Developmental Cell, Vol. 3, 499–510, October, 2002, Copyright 2002 by Cell Press
Plzf Mediates Transcriptional Repression
of HoxD Gene Expression
through Chromatin Remodeling
SMRT, N-CoR, Sin-3, and, in turn, class I and II histone
deacetylases to the transcriptional complex (Hong et
al., 1997; He et al., 1998; Grignani et al., 1998; Lin et al.,
1998; David et al., 1998; Lemercier et al., 2002).
We have generated mice with a null mutation in Plzf,
which show striking patterning defects in both the limb
and axial skeleton, including homeotic transformations
of anterior skeletal elements in the developing limb into
posterior structures (Barna et al., 2000). These transfor-
mations are accompanied by the anteriorization and ec-
topic expression of each member of the 5? AbdB HoxD
gene complex in the developing hindlimb. These results
gene expression; however, the molecular mechanisms
by which Plzf would restrict the posterior boundaries of
expression of the HoxD genecomplex are still unknown.
Several models have been proposed to account for reg-
ulation of Hox gene expression which illustrate (1) the
importance of cis elements within these genes which
would regulate their transcription in space and time and
vent posterior Hox genes from being activated at an
earlier stage through a repressive chromatin configura-
tion (Dolle et al., 1989; van der Hoeven et al., 1996;
been , so far, implicated in this process.
activation of the AbdB HoxD gene complex through
binding to cis elements within Hox genes and recruit-
ment of histone deacetylases as well as Polycomb pro-
teins, in turn favoring the transition from a euchromatic
to a heterochromatic chromatin state. These results will
be discussed in the context of previous models of Hox
Maria Barna,1Taha Merghoub,1Jose ´ A. Costoya,1
Davide Ruggero,1Matthew Branford,1
Anna Bergia,2Bruno Samori,2
and Pier Paolo Pandolfi1,3
1Molecular Biology Program
Department of Pathology
Memorial Sloan-Kettering Cancer Center
New York, New York 10021
2Department of Biochemistry
University of Bologna and
The molecular mechanisms that regulate coordinated
and colinear activation of Hox gene expression in
space and time remain poorly understood. Here we
demonstrate that Plzf regulates the spatial expression
of the AbdB HoxD gene complex by binding to regula-
sion and can recruit histone deacetylases to these
sites. We show by scanning forced microscopy that
Plzf, via homodimerization, can form DNA loops and
bridge distant Plzf binding sites located within HoxD
gene regulatory elements. Furthermore, we demon-
teins on DNA. We propose a model by which the bal-
ance between activating morphogenic signals and
transcriptional repressors such as Plzf establishes
proper Hox gene expression boundaries in the limb
The spatial and temporal order of Hox gene activation
is colinear with the physical position of the genes along
their respective clusters (Gaunt et al., 1989; Dolle et al.,
1989; Izpisua-Belmonte et al., 1991). In vertebrates, the
transcriptional mechanisms involved in Hox gene regu-
lation are largely unknown. The PLZF (promyelocytic
leukemia zinc finger) gene was identified by virtue of its
involvement in chromosomal translocations associated
with acute promyelocytic leukemia (APL) (Chen et al.,
1993). PLZF is a nuclear protein (Reid et al., 1995) con-
taining at the C terminus nine Kru ¨ppel-type zinc-finger
domains, which recognize specific DNA sequences (Li
et al., 1997; Sitterlin et al., 1997). At the N terminus,
association and transcriptional repression when fused
to a heterologous DNA binding region (Bardwell and
Treisman, 1994; Dong et al., 1996). PLZF functions as
a transcriptional repressor through its ability to recruit,
via the BTB/POZ domain, nuclear corepressors such as
Selective Deregulation of Spatial but Not
Temporal Expression of the Abdb HoxD
Complex in Plzf?/?Mice
Throughout normal limb development, 5? HoxD genes
display restricted patterns of expression which corre-
late with both the temporal and spatial colinearity of
the complex. Whole-mount in situ hybridization of
Plzf?/?embryos did not reveal any premature temporal
activation of 5? HoxD genes, as Hoxd11 transcripts were
not expressed at an earlier time than wild-type embryos
(Figure 1A). However, when 5? HoxD transcripts are first
expressed in spatially restricted posterior domains of
the limb, their expression was anteriorized in Plzf?/?
embryos, showing a uniform expression of transcripts
along the entire extent of the hindlimb bud (Figure 1B;
and data not shown). As previously reported (Barna et
al., 2000), the Plzf?/?phenotype is largely restricted to
the hindlimb, which may be due to redundancy of Plzf
function in the forelimb because of the presence of a
Figure 1. Aberrant HoxD Gene Expression in
Plzf?/?Mice and Its Rescue in Plzf?/?Limb
(A) Expression of Hoxd11 in Plzf?/?and wild-
type embryos at 9.0 and 9.5 dpc.
ing hindlimb of Plzf?/?and wild-type controls
at 10.5 dpc.
(C) Northern blot analysis of Hoxd13 and
Hoxb2 in Plzf?/?and wild-type limb cells as
wellas inPlzf?/?limbcells followingtransfec-
tion of a Plzf expression vector or an empty
Plzf homolog. Similarly, these changes in Hox gene ex-
pression are limited to the hindlimb. Anteriorization of
HoxD expression occurred prior to any visible morpho-
logical defects in Plzf?/?embryos and is consistent with
the misexpression of 5?HoxD genes at later stages of
Plzf inactivation therefore results in loss of spatial colin-
ear expression of 5?HoxD genes, while their temporal
sequential activation does not appear to be affected.
1997; Sitterlin et al., 1997), as cis regulatory regions of
this gene have been previously identified (Gerard et al.,
1993). We identified five putative Plzf binding sites. Two
of the binding sites were located in the promoter of
highly conserved regulatory regions, known as region
VI and IX, present in the 3? UTR of the gene (Figure 2A).
We employedlimb extracts from Plzf?/?and wild-type
embryos to perform gel shift retardation assays utilizing
labeled oligonucleotide probes spanning the Plzf bind-
ing sites present within the Hoxd11 gene (Plzf binding
sites 1–5). A shift is present only in wild-type extracts,
but not in extracts from Plzf?/?mutants, is competed
by a cold oligonucleotide, and is absent with a mutant
oligonucleotide in all cases (Figure 2B). As a control,
of shifting a SP3 oligonucleotide (data not shown). The
presence of Plzf in the retarded DNA protein complex
was confirmed by Western blot analysis of the proteins
eluted from the region of the gel encompassing the
shifted band observed utilizing wild-type extracts (Fig-
ure 2C). We confirmed that this interaction is direct by
the ability of GST-Plzf to bind to an oligo spanning
Hoxd11 RRIX (Figure 2E).
To assess whether Plzf binds to Hox regulatory ele-
ments in vivo, we performed DNA immunoprecipitation
limb cells. We specifically detected Hoxd11 RRIX in the
DNA that was bound by Plzf in vivo (Figure 2D).
Plzf possesses an evolutionarily conserved BTB/POZ
domain that has been shown to mediate homodimeriza-
tion (Minucci et al., 2000). In order to assess if Plzf
the HoxD locus, we utilized scanning force microscopy
(SFM) to visualize stretches of Hoxd11 genomic DNA in
the presence of GST-Plzf. To this end, purified GST-
Plzf was incubated with different DNA fragments within
Hoxd11 (Figures 2A and 2G). The samples were depos-
ited on freshly cleaved mica and imaged by SFM. The
position of GST-Plzf along the DNA was determined by
Elevated HoxD Gene Expression Is Rescued by
the Reintroduction of Plzf in Limb Cultures
Derived from Plzf?/?Embryos
As alterations in limb development may result in the
ectopic activation of 5?HoxD genes, whose elevated
expression are a response to these morphological
changes, we sought to test whether the anteriorization
of HoxD transcripts in Plzf?/?embryos was a direct or
indirect consequence of Plzf activity. To this end, we
utilized low-density primary limb cells from wild-type
and Plzf?/?embryos to assay the level of expression
of one of the 5?HoxD genes that showed anteriorized
expression in the hindlimb bud, Hoxd13. Plzf?/?limb
cultures showed a marked increase in Hoxd13 tran-
scripts with respect to wild-type cultures, by Northern
blot analysis (Figure 1C; and data not shown). The rein-
troduction of Plzf in Plzf?/?limb cultures (Experimental
Procedures) reduced the level of Hoxd13 transcripts to
while it had no effect on the expression of Hoxb2, a Hox
therefore suggest that Plzf directly modulates the expres-
sion of 5?HoxD genes in primary limb cells.
Plzf Binds to Multiple Sites within the 5?HoxD
Gene Cluster and Mediates Long-Range
To understand the mechanisms by which Plzf mediates
for the presence of putative Plzf binding sites (Li et al.,
Role of Plzf in Hox Gene Regulation
total body embryo extracts with a Plzf monoclonal antibody (Onco-
gene) at 2 ?g/ml concentration.
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Oligo Affinity Binding Assay
Theoligo affinitybindingassay (OABA)wasperformed aspreviously
described (Zhong et al., 1999). Biotinylated RRIX oligo and mutated
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For injection, the pGemE/ElacZpA (Gerard et al., 1993), Hoxd11/
lacZ mut, and Hoxd11/lacZ bs5 mut constructs were excised with
NsiI, and a fragment of 10.7 kb was injected in all cases. A Plzf?/?
transgenic line carrying the Hoxd11/lacZ transgene was obtained
injecting this construct into Plzf?/?eggs. In addition, all the con-
structs were tested in transient assays using F1 eggs. Eleven em-
transient assays, and all embryos showed a posteriorly restricted
expression pattern in the hindlimb as previously described (Gerard
et al., 1993). Four embryos expressing the Hoxd11/lacZ mut trans-
gene were obtained, and two embryos showed an anteriorization
of ?-gal expression throughout the hindlimb that was never ob-
served in the Hoxd11/lacZ transgene. Eight embryos expressing the
Hoxd11/lacZ bs5 mut transgene were obtained, and three embryos
showed a partial anteriorization of ?-gal expression in the hindlimb
that was never observed in the Hoxd11/lacZ transgene.
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We thank Lee Niswander foruseful discussions and for critical read-
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Hoxd11 genomic region, the Hoxd11-LacZ expression vector, and
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