Histone and DNA methylation defects at Hox genes in mice expressing a SET domain-truncated form of Mll.
ABSTRACT The Mll gene is a member of the mammalian trithorax group, involved with the antagonistic Polycomb group in epigenetic regulation of homeotic genes. MLL contains a highly conserved SET domain also found in various chromatin proteins. In this study, we report that mice in which this domain was deleted by homologous recombination in ES cells (DeltaSET) exhibit skeletal defects and altered transcription of particular Hox genes during development. Chromatin immunoprecipitation and bisulfite sequencing analysis on developing embryo tissues demonstrate that this change in gene expression is associated with a dramatic reduction in histone H3 Lysine 4 monomethylation and DNA methylation defects at the same Hox loci. These results establish in vivo that the major function of Mll is to act at the chromatin level to sustain the expression of selected target Hox genes during embryonic development. These observations provide previously undescribed evidence for the in vivo relationship and SET domain dependence between histone methylation and DNA methylation on MLL target genes during embryonic development.
- [show abstract] [hide abstract]
ABSTRACT: Distinct modifications of histone amino termini, such as acetylation, phosphorylation and methylation, have been proposed to underlie a chromatin-based regulatory mechanism that modulates the accessibility of genetic information. In addition to histone modifications that facilitate gene activity, it is of similar importance to restrict inappropriate gene expression if cellular and developmental programmes are to proceed unperturbed. Here we show that mammalian methyltransferases that selectively methylate histone H3 on lysine 9 (Suv39h HMTases) generate a binding site for HP1 proteins--a family of heterochromatic adaptor molecules implicated in both gene silencing and supra-nucleosomal chromatin structure. High-affinity in vitro recognition of a methylated histone H3 peptide by HP1 requires a functional chromo domain; thus, the HP1 chromo domain is a specific interaction motif for the methyl epitope on lysine9 of histone H3. In vivo, heterochromatin association of HP1 proteins is lost in Suv39h double-null primary mouse fibroblasts but is restored after the re-introduction of a catalytically active SWUV39H1 HMTase. Our data define a molecular mechanism through which the SUV39H-HP1 methylation system can contribute to the propagation of heterochromatic subdomains in native chromatin.Nature 04/2001; 410(6824):116-20. · 38.60 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Heterochromatin protein 1 (HP1) is localized at heterochromatin sites where it mediates gene silencing. The chromo domain of HP1 is necessary for both targeting and transcriptional repression. In the fission yeast Schizosaccharomyces pombe, the correct localization of Swi6 (the HP1 equivalent) depends on Clr4, a homologue of the mammalian SUV39H1 histone methylase. Both Clr4 and SUV39H1 methylate specifically lysine 9 of histone H3 (ref. 6). Here we show that HP1 can bind with high affinity to histone H3 methylated at lysine 9 but not at lysine 4. The chromo domain of HP1 is identified as its methyl-lysine-binding domain. A point mutation in the chromo domain, which destroys the gene silencing activity of HP1 in Drosophila, abolishes methyl-lysine-binding activity. Genetic and biochemical analysis in S. pombe shows that the methylase activity of Clr4 is necessary for the correct localization of Swi6 at centromeric heterochromatin and for gene silencing. These results provide a stepwise model for the formation of a transcriptionally silent heterochromatin: SUV39H1 places a 'methyl marker' on histone H3, which is then recognized by HP1 through its chromo domain. This model may also explain the stable inheritance of the heterochromatic state.Nature 04/2001; 410(6824):120-4. · 38.60 Impact Factor
- EXS 02/1993; 64:523-68.
Histone and DNA methylation defects at Hox genes in
mice expressing a SET domain-truncated form of Mll
Re ´mi Terranova*†‡, Hanane Agherbi*‡, Annie Boned*, Ste ´phane Meresse*, and Malek Djabali*§
*Centre d’Immunologie de Marseille-Luminy, Institut National de la Sante ´ et de la Recherche Me ´dicale–Centre National de la Recherche Scientifique,
Case 906, 13288 Marseille Cedex 9, France; and†Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland
Edited by Mark T. Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved March 1, 2006 (received for review August 25, 2005)
The Mll gene is a member of the mammalian trithorax group,
involved with the antagonistic Polycomb group in epigenetic
domain also found in various chromatin proteins. In this study, we
report that mice in which this domain was deleted by homologous
transcription of particular Hox genes during development. Chro-
matin immunoprecipitation and bisulfite sequencing analysis on
developing embryo tissues demonstrate that this change in gene
expression is associated with a dramatic reduction in histone H3
Lysine 4 monomethylation and DNA methylation defects at the
same Hox loci. These results establish in vivo that the major
function of Mll is to act at the chromatin level to sustain the
expression of selected target Hox genes during embryonic devel-
opment. These observations provide previously undescribed evi-
dence for the in vivo relationship and SET domain dependence
between histone methylation and DNA methylation on MLL target
genes during embryonic development.
histone methyltransferase ? MLL-SET domain ? homeotic transformations
homeobox-containing (Hox) genes in specific segments of the
embryo (1). The trithorax and polycomb groups (trx-G and PcG)
were identified for their role in faithfully maintaining the transcrip-
tional states of these key developmental regulators, providing an
epigenetic mechanism of cellular memory (2–4).
The gene expression maintenance function of the trxG and PcG
proteins is highly conserved. Mixed lineage leukemia (Mll), a
human homolog of Drosophila trithorax and a member of the trxG
family, was identified first for its involvement in chromosomal
translocations associated with lymphoid and myeloid acute leuke-
mia in infants and adults (5, 6). Mll encodes a 3,969-aa nuclear
protein with multiple domains, including three AT-hook motifs, a
DNA methyltransferase homology domain (DNMT) in the amino-
a highly conserved 130-aa carboxyl-terminal SET domain. The
MLL protein was shown to be proteolytically processed into two
portions (MLLNand MLLC) with antagonistic transcriptional
effector properties, that reassociate and stabilize each other (7–9).
The MLL protein is critical for proper regulation of the Hox genes
during embryonic development (10). In Mll null mutant mice
(Mll???), Hox gene expression is correctly initiated but is not
sustained as the function of Mll becomes necessary (11), leading to
It is strongly believed that maintenance of the transcriptional
status of target genes by PcG and trxG proteins is achieved through
chromatin modifications (12). The structure similarity between
some trxG?PcG and suppressors or enhancers of position effect
variegation (PEV) further substantiates this point. One of the most
remarkable shared domains within these chromatin proteins is the
SET domain. Present at the C terminus of the Trithorax and MLL
including the PEV modifier SU(VAR)3–9 and the PcG protein
E(z) (13, 14). Many SET domain-containing proteins have been
he control of cell identity during development is specified, in
large part, by the unique expression patterns of multiple
demonstrated to mediate lysine-directed histone methylation (15–
19). These proteins decorate the histones at specific position,
providing a recognition site for activating or repressing proteins. In
particular, the methylation on lysine-9 of histone H3 is associated
with transcriptional repression (20, 21). Di- and trimethylation on
lysine-4 of histone H3 (H3K4me2 and H3K4me3), on the other
hand, are associated with a permissive and transcriptionally active
to bind to the promoter regions of some active Hox genes, where it
recruits a very large multiprotein complex carrying several chro-
matin modifying and remodeling activities. They include a histone
recruitment of MOF, a member of the MYST family of histone
acetyltransferases (HATs), which acetylates histone H4 at Lys-16
To gain more insight into the in vivo function of the MLL-SET
domain during development, we have generated mice in which the
SET domain of Mll was deleted by homologous recombination
(?SET). In these conditions a SET domain-truncated allele of Mll
show that they exhibit developmental skeletal defects and an
alteration in the maintenance of the proper transcription levels of
several target Hox loci during development. Importantly, these
changes in gene expression levels are associated with a reduction of
histone H3K4 monomethylation (H3K4me1) and altered DNA
methylation patterns at the same Hox loci. These results demon-
strate in vivo an essential role for the MLL-SET domain on
chromatin structure and Hox gene regulation. They provide evi-
dence for epigenetic relationship in the maintenance of Hox genes
activation during embryonic development.
Targeted Disruption of the Mll-SET Domain. To investigate the func-
tion of the SET domain of the Mll gene during development, we
(neo) gene. A ‘‘stop’’ codon was introduced in frame to allow
appropriate termination of protein synthesis in the absence of the
SET domain (Fig. 1A). Homologous recombinant clones were
obtained and transfected in vitro with a CRE expressing vector to
excise the neo cassette. Two independent ?SET??? ES cell clones
were at the origin of several chimeras, which transmitted the
truncated Mll allele to their offsprings. Southern analysis on
BamHI- and HindIII-digested tail DNA from selected mice, using
the appropriate DNA probes (Fig. 1A), confirmed that the SET
domain was deleted in mutant mice (Fig. 6A, which is published as
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Freely available online through the PNAS open access option.
Abbreviations: dpc, days postcoitum; MEF, mouse embryonic fibroblast.
‡R.T. and H.A. contributed equally to this work.
§To whom correspondence should be addressed. E-mail: email@example.com.
© 2006 by The National Academy of Sciences of the USA
April 25, 2006 ?
vol. 103 ?
no. 17 ?
supporting information on the PNAS web site). RT-PCR by using
RNA extracted from WT (???), ?SET???, and ??? mouse
embryonic fibroblasts [MEFs; derived from 12.5 days postcoitum
(dpc) embryos] demonstrated that the ?SET allele was transcribed
at similar level than the WT allele (Fig. 1B). Immunofluorescence
studies on WT and ?SET ??? MEFs confirmed that the deletion
of the SET domain did not affect the level of expression or
distribution of the truncated MLL protein in the nucleus. Consis-
tent with previous studies (24), a punctuate pattern was observed
(Fig. 6B). The biochemical purification of MLL complex identified
multiple components, including the tumor suppressor Menin, an
Hox gene expression. This complex is highly similar to that of the
yeast and human SET1 complex (COMPASS) (9). Upon deletion
of the SET domain of MLL, the global levels of MLLCand
MLL-associated proteins WDR5 and Menin were not significantly
affected (Fig. 1C). Total protein extract were prepared from WT
and ?SET MEFs for Western blot analysis. MLLC, WDR5, and
Menin levels were assessed while Actin estimated the equivalence
by using an antibody directed against MLLNfurther demonstrated
that the SET domain truncated MLLCis still associated in a
complex with MLLNand Menin (Fig. 1D). Altogether these
analyses show that the loss of the SET domain of MLL does not
significantly alter the stability of the MLL complex.
Homeotic Transformations in ?SET Mice.ThreeMll-disruptionalleles
have been previously generated (10, 28, 29); all result in embryonic
lethality in homozygotes. In sharp contrast, in the ?SET mutant
mice, the mating of heterozygous mice yielded WT, heterozygous,
and homozygous offspring at the expected Mendelian ratios, indi-
cating no significant embryonic lethality. At birth, homozygotes
1.5 g (n ? 28) and 12.8 ? 0.5 g (n ? 35), for 3-week-old WT and
?SET mice, respectively].
Mice heterozygous for the Mll-lacZ allele displayed bidirectional
homeotic transformations of the axial skeleton and sternal malfor-
mations. These homeotic transformations were associated with a
posterior shift of the anterior boundaries of expression of several
3-week-old homozygous ?SET mutant skeletons showed defects in
the formation of the vertebral column (Fig. 2). Mutant mice
displayed an additional ossification center between sternebra 5 and
typical sternal malformations included uni- or bilateral fusions
between ribs (Fig. 2 C and D, arrows). The cervical region of the
?SET mutant mice was also affected; the atlas (C1) and the axis
whereas the anterior arch of the atlas (aaa) was often missing or
reduced in size (Fig. 2F, compare with WT, Fig. 2E). Moreover
C7 revealed by the presence of rudimentary ribs. These observa-
role of Mll in defining the proper identity of cells in the segments
along the anteroposterior axis. With the exception of the sternal
with homology arms, LoxP sites (triangles) flanking the neomycin resistance
cassette. Partial restriction map of the Mll locus (A, ApaI; B, BamHI; H, HindIII)
is shown before and after targeted replacement of the SET domain encoding
region by the neomycin resistance cassette. Southern strategy to detect
homologous recombination (BamHI digestion and ‘‘ext5’’ probe) and CRE
deletion of the neomycin resistance cassette (HindIII digestion and ‘‘int3’’
probe) is indicated. RT-PCR primers encompassing the SET domain region are
indicated by arrows. (B) The expression of the WT and MLL SET truncated
alleles was evaluated by RT-PCR analysis in embryonic fibroblasts generated
the WT and truncated alleles. Equivalent loading of RNA was controlled by
using GAPDH primers. (C) Western blot analysis of selected MLL-associated
factors: MLLc, Menin, and WDR5 in WT and MLL?SET MEFs. Molecular sizes of
marker proteins are shown on the right. (D) Coimmunoprecipitation of MLL
were subjected to immunoprecipitation (IP) by using an antibody specific for
MLLN. The immunoprecipitates were fractionated in SDS?PAGE and immuno-
blotted with the antibodies indicated to the left of the panels (anti-MLLCand
mutant mice (B–D and F). Typical sternal abnormalities were detected in mutant mice. The additional ossification center is indicated by an arrow in B. Two
different fusions between ribs are shown (C and D, indicated by arrows). Lateral view of the cervical region of a WT (E) and ?SET mouse (F) is shown. ?SET mice
present bone abnormalities in the cervical region. The C2 vertebra is broadened (C2*). The anterior arch of the atlas is severely reduced (aaa*). C6 (black arrow)
show posterior transformation to C7.
Skeletal abnormalities in ?SET mice. Views are shown of the thoracic (A–D) and cervical (E and F) regions of cleared skeletons of WT (A and E) and ?SET
www.pnas.org?cgi?doi?10.1073?pnas.0507425103Terranova et al.
malformations, the defects are specific for the ?SET allele, sug-
gesting that several domains of MLL are involved in establishing a
proper segment identity in mice.
Hox Gene Expression Defects in ?SET Mutant Embryos. Regulationof
both Hox gene expression boundaries and dosage are important for
correct specification of segment identity (30). Knockout mice have
revealed that Mll is required for maintenance rather than estab-
lishment of Hox gene expression early in embryogenesis. Down-
stream targets of Mll are activated appropriately in the absence of
Mll but require Mll for sustaining their expression between 8.5 and
of some representative Hox genes, known to be regulated by trxG
and PcG genes, in 9.5 dpc ?SET embryos (24, 31). RNA in situ
normal anterior boundaries both in mesodermal and neuroecto-
dermal tissues (Fig. 3A). This observation suggests that the SET
domain function is not required for the spatial regulation of Hox
genes expression along the anteroposterior axis. However, the
general level of expression of those genes was consistently reduced
in mutants as compared with WT mice (Fig. 3A). To confirm the
reduction of Hox genes expression, real-time quantitative RT-PCR
was used on RNA extracted from 9.5 dpc embryos and from
thymocytes (number of thymocytes were equivalent in WT and
mutant mice). In these experiments, 9.5 dpc WT and ?SET
embryos were divided at the level of the otic vesicle (indicated by
asterisks in Fig. 3A Top). The head (Hoxd4 and c8 negative) and
trunk (Hoxd4 and c8 positive) were used for RNA extraction. As
shown on Fig. 3B, Mll?SET trunk presented a 4-fold decrease of
Hoxc8 mRNA level as compared with WT embryos and a 6-fold
reduction in thymus. Similarly, expression levels of Hoxd4, Hoxa7,
and Hoxa5 were significantly reduced in thymocytes from 3-week-
old mutant mice (Fig. 3C). However, some Hox genes, including
Hoxb9 and Hoxd11, did not show any significant reduction in their
level of transcription (data not shown), suggesting a specific rather
than global alteration of Hox gene expression in ?SET mice.
Altogether, these results indicate that the SET domain may be
involved in maintaining proper Hox gene expression levels during
Reduced Histone H3K4 Monomethylation at Target Hox Loci in ?SET
Mutant Embryos. Recently it was shown that the MLL-SET domain
possesses an intrinsic H3K4 methyltransferase activity (24, 25).
Using cell transfection and biochemical assays, Milne et al. (24)
demonstrated that this methyltransferase activity is associated with
Hox gene activation and is stimulated by acetylation of Lys-9 or -14
of H3 peptides.
We first examined by immunofluorescence on WT and ?SET
embryonic fibroblasts the abundance and distribution of specific
hallmarks of euchromatin. In these experiments, the relative abun-
dance of histone modifications typical of transcriptionally active
(H3K4me2 and H3K9ac) and inactive (H3K9me2) chromatin was
compared by using a TCS Leica laser-scanning confocal micro-
scope. The microscope settings and laser power were kept constant
so that the relative abundance of each modification within 100 WT
and ?SET MEF nuclei could be directly compared and quantified
by using METAMORPH 4.0. No overall change in histone methylation
levels or distribution was detected (Fig. 7, which is published as
supporting information on the PNAS web site), and Western blot
protein extracts were prepared from WT and ?SET MEFs.
H3K4me1 (Fig. 4Cb), H3K4me2, and H3K9me2 levels were as-
sessed, and Histone H1 estimated the equivalence of protein
loading. This result is consistent with a locus specific effect of the
Hoxc9 whole-mount in situ hybridization on 9.5 dpc, somite-matched, ?SET
and WT embryos. Red arrows mark the anterior limit of neurectoderm ex-
pression, and black arrows mark the anterior limit of mesodermal expression.
The numbers of ?SET ??? embryos presenting decreased levels of Hox gene
expression are indicated. (B) Quantitative PCR analysis of Hoxc8 expression in
9.5 dpc embryos (a) and thymocytes (b). (C) Semiquantitative RT-PCR analysis
of Hox gene expression in thymocytes (Hoxd4, Hoxa7, and Hoxa5). Actin was
of RNA used to compare expression levels.
Altered expression of Hox genes in ?SET mice. (A) Hoxd4, Hoxc8, and
(A) Western blots confirming the steady levels of H3K4me2 and H3K9me2 in
the ?SET mutant MEFs. (B) Chromatin immunoprecipitation analysis in which
the abundance of histone modifications (H3K4me1, H3K4me2, H3K9ac, and
H3K9me2) were compared at Hoxd4 P1 and Hoxc8 promoters in the head and
trunk of WT (black histograms) and ?SET (gray histograms) 9.5 dpc embryos.
Values shown are the mean of five independent experiments with average
deviation. (C) Immunofluorescence (a) and Western blots (b) confirming the
steady levels of H3K4me1 in the ?SET mutant MEFs. Whole-cell extracts were
prepared from MEFs cultures, and histone H1 was used as a control to
standardize protein loading.
H3 lysine-4 methylation pattern alteration at Hox loci in ?SET mice.
Terranova et al.
April 25, 2006 ?
vol. 103 ?
no. 17 ?
?SET mutation at selective target genes and suggests that most
features of euchromatin are probably preserved in ?SET MEFs.
pare the chromatin structure at the promoter of Hox genes whose
expression was altered in ?SET thymocytes and embryos. To
evaluate chromatin changes more accurately during embryonic
development, chromatin immunoprecipitation was performed on
developing embryo tissue. In these experiments, 9.5 dpc WT and
?SET embryos were divided at the level of the otic vesicle (indi-
and trunk (Hoxd4 and c8 positive) were used for the extraction of
chromatin samples, and the relative abundance of H3K4me1,
H3K4me2, H3K4me3, H3K9ac, and H3K9me2 was compared at
the promoter regions of Hoxd4 (P1) and Hoxc8. As shown in Fig.
4B, chromatin immunoprecipitation analysis revealed a very sig-
nificant decrease in H3K4me1 in ?SET mutant trunks at both
Hoxd4 and Hoxc8 promoters (?10-fold). H3K4me2 and H3K9ac,
two other marks associated with transcriptional activation, were
mildly affected at both Hox genes (20–40% reduction), whereas
H3K4me3 levels were not affected at these Hox genes in the trunk
(data not shown). H3K9me2 levels, associated with gene silencing,
and no significant changes in histone modification was detected in
the head in the ?SET mutant embryos. Interestingly, in WT
animals, both H3K4me2 and H3K9ac levels were significantly
higher in the trunk, where Hoxd4 and c8 genes are expressed.
H3K4me1 levels were reduced but comparable, and H3K9me2
levels were low both in the head and trunk. Consistent with
transcriptional activation in the trunk, RNA Pol II was associated
with Hoxd4 and Hoxc8 promoters in the trunk but was absent from
the head region (data not shown).
Our results establish in vivo the specific histone H3 lysine-4
methylation activity of the MLL-SET domain at target loci. In
addition, we show that H3K4 mono-, di- and trimethylation are
differentially affected, suggesting a role for the SET domain of
MLL in monomethylation at lysine-4 of histone H3 in vivo at the
Hoxd4 and Hoxc8 loci during embryonic development. The chro-
matin state in the head and trunk is consistent with gene activity in
9.5 dpc embryos.
DNA Methylation Defects in ?SET Mutant Mice. DNA methylation at
of gene expression (32–34). To verify the status of DNA methyl-
ation in ?SET mutants, we performed PCR amplification of
sodium bisulfite-treated DNA isolated from embryonic fibroblasts.
The CpG-rich region of Hoxd4, in the vicinity of exon 2 (Fig. 5A)
was abnormally methylated, with 10 of 14 CpGs found to be hyper-
or hypomethylated in ?SET MEFs (compare with WT MEFs, Fig.
5A). Changes in DNA methylation pattern were confirmed by
(Fig. 5B). Similar results were consistently obtained on a different
portion of Hoxd4, on promoter P2 (Fig. 8A, which is published as
supporting information on the PNAS web site). These modifica-
tions are gene-specific rather than global because the analysis of a
in the DNA methylation pattern (Fig. 8B). In addition, immuno-
fluorescence stainings by using an antibody directed against
SET domain has a significant effect on DNA methylation at the
Hoxd4 locus. However, the finding that both DNA methylation and
demethylation alterations coexist suggests that the SET domain is
not directly responsible for these modifications. Altogether, these
epigenetic changes at Hox genes could account for decreased level
of expression, and they demonstrate that the SET domain, pre-
sumably through histone H3K4 methylation, influences DNA
We report here the generation and characterization of a previously
undescribed mutant allele of Mll, the mammalian homolog of the
Drosophila trithorax gene. The ?SET mutation deleted the two last
exons of the Mll gene, which encodes the highly conserved SET
domain. In contrast to Mll-null mutant mice, ?SET??? animals
survive embryogenesis. Three previously reported Mll alleles have
suggested that the N terminus of MLL protein is functionally
important, the phenotype being less severe as a larger segment of
the N terminus of the MLL protein is expressed (10, 28). Recently,
several studies showed that MLL can be proteolytically processed
into two fragments and that postcleavage association of the N-
terminal (p320) and C-terminal (p180) fragments confers its sta-
bility and subnuclear localization to MLL (8, 25). This process,
unaffected by the deletion of the SET domain, may confer normal
stability and subnuclear localization to the truncated MLL protein,
allowing it to achieve important SET-independent functions nec-
essary for survival.
We show that ?SET mutant mice present homeotic skeletal
defects and that the level of expression of some Hox genes is
reduced during development. On the other hand, the anterior limit
in mutant embryos. We propose that the SET domain function of
Mll is required for the maintenance of optimal levels of Mll target
gene expression levels (24). The spatial regulation of Hox genes
expression may be achieved through an Mll-SET-independent
mechanism, probably mediated by the N terminus of the protein.
Proper levels of Hox genes expression are important for specific
developmental processes (35) and could account for the homeotic
transformations detected in ?SET mutant skeleton.
It was shown previously that the MLL protein is responsible for
36). In Mll??? embryos, the pattern of Hox genes expression is
established normally and lost between embryonic days 8.5 and 9
when the MLL protein is required to maintain this pattern (11).
This development stage may be indicative of a period of epigenetic
transition at Mll target genes, leading to either silencing or main-
a H3K4 histone methyltransferase activity (24). H3K4 mono?
of the mouse Hoxd4 gene is shown. Methylation of cytosines was assessed by
A summary of methylation data from MEFs is shown where each line repre-
sents a separate clone. Methylated CpGs are represented by filled beads, and
of DNA methylation changes at Hoxd4 in ?SET embryonic cells (trunk from
DNA methylation changes at Hox loci in ?SET mice. (A) A partial map
www.pnas.org?cgi?doi?10.1073?pnas.0507425103 Terranova et al.
transcriptionally active genes (37, 38). It is therefore the presence
whereas mono?dimethylated K4 may distinguish transcriptionally
permissive chromatin (23). The SET domain of MLL is important
for H3K4 HMT activity and transcriptional activation at MLL
target loci. Recombinant MLL SET domain was shown to have
methyltransferase activity toward unmodified H3 peptides but
appeared to lack the ability to catalyze the conversion from di- to
trimethylation (24). Recently, a stable complex containing both
MLL and the histone acetyltransferase MOF was purified from
HeLa cells (26). This complex was shown to effect H3K4 methyl-
ation (mono-, di-, and tri-) and H4K16 acetylation, resulting in
transcriptional activation of MLL target Hox genes. In these
experiments, dimethylated H3K4 peptide is a better substrate than
at Hox genes. A reduction in H3K9 acetylation levels mirrors the
reduction in H3K4 dimethylation and no increase in H3K9 meth-
ylation levels could be detected. This altered histone modification
pattern at both Hoxd4 and Hoxc8 loci may be responsible for the
sustained but reduced levels of transcriptional gene expression in
the ?SET embryos. Our results favor a model in which the SET
domain has mono-H3K4 histone methyltransferase activity. A SET
domain-dependent mechanism?interaction may actively dimethyl-
ate H3K4, whereas a SET domain-independent mechanism would
mediate the conversion from di- to trimethylation via the recruit-
ment of specific enzymatic activity in the same way that the
a SET domain-independent interaction with MOF (26). However,
we cannot formally exclude that another trithorax-group protein
with a redundant function is compensating for the loss of the
Mll-SET domain and is able to di- and trimethylate H3K4.
Interestingly, appropriate chromatin modifications distinguish
the transcriptional status of Hox genes in regions where they are
active (trunk) and silent (head). Hoxd4 and Hoxc8 loci in the trunk
bear modifications associated with transcriptional activation (di-
and trimethylation of H3K4 and acetylation of H3K9), and these
features are reduced to absent in the head at the same loci. These
results are consistent with previously reported study of chromatin
states in anterior and posterior compartments during embryonic
development (39). The difference in monomethylation of lysine-4
of H3 between the head and trunk are not significant, suggesting
that H3K4me1 is not directly involved in transcriptional activation.
In addition, transcriptional expression of Hox genes is maintained
in the trunk of ?SET embryos despite reduced H3K4me1 levels.
We propose that H3K4me1 could be a mark for poised chromatin,
which is established at Hoxd4 and Hoxc8 loci through a MLL-
the nonexpressing, anterior region of the embryo. In this context,
the SET domain of MLL would be involved in the maintenance of
establishment?maintenance of H3K4me1 marks), whereas other
factors recruited within the MLL complex are required to generate
further chromatin modifications and proper transcriptional activa-
tion of target loci (9, 26).
An increasing number of studies suggest that DNA methylation
could be regulated, at least partly, by H3K9 modifications, possibly
methylation (40–43). Milne et al. (24) have shown that the Hoxc-8
locus is extensively DNA methylated in Mll??? cells, and the
bisulfite sequencing data we present suggest that in the absence of
Mll-SET domain, reduced levels of H3K4me1 are associated with
abnormal DNA methylation patterns. The loss of histone H3K4
monomethylation in Mll?SET could affect the recruitment or
exclusion of proteins involved in the regulation of DNA methyl-
ation. It was demonstrated that the PcG SET domain containing
protein EZH2 interacts with DNA methyltransferases and that
(44). In the same way, MLL could recruit DNA methyltransferases
at target genes; Alternatively, the N terminus of MLL contains a
region with sequence similarity to the DNA methyltransferase
DNMT1, which binds in vitro to unmethylated CpG-rich DNA
(45–47), suggesting a more direct role of MLL in the regulation of
DNA methylation at target genes. The biochemical mechanism
mice remains an open question.
Taken together, our data show that the loss of the MLL SET
at the promoter of several Hox genes in mutant mice and that
expression of these same Hox genes is down-regulated. Viability
despite the altered genomic methylation and reduction of methyl-
an ideal model system for the study of epigenetic regulation during
developmental processes and tumorigenesis.
Materials and Methods
Generation of ?SET Mutant Mice. Targeting vector DNA (Fig. 1A;
see also Supporting Materials and Methods, which is published as
supporting information on the PNAS web site, for a detailed
description of the Mll-SET targeting vector generation) was elec-
troporated in 3 ? 107129?ola ES cells cultured in DMEM
(Invitrogen) supplemented with 15% FCS?1,000 units/ml of LIF
and antibiotics. The transfected cells were selected with 0.3 mg?ml
G418, and resistant colonies were screened for homologous re-
(Mll?SET???neo) were transiently transfected with a vector
encoding the CRE recombinase (pMC-CRE) and expanded in the
absence of G418 selection for 7 days. Individual clones were
screened by PCR and Southern blot for the deletion of the
neomycin resistance gene (Mll?SET??? ES cells). Three inde-
3.5 blastocysts and transferred into the uteri of pseudopregnant
mice. Resulting chimeric mice were mated with BALB?c mice, and
germ-line transmission was confirmed by PCR and Southern blot
analysis of tail DNA.
Establishment of MEFs. MEFs were obtained from WT,
Mll?SET??? and ??? mutant embryos at developmental stage
FCS and antibiotics. Cells were frozen as stocks at the second
passage and used for the subsequent studies at the third passage.
RT-PCR Analysis. Total RNA was extracted with RNAzol B, and
cDNA synthesis was performed by using oligo (dT)12–18(Invitro-
gen). PCR amplification was performed by using Standard
TaqDNA polymerase (Qiagen, Valencia, CA) and the following
cycles: (94°C for 30 s, Tmfor 30 s, and 72°C for 60 s). For Analysis
of Hox gene expression, OneStep RT-PCR kit (Qiagen) was used
according to manufacturer’s instructions. The primers used for
PCR amplification are available in Table 1, which is published as
supporting information on the PNAS web site.
Coimmunoprecipitation and Western Blot Analysis. Indirect immu-
nofluorescence labeling was performed by using the following
primary antibodies: rabbit anti-MLL (kind gift from A. G. Fisher,
Medical Research Council, London), rabbit anti-H3K9ac, anti-
H3K4me2, and anti-H3K9me2 (Upstate Biotechnology). Second-
ary antibodies were purchased from Molecular Probes. For West-
ern blot and coimmunoprecipitation analysis, the primary
antibodies used were as follows: anti-MLLN[Abcam, Inc. (Cam-
bridge, U.K.) ab17959], anti-MLLCand anti-Menin (Bethyl Lab-
oratories, A300–086A; A300–374A; A300–105A), anti-H3K4me2,
anti-H3K9me2, anti-H1 (Upstate Biotechnology), and WDR5
(kind gift of W. Herr, Cold Spring Harbor Laboratory, Plainview,
NY). Whole-cell extracts were prepared by direct lysis of cells in
SDS-loading buffer. After electrophoresis, blots were probed with
Terranova et al.
April 25, 2006 ?
vol. 103 ?
no. 17 ?
primary antibodies and visualized by using an Amersham Pharma-
using Nuclear Complex Co-IP Kit (active motif).
Immunostaining and Microscopy. Immunostaining samples were
fixed for 20 min in 2% paraformaldehyde, permeabilized in 0.4%
Triton, and incubated 30 min in a blocking solution (2.5% BSA?
0.05% Tween 20 in PBS), incubated in primary antibody (1.5 h at
room temperature), washed, and incubated in fluorochrome-
labeled secondary antibody (45 min at room temperature). Nuclei
were counterstained by using DAPI (1 ?g?ml), and slides were
mounted in Vectashield before analysis by using a TCS Leica
laser-scanning confocal microscope and quantification by using
Whole-Mount RNA in Situ Hybridization. Whole-mountinsituhybrid-
ization was performed as described in refs. 48 and 49. See Sup-
porting Materials and Methods for a detailed protocol.
Skeletal Analysis. Dissected animals were fixed 12 h in 100%
rinsed for 1 h in 95% ethanol and incubated for 24 h in a 2% KOH
solution for 12–24 h. Stained skeletons were stored in a 20%
glycerol?1% KOH solution and preserved in a glycerol?ethanol
Chromatin Immunoprecipitation on Embryos and Real-Time PCR. The
9.5 dpc embryos were carefully dissected into anterior (head) and
posterior (trunk) regions. A section was made by using fine forceps
Chromatin immunoprecipitation experiments were performed ac-
lysis buffer for 20 min on ice to release the chromatin. After
protein A-agarose beads for 1 h and incubated overnight with
specific antibodies (5 ?g per experiment). Chromatin was eluted
from the beads, and cross-links were reversed at 65°C for 6 h.
Phenol?chloroform-extracted DNA was ethanol precipitated and
used as a template for real-time PCR (in triplicate) by using the
SYBR Green Taq ReadyMix kit for quantitative PCR (Sigma) and
the 5700 detection system from Applied Biosystems. Data are
expressed as a percentage of the input.
Bisulphite Sequencing. Genomic DNAs from embryonic WT and
?SET embryonic fibroblasts were modified by sodium bisulfite
treatment as described in ref. 50. The primers used for PCR
denaturation at 94°C, 45 sec annealing at 54°C, 45 sec of extension
at 72°C, with final extension at 72°C for 10 min.
We thank J. Hess and P. Ernst for critical reading of the manuscript and
for helpful discussions. This work was supported by grants from Centre
National de la Recherche Scientifique, Association pour la Recherche
sur le Cancer, Institut National du Cancer, and Fondation de France.
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