1 Department of Molecular, Cell, and Developmental Biology, University of
California, Santa Cruz, Santa Cruz, CA 95064, USA
2 Cancer Research UK, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK
Author for correspondence (e-mail:
tamkun{at}biology.ucsc.edu)
Accepted 26 January 2005
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SUMMARY |
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Key words: Kismet, BRM complex, Polycomb, Trithorax, RNA Polymerase II, Chromatin, Transcription
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Introduction |
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Although the molecular mechanisms used to maintain heritable states of Hox
transcription remain relatively mysterious, a growing body of evidence
suggests that they involve changes in chromatin structure
(Francis and Kingston, 2001;
Simon and Tamkun, 2002
). PcG
proteins silence the transcription of their target genes via cis-regulatory
elements known as Polycomb-response elements (PREs). Two complexes of
Drosophila PcG proteins have been identified: PRC1 (which contains PC
and other PcG proteins) and the ESC/E(Z) complex
(Levine et al., 2004
). The
ESC/E(Z) complex methylates lysine 27 of histone H3; this modification is
required for PcG silencing in vivo and may help recruit PRC1 or stabilize the
binding of PRC1 to PREs (Cao and Zhang,
2004
).
How does PRC1 silence transcription once targeted to a PRE? One popular
model is that PRC1 packages chromatin into a configuration that is
inaccessible to transcription factors or the general transcription machinery
(Francis and Kingston, 2001;
Ringrose and Paro, 2004
).
However, recent studies have indicated that PRC1 may repress transcription via
more selective mechanisms. For example, PRC1 may block transcription via
direct interactions with components of the basal transcription machinery, as
evidenced by the presence of TFIID subunits in PRC1
(Breiling et al., 2001
;
Saurin et al., 2001
).
Furthermore, DNA-binding activators TBP and Pol II are present at promoters
repressed by PRC1, suggesting that PRC1 selectively interferes with events
downstream of Pol II recruitment (Breiling
et al., 2001
; Dellino et al.,
2004
).
Other potential targets of PRC1 include the members of the trxG of
activators. Mutations in many trxG genes suppress Pc mutations and
cause homeotic transformations because of the failure to maintain active
states of Hox transcription (Simon and
Tamkun, 2002). The majority of trxG proteins characterized to date
have been implicated in either chromatin remodeling or the covalent
modification of nucleosomal histones. For example, the trxG genes
trithorax (trx) and absent, small or homeotic 1
(ash1) encode SET domain proteins with histone methyltransferase
activity (Beisel et al., 2002
;
Milne et al., 2002
;
Nakamura et al., 2002
;
Smith et al., 2004
); these
histone-modifying enzymes counteract silencing by PcG proteins in vivo
(Klymenko and Müller,
2004
). Another trxG gene, brahma (brm), encodes
a member of the SWI2/SNF2 family of ATPases
(Tamkun et al., 1992
). The BRM
ATPase, together with the trxG proteins Moira (MOR) and OSA
(Collins et al., 1999
;
Crosby et al., 1999
), are
subunits of a 2 MDa chromatin-remodeling complex that is highly related to the
yeast SWI/SNF and RSC, and the human BAF and PBAF complexes
(Mohrmann et al., 2004
;
Papoulas et al., 1998
). By
altering the structure or positioning of nucleosomes, these complexes
facilitate the binding of transcription factors and other regulatory proteins
to chromatin (Flaus and Owen-Hughes,
2004
). The BRM complex plays a global role in transcription by Pol
II (Armstrong et al., 2002
) and
is therefore an excellent candidate for a target of PRC1 repression.
Consistent with this possibility, PRC1 strongly inhibits chromatin remodeling
by human SWI/SNF in vitro (Francis et al.,
2001
; Levine et al.,
2002
; Shao et al.,
1999
).
Like brm, mor and osa, the trxG gene kismet
(kis) was identified in a screen for extragenic suppressors of
Pc, suggesting that it acts antagonistically to Pc to
activate homeotic gene expression
(Kennison and Tamkun, 1988).
Loss of maternal kis function causes segmentation defects identical
to those caused by mutations in the pair-rule gene even-skipped
(eve) (Daubresse et al.,
1999
). Loss of zygotic kis function causes homeotic
transformations, including the transformation of first leg to second leg and
the fifth abdominal segment to a more anterior identity
(Daubresse et al., 1999
).
These phenotypes are identical to those resulting from the decreased
transcription of the Hox genes Sex combs reduced (Scr) and
Abdominal-B (Abd-B). Thus, kis plays a dual role
during development; maternal kis activity is required for embryonic
segmentation and zygotic kis activity is required for the control of
cell fate. Mutations in kis have also been recovered in screens for
genes involved in the Notch and Ras signaling pathways, suggesting that its
function is not limited to segmentation and the determination of body segment
identities (Go and Artavanis-Tsakonas,
1998
; Therrien et al.,
2000
; Verheyen et al.,
1996
).
kis encodes two major nuclear proteins with molecular weights of
574 kDa (KIS-L) and 225 kDa (KIS-S) (Fig.
1) (Daubresse et al.,
1999; Therrien et al.,
2000
). KIS-L contains an ATPase domain that is highly related to
those found in chromatin-remodeling factors, suggesting that KIS-L, like BRM,
catalyzes ATP-dependent alterations in chromatin structure. KIS-L also
contains two chromodomains and a BRK domain. Chromodomains mediate
protein-protein or protein-RNA interactions, and are found in members of the
CHD subfamily of ATPases (including Mi-2 and CHD1) and other proteins that
interact with chromatin (Brehm et al.,
2004
). Some chromodomains are involved in the selective
recognition of methylated histone tails
(Brehm et al., 2004
). The BRK
domain is a 41 amino acid segment of unknown function that is conserved in BRM
and its human homologs BRG1 and HBRM
(Daubresse et al., 1999
). The
KIS-L protein lacks PHD fingers, a domain characteristic of CHD ATPases
(Aasland et al., 1995
), and a
bromodomain, a domain conserved in SWI2/SNF2 ATPases that mediates
interactions with acetylated histone tails
(Zeng and Zhou, 2002
). Thus,
KIS-L is unusual in that it has characteristics of both the CHD and SWI2/SNF2
subfamilies of ATPases, but is a clear member of neither class. Potential
orthologs of kis are present in nematodes, mice and humans, but not
yeast, suggesting that it may play a specialized role in transcription or
development in higher eukaryotes
(Daubresse et al., 1999
;
Schuster and Stoger, 2002
;
Therrien et al., 2000
).
|
To clarify the mechanism of action of kis, we compared the distribution of KIS-L and other proteins involved in chromatin remodeling and transcription on salivary gland polytene chromosomes. Here, we report that KIS-L, like BRM, is associated with virtually all transcriptionally active regions of the Drosophila genome. The levels of elongating Pol II and the elongation factors SPT6 and CHD1 are dramatically reduced on polytene chromosomes from kis mutant larvae. By contrast, the loss of KIS-L function does not affect the binding of PC to chromatin or the recruitment of Pol II to promoters. These findings suggest that KIS-L facilitates an early step in transcriptional elongation by Pol II.
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Materials and methods |
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Antibody generation
To generate an antibody that specifically recognizes KIS-L, a DNA fragment
was PCR amplified from the kis 2 cDNA
(Therrien et al., 2000) using
the primers 5'-CGCGGATCCGTCACTCAACGATCAATTGC-3' and
5'-CGGAATTCGAGAATGCTGCTCAGGTGATG-3'; digested with BamHI
and EcoRI; and subcloned in pGEX 3X (Pharmacia). Antibodies against
GST fusion proteins were generated in rabbits and rats and purified using
columns containing GST-KIS-L coupled to Affigel 15 (BioRad, Richmond, CA)
(Harlow and Lane, 1988
).
Immunostaining of polytene chromosomes and embryos
Salivary gland chromosomes from third instar larvae were fixed for 5
minutes in 45% acetic acid/1.85% formaldehyde and stained as described
previously (Armstrong et al.,
2002). Antibodies used in this study include goat antibodies
against Pol IIa and Pol IIc (Skantar and
Greenleaf, 1995
; Weeks et al.,
1993
); rabbit antibodies against PC
(Strutt et al., 1997
), Mi-2
(Brehm et al., 2000
), ISWI
(Tsukiyama et al., 1995
), CHD1
(Stokes et al., 1996
) and BRM
(Elfring et al., 1998
); guinea
pig antibodies against SPT6 (Kaplan et
al., 2000
); and mouse antibodies against PoI IIoser2,
Pol IIoser5 (Covance, Berkeley, CA) and dynein heavy chain
(McGrail and Hays, 1997
). The
specificities of secondary antibodies (Jackson ImmunoResearch Laboratories,
West Grove, PA) were verified as described previously
(Corona et al., 2004
). Slides
were mounted in Vectashield containing DAPI (Vector Laboratories). Images were
captured on a Zeiss Axioskop 2 plus microscope using an Axioplan HRm camera
and Axiovision 4 software (Carl Zeiss, Germany) and processed using Adobe
PhotoShop 7.0 software. Merged and split images were generated as described
previously (Corona et al.,
2004
). To compare the chromosomal levels of proteins in wild-type
and mutant larvae, squashes were prepared, stained and photographed at the
same time using identical conditions. The images shown are representative of
multiple experiments. Embryos (0-12 hour) were fixed and stained with
antibodies as described previously
(Papoulas et al., 2001
).
Protein biochemistry
Native protein extracts were prepared from 0-16 hour embryos as described
(Elfring et al., 1998) using
an equal volume of ice-cold extraction buffer (20 mM Tris, pH 7.6, 150 mM
NaCl, 0.55% Tween-20, 0.125 mM EGTA, 1 mM MgCl2 and 10% glycerol)
containing protease inhibitors [1 µg/ml of each aprotinin, leupeptin,
chymostatin and Pepstatin A, 1 mM PMSF and 1 mg/ml of complete protease
inhibitor cocktail (Roche, Germany)]. For western blotting, proteins were
electrophoresed on a 5% SDS-polyacrylamide gel, transferred to nitrocellulose
overnight at 4°C, and incubated with primary and HRP-conjugated secondary
antibodies (Harlow and Lane,
1988
). Secondary antibodies were detected using the Super Signal
West Pico chemiluminescent substrate (Pierce, Rockford, IL) and a BioRad
GS-525 Molecular Imager. For gel filtration chromatography, 5 mg of protein
extract was fractionated on a Superose 6 HR 10/30 FPLC column (Pharmacia)
equilibrated with 50 mM HEPES, pH 7.6, 375 mM NaCl, 0.55% Tween-20, 0.125 mM
EGTA, 1 mM MgCl2 and 10% glycerol. Fractions (0.5 ml) were
collected and analyzed by western blotting. Co-immunoprecipitations were
carried out using affinity-purified rabbit anti-KIS-L or normal rabbit IgG
(Santa Cruz Biotech, Santa Cruz, CA) as described previously
(Papoulas et al., 1998
).
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Results |
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To monitor specifically the expression of KIS-L, we raised antibodies
against a region unique to this protein (residues 100-300;
Fig. 1). Affinity-purified
rabbit polyclonal antibodies against this segment, but not preimmune serum,
specifically recognize the 574 kDa KIS-L protein in Drosophila embryo
extracts by western blotting (Fig.
2A; data not shown). By contrast, antibodies against the
C-terminal segment common to KIS-L and KIS-S detect both the 574 kDa KIS-L and
225 kDa KIS-S proteins (Fig.
2A). We next used our antibodies against KIS-L to monitor its
temporal and spatial expression. As previously observed for other trxG
proteins and KIS-S (Daubresse et al.,
1999; Simon,
1995
), KIS-L is ubiquitously expressed in nuclei throughout
embryogenesis (Fig. 2B).
|
KIS-L is associated with virtually all transcriptionally active regions of chromatin
To directly visualize interactions between KIS-L and chromatin, we examined
its distribution on salivary gland polytene chromosomes. Antibodies against
KIS-L recognize 300 sites in euchromatin
(Fig. 3A). The vast majority of
these sites reside in interbands: regions of less condensed DNA that stain
lightly with DAPI (Fig. 3B). By
contrast, KIS-L is not associated with the heterochromatic chromocenter of
polytene chromosomes (Fig. 3A).
These findings suggest that KIS-L plays a relatively general role in
transcription or other processes, perhaps by creating open regions of
chromatin. We could not examine the distribution of KIS-S on polytene
chromosomes because it is not expressed in the salivary gland
(Fig. 2A).
|
|
CHD1, which is related to KIS-L, has been implicated in transcriptional
elongation in yeast and mammals. CHD1 associates with transcriptional
elongation factors in yeast (Krogan et
al., 2003; Simic et al.,
2003
) and mammalian cell lines
(Kelley et al., 1999
). CHD1 is
also associated with interband regions of Drosophila polytene
chromosomes (Stokes et al.,
1996
) and the body of actively transcribed genes in yeast
(Simic et al., 2003
).
Consistent with a role in transcriptional elongation, CHD1 and Pol
IIoser2 have identical patterns on polytene chromosomes
(Fig. 5A). By contrast, the
distribution of KIS-L is not identical to any one form of Pol II. Instead,
staining of KIS-L extensively overlaps with that of Pol IIoser2 and
Pol IIa (Fig. 5B,C) and to a
lesser extent with Pol IIoser5
(Fig. 5D). These findings
suggest that KIS-L is required for an earlier step in transcriptional
initiation or elongation than CHD1.
|
|
Based on its association with histone deacetylases and transcriptional
repressors (Brehm et al., 2000;
Kehle et al., 1998
;
Zhang et al., 1998
), Mi-2 is
thought to be involved in transcriptional repression. Genetic studies in
Drosophila also suggest that Mi-2 acts in concert with PcG proteins
to repress Hox transcription (Kehle et
al., 1998
). We therefore anticipated that the chromosomal
distributions of KIS-L and Mi-2 would be very different, if not mutually
exclusive. Much to our surprise, we found that the patterns of KIS-L and Mi-2
are actually very similar (Fig.
6C). Although the relative levels of KIS-L and Mi-2 vary from site
to site, only 1 to 2% of the binding sites of the two proteins fail to
overlap. These data suggest that Mi-2 plays an unanticipated and relatively
general role in transcription by Pol II.
KIS-L is not physically associated with Pol II and other chromatin-remodeling factors
Physical interactions between KIS-L and BRM, Mi-2 or Pol II could account
for their similar chromosomal distributions. Indeed, physical interactions
between yeast SWI/SNF complexes and Pol II holoenzyme have been reported
(Neish et al., 1998;
Wilson et al., 1996
), but
these findings remain somewhat controversial. To investigate this possibility,
we attempted to co-immunoprecipitate KIS-L, other chromatin-remodeling factors
and Pol II from embryo extracts using antibodies against KIS-L. KIS-L, but not
KIS-S, could be efficiently immunoprecipitated from embryo extracts using
affinity-purified antibodies against the N-terminal segment unique to the
KIS-L protein (Fig. 7). In
addition to confirming the specificity of this antibody, this result suggests
that KIS-L and KIS-S do not stably interact with each other. We were also
unable to co-immunoprecipitate KIS-L with other chromatin-remodeling factors
(including BRM, Mi-2 and ISWI) or Pol II, even when very mild conditions were
used (Fig. 7). These findings
are consistent with our gel filtration data, suggesting that KIS-L, BRM and
Mi-2 are subunits of distinct protein complexes. Thus, physical interactions
between KIS-L, BRM, Mi-2 and Pol II are probably not responsible for their
similar chromosomal distributions.
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Discussion |
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Our findings strongly suggest that KIS-L plays a global role in transcription by Pol II. Consistent with this view, KIS-L is associated with virtually all transcriptionally active regions of chromatin in salivary gland nuclei. The initial stages of transcription are normal in kis mutant larvae; Pol II is efficiently recruited to promoters and the phosphorylation of serine 5 of the CTD is not affected. However, the absence of Pol IIoser2 and the elongation factors SPT6 and CHD1 on the polytene chromosomes of kis mutant larvae strongly suggests that KIS-L is required for an early step in transcriptional elongation.
The discovery that KIS-L plays a global role in transcription by Pol II was
unanticipated, as previous genetic studies suggested that kis plays
relatively specialized roles in development
(Daubresse et al., 1999). The
limited phenotypes resulting from the loss of zygotic kis function
may be due to the high maternal contribution of kis gene products. It
is also possible that the mutations used in previous genetic studies of
kis are not null alleles, or that other factors can partially
compensate for the loss of kis function in tissues other than the
salivary gland.
How might KIS-L facilitate an early step in transcriptional elongation by
Pol II? Based on its similarity to chromatin-remodeling factors, it is likely
that KIS-L promotes transcription by altering chromatin structure. Nucleosomes
and other components of chromatin can repress transcription at many different
levels (Narlikar et al.,
2002). For example, nucleosomes can interfere with the assembly of
the preinitiation complex by blocking access of gene-specific and general
transcription factors to promoter regions. Nucleosomes also present a physical
barrier to Pol II during transcriptional elongation. Histone-modifying
enzymes, chromatin-remodeling factors and numerous other factors are therefore
crucial for transcriptional initiation and elongation in a chromatin
environment (Berger, 2002
;
Flaus and Owen-Hughes,
2004
).
Recent studies of the mammalian hsp70 gene have suggested that the
remodeling of nucleosomes near promoters is important for early stages of
transcriptional elongation. Prior to induction, a paused polymerase is located
just downstream of the hsp70 promoter
(Brown et al., 1996).
Following induction, heat shock factor 1 targets mammalian SWI/SNF to the
hsp70 promoter resulting in the disruption of this nucleosome, thus
allowing elongation to proceed (Corey et
al., 2003
). By analogy, KIS-L may promote elongation by remodeling
nucleosomes immediately downstream of promoters.
The presence of two chromodomains in KIS-L suggests that the methylation of
N-terminal histone tails may also be important for its targeting or function.
This possibility is intriguing in light of recent studies suggesting that
histone methyltransferases modulate distinct stages of transcriptional
elongation by Pol II. The phosphorylation of serine 5 of the CTD promotes
interactions between Pol II and the SET1 methyltransferase
(Ng et al., 2003), resulting
in the methylation of lysine 4 of histone H3 in the vicinity of promoters
(Nagy et al., 2002
;
Roguev et al., 2001
;
Santos-Rosa et al., 2002
). The
subsequent phosphorylation of serine 2 of the CTD promotes interactions with
the SET2 methyltransferase (Krogan et al.,
2003
; Xiao et al.,
2003
), resulting in the methylation of lysine 36 of histone H3 in
the body of transcribed genes. The pattern of histone methylation resulting
from the dynamic interactions between Pol II and histone methyltransferases
may facilitate the transition from early to late stages of elongation by
regulating interactions between chromatin-remodeling factors and nucleosomes
near promoters.
The yeast SET1 histone methyltransferase is a subunit of a large protein
complex known as COMPASS (Miller et al.,
2001). Functional counterparts of yeast COMPASS have been
identified in humans; these complexes contain subunits related to
Drosophila TRX (human MLL1 and MLL2) and ASH2 (human ASH2L)
(Hughes et al., 2004
;
Yokoyama et al., 2004
). Human
MLL1 and MLL2 methylate lysine 4 of histone H3
(Hughes et al., 2004
;
Milne et al., 2002
;
Nakamura et al., 2002
;
Yokoyama et al., 2004
), as
does Drosophila TRX (Smith et
al., 2004
), suggesting that they are functional counterparts of
SET1. Another Drosophila trxG protein, ASH1, also methylates lysine 4
of histone H3 both in vitro and in vivo
(Beisel et al., 2002
;
Byrd and Shearn, 2003
).
The above findings suggest a plausible model for how KIS-L interacts with
other trxG proteins to activate transcription
(Fig. 12). Perhaps KIS-L, like
SWI/SNF and other chromatin-remodeling factors, is targeted to promoters via
interactions with transcriptional activators or components of the general
transcription machinery (Hassan et al.,
2001; Peterson and Logie,
2000
). Once targeted to the vicinity of a promoter, KIS-L may
recognize promoter-proximal nucleosomes methylated on lysine 4 of histone H3
(by TRX or ASH1) via its chromodomains, leading to the localized remodeling of
nucleosomes that pose a barrier to elongation by Pol II.
Our findings may help explain the functional antagonism between
kis and PcG proteins. PcG proteins do not merely render chromatin
inaccessible to the general transcription machinery, as transcriptional
activators, basal transcription factors (including TFIID and TFIIF) and even
Pol II are associated with targets of PcG repression
(Breiling et al., 2004;
Breiling et al., 2001
;
Dellino et al., 2004
). Thus,
PcG proteins may act directly on components of the general transcription
machinery assembled at promoters. Consistent with this possibility, Pol II is
efficiently recruited to an hsp26 promoter silenced by the
bxd PRE, but is unable to melt the promoter and initiate
transcription (Dellino et al.,
2004
). A separate study of a promoter silenced by PcG proteins in
its natural context (the Ubx promoter in wing imaginal discs)
revealed that PcG proteins bind to both PREs and a very narrow region just
downstream of the start of transcription
(Wang et al., 2004
). Although
their precise mechanism of action remains to be determined, the above studies
suggest that PcG proteins exert their influence during the later stages of
transcriptional initiation or early stages of elongation. It is therefore
tempting to speculate that PcG proteins may repress transcription by blocking
KIS-L activity. Further analysis of the role of KIS-L in transcription,
together with the development of systems for analyzing its function in vitro,
will be necessary to test this hypothesis and clarify the role of KIS-L in
gene expression and development.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Present address: Adolf-Butenandt-Institut, Molekularbiologie,
Schillerstrasse 44, D-80336 München, Germany
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