Interactive Fly, Drosophila

A mouse protein has been found which bears homology to Polyhomeotic. Although the function of the mammalian protein is unknown, the proteins share a single zinc finger motif classified as a C4 domain. They also share a glutamine rich sequnce and two other regions of homology (Nomura, 1994).

Bmi1-interacting proteins are constituents of a multimeric mammalian Polycomb complex. Bmi, and the closely related Mel18 gene share sequence homology to Drosophila PSC and SU(Z)2. M33 is a murine homolog of Drosophila Polycomb protein. A Bmi binding protein, Mph1 has sequence similarity to Drosophila Polyhomeotic. Although the overall identity of the Mph1 protein to Polyhomeotic is only 31%, Mph1 and PH share significant similarity at the C-terminal region. A C-terminal homology domain shares weak similarity to members of the Ets family of transcription factors. This domain is most similar to the Drosophila protein Lethal(3)malignant brain tumor, Bmi1 and Mph1, as well as the Mel18 and M33 proteins are constituents of a multimeric protein complex in mouse embryos and human cells. A central domain of Bmi1 interacts with the carboxyl terminus of Mph1, a conserved alpha-helical domain in the Mph1 protein is required for its homodimerization. Transgenic mice overexpressing various mutant Bmi1 proteins demonstrate that the central domain of Bmi1 is required for the induction of anterior transformations of the axial skeleton. Bmi1, M33 and Mph1 show an overlapping speckled distribution in interphase nuclei. Localization of Bmi1 to subnuclear domains depends on an intact RING finger (Alkema, 1997).

Two human proteins, HPH1 and HPH2 coimmunoprecipitate and cofractionate with each other and BMI1, a homolog of Drosophila Posterior sex combs. They also colocalize with BMI1 in interphase nuclei of human cultured cells. HPH1 and HPH2 have little sequence homology with each other, except in two highly conserved domains, designated homology domains I and II. They share these homology domains I and II with the Drosophila PcG protein Polyhomeotic. Homology domain II is C terminal in the two human proteins and Polyhomeotic. Homology domain I is located about 230 amino acids upstream of Homology domain II in all three proteins. HPH1, HPH2 and BMI1 show distinct, although overlapping expression patterns in different tissues and cell lines. The highest level of expression of HPH1 is found in thymus, testis and ovary. Low expression levels are detected in spleen, prostate, small intestine, colon and peripheral blood leukocytes. In contrast, HPH2, like BMI1, is expressed ubiquitously. Homology domain II of HPH1 interacts with both homology domains I and II of HPH2. In contrast, homology domain I of HPH1 interacts with homology domain II of HPH2, but not with homology domain I of HPH2. Furthermore, BMI1 does not interact with the individual homology domains. Instead, both intact homology domains I and II need to be present for interactions with BMI1. HPH1 and HPH2 colocalize with BMI1 in large domains in the nuclei of cultured cells. The occurrence of two proteins with homology to PH may also point towards the interesting possibility of the existence of different mammalian multimeric PcG complexes. This idea is supported by the differential expression of HPH1 and of HPH2 and BMI1. This idea is reinforced by the observation that PSC shares many, but not all binding sites with the PcG proteins PC, PH and Polycomblike. This provides flexibility, and it increases the possibilities of regulating a wide range of target genes with only a limited number of components. In conclusion, homology domains I and II are involved in protein-protein interactions and the results indicate that HPH1 and HPH2 are able to heterodimerize (Gunster, 1997).

The rae28 gene is a mouse homolog of the Drosophila polyhomeotic gene, which is a member of the Polycomb group (Pc-G) of genes. The Pc-G genes are required for the correct expression of the Homeotic complex genes and segment specification during Drosophila embryogenesis and larval development. To study the role of the rae28 gene in mouse development, rae28-deficient mice were generated by gene targeting in embryonic stem cells. The rae28-/- homozygous mice exhibit perinatal lethality, posterior skeletal transformations and defects in neural crest-related tissues, including ocular abnormalities, cleft palate, parathyroid and thymic hypoplasia and cardiac anomalies. The anterior boundaries of Hoxa-3, a-4, a-5, b-3, b-4 and d-4 expression are shifted rostrally in the paraxial mesoderm of the rae28-/- homozygous embryos, and those of Hoxb-3 and b-4 expression are also similarly altered in the rhombomeres and/or pharyngeal arches. These altered Hox codes are presumed to be correlated with the posterior skeletal transformations and neural crest defects observed in the rae28-/- homozygous mice. These results indicate that the rae28 gene is involved in the regulation of Hox gene expression and segment specification during paraxial mesoderm and neural crest development (Takihara, 1997).

A retinoic acid (RA)-inducible clone was isolated from a cDNA library prepared from mouse embryonal carcinoma cells. The deduced RAE-28 protein shares several motifs and highly homologous regions with a Drosophila Polyhomeotic protein. As the Drosophila polyhomeotic gene is involved in regulating morphogenesis, the rae-28 gene may participate in regulating early mammalian development (Nomura, 1994).

In a two-hybrid screen with a vertebrate Polycomb homolog as a target (Xenopus Pc), the human RING1 protein was identified as interacting with Pc. RING1 is a protein that contains the RING finger motif, a specific zinc-binding domain, which is found in many regulatory proteins. So far, the function of the RING1 protein has remained enigmatic. RING1 coimmunoprecipitates with a human Pc homolog, the vertebrate PcG protein BMI1, and HPH1, a human homolog of the PcG protein Polyhomeotic (Ph). The human polycomb homolog, (hPc2) shows an overall identity of 70% with XPc but a mere 24% identity with M33, a murine Pc homolog closely related to hPc1. Interaction between RING1 and XPc does not involve the RING finger motif. RING1 colocalizes with the vertebrate PcG proteins in nuclear domains of SW480 human colorectal adenocarcinoma and Saos-2 human osteosarcoma cells. RING1, like Pc, is able to repress gene activity when targeted to a reporter gene. These findings indicate that RING1 is associated with the human PcG protein complex and that RING1, like PcG proteins, can act as a transcriptional repressor. It is possible that RING1 is a vertebrate homolog of the product of a Drosophila PcG gene that has not yet been characterized. No Drosophila RING1 homolog has yet been described (Satijn, 1997).

The rae28 gene, a mouse homolog of the Drosophila polyhomeotic gene, is involved in the maintenance of the transcriptional repression states of Hox genes. A glutathione S transferase-RAE28 (GST-RAE28) fusion protein has been synthesized and sequence-specific DNA binding activity in the RAE28 protein was examined by using the selected and amplified binding site method. After five rounds of enrichment, the eluted DNAs were amplified, cloned and sequenced. The sequences of individual oligonucleotides included the following consensus sequences; 5'-ACCA-3', 5'-ACCCA-3', 5'-CTATCA-3' and 5'-TGCC-3'. The oligonucleotides including these consensus sequences have significant affinity with the GST-RAE28 fusion protein. The RAE28 protein forms multimeric protein complexes with other members of mouse Pc-G proteins in the nucleus. These findings strongly suggest that the RAE28 protein constitutes a sequence-specific DNA binding domain in multimeric Pc-G protein complexes (Nomura, 1998).

The Polycomb group loci in Drosophila encode chromatin proteins required for repression of homeotic loci in embryonic development. Mouse Polycomb group homologs, RAE28, BMI1 and M33, have overlapping but not identical expression patterns during embryogenesis and in adult tissues. These three proteins coimmunoprecipitate from embryonic nuclear extracts. Gel filtration analysis of embryonic extracts indicates that RAE28, BMI1 and M33 exist in large multimeric complexes. M33 and RAE28 coimmunoprecipitate and copurify as members of large complexes from F9 cells, which express BMI1 at very low levels, suggesting that different Polycomb group complexes can form in different cells. RAE28, BMI1 and M33 interact homotypically, and both RAE28 and M33 interact with BMI1, but not with each other. The domains required for interaction have been localized. Together, these studies indicate that murine Polycomb group proteins are developmentally regulated and function as members of multiple, heterogeneous complexes (Hashimoto, 1998).

The E2F transcription factors play a key role in the regulation of cellular proliferation and terminal differentiation. E2F6 is the most recently identified and the least well understood member of the E2F family. It is only distantly related to the other E2Fs and lacks the sequences responsible for both transactivation and binding to the retinoblastoma protein. Consistent with this finding, E2F6 can behave as a dominant negative inhibitor of the other E2F family members. In this study, the possible role(s) of E2F6 in vivo have been investigated. The isolation of RYBP, a recently identified member of the mammalian polycomb complex, as an E2F6-interacting protein, is reported in this study. Mapping studies indicate that RYBP binds within the known 'repression domain' of E2F6. Moreover, endogenous E2F6 and polycomb group proteins, including RYBP, Ring1, MEL-18, mph1, and the oncoprotein Bmi1 (Drosophila homolog: Polyhomeotic), associate with one another. These findings suggest that the biological properties of E2F6 are mediated through its ability to recruit the polycomb transcriptional repressor complex (Trimarchi, 2001).

Polycomb group (PcG) genes were initially described in Drosophila melanogaster as regulators of the homeobox gene. Four mammalian homologs, mel-18, bmi-1, M33 and rae-28, have been analyzed in this study. They not only regulate mammalian homeotic genes by analogy with their Drosophila counterparts, but also have some influence on the growth and differentiation of B lymphocytes. These four mammalian PcG genes are rapidly induced after antigen-receptor cross-linking in B cells. Thus it is proposed that mammalian PcG genes can be categorized as a new type of immediate early gene (Hasegawa, 1998).

Using exon trapping, a new human gene in Xp22 has been identified encoding a 3-kb mRNA. Expression of this RNA is detectable in a range of tissues but is most pronounced in skeletal muscle and heart. The gene, designated 'sex comb on midleg-like-1' (SCML1), maps 14 kb centromeric of marker DXS418, between DXS418 and DXS7994, and is transcribed from telomere to centromere. SCML1 spans 18 kb of genomic DNA, consists of six exons, and has a 624-bp open reading frame. The predicted 27-kDa SCML1 protein contains two domains that each have a high homology to two Drosophila transcriptional repressors of the polycomb group (PcG) genes and their homologues in mouse and human. PcG genes are known to be involved in the regulation of homeotic genes, and the mammalian homologs of the PcG genes repress the expression of Hox genes. SCML1 appears to be a new human member of this gene group and may play an important role in the control of embryonal development (van de Vosse, 1998).

A novel gene with homologies to the Drosophila Sex combs on midleg (Scm) gene from the short arm of the X chromosome has been identified. Scm is a member of the Polycomb group (PcG) genes, which encode transcriptional repressors essential for appropriate development in the fly and in mammals. The newly identified transcript named SCML2 (sex comb on midleg like-2, HGMW-approved symbol) is ubiquitously expressed and encodes a protein of 700 amino acids. SCML2 maps very close to the recently identified SCML1, revealing the presence of a new gene cluster in Xp22. The homology and map location identify SCML2 as a candidate gene for Xp22-linked developmental disorders, including the oral-facial-digital type I (OFDI) syndrome. A study of the SCML1-SCML2 cluster in primates indicates that the two genes are localized to the same region in Old World monkeys, New World monkeys, and prosimians, suggesting that the duplication event leading to the formation of the SCML cluster on Xp22 occurred before primate divergence (Montini, 1999).

In Drosophila, the Polycomb-group constitutes a set of structurally diverse proteins that act together to silence target genes. Many mammalian Polycomb-group proteins have also been identified and show functional similarities with their invertebrate counterparts. To begin to analyze the function of Polycomb-group proteins in Xenopus development, a Xenopus homolog of Drosophila Polycomblike, XPcl1, has been cloned. XPcl1 mRNA is present both maternally and zygotically, with prominent zygotic expression in the anterior central nervous system. Misexpression of Pcl1 by RNA injection into embryos produces defects in the anterior central nervous system. The forebrain and midbrain contain excess neural tissue at the expense of the ventricle and include greatly thickened floor and roof plates. The eye fields are present but Rx2A, an eye-specific marker, is completely repressed. Overexpression of Pcl1 in Xenopus embryos alters two hindbrain markers, repressing En-2 and shifting it and Krox-20 in a posterior direction. Similar neural phenotypes and effects on the En-2 expression pattern were produced by overexpression of three other structurally unrelated Polycomb-group proteins: M33 (homolog of Drosophila Polycomb), XBmi-1 (homolog of Drosophila Polycomb), and mPh2 (homolog of Drosophila Polyhomeotic). These observations indicate an important role for the Polycomb-group in regulating gene expression in the developing anterior central nervous system (Yoshitake, 1999).

E2F is a family of transcription factors that regulate both cellular proliferation and differentiation. To establish the role of E2F3 in vivo, an E2f3 mutant mouse strain was generated. E2F3-deficient mice arise at one-quarter of the expected frequency, demonstrating that E2F3 is important for normal development. To determine the molecular consequences of E2F3 deficiency, the properties of embryonic fibroblasts derived from E2f3 mutant mice were analyzed. Mutation of E2f3 dramatically impairs the mitogen-induced, transcriptional activation of numerous E2F-responsive genes. A number of genes, including B-myb, cyclin A, cdc2, cdc6, and DHFR, could be identified whose expression is dependent on the presence of E2F3 but not E2F1. The E2Fs regulate the expression of several proteins that are involved in early development, including homeobox proteins, transcription factors involved in cell fate decisions, a number of proteins that determine homeotic gene transcription, and signaling pathways such as the TGFbeta and Wnt pathways that are essential for early development. As an example of the relevance of these findings, it has been reported that position-effect variegation (PEV) in Drosophila depends on E2F activity. Loss of E2F activity enhances PEV, whereas overexpression of E2F activity suppresses PEV in Drosophila. These data suggested that the E2Fs themselves have an epigenetic effect by regulating chromatin structure or, more likely, that the E2Fs control PEV by regulating genes of the Polycomb group (PcG) family. In this screen, several PcG genes have been identified, like Enhancer of Zeste 2 (EZH2), Embryonic Ectoderm Development protein (EED) and Homolog of Polyhomeotic (EDR2/HPH2). The E2F-induced expression of these genes may provide an explanation for the role of E2F in the regulation of PEV and, more importantly, in development (Muller, 2001).

Mammalian polyhomeotic homologues Phc2 and Phc1 act in synergy to mediate Polycomb-repression of Hox genes

The Polycomb group (PcG) gene products form multimeric protein complexes and contribute to anterior-posterior (A-P) specification via the transcriptional regulation of Hox cluster genes. The Drosophila polyhomeotic genes and their mammalian orthologues, Phc1, Phc2, and Phc3, encode nuclear proteins that are constituents of evolutionarily conserved protein complexes designated class II PcG complexes. This study describes the generation and phenotypes of Phc2-deficient mice. Posterior transformations of the axial skeleton and premature senescence of mouse embryonic fibroblasts are associated with derepression of Hox cluster genes and Cdkn2a genes, respectively. Synergistic actions of a Phc2 mutation with Phc1 and Rnf110 mutations during A-P specification, coimmunoprecipitation of their products from embryonic extracts, and chromatin immunoprecipitation by anti-Phc2 monoclonal antibodies suggest that Hox repression by Phc2 is mediated through the class II PcG complexes, probably via direct binding to the Hox locus. The genetic interactions further reveal the functional overlap between Phc2 and Phc1 and a strict dose-dependent requirement during A-P specification and embryonic survival. Functional redundancy between Phc2 and Phc1 leads to the hypothesis that the overall level of polyhomeotic orthologues in nuclei is a parameter that is critical in enabling the class II PcG complexes to exert their molecular functions (Isono, 2005).

Mammalian Polycomb Scmh1 mediates exclusion of Polycomb complexes from the XY body in the pachytene spermatocytes

The product of the Scmh1 gene, a mammalian homolog of Drosophila Sex comb on midleg, is a constituent of the mammalian Polycomb repressive complexes 1 (Prc1). Scmh1 has been identified as an indispensable component of the Prc1. During progression through pachytene, Scmh1 was shown to be excluded from the XY body at late pachytene, together with other Prc1 components such as Phc1 and Phc2 (Polyhomeotic homologs), Rnf110 (Pcgf2), Bmi1 [Drosophila homologs Psc and Su(z)2] and Cbx2 (Polycomb homolog). The role of Scmh1 in mediating the survival of late pachytene spermatocytes has been identified. Apoptotic elimination of Scmh1^-/- spermatocytes is accompanied by the preceding failure of several specific chromatin modifications at the XY body, whereas synapsis of homologous autosomes is not affected. It is therefore suggested that Scmh1 is involved in regulating the sequential changes in chromatin modifications at the XY chromatin domain of the pachytene spermatocytes. Restoration of defects in Scmh1^-/- spermatocytes by Phc2 mutation indicates that Scmh1 exerts its molecular functions via its interaction with Prc1. Therefore, for the first time, it is possible to indicate a functional involvement of Prc1 during the meiotic prophase of male germ cells and a regulatory role of Scmh1 for Prc1, which involves sex chromosomes (Takada, 2007).

Based on the present observations, it is postulated that Scmh1 could primarily promote the exclusion of Prc1 components from the XY body in the pachytene spermatocytes because Scmh1 itself is a functional component of Prc1. By contrast, failure to maintain exclusion of trimethylated H3-K27 and to undergo H3-K9 methylation at the XY body in Scmh1^-/- spermatocytes may occur secondarily to the failure to exclude Prc1 from the XY body. At many loci, epistatic engagement of Prc1 by Prc2 has been shown to be essential for the mediation of transcriptional repression. Preceding exclusion of trimethylated H3-K27, which represents Prc2 actions, for Prc1 exclusion from the XY body, is consistent with epistatic roles of Prc2 for Prc1 at the XY body. Therefore, Scmh1 may affect H3-K27 trimethylation at the XY body through the Prc1-Prc2 engagement. It is noteworthy that H3-K27 trimethylation has been shown to be regulated by Prc1 at the XY body. This may imply that Prc1-Prc2 engagement is a reciprocal rather than epistatic process at the XY body. This possibility should be addressed by using conditional mutants for Prc2 components. A functional correlation between Prc1 exclusion and H3-K9 methylations at the XY body is also hypothesized because the indispensable H3-K9 methyltransferase complex, composed of G9a and GLP, is constitutively associated with E2F6 complexes, which share at least Rnf2 and Ring1 components with Prc1. Moreover, several components of respective complexes are structurally related to each other. Intriguingly, although Prc1 components, apart from Rnf2, have been shown to be excluded from the XY body at late pachytene stage, components of E2F6 complexes including Rnf2, RYBP, HP1gamma and G9a are retained. The most attractive scenario would be that exclusion of Prc1 is a prerequisite for the functional manifestation of E2F6 complexes to mediate the hypermethylation of H3-K9 at the XY body. It is thus proposed that Scmh1-mediated exclusion of Prc1 from the XY body might be a prerequisite for maintaining appropriate chromatin structure to undergo subsequent sequential chromatin remodeling of the XY chromatin in pachytene spermatocytes (Takada, 2007).

It is also suggested that sequential changes in chromatin modifications of the sex chromosomes in the pachytene spermatocytes might be monitored by some meiotic checkpoint mechanisms. This is supported by the temporal concurrence of Prc1 exclusion from the XY body and apoptotic depletion of meiotic spermatocytes, their coincidental restorations by Phc2 mutation, and normal oogenesis and fertility in Scmh1^-/- females. In addition, defects in the XY body formations have been shown to correlate with apoptotic depletion of meiotic spermatocytes by studies using H2A.X and Brca1 mutants, although developmental arrests occurred by early pachytene stage. However, this link has not been substantially demonstrated (Takada, 2007).

Although Scmh1 has been shown to act together with Prc1, the role of Scmh1 for Prc1 might be modified in a tissue- or locusspecific manner because spermatogenic defects by Scmh1 mutation are restored by Phc2 mutation, whereas premature senescence of MEFs is enhanced mutually by both mutations. This is supported by an immunofluorescence study revealing the co-localization of Scmh1 with other class 2 PcG proteins in subnuclear speckles in U2OS cells, whereas in female trophoblastic stem (TS) cells it is excluded from the inactive X chromosome domain, which is intensely decorated by Rnf2, Phc2 and Rnf110. It may be possible to postulate some additional factors that modify the molecular functions or subnuclear localization of Scmh1. Indeed, most of the soluble pool of SCM in Drosophila embryos is not stably associated with Prc1, although SCM is capable of assembling with the Polyhomeotic protein by their respective SPM domains in the Polycomb core complex. As the SPM domain is shared, not only by polyhomeotic homologs, but also by multiple paralogs of the Drosophila Scm gene, namely Scml1, Scml2, Sfmbt, l(3)mbt3 and others in mammals, these structurally related gene products may potentially interact with Scmh1 and modulate its functions. Conservations of crucial amino acid residues required for the mutual interaction of SPM domains and multiple mbt repeats in these proteins may further suggest functional overlap with Scmh1. It is notable that phenotypic expressions of Scmh1 mutation are quite variable during spermatogenesis and axial development even after more than five times backcrossing to a C57Bl/6 background. This incomplete penetrance might involve multiple paralogs of the canonical Scm proteins, which may act in compensatory manner for Scmh1 mutation, as revealed between Rnf110 and Bmi1 or Phc1 and Phc2 (Takada, 2007).

polyhomeotic: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

The Interactive Fly resides on the
Society for Developmental Biology's Web server.