extra sexcombs
The yeast transcriptional repressor Tup1, a potential homolog of Drosophila Extra sexcombs, contains seven WD repeats that interact with the
DNA-binding protein alpha2. Mutations have been identified in Tup1 that disrupt this interaction. The
positions of the amino acids changed by these mutations are consistent with Tup1 being folded into a
seven-bladed propeller like that formed by another WD repeat-containing protein, the beta subunit of
the heterotrimeric G protein used in signal transduction. These results also indicate that the interaction
between Tup1 and alpha2 resembles the interaction between Gbeta and G alpha, suggesting that a
similar structural interface is formed by WD repeat proteins used in both transcriptional
regulation and signal transduction (Komachi, 1997).
Repression of a-cell specific gene expression in yeast alpha cells requires MAT alpha 2 and
MCM1, as well as two global repressors, SSN6 and TUP1, the yeast homolog of ESC. Previous studies demonstrated that
nucleosomes positioned adjacent to the alpha 2/MCM1 operator in alpha cells directly contribute
to repression. To investigate the possibility that SSN6 and TUP1 provide a link between MAT
alpha 2/MCM1 and neighboring histones, nucleosome locations were examined in mutant ssn6 and tup1
alpha cells. In both cases, nucleosome positions downstream of the operator were disrupted, and
the severity of the disruption correlated with the degree of derepression. Nevertheless, the
observed changes in chromatin structure were not dependent on transcription. These data strongly
indicate that SSN6 and TUP1 directly organize repressive regions of chromatin (Cooper, 1994).
Based on the results of mutational studies, the WD repeats appear to be essential for ESC protein to function as a repressor of homeotic genes. WD repeats may
mediate interactions between ESC and other Polycomb Group proteins, recruiting still other proteins to their
target genes, perhaps by additional interactions with transiently expressed repressors such as
hunchback. The ESC protein is highly conserved between D. melanogaster and D. virulis, particularly its
WD motifs. The high degree of conservation suggests that each of the WD
repeats in the ESC protein is functionally specialized and that this specialization has been highly
conserved during evolution. The highly charged N-terminus of ESC exhibits the greatest divergence, but
even these differences are conservative of its predicted physical properties. These observations
suggest that the ESC protein is functionally compact, nearly every residue making an important
contribution to its function (Sathe, 1995b).
Four Caenorhabditis elegans genes, mes-2, mes-3, mes-4 and mes-6, are essential for the normal proliferation and viability of the germline. Mutations in these genes cause a maternal-effect sterile (i.e. mes) or grandchildless phenotype. The mes-6 gene is in an unusual operon, the second example of this type of operon found in C. elegans: mes-6 encodes the nematode homolog of Extra sex combs, a WD-40 protein in the Polycomb group in Drosophila. mes-2 encodes another Polycomb group protein, Enhancer of zeste. Consistent with the known role of Polycomb group proteins in regulating gene expression, MES-6 is a nuclear protein. It is enriched in the germline of larvae and adults and is present in all nuclei of early embryos. Molecular epistasis results predict that the MES proteins, like Polycomb group proteins in Drosophila, function as a complex to regulate gene expression. Database searches reveal that there are considerably fewer Polycomb group genes in C. elegans than in Drosophila or vertebrates. MES-6 and MES-2 are the only recognizable Pc-G products in the C. elegans genome. These studies suggest that their primary function is in controlling gene expression in the germline and ensuring the survival and proliferation of that tissue (Korf, 1998).
Sequence analysis of the mes-6 cDNA has revealed that it encodes a 459 amino acid protein similar to Drosophila Extra sex combs (Esc) and the murine homolog of Esc, termed Eed for Embryonic ectodermal development. Similarity searches involving ~90% of the entire worm genomic sequence indicates that Esc is more similar to MES-6 than to any other sequence in the worm genome, and therefore, MES-6 likely represents the worm ortholog of Esc. MES-6, Esc and Eed share regions of sequence similarity that line up in register along the entire length of the proteins. These regions contain WD-40 repeats, which are thought to be involved in protein-protein interactions. WD-40 repeats within a protein are often very dissimilar. In contrast, positionally equivalent repeats in homologous proteins are more highly conserved. Indeed, positionally equivalent WD-40 repeats in MES-6, Esc and Eed are more similar to each other than to any non-equivalent repeats compared within or between proteins. MES-6 also appears to contain seven WD-40 repeats, so it also may adopt a propeller-like tertiary stucture. Interestingly, the gly-to-glu change found in the bn66 allele of mes-6 maps to one of the loops that is predicted to project from the top of the Esc propeller. The this region is likely to be important for protein-protein interactions based on sequence conservation among insect esc genes. (Korf, 1998).
As predicted by the similarity of MES-6 to Esc, MES-6 is localized in nuclei. In wild-type adults, MES-6 staining is most prominent in the germline, but is also detectable in intestinal nuclei. A maternal load of protein is seen in the nuclei of oocytes. In early embryos, MES-6 is present in the nuclei of all cells. As embryogenesis proceeds, staining gradually fades in somatic cells. In late embryos and L1 larvae, MES-6 remains faintly visible in a number of cell types, including the intestine, but is most prominent in Z2 and Z3, the primordial germ cells. Nuclear staining is diminished or reduced below detectability in worms bearing any of the four mutant alleles of mes-6 (Korf, 1998).
Do Pc-G genes serve an essential role in germline development in Drosophila, in addition to their well-known roles in somatic development? Since Pc-G mutations are generally lethal, addressing this question has required generating mutant germline clones by pole cell transplantation or by induction of mitotic recombination in the germline. Most of the Pc-G genes tested appear not to be essential for female germline development. E(z) does appear to be essential in the germline, since certain temperature-sensitive alleles of E(z) are sterile: transplanted pole cells mutant for a null allele of E(z) generate germlines indistinguishable from those in the temperature-sensitive alleles, indicating that the defect is germline autonomous (A. Shearn, personal communication to Korf, 1998). The strict maternal-effect sterility caused by mes mutations reveals that a maternal supply of wild-type mes gene product is both necessary and sufficient for normal germline development in the next generation. With this in mind, it has been hypothesized that the MES proteins are required to maintain a germline-specific organization of chromatin from one generation to the next, and that this chromatin state is essential to initiate the correct pattern of gene expression in the germline. This hypothesis integrates both the maintenance function of Pc-G proteins and the initiation function and germline specificity suggested by the maternal-effect sterile phenotype of mes mutants (Korf, 1998).
Recent results from Kelly and Fire (1998) provide strong support for this hypothesis. Kelly and Fire observed that ëhousekeepingí genes introduced into worms as transgenes and present in many tandem copies in extrachromosomal arrays are efficiently expressed in somatic cells but are specifically silenced in the germline of wild-type worms. Remarkably, gene expression is desilenced in the germline of animals mutant for any of the four mes genes. Gene expression also can be desilenced in the germline of wild-type worms by placing the test genes in the context of complex DNA in the array. These findings indicate that gene expression in the germline is dependent on chromatin context and wild-type MES function, supporting the notion that the MES proteins repress gene expression through an influence on chromatin state. The following model of germline establishment and protection of immortality is presented (Korf, 1998).
The early germline requires the silencing of gene expression that takes two forms. As the germline is being set apart from the soma in the early embryo, maternally supplied PIE-1, a transcriptional repressor that keeps somatically expressed genes turned off in the germline of early PIE-1 embryos keeps the germline blastomeres transcriptionally quiescent, while the surrounding somatic cells respond to differentiation factors and become transcriptionally active. During this period, maternally supplied MES proteins provide an underlying 'memory' of the germline state of chromatin, by maintaining nucleosomes or higher order chromatin in a particular conformation. When PIE-1 decays and the primordial germ cells become transcriptionally active, the MES-regulated chromatin conformation maintains repression of certain genes or regions of the genome and selectively allows initiation of expression of germline-required genes. In the absence of a functional MES system, death of the germline could be due to ectopic expression of genes that are normally kept silent in the germline, or alternatively, could be due to altered levels or timing of expression of genes that are normally expressed in the germline. The chromosomal binding sites for the predicted MES complex are currently unknown. However, several observations suggest that a key feature of these sites may be the number of copies of target sequences. MES+ function participates in keeping repetitive arrays of transgenes silenced in the germline, while non-repetitive arrays escape this silencing (Kelly, 1998). Somewhat analogously, Drosophila Pc-G proteins repress expression of transgenes that are present in multiple copies (Pal-Bhadra, 1997). Interestingly, the requirement for MES+ function is sensitive to chromosome dosage: animals with three X chromosomes absolutely require MES+ function for fertility, while animals with one X show a reduced requirement. This sensitivity to X-chromosome dosage may indicate participation of the MES proteins in dosage compensation in the germline. Future identification of MES targets will enhance understanding of the mechanism of Pc-G/MES control of gene expression and how this control contributes to the germline/soma dichotomy in C. elegans (Korf, 1998 and references).
Evolutionary conservation of the Esc-Enhancer of zeste partner relationship is supported by recent studies of two Caenorhabditis elegans maternal-effect sterile genes, mes-2 and mes-6. Mutant alleles of either mes-2 or mes-6 produce grandchildless phenotypes that result from the limited proliferation and death of germ cells. MES-2 and MES-6 share sequence similarity with E(z) and Esc, respectively, across extensive portions of these proteins. However, unlike E(z) and Esc functions, mes-2 and mes-6 functions appear to be restricted to the germ line; thus far, there is no evidence for their involvement in Hox gene control. Thus, the developmental roles of MES-2 and MES-6 in worms are distinct from those of their E(z) and Esc homologs in flies and mammals. Nevertheless, there is evidence that the basic biochemical partnership between these proteins has been conserved. The spatial and temporal patterns of MES-2 and MES-6 accumulation in nuclei are identical, and mutations in either gene disrupt the spatial distribution or stability of the other protein. One of these mutations, mes-6bn66, substitutes a residue at a position that aligns with the predicted loop region of Esc that is required for binding to E(z) protein. Taken together, these results are consistent with a direct physical interaction between MES-2 and MES-6 in vivo. The conservation of these worm homologs, together with the evolutionary divergence of their developmental functions, may reflect a common biochemical role in chromatin that has been adapted for use in different cell lineages in worms and flies (Jones, 1998 and references).
The C. elegans maternal-effect sterile genes, mes-2, mes-3, mes-4, and mes-6, encode nuclear proteins that are essential for germ-line development. They are thought to be involved in a common process because their mutant phenotypes are similar. MES-2 and MES-6 are homologs of Enhancer of zeste and extra sex combs, both members of the Polycomb group of chromatin regulators in insects and vertebrates. MES-3 is a novel protein, and MES-4 is a SET-domain protein. To investigate whether the MES proteins interact and likely function as a complex, biochemical analyses were performed on C. elegans embryo extracts. Results of immunoprecipitation experiments indicate that MES-2, MES-3, and MES-6 are associated in a complex and that MES-4 is not associated with this complex. Based on in vitro binding assays, MES-2 and MES-6 interact directly, via the amino terminal portion of MES-2. Sucrose density gradient fractionation and gel filtration chromatography were performed to determine the Stokes radius and sedimentation coefficient of the MES-2/MES-3/MES-6 complex. Based on those two values, it is estimated that the molecular mass of the complex is ~255 kDa, close to the sum of the three known components. These results suggest that the two C.
elegans Polycomb group homologs (MES-2 and MES-6) associate with a novel partner (MES-3) to regulate germ-line development in C. elegans (Xu, 2001).
Twelve PcG proteins have been cloned so far in Drosophila, but only two of them, E(Z) and ESC, are conserved in worms and also in plants. These data suggest that these two proteins might be distinctive PcG members, which function independently of the other PcG proteins. In Drosophila, E(Z) and ESC are
associated in a complex in vivo and interact with each other directly in vitro. This association is conserved for their mammalian homologs, ENX and EED, and also for their worm homologs, MES-2 and MES-6 (Xu, 2001).
The portion of MES-2 that is important for its interaction with MES-6 in vitro is the N-terminal 194 aa. Similarly, sequences within the N-terminal region of fly E(Z) (amino acids 34-66) and the mouse E(Z) homolog (amino acids 132-160) are responsible for their interactions with ESC homologs. Although there is very
little sequence similarity between the N termini of MES-2 and E(Z) homologs, their tertiary structure and the nature of the interaction between the partners might be conserved. The ESC-binding region of E(Z) is predicted to include a long stretch of helix, and the N-terminal 194 aa of MES-2 is predicted to form multiple long helices (Xu, 2001).
The conservation of the interaction between E(Z) and ESC among worms, flies, and mammals suggests that the molecular mechanism by which these protein partners
function has been maintained throughout evolution. The MES-2/MES-6 complex therefore might regulate gene expression in C. elegans by the same mechanism
used by the E(Z)/ESC complex in Drosophila. Indeed, MES-2 and MES-6 participate in repressing gene expression in C. elegans, as E(Z) and ESC are known to do in Drosophila (Xu, 2001).
Polycomb group (PcG) chromatin proteins regulate homeotic genes in both animals and plants. In Drosophila and vertebrates, PcG proteins form complexes and maintain early patterns of Hox gene repression, ensuring fidelity of developmental patterning. PcG proteins in C. elegans form a complex and mediate transcriptional silencing in the germline, but no role for the C. elegans PcG homologs in somatic Hox gene regulation has been demonstrated. Surprisingly, it is found that the PcG homologs MES-2 [E(Z)] and MES-6 (ESC), along with MES-3, a protein without known homologs, do repress Hox expression in C. elegans. mes mutations cause anteroposterior transformations and disrupt Hox-dependent neuroblast migration. Thus, as in Drosophila, vertebrates, and plants, C. elegans PcG proteins regulate key developmental patterning genes to establish positional identity (Ross, 2003).
The three mes genes act upstream of the Hox genes mab-5 and egl-5 during V ray differentiation, and loss of mes activity can restore normal ray development and mating ability to males mutant in the mab-5 activator pal-1. Males lacking mes activity display anterior expansions of tail structures and ectopic expression of the Hox reporter egl-5::gfp and the Hox target lin-32::gfp. This regulation is not restricted to the male tail: mes-2, -3, and -6 also repress lin-39::lacZ expression in the midbody and head and mab-5 activity in a migrating neuroblast. Consistent with a general somatic regulatory function, MES protein expression is widespread in larvae, particularly males. These findings suggest that the regulatory relationship between PcG chromatin proteins and the Hox genes has been conserved in nematodes (Ross, 2003).
Polycomb group (PcG) genes are required for stable inheritance of epigenetic states throughout development, a phenomenon termed cellular memory. In Drosophila and mice, the product of the E(z) gene, one of the PcG genes, constitutes the ESC-E(Z) complex and specifically methylates histone H3. It has been argued that this methylation sets the stage for appropriate repression of certain genes. This study reports the isolation of a well-conserved homolog of E(z), olezh2, in medaka. Hypomorphic knock-down of olezh2 resulted in a cyclopia phenotype and markedly perturbed hedgehog signaling, consistent with a previous report on oleed, a medaka esc. Cyclopia was also found in embryos treated with trichostatin A, an inhibitor of histone deacetylase, which is a transient component of the ESC-E(Z) complex. The level of tri-methylation at lysine 27 of histone H3 is substantially decreased in both olezh2 and oleed knock-down embryos, and in embryos with hedgehog signaling perturbed by forskolin. It is concluded that the ESC-E(Z) complex per se participates in hedgehog signaling (Shindo, 2005).
A classic mouse mutation, eed (embryonic ectoderm development) is a highly conserved homolog of esc. Mutants for a null allele of eed display disrupted anterior-posterior patterning of the primitive streak during gastrulation. Mutant embryos lack a node, notochord and somites, and there is no neural induction. In contrast to absence of anterior structures, extra-embryonic mesoderm is abundant. Mice carrying a hypomorphic eed mutation exhibit posterior transformations along the axial skeleton. EED consists of 441 amino acids and contains five tandemly repeated WD motifs. Sequence comparison with ESC reveals a 55% identity, with conservation between WD motifs almost as high as that within the WD motifs. The high evolutionary conservation spans 83% of the EED sequence, and is not interrupted by a single gap or insertion. Mutation in eed causes spatial derepression of Hox genes. Disruption of the unsegmented primitive streak during gastrulation reflects eed involvement in regulating anterior-posterior patterning before segmentation. Homeodomain transcription factor Evx1 (the mouse homolog of Even-skipped) is downstream of eed. It is conceivable the eed may be required to establish and/or maintain an Evx1 expression gradient, which in turn may impart positional information to mesodermal cells leaving the primitive streak (Schumacher, 1996)
Similar to Drosophila, murine Polycomb-group (PcG) genes regulate
anterior-posterior patterning of segmented axial structures by transcriptional
repression of homeotic gene expression. The murine PcG gene eed (embryonic
ectoderm development) encodes a 441-amino-acid protein with five WD motifs
which, except for the amino terminus, is highly homologous to Drosophila
Extra Sex Combs (Esc). Sequence and expression analysis as well as chromosomal
mapping of the human ortholog of eed are described in this study. Absolute conservation of
the human eed protein along with significant divergence at the nucleotide level
reveals functional constraints operating on all residues. The human orthologue
appears to be ubiquitously expressed and maps to chromsome 11q14.2-q22.3. Using
the first WD motif of the beta-subunit of the bovine G protein as a structural
reference, the predicted locations of two previously identified eed point
mutations are reported in this study. The proline substitution (L196P) in the second WD motif of the
l7Rn5(3354SB) null allele maps to the internal core of the inner end of the
beta-propeller blade and is likely to disrupt protein folding. In contrast, the
asparagine substitution (I193N) in the second WD motif of the hypomorphic
l7Rn5(1989SB) allele maps onto the surface of the beta-propeller blade near the
central cavity and may affect surface interactions without compromising
propeller packing. These results illustrate the critical importance of all
residues for eed function in mammals and support a model whereby the amino
terminus might implement function(s) related to embryonic development in higher
organisms (Schumacher, 1998).
An induced mutation, embryonic ectoderm development or eed, has been characterized: it disrupts A-P patterning of the
mouse embryo during gastrulation. Positional cloning of this gene reveals it to be the highly conserved homolog of the
Drosophila gene extra sex combs, which is required for maintenance of long-term transcriptional repression of homeotic gene
expression. Mouse embryos homozygous for loss-of-function alleles of eed initiate gastrulation but display abnormal
mesoderm production. Very little embryonic mesoderm is produced; in contrast, extraembryonic mesoderm is relatively
abundant. These observations, along with mRNA in situ hybridization analyses, suggest a defect in the anterior primitive
streak, from which much of the embryonic mesoderm of the wild-type embryo is derived. Clonal analysis of the pre-streak epiblast was initiated in eed mutant embryos, using the lineage tracer horseradish peroxidase (HRP). The
results of these studies indicate that epiblast cells ingress through the anterior streak, but the newly formed mesoderm does not
migrate to the anterior and is mislocalized to the extraembryonic compartment. Abnormal localization of mesoderm to the
extraembryonic region does not appear to be due to a restriction and alteration of distal epiblast cell fate, since the majority of
clones produced from regions fated to ingress through the anterior streak are mixed, displaying descendants in both
embryonic and extraembryonic derivatives. eed mutant embryos also fail to display proper epiblast expansion, particularly with
respect to the A-P axis. Based on patterns of clonal spread and calculated clone doubling times for the epiblast, this does not
appear to be due to decreased epiblast growth. Rather, epiblast, which is normally fated to make a substantial contribution to
the axial midline, appears to make mesoderm preferentially. The data are discussed in terms of global morphogenetic
movements in the mouse gastrula and a disruption of signalling activity in the anterior primitive streak (Faust, 1998).
It has been proposed that Drosophila Esc interacts
with the transcriptional machinery through the WD-40 domains. This model is based on the homology that is
found between Esc and Tup1, a yeast protein that also contains seven WD-40 domains. These WD-40 domains are important for the involvement of the Tup1 protein in the repression of gene activity and in its binding to the DNA-binding homeodomain protein 2. Point mutations in the
WD-40 domains have also been found in several esc mutants. Either one of two point mutations in the second WD-40 domain completely abolishes the interaction in the two-hybrid system between Enx1 and EED. Precisely these two point mutations are responsible for the severe developmental defects in eed mutant mice. It is significant that the ability of the eed535 protein to repress gene activity is also completely abolished by these point mutations. It is therefore tempting to speculate that both the interference with the binding capacity and the repressing abilities of the eed/EED protein through these point mutations contribute to the developmental defects in eed mutant mice. One
immediate consequence of these point mutations can be that the Enx1 protein is no longer able to bind to eed; this leads to a subsequent loss of integrity for the protein complex formed in part by both Enx1/EZH2 and eed (Sewalt, 1998).
The murine Polycomb-Group (PcG) proteins Bmi1 (B cell-specific Mo-MLV
integration site 1) and eed
(embryonic ectoderm development)
govern axial
patterning during embryonic development by segment-specific repression of Hox
gene expression. The two proteins engage in distinct multimeric complexes that
are thought to use a common molecular mechanism to render the regulatory
regions of Hox and other downstream target genes inaccessible to transcriptional
activators. Beyond axial patterning, Bmi1 is also involved in hemopoiesis because a loss-of-function allele
causes a profound decrease in bone marrow progenitor cells. Here, evidence is presented that is consistent
with an antagonistic function of eed and Bmi1 in hemopoietic cell proliferation. Heterozygosity for an eed null
allele causes marked myelo- and lympho-proliferative defects, indicating that eed is involved in the negative
regulation of the pool size of lymphoid and myeloid progenitor cells. This antiproliferative function of eed does
not appear to be mediated by Hox genes or the tumor suppressor locus p16INK4a/p19ARF because
expression of these genes was not altered in eed mutants. Intercross experiments between eed and Bmi1
mutant mice reveal that Bmi1 is epistatic to eed in the control of primitive bone marrow cell proliferation.
However, the genetic interaction between the two genes is cell-type specific, because the presence of one or two
mutant alleles of eed trans-complements the Bmi1-deficiency in pre-B bone marrow cells. Thus, these studies
suggest that hemopoietic cell proliferation is regulated by the relative contribution of repressive
(Eed-containing) and enhancing (Bmi1-containing) PcG gene complexes.
Biochemical studies have
indicated that the PcG proteins Bmi1, Mel18, M33, and Mph1/Rae28 are constituents of a multimeric protein
complex A, which localizes to discrete nuclear foci in U-2 OS osteosarcoma cells. Importantly, Eed neither interacts physically with Bmi1 nor engages
in this protein complex. Instead, Eed forms a
complex B with the PcG proteins Enx1/EzH2 and Enx2/EzH1, which lack signs of a discrete subnuclear
distribution and are found rather uniformly throughout the nucleoplasm of U-2 OS osteosarcoma cells (Lessard, 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).
X inactivation in female mammals is one of the best studied examples of heritable gene silencing and provides an important model for studying maintenance of patterns of gene expression during differentiation and development. The process is initiated by a cis-acting RNA, the X inactive specific transcript (Xist). Xist RNA is thought to recruit silencing complexes to the inactive X, that then serve to establish and maintain the inactive state in all subsequent cell divisions. Most lineages undergo random X inactivation, there being an equal probability of either the maternally (Xm) or paternally (Xp) inherited X chromosome being inactivated in a given cell. In the extraembryonic trophectoderm and primitive endoderm lineages of mouse embryos, however, there is imprinted X inactivation of Xp. This process is also Xist dependent. A recent study has shown that imprinted X inactivation in trophectoderm is not maintained in embryonic ectoderm development (eed) mutant mice. Eed, the mammalian homolog of Drosophila Extra sex combs, and Enx1, the mammalian homolog of enhancer of Zeste, are directly localized to the inactive X chromosome in XX trophoblast stem (TS) cells. The association of Eed/Enx1 complexes is mitotically stable, suggesting a mechanism for the maintenance of imprinted X
inactivation in these cells (Mak, 2002).
The Polycomb group (PcG) protein Eed is implicated in regulation of imprinted X-chromosome inactivation in extraembryonic cells but not of random X inactivation in embryonic cells. The Drosophila homolog of the Eed-Ezh2 PcG protein complex achieves gene silencing through methylation of histone H3 on lysine 27 (H3-K27), which suggests a role for H3-K27 methylation in imprinted X inactivation. This study demonstrates that transient recruitment of the Eed-Ezh2 complex to the inactive X chromosome (Xi) occurs during initiation of X inactivation in both extraembryonic and embryonic cells and is accompanied by H3-K27 methylation. Recruitment of the complex and methylation on the Xi depend on Xist RNA but are independent of its silencing function. Together, these results suggest a role for Eed-Ezh2-mediated H3-K27 methylation during initiation of both imprinted and random X inactivation and demonstrate that H3-K27 methylation is not sufficient for silencing of the Xi (Plath, 2003).
The heterogeneous nuclear ribonucleoprotein K protein represents a novel class of
proteins that may act as docking platforms that orchestrate cross-talk among
molecules involved in signal transduction and gene expression. Using a fragment of K
protein as bait in the yeast two-hybrid screen, a cDNA was isolated that encodes a
protein whose primary structure has extensive similarity to the Drosophila
melanogaster extra sexcombs (esc) gene product, Esc, a putative silencer of
homeotic genes. The cDNA that was isolated is identical to the cDNA of the recently
positionally cloned mouse embryonic ectoderm development gene, eed. Like Esc, Eed
contains six WD-40 repeats in the C-terminal half of the protein and is thought to
repress homeotic gene expression during mouse embryogenesis. Eed binds to K
protein through a domain in its N terminus, but interestingly, this domain is not found in
the Drosophila Esc. Gal4-Eed fusion protein represses transcription of a reporter gene
driven by a promoter that contains Gal4-binding DNA elements. Eed also represses
transcription when recruited to a target promoter by Gal4-K protein. Point mutations
within the eed gene, responsible for severe embryonic development
abnormalities, abolish the transcriptional repressor activity of Eed. Results of this
study suggest that Eed-restricted homeotic gene expression during embryogenesis
reflects the action of Eed as a transcriptional repressor. The Eed-mediated
transcriptional effects are likely to reflect the interaction of Eed with multiple
molecular partners, including K protein (Denisenko, 1997).
The Polycomb group proteins are involved in maintenance of the silenced state of several
developmentally regulated genes. These proteins form large aggregates with different subunit
compositions. To explore the nature of these complexes and their function(s), the full-length Eed
(embryonic ectoderm development) protein, a mammalian homolog of the Drosophila Polycomb
group protein Esc, was used as a bait in a yeast two-hybrid screen. Several strongly interacting cDNA clones
were isolated. The cloned cDNAs all encode the 150- to 200-amino-acid N-terminal fragment of the
mammalian homolog of the Drosophila Enhancer of zeste [E(z)] protein, Ezh2. The full-length Ezh2
binds strongly to Eed in vitro, and Eed coimmunoprecipitates with Ezh2 from murine 70Z/3 cell
extracts, confirming the interaction between these proteins that was observed in yeast. Mutations T1031A and
T1040C in one of the WD40 repeats of Eed, which account for the hypomorphic and lethal phenotype
of eed in mouse development, block binding of Ezh2 to Eed in a two-hybrid interaction in both yeast and
mammalian cells. These mutations also blocked the interaction between these proteins in vitro. In
mammalian cells, the Gal4-Eed fusion protein represses the activity of a promoter bearing Gal4 DNA
elements. The N-terminal fragment of the Ezh2 protein abolishes the transcriptional repressor activity
of Gal4-Eed protein when these proteins are coexpressed in mammalian cells. Eed and Ezh2 are also found
to bind RNA in vitro, and RNA alters the interaction between these proteins. These findings suggest
that Polycomb group proteins Eed and Ezh2 functionally interact in mammalian cells, an interaction that
is mediated by the WD40-containing domain of Eed protein (Denisenko, 1998).
Several lines of evidence suggest a functional interaction
between the PcG and trxG proteins. For example, genetic evidence indicates that the Enhancer of zeste [E(z)] gene can be considered both a PcG and a trxG gene. To better understand the molecular interactions in which the E(z) protein is involved, a two-hybrid screen was performed with Enx1/EZH2, a mammalian homolog of E(z), as the target. The human EED protein is shown to interact with Enx1/EZH2. EED is the human homolog of eed, a murine PcG gene that has extensive homology with the Drosophila PcG gene extra sex combs (esc). Enx1/EZH2 and EED coimmunoprecipitate, indicating that they also interact in vivo. However, Enx1/EZH2 and EED do not coimmunoprecipitate with other human PcG proteins, such as HPC2 and BMI1. Furthermore, unlike HPC2 and BMI1, which colocalize in nuclear domains of U-2 OS osteosarcoma cells, Enx1/EZH2 and EED do not colocalize with HPC2 or BMI1. These findings indicate that Enx1/EZH2 and EED are members of a class of PcG proteins that is distinct from previously described human PcG proteins (Sewalt, 1998).
To define the domains that are responsible for the interaction between Enx1 and EED, different parts of Enx1 and EED were subcloned in frame with
the GAL4-DBD and to see whether these proteins could still interact with full-length EED or full-length Enx1. Enx1 comprises two N-terminal domains that show strong homology between Drosophila E(z) and its mammalian homologs. These domains have been designated domains I and II. Furthermore, Enx1 contains a C-terminal cysteine-rich domain and a SET domain. This last domain is found in a number of different proteins such as the Trithorax protein. The region encompassing both the cysteine-rich domain and the SET domain
(aa 498 to 746) does not interact with EED. A region extended toward the N terminus (aa 285 to 746) also
does not interact with EED. In contrast, the region encompassing domain I and a part of domain II (aa 1 to 285) does interact with EED. To analyze this region in more detail, two constructs were made, one containing homology domain I (aa 1 to 195) and the other containing homology domain II (aa 172 to 335). Only the region of Enx1 that contains homology domain I interacts with the EED protein. It is concluded that EED binds to the N-terminal region of Enx1 that encompasses homology domain I (Sewalt, 1998).
EED contains five WD-40 domains which are thought to be involved in protein-protein interactions. The importance of these WD-40 domains for the interaction between Enx1 and EED was tested. Truncated EED
protein constructs were made that contain an increasing number of WD-40 domains. None of the truncated EED proteins, which contain up to four WD-40 domains, interact with Enx1. Only when all five WD-40
domains are present is this truncated EED protein (aa 184 to 535) able to interact with Enx1. The
most N-terminal region of EED, which does not contain WD-40 domains, is not important for
mediating the interaction between Enx1 and EED (Sewalt, 1998).
Drosophila Extra sex combs (Esc) and its mammalian homolog, embryonic ectoderm development (eed), are special Pc-G members; they are required early during development when
Pc-G repression is initiated, a process that is still poorly understood. To gain insight into the molecular function of Eed, the yeast two-hybrid method was used to find Eed-interacting proteins. Described is the specific in vivo binding of Eed to Enx1 and Enx2, two mammalian homologs of the essential Drosophila Pc-G gene Enhancer-of-zeste [E(z)]. No direct biochemical interactions are found between Eed/Enx and a previously characterized mouse Pc-G protein complex containing
several mouse Pc-G proteins, including mouse polyhomeotic (Mph1). This suggests that different Pc-G complexes with distinct functions may exist. However, and in support of a bridging role for Enx1, partial colocalization of Enx1 and Mph1 to subnuclear domains may point to more transient
interactions between these complexes (van Lohuizen, 1998).
If the observed interactions between Enx1, Enx2, and Eed are relevant for their in vivo function, one would expect the expression patterns to coincide during development. Since the onset of Eed expression possibly occurs earlier than for most other Pc-G genes, a sensitive, semiquantitative RT-PCR assay was used to monitor the expression patterns of Enx1 and Enx2 during development and in adult mice. Whereas no expression is detected in blastocysts, Enx1 is already highly expressed in day 7.3 mouse embryos and becomes gradually more restricted to specific organs during development, with the highest expression being in the testis. In contrast, Enx2 expression was first detected on day 9 and continues to be expressed at moderate levels in most tissues. These results indicate that there is indeed extensive overlap in onset and tissue distribution between Enx1, Enx2, and Eed: the onset of Enx1 expression parallels that of Eed, whereas in adult mice Enx2 and Eed are expressed in most tissues (van Lohuizen, 1998).
Polycomb-group (PcG) proteins form multimeric protein complexes, which are
involved in maintaining the transcriptional repressive state of genes over
successive cell generations. Components of PcG complexes and their mutual
interactions have been identified and analysed through extensive genetic and
biochemical analyses. However, molecular mechanisms underlying PcG-mediated repression of
gene activity have remained largely unknown. There are two distinct human PcG protein complexes. The EED/EZH protein
complex contains the embryonic ectoderm development (EED) and enhancer of zeste
2 (EZH2) PcG proteins. The HPC/HPH PcG complex contains the human
polycomb 2 (HPC2), human polyhomeotic (HPH), BMI1 and RING1 proteins. In this study, EED has been shown to interact,
both in vitro and in vivo, with histone deacetylase (HDAC) proteins. This
interaction is highly specific because the HDAC proteins do not interact with
other vertebrate PcG proteins. Histone deacetylation
activity co-immunoprecipitates with the EED protein. The histone
deacetylase inhibitor trichostatin A relieves transcriptional
repression mediated by EED, but not by HPC2, a human homolog of polycomb. These
data indicate that PcG-mediated repression of gene activity involves histone
deacetylation. This mechanistic link between two distinct, global gene
repression systems is accomplished through the interaction of HDAC proteins with
a particular PcG protein, EED (van der Vlag, 1999).
Polycomb group (PcG) proteins form multimeric protein complexes that are involved in the heritable stable repression of genes. Two distinct human PcG protein complexes have been identified. The EED-EZH protein complex contains the EED and EZH2 PcG proteins, and the HPC-HPH PcG complex contains the HPC, HPH, BMI1, and RING1 PcG proteins. YY1, a homolog of the Drosophila PcG protein Pleiohomeotic (Pho), interacts specificially with the human PcG protein EED (Drosophila homolog: Extra sexcombs) but not with proteins of the HPC-HPH PcG complex. Since YY1 and Pho are DNA-binding proteins, the interaction between YY1 and EED provides a direct link between the chromatin-associated EED-EZH PcG complex and the DNA of target genes. To study the functional significance of the interaction, the Xenopus homologs of EED and YY1 were expressed in Xenopus embryos. Both Xeed and XYY1 induce an ectopic neural axis but do not induce mesodermal tissues. In contrast, members of the HPC-HPH PcG complex do not induce neural tissue. The exclusive, direct neuralizing activity of both the Xeed and XYY1 proteins underlines the significance of the interaction between the two proteins. These data also indicate a role for chromatin-associated proteins, such as PcG proteins, in Xenopus neural induction (Satijn, 2001).
Esc, the Drosophila homolog of EED, is distinguished from other PcG proteins
in Drosophila in that it is primarily required only during
embryogenesis. It has been speculated that deacetylation
of histones by HDACs and the recruitment of EED to the HDAC proteins
may be among the initial repressive events during embryogenesis that eventually lead to stable and heritable PcG-mediated repression of
target genes. Now it has been found that YY1 is part of or associated with the EED-EZH PcG complex, which displays HDAC activity. Since Pho and YY1 display specific DNA-binding properties, this finding suggests a model in which YY1 directs the EED-EZH PcG
complex to target genes. This first step is consistent with
the early developmental role of the EED-EZH complex, as has been
defined genetically. It is also consistent with a role for histone
deacetylation, mediated by the HDACs, which are associated with both
EED and YY1, as an early event by which PcG proteins set up stable
repression of target genes (Satijn, 2001).
The existence of two distinct PcG protein complexes has also been
observed in Drosophila. Using a two-hybrid analysis, the Esc and E(z) proteins have been found to interact. Furthermore, two distinct Drosophila PcG protein complexes have been characterized biochemically. One complex contains
the Pc, Psc, and Ph proteins; the other contains the Esc and E(z) proteins. These findings are very similar to the observations in the human system.
There are, however, significant differences between the two
developmental systems. For instance, the Drosophila Pho
protein lacks a domain that mediates histone deacetylation activity. This domain is present in the YY1 protein. It is possible that this constitutes a fundamental difference between the Drosophila and the vertebrate systems, indicating that histone deacetylation plays a less significant role in the Drosophila system. Further, the Pho protein has not been detected in the Pc-Ph complex. However, the Pho protein could not be detected in the biochemically purified ESC-E(Z) complex either. These puzzling findings may reflect a more transient nature of interactions between Pho and other proteins, which precludes biochemical purification as part of a stable protein complex. The observation that even an in vitro interaction between EED and the Drosophila Pho protein exists at least suggests a highly conserved nature of the interaction between EED and
YY1. Also, the virtually identical phenotypes that are induced by Xeed,
XYY1, and Pho in Xenopus embryos suggest that YY1 or Pho is
either a stable component of or at least transiently associated with
the EED-EZH PcG complex and not the HPC-HPH PcG complex (Satijn, 2001).
To study the functional significance of
the interaction between EED and YY1, the expression
levels of the Xenopus homologs of these proteins, Xeed and
XYY1, were manipulated. Both proteins, but no other PcG proteins, induce an ectopic neural axis in Xenopus embryos, but neither Xeed nor XYY1
is able to induce mesodermal tissue, such as muscle or notochord.
Importantly, the Drosophila Pho protein induces the same
phenotype. The similarity of effects underlines the significance of the
EED-YY1 interaction. The fact that Pho induces the same phenotype and
neural tissue in ectoderm explants also substantiates the notion that
YY1 is indeed a functional homolog of the Drosophila Pho protein (Satijn, 2001).
These data point towards an early developmental role for the EED-EZH
complex. Also, in homozygous eed minus mice
the earliest developmental decisions are affected, pointing towards an
early role for EED in setting up vertebrate PcG-mediated repression. It
may be significant that homozygous eed minus
mice lack a node and, probably as a consequence of this, also neural
tissue. Whereas the homozygous
YY1 minus mutation is embryonic lethal, in
heterozygote YY1/+ mice the formation of
a proper neural tube is seriously hampered. Both
phenotypes are complementary to the phenotypes observed after
overexpression of both Xeed and XYY1 proteins in Xenopus
embryos. Although a detailed comparison between the
loss-of-function data in mice and overexpression of proteins in
Xenopus is not possible, the opposing effects on neural
tissue are compatible with each other. The results reinforce one another and both point towards an early role of these PcG proteins in developmental decisions, such as the induction of embryonic tissues (Satijn, 2001).
The following questions remain: which are the target genes of Xeed and
XYY1, and how does the modulation of the activity of these target genes
result in the induction of neural tissue? Since EED is a repressor of
gene activity, it is likely that Xeed is also a
repressor of gene activity. Furthermore, both XYY1 and
XYY1-EnR directly induce neural tissue, and by
virtue of the EnR domain, XYY1-EnR is a transcriptional repressor. It is, therefore, likely that the target genes of Xeed and XYY1 are repressed by these proteins and that this repression results in the induction of
neural tissue. It will be of considerable interest to identify these
target genes. Since the effects of Xeed and XYY1 occur early in
development, these target genes may well represent a class of PcG
target genes other than the known PcG target genes in
Drosophila that are affected relatively late during
development. Also, identification of such target genes may reveal
pathways, distinct from the known ones, that are involved in mediating
neural induction in Xenopus (Satijn, 2001).
heed, the human homolog of mouse eed and Drosophila esc, two members of the
trithorax (trx) and Polycomb group (Pc-G) of genes, were 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) has 99.5% identity with the mouse EED protein and contains
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, originating from
the unspliced message, lacks the seventh WD repeat. HEED binds to
MA protein in vitro, as efficiently as in vivo in yeast cells. Site-directed
mutagenesis and phage biopanning suggest 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, is 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-localize 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 suggests a role of HEED at the early
stages of virus infection, rather than late in the virus life cycle (Peytavi, 1999).
The mouse eed
locus encodes the highly conserved ortholog of the Drosophila ESC protein. To
test the functional conservation between the two genes, eed was introduced into the fly to determine whether it could rescue the esc mutant phenotype. eed exerts a dominant negative effect on the leg transformation phenotype
associated with the esc mutation. This result is interpreted in light of in
vitro protein-protein binding data and in vivo polytene chromosome staining
indicating the lack of significant interaction between Eed and fly E(Z), a
molecular partner of ESC (Wang, 2000).
Polycomb group (PcG) proteins play important roles in
maintaining the silent state of HOX genes. Recent studies have
implicated histone methylation in long-term gene silencing. However, a
connection between PcG-mediated gene silencing and histone methylation
has not been established. This study reports the purification and
characterization of an EED-EZH2 complex, the human counterpart of the Drosophila ESC-E(Z) complex. The
complex specifically methylates nucleosomal histone H3 at lysine 27 (H3-K27). Using chromatin immunoprecipitation assays, it is shown that
H3-K27 methylation colocalizes with, and is dependent on, E(z)
binding at an Ultrabithorax (Ubx) Polycomb
response element (PRE), and that this methylation correlates with
Ubx repression. Methylation on H3-K27 facilitates binding of
Polycomb (Pc), a component of the PRC1 complex, to histone H3
amino-terminal tail. Thus, these studies establish a link between
histone methylation and PcG-mediated gene silencing (Cao, 2002).
To understand the function of histone methylation, attempts were made to
identify histone methyltransferase (HMTase) using a systematic biochemical approach. Certain fractions derived from HeLa cell nuclear
pellet contained high levels of HMTase activity toward nucleosomal
histone H3. To identify the enzyme(s) present in these fractions, the
proteins were further fractionated in a DEAE5PW column, which separated the HMTase activities into two peaks. The present study focuses on the second peak.
After fractionation on phenyl sepharose and hydroxyapatite columns, the active fractions were further purified
through a gel filtration Superose 6 column. Analysis of the fractions
derived from this column indicates that the HMTase activity elutes
between fraction 47 and 50 with an estimated mass of about 500 kD. Silver staining of an SDS-polyacrylamide gel containing these fractions revealed that six major polypeptides copurify with the enzymatic activity. Because the largest protein band neither
cofractionates with the HMTase activity in the hydroxyapatite column, nor coimmunoprecipitates with the other components, it is concluded that this largest band is not a part of the HMTase protein complex (Cao, 2002).
To identify the proteins that copurify with the HMTase activity, the protein bands were excised and analyzed by a combination of peptide
mass fingerprinting and mass spectrometric sequencing.
In addition to RbAp48, a polypeptide present in many protein complexes
involved in histone metabolism, several human PcG proteins, including
EZH2, SUZ12, and EED, were identified in the HMTase complex. A zinc finger transcriptional repressor named AEBP2 was also
identified. Whether this protein is involved in
targeting the complex remains to be determined. EZH2 contains a SET
domain, a signature motif for all known histone lysine
methyltransferases, except the H3-K79 methyltransferase DOT1, and is therefore likely to be the
catalytic subunit. However, recombinant EZH2 made in Escherichia coli or baculovirus-infected SF9 cells has no detectable HMTase activity, indicating that either a posttranslational modification or
other components in the complex are required for the HMTase activity.
This is consistent with previous results in which a partial EZH2
protein containing the SET domain was used (Cao, 2002).
Although mammalian EZH2 and EED, and their respective homologs in
Drosophila and Caenorhabditis elegans, are known to interact directly, the presence of SUZ12 in such a complex has not been previously reported. To verify that these proteins are components of the same protein complex, antibodies against each of these proteins were generated. Western blot analysis of the column fractions derived from the last two columns indicates that these proteins copurify with the HMTase activity. To further confirm that the copurified proteins exist as a single protein complex, the last column fractions were immunoprecipitated with an antibody to SUZ12. All five proteins coimmunoprecipitate. Because a protein complex containing Drosophila Esc and E(z), respective homologs of EED and EZH2, has been previously named the ESC-E(Z) complex, the human counterpart is referred to as the EED-EZH2 complex. Although both EED-EZH2 and Esc-E(z) complexes physically associate with HDACs, the purified complex neither contains any HDAC polypeptide nor possesses detectable HDAC activity. It is possible that a different protein complex containing EED, EZH2, and HDAC may exist. Alternatively, HDACs may be recruited to target sites through direct interaction with EED, yet may not exist as a stable subunit of EED-EZH2 complexes. Further work is needed to differentiate these possibilities (Cao, 2002).
To characterize the substrate specificity of the EED-EZH2
complex, equivalent amounts of histone H3 that exist alone, in complex with other core histones, and in mono- or
oligo-nucleosome forms were subjected to methylation by equal amounts of the enzyme. The EED-EZH2 complex
is capable of methylating all forms of histone H3, but shows a strong
preference for H3 in oligonucleosome forms (Cao, 2002).
Attempts were made to identify the residue methylated by the EED-EZH2
complex. Because oligonucleosomes are preferred substrates, they were
subjected to methylation by the EED-EZH2 complex in the presence of
S-adenosyl-L-[methyl-3H]methionine
(3H-SAM). After purification, the labeled H3 was subjected
to microsequencing followed by liquid scintillation counting. Neither
K4 nor K9 released numbers of counts clearly greater than background.
However, a small radioactive peak was detected in cycle 27.
Given that the recovery efficiency decreases with each microsequencing cycle, the detection of a small peak on cycle 27 indicates that K27 is
likely to be the site targeted by the EED-EZH2 complex. To confirm this
possibility, each of the five potential methylation sites on
H3 were mutated and the effect of the mutation on the ability of H3 to
serve as a substrate for the enzyme was compared. As a control, the ability of these H3 mutants to be methylated by SUV39H1 was also analyzed. Mutation of
K27 completely abolishes the ability of H3 to serve as a substrate,
whereas mutations of other sites have little effect. As expected, only mutation of K9 affects the SUV39H1-mediated H3 methylation. These data, led to the conclusion that K27 is the predominant site, if not the only site, that is targeted for methylation by the EED-EZH2 complex (Cao, 2002).
To gain insight into the function of H3-K27 methylation in vivo, a polyclonal antibody was generated against a dimethyl-K27 H3 peptide. This
antibody is highly specific for mK27 when evaluated by peptide
competition and enzyme-linked immunosorbent assay. Western
blot analysis with the H3-mK27-specific antibody demonstrates
that H3-K27 methylation occurs in a variety of multicellular organisms,
including human, chicken, and Drosophila . However, it does
not appear to occur in the budding yeast Saccharomyces cerevisiae (Cao, 2002).
Given that both H3-K27 methylation as well as the EED-EZH2 counterpart
exist in Drosophila , whether the ESC-E(Z) complex is responsible for H3-K27 methylation was examined in this organism. Several E(z)
temperature-sensitive mutant alleles have been characterized, one of which,
E(z)61, contains a
Cys-to-Tyr substitution (C603Y) in the cysteine-rich region immediately
preceding the SET domain. When reared continuously at
18°C (permissive temperature), E(z)61 homozygotes exhibit no detectable mutant phenotype and maintain wild-type expression patterns of HOX genes, such as Ubx. However, at 29°C (restrictive temperature), E(z)61 produces multiple homeotic phenotypes due to derepression of HOX genes,
which correlates with loss of polytene chromosome binding by the
E(Z)61 protein and disruption of chromosome
binding by Polycomb (PC) and other PRC1 components. Given that chromosome binding by E(Z)61 protein is abolished at 29°C, H3-K27 methylation should be correspondingly reduced in the mutants at 29°C, if E(Z) is responsible for H3-K27 methylation. Western blot analysis of
the histones from wild-type and E(z)61 fly embryos at 18° and 29°C demonstrate that the H3-K27 methylation is abolished in the E(z)61 embryos at
29°C. However, these conditions do not have a
detectable effect on H3-K9 methylation. It is therefore concluded that functional E(Z) protein is required for H3-K27 methylation in vivo (Cao, 2002).
To understand the functional relation between E(z)-mediated H3-K27
methylation and HOX gene silencing, a study was carried out of E(z) binding, H3-K27 methylation, and recruitment of PC, a core component of the PRC1
complex, to the major PRE of the Ubx gene in
S2 tissue culture cells by chromatin immunoprecipitation (ChIP). Consistent with the involvement of E(z) in H3-K27 methylation, ChIP
analysis of a 4.4-kb region that includes this PRE showed
precise colocalization of E(z) binding and H3-K27 methylation. In contrast, similar colocalization was not observed for
mK9, indicating that H3-K9 methylation, or at least K9-dimethylation,
is independent of E(z) binding. To further verify the importance of E(z)
binding for H3-K27 methylation, attempts were made to disrupt Esc-E(z)
complex activity using RNA interference (RNAi). It was reasoned that
depletion of the Esc protein, a direct binding partner of E(z) and a
component of the Esc-E(z) complex, would result in disruption of PRE
binding by E(z). Depletion of Esc with RNAi results in greatly reduced PRE binding by E(z), loss of H3-K27 methylation, and concomitant loss
of PC binding. Depletion of PC in S2 cells has
been shown to result in derepression of Ubx.
Therefore, these data collectively suggest that the Esc-E(z) complex is
critical not only for H3-K27 methylation, but also for PC binding to
the PRE region, and that H3-K27 methylation is associated with
Ubx repression (Cao, 2002).
To examine the relation between E(z) binding, H3-K27 methylation, and
Ubx gene repression in vivo, wing imaginal discs were dissected from homozygous E(z)61 larvae that had
been either reared continuously at 18°C or shifted from 18° to
29°C ~48 hours before dissection, and analyzed E(z) binding and
H3-K27 methylation in the same Ubx PRE region by ChIP. Consistent with previous studies demonstrating disruption of polytene chromosome binding by both E(z)61 and PC proteins at 29°C, ChIP analysis showed loss of E(z)61 and PC binding to this PRE at restrictive temperature.
In addition, H3-K27 methylation colocalizes with E(z) binding at
permissive temperature, but is lost along with E(z) binding at 29°C.
In contrast, similar changes in H3-K9 methylation were not observed
under the same conditions. Under normal conditions,
Ubx is not expressed in wing discs due to PcG-mediated
silencing. Similar inactivation of an
E(z) temperature-sensitive allele during larval
development has been shown to result in derepression of Ubx
in wing discs. Thus, Ubx PRE-associated
nucleosomes appear to be targeted by E(z)-mediated H3-K27 methylation,
which correlates with PC binding and repression of Ubx.
Collectively, these data suggest that H3-K27 methylation plays an
important role in the maintenance of Ubx gene silencing (Cao, 2002).
The chromodomain of the heterochromatin protein HP1 specifically
binds to H3 tails that are methylated at K9 by the HMTase SUV39H1. Given that PC contains a chromodomain and that loss of E(z) function abolishes H3-K27
methylation as well as Pc binding to the Ubx PRE, it is possible that methylation of H3-K27 by Esc-E(z)
facilitates PRE binding by PC, analogous to the effect of H3-K9
methylation on nucleosome binding by HP1. To test this possibility,
Drosophila PC was generated using the rabbit reticulocyte
transcription/translation-coupled system and it was incubated with
biotinylated H3 peptides with or without K27 methylation in the
presence of streptavidin-conjugated Sepharose beads. Analysis by
peptide pull-down assay indicated that methylation on K27 facilitates binding of Pc to the H3 peptide. Binding of Pc to the peptides is
specific because the chromodomain-containing protein HP1 fails to bind to the same peptides under the same conditions (Cao, 2002).
Previous studies strongly suggest that the chromodomain of PC is
necessary and sufficient for targeting PC to specific chromosomal locations in vivo because mutations in the PC chromodomain abolish the
ability of PC to bind to chromatin in vivo. In
addition, a chimeric PC/HP1 protein, in which the HP1 chromodomain is
replaced by the PC chromodomain, binds to both heterochromatin and PcG
target sites in euchromatin. To evaluate the
contribution of the chromodomain in the preferential binding of PC to
K27 methylated peptide, a PC mutant was generated in which two of the
highly conserved amino acids Trp-47 and Trp-50 were changed to Ala.
These two amino acids were chosen because the corresponding amino acids
in the HP1 chromodomain have been shown to directly contact the methyl
group of an H3-mK9 peptide. The mutant
PC does not preferentially bind to the K27 methylated peptide, suggesting that the chromodomain of PC is responsible
for the preferential binding to the H3-mK27 (Cao, 2002).
Collectively, these studies support a model in which
Esc-E(z)-mediated H3-K27 methylation serves as a signal for the
recruitment of the PRC1 complex by facilitating PC binding. Recruitment of PRC1 in turn prevents the access of nucleosome remodeling factors, such as SWI/SNF, leading to the formation of a repressive chromatin state. Although this model is attractive, it does not exclude the possibility that protein-protein interaction also contributes to the recruitment of
PRC1 to PREs. Indeed, a recent study indicates that PC transiently associates with the Esc-E(z) complex during early embryogenesis. These studies established a correlation between H3-K27 methylation and PcG silencing. Further work is needed to establish the
exact role of H3-K27 methylation in PcG silencing (Cao, 2002).
The Eed-Enx1 Polycomb group complex has been implicated in the maintenance of imprinted X inactivation in the trophectoderm lineage in mouse. Recruitment of Eed-Enx1 to the inactive X chromosome (Xi) also occurs in random X inactivation in the embryo proper. Localization of Eed-Enx1 complexes to Xi occurs very early, at the onset of Xist expression, but then disappears as differentiation and development progress. This transient localization correlates with the presence of high levels of the complex in totipotent cells and during early differentiation stages. Functional analysis demonstrates that Eed-Enx1 is required to establish methylation of histone H3 at lysine 9 and/or lysine 27 on Xi and that this, in turn, is required to stabilize the Xi chromatin structure (Silva, 2003).
In eed-/- XX embryos Enx1 protein does not localize to Xi. This result is consistent with in vitro analysis demonstrating that the eed3354SB mutation disrupts a WD40 domain required for the interaction of Eed with Enx1. Thus, in the absence of functional Eed protein, the Enx1 HMTase cannot be directed toward specific targets. Eed/Enx1 complexes methylate H3-K9 and K27 in vitro, with a strong preference for K27. Failure to localize Enx1 clearly accounts for the absence of H3-K9/K27 methylation on Xi in eed-/- embryos (Silva, 2003).
It should be noted that, while previous studies have identified H3-K9 methylation as an early mark of silent chromatin on the inactive X chromosome, more recent data indicates that this could be attributable to cross-reactivity of di/tri-meH3-K9 antisera toward di/tri-meH3-K27. New antisera highly specific for di/tri-meH3-K9 detect pericentromeric heterochromatin, but not Xi, while the tri-meH3-K27 antibody used in this study detects Xi, but not pericentromeric heterochromatin. It is also possible that Xi is di/trimethylated both at K9 and K27 and that this configuration is not recognized by the novel di/trimethylH3-K9 antisera (Silva, 2003).
H3-K9/K27 methylation is shown to serve to stabilize the Xi chromatin structure. Thus, in eed-/- embryos, H3-K9 hypoacetylation and loss of H3-K4 methylation on Xi are not seen in a significant number of cells. Moreover, both the Xa and Xi alleles of two X-linked genes were seen to be expressed in a similar proportion of cells. These observations provide a basis for explaining reactivation of the X-linked GFP transgene in trophectoderm cells of eed-/- embryos. However, other studies have not observe reactivation of the GFP transgene in cells of the embryo proper, leading to the conclusion that Eed is required for the maintenance of imprinted, but not random, X inactivation. This difference can be accounted for by two factors. First, it is probable that reactivation of any given locus on Xi is sporadic and progressive. Since embryos exhibit mosaic expression of the XGFP transgene because of random X inactivation, a relatively small increase in the proportion of cells expressing the transgene, as observed for endogenous X-linked genes in this study, would be difficult to quantify. A second factor, is that additional levels of epigenetic silencing, for example, DNA methylation, play a more significant role in maintenance of X inactivation in cells of the embryo proper compared with the trophectoderm, potentially masking the effects of failure to establish H3-K9/K27 methylation (Silva, 2003 and references therein).
Initiation and propagation of X inactivation occurs coincident with cellular differentiation and involves a large nonprotein-coding RNA, the X inactive-specific transcript (Xist). Xist RNA spreads over the X chromosome and is thought to induce chromosome inactivation through recruitment of as yet unidentified silencing factors. The silencing function of Xist RNA has been shown to map to a short tandemly repeated element at the 5' end of the transcript (Silva, 2003 and references therein).
The banded localization of Eed-Enx1 complexes on Xi parallels that observed for Xist RNA. There could be an interaction, either direct or indirect, between Xist and the Eed-Enx1 complex. This view is further supported by observations reported in this study. (1) Recruitment of Eed-Enx1 complexes occurs extremely rapidly after the onset of stable Xist RNA accumulation in differentiating XX ES cells. (2) Eed-Enx1 recruitment occurs in response to expression of ectopic Xist RNA transgenes, both in undifferentiated XY ES cells and also in XYTg15 blastocysts. (3) Eed-Enx1 complexes are not recruited in response to expression of Xistinv mutant RNA, which fails to elicit X inactivation (Silva, 2003).
While the data support the idea that Eed-Enx1 complexes are recruited by Xist RNA, they do not prove that this interaction is direct. In fact, localization of Eed-Enx1 to Xi is first seen to occur at the morula stage, when imprinted X inactivation is initiated, whereas Xist RNA expression is detected earlier, in cleavage stage embryos. High levels of Eed-Enx1 complexes are available during the cleavage stages, suggesting that a factor(s) required for the interaction of the complex with Xist is absent (Silva, 2003).
Also relevant to the question of whether or not the Eed-Enx1 complex interacts directly with Xist RNA is the observation that eed-/- embryos are able to initiate X inactivation. This is, presumably, at least in part, attributable to recruitment of an HDAC complex that can deacetylate H3-K9. It is possible that components of the PcG complex other than Eed and Enx1 are still assembled in eed-/- embryos and that these confer H3-K9 HDAC activity. In the Drosophila ESC (Eed) protein, an equivalent mutation disrupts EZ (Enx1) recruitment but does not appear to ablate complex formation. An alternative scenario is that the complex responsible for H3-K9 deacetylation is a direct target of Xist RNA and that the Eed-Enx1 HMTase is then recruited by this complex. In support of this idea, heritable silencing at Drosophila homeotic genes is initiated by recruitment of the dMi2 protein, a component of an HDAC and chromatin remodeling complex, by Hunchback. Moreover, Eed protein can interact with type 1 HDACs, both in mammalian cells and in Drosophila. Thus, HDAC complexes may recruit Eed-Enx1 to target sites, including Xi, rather than vice versa (Silva, 2003).
It is interesting to note that the time during which high levels of Eed-Enx1 complex are available corresponds closely to the window of opportunity during which cells are responsive to Xist RNA. On the basis of this consideration, a model is proposed that speculates that Xist RNA recruits HDAC and Eed-Enx1 complexes, which lead to establishment of a primary level of chromatin silencing. Only during early differentiation stages would levels of these complexes be sufficient to establish chromosome-wide primary silencing. This would explain why expression of Xist in more differentiated cell types cannot induce X inactivation. It is further suggested that maintaining localization of the HDAC/Eed-Enx1 complexes is Xist RNA dependent. This would account for reversibility and Xist dependence of silencing in undifferentiated ES cells or during early differentiation stages. Extinguishing Xist expression would result in delocalization of HDAC/Eed-Enx1 complexes, loss of H3-K9/K27 methylation, increased H3-K9 acetylation, and, hence, reactivation of Xi (Silva, 2003).
To account for the fact that X inactivation does subsequently become stabilized and Xist independent, it is suggested that the chromatin modifications induced by the HDAC and Eed-Enx1 complex provide a template for recruitment of other silencing components. These could be responsible for further histone N-terminal modifications, for example, global H4 deacetylation, and also for DNA methylation at CpG islands and recruitment of macroH2A1.2. It should be noted that proteins of the PRC1 PcG group complex are not localized to Xi at any stage and are therefore unlikely to be involved in maintaining X inactivation in late development (Silva, 2003).
A key finding from these experiments is that recruitment of Eed-Enx1 to Xi is temporally regulated, rather than lineage specific, and that this, in turn, appears to relate to temporal regulation of overall levels of Eed and, to a lesser extent, Enx1 proteins. A similar expression profile has been reported for ESC (Eed) protein in Drosophila embryogenesis. It is striking that these factors are expressed at highest levels in totipotent or multipotent precursors and during early stages of differentiation. One interpretation of this observation is that Eed-Enx1 complexes are components of the machinery required to confer genome plasticity. Thus, like X inactivation during the window of opportunity, silent chromatin at other Eed-Enx1 targets may be reversible if the primary signal (for example Hunchback at homeotic loci in Drosophila) is removed. This would provide cells of the early embryo with the capacity to activate regions of the genome in response to specific differentiation signals, contrasting with the situation in differentiated cells, where heritable silencing is highly stable and is normally irreversible (Silva, 2003).
Polycomb group complexes 2 and 3 are involved in transcriptional silencing. These complexes contain a histone lysine methyltransferase (HKMT) activity that targets different lysine residues on histones H1 or H3 in vitro. However, it is not known if these histones are methylation targets in vivo because the human PRC2/3 complexes have not been studied in the context of a natural promoter because of the lack of known target genes. RNA expression arrays and CpG-island DNA arrays were used to identify and characterize human PRC2/3 target genes. Using oligonucleotide arrays, a cohort of genes were identified whose expression changes upon siRNA-mediated removal of Suz12 [Drosophila homolog Su(z)12], a core component of PRC2/3, from colon cancer cells. To determine which of the putative target genes are directly bound by Suz12 and to precisely map the binding of Suz12 to those promoters, a high-resolution chromatin immunoprecipitation (ChIP) analysis was combined with custom oligonucleotide promoter arrays. Additional putative Suz12 target genes were identified by using ChIP coupled to CpG-island microarrays. HKMT-Ezh2 and Eed, two other components of the PRC2/3 complexes, colocalize to the target promoters with Suz12. Importantly, recruitment of Suz12, Ezh2 and Eed to target promoters coincides with methylation of histone H3 on Lys 27 (Kirmizis, 2004).
Identification of mammalian PcG target genes has remained elusive for two main reasons. First, the majority of the previous PcG studies focused mostly on the biochemical purification and in vitro characterization of the activities of the PcG complexes and second, the lack of DNA-binding domains within PcG proteins makes the search for their target loci difficult. In this present study, the first known direct target genes of mammalian PcG complexes has been identified. To do so, RNAi was first used to identify genes deregulated by the loss of Suz12 protein in colon cancer cells. Next, Suz12 was shown to bind directly to the promoter of one of these genes (MYT1). Other members of the PRC2/3 complexes were shown to colocalize with Suz12 at the MYT1 promoter. Most importantly, recruitment of Suz12, Ezh2, and Eed to the MYT1 promoter was shown to correlate with methylation of H3-K27. To demonstrate that this silencing mechanism is not unique to MYT1, other Suz12 target genes were identified using a ChIP assay coupled to a CpG island microarray. Similarly to MYT1, the other target promoters of Suz12 are bound by the PRC2/3 components and are characterized by H3-K27 methylation. Thus, the first identified human PcG target genes all appear to be regulated by the histone methylase activity of the PRC complexes (Kirmizis, 2004).
The Suz12 target gene MYT1 was originally cloned from a human brain cDNA library on the basis of its ability to bind cis-regulatory elements of the glia-specific myelin proteolipid protein (PLP) gene and is suggested to be the prototype of the C2HC-type zinc finger protein family. More recently, the Xenopus ortholog of MYT1 (X-MYT1) was identified as a transcriptional activator because it could induce expression of an N-tubulin promoter reporter construct in transient transfection assays. Dominant-negative forms of X-MYT1 inhibited normal neurogenesis, suggesting that X-MYT1 is essential for inducing neuronal differentiation. Intriguingly, a recent report shows that the Xenopus ortholog of Ezh2 (XEZ) is expressed exclusively in the anterior neural plate during early Xenopus embryogenesis, and it was postulated that XEZ might be involved in delaying anterior neuronal differentiation (Barnett, 2001). Based on the current findings, it is possible that Ezh2 delays neuronal differentiation, via the PRC2/3 complexes, by repressing the activity of the MYT1 gene. In addition to MYT1, four additional promoters were identified as being robustly bound by components of the PRC2/3 complexes; each of these promoters is also characterized by high levels of H3-K27. Although a link between components of the PRC2/3 complexes and Wnt1, the cannabinoid receptor (CNR1), or the potassium channel KCNA1 have not been previously reported, it is intriguing to note that these mRNAs are expressed at very low levels in most human tissues, suggesting that they may be generally silenced by the PRC complexes. In support of this hypothesis, some of these target genes were shown to be bound by PRC2/3 components in other cell lines, such as the human MCF7 and mouse F9 (Kirmizis, 2004).
Polycomb group proteins are essential for early development in metazoans, but their contributions to human development are not well understood. The Polycomb Repressive Complex 2 (PRC2) subunit SUZ12 has been mapped across the entire nonrepeat portion of the genome in human embryonic stem (ES) cells. SUZ12 is distributed across large portions of over two hundred genes encoding key developmental regulators. These genes are occupied by nucleosomes trimethylated at histone H3K27, are transcriptionally repressed, and contain some of the most highly conserved noncoding elements in the genome. PRC2 target genes are preferentially activated during ES cell differentiation and the ES cell regulators OCT4, SOX2, and NANOG cooccupy a significant subset of these genes. These results indicate that PRC2 occupies a special set of developmental genes in ES cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation (Lee, 2006).
It was striking that SUZ12 occupied many families of genes that control development and transcription. These included 39 of 40 of the homeotic genes found in the HOX clusters and the majority of homeodomain genes. SUZ12 bound homeodomain genes included almost all members of the DLX, IRX, LHX, and PAX gene families, which regulate early developmental steps in neurogenesis, hematopoiesis, axial patterning, tissue patterning, organogenesis, and cell-fate specification. SUZ12 also occupied promoters for large subsets of the FOX, SOX, and TBX gene families. The forkhead family of FOX genes is involved in axial patterning and tissue development from all three germ layers. Mutations in members of the SOX gene family alter cell-fate specification and differentiation and are linked to several developmental diseases. The TBX family of genes regulates a wide variety of developmental processes such as gastrulation, early pattern formation, organogenesis, and limb formation. Thus, the genes preferentially bound by SUZ12 have functions that, when expressed, promote differentiation. This is likely to explain, at least in part, why PRC2 is essential for early development and ES cell pluripotency (Lee, 2006).
A remarkable feature of PRC2 binding at most genes encoding developmental regulators was the extensive span over which the regulator occupied the locus. For the majority (72%) of bound sites across the genome, SUZ12 occupied a small region of the promoter similar in size to regions bound by RNA polymerase II. For the remaining bound regions, SUZ12 occupancy encompassed large domains spanning 2–35 kb and extending from the promoter into the gene. A large portion of genes encoding developmental regulators (72%) exhibited these extended regions of SUZ12 binding. In some cases, binding encompassed multiple contiguous genes. For instance, SUZ12 binding extended ~100 kb across the entire HOXA, HOXB, HOXC, and HOXD clusters but did not bind to adjacent genomic sequences, yielding a highly defined spatial pattern. In contrast, clusters of unrelated genes, such as the interleukin 1-β cluster, were not similarly bound by SUZ12. Thus, genes encoding developmental regulators showed an unusual tendency to be occupied by PRC2 over much or all of their transcribed regions (Lee, 2006).
Previous studies have noted that many highly conserved noncoding elements of vertebrate genomes are associated with genes encoding developmental regulators. Given SUZ12's strong association with this class of genes, the possibility that SUZ12 bound regions are associated with these highly conserved elements was investigated. Inspection of individual genes suggested that SUZ12 occupancy was associated with regions of sequence conservation. Eight percent of the approximately 1,400 highly conserved noncoding DNA elements were found to be associated with the SUZ12 bound developmental regulators. Using entries from the PhastCons database of conserved elements (Siepel, 2005), it was found that SUZ12 occupancy of highly conserved elements was highly significant. Since PRC2 has not been shown to directly bind DNA sequences, it is expected that specific DNA binding proteins occupy the highly conserved DNA sequences and may associate with PRC2, which spreads and occupies adjacent chromatin. Thus, the peaks of SUZ12 occupancy might not be expected to precisely colocate with the highly conserved elements, even if these elements are associated with PRC2 recruitment (Lee, 2006).
The observation that OCT4, SOX2, and NANOG are bound to a significant subset of developmental genes occupied by PRC2 supports a link between repression of developmental regulators and stem cell pluripotency. Like PRC2, OCT4 and NANOG have been shown to be important for early development and ES cell identity. It is possible, therefore, that inappropriate regulation of developmental regulators that are common targets of OCT4, NANOG, and PRC2 contributes to the inability to establish ES cell lines in OCT4, NANOG, and EZH2 mutants (Lee, 2006).
Best known as epigenetic repressors of developmental Hox gene transcription, Polycomb complexes alter chromatin structure by means of post-translational modification of histone tails. Depending on the cellular context, Polycomb complexes of diverse composition and function exhibit cooperative interaction or hierarchical interdependency at target loci. The present study interrogated the genetic, biochemical and molecular interaction of BMI1 [Drosophila homologs Psc and Su(z)2] and EED (Drosophila homolog; Esc), pivotal constituents of heterologous Polycomb complexes, in the regulation of vertebral identity during mouse development. Despite a significant overlap in dosage-sensitive homeotic phenotypes and co-repression of a similar set of Hox genes, genetic analysis implicated eed and Bmi1 in parallel pathways, which converge at the level of Hox gene regulation. Whereas EED and BMI1 formed separate biochemical entities with EzH2 and Ring1B, respectively, in mid-gestation embryos, YY1 engaged in both Polycomb complexes. Strikingly, methylated lysine 27 of histone H3 (H3-K27), a mediator of Polycomb complex recruitment to target genes, stably associated with the EED complex during the maintenance phase of Hox gene repression. Juxtaposed EED and BMI1 complexes, along with YY1 and methylated H3-K27, were detected in upstream regulatory regions of Hoxc8 and Hoxa5. The combined data suggest a model wherein epigenetic and genetic elements cooperatively recruit and retain juxtaposed Polycomb complexes in mammalian Hox gene clusters toward co-regulation of vertebral identity (Kim, 2006).
At least two PcG complexes with diverse composition and function in chromatin remodeling have been identified in mammals. The Polycomb repressive complex 1 (PRC1) involves the paralogous PcG proteins BMI1/MEL18, M33/PC2, RAE28, and RING1A. Evidence for PRC1-mediated chromatin modification derived from ubiquitylation at lysine 119 of histone H2A (H2A-K119). A second PcG complex, PRC2, encompasses EED, the histone methyltransferase EZH2, the zinc finger protein SUZ12, the histone-binding proteins RBAP46/RBAP48, and the histone deacetylase HDAC1. Several EED isoforms, generated by alternate translation start site usage of eed mRNA, differentially engage in PRC2-related complexes (PRC2/3/4), targeting the histone methyltransferase activity of EZH2 to H3-K27 or H1-K26. PcG complexes bind to cis-acting Polycomb response elements (PREs), which encompass several hundred base pairs and are necessary and sufficient for PcG-mediated repression of target genes. Whereas the function of several PREs has been delineated in Drosophila, similar elements await characterization in mammals (Kim, 2006 and references therein).
An antibody raised against residues 123-140 of the EED amino terminus
precipitated three distinct isoforms of approximately 50 and 75 kDA from E12.5
trunk, representing three of the four EED isoforms previously reported in 293 cells. In
addition to EZH2 and YY1, dimethylated H3-K27 co-immunoprecipitated with EED. Immunoprecipitation identified three BMI1 isoforms of approximately 39-41 kDA. BMI1 was found in a complex with RING1B, but not dimethylated H3-K27. Similar to the EED complex,
the BMI1 complex also contained YY1. It should be emphasized that all (co-)immunoprecipitating bands were detected by at least two antibodies against different epitopes. Strikingly, while dimethylated H3-K27 engaged in the EED complex,
trimethylated H3-K27 did not appear to associate with either the EED or the
BMI1 complex. Importantly, reciprocal co-immunoprecipitation detected EED and
BMI1 in separate protein complexes (Kim, 2006).
Ectopic expression in mutant embryos revealed Hoxc8 and
Hoxa5 as downstream targets of EED and BMI1 function. ChIP detected EED and
BMI1 binding immediately upstream of the Hoxc8 transcribed region
near putative promoter elements. The binding sites could not be separated, indicating close proximity of the complexes. EED and BMI1 binding also
clustered within a small fragment 1.5 kb upstream of the Hoxc8
transcription start site, suggesting long-range juxtaposition of heterologous PcG
complexes. Similar to EED and BMI1, YY1 localized to both regions. In support
of YY1 binding to Hox regulatory regions, inspection of the mouse genome
sequence revealed clusters of putative YY1 binding sites in
both regions a and b, including TGTCCATTAG and
CCCCCATTCC (region a), as well as ACACCATGGC,
TTTCCATTAG and TCCCCATAAA (region b). CCAT represents
the core of the YY1 consensus binding site, while flanking sequences exhibited
significant tolerance for multiple nucleotides. EED,
BMI1 and YY1 also co-localized approximately 1.5 kb upstream of the
transcription start site of Hoxa5. In addition to PcG binding, ChIP detected trimethylated H3-K27 throughout the regulatory regions of Hoxc8 and Hoxa5. Furthermore, dimethylated H3-K27 localized to region b of Hoxc8 (Kim, 2006).
Spatial regulation of EED and BMI1 binding to Hox regulatory regions was
evident from ChIP analysis of dissected anterior and posterior regions of
E12.5 trunk. In agreement with transcriptional silencing of Hoxc8 and
Hoxa5, EED and BMI1 binding was detected upstream of these loci in
anterior regions of the trunk. By contrast, EED and BMI1 binding was absent from posterior regions of the trunk, where Hoxc8 and Hoxa5 are transcribed.
These findings implicate PcG complexes in Hox gene repression in anterior
regions of the AP axis (Kim, 2006).
The combined interpretation of the co-immunoprecipitation and ChiP results
indicates that trimethylated H3-K27 did not form a complex with EED or BMI1,
despite co-localization of the three proteins in Hox regulatory regions. By
contrast, co-immunoprecipitation demonstrated physical association of the EED
complex with dimethylated H3-K27. In aggregate, the results support a model in
which EED- and BMI1-containing chromatin remodeling complexes exist as
separate, but juxtaposed, biochemical entities at Hox target loci (Kim, 2006).
The mechanisms by which embryonic stem (ES) cells self-renew while maintaining the ability to differentiate into virtually all adult cell types are not well understood. Polycomb group (PcG) proteins are transcriptional repressors that help to maintain cellular identity during metazoan development by epigenetic modification of chromatin structure. PcG proteins have essential roles in early embryonic development and have been implicated in ES cell pluripotency, but few of their target genes are known in mammals. This study shows that PcG proteins directly repress a large cohort of developmental regulators in murine ES cells, the expression of which would otherwise promote differentiation. Using genome-wide location analysis in murine ES cells, it was found that the Polycomb repressive complexes PRC1 and PRC2 co-occupied 512 genes, many of which encode transcription factors with important roles in development. All of the co-occupied genes contained modified nucleosomes (trimethylated Lys 27 on histone H3). Consistent with a causal role in gene silencing in ES cells, PcG target genes were de-repressed in cells deficient for the PRC2 component Eed (homolog of Drosophila Extra sexcombs), and were preferentially activated on induction of differentiation. These results indicate that dynamic repression of developmental pathways by Polycomb complexes may be required for maintaining ES cell pluripotency and plasticity during embryonic development (Boyer, 2006).
Noncoding RNAs (ncRNA) participate in epigenetic regulation but are poorly understood. This study characterized the transcriptional landscape of the four human HOX loci at five base pair resolution in eleven anatomic sites, and identified 231 HOX ncRNAs that extend known transcribed regions by more than 30 kilobases. HOX ncRNAs are spatially expressed along developmental axes, possess unique sequence motifs, and their expression demarcate broad chromosomal domains of differential histone methylation and RNA polymerase accessibility. A 2.2 kilobase ncRNA was identified residing in the HOXC locus, termed HOTAIR, which represses transcription in trans across 40 kilobases of the HOXD locus. HOTAIR interacts with Polycomb Repressive Complex 2 (PRC2) and is required for PRC2 occupancy and histone H3 lysine-27 trimethylation of HOXD locus. Thus, transcription of ncRNA may demarcate chromosomal domains of gene silencing at a distance; these results have broad implications for gene regulation in development and disease states (Rinn, 2007).
By analyzing the transcriptional and epigenetic landscape of the HOX loci at high resolution in cells with many distinct positional identities, a panoramic view was obtained of multiple layers of regulation involved in maintenance of site-specific gene expression. The HOX loci are demarcated by broad chromosomal domains of transcriptional accessibility, marked by extensive occupancy of RNA polymerase II and H3K4 dimethylation and, in a mutually exclusive fashion, by occupancy of PRC2 and H3K27me3. The active, PolII-occupied chromosomal domains are further punctuated by discrete regions of transcription of protein-coding HOX genes and a large number of long ncRNAs. These results confirm the existence of broad chromosomal domains of histone modifications and occupancy of HMTases over the Hox loci, and extend on those observation in several important ways (Rinn, 2007).
First, by comparing the epigenetic landscape of cells with distinct positional identities, it was showm that the broad chromatin domains can be programmed with precisely the same boundary but with diametrically opposite histone modifications and consequences on gene expression. The data thus functionally pinpoint the locations of chromatin boundary elements in the HOX loci, the existence of some of which have been predicted by genetic experiments. One such boundary element appears to reside between HOXA7 and HOXA9. This genomic location is also the switching point in the expression of HOXA genes between anatomically proximal versus distal patterns and is the boundary of different ancestral origins of HOX genes, raising the possibility that boundary elements are features demarcating the ends of ancient transcribed regions. Second, the ability to monitor 11 different HOX transcriptomes in the context of the same cell type conferred the unique ability to characterize changes in ncRNA regulation that reflect their position in the human body. This unbiased analysis identified more than 30 kb of new transcriptional activity, revealed ncRNAs conserved in evolution, mapped their anatomic patterns of expression, and uncovered enriched ncRNA sequence motifs correlated with their expression pattern -- insights which could not be gleamed from examination of EST sequences alone. The finding of a long ncRNA that acts in trans to repress HOX genes in a distant locus is mainly due to the ability afforded by the tiling array to comprehensively examine the consequence of any perturbation over all HOX loci. The expansion of a handful of Hox-encoded ncRNAs in Drosophila to hundreds of ncRNAs in human HOX loci suggests increasingly important and diverse roles for these regulatory RNAs (Rinn, 2007).
An important limitation of the tiling array approach is that while improved identification of transcribed regions is obtained, the data does not address the connectivity of these regions. The precise start, end, patterns of splicing, and regions of double-stranded overlap between ncRNAs will need to be addressed by detailed molecular studies in the future (Rinn, 2007).
The results uncovered a new mechanism whereby transcription of ncRNA dictates transcriptional silencing of a distant chromosomal domain. The four HOX loci demonstrate complex cross regulation and compensation during development. For instance, deletion of the entire HOXC locus exhibits a milder phenotype than deletion of individual HOXC genes, suggesting that there is negative feedback within the locus. Multiple 5' HOX genes, including HOXC genes, are expressed in developing limbs, and deletion of multiple HOXA and HOXD genes are required to unveil limb patterning defects. The results suggest that deletion of the 5' HOXC locus, which encompass HOTAIR, may lead to transcriptional induction of the homologous 5' HOXD genes, thereby restoring the total dosage of HOX transcription factors. How HOX ncRNAs may contribute to cross-regulation among HOX genes should be addressed in future studies (Rinn, 2007).
HOTAIR ncRNA is involved in Polycomb Repressive Complex 2-mediated silencing of chromatin. Because many HMTase complexes lack DNA binding domains but possess RNA binding motifs, it has been postulated that ncRNAs may guide specific histone modification activities to discrete chromatin loci. This study has shown that HOTAIR ncRNA binds PRC2 and is required for robust H3K27 trimethylation and transcriptional silencing of the HOXD locus. HOTAIR may therefore be one of the long sought after RNAs that interface the Polycomb complex with target chromatin. A potentially attractive model of epigenetic control is the programming of active or silencing histone modifications by specific noncoding RNAs. Just as transcription of certain ncRNA can facilitate H3K4 methylation and activate transcription of the downstream Hox genes (Sanchez-Elsner, 2006; Schmitt, 2005), distant transcription of other ncRNAs may target the H3K27 HMTase PRC2 to specific genomic sites, leading to silencing of transcription and establishment of facultative heterochromatin. In this view, extensive transcription of ncRNAs is both functionally involved in the demarcation of active and silent domains of chromatin as well as being a consequence of such chromatin domains (Rinn, 2007).
Several lines of evidence suggest that HOTAIR functions as a bona fide long ncRNA to mediate transcriptional silencing. First, full length HOTAIR is detected in vivo and in primary cells, but not small RNAs derived from HOTAIR indicative of miRNA or siRNA production. Second, depletion of full length HOTAIR led to loss of HOXD silencing and H3K27 trimethyation by PRC2, and third, endogenous or in vitro transcribed full length HOTAIR ncRNA physically associated with PRC2. While these results do not rule out the possibility that RNA interference pathways may be subsequently involved in PcG function, they support the notion that the long ncRNA form of HOTAIR is functional. The role of HOTAIR is reminiscent of XIST, another long ncRNA shown to be involved in transcriptional silencing of the inactive X chromosome. An important difference between HOTAIR and XIST is the strictly cis-acting nature of XIST. HOTAIR is the first example of a long ncRNA that can act in trans to regulate a chromatin domain. While a trans repressive role for HOTAIR was observed, the data do not permit ruling out a cis-repressive role in the HOXC locus. siRNA-mediated depletion of HOTAIR was substantial but incomplete; further, the proximity between the site of HOTAIR transcription and the neighboring HOXC locus may ensure significant exposure to HOTAIR even if the total pool of HOTAIR in the cell were depleted. The precise location of HOTAIR at the boundary of a silent chromatin domain in the HOXC locus makes a cis-repressive role a tantalizing possibility. Judicious gene targeting of HOTAIR may be required to address its role in cis-regulation of chromatin (Rinn, 2007).
The discovery of a long ncRNA that can mediate epigenetic silencing of a chromosomal domain in trans has several important implications. First, ncRNA guidance of PRC2-mediated epigenetic silencing may operate more globally than just in the HOX loci, and it is possible that other ncRNAs may interact with chromatin modification enzymes to regulate gene expression in trans. Second, PcG proteins are important for stem cell pluripotency and cancer development; these PcG activities may also be guided by stem cell or cancer-specific ncRNAs. Third, Suz12 contains a zinc finger domain, a structural motif that can bind RNA, and EZH2 and EED both have in vitro RNA binding activity. The interaction between HOTAIR and PRC2 may also be indirect and mediated by additional factors. Detailed studies of HOTAIR and PRC2 subunits are required to elucidate the structural features that establish the PRC2 interaction with HOTAIR. As is illustrated in this study, high throughput approaches for the discovery and characterization of ncRNAs may aid in dissecting the functional roles of ncRNAs in these diverse and important biological processes (Rinn, 2007).
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