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PubMed1999

Maintenance of genomic imprinting at the Arabidopsis medea locus requires zygotic DDM1 activity.

In higher plants, seed development requires maternal gene activity in the haploid (gametophytic) as well as diploid (sporophytic) tissues of the developing ovule. The Arabidopsis thaliana gene MEDEA (MEA) encodes a SET-domain protein of the Polycomb group that regulates cell proliferation by exerting a gametophytic maternal control during seed development. Seeds derived from female gametocytes (embryo sacs) carrying a mutant mea allele abort and exhibit cell proliferation defects in both the embryo and the endosperm. In this study we show that the mea mutation affects an imprinted gene expressed maternally in cells of the female gametophyte and after fertilization only from maternally inherited MEA alleles. Paternally inherited MEA alleles are transcriptionally silent in both the young embryo and endosperm. Mutations at the decrease in DNA methylation1 (ddm1) locus are able to rescue mea seeds by functionally reactivating paternally inherited MEA alleles during seed development. Rescued seeds are larger than the wild type and exhibit some of the abnormalities found in aborting mea seeds. Our results indicate that the maintenance of the genomic imprint at the mea locus requires zygotic DDM1 activity. Because DDM1 encodes a putative chromatin remodeling factor, chromatin structure is likely to be interrelated with genomic imprinting in Arabidopsis.

Alleles↗

PubMed1989

The molecular genetics of the bithorax complex of Drosophila: characterization of the products of the Abdominal-B domain.

In Drosophila the Abdominal-B (Abd-B) domain of the bithorax complex specifies the identities of several posterior abdominal segments, comprises homeo-protein-coding regions and cis-regulatory regions, and extends from infra-abdominal-5 (iab-5) to iab-8, inclusive. Mutations that eliminate the Abd-B domain act as late embryonic lethals and result in transformations of posterior abdominal segments toward more anterior ones. The Abd-B domain gives rise to a minimum of five homeo-box-containing transcripts, 7.8, 4.7, 4.3, 3.7, and 3.3 kb in length. We examined the structure of the Abd-B domain by sequencing two Abd-B cDNA clones derived from the 4.3- and the 4.7-kb transcripts and the corresponding genomic DNA. The domain spans approximately 100 kb and contains at least eight exons. The 4.7- and 4.3-kb transcripts contain an open reading frame capable of encoding a 54-kD protein. A portion of the deduced protein-coding sequence common to all of the Abd-B transcripts was cloned into an expression vector. The resultant fusion protein then was used to derive a monoclonal antibody specific to Abd-B. By use of that antibody, we identified two embryonic Abd-B proteins, 54 and 36 kD and determined the sum of their segmental distribution by immunohistochemical analysis of whole-mounted embryos and immunofluorescent analysis of dissected embryonic nervous systems. The proteins are distributed in the fourth to the ninth abdominal segments [parasegments (PS) 10-15] inclusive. Embryos homozygous for Polycomb (Pc) show labeling over almost the entire embryo, whereas embryos deficient for the Abd-B domain show no detectable labeling.

Amino Acid Sequence↗

PubMed1993

polyhomeotic regulatory sequences induce developmental regulator-dependent variegation and targeted P-element insertions in Drosophila.

Variegation of the miniwhite gene is observed in a euchromatic context in transformant lines that contain a P transposon including regulatory sequences of the polyhomeotic (ph) gene upstream of the resident miniwhite gene (P[ph]). This variegated phenotype is not affected by most of the genetic modifiers of heterochromatic position-effect variegation (PEV) nor by removal of the Y chromosome. Interestingly, it is sensitive to ph and Polycomb (Pc) mutations, which are known to affect homeotic gene regulation. Regulatory DNA of ph can also mediate transvection of the miniwhite gene. This transvection is abolished in a ph but not in a zeste mutant background. In addition, P[ph] inserts preferentially in sites corresponding to PH/PC protein-binding sites as defined at the polytene chromosome level. These insertions induce an unusually high proportion of mutations in genes affecting homeotic gene regulation. In particular, one insertion is located within the tramtrack locus, which is thought to regulate fushi tarazu, an Ultrabithorax activator. We suggest that a multimeric complex containing PH and PC proteins, at a minimum, causes a local and clonally inherited heterochromatinization, which maintains the repressed state of transcription of the homeotic genes.

Animals↗

PubMed2002

Transcriptional cosuppression of yeast Ty1 retrotransposons.

Cosuppression, the silencing of dispersed homologous genes triggered by high copy number, may have evolved in eukaryotic organisms to control molecular parasites such as viruses and transposons. Ty1 retrotransposons are dispersed gene repeats in Saccharomyces cerevisiae, where no cosuppression has been previously observed. Ty1 elements are seemingly expressed undeterred to a level as high as 10% of total mRNA. Using Ty1-URA3 reporters and negative selection with 5-fluoroorotic acid, it is shown that Ty1 genes can undergo transcriptional cosuppression that is independent of DNA methylation and polycomb-mediated repression. Expression of Ty1-related genes was shown to be in one of two states, the coexpressed state with all Ty1-related genes transcribed or the cosuppressed state with all Ty1-related genes shut off, without uncoordinated or mosaic expression in any individual cell. Rapid switches between the two states were observed. A high copy number of Ty1 elements was shown to be required for the initiation of Ty1 homology-dependent gene silencing, implying that Ty1 gene expression is under negative feedback control. Ty1 transcriptional repressors facilitated the onset of Ty1 cosuppression, and the native Ty1 promoters were required for Ty1 cosuppression, indicating that Ty1 cosuppression occurs at the transcriptional level.

Base Sequence↗

PubMed1995

An expression-independent catalog of genes from human chromosome 22.

To accomplish large-scale identification of genes from a single human chromosome, exon amplification was applied to large pools of clones from a flow-sorted human chromosome 22 cosmid library. Sequence analysis of more than one-third of the 6400 cloned products identified 35% of the known genes previously localized to this chromosome, as well as several unmapped genes and randomly sequenced cDNAs. Among the more interesting sequence similarities are those that represent novel human genes that are related to others with known or putative functions, such as one exon from a gene that may represent the human homolog of Drosophila Polycomb. It is anticipated that sequences from at least half of the genes residing on chromosome 22 are contained within this exon library. This approach is expected to facilitate fine-structure physical and transcription mapping of human chromosomes, and accelerate the process of disease gene identification.

Amino Acid Sequence↗

PubMed2003

EMF genes maintain vegetative development by repressing the flower program in Arabidopsis.

The EMBRYONIC FLOWER (EMF) genes EMF1 and EMF2 are required to maintain vegetative development and repress flower development. EMF1 encodes a putative transcriptional regulator, and EMF2 encodes a Polycomb group protein homolog. We examined expression profiles of emf mutants using GeneChip technology. The high degree of overlap in expression changes from the wild type among the emf1 and emf2 mutants was consistent with the functional similarity between the two genes. Expression profiles of emf seedlings before flower development were similar to that of Arabidopsis flowers, indicating the commitment of germinating emf seedlings to the reproductive fate. The germinating emf seedlings ectopically expressed flower organ genes, suggesting that vegetative development in wild-type plants results from EMF repression of the flower program, directly or indirectly. In addition, the seed development program is derepressed in the emf1 mutants. Gene expression analysis showed no clear regulation of CONSTANS (CO), FLOWERING LOCUS T (FT), LEAFY (LFY), and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 by EMF1. Consistent with epistasis results that co, lfy, or ft cannot rescue rosette development in emf mutants, these data show that the mechanism of EMF-mediated repression of flower organ genes is independent of these flowering genes. Based on these findings, a new mechanism of EMF-mediated floral repression is proposed.

Arabidopsis↗

PubMed2004

Detection of protein-protein interactions in plants using bimolecular fluorescence complementation.

Protein function is often mediated via formation of stable or transient complexes. Here we report the determination of protein-protein interactions in plants using bimolecular fluorescence complementation (BiFC). The yellow fluorescent protein (YFP) was split into two non-overlapping N-terminal (YN) and C-terminal (YC) fragments. Each fragment was cloned in-frame to a gene of interest, enabling expression of fusion proteins. To demonstrate the feasibility of BiFC in plants, two pairs of interacting proteins were utilized: (i) the alpha and beta subunits of the Arabidopsis protein farnesyltransferase (PFT), and (ii) the polycomb proteins, FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) and MEDEA (MEA). Members of each protein pair were transiently co-expressed in leaf epidermal cells of Nicotiana benthamiana or Arabidopsis. Reconstitution of a fluorescing YFP chromophore occurred only when the inquest proteins interacted. No fluorescence was detected following co-expression of free non-fused YN and YC or non-interacting protein pairs. Yellow fluorescence was detected in the cytoplasm of cells that expressed PFT alpha and beta subunits, or in nuclei and cytoplasm of cells that expressed FIE and MEA. In vivo measurements of fluorescence spectra emitted from reconstituted YFPs were identical to that of a non-split YFP, confirming reconstitution of the chromophore. Expression of the inquest proteins was verified by immunoblot analysis using monoclonal antibodies directed against tags within the hybrid proteins. In addition, protein interactions were confirmed by immunoprecipitations. These results demonstrate that plant BiFC is a simple, reliable and relatively fast method for determining protein-protein interactions in plants.

Amino Acid Sequence↗

PubMed2006

Genomic imprinting, methylation and molecular evolution of maize Enhancer of zeste (Mez) homologs.

Imprinted gene expression refers to differential transcription of alleles depending on their parental origin. To date, most examples of imprinted gene expression in plants occur in the triploid endosperm tissue. The Arabidopsis gene MEDEA displays an imprinted pattern of gene expression and has homology to the Drosophila Polycomb group (PcG) protein Enhancer-of-zeste (E(z)). We have tested the allele-specific expression patterns of the three maize E(z)-like genes Mez1, Mez2 and Mez3. The expression of Mez2 and Mez3 is not imprinted, with a bi-allelic pattern of transcription for both genes in both the endosperm and embryonic tissue. In contrast, Mez1 displays a bi-allelic expression pattern in the embryonic tissue, and a mono-allelic expression pattern in the developing endosperm tissue. We demonstrate that mono-allelic expression of the maternal Mez1 allele occurs throughout endosperm development. We have identified a 556 bp differentially methylated region (DMR) located approximately 700 bp 5' of the Mez1 transcription start site. This region is heavily methylated at CpG and CpNpG nucleotides on the non-expressed paternal allele but has low levels of methylation on the expressed maternal allele. Molecular evolutionary analysis indicates that conserved domains of all three Mez genes are under purifying selection. The common imprinted expression of Mez1 and MEDEA, in concert with their likely evolutionary origins, suggests that there may be a requirement for imprinting of at least one E(z)-like gene in angiosperms.

Alleles↗

PubMedDate not supplied

Epigenetics of prostate cancer: beyond DNA methylation.

Epigenetic mechanisms permit the stable inheritance of cellular properties without changes in DNA sequence or amount. In prostate carcinoma, epigenetic mechanisms are essential for development and progression, complementing, amplifying and diversifying genetic alterations. DNA hypermethylation affects at least 30 individual genes, while repetitive sequences including retrotransposons and selected genes become hypomethylated. Hypermethylation of several genes occurs in a coordinate manner early in carcinogenesis and can be exploited for cancer detection, whereas hypomethylation and further hypermethylation events are associated with progression. DNA methylation alterations interact with changes in chromatin proteins. Prominent alterations at this level include altered patterns of histone modification, increased expression of the EZH2 polycomb histone methyltransferase, and changes in transcriptional corepressors and coactivators. These changes may make prostate carcinoma particularly susceptible to drugs targeting chromatin and DNA modifications. They relate to crucial alterations in a network of transcription factors comprising ETS family proteins, the androgen receptor, NKX3.1, KLF, and HOXB13 homeobox proteins. This network controls differentiation and proliferation of prostate epithelial cells integrating signals from hormones, growth factors and cell adhesion proteins that are likewise distorted in prostate cancer. As a consequence, prostate carcinoma cells appear to be locked into an aberrant state, characterized by continued proliferation of largely differentiated cells. Accordingly, stem cell characteristics of prostate cancer cells appear to be secondarily acquired. The aberrant differentiation state of prostate carcinoma cells also results in distorted mutual interactions between epithelial and stromal cells in the tumor that promote tumor growth, invasion, and metastasis.

Animals↗

PubMed2006

The epigenetic magic of histone lysine methylation.

Epigenetic mechanisms control eukaryotic development beyond DNA-stored information. There are several pathways, including histone tail modifications, histone variant incorporation, nucleosome remodelling, DNA methylation and noncoding RNAs that together all contribute to the dynamic 'make-up' of chromatin under distinct developmental options. The histone tail modifications are most variable and over 50 marks have by now been mapped. While the majority of these modifications are transient, histone lysine methylation and, in particular, a histone lysine tri-methyl state has been regarded as a more robust signal, consistent with proposed roles to impart long-term epigenetic memory. Based on the paradigm of SET-domain histone lysine methyltransferases (HMTases) and chromo-domain adaptor proteins, and in conjunction with the Sir Hans Krebs Medal 2005, I describe here my personal view on the discovery of the first HMTase in 2000, and the subsequent advances on the biology of histone lysine methylation. This discovery has changed my scientific career and significantly contributed to a better understanding of epigenetic control, with important implications for heterochromatin formation, X inactivation, Polycomb group silencing and novel insights into stem cell research, nuclear reprogramming and cancer.

Animals↗

PubMed1998

Regulation of the Sex combs reduced gene in Drosophila.

The Sex combs reduced gene of the Antennapedia complex specifics the identities of the anterior thoracic and posterior head segments, including the primordium of the larval salivary gland. The Sex combs reduced transcription unit spans over 30 kb of genomic DNA, with another 40 kb of upstream cis-regulatory sequences. The pattern of Sex combs reduced transcription is set in the early embryo by the segmentation genes and is then maintained by two competing sets of proteins, the Polycomb group and the trithorax group. One of the trithorax group genes required for activation, the brahma gene, encodes an evolutionarily conserved DNA-stimulated ATPase that is part of a large protein complex. This complex facilitates the action of sequence-specific, DNA-binding proteins in regulating target genes, possibly by altering chromatin structure.

Animals↗

PubMed2002

A complex with chromatin modifiers that occupies E2F- and Myc-responsive genes in G0 cells.

E2F-6 contributes to gene silencing in a manner independent of retinoblastoma protein family members. To better elucidate the molecular mechanism of repression by E2F-6, we have purified the factor from cultured cells. E2F-6 is found in a multimeric protein complex that contains Mga and Max, and thus the complex can bind not only to the E2F-binding site but also to Myc- and Brachyury-binding sites. Moreover, the complex contains chromatin modifiers such as a novel histone methyltransferase that modifies lysine 9 of histone H3, HP1gamma, and Polycomb group (PcG) proteins. The E2F-6 complex preferentially occupies target promoters in G0 cells rather than in G1 cells. These data suggest that these chromatin modifiers contribute to silencing of E2F- and Myc-responsive genes in quiescent cells.

Amino Acid Sequence↗

PubMed2003

Epigenetic dynamics of imprinted X inactivation during early mouse development.

The initiation of X-chromosome inactivation is thought to be tightly correlated with early differentiation events during mouse development. Here, we show that although initially active, the paternal X chromosome undergoes imprinted inactivation from the cleavage stages, well before cellular differentiation. A reversal of the inactive state, with a loss of epigenetic marks such as histone modifications and polycomb proteins, subsequently occurs in cells of the inner cell mass (ICM), which give rise to the embryo-proper in which random X inactivation is known to occur. This reveals the remarkable plasticity of the X-inactivation process during preimplantation development and underlines the importance of the ICM in global reprogramming of epigenetic marks in the early embryo.

Acetylation↗

PubMed2002

RGS12TS-S localizes at nuclear matrix-associated subnuclear structures and represses transcription: structural requirements for subnuclear targeting and transcriptional repression.

RGS12TS-S, an 1,157-amino-acid RGS protein (regulator of G protein signaling), is a nuclear protein that exhibits a unique pattern of subnuclear organization into nuclear foci or dots when expressed endogenously or ectopically. We now report that RGS12TS-S is a nuclear matrix protein and identify structural determinants that target this protein to the nuclear matrix and to discrete subnuclear sites. We also determine the relationship between RGS12TS-S-decorated nuclear dots and known subnuclear domains involved in control of gene expression and provide the first evidence that RGS12TS-S is functionally involved in the regulation of transcription and cell cycle events. A novel nuclear matrix-targeting sequence was identified that is distinct from a second novel motif needed for targeting RGS12TS-S to nuclear dots. RGS12TS-S nuclear dots were distinct from Cajal bodies, SC-35 domains, promyelocytic leukemia protein nuclear bodies, Polycomb group domains, and DNA replication sites. However, RGS12TS-S inhibited S-phase DNA synthesis in various tumor cell lines independently of Rb and p53 proteins, and its prolonged expression promoted formation of multinucleated cells. Expression of RGS12TS-S dramatically reduced bromo-UTP incorporation into sites of transcription. RGS12TS-S, when tethered to a Gal4 DNA binding domain, dramatically inhibited basal transcription from a Gal4-E1b TATA promoter in a histone deacetylase-independent manner. Structural analysis revealed a role for the unique N-terminal domain of RGS12TS-S in its transcriptional repressor and cell cycle-regulating activities and showed that the RGS domain was dispensable for these functions. These results provide novel insights into the structure and function of RGS12TS-S in the nucleus and demonstrate that RGS12TS-S possesses biological activities distinct from those of other members of the RGS protein family.

Animals↗

PubMed2004

Differential targeting of two distinct SWI/SNF-related Drosophila chromatin-remodeling complexes.

The SWI/SNF family of ATP-dependent chromatin-remodeling factors plays a central role in eukaryotic transcriptional regulation. In yeast and human cells, two subclasses have been recognized: one comprises yeast SWI/SNF and human BAF, and the other includes yeast RSC and human PBAF. Therefore, it was puzzling that Drosophila appeared to contain only a single SWI/SNF-type remodeler, the Brahma (BRM) complex. Here, we report the identification of two novel BRM complex-associated proteins: Drosophila Polybromo and BAP170, a conserved protein not described previously. Biochemical analysis established that Drosophila contains two distinct BRM complexes: (i) the BAP complex, defined by the presence of OSA and the absence of Polybromo and BAP170, and (ii) the PBAP complex, containing Polybromo and BAP170 but lacking OSA. Determination of the genome-wide distributions of OSA and Polybromo on larval salivary gland polytene chromosomes revealed that BAP and PBAP display overlapping but distinct distribution patterns. Both complexes associate predominantly with regions of open, hyperacetylated chromatin but are largely excluded from Polycomb-bound repressive chromatin. We conclude that, like yeast and human cells, Drosophila cells express two distinct subclasses of the SWI/SNF family. Our results support a close reciprocity of chromatin regulation by ATP-dependent remodelers and histone-modifying enzymes.

Amino Acid Sequence↗

PubMed2005

In vivo HP1 targeting causes large-scale chromatin condensation and enhanced histone lysine methylation.

Changes in chromatin structure are a key aspect in the epigenetic regulation of gene expression. We have used a lac operator array system to visualize by light microscopy the effect of heterochromatin protein 1 (HP1) alpha (HP1alpha) and HP1beta on large-scale chromatin structure in living mammalian cells. The structure of HP1, containing a chromodomain, a chromoshadow domain, and a hinge domain, allows it to bind to a variety of proteins. In vivo targeting of an enhanced green fluorescent protein-tagged HP1-lac repressor fusion to a lac operator-containing, gene-amplified chromosome region causes local condensation of the higher-order chromatin structure, recruitment of the histone methyltransferase SETDB1, and enhanced trimethylation of histone H3 lysine 9. Polycomb group proteins of both the HPC/HPH and the EED/EZH2 complexes, which are involved in the heritable repression of gene activity, are not recruited to the amplified chromosome region by HP1alpha and HP1beta in vivo targeting. HP1alpha targeting causes the recruitment of endogenous HP1beta to the chromatin region and vice versa, indicating a direct interaction between the two HP1 homologous proteins. Our findings indicate that HP1alpha and HP1beta targeting is sufficient to induce heterochromatin formation.

Cells, Cultured↗

PubMed2005

A motif within SET-domain proteins binds single-stranded nucleic acids and transcribed and supercoiled DNAs and can interfere with assembly of nucleosomes.

The evolutionary conserved SET domain is present in many eukaryotic chromatin-associated proteins, including some members of the trithorax (TrxG) group and the polycomb (PcG) group of epigenetic transcriptional regulators and modifiers of position effect variegation. All SET domains examined exhibited histone lysine methyltransferase activity, implicating these proteins in the generation of epigenetic marks. However, the mode of the initial recruitment of SET proteins to target genes and the way that their association with the genes is maintained after replication are not known. We found that SET-containing proteins of the SET1 and SET2 families contain motifs in the pre-SET region or at the pre-SET-SET and SET-post-SET boundaries which very tightly bind single-stranded DNA (ssDNA) and RNA. These motifs also bind stretches of ssDNA generated by superhelical tension or during the in vitro transcription of duplex DNA. Importantly, such binding withstands nucleosome assembly, interfering with the formation of regular nucleosomal arrays. Two representatives of the SUV39 SET family, SU(VAR)3-9 and G9a, did not bind ssDNA. The trxZ11 homeotic point mutation, which is located within TRX SET and disrupts embryonic development, impairs the ssDNA binding capacity of the protein. We suggest that the motifs described here may be directly involved in the biological function(s) of SET-containing proteins. The binding of single-stranded nucleic acids might play a role in the initial recruitment of the proteins to target genes, in the maintenance of their association after DNA replication, or in sustaining DNA stretches in a single-stranded configuration to allow for continuous transcription.

Amino Acid Motifs↗

PubMed2005

The Mcp element from the bithorax complex contains an insulator that is capable of pairwise interactions and can facilitate enhancer-promoter communication.

Chromatin insulators, or boundary elements, appear to control eukaryotic gene expression by regulating interactions between enhancers and promoters. Boundaries have been identified in the 3' cis-regulatory region of Abd-B, which is subdivided into a series of separate iab domains. Boundary elements such as Mcp, Fab-7, and Fab-8 and adjacent silencers flank the iab domains and restrict the activity of the iab enhancers. We have identified an insulator in the 755-bp Mcp fragment that is linked to the previously characterized Polycomb response element (PRE) and silences the adjacent genes. This insulator blocks the enhancers of the yellow and white genes and protects them from PRE-mediated repression. The interaction between the Mcp elements, each containing the insulator and PRE, allows the eye enhancer to activate the white promoter over the repressed yellow domain. The same level of white activation was observed when the Mcp element combined with the insulator alone was interposed between the eye enhancer and the promoter, suggesting that the insulator is responsible for the interaction between the Mcp elements.

ATP-Binding Cassette Transporters↗