation, and skeletal and other anomalies. Two loci associated with this syndrome, MAA (microphthalmia with associated anomalies) and MAA2, are situated respectively at Xq27–q28 (refs. 1,2) and Xp11.4–p21.2 (ref. 3). We identified a substitution, nt 254C→T; P85L, in BCOR (encoding BCL-6-interacting corepressor, BCOR) in affected males from the family with Lenz syndrome previously used to identify the MAA2 locus. Oculofaciocardiodental syndrome (OFCD; OMIM 300166) is inherited in an X-linked dominant pattern with presumed male lethality and comprises microphthalmia, congenital cataracts, radiculomegaly, and cardiac and digital abnormalities. Given their phenotypic overlap, we proposed that OFCD and MAA2-associated Lenz microphthalmia were allelic, and we found different frameshift, deletion and nonsense mutations in BCOR in seven families affected with OFCD. Like wild-type BCOR, BCOR P85L and an OFCD-mutant form of BCOR can interact with BCL-6 and efficiently repress transcription. This indicates that these syndromes are likely to result from defects in alternative functions of BCOR, such as interactions with transcriptional partners other than BCL-6. We cloned the zebrafish (Danio rerio) ortholog of BCOR and found that knock-down of this ortholog caused developmental perturbations of the eye, skeleton and central nervous system consistent with the human syndromes, confirming that BCOR is a key transcriptional regulator during early embryogenesis.

Critical Residues within the BTB Domain of PLZF and Bcl-6 Modulate Interaction with Corepressors

ABSTRACT The PLZF (promyelocytic leukemia zinc finger) transcriptional repressor, when fused to retinoic acid receptor alpha (RARα), causes a refractory form of acute promyelocytic leukemia. The highly conserved N-terminal BTB (bric a brac, tramtrack, broad complex)/POZ domain of PLZF plays a critical role in this disease, since it is required for transcriptional repression by the PLZF-RARα fusion protein. The crystal structure of the PLZF BTB domain revealed an obligate homodimer with a highly conserved charged pocket formed by apposition of the two monomers. An extensive structure-function analysis showed that the charged pocket motif plays a major role in transcriptional repression by PLZF. We found that mutations of the BTB domain that neutralize key charged pocket residues did not disrupt dimerization, yet abrogated the ability of PLZF to repress transcription and led to the loss of interaction with N-CoR, SMRT, and histone deacetylases (HDACs). We extended these studies to the Bcl-6 protein, which is linked to the pathogenesis of non-Hodgkins lymphomas. In this case, neutralizing the charged pocket also resulted in loss of repression and corepressor binding. Experiments with purified protein showed that corepressor-BTB interactions were direct. A comparison of the PLZF, Bcl-6, and the FAZF (Fanconi anemia zinc finger)/ROG protein shows that variations in the BTB pocket result in differential affinity for corepressors, which predicts the potency of transcriptional repression. Thus, the BTB pocket represents a molecular structure involved in recruitment of transcriptional repression complexes to target promoters.

The Mammalian Doublesex Homolog DMRT1 Is a Transcriptional Gatekeeper that Controls the Mitosis versus Meiosis Decision in Male Germ Cells

The switch from mitosis to meiosis is a unique feature of germ cell development. In mammals, meiotic initiation requires retinoic acid (RA), which activates meiotic inducers, including Stra8, but how the switch to meiosis is controlled in male germ cells (spermatogonia) remains poorly understood. Here we examine the role of the Doublesex-related transcription factor DMRT1 in adult spermatogenesis using conditional gene targeting in the mouse. Loss of Dmrt1 causes spermatogonia to precociously exit the spermatogonial program and enter meiosis. Therefore, DMRT1 determines whether male germ cells undergo mitosis and spermatogonial differentiation or meiosis. Loss of Dmrt1 in spermatogonia also disrupts cyclical gene expression in Sertoli cells. DMRT1 acts in spermatogonia to restrict RA responsiveness, directly repress Stra8 transcription, and activate transcription of the spermatogonial differentiation factor Sohlh1, thereby preventing meiosis and promoting spermatogonial development. By coordinating spermatogonial development and mitotic amplification with meiosis, DMRT1 allows abundant, continuous production of sperm.

The DM domain protein DMRT1 is a dose-sensitive regulator of fetal germ cell proliferation and pluripotency

Dmrt1 (doublesex and mab-3 related transcription factor 1) is a conserved transcriptional regulator of male differentiation required for testicular development in vertebrates. Here, we show that in mice of the 129Sv strain, loss of Dmrt1 causes a high incidence of teratomas, whereas these tumors do not form in Dmrt1 mutant C57BL/6J mice. Conditional gene targeting indicates that Dmrt1 is required in fetal germ cells but not in Sertoli cells to prevent teratoma formation. Mutant 129Sv germ cells undergo apparently normal differentiation up to embryonic day 13.5 (E13.5), but some cells fail to arrest mitosis and ectopically express pluripotency markers. Expression analysis and chromatin immunoprecipitation identified DMRT1 target genes, whose missexpression may underlie teratoma formation. DMRT1 indirectly activates the GDNF coreceptor Ret, and it directly represses the pluripotency regulator Sox2. Analysis of human germ cell tumors reveals similar gene expression changes correlated to DMRT1 levels. Dmrt1 behaves genetically as a dose-sensitive tumor suppressor gene in 129Sv mice, and natural variation in Dmrt1 activity can confer teratoma susceptibility. This work reveals a genetic link between testicular dysgenesis, pluripotency regulation, and teratoma susceptibility that is highly sensitive to genetic background and to gene dosage.

Evolutionary dynamics of the DM domain gene family in metazoans.

The DM domain gene family encodes putative transcription factors related to the sexual regulators Doublesex from Drosophila melanogaster and MAB-3 from Caenorhabditis elegans. While some DM domain proteins are involved in sexual development in very distant metazoan phyla and one in somite development, the function of the great majority of them remains unclear. DM domain genes underwent frequent independent events of gene duplication during the course of evolution and the number of DM domain genes differs between phyla. Variation is even observed within the vertebrate lineage, where some genes present in mammals are absent from fish and vice versa. Of particular interest is the very recent duplication of the DM domain gene dmrt1 that apparently led to the formation of the master male-determining gene in the medaka fish but not in more divergent fish species. Hence, the DM domain gene family undergoes an important evolutionary turnover probably associated in some cases with novel expression patterns and possibly with new functions. Here we examine the current classification of vertebrate DM domain dmrt genes based on structural features, and propose a simpler nomenclature for dmrt genes.

Sexually dimorphic expression of multiple doublesex-related genes in the embryonic mouse gonad

The only molecular similarity shown so far for sexual regulatory genes among different phyla involves doublesex (dsx) of Drosophila, mab-3 and mab-23 of Caenorhabditis elegans, and Dmrt1 of vertebrates. These genes encode DM domain transcription factors (DM = dsx and mab-3) and are required for sexual differentiation. In the case of dsx and mab-3, the two genes control analogous aspects of sexual development, bind similar DNA sequences, and are capable of functional substitution in vivo. All three phyla have multiple DM domain genes, but it is unknown how many of these are involved in sexual development. Mammals, for example, have at least seven DM domain genes, but embryonic expression has only been examined in detail for Dmrt1(dsx- and mab-3 related transcription factor 1). We have identified additional murine DM domain genes and have examined their expression in the mouse embryo, with emphasis on the developing gonad. At least three murine DM domain genes in addition to Dmrt1 are expressed in the embryonic gonad: Dmrt4 is expressed at similar levels in gonads of both sexes; Dmrt3 is more highly expressed in males; and Dmrt7 is more highly expressed in females. Expression of three other genes is low or absent in the embryonic gonad. Two of these, Dmrt5 and Dmrt6, are expressed primarily in the brain, and the third, Dmrt2, is expressed in presomitic mesoderm and developing somites. Our data suggest that multiple DM domain genes may be involved in mammalian sexual development, and that they may function in both testis and ovary development.