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PubMed1997

A gene trap approach to identify genes that control development.

One methodology called gene trap represents a versatile strategy by which murine genes that control developmental events can be captured and identified with corresponding mutants produced at the same time. Gene trap methodology has been developed and several genes and their mutants have been analyzed, but almost all of the genes reported are those already known or murine homologs of other species. In this study, the efficiency of the gene trap methodology was improved and a novel mutant mouse strain named jumonji established which displayed an intriguing defect. Homozygous fetal mice died in utero and a significant proportion of the homozygotes showed abnormal groove formation on the neural plate and a defect in neural tube closure with a mixed genetic background of 129/Ola and BALB/c. The trapped gene believed to be responsible for these phenotypes encodes a novel nuclear protein. The results reveal that the gene trap approach can identify unknown interesting genes in murine development. The gene trap strategy, however, has several problems, the greatest of which is the difficulty in prescreening embryonic stem (ES) cells for interesting trapped genes. Recent studies are solving this problem and show that the prescreening of ES cells for genes with several characteristics is possible.

Amino Acid Sequence↗
PubMed2000

Gene expression profiles in normal and Otx2-/- early gastrulating mouse embryos.

The mouse Otx2 gene is a homeobox transcription factor required as early as gastrulation for the proper development of the head. We compared gene expression profiles in wild-type and Otx2(-/-) 6.5 days postcoitum embryos by using a serial analysis of gene expression assay adapted to microdissected structures. Among a broader list, the study of six genes found to be differentially expressed allows defining a role for Otx2 in the orchestration of cell movements leading to the adequate organization of the embryo before gastrulation.

Animals↗
PubMed1997

Zeste-mediated activation by an enhancer is independent of cooperative DNA binding in vivo.

It is not clear how transcription factors bound at distal enhancer and proximal promoter sequences cooperate to stimulate transcription in vivo. To distinguish between different models for the action of enhancer elements, we have directly measured DNA binding of the Drosophila activator zeste by in vivo UV crosslinking. Experiments in Drosophila embryos show that binding of zeste protein to either the proximal promoter of the Ultrabithorax (Ubx) gene or to a Ubx enhancer element does not require the presence of the other element. However, significant transcription is observed only when both elements are present and bound by zeste. The results indicate that stimulation by an enhancer can occur by a mechanism other than increasing the occupancy of an activator to binding sites near the start site of transcription.

Animals↗
PubMed1999

Genes controlling fertilization-independent seed development in Arabidopsis thaliana.

We have cloned two genes, FIS1 and FIS2, that control both fertilization independent seed development and postpollination embryo development in Arabidopsis. These genes confer female gametophytic phenotypes. FIS2 encodes a protein with a C2H2 zinc-finger motif and three putative nuclear localization signals, indicating that it is likely to be a transcription factor. FIS1 encodes a protein with homology to the Drosophila Polycomb group gene Enhancer-of-zeste and is identical to the recently described Arabidopsis gene MEDEA. FIS1 is a protein with a number of putative functional domains, including the SET domain present in Enhancer-of-zeste-related proteins. Comparison of the position of the lesions in the fis1 and medea mutant alleles indicates that fis1 is a null allele producing a truncated polypeptide lacking all the protein domains whereas the deduced protein from medea lacks only the SET domain. We present a model of the role of FIS1 and FIS2 gene products in seed development.

Alleles↗
PubMed1998

The human WD repeat protein WAIT-1 specifically interacts with the cytoplasmic tails of beta7-integrins.

Integrins of the beta7 subfamily, alpha4 beta7 and alphaE beta7, contribute to lymphocyte homing and to the development of protective or autoreactive immune responses at mucosal sites. The beta subunits of integrins are considered important for regulation of stimulated cell adhesion and adhesion-dependent signal transduction. Using a yeast interaction trap screen, a human WD repeat protein, termed WAIT-1, was isolated that interacts with the integrin beta7 cytoplasmic tail and is homologous to mouse EED and Drosophila ESC proteins. WAIT-1 also binds to the cytoplasmic domains of alpha4 and alphaE but not to those of integrin beta1, beta2, and alphaL subunits. Association of WAIT-1 and beta7-integrin was confirmed by coprecipitation from transiently transfected 293 cells. The binding site for WAIT-1 was mapped to a short membrane-proximal region of the beta7 cytoplasmic tail with Tyr-735 being of critical importance. Northern blot analysis revealed multiple WAIT-1-related transcripts with differential expression in circulating leukocytes, tissue-resident cells of diverse origin, and lymphoid malignancies. These results suggest that WAIT-1, together with the recently identified RACK1, may define a novel subfamily of WD repeat proteins that interact with distinct subsets of integrin cytoplasmic tails and may act as specific regulators of integrin function.

Amino Acid Sequence↗
PubMed1999

HEED, the product of the human homolog of the murine eed gene, binds to the matrix protein of HIV-1.

heed, the human homolog of mouse eed and Drosophila esc, two members of the trithorax (trx) and Polycomb group (Pc-G) of genes, was isolated by screening an activated lymphocyte cDNA library versus the immunodeficiency virus type 1 (HIV-1) MA protein used as a bait in a two-hybrid system in yeast. The human EED protein (HEED) had 99. 5% identity with the mouse EED protein and contained seven WD repeats. Two heed gene transcripts were identified, with a putative 407-nucleotide-long intron, giving rise to two HEED protein isoforms of 535 and 494 residues in length, respectively. The shorter HEED isoform, originated from the unspliced message, lacked the seventh WD repeat. HEED was found to bind to MA protein in vitro, as efficiently as in vivo in yeast cells. Site-directed mutagenesis and phage biopanning suggested that the interaction between HEED and MA involved the N-terminal region of the MA protein, including the first polybasic signal, in a MA conformation-dependent manner. In the HEED protein, however, two discrete linear MA-binding motifs were identified within residues 388-403, overlapping the origin of the fifth WD repeat. Deletion of the C-terminal 41 residues of HEED, spanning the seventh WD repeat, as in the 494-residue HEED protein, was detrimental to HEED-MA interaction in vivo, suggesting the existence of another C-terminal binding site and/or a conformational role of the HEED C-terminal domain in the MA-HEED interaction. MA and HEED proteins co-localized within the nucleus of co-transfected human cells and of recombinant baculovirus co-infected insect cells. This and the failure of HEED to bind to uncleaved GAG precursor suggested a role of HEED at the early stages of virus infection, rather than late in the virus life cycle.

Amino Acid Sequence↗
PubMed2001

Polycomblike PHD fingers mediate conserved interaction with enhancer of zeste protein.

The products of Polycomb group (PcG) genes are required for the epigenetic repression of a number of important developmental regulatory genes, including homeotic genes. Enhancer of zeste (E(Z)) is a Drosophila PcG protein that previously has been shown to bind directly to another PcG protein, Extra Sex Combs (ESC), and is present along with ESC in a 600-kDa complex in Drosophila embryos. Using yeast two-hybrid and in vitro binding assays, we show that E(Z) binds directly to another PcG protein, Polycomblike (PCL). PCL.E(Z) interaction is shown to be mediated by the plant homeodomain (PHD) fingers domain of PCL, providing evidence that this motif can act as an independent protein interaction domain. An association was also observed between PHF1 and EZH2, human homologs of PCL and E(Z), respectively, demonstrating the evolutionary conservation of this interaction. E(Z) was found to not interact with the PHD domains of three Drosophila trithorax group (trxG) proteins, which function to maintain the transcriptional activity of homeotic genes, providing evidence for the specificity of the interaction of E(Z) with the PCL PHD domain. Coimmunoprecipitation and gel filtration experiments demonstrate in vivo association of PCL with E(Z) and ESC in Drosophila embryos. We discuss the implications of PCL association with ESC.E(Z) complexes and the possibility that PCL may either be a subunit of a subset of ESC.E(Z) complexes or a subunit of a separate complex that interacts with ESC.E(Z) complexes.

Amino Acid Motifs↗
PubMed2003

JUMONJI, a critical factor for cardiac development, functions as a transcriptional repressor.

JUMONJI (JMJ) is a nuclear factor that is critical for normal cardiovascular development, evidenced by the analysis of jmj homozygous mutant mice. However, the molecular function of JMJ remains to be elucidated. In the present study, we investigated whether JMJ is a transcriptional modulator. Reporter gene assays using the GAL4-DNA binding domain fused to JMJ and a reporter gene consisting of the GAL4 binding sites upstream of a luciferase reporter gene indicated that JMJ functions as a powerful transcriptional repressor. The DNA binding motif of JMJ was determined using CASTing experiments by incubating a random oligonucleotide library with the GST-JMJ fusion protein coupled to agarose beads. Among the selected binding oligonucleotides, the high affinity DNA binding sequences were identified by gel retardation assays. JMJ repressed expression of the reporter genes containing the high affinity JMJ binding sequences, indicating that JMJ is a DNA-binding transcriptional repressor. The domains for transcriptional repression, DNA binding, and nuclear localization signal were mapped by mutational analyses using reporter gene assays, gel retardation assays, and immunostaining experiments, respectively. The present data demonstrate for the first time that JMJ functions as a DNA-binding transcriptional repressor. Therefore, JMJ may play a critical role in transcription factor cascade to regulate expression of heart-specific genes and normal cardiac development.

Animals↗
PubMed2005

Jumonji regulates cardiomyocyte proliferation via interaction with retinoblastoma protein.

Jumonji (JMJ) can function as a transcriptional repressor and plays critical roles in embryonic development including heart development in mice. Although JMJ has been suggested to play a role in cell growth, the molecular mechanisms have not been resolved. The present data demonstrate that JMJ interacts with the retinoblastoma protein (Rb), one of the master regulatory genes of cell cycle. JMJ potentiates the repression function of Rb on E2F activities, leading to reduced cell cycle progression. The transcriptional repression domain of JMJ is critical for the interaction with Rb as well as repression of cell cycle. The physiological relevance of the association between Rb and JMJ was assessed in cardiomyocytes. Primary cardiomyocytes cultured from homozygous jmj knock-out mouse embryos (jmj mutants) show increased cell mitosis in a cardiomyocyte-specific manner. Reporter gene analyses demonstrate that promoter activities of cyclin D1, cyclin D2, and Cdc2 are up-regulated in jmj mutant cardiomyocytes. These data suggest that JMJ down-regulates the cell growth via interaction with Rb, which would provide important insights into the cardiac defects observed in jmj mutant mice.

Animals↗
PubMed1997

Mammalian homologues of the Polycomb-group gene Enhancer of zeste mediate gene silencing in Drosophila heterochromatin and at S. cerevisiae telomeres.

Gene silencing is required to stably maintain distinct patterns of gene expression during eukaryotic development and has been correlated with the induction of chromatin domains that restrict gene activity. We describe the isolation of human (EZH2) and mouse (Ezh1) homologues of the Drosophila Polycomb-group (Pc-G) gene Enhancer of zeste [E(z)], a crucial regulator of homeotic gene expression implicated in the assembly of repressive protein complexes in chromatin. Mammalian homologues of E(z) are encoded by two distinct loci in mouse and man, and the two murine Ezh genes display complementary expression profiles during mouse development. The E(z) gene family reveals a striking functional conservation in mediating gene repression in eukaryotic chromatin: extra gene copies of human EZH2 or Drosophila E(z) in transgenic flies enhance position effect variegation of the heterochromatin-associated white gene, and expression of either human EZH2 or murine Ezh1 restores gene repression in Saccharomyces cerevisiae mutants that are impaired in telomeric silencing. Together, these data provide a functional link between Pc-G-dependent gene repression and inactive chromatin domains, and indicate that silencing mechanism(s) may be broadly conserved in eukaryotes.

Amino Acid Sequence↗
PubMed1994

A role of polycomb group genes in the regulation of gap gene expression in Drosophila.

Anteroposterior polarity of the Drosophila embryo is initiated by the localized activities of the maternal genes, bicoid and nanos, which establish a gradient of the hunchback (hb) morphogen. nanos determines the distribution of the maternal Hb protein by regulating its translation. To identify further components of this pathway we isolated suppressors of nanos. In the absence of nanos high levels of Hb protein repress the abdomen-specific genes knirps and giant. In suppressor-of-nanos mutants, knirps and giant are expressed in spite of high Hb levels. The suppressors are alleles of Enhancer of zeste (E(z)) a member of the Polycomb group (Pc-G) of genes. We show that E(z), and likely other Pc-G genes, are required for maintaining the expression domains of knirps and giant initiated by the maternal Hb protein gradient. We have identified a small region of the knirps promoter that mediates the regulation by E(z) and hb. Because Pc-G genes are thought to control gene expression by regulating chromatin, we propose that imprinting at the chromatin level underlies the determination of anteroposterior polarity in the early embryo.

Abdomen↗
PubMed1998

Trans-silencing by P elements inserted in subtelomeric heterochromatin involves the Drosophila Polycomb group gene, Enhancer of zeste.

Drosophila P-element transposition is regulated by a maternally inherited state known as P cytotype. An important aspect of P cytotype is transcriptional repression of the P-element promoter. P cytotype can also repress non-P-element promoters within P-element ends, suggesting that P cytotype repression might involve chromatin-based transcriptional silencing. To learn more about the role of chromatin in P cytotype repression, we have been studying the P strain Lk-P(1A). This strain contains two full-length P elements inserted in the heterochromatic telomere-associated sequences (TAS elements) at cytological location 1A. Mutations in the Polycomb group gene (Pc-G gene), Enhancer of zeste (E(z)), whose protein product binds at 1A, resulted in a loss of Lk-P(1A) cytotype control. E(z) mutations also affected the trans-silencing of heterologous promoters between P-element termini by P-element transgenes inserted in the TAS repeats. These data suggest that pairing interactions between P elements, resulting in exchange of chromatin structures, may be a mechanism for controlling the expression and activity of P elements.

Animals↗
PubMed2001

Long-range repression by multiple polycomb group (PcG) proteins targeted by fusion to a defined DNA-binding domain in Drosophila.

A tethering assay was developed to study the effects of Polycomb group (PcG) proteins on gene expression in vivo. This system employed the Su(Hw) DNA-binding domain (ZnF) to direct PcG proteins to transposons that carried the white and yellow reporter genes. These reporters constituted naive sensors of PcG effects, as bona fide PcG response elements (PREs) were absent from the constructs. To assess the effects of different genomic environments, reporter transposons integrated at nearly 40 chromosomal sites were analyzed. Three PcG fusion proteins, ZnF-PC, ZnF-SCM, and ZnF-ESC, were studied, since biochemical analyses place these PcG proteins in distinct complexes. Tethered ZnF-PcG proteins repressed white and yellow expression at the majority of sites tested, with each fusion protein displaying a characteristic degree of silencing. Repression by ZnF-PC was stronger than ZnF-SCM, which was stronger than ZnF-ESC, as judged by the percentage of insertion lines affected and the magnitude of the conferred repression. ZnF-PcG repression was more effective at centric and telomeric reporter insertion sites, as compared to euchromatic sites. ZnF-PcG proteins tethered as far as 3.0 kb away from the target promoter produced silencing, indicating that these effects were long range. Repression by ZnF-SCM required a protein interaction domain, the SPM domain, which suggests that this domain is not primarily used to direct SCM to chromosomal loci. This targeting system is useful for studying protein domains and mechanisms involved in PcG repression in vivo.

ATP-Binding Cassette Transporters↗
PubMed2001

The Trithorax-mimic allele of Enhancer of zeste renders active domains of target genes accessible to polycomb-group-dependent silencing in Drosophila melanogaster.

Two antagonistic groups of genes, the trithorax- and the Polycomb-group, are proposed to maintain the appropriate active or inactive state of homeotic genes set up earlier by transiently expressed segmentation genes. Although some details about the mechanism of maintenance are available, it is still unclear how the initially active or inactive chromatin domains are recognized by either the trithorax-group or the Polycomb-group proteins. We describe an unusual dominant allele of a Polycomb-group gene, Enhancer of zeste, which mimics the phenotype of loss-of-function mutations in trithorax-group genes. This mutation, named E(z)(Trithorax mimic) [E(z)(Trm)], contains a single-amino-acid substitution in the conserved SET domain. The strong dominant trithorax-like phenotypes elicited by this E(z) allele suggest that the mutated arginine-741 plays a critical role in distinguishing between active and inactive chromatin domains of the homeotic gene complexes. We have examined the modification of E(z)(Trm) phenotypes by mutant alleles of PcG and trxG genes and other mutations that alter the phosphorylation of nuclear proteins, covalent modifications of histones, or histone dosage. These data implicate some trxG genes in transcriptional repression as well as activation and provide genetic evidence for involvement of histone modifications in PcG/trxG-dependent transcriptional regulation.

Acetylation↗
PubMed1996

Characterization of the human jumonji gene.

While constructing a cDNA library of human embryos, we have isolated a clone homologous to jumonji, a mouse gene required for neural tube formation. We have determined the complete coding sequence of the human homologue (JMJ) and deduced the amino acid sequence of the putative protein. We show here that human and mouse jumonji putative proteins are homologous and present 90% identity. During human embryogenesis, JMJ mRNAs are predominantly expressed in neurons and particularly in dorsal root ganglion cells. They are also expressed in neurons of human adult cerebral cortex. In view of these observations, we propose JMJ as a candidate gene for developmental defects of the central nervous system in the human. The human JMJ gene maps at position 6p24-6p23.

Amino Acid Sequence↗
PubMed2003

The Drosophila Corto protein interacts with Polycomb-group proteins and the GAGA factor.

In Drosophila, PcG complexes provide heritable transcriptional silencing of target genes. Among them, the ESC/E(Z) complex is thought to play a role in the initiation of silencing whereas other complexes such as the PRC1 complex are thought to maintain it. PcG complexes are thought to be recruited to DNA through interaction with DNA binding proteins such as the GAGA factor, but no direct interactions between the constituents of PcG complexes and the GAGA factor have been reported so far. The Drosophila corto gene interacts with E(z) as well as with genes encoding members of maintenance complexes, suggesting that it could play a role in the transition between the initiation and maintenance of PcG silencing. Moreover, corto also interacts genetically with Trl, which encodes the GAGA factor, suggesting that it may serve as a mediator in recruiting PcG complexes. Here, we show that Corto bears a chromo domain and we provide evidence for in vivo association of Corto with ESC and with PC in embryos. Moreover, we show by GST pull-down and two-hybrid experiments that Corto binds to E(Z), ESC, PH, SCM and GAGA and co-localizes with these proteins on a few sites on polytene chromosomes. These results reinforce the idea that Corto plays a role in PcG silencing, perhaps by confering target specificity.

Amino Acid Sequence↗