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First published online 15 March 2006
doi: 10.1242/dev.02302
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1 Genomics, BioInnovationsZentrum, Dresden University of Technology, Am Tatzberg
47, Dresden 01307, Germany.
2 Department of Molecular Biology and Computational Biology Unit at BCCS,
University of Bergen, HiB, Bergen N5020, Norway.
3 National Institute for Medical Research, The Ridgeway Mill Hill, London NW7
1AA, UK.
Author for correspondence (e-mail:
stewart{at}biotec.tu-dresden.de)
Accepted 7 February 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Epigenetics, Histone methylation, SET domain
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Egger et al., 2004). Evidence
that transcriptionally active states of chromatin can also be epigenetically
maintained is now accumulating (Roguev et
al., 2001; Noma and Grewal,
2002; Jaenisch and Bird,
2003; Wysocka et al.,
2005). This paradigm shift began with the realization that DNA
methylation is secondary to a more universal mechanism for epigenetic
silencing based on methylation of histone 3 lysine 9 (H3 K9) for constitutive
heterochromatin (Rea et al.,
2000; Lachner et al.,
2001; Bannister et al.,
2001) and on H3 K27 for facultative heterochromatin
(Cao et al., 2002;
Czermin et al., 2002;
Kuzmichev et al., 2002;
Muller et al., 2002).
Active chromatin states may also be epigenetically maintained because
alternative histone lysine methylations, mainly at H3 K4, characterize active
chromatin and preclude the lysine methylations that characterize inactive
chromatin (Noma et al., 2001;
Litt et al., 2001). The
functional opposition of these two classes of histone lysine methylation was
further supported by linkage to the antagonism between Polycomb- and
trithorax-Group (PcG and trxG) action
(Brock and Fisher, 2005;
Ringrose and Paro, 2004). The
association of trxG action with H3 K4 methylation
(Roguev et al., 2001;
Krogan et al., 2002;
Milne et al., 2002;
Nagy et al., 2002;
Nakamura et al., 2002;
Beisel et al., 2002) and PcG
action with H3 K27 methylation (Cao et
al., 2002; Czermin et al.,
2002; Kuzmichev et al.,
2002; Muller et al.,
2002) provides a molecular explanation for this antagonism.
The silencing methylations at both H3 K9 and H3 K27 are epigenetically maintained via positive-feedback loops. The relevant methyltransferases [SUV39 and E(Z), respectively] associate with a protein (HP1 and Polycomb, respectively) that recognizes the methylated epitope. The enzyme is thereby associated with the chromatin that it has methylated, to methylate it further and propagate the silent state.
Recent evidence indicates that maintenance of active chromatin occurs in
the same way, because a constituent protein of all H3 K4 methyltransferase
complexes, WDR5/SWD3 (Roguev et al.,
2001; Krogan et al.,
2002; Nagy et al.,
2002; Wysocka et al.,
2003; Hughes et al.,
2004; Yokoyama et al.,
2004; Dou et al.,
2005; Lee and Skalnik,
2005), binds to methylated H3 K4
(Wysocka et al., 2005).
These observations support the polarization model, which is based on
opposing positive feedback loops (Jaenisch
and Bird, 2003). Both active and heterochromatic states involve
several positive feedback loops that reinforce the local status quo. Hence,
each state has an implicit epigenetic status that adds stability and reduces
the chances of inadvertent transitions to the other state.
The polarization model poses questions that can now be addressed. Do
uncommitted neutral chromatin states exist? How are active and silenced states
specified? Are transitions between active and heterochromatic states used to
regulate gene expression and if so, how? These questions are particularly
relevant to the study of development because the transition from the
totipotency of the zygote to differentiated states in the adult is reflected
by changes in the epigenetic status of chromatin
(Jaenisch and Bird, 2003).
S. pombe has only one enzyme for each of the active and
heterochromatic lysine methylation sites
(Roguev et al., 2003;
Sanders et al., 2004;
Cam et al., 2005). To date, no
evidence for specific gene regulation by histone methylation in either S.
pombe or S. cerevisiae has been observed. Higher eukaryotes have
several enzymes for each site of lysine methylation. SET domain sequence
alignments suggest that the mouse genome encodes at least six H3 K4
methyltransferases and at least seven H3 K9 methyltransferases
(Fig. 1). Because gene-specific
regulation by epigenesis during development has been anticipated but not yet
defined, we decided to look among these SET domain factors in mouse
development.
|
; Hess et al.,
1997; Yu et al.,
1998; Hanson et al.,
1999). This mutant allele expresses the first 458 amino acids of
Mll, including the conserved AT hooks region, both protein phosphatase 2A
interaction sites and SNL1 (speckled nuclear localization signal) fused to the
tetramerizing protein, ß-galactosidase (see Fig. S1 in the supplementary
material). Heterozygotes showed a hypofertile and mildly homeotic phenotype.
By contrast, truncation at exon 5, which permits expression of the above
regions plus the MT (CxxC methyltransferase homology) domain, was lethal at
the two-cell stage in homozygotes, with a mildly homeotic heterozygous
phenotype without reported hypofertility
(Ayton et al., 2001).
Replacement of exons 12-14 truncated the transcript in the middle of the
conserved PHD finger region and caused embryonic lethality around E13.5,
without a reported heterozygous phenotype
(Yagi et al., 1998). It is not
clear whether any of these mutations resulted in a complete loss of function.
However, it is clear that Mll is required for definitive hematopoiesis
(Ernst et al., 2004a) and
sustains expression of certain Hox genes, particularly Hoxa7, Hoxa9
and Hoxc8 (Yu et al.,
1998; Hanson et al.,
1999; Ernst et al.,
2004b).
Mammals have a second trithorax homologue, Mll2, as well
as two other similar genes Mll3 and Mll4, and two more
genes, Set1a and Set1b, which contain very similar SET
domains (Fig. 1). Mll
and Mll2 are closely related proteins (see Fig. S1 in the
supplementary material), having arisen from a duplication that includes the
upstream [Plzf (Zbtb16 – Mouse Genome Informatics) and
Plzf2] and downstream (U2af1 and U2af1l4) genes
(FitzGerald and Diaz, 1999).
The functional relationship between Mll and Mll2 is not
known. Both proteins are very large, share the same architecture
(Fig. 2A; see Fig. S1 in the
supplementary material) and are expressed from CpG islands in a nearly, but
not completely, ubiquitous manner, including in ES cells and all major
tissues, as determined by northern analysis
(FitzGerald and Diaz, 1999)
(data not shown). Partial characterizations of associated proteins indicate
that they both reside in similar complexes
(Hughes et al., 2004;
Yokoyama et al., 2004;
Dou et al., 2005), as do
Set1a/b (Wysocka et al., 2003;
Lee and Skalnik, 2005).
Because Mll and Mll2 are orthologous, a comparison of their functional roles in mouse development represents a good way to look for evidence of epigenetic regulation in specific gene expression. Therefore we created a multipurpose allele for Mll2. By conversion of the allele from one state to another, we established the null phenotype in mouse development and in ES cells.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) into
intron 1 by recombineering (Angrand et al.,
1999). The cassette is flanked on the 3' end by a loxP site
and a second loxP site replaced an ApaL1 site in intron 2. Two correctly
targeted E14 ES cell clones were used to generate chimeric mice that were
subsequently bred to C57BL/6 mice. Offspring from
Mll2+/– crosses and individual embryos were
genotyped by PCR with the following primers that produced a 1.7 kb product
from the targeted allele: primer 34,
5'-GGGCTGACCGCTTCCTCGTGCTTTAC-3'; primer 36,
5'-GGAGAACAGTTGTGGGGAGATGGGTC-3'. Homozygote Mll2 embryos
were distinguished from heterozygotes with the following primers that produced
a 914 bp product from the wild-type allele and no product from the targeted
allele: primer 31, 5'-CTCTCTGGTTCTAAGGTAGAGTG-3' and primer 36. To
generate Mll2 F/+ mice, we crossed Mll2+/–
mice to the hATPC-Flpe line (Rodriguez et
al., 2000). Subsequent crossing to the PGK-Crem line
(Lallemand et al., 1998)
produced Mll2 FC/+ mice. Mll2 F/+ and Mll2 FC/+
mice were genotyped by PCR with the following primers: primer 145,
5'-CGGAGGAAGAGAGCAGTGACG-3'; primer 147,
5'-GGACAGGAGTCACATCTGCTAGG-3'. The products were 1554 bp, 1448 bp
and 737 bp for the Mll2F, Mll2+ and Mll2FC alleles,
respectively. For double-targeted ES cells, the targeting construct was
changed from a lacZ-neo fusion to a lacZ-hygro (hygromycin
resistance) fusion by recombineering
(Testa et al., 2004).
Double-targeted cells were selected for G418 (350 µg/ml) and hygromycin
(180 µg/ml) resistance.
Expression analysis by RT-PCR and western blotting
Total RNA from individual embryos or cells was extracted using TriReagent
(Sigma) according to the manufacturer's instructions. First-strand cDNA was
synthesized from 1 µg of total RNA using M-MLV reverse transcriptase
(Promega). The primer pairs that produced a 1228 bp product were: mll2se,
5'-GCAGCAGAGGAGAACCAGACC-3'; mll2as,
5'-GGAGGAACCTCCCCTGCCATC. LacZse (5'-AAGTTCAGATGTGCGGCGAGTT) and
LacZas (5'-GGCTTCATCCACCACATACAGG) produced a 511 bp product. Actin
primers were described previously (Testa
et al., 2004). Q-PCR was performed using a Stratagene MX4000
according to the manufacturer's instructions. Cells were homogenized in buffer
E (20 mM HEPES, 350 mM NaCl, 10% Glycerol, 0.1% Tween, 1 µg/ml pepstatin A,
0.5 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM PMSF) and snap-frozen three
times for protein extraction. Protein extracts were fractionated by 5%
SDS-PAGE, transferred to nitrocellulose membranes and revealed with a rabbit
IgG polyclonal antibody raised against the Mll2 amino acids between SNL1 and
SNL2 (see Fig. S1 in the supplementary material), and a polyclonal CBP
antibody (Santa Cruz). The primer pairs for Q-PCR amplification of Hox cluster
genes were: Hoxa2, TTCCCAGTTTCGCCTTTAACC and CAGTTCTGGCCCATTGTTGAC; Hoxa3,
CCTTTCCCTTTTCTCCTCTGC and ACTGACAGCCTTTCCAGCAAC; Hoxa5, TATAGACGCACAAACGACCGC
and CATTTGGATAGCGACCGCA; Hoxb2, CCCGCTGTCTTGGAGACATTT and
TTTTGGCTCCCTGGTCTCTGA; Hoxb4, CGGAAACAGGAAAACGAGTCA and TGTGAATACTCCTCGCACGGA;
Hoxb5, CCCCAAGTTGCCAGTGTTTCT and AACCTCAACTGCTGCCCCTTA; Pbx1,
GCGCCGGGAGCCCATTTCTGC and GGTCCCTCCGGCCCCATCCTG.
Whole mount X-gal staining, immunohistochemistry and TUNEL assay
Embryos were dissected in PBS containing 0.4% BSA. Fixation was carried out
at 4°C in 4% PFA for 20 minutes for E7.5 embryos and 1 hour for later
stages. Embryos were washed three times for 5 minutes in PBS and stained
overnight at 37°C in PBS containing 0.8 mg/ml X-Gal, 0.2 mg/ml sodium
deoxycholate, 5 mM K3FE(CN)6, 5 mM
K4Fe(CN)6, 2 mM MgCl2 and 0.02% NP-40.
Stained embryos were washed twice in PBS and fixed for 1 hour at room
temperature in 4% PFA. Embryos were fixed in 4% paraformaldehyde, dehydrated,
embedded in paraffin and sectioned at 6 µm. Sections were processed for
immunohistochemistry using the monoclonal Ki67 antigen antibody (Novo Castra)
and a universal detection kit (Novostain) according to the manufacturer's
instructions. Apoptotic cells on paraffin-embedded sections of three mutant
embryos and three heterozygous littermates were detected by using an in situ
Cell Death Detection Kit (Roche). Total cells numbers were determined on
adjacent sections by DAPI staining. Embryos were collected and fixed in 4% PFA
in PBS for 1 hour on ice. After three washes in PBS plus 0.1% Tween 20,
embryos were stored at –20°C in methanol. Mutant embryos were
identified by yolk-sac PCR. Whole-mount in situ hybridization was performed as
described (Wilkinson and Nieto,
1993). An Mll2 probe was cloned with the following
primers: 5-TAGAAGCAGCAGAGGAGAACC-3' and
5'-GGAGGAACCTCCCCTGCCATC-3'.
Chimera analysis and ES cell differentiation
Blastocysts were isolated at embryonic day E4.5. After injection of 20-25
targeted ES cells per blastocyst, 14 blastocysts were reintroduced into the
uterus of pseudopregnant foster mothers. Injected
Mll2+/– and Mll2–/–
cells were detected in embryos by ß-galactosidase staining as whole mount
(E8.5 and E9.5) or in sections (E10.5, E18.5). ES cells were differentiated on
mass by plating onto bacterial plates in the absence of LIF or retinoic
acid.
Database searches and classification of SET domains
The murine SET domain proteins were identified by searching the murine
proteome (ENSEMBL release 30.33f) using a set of 11 profile HMMs
(Eddy, 1998) based on a
classification of the human SET domain proteins. All proteins included had
E-values lower than 10–9. Each murine SET protein was
classified by the highest scoring profile. The SET domain profile HMMs are
available at
http://www.uib.no/aasland/chrab/.
In brief, they were obtained as follows: initially, a non-redundant set of
human SET proteins was obtained with SMART
(http://smart.embl-heidelberg.de/).
After alignment and clustering the SET domains using Clustal X (Thomspon et
al., 1997), nine groups were identified. Clusters of related sequences for
each group were obtained by Blastp searches in all of SwissProt/Trembl. For
each group, six to eight of the closest sequences were used to build profile
HMMs that were subsequently used in iterative searches in SwissProt/Trembl.
During this process, the groups SET7/9 and SET8 were separated from the larger
Suv3-9 group, resulting in a total of 11 groups and corresponding profile
HMMs. An alignment of SET domain sequences from mouse, human, zebrafish and
Drosophila corresponding to the 11 groups described above was
obtained by progressive profile-profile alignments using Clustal_X guided by a
structure mask based on information from the structures of SET7/9, DIM-5, Clr4
and LSMT (pdb: 1O9S, 1PEG, 1MVH, 1P0Y). The mouse subset of this alignment
(available at
http://www.uib.no/aasland/chrab/)
was used to generate a phylogenetic tree using the MrBayes software
(Huelsenbeck and Ronquist,
2001).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). An FRT
flanked, genetrap-type, stop cassette
(Friedrich and Soriano, 1991)
was inserted into the first intron. As determined by RT-PCR
(Fig. 2B), western blotting
using an Mll2 specific antibody (Fig.
2C), in situ hybridization
(Fig. 2D) and
ß-galactosidase expression (Fig.
3), this cassette captures and truncates the transcript before the
second exon. Because the Mll2 initiating methionine is in the first exon, a
transcript encoding the first 121 amino acids of Mll2 fused to 40 frameshifted
amino acids encoded by the second exon of the engrailed 2 gene could be
expressed. The retained first 121 amino acids of Mll2 do not appear to contain
any conserved residues or motifs (see Fig. S1 in the supplementary
material). Removal of one or both FRT cassettes either in homozygously mutated ES cells or in the germline fully restored wild-type function (data not shown). The targeted allele also included loxP sites flanking the small 73 bp second exon (Fig. 2A). Removal of exon 2 by Cre recombination invokes a frameshift in the mRNA. Embryos homozygous for deletions of both the FRT cassette and exon 2 displayed a phenotype indistinguishable from embryos homozygous for the targeted allele (Fig. 2E, Table 1). This phenotype was also observed in embryos homozygous for the removal of the second exon while leaving the FRT cassette in the gene (data not shown). Therefore we conclude that the mutant alleles are most probably nulls. For clarity we term the targeted allele `-', the FLP recombined allele `F', and the FLP and Cre recombined allele `FC' (Fig. 2A). As determined by ß-galactosidase expression, Mll2 is expressed widely from both alleles, except in the extra-embryonic tissues (Fig. 3).
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; Nagy and Rossant,
2001). Therefore we used Mll2–/–
ES cells in chimera experiments. The Mll2–/–
cells were made by exchanging the selectable gene in the targeting construct
from neomycin resistance to hygromycin resistance by recombineering
(Testa et al., 2004), followed
by targeting in the Mll2+/– ES cells and double
selection for G418 and hygromycin resistance. Targeting of the second allele
occurred at a frequency compatible with the first targeting frequency (3%
versus 8%, respectively), hence Mll2 is not required for ES cell survival.
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),
was used to examine this issue more closely
(Fig. 6). Up to about the
eight-somite stage, which is E9.5 for the mutant embryos
(Table 2), Mox1
expression was initiated normally. Thereafter it was lost from the
anteriormost somites (Fig.
6B,C) before decaying entirely
(Fig. 6D). Loss of
Mox1 expression appears to precede the disappearance of the somites
for two reasons. First, the observed generalized apoptosis does not explain
why Mox1 expression is lost first from the anteriormost somites.
Second, inspection of sections showed that Mox1 expression is lost
even though some somitic cells remain (Fig.
6F). Later, cells in this region show intense apoptosis
(Fig. 6G). By E9.5, Mll2–/– embryos are moribund, so the loss of Mox1 expression could be due to a generalized catastrophe rather than to a cell type-specific effect. Evidence in favor of cell type specificity was found because of the persistence of Wnt1 expression along the neural tube (Fig. 7B). As can be seen from this staining, the neural tube becomes highly buckled after the disappearance of the somites.
Trithorax in flies is a known regulator of HOM-C expression, and Mll regulates at least several Hox genes in mouse. Therefore, we looked at Hox complex gene expression. The developmental retardation and embryonic lethality prevented any meaningful analysis of middle to late Hox complex genes. However, a collapse of expression of Hoxb1 occurred sufficiently early in the mutant embryos to permit some confidence (Fig. 8). The correct expression domains of Hoxb1 were established in mutant embryos; however, expression decayed after establishment. Notably, expression decayed at the same time in all three main expression areas. By contrast, expression of Otx2, brachyury (Fig. 7C-E) and Hoxa1 (not shown) appeared normal.
Using Mll–/– ES cells in embryoid body
differentiation experiments, Ernst et al.
(Ernst et al., 2004b) showed
that the induction of several Hoxa genes was severely impaired; however, Hoxb
gene induction was not. Therefore, we performed similar experiments with
Mll2–/– ES cells, using parental E14 cells and
the doubly targeted Mll2–/– cells rescued by
FLPe transfection to restore Mll2 expression, as controls
(Fig. 8). Loss of Mll2 had no
significant effect on expression of several Hoxa genes; however it did have a
strong effect on expression of Hoxb2 and Hoxb5. These data
strengthen the conclusion that Mll and Mll2 regulate different target
genes.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Czermin et al., 2002;
Kuzmichev et al., 2002;
Muller et al., 2002;
Levine et al., 2004). Whether
the trxG has biochemical coherence is not clear. Based on finding a linkage
between the yeast homolog of trxG member Ash2 and the first
identified H3 K4 methyltransferase Set1, we proposed that a part of trxG
action is linked to histone lysine methylation at H3 K4
(Roguev et al., 2001). Because
active chromatin is characterized by methylation at H3 K4, and inactive
chromatin by methylations at H3 K9 and/or H3 K27, the opposition between PcG
and trxG lies, in part, at the level of histone methylation.
|
According to several criteria, the Mll2 alleles studied here show
complete loss of function and we report the following observations. Mll2 is
expressed in ES cells but is not required for ES cell viability.
Mll2–/– ES cells or embryos show no detectable
decrease in global H3 K4 dimethylation level, indicating that Mll2 is not a
major source of this modification (data not shown). The first manifestation of
loss of Mll2 in development is widespread growth retardation apparent by E7.5.
The growth retarded embryos display widespread apoptosis and an increasing
delay of developmental progress. By E9.5 the developmental delay is 
1
day. At this time, the first evidence for cell type specificity, involving
specific loss of expression of Mox1 and Hoxb1, was
observed.
Previous studies with H3 K4 methyltransferases in development have focused
upon gene-specific regulation, including regulation of HOM-C and Hox complex
genes, as well as other transcription factors such as the ecdysone receptor
(Breen, 1999;
Yu et al., 1998;
Sedkov et al., 2003). Although
we uncovered evidence for gene specificity, the loss of expression of
Hoxb1 and Mox1 occurred well after other, widespread,
cell-autonomous aspects of the phenotype. Growth retardation, apoptosis and
developmental retardation all preceded noticeable cell type-specific effects.
These defects were not due to inadequate function of extra-embryonic cells.
Because both Hoxb1 and Mox1 expression patterns are
established properly in the absence of Mll2, and then decay shortly before
death, it is possible that the collapse of expression is due to secondary
effects in the dying embryo. However, we found that expression of
Wnt1 in cells neighboring those affected by the loss of Mll2 is
maintained well after the loss of Mox1 expression. This observation
lends some reason to conclude that Mll2 is a specific maintenance factor for
Mox1 expression. It is also concordant with the existing propositions
regarding its homolog in flies, Trithorax, and sister, Mll, which are both
believed to act as maintenance factors for specific gene expression
(Sedkov et al., 1994;
Yu et al., 1998;
Klymenko and Muller,
2004).
The roles for H3 K4 methyltransferases in gene expression remain enigmatic.
In yeast, there is no evidence so far that the H3 K4 methyltransferase Set1
regulates specific gene expression
(Santos-Rosa et al., 2002).
Rather, Set1 and H3 K4 methylation play general roles in the maintenance of
chromatin status, which includes an association with transcriptional activity
(Ng et al., 2003;
Krogan et al., 2003) and an
opposition to H3 K9 methylation (Noma and
Grewal, 2002). In development, the action of H3 K4
methyltransferases as maintenance factors is related to the opposition of PcG
repression. That is, specific genes require trxG factors to maintain
expression because otherwise PcG action will extinguish expression
(Klymenko and Muller, 2004).
This opposition may be due to the control of opposing nucleosomal methylations
at H3 K4 and H3 K27. However, Trithorax and homologs appear to be
transcriptional co-factors, both biochemically, because of associations with
CBP (Ernst et al., 2001;
Petruk et al., 2001) and the
elongating RNAP II (Ng et al.,
2003; Krogan et al.,
2003; Smith et al.,
2004; Guenther et al.,
2005), and functionally (Milne
et al., 2002; Sedkov et al.,
2003; Smith et al.,
2004; Milne et al.,
2005). Because Trithorax and homologs are invariably large
multi-domain proteins, it is likely that they act in both roles: as epigenetic
factors to maintain active chromatin and as transcriptional co-factors that
interact with the transcriptional machinery.
The Mll2–/– phenotype displays
characteristics consistent with a general role implicit to all cells of the
embryo, as well as gene-specific regulation in some cell types. The general
role becomes evident after gastrulation and relates to widespread growth
retardation, which is probably due to elevated apoptosis. If so, Mll2 may
regulate an apoptotic component. Alternatively, increased apoptosis may be a
consequence of complications caused by disorganized gene expression resulting
from the loss of Mll2. If so, this may reflect a general role for
Mll2 in gene expression, such as an association with RNA polymerase, as has
been suggested for Mll (Guenther et al.,
2005).
|
) and Yagi
(Yagi et al., 1998) alleles
share specific homozygous phenotypic features that are distinct from those
reported here for Mll2. When homozygous, neither of those alleles
provokes obvious growth and developmental retardation, and somitic development
appears to proceed normally at least until E10.5. Thereafter, apoptosis in the
somites, but not the neural tube, was observed
(Yu et al., 1998). Similarly
we observed apoptosis confined to the somites
(Fig. 6), but at an earlier
developmental stage (Table 2).
This represents another difference between the developmental roles of these
sister genes. Furthermore, we suggest that the Yagi allele is the null
phenotype because of the lack of polyadenylation, nuclear export of truncated
Mll transcripts and protein expression. By contrast, it is known that
a conserved part of Mll is expressed as a fusion protein with
ß-galactosidase in the more aggressive Yu allele, which also shows a
heterozygous phenotype as expected for a dominant-negative effect. If this
suggestion is correct, then the differences between the Mll and
Mll2 loss-of-function phenotypes in mouse development are
considerable. The conclusion that Mll and Mll2 regulate different processes in
development, including differences in Hox gene regulation, is strengthened by
the observation that Mll2–/– ES cells show
severely impaired Hoxb gene inductions
(Fig. 9), whereas
Mll–/– ES cells do not
(Ernst et al., 2004b). Whether
similar conclusions regarding specificities will also apply to the other four
members of the H3 K4 methyltransferase group, or indeed to the other classes
of epigenetic maintenance or silencing factors
(Fig. 1), remains to be
determined. Our findings indicate that mammalian development not only relies
on networks of transcriptional regulation mediated by sequence specific
transcription factors, but also on networks of epigenetic specificities. |
ACKNOWLEDGMENTS |
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|
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/8/1423/DC1
* Present address: Sydney IVF, 4 O'Connell Street, Sydney 2000, Australia 
Present address: Samuel Lunenfeld Research Institute, 600 University
Avenue, Toronto, Ontario M5G 1X5, Canada
Present address: German Cancer Research Centre, Im Neuenheimer Feld 280,
Heidelberg, Germany
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