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J. Biol. Chem., Vol. 276, Issue 46, 43065-43073, November 16, 2001
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From the
Received for publication, May 11, 2001, and in revised form, September 17, 2001
The products of Polycomb group (PcG) genes are
required for the epigenetic repression of a number of important
developmental regulatory genes, including homeotic genes. Enhancer of
zeste (E(Z)) is a Drosophila PcG protein that previously
has been shown to bind directly to another PcG protein, Extra Sex Combs
(ESC), and is present along with ESC in a 600-kDa complex in
Drosophila embryos. Using yeast two-hybrid and in
vitro binding assays, we show that E(Z) binds directly to another
PcG protein, Polycomblike (PCL). PCL·E(Z) interaction
is shown to be mediated by the plant homeodomain (PHD) fingers domain
of PCL, providing evidence that this motif can act as an independent
protein interaction domain. An association was also observed between
PHF1 and EZH2, human homologs of PCL and E(Z), respectively,
demonstrating the evolutionary conservation of this interaction. E(Z)
was found to not interact with the PHD domains of three
Drosophila trithorax group (trxG) proteins, which function
to maintain the transcriptional activity of homeotic genes, providing
evidence for the specificity of the interaction of E(Z) with the PCL
PHD domain. Coimmunoprecipitation and gel filtration experiments
demonstrate in vivo association of PCL with E(Z) and ESC in
Drosophila embryos. We discuss the implications of PCL
association with ESC·E(Z) complexes and the possibility that PCL may
either be a subunit of a subset of ESC·E(Z) complexes or a subunit of
a separate complex that interacts with ESC·E(Z) complexes.
Regulated homeotic gene expression is responsible for the
specification of segmental identity along the anterior/posterior axis
of developing metazoans. In Drosophila melanogaster this spatially restricted pattern of homeotic gene expression is initiated early in embryogenesis by the products of the gap and pair-rule genes
(see Ref. 1 for a review). The early patterns of gap and pair-rule gene
expression cease at approximately stage 10, yet correct expression of
homeotic genes needs to be maintained throughout development. Two
groups of genes, the trithorax group (trxG) and Polycomb group
(PcG),1 act to maintain the
correct expression of the homeotic genes after the degradation of the
gap and pair-rule proteins. The trxG acts to maintain the expression of
homeotic genes, whereas the PcG acts to maintain their repression (see
Refs. 2 and 3 for reviews).
The PcG genes were initially identified in Drosophila on the
basis of mutant phenotypes that resemble gain-of-function homeotic mutations. These phenotypes result from derepression of homeotic genes,
outside of their normal expression domains, ~5-6 h after fertilization (see Ref. 4 for a review). Derepression occurred after
normal initiation of expression, indicating that the PcG is required
specifically for the maintenance of repression and not its initiation.
Fourteen PcG members have so far been identified from
Drosophila, 10 of which have been characterized at the
molecular level (5, 6; for review see Ref. 3). Mammalian homologs have
also been identified for several members of the group, and mutant
analysis of some of these members demonstrates a role in the regulation of HOX gene expression, analogous to that of the Drosophila
PcG (see Ref. 7 for a review).
The mechanisms through which the PcG carries out its repressive
function is unknown. Several models have been postulated, including the
creation of a higher order chromatin structure that is inaccessible to
transcription factors, or the sequestering of target genes into nuclear
compartments that exclude the transcription machinery (3, 8). Despite
the limited understanding of the mechanism of action, there is
considerable evidence that PcG proteins act in multimeric protein
complexes to repress target gene expression. To date, two
Drosophila PcG complexes have been isolated and several of
their components have been identified. PRC1 is a 2-MDa complex that
includes the PcG proteins Polycomb (PC), Polyhomeotic (PH), Posterior
Sex Combs, and Sex Combs on Midlegs along with several other proteins
(9). PRC1 has been shown to inhibit ATP-dependent chromatin
remodeling by the human SWI/SNF protein complex in vitro (9). Several members of the Drosophila trxG are subunits of the Brahma complex, which is related to SWI/SNF (10-12). Thus, PRC1
may contribute to PcG-dependent silencing by directly
inhibiting chromatin remodeling that is necessary for transcriptional
activation. ESC, E(Z), and PCL were found to not be part of the PRC1
complex. Recently, a separate 600-kDa complex containing ESC and E(Z)
was identified (13, 14). Analysis of the isolated ESC·E(Z) complex has identified two other subunits, the histone binding p55 protein and
the histone deacetylase RPD3 (14), supporting the model that chromatin
modification, including histone deacetylation, may play a central role
in PcG-depending silencing. Composition of the ESC·E(Z) complex
appears to be at least partially conserved in mammals (14, 15). PCL is
yet to be assigned to a protein complex.
In this study, we provide additional insight into the molecular
activities of the PCL and E(Z) proteins. The PCL protein contains two
PHD finger domains (16, 17). These domains are characterized by a
highly conserved Cys4-His-Cys3 motif identified
in a number of proteins involved in modulating transcription, including
the trxG proteins TRX, Absent, small, or homeotic discs 1 (ASH1) and Absent, small, or homeotic discs 2 (ASH2) (17-19). It has been suggested that PHD fingers may mediate either protein·DNA
interactions or protein·protein interactions (17). Two recent reports
provide evidence that PHD fingers are capable of mediating
protein·protein interactions. Adjacent PHD finger and bromodomains of
the KAP-1 corepressor have been shown to work in concert to mediate
interaction with a subunit of the NuRD complex (20). In addition, the
PHD fingers of MLL, a human homolog of TRX, homodimerize and bind to
Cyp33 (21). A PCL region encompassing the PHD fingers, the 40 amino
acids separating them, and 40 flanking amino acids C-terminal to the
second PHD finger shares 34% identity with two mammalian homologs,
MTF2 (murine) and PHF1 (human) (22). Conservation of this region
suggests that it is important in PCL function. Prior to this study, no
interactions between PCL and other PcG members had been identified.
E(Z) contains several domains that are highly conserved in mammalian
homologs (23). However, with the exception of the Cys-rich domain,
which is required for chromosome binding (24), the functions of these
domains are not known. The ESC-binding domain has been mapped to a
relatively unconserved 33-amino acid region near the N terminus
(25).
Here we report yeast two-hybrid and in vitro binding data
demonstrating direct interaction between PCL and E(Z). Significantly, this interaction is shown to be mediated by the PHD finger domain of
PCL, providing supporting evidence that a PHD finger domain can
function alone as a protein·protein interaction domain. Yeast two-hybrid analysis using human homologs also suggests that the PCL·E(Z) interaction is evolutionarily conserved.
Coimmunoprecipitation and gel filtration chromatography experiments
provide evidence for in vivo association of PCL with E(Z)
and ESC.
Yeast Two-hybrid Constructs--
All constructs, unless
otherwise stated, were cloned using linker PCR to generate the
appropriate restriction enzyme site at the 5' and 3' ends of fragments,
which were then cloned into the pEG202 or pJG4-5 vectors (26). The
template cDNA used for PCL constructs was the full-length
Pcl cDNA AL15 (16). The template cDNA used for the
LexA-PHF1 construct was pBS-PHF1 (22). The coding sequence for PHF1
residues 86-240 were amplified and cloned as an
EcoRI-XhoI fragment into pEG202. The template
cDNA used for the LexA-hMTF2 and AD-hMTF2 constructs was human
cDNA clone (ID number 589332; obtained from IMAGE, National
Institutes of Health (27)). The PHD finger coding region (residues
102-256) was cloned as an EcoRI fragment into pJG4-5 and
pEG202. The LexA-EZH1 and AD-EZH1 constructs were generated by dropping
out a full-length EZH1 fragment (XhoI) from pGEX-EZH1 (25)
and cloning it in-frame into pEG202 and pJG4-5, respectively. The
full-length LexA-EZH2 and AD-EZH2 constructs were generated by PCR
using pBS-EZH2 cDNA (28) as a template and cloned as an
XhoI fragment. The LexA-TRX-PHDF construct was generated by
PCR amplifying the coding sequence for TRX residues 1266-1481 from the
pTM15'-Trx PHD finger domains 1-3 template and ligating the PCR
product into the EcoRI site of pEG202. LexA-ASH1-PHDF and
AD-ASH1-PHDF constructs were generated by PCR-amplifying the coding
sequence for ASH1 residues 1778-1818 from a full-length
ash1 cDNA and ligating the PCR product into the
EcoRI-XhoI sites of pEG202 and pJG4-5,
respectively. LexA-ASH2-PHDF and AD-ASH2-PHDF constructs were generated
by ligating the PCR-amplified coding sequence for ASH2 residues 50-131
from ash2 cDNA LD31680 into the
EcoRI-XhoI sites of pEG202 and pJG4-5,
respectively. Both ash1 and ash2 cDNAs were
kindly provided by Allen Shearn. The AD-PcG constructs AD-PC,
AD- Site-directed Mutagenesis--
Site-directed mutagenesis of
Pcl was performed using the QuikChange site-directed
mutagenesis kit (Stratagene), with the exception of the
C430S-PCLCR mutation. A 2.54-kb open reading frame fragment
of Pcl was cloned into pBSKS+ and used as a template for the
PCLCR mutagenesis. Specific oligonucleotides were used to
generate C518S, C430A, C518A, PHDF1-QQQA, and
PHDF2-QQQA mutations. The C430S-PCLCR mutation
was generated by performing two high fidelity PCRs (using Pfu DNA polymerase, Stratagene), using the primer pairs
Pcl5' Bam (5'-GGAGGATCCTGATGAACAACCATT-3')/Nhe bottom
(5'-AAAGCTAGCCACGCACATGGGTCCACT-3') and Nhe top
(5'-AAAGCTAGCAAGCGATCGGATATCGAA-3')/Pcl3'
(5'-GGAGGATCCTTATGATGCCATTTAC-3'). The products were restricted with
NheI and then ligated to each other. All mutations were
confirmed by sequence analysis and the PCLCR of each mutant
(aa 424-605) was PCR-amplified and cloned into pEG202 to generate
LexA-mutant-PCLCR fusions. The stability of each
LexA-PCLCR mutation was analyzed in yeast by performing
Western blot analysis with the LexA antisera (provided by Roger Brent).
Site-directed mutagenesis of E(z) was performed using the
Altered Sites mutagenesis kit (Promega) and the pAlter-e32-55.26 plasmid template, as previously described (25) to create the E(Z)EDE116AAA, E(Z)H122A, E(Z)L132A, G136A, E(Z)K144A, and
E(Z)DGK147AAA mutations. All mutations were confirmed by sequence
analysis, and the indicated coding regions were PCR-amplified using
Pfu DNA polymerase and inserted into the pGEX-BgRP3i vector
(25).
Yeast Two-hybrid Assays--
The starting yeast strain used for
the two-hybrid assay, EGY48 (MAT In Vitro Pull-down Assays--
Radiolabeled PCL and E(Z)
proteins were synthesized by in vitro
transcription-translation with the TnT-coupled reticulocyte lysate
system (Promega) and [35S]methionine. 35S-PCL
was produced from Pcl cDNA AL15 (16) in the vector
pBluescript-SK using T7 RNA polymerase. 35S-E(Z) was
produced from cDNA clone e32-55.26 in the vector pBC-SK using T7
RNA polymerase. Isolation of GST fusion proteins and GST pull-down
assays were performed as previously described (25). pGEX-E(z) constructs were made by PCR-amplifying the
indicated coding regions and ligating the PCR products into the
pGEX-BgRP3i vector. His6 fusion protein pull-down assays
were performed in a manner similar to GST pull-downs, except that
His6 fusion proteins were purified from Escherichia
coli according to Qiagen's protocol for purifying insoluble
proteins under denaturing conditions, and immobilized on Ni-NTA-agarose
instead of glutathione agarose. Binding and wash buffers were the same
as those used for GST pull-downs, except that 20 mM
imidazole was added to both buffers to reduce nonspecific sticking of
radiolabeled proteins. Prior to using affinity-purified GST or
His6 fusion proteins in binding assays, aliquots of each
protein sample were run on SDS-polyacrylamide gel electrophoresis gels,
stained with Coomassie Blue to estimate protein concentrations, and
sample volumes were adjusted to equalize the amounts of fusion proteins
used in each assay. In GST and His6 pull-downs, bound
proteins were eluted in wash buffer supplemented with 100 mM free glutathione or 250 mM imidazole,
respectively. pQE-PCL403-605 was constructed by inserting the
PCR-amplified coding sequence for Pcl residues 403-605 into
the pQE30 vector.
Antibody Production and Immunoprecipitations--
Rabbit
anti-PCL antibodies were generated against His6-PCL113-537
and affinity-purified essentially as previously described (24). The
region encoding PCL residues 113-537 was amplified from Pcl
cDNA AL15 and inserted into the pQE32 bacterial expression vector
(Qiagen). His6-PCL113-537 fusion protein was affinity
purified from E. coli using Ni-NTA-agarose and injected into
rabbits (all injections and bleeds were performed at Covance Research
Products, Inc.). Antibodies were affinity-purified by first mixing the
antiserum with total E. coli extract that included
His6-MDH (E. coli malate dehydrogenase; kindly
provided by Bilal Armeneh and Steven Vik) that had been covalently
attached to CNBr-activated Sepharose. Unbound antiserum was then
incubated with His6-PCL113-537 (attached to CNBr-activated
Sepharose). Following thorough washing, bound antibodies were eluted in
low pH buffer. These antibodies were found to cross-react with numerous
proteins from Drosophila embryo extracts on Western blots.
Therefore, a portion of these antibodies was re-affinity-purified
against a smaller fusion protein, His6-PCL113-371. Both
antibody fractions that bound to His6-PCL113-371 and those that were depleted of His6-PCL113-371-binding antibodies,
which are assumed to recognize epitopes within PCL372-537, were tested on embryo Western blots. The anti-PCL372-537 antibody fraction displayed much greater specificity for PCL and was used for all immunoprecipitations and immunoblot assays. 10 µl was used in immunoprecipitations, and 1:2000 dilutions were used for Western blots.
Immunoprecipitations and Western blots were performed essentially as
previously described (25). 20 and 15 µl, respectively, of affinity-purified rabbit anti-E(Z) antibodies described before (24) and
mouse HA.11 anti-HA monoclonal antibodies (Covance) were used for
immunoprecipitations. For Western blots, anti-E(Z) and anti-HA
antibodies were used at 1:800 and 1:10,000 dilutions, respectively.
Gel Filtration Chromatography--
Nuclear extracts were
prepared from 0-24 h HA-esc embryos as described by Ng
et al. (13) and stored at The Conserved Region of PCL Interacts with E(Z) in the Yeast
Two-hybrid System--
To determine whether PCL is able to interact
with cloned members of the PcG, yeast two-hybrid assays were performed.
Two constructs were generated in the LexA-yeast two-hybrid vector
pEG202 (26), a full-length PCL construct, LexA-PCL, and a construct,
LexA-PCLCR, containing the region of PCL that is conserved
in its mammalian homologs, here denoted the PCL conserved region
(PCLCR, aa 424-605). This conserved region contains two
PHD finger motifs and a region C-terminal to the second PHD finger (aa
566-605, see Fig. 1A). Both
the LexA-PCL and LexA-PCLCR constructs were tested for
interaction with PcG members that had been cloned into the yeast
two-hybrid activation domain vector, pJG4-5, to generate AD-PcG fusion
proteins (see "Experimental Procedures" for a list of PcG members
tested). Two-hybrid interactions were assayed in the
Saccharomyces cerevisiae strain EGY48, which contains six
LexA binding sites upstream of an endogenous LEU2 reporter
gene. Transformed colonies containing both a LexA and AD construct were
tested for growth on leucine-deficient medium. Here, growth would only
occur if the AD fusion interacted with the LexA fusion to activate
the LEU2 reporter gene.
The LexA-PCL fusion protein did not interact with any PcG members in
this assay (data not shown), but the LexA-PCLCR interacted
with AD-E(Z) (Fig. 1B). In yeast containing both the LexA-PCLCR and AD-E(Z) constructs, growth was only observed
on plates containing galactose, indicating that induction of the
AD-E(Z) fusion protein (which is under the control of a galactose
inducible promoter) is required for the interaction (Fig.
1B). Additional controls showed that growth required the
presence of both the LexA-PCLCR and the AD-E(Z) fusion
proteins (data not shown). The inability of the full-length PCL fusion
protein to interact with E(Z) in this assay could reflect a limitation
of using a transcriptional assay as a basis for identifying
interactions between transcriptional repressors. Previous experiments
showed that full-length Posterior Sex Combs and PH proteins appeared
not to interact in the yeast two-hybrid assay despite the fact that an
interaction was observed between smaller fragments of the proteins
(29).
The Interaction between PCL and E(Z) Is Direct--
To test
whether the interaction between PCL and E(Z) is direct, in
vitro binding assays were performed. His6-PCL403-605
fusion protein, which includes the PCL conserved region, and
His6-MDH (E. coli-malate dehydrogenase, which
served as a negative control in these assays) were purified from
E. coli and tested for binding to full-length radiolabeled
E(Z) protein produced by in vitro translation. Equal amounts
of His6-PCL-(403-605) and His6-MDH were
immobilized on Ni-NTA-agarose and incubated with
35S-E(Z). After extensive washing, bound proteins were
eluted and binding assessed by SDS-polyacrylamide gel electrophoresis.
Radiolabeled E(Z) bound to His6-PCL-(403-605) (Fig.
2A, lane 2) but not
to His6-MDH (lane 3) or Ni-NTA beads alone
(lane 4).
Reciprocal pull-down experiments confirmed direct PCL·E(Z)
interaction and were used to map the PCL-interacting domains of E(Z)
protein. The assays were performed as described above, except that
full-length radiolabeled PCL protein was tested for binding to GST
alone or to GST-E(Z) fusion proteins that were purified from E. coli and immobilized on glutathione-agarose. PCL does not bind to
the highly conserved E(Z) Cys-rich or SET domains, which are included
in GST-E(Z)-(512-760) (Fig. 2B, lane 3),
but rather binds to E(Z) domains included in GST-E(Z)-(1-511) (Fig. 2B, lane 2). Attempts to further delimit the
region of E(Z) responsible for PCL binding revealed two separate
PCL-interacting domains, E(Z)-(86-171) (Fig. 2B, lane
7) and E(Z)-(377-446) (Fig. 2C, lane 2).
All GST-E(Z) fusion proteins that were able to bind to PCL included one
or both of these regions. Essentially identical results were obtained
in yeast two-hybrid assays in which the same E(Z) regions were tested
as AD-E(Z) or LexA-E(Z) fusions for interaction with
LexA-PCLCR or AD-PCLCR, respectively (data not
shown). Therefore, two separate E(Z) regions are able to bind
independently to PCL in vitro and in yeast cells.
E(Z)-(86-171) includes the highly conserved Domain I (aa 95-170),
which is 70.9 and 60.5% identical to the homologous regions of the
human EZH2 and EZH1 proteins, respectively (23), but E(Z)-(377-446) is
much less well conserved. On the basis of their newly defined
functions, we will refer to the 86-171 region as PBD1 and the 377-446
region as PBD2 (for PCL Binding
Domains 1 and 2). Alignments of these E(Z), EZH2, and EZH1
sequences are shown in Fig. 3
(B and C). The positions of these and other E(Z) domains, which were identified on the basis of evolutionary sequence conservation (23, 30) or defined by function (25), are shown in Fig.
3A.
Effects of E(Z) Point Mutations on PCL Binding--
To identify
E(Z) residues within the two PCL binding regions that are required for
the interaction with PCL, and to provide additional evidence for the
specificity of the interaction, selected E(Z) residues within the PCL
binding regions were replaced by alanine by site-directed mutagenesis.
Five mutations in PBD1 and two mutations in PBD2 were tested. The
locations of these mutations are shown in Fig. 3 (B and
C). Two of the PBD1 mutations, EDE116AAA and DGK147AAA, are
substitutions of adjacent residues that are conserved in E(Z) and both
human homologs, EZH1 and EZH2. Three PBD1 mutations, H122A, G136A, and
K144A, are single-amino acid substitutions of residues that are
conserved in E(Z) and EZH2, but not EZH1. These were selected because
EZH2, but not EZH1, interacts with PHF1, a human homolog of PCL
(described below). On the basis of this same reasoning, the two PBD2
mutations are substitutions of adjacent residues at which E(Z) and EZH2
are either identical or contain conservative substitutions. Initially, the effect of each mutation was tested within the context of the isolated respective regions, 86-171 or 377-446. Of the five PBD1 mutants, only EDE116AAA exhibited strongly reduced PCL binding (Fig.
3D, lane 3). Of the two PBD2 mutants, EIN429AAA
is essentially unable to bind PCL (Fig. 3E, lane
4), whereas the HEN412AAA mutation has no effect on PCL binding
(Fig. 3E, lane 3). Either mutation alone has
little, if any, effect on PCL binding when expressed as a larger fusion
protein that includes a wild type version of the other PCL-interacting
domain (Fig. 3F, lanes 5 and 6). However, GST-E(Z)-(1-511) fusion protein containing both mutations is unable to
bind to PCL (Fig. 3F, lane 4). Thus, either PBD1
or the less conserved PBD2 is capable of binding to PCL in
vitro.
The Highly Conserved PHD Finger Domains of PCL Are Responsible for
the Interaction with E(Z)--
To ascertain which sequences within the
PCL conserved region are responsible for the interaction between PCL
and E(Z), a LexA-PCLCR
This refined the interaction domain to the two PHD finger domains and
the conserved 40 amino acids separating them. To determine if the PHD
finger domains mediated the interaction between PCL and E(Z), PHD
finger mutants were generated using in vitro site-directed mutagenesis (Fig. 4A). As previously discussed, the PHD
finger contains a Cys4-His-Cys3 motif, which is
thought to coordinate Zn2+ (17). No structural studies have
been performed on this recently defined domain, and it is not known
whether the conserved spacing of the Cys and His residues is important.
It is also not known whether the Cys residues form hydrogen bonds to
coordinate Zn2+ binding or covalently bind to
Zn2+. Presumably, these Cys and His residues have a
structural role. Mutating one of these highly conserved residues would
therefore be expected to destroy the ability to coordinate
Zn2+.
Three mutagenesis experiments were designed to test the role of either
PHD finger in the PCL·E(Z) interaction. The first mutagenesis converted the second conserved Cys in each PHD finger (residue 430 in
PHDF1 and residue 518 in PHDF2) to a Ser, which is likely to destroy
the ability of that cysteine to covalently coordinate Zn2+
but may not destroy the ability to form a hydrogen bond (Fig. 4A). These constructs were named
LexA-C430S-PCLCR and LexA-C518S-PCLCR for
mutations in PHDF1 and PHDF2, respectively. The second mutagenesis converted the second conserved Cys to an Ala in both PHDF1 and PHDF2
(residues 430 and 518, respectively). These constructs were called
LexA-C430A-PCLCR and LexA-C518A-PCLCR,
respectively (Fig. 4A). The conversion of a conserved Cys to an Ala would be expected to destroy the ability of the PHD finger to
chelate Zn2+ and the ability to form hydrogen bonds.
Apart from the conserved Cys and His residues, Aasland et
al. (17) also noticed a region of highly conserved hydrophobicity adjacent to the third conserved Cys. This region corresponds to residues 439-442 in PHDF1 and residues 527-530 in PHDF2 (Fig. 4A). The significance of this stretch of residues is not
known, and, in the PHD finger domains of PCL, only two of the three
residues are hydrophobic. But the conservation of this stretch of amino acids in many PHD finger-containing proteins suggests that it is of
importance. The third mutagenesis was therefore designed to convert
this stretch of hydrophobic residues to hydrophilic Gln, residues that
would be expected to destroy the function of this stretch of amino
acids. In addition to altering the hydrophobic residues to Gln, the
adjacent Cys was changed to an Ala to generate LexA-PHDF1-QQQA-PCLCR and
LexA-PHDF2-QQQA-PCLCR, respectively.
All six constructs, LexA-C430S-PCLCR,
LexA-C518S-PCLCR, LexA-C430A-PCLCR,
LexA-C518A-PCLCR,
LexA-PHDF1-QQQA-PCLCR, and
LexA-PHDF2-QQQA-PCLCR were shown by Western
analysis using anti-LexA antisera to be expressed at a level similar to
LexA-PCLCR (data not shown), and all six were tested
individually for their ability to interact with AD-E(Z). All six PHD
finger mutants failed to interact with AD-E(Z), indicating that the PHD
finger domains are required to mediate the interaction between PCL and
E(Z) and that both PHDF1 and PHDF2 are required for this interaction
(Fig. 4C).
PCL Is Associated with E(Z) and ESC in Drosophila
Embryos--
To test whether the interactions observed in the
yeast two-hybrid and in vitro binding assays reflect
in vivo association of PCL and E(Z), coimmunoprecipitation
experiments were performed. Anti-E(Z) and anti-PCL antibodies
separately were used to immunoprecipitate proteins from extracts
prepared from Drosophila embryos expressing epitope-tagged
ESC protein, HA-ESC, which provides wild type esc function
in vivo (25). Precipitated proteins were electrophoresed and
immunoblotted. Duplicate Western blots were probed with either anti-E(Z) (Fig. 5, top panel)
or anti-PCL (Fig. 5, middle panel) antibodies. Lane
3 shows that the E(Z) protein was immunoprecipitated (top
panel) and that the PCL protein was coimmunoprecipitated (middle panel) in the same sample. Reciprocal
immunoprecipitations were performed using anti-PCL antibodies.
Lane 4 shows that PCL was immunoprecipitated by anti-PCL
antibodies (middle panel) and that E(Z) was
coimmunoprecipitated (top panel). Neither protein was
precipitated by protein A-Sepharose alone (lane 2,
top and middle panels). From these results, we
conclude that PCL and E(Z) are associated in Drosophila
embryo extracts.
E(Z) previously has been shown to bind directly to another PcG protein,
ESC (25, 31). Recently, E(Z) and ESC have been shown to be subunits of
a 600-kDa complex in Drosophila embryos, which is distinct
from the 2-MDa PRC1 complex (9, 13, 14). If PCL is associated with the
ESC·E(Z) complex, then PCL would be expected to also
coimmunoprecipitate with ESC. To test this, additional aliquots of the
same immunoprecipitations described above were immunoblotted and probed
with anti-HA antibodies (Fig. 5, bottom panel). Lanes
3 and 4 show HA-ESC was coimmunoprecipitated by both
anti-E(Z) and anti-PCL antibodies, respectively. HA-ESC was not
precipitated by Protein A alone (lane 2, bottom
panel). Reciprocal coimmunoprecipitations were performed using
anti-HA antibodies. Comigration of ESC with immunoglobulins precludes detection of HA-ESC in these samples (25). Nevertheless, E(Z) and PCL
were coimmunoprecipitated by anti-HA (lane 6, top
and middle panels, respectively). Neither protein was
precipitated by Protein G alone (lane 5). Although the
anti-HA coimmunoprecipitated E(Z) and PCL signals appear to be
approximately equal, it should be noted that lanes 5 and
6 of the middle panel were derived from x-ray
film exposed to the Western blot for 2 h, whereas a 5-min exposure
of the same Western blot produced the signals in lanes 1-4
of that panel. All lanes in the top and bottom
panels were derived from single exposures of the respective
Western blots. This suggests that a relatively lower percentage of
total PCL protein (compared with E(Z)) is coimmunoprecipitated by
anti-HA. To rule out possible artifactual explanations for these
results, such as gel loading errors or unequal transfer of proteins to the immunoblots, the blots in the top and middle
panels were stripped and re-probed with the reciprocal antibodies.
When the blot originally probed with anti-PCL (middle panel)
was re-probed with anti-E(Z), results identical to those in the
top panel were obtained (data not shown). Likewise, when the
blot originally probed with anti-E(Z) (top panel) was
re-probed with anti-PCL, the PCL signal from the anti-HA
immunoprecipitated sample was again weak relative to the signals in
other lanes (data not shown).
The size(s) of native PCL-containing complexes were determined by gel
filtration chromatography. Nuclear extract from 0- to 24-h
HA-esc embryos was size-fractionated on a Superose 6 column, 0.5-ml fractions were collected, and aliquots from even-numbered fractions were subjected to Western blot analysis. As previously described, E(Z) and HA-ESC cofractionate within a complex of ~600 kDa, which is distinct from the higher molecular mass (2 MDa) PRC1 complex (13, 14; and Fig. 6,
top and middle panels, respectively). An
identical Western blot was probed with anti-PCL antibodies (bottom panel), revealing cofractionation of PCL with HA-ESC
and E(Z). However, the relative abundance of PCL in these fractions differs from that of HA-ESC and E(Z). For example, PCL signal is
noticeably stronger in fraction 26 than in fraction 28, whereas HA-ESC
and E(Z) signals are approximately equal in fractions 26, 28, and 30. These data confirm that PCL is not a component of the PRC1 complex and
are consistent with inclusion of PCL in at least a subset of ESC·E(Z)
complexes.
The Interaction between PCL and E(Z) Is Conserved in Their Human
Homologs--
Many interactions that have been identified between
Drosophila PcG members have also been observed with their
mammalian counterparts. For example, two human homologs of E(Z), EZH1
and EZH2, both interact with a human ESC homolog, EED (25, 32). It was
therefore of interest to determine whether the interaction between PCL
and E(Z) was conserved in their human counterparts. Both EZH1 and EZH2
show a high level of sequence similarity in four domains spanning the
open reading frame (23). One potential PCL homolog, PHF1, shows 34%
identity over the conserved region (22). A second human homolog was
identified using the BLAST program (AJ010014, available at
www.ncbi.nlm.nih.gov (33)), here termed hMTF2, because it displays a
high level of sequence identity (96%) with MTF2, the murine homolog of
PCL, over the central region of the protein (22). hMTF2 displays 37%
identity (57% similarity) to PCL over the conserved region (Fig.
7A).
To determine whether the interaction between PCL and E(Z) is conserved
in their mammalian counterparts, the region spanning the two PHD finger
domains of PHF1 and hMTF2 were cloned into pEG202 to generate LexA-PHF1
and LexA-hMTF2, respectively. Full-length AD-EZH1 and AD-EZH2 were also
generated. LexA-hMTF2 alone was able to strongly activate reporter gene
expression in yeast strains containing as few as two LexA binding
sites. To overcome the problem of auto-activation, the reciprocal
constructs, LexA-EZH1, LexA-EZH2, and AD-hMTF2, were generated. Both
LexA-EZH1 and LexA-EZH2 were tested for auto-activation in EGY48.
LexA-EZH1 did not self-activate the reporter gene, whereas LexA-EZH2
did. However, when transformed into EGY191 (which carries only 2 LexA
binding sites), activation by LexA-EZH2 did not occur.
Because LexA-PHF1 alone did not activate, it was tested for an
interaction with AD-EZH1 and AD-EZH2, whereas AD-hMTF2 was tested for
an interaction with LexA-EZH1 and LexA-EZH2. An interaction was
observed between LexA-PHF1 and AD-EZH2 (Fig. 7B). No
interaction was observed with any other combination (Fig.
7B). Western analysis using anti-LexA antiserum and anti-HA
antisera confirmed the expression of all fusion proteins (data not shown).
PHD Finger Domains of trxG Proteins Appear Not to Interact with
E(Z)--
There are several lines of evidence suggesting that E(Z) is
a member of both the PcG and trxG (see "Discussion"). Three trxG proteins, TRX, Abnormal, Small or Homeotic discs 1 (ASH1) and Abnormal,
Small or Homeotic discs 2 (ASH2), contain PHD finger motifs (17-19).
The ability of E(Z) to bind to the PHD domain of PCL and the presence
of PHD finger motifs in these trxG proteins, raised the possibility
that the role of E(Z) in trxG-dependent transcriptional
activation might involve interaction with the PHD finger domains of one
or more of these trxG proteins. However, in reciprocal yeast two-hybrid
assays, none of these trxG PHD finger domains were found to interact
with E(Z) (data not shown). The absence of interaction between these
trxG PHD finger domains and E(Z) further demonstrates the specificity
of the interaction between E(Z) and the PHD finger domains of PCL.
There is now overwhelming evidence that PcG proteins act in
multimeric complexes to repress transcription. Detailed elucidation of
PcG-mediated repression therefore requires characterization of the
interactions that establish and maintain these complexes. Using both
yeast two-hybrid and in vitro binding assays, direct interaction was observed between the conserved PHD finger-containing region of PCL and E(Z). Deletion analysis and site-directed mutagenesis demonstrated that both PCL PHD fingers, PHDF1 and PHDF2, are important in mediating the interaction of PCL and E(Z), because mutations in
conserved residues of either finger abolishes the interaction. Recently, adjacent PHD fingers and the bromodomain of KAP-1 were shown
to function in a cooperative manner to mediate direct interaction with
an isoform of Mi-2 Coimmunoprecipitation of PCL with E(Z) from embryonic extracts
demonstrates in vivo association of PCL and E(Z). PCL also coimmunoprecipitates with HA-ESC, another component of E(Z) complexes. Gel filtration analysis of native protein complexes from embryo nuclear
extracts show partial cofractionation of PCL with E(Z) and ESC.
Cumulatively, our data are consistent with inclusion of PCL in at least
a subset of ESC·E(Z) complexes. However, it is also possible that PCL
may be a component of a distinct ~600-kDa complex and that PCL may
mediate interaction between this complex and the ESC·E(Z) complex.
Physical interaction of PCL with, and possible inclusion in, ESC·E(Z)
complexes in Drosophila embryos is consistent with genetic interactions of Pcl and E(z) with esc.
A 50% reduction in zygotic dosage of either
Pcl+ or E(z)+
significantly enhances the mutant phenotypes of embryos produced by
esc Why is it, then, that the lack of PCL in Drosophila embryos
produces such mild homeotic phenotypes? It should be remembered that
the cuticular phenotypes described above result from misexpression of
homeotic genes in epidermal cells. Soto et al. (40)
presented evidence for tissue-specific regulation of homeotic genes by
some members of the PcG. Embryos lacking maternal and zygotic
Pcl show extensive ectopic expression of the homeotic gene
AbdB in the central nervous system and visceral mesoderm but
only moderate ectopic expression in a subset of epidermal cells.
Embryos that are homozygous for a null Pcl allele, but which
were derived from heterozygous mothers, exhibit similar tissue-specific
derepression of the homeotic gene abdA (41). In striking
contrast, embryos lacking maternal and zygotic esc or
E(z) activities show extensive ectopic expression of
abdA and AbdB in all tissues (40, 41). Moderate
misexpression of abdA and AbdB in epidermal cells
is consistent with the relatively weak posteriorly directed
transformations of embryonic cuticles in Pcl mutants.
Collectively, the embryonic phenotypes produced by Pcl,
E(z), and esc mutant alleles, the effects of
these mutations on misregulation of homeotic genes, and our
coimmunoprecipitation and gel filtration data suggest that PCL may be
necessary for ESC·E(Z) complex activity only in a subset of embryonic
tissues (i.e. central nervous system and visceral mesoderm
cells and, possibly, a small subset of epidermal cells in
Drosophila embryos, but not in the vast majority of
epidermal cells). Alternatively, PCL may be required in a
temporal-specific manner for ESC·E(Z) complex activity, and the
tissue-specific misregulation of homeotic genes in Pcl
mutants may be due to differences in the times when genes in different
tissues require PcG-dependent repression. These two models
are not necessarily mutually exclusive.
The interaction between Drosophila PCL and E(Z) proteins was
found to be conserved through evolution, as PHF1 and EZH2, human homologs of PCL and E(Z), respectively, interact in yeast two-hybrid assays. Failure to detect an interaction between other combinations of
human PCL and E(Z) homologs, PHF1 and EZH1, hMTF2 and EZH1, or hMTF2
and EZH2, could indicate an in vivo specificity between PHF1
and EZH2 or improper folding of some of the LexA and AD fusion proteins. If folding is not the explanation, hMTF2 may interact in vivo with an as yet unidentified E(Z) homolog. Another
possibility is that some PCL PcG functions may not require interaction
with E(Z). If so, hMTF2 may be specific for those functions.
Alternatively, hMTF2 may have a PcG-independent function that does not
involve interaction with an E(Z) homolog or other PcG proteins. The
specificity of the interaction of PHF1 with EZH2, but not with EZH1,
also suggests distinct functions for these E(Z) homologs.
The conservation of the PCL·E(Z) interaction in humans strongly
suggests that this interaction plays an important role in PcG-dependent silencing in both mammals and
Drosophila. Recent experiments have demonstrated that
mammalian PcG proteins function in manners analogous to that of their
Drosophila counterparts. Mice mutant for mammalian PcG
homologs eed (the esc homolog (42)) or
bmi-I or mel-18 (the Psc and
Su(z)2 homologs (43, 44)) display posterior homeotic
transformations of axial skeletal structures (42, 45, 46). This
phenotype is analogous to the phenotype of the corresponding
Drosophila mutants and reflects derepression of homeotic
genes along the anterior/posterior axis. More striking is the
observation that M33, the murine homolog of Drosophila PC,
partially rescues Pc mutant phenotypes (47). Similarly, overexpression of either Drosophila E(Z) or human EZH2 in
Drosophila enhances the phenomenon of position effect
variegation (23), again demonstrating a conservation of function
between the mammalian and Drosophila genes. Given the
functional similarity of several components of the PcG in both mammals
and Drosophila, it is likely that similar biochemical
mechanisms exist for the heritable repression of homeotic genes
throughout development.
Ample evidence classifies E(z) as a member of the PcG. Male
flies homozygous for a hypomorphic E(z) allele display extra
sex combs on the second and third legs, a characteristic phenotype of
PcG mutants (48). This phenotype is caused by ectopic expression of the
homeotic gene Sex combs reduced (Scr) in second
and third thoracic leg imaginal discs (37). Larvae with reduced
E(z) activity also exhibit ectopic expression of the
homeotic gene Ultrabithorax (Ubx) in wing
imaginal discs, and ectopic expression of Scr and Ubx in the larval central nervous system (37). Embryos
lacking maternally contributed E(z) activity display
posteriorly directed homeotic transformations (36, 37) and show
derepression of homeotic genes (37, 41). E(z) mutant alleles
enhance the homeotic phenotypes produced by mutant alleles of some
other PcG genes. For example, flies heterozygous for mutations in both
E(z) and ph display a more severe extra sex combs
phenotype when compared with single mutants alone (49). Both decreased
and increased E(z)+ dosage enhance the homeotic
phenotypes of embryos derived from homozygous esc females.
Furthermore, E(Z) protein directly interacts with two other PcG
proteins, ESC and PCL, and exists as a component of a multimeric
complex in Drosophila embryos that includes ESC, p55, RPD3,
and possibly PCL (13, 14, 25, 31; Figs. 5 and 6).
However, LaJeunesse and Shearn (50) provided evidence that E(Z) also is
involved in trxG-dependent activation of homeotic genes.
Double-heterozygous combinations of recessive loss of function E(z) and ash1 alleles result in homeotic
transformation phenotypes similar to that observed in
double-heterozygous combinations of recessive loss of function alleles
in trx and ash1 (50, 51). Loss of expression of
the homeotic genes Scr, Ubx, and
Antennapedia (Antp) was also observed in the
thoracic imaginal discs of larvae hemizygous for a null E(z)
allele. This phenotype is also seen in the imaginal discs of larvae
mutant for null ash1 alleles (52). Gildea et al.
(53) have provided evidence that several additional PcG and trxG genes
may also contribute to both repression and activation. The presence of
PHD finger domains in PCL as well as the trxG proteins TRX, ASH1, and
ASH2 and the observation that the PHD finger domains of PCL mediate
binding to E(Z), raised the possibility that the trxG function of E(Z)
might involve binding to the PHD finger domains of TRX, ASH1, and/or
ASH2, perhaps in competition with binding to the PHD finger domains of
PCL. However, no interaction was observed between the PHD finger
domains of these trxG proteins and E(Z) in yeast two-hybrid assays. An
as yet unidentified interaction between E(Z) and a trxG protein may account for its role in trxG-dependent activation. It is
also likely that the PHD domains of these trxG proteins mediate
interactions with other proteins, such as Cyp33 (21), and others that
have not yet been identified.
We thank Michael Kyba and Hugh Brock for
generously providing AD-PcG constructs prior to publication, Michelle
Coulson, Peter Harte, Judith Kassis, and Roger Brent for
Drosophila and yeast strains and antibodies, and Allen
Shearn for ash1 and ash2 cDNA clones. We also
thank Michelle Coombe for excellent technical assistance.
*
This work was supported in part by Australian Research
Council grants (to R. S.) and by National Institutes of Health Grant GM46567 (to R. S. J.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Supported by an Australian Postgraduate Research Award.
¶
Both authors contributed equally to this work.
**
Current address: CSIRO Marine Research, G. P. O. Box 1538, Hobart, Tasmania 7001, Australia.
§§
To whom correspondence may be addressed: Dept. of Biological
Sciences, Southern Methodist University, Dallas, TX 75275-0376. Tel.:
214-768-3810; Fax: 214-768-3955; E-mail: rjones@mail.smu.edu.
Published, JBC Papers in Press, September 24, 2001, DOI 10.1074/jbc.M104294200
2
H. Brock, personal communication.
The abbreviations used are:
trxG, trithorax
group;
PcG, Polycomb group;
PC, Polycomb;
PH, Polyhomeotic;
ESC, Extra
Sex Combs;
PCL, Polycomb-like;
ASH1, Absent, small, or homeotic discs
1;
ASH2, Absent, small, or homeotic discs 2;
kilobase(s), PCR,
polymerase chain reaction;
aa, amino acid(s);
GST, glutathione
S-transferase;
Ni-NTA, nickel-nitrilotriacetic acid;
HA, hemagglutinin;
PBD1, -2, PCL binding domains 1 and 2;
PHD, plant
homeodomain.
Polycomblike PHD Fingers Mediate Conserved Interaction
with Enhancer of Zeste Protein*
§¶,
,**,,
, and§§
Centre for the Molecular Genetics of
Development and Department of Genetics, University of Adelaide,
Adelaide, South Australia 5005, Australia and the Department of
Biological Sciences, Southern Methodist University, Dallas, Texas
75275-0376
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chrPC, AD-PC3', AD-ph, AD-phHD, AD-phN, AD-phS, AD-Psc,
AD-PscB, AD-Su(z)2, AD-Su(z)2B, AD-AsxA, AD-AsxZn, AD-AsxQ,
AD-Scm2, AD-E(Pc)Ybox, and AD-E(Z) were generously provided by Hugh
Brock and Michael Kyba (University of British Columbia)
(29).2 AD-PHO (full-length)
was generated by PCR and cloned as an EcoRI fragment into
pJG4-5 using pET-PHO (provided by Judy Kassis) as a template. AD-ESC
(full-length) was generated by PCR using pact-esc (provided by H. Brock) as a template and cloned into pJG4-5 as an XhoI fragment.
his3 trp1 ura3
6lexAop-LEU2), was transformed with pEG202 and pJG4-5 plasmids,
encoding the DNA binding domain and activator fusion proteins,
respectively. Three individual transformant colonies were picked and
streaked out on both glucose and galactose plates lacking leucine,
uracil, histidine, and tryptophan. Growth was scored after 4 days at
30 °C. Colonies of ~1 mm in diameter were scored as having strong
interactions. Absence of growth on galactose medium indicated no interaction.
80 °C. 1.0 mg of the nuclear
extract was fractionated on a Superose 6 column equilibrated in 45 mM HEPES (pH 7.6), 360 mM NaCl, 10% glycerol,
0.1% Tween-20, 0.1 mM EGTA, 1 mM
MgCl2, 1 mM ammonium molybdate, 10 mM NaF, 0.1 mM dithiothreitol, 1 µg/ml
aprotinin, 5 µg/ml leupeptin, 5 µg/ml antipain, 5 µg/ml pepstatin
A, and 5 µg/ml chymostatin. Chromatography was performed under fast
performance liquid chromatography conditions using a P-500 pump
(Amersham Pharmacia Biotech). 0.5-ml fractions were collected, and
20-µl aliquots from even-numbered fractions were examined by Western
blotting. Molecular masses of eluted proteins were determined by
pre-calibrating the column with the following size standards (Sigma):
blue dextran (2000 kDa), thyroglobulin (670 kDa), apoferritin (440 kDa), 
-amylase (200 kDa), and bovine serum albumin (68 kDa).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (84K):
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Fig. 1.
The conserved domain of PCL
(PCLCR) interacts with E(Z) in the yeast two-hybrid
assay. A, schematic representation of the PCL protein
showing the conserved region that encodes the two PHD finger domains,
depicted here by hatched boxes, the region between the PHD
finger domains and the Polycomblike extended homology
(EH) domain, which is represented by a shaded
box. The amino acid position of each motif is stated above
the respective boxes. B, growth assay with EGY48 yeast
containing the following combinations of fusion proteins;
left, LexA-PCLCR and AD alone;
middle, LexA-PCLCR and AD-E(Z);
right, LexA-alone and AD-E(Z). Yeast strains were streaked
in parallel on medium lacking leucine but containing galactose
(GAL, top) or glucose (GLU,
bottom). Expression of the AD fusion proteins were induced
on GAL but not GLU medium. Yeast strains were grown for 4 days at
30 °C. Only yeast containing both LexA-PCLCR and AD-E(Z)
were able to grow.
View larger version (26K):
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Fig. 2.
In vitro binding of the PCL and
E(Z) proteins and localization of the E(Z) domains that bind to
PCL. Depicted are autoradiographs of SDS gels. The input
lanes contain 20% of the amount of radiolabeled protein used in
each binding assay. A, radiolabeled E(Z) protein was tested
for binding to His6-PCL-(403-605) protein, which includes
PCLCR. B and C, radiolabeled PCL was
tested for binding to GST or GST-E(Z) fusion proteins containing the
indicated E(Z) residues.
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Fig. 3.
Effects of E(Z) point mutations
on PCL binding. A, diagram of E(Z) protein illustrating
the locations of domains defined either on the basis of function
(ESC-binding domain, PBD1 and PBD2) or evolutionary conservation
(Domain II, Cys-rich region and SET domain). B and
C, alignments of E(Z) PCL binding domains PBD1
(B) and PBD2 (C) with homologous regions of human
EZH2 and EZH1 proteins. The numbers above the sequences
indicate the locations of residues that were substituted by alanine in
this study. D-F, autoradiographs of SDS gels illustrating
the effects of E(Z) alanine substitutions on binding to in
vitro translated PCL protein. GST-E(Z) fusion proteins containing
E(Z) residues 86-171 (D), 377-446 (E), or
1-511 (F) and the indicated alanine substitutions were
incubated with radiolabeled PCL protein. Equal amounts of bound protein
samples were electrophoresed on SDS gels.
EH (aa 424-565) construct was
generated containing a truncated conserved region lacking the extended
homology region (aa 566-605). When LexA-PCLCREH was
tested for its ability to interact with AD-E(Z), growth was observed on
leucine-deficient medium, indicating that the region of extended
homology is not required for the interaction between E(Z) and PCL (Fig.
4B).
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Fig. 4.
The PHD finger motifs of PCL are responsible
for mediating the interaction with E(Z). A, an
alignment of PHD finger domains 1 and 2 with the consensus PHD finger
motif (17) and a schematic of the in vitro mutagenesis
strategy. The conserved Cys and His residues are shown in
boldface, and the highly conserved hydrophobic residues,
including the adjacent Cys are underlined. Residues that
were mutated for the analysis shown in C are indicated by an
asterisk. The consensus symbols are: #, strongly conserved
hydrophobicity; §, highly conserved hydrophobicity. B,
growth assay with yeast containing AD-E(Z) and either
LexA-PCLCR (positive control, left) or
LexA-PCLCR EH (right). Yeast were streaked
onto medium lacking leucine but containing galactose (GAL,
top) or glucose (GLU, bottom). Plates
were incubated at 30 °C for 4 days. C, growth assay with
yeast containing AD-E(Z) and a LexA-PCLCR mutant. The
schematic on the left shows the arrangement of
yeast on adjacent plates. Yeast medium lacks leucine and contains
galactose (GAL, top) or glucose (GLU,
bottom). Yeast plates on left contain LexA-PCLCR
mutants in PHD finger 1 along with normal LexA-PCLCR. Yeast
plates on the right contain LexA-PCLCR mutants in PHD
finger 2 along with normal LexA-PCLCR. C 
A
indicates mutation of the second conserved Cys to an Ala residue.
C S indicates mutation of the second conserved Cys to a
Ser residue. QQQA indicates mutation of hydrophobic region
and adjacent Cys to Gln and Ala, respectively.
View larger version (39K):
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Fig. 5.
Coimmunoprecipitation of E(Z), PCL, and
HA-ESC proteins from Drosophila embryo extracts.
Proteins were immunoprecipitated from 300 µg of
HA-esc embryo extracts with anti-E(Z), anti-PCL, or anti-HA
antibodies, as indicated. Equal amounts of immunoprecipitated proteins
(one-third of each sample) were run separately on 8% SDS gels,
transferred to nitrocellulose filters, and incubated with
(top) anti-E(Z), (middle) anti-PCL, or
(bottom) anti-HA antibodies. Lanes: 1,
30 µg total embryo extract; 2, mock immunoprecipitation by
protein A-Sepharose without antibody; 3, immunoprecipitation
with anti-E(Z) antibody; 4, immunoprecipitation with
anti-PCL antibody; 5, mock immunoprecipitation by protein
G-Sepharose without antibody; 6, immunoprecipitation with
anti-HA antibody.
View larger version (29K):
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Fig. 6.
Gel filtration analysis of E(Z), HA-ESC, and
PCL proteins in Drosophila nuclear extracts.
Nuclear extract from HA-esc embryos was fractionated by
Superose 6 chromatography. 20-µl aliquots of even-numbered 500-µl
fractions were run on separate 8% SDS gels, transferred to
nitrocellulose filters, and incubated with anti-E(Z) antibodies
(top), anti-HA antibodies (middle), or anti-PCL
antibodies (bottom). Elution positions of molecular mass
standards are indicated with arrows above the numbers of the
appropriate fractions.
View larger version (76K):
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Fig. 7.
Interactions between the human homologs of
PCL and E(Z). A, protein alignment of the region of
similarity between PHF1 (GenBankTM accession number
AF029678), hMTF2 (GenBankTM accession number AJ010014), and
PCL (GenBankTM accession number Q24459). Over this region
PHF1 and hMTF2 share 58% identity (71% similarity), PHF1 and PCL
share 36% identity (48% similarity), and hMTF2 and PCL share 37%
identity (57% similarity). Conserved residues are black,
and similar residues are gray. B, growth assay
with yeast containing human homologs of PCL and E(Z). The left
panel is a schematic of the arrangement of yeast on the
middle and right panels. Yeast were streaked onto
leucine-deficient medium, which contained either galactose
(GAL, right panel) or glucose (GLU,
middle panel).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(20). The PHD fingers domain of myeloid lymphoid
leukemia protein has been shown to both homodimerize and bind to
Cyp33 (21). Our data demonstrate that a PHD finger domain can function
as an independent protein interaction domain. Two separate E(Z) regions
bind to the PCL PHD fingers in vitro. One, PBD1, is highly
conserved in mammalian homologs; the other, PBD2, is not. Each region
is capable of independently binding to PCL in vitro.
However, it is not known whether this is also true in
vivo.
females. Among PcG loci, Pcl is
second only to E(z) in the strength of enhancement of the
esc maternal effect embryonic phenotype (34). However, if
ESC·E(Z) complexes required PCL activity for all embryonic functions,
one would expect mutant alleles of Pcl, E(z), and
esc to produce very similar, if not identical, phenotypes. This is not the case. Embryos lacking maternally and zygotically produced E(Z) or ESC do exhibit essentially identical extreme homeotic
phenotypes in which all thoracic and abdominal segments are transformed
into copies of the eighth abdominal segment (35-37). In contrast,
embryos lacking both maternal and zygotic Pcl activity display relatively weak posteriorly directed cuticular transformations; abdominal segments are slightly more posterior-like in appearance, but
thoracic segments are essentially unaffected (38). Indeed, the
Pcl mutant embryonic cuticular transformations are
relatively weak with respect to most other PcG mutations. For example,
embryos that lack both maternal and zygotic Pc activity also
display a severe homeotic phenotype similar to the E(z) and
esc phenotypes described above (39). Therefore, lack of
individual subunits of either the ESC·E(Z) complex or the PRC1
complex (e.g. PC) can effectively disrupt
PcG-dependent repression of homeotic genes in embryos.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence may be addressed: Center for the
Molecular Genetics of Development and Dept. of Molecular Biosciences, University of Adelaide, Adelaide South Australia 5005, Australia. Tel.:
61-8-8303-5563; Fax: 61-8-8303-4399; E-mail:
robert.saint@adelaide. edu.au.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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