1 Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis
University School of Medicine, 1402 South Grand Boulevard, Saint Louis, MO
63104, USA
2 Waksman Institute at Rutgers University, Piscataway, NJ 08854, USA
* Author for correspondence (e-mail: dorsettd{at}slu.edu)
Accepted 26 August 2005
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SUMMARY |
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Key words: Eco1/deco, HEAT Repeat, Insulator, Scc3/stromalin, Smc1, Spo76
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Introduction |
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In addition to gene-specific sequences, there are likely to be general
factors that act to support long-range activation in many genes (reviewed by
Dorsett, 1999). One origin of
this idea is that insulator sequences, such as the one in the Drosophila
gypsy transposon, block diverse enhancers in many genes. Insulators only
block when between an enhancer and a promoter, and thus it has been postulated
that they interfere with general factors that function between many enhancers
and promoters to facilitate enhancer-promoter communication
(Dorsett, 1999
).
To identify general facilitators of enhancer-promoter communication,
genetic screens were conducted to isolate factors that support activation of
the cut gene by a wing margin-specific enhancer located 85 kbp
upstream of the promoter (Morcillo et al.,
1996; Morcillo et al.,
1997
; Rollins et al.,
1999
). The region between this enhancer and the promoter contains
many enhancers that activate cut in specific tissues during
embryogenesis and larval development (Jack
and DeLotto, 1995
). In addition to tissue-specific activators that
bind to the wing margin enhancer, these screens identified two proteins, Chip
and Nipped-B, that are expressed in virtually all cells, and facilitate the
expression of diverse genes. Chip interacts with many DNA-binding proteins,
and likely supports the cooperative binding of proteins to enhancers and to
sites between enhancers and promoters
(Morcillo et al., 1997
;
Torigoi et al., 2000
;
Gause et al., 2001
).
Nipped-B functions by a different mechanism. Unlike other cut
regulators, Nipped-B is more limiting for cut expression when
enhancer-promoter communication is partially compromised by a weak
gypsy insulator than it is when the enhancer is partially inactivated
by a small deletion, leading to the idea that Nipped-B specifically
facilitates enhancer-promoter communication
(Rollins et al., 1999).
Nipped-B homologs in Saccharomyces cerevisiae, S. pombe and
Xenopus (Scc2, Mis4 and Xscc2), known collectively as adherins, load
the cohesin protein complex onto chromosomes
(Ciosk et al., 2000
;
Tomonaga et al., 2000
;
Gillespie and Hirano, 2004
;
Takahashi et al., 2004
)
(reviewed by Dorsett, 2004
).
Nipped-B is required for sister chromatid cohesion, and thus is a functional
adherin (Rollins et al.,
2004
). The fact that Nipped-B is an adherin raises the critical
question, addressed here, of whether or not cohesin plays a role in
enhancer-promoter communication. In all metazoans examined, cohesin loading
starts in late anaphase, and it is not removed from the chromosome arms until
prophase. Cohesin, therefore, is a structural component of chromosomes during
interphase, when gene expression occurs.
Cohesin consists of two Smc proteins, Smc1 and Smc3, and two accessory
subunits, Rad21 (Mcd1/Scc1) and Stromalin (Scc3/SA)
(Fig. 1) (Chan et al., 2003;
Losada et al., 1998
;
Losada et al., 2000
;
Sumara et al., 2000
;
Tomonaga et al., 2000
;
Toth et al., 1999
;
Vass et al., 2003
). Cohesin
forms a ring-like structure (Anderson et
al., 2002
; Gruber et al.,
2003
; Haering et al.,
2002
; Losada et al.,
2000
; Weitzer et al.,
2003
). One idea is that adherins, such as Nipped-B, temporarily
open the ring and allow it to encircle the chromosome
(Arumugam et al., 2003
). It is
proposed that cohesin encircles both sister chromatids after DNA replication
to establish cohesion. Cohesin binds every 10 kbp or so along the chromosome
arms in yeast (Blat and Kleckner,
1999
; Glynn et al.,
2004
; Laloraya et al.,
2000
; Lengronne et al.,
2004
; Tanaka et al.,
1999
). If it binds at a similar density in metazoans, it could
potentially affect the expression of many genes.
Determining if the effects of Nipped-B on gene expression are mediated
through cohesin is pertinent to Cornelia de Lange syndrome (CdLS, OMIM
#122470), which is caused by heterozygous loss-of-function mutations in the
human homolog of Nipped-B, Nipped-B-Like (NIPBL, GenBank
Accession Number NM_133433) (Krantz et
al., 2004; Tonkin et al.,
2004
). CdLS results in numerous birth defects, including slow
physical and mental growth, upper limb deformities, gastroesophageal and
cardiac abnormalities. These developmental deficits likely reflect changes in
gene expression similar to those caused by heterozygous Nipped-B
mutations.
Here, we examine binding of cohesin to the cut gene, and the effects that the Pds5 sister chromatid cohesion factor has on cut expression and cohesin binding to chromosomes. Our results are consistent with the idea that cohesin inhibits the activation of cut by the wing margin enhancer.
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Materials and methods |
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Anti-Smc1 and anti-Stromalin antibodies
His6-tagged N-terminal fragments of Smc1 and Stromalin were
expressed in bacteria using the pMCSG7 vector
(Stols et al., 2002), and
purified under denaturing conditions using Qiagen NTA beads. Denatured protein
was precipitated using 30% polyethylene glycol, suspended in
phosphate-buffered saline (PBS) and then sent to the Pocono Rabbit Farm and
Laboratory (Canadensis, PA) for immunization of a rabbit (Smc1) and a guinea
pig (Stromalin). Inserts for protein expression were generated by PCR from
cDNA clones (Stapleton et al.,
2002
) (Open Biosystems). Primers for Smc1 were
5'-TACTTCCAATCCAATGCCATGACCGAAGAGGACGACGAT-3' and
5'-TTATCCACTTCCAATGCTAGATTTTGGCCAGGTCTCGGGT-3', which amplify
sequences encoding amino acids 1 to 303. The primers for Stromalin were
5'-TACTTCCAATCCAATGCCATGGATGATCCGCCGCCGGAC-3' and
5'-TTATCCACTTCCAATGCTACATATTCTCTTTCAATTCGGA-3', which amplify
sequences encoding amino acid residues 1 to 300.
Immunostaining of polytene chromosomes
Salivary glands were fixed for 30 seconds in 2% formaldehyde, then in 45%
acetic acid and 2% formaldehyde for 3 minutes
(Lis et al., 2000), before
storage in 67% glycerol and 33% PBS at 20°C. Anti-stromalin serum
was used at 1:100, anti-SMC1 at 1:100, donkey anti-guinea pig Cy3 serum
(Jackson ImmunoResearch) at 1:200, and donkey anti-rabbit FITC serum (Sigma)
at 1:200. Pre-immune serum was used at the same dilution as the primary
antibodies. Primary antibody staining was done for 3 hours at room temperature
or overnight at 4°C. Secondary antibody staining was performed for one
hour at room temperature. Epifluorescence microscopy was performed with a
Nikon microscope equipped with a digital camera and Northern Eclipse software.
Micrographs were adjusted using Adobe Photoshop software.
Chromatin immunoprecipitation
Chromatin immunoprecipitation was performed as described by others
(Orlando et al., 1997;
Schwartz et al., 2005
), with
modifications. Kc cells were cultured in Schneider's media to
6x106 cells/ml and fixed with 1% formaldehyde for 10 minutes.
Fixation was stopped with 0.125 M glycine (pH 7.0). Fixed cells were washed
once in 200 ml PBS, once in buffer A [10 mM Hepes (pH 7.9), 10 mM EDTA, 0.5 mM
EGTA, 0.25% Triton X-100] and once in buffer B [10 mM Hepes (pH 7.9), 200 mM
NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.01% Triton X-100]. The cells were sonicated in
the presence of glass beads (400 to 600 microns) in buffer [10 mM Hepes (pH
7.9), 1 mM EDTA, 0.5 mM EGTA]. Sarkosyl was added to 0.5%, followed by
centrifugation in an Eppendorf microfuge at top speed for 10 minutes at
4°C. The supernatant was stored at 80°C.
For immunoprecipitation, a 200 µl chromatin aliquot containing 100 µg
of DNA was adjusted to a total volume of 500 µl in immunoprecipitation
buffer [10 mM Hepes (pH 7.9), 140 mM NaCl, 1 mM EDTA (pH 8.0), 1% (v/v) Triton
X-100, 0.1% (w/v) sodium deoxycholate, 0.2% (w/v) SDS], and pre-cleared by
incubation with 30 µl of protein A agarose beads (Pierce) for 3 hours at
4°C, followed by centrifugation to remove the beads. Each aliquot was
incubated with 20 µl of the appropriate pre-immune or immune serum
overnight at 4°C. Immunocomplexes were bound to 30 µl of protein A
beads at 4°C for 4 hours. Beads were collected by centrifugation for 15
seconds at top speed in an Eppendorf microfuge, washed 5 times with 1 ml of
immunoprecipitation buffer, once with 1 ml of LiCl buffer [250 mM LiCl, 10 mM
Tris-HCl (pH 8.0), 1 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate], and twice
with 1 ml of TE at 4°C. Beads were suspended in 100 µl of TE, and
crosslinks reversed by RNase A and proteinase K treatment
(Orlando et al., 1997). The
samples were extracted once with phenol-chloroform and twice with chloroform.
DNA was precipitated with 0.3 M sodium acetate (pH 5.3) and ethanol, with 30
µg of glycogen carrier. Precipitates were washed with 70% ethanol,
dissolved in 200 µl of TE, and stored at 20°C.
The amount of cut regulatory region DNA recovered in the immunoprecipitates was determined by PCR using amplicons (see Table S1 in the supplementary material) ranging from 150 to 236 bp in size, spaced at intervals of 1 kbp, starting 0.6 kbp upstream of the wing margin enhancer and extending 2.8 kbp downstream of the transcription start site. PCR was conducted for 30 cycles with 1 µl of the sample as template. PCR products were quantified after gel electrophoresis using an Alpha Innotech FluorChem imager. For each amplicon, the amount of product obtained from the immune serum precipitation was divided by the amount obtained with pre-immune precipitation to calculate enrichment. Most amplicons were amplified two to three times and the amounts averaged.
P element excision alleles of pds5
P{EPgy2}CG17509EY06473 is an insertion in the first exon of the
Drosophila pds5 homolog. Excisions of
P{EPgy2}CG17509EY06473 were generated using transposase
(2-3) and selecting for flies that lost the P element
mini-white eye color marker. These were screened by PCR using a
P-specific primer and a second primer either 5' or 3' to the
insertion site. The pds5e3 excision deleted sequence
5' to the insertion site, whereas pds5e6 deleted
sequences 3' to the insertion site.
Neuroblast squashes
Squashes of pds5 mutant third instar neuroblasts were performed
after colchicine treatment (Gatti et al.,
1994). Homozygous mutant larvae were selected using mouthpart
pigmentation from y w; pds5e3/CyO,
Df(2R)Kr Tp(1;3)y+ and y
w; pds5e6/CyO, Df(2R)Kr
Tp(1;3)y+ cultures.
Northern blots and 5' RACE
RNA was isolated using Trizol (Gibco BRL) and northern blots were performed
using radioactively labeled single-stranded RNA probes
(Dorsett et al., 1989).
pds5 probe vectors were prepared by cloning PCR products of exon 9
from genomic DNA into the BamHI and EcoRI sites of pGEM-1
(Promega). The primers were
5'-ATTAGATCTCGTCTTTTCGGCTCATTTCTTCAC-3' and
5'-ATTAGAATTCGCGGTAGTTTCTCTTGGGCAC-3'.
5' RACE of pds5e6 transcripts was performed using BD SMARTTM RACE cDNA amplification and the products were cloned into a TOPO TA pCRII vector (Invitrogen). Clones were sequenced by Retrogen (San Diego, CA), and a consensus sequence was generated using CodonCode Aligner software (CodonCode). Random hexamer primers were used for reverse transcription, and the pds5 gene-specific primer was 5'-CTGAAGACTTGGGTGATTGAGCAGGAAG-3'.
PCR analysis of pds5 mutations
Genomic DNA was prepared from pds5e3 and
pds5e6 larvae using squishing buffer [10 mM Tris-HCl (pH
8.2), 1 mM EDTA, 25 mM NaCl and 200 µg/ml Proteinase K]
(Gloor et al., 1993), and
subjected to PCR using several primer pairs to determine the extent of
deletions caused by P excision. Primer sequences can be found in Table S2 in
the supplementary material.
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Results |
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The effects of smc1exc46 on the mutant phenotype
displayed by the ctK gypsy transposon insertion
allele of cut were used to determine changes in cut
expression (Fig. 1). The
ctK gypsy insulator partially blocks activation
of cut by the wing margin enhancer, causing a scalloped wing
phenotype (Fig. 1) sensitive to
the dosage of factors that regulate cut
(Gause et al., 2001;
Rollins et al., 2004
). A
decrease in cut expression increases nicks in the wing margin, and an
increase in expression leads to fewer nicks. The wing-nicking assay is a
highly specific and sensitive measure of the activation of cut by the
wing margin enhancer, in the developing margin cells of the wing discs during
the 24-hour period centered around pupariation
(Dorsett, 1993
;
Jack et al., 1991
).
In repeated experiments, the heterozygous smc1exc46 mutation reduced the number of ctK wing margin nicks relative to the number observed with the heterozygous parental chromosome (Fig. 1). The difference was significant (P<0.0001). We conclude that, similar to the effects of reducing the Stromalin (Scc3) and Rad21 (Mcd1/Scc1) cohesin subunits, reducing the levels of the Smc1 subunit increases cut expression. Because all three cohesin subunits have a similar effect, we conclude that the cohesin complex inhibits cut expression.
We also tested mutations in genes that modulate cohesin activity for
effects on cut expression. The separation anxiety
(san) and deco (eco FlyBase) genes encode
putative acetyltransferase proteins that are required for sister chromatid
cohesion and the association of cohesin with centromeric regions
(Williams et al., 2003).
Separase (Sse) encodes a protease that cleaves cohesin to
permit sister chromatid separation
(Jäger et al., 2001
).
Mutations in these genes did not have significant effects on the
ctK mutant phenotype
(Fig. 2). It is possible that
heterozygosity for these mutations did not sufficiently alter cohesin activity
to change cut expression. Alternatively, these proteins may affect
cohesin only at the centromere.
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The examination of several nuclei showed that the chromosome band
containing the cut locus (7B3-4) consistently displayed cohesin
staining (Fig. 6B). This band
contains 150 kbp of DNA, and four genes other than cut, in the
regulatory region between the wing margin enhancer and the cut
promoter (Drysdale et al.,
2005). At least three of these genes are testis specific
(Andrews et al., 2000
).
Salivary glands do not express cut, but because cohesin is a
constitutive chromosomal component, and probably binds to cut in most
cells, these data are consistent with the view that the effects of cohesin on
cut expression in the wing margin are direct.
Further support is provided by chromatin immunoprecipitation experiments (Fig. 4), which show that cohesin binds to the regulatory region of cut in Drosophila cultured Kc cells of embryonic origin. We examined an 85-kbp region encompassing the wing margin enhancer and the promoter, which revealed four cohesin-binding sites. The amounts of cut DNA precipitated by the immune and pre-immune sera were determined by PCR at 1 kb intervals, and enrichment of each amplicon was measured by the ratio of the amount of immune PCR product to amount of pre-immune PCR product. As expected, the baseline approached an immune to pre-immune ratio of 1, and peaks of cohesin binding were recognized by increases in the ratio over the baseline. Two binding sites were centered 0.5- and 4-kbp upstream of the promoter, one was centered about 30.5-kbp upstream of the promoter, and another small broad peak was 68-kbp upstream of the promoter. The same sites were seen with both antisera, and neither pre-immune serum showed enrichment of any sequences. Thus, in addition to the non-dividing polytene salivary cells, cohesin also binds cut in predominantly diploid dividing cells of embryonic origin. Based on the assumption that cohesin is a constitutive chromosomal component, and the finding that it binds to the cut locus in two very different cell types, we posit that it also binds cut in the developing wing margin cells and that the effects of cohesin on cut expression in the developing wing are direct.
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By sequence analysis, the CG17509 gene
(Celniker et al., 2002) was
identified as the likely pds5 homolog
(Fig. 5B). The
P{EPgy2}CG17509EY06473 transposon insertion in the first exon is
homozygous viable. It was mobilized to generate two recessive lethal
mutations, pds5e3 and pds5e6, that
fail to complement each other, and a deletion of the region [Df(2R)BSC39].
Both homozygous mutants and the heteroallelic combination are lethal in late
third instar to early pupal stages of development. Late third instar larvae of
both mutants display small or missing imaginal discs, and the larval brains
are approximately half the volume of wild type, consistent with a mitotic
defect.
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pds5e6 produces an altered transcript
We examined pds5 expression in the two pds5 mutants to
determine why they have different effects on cut expression. Northern
blots revealed a pds5 transcript of the expected size (4.6 kb,
Fig. 7A) in embryos prior to
zygotic gene expression (Fig.
7A, lane 1), indicating that it is maternal. The transcript was
present at 10- to 25-fold lower levels in larvae. To avoid detecting maternal
pds5 mRNA, we examined the transcripts produced in
pds5e3 and pds5e6 second instar
larvae. We were unable to detect pds5 transcripts in homozygous
pds5e3 mutants (<2% of wild type), but saw a shorter
transcript (3.65 kb) at levels similar to those of wild type in both
heterozygous and homozygous pds5e6 mutants
(Fig. 7B). A wild-type sized
transcript was present in heterozygous pds5e6 mutants, but
was undetectable in homozygotes. The viable parental P insertion line used to
generate both lethal pds5 alleles produced wild-type levels of a
wild-type sized transcript (Fig.
7B).
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The presence of a new transcript in the pds5e6 mutant
suggested that it could produce a mutant protein lacking an activity crucial
for sister chromatid cohesion that somehow interferes with the inhibition of
cut by cohesin. PCR analysis of pds5e6 genomic
DNA revealed that the region from the P insertion site through exon 5 is
missing. 5' RACE analysis of the pds5e6 transcript
shows that it starts 67 nucleotides upstream of the wild-type start site
predicted by EST analysis (Fig.
7C). The pds5e6 transcript extends from the
start site to the P insertion site. The next 17 nucleotides are from the end
of the P insertion, followed by 12 nucleotides of internal P sequence fused to
the pds5 sequence 11 nucleotides downstream of the exon 6 5'
splice site (Fig. 7C). The exon
6 sequences present in the mutant transcript contain six in-frame AUG codons,
two of which match a consensus (RNVATGR) for Drosophila
translation initiation sites (Cavener and
Ray, 1991). Thus the pds5e6 mutant transcript
encodes a protein lacking the N terminus.
pds5e6 reduces association of cohesin with chromosomes
The effects of the two pds5 mutations on cut expression
correlate with a difference in cohesin binding to chromosomes
(Fig. 8). Although the salivary
glands of the homozygous pds5 mutants are substantially reduced, we
obtained polytene chromosomes from both. Morphology was altered enough to make
it difficult to identify specific loci. Nevertheless, individual chromosome
tips could be identified, and developmental puffs, including the puff at 2B,
were present in both mutants, indicating that the chromosomes are transcribed.
In size-matched third instars, the pds5e3 mutant
chromosomes were thicker than wild type, and the pds5e6
mutant chromosomes were thinner (Fig.
7). The pds5e3 null allele did not reduce
staining for Smc1 or Stromalin, although the pattern appeared less discrete
(Fig. 8). By contrast,
pds5e6 mutant chromosomes showed strongly reduced staining
for Smc1 and Stromalin (Fig.
8). Loss of cohesin staining was observed in multiple nuclei from
multiple pds5e6 salivary glands. These results indicate
that the pds5e6 mutant either blocks loading of cohesin
onto chromosomes, or facilitates removal. The reduction of cohesin binding
caused by pds5e6, which dominantly increases cut
expression, is consistent with the hypothesis that cohesin inhibits
cut expression.
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Discussion |
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The demonstration that cohesin binds to the cut locus in polytene
chromosomes, and to multiple sites between the remote wing margin enhancer and
the promoter in cultured cells, supports the hypothesis that the effects of
cohesin on cut expression in the wing margin are direct. The wing
margin enhancer does not activate cut in salivary glands or Kc cells,
but it is not technically feasible to examine the association of cohesin with
cut in the developing margin cells in which the wing margin enhancer
functions. Based on the association of cohesin with cut in two
diverse cell types, we posit that cohesin also binds cut in the
developing wing margin cells, and inhibits activation by the wing margin
enhancer. Such a direct effect of cohesin could explain why small reductions
(<20%) in cohesin subunits induced by RNAi have detectable effects on
cut expression (Rollins et al.,
2004).
Both pds5 mutations tested cause similar sister chromatid cohesion defects, but only pds5e6 reduces the binding of cohesin to chromosomes and increases cut expression. This provides additional evidence that binding of cohesin to chromosomes is required for it to inhibit cut activation, and also shows that Pds5 itself is not required to inhibit gene expression.
The negative effects of cohesin on cut expression raise the
possibility that cohesin contributes to the silencing of euchromatic genes
placed in heterochromatin. Cohesin binds more densely in centromeric
heterochromatin in yeasts and metazoans, and, in S. pombe,
heterochromatin proteins recruit cohesin
(Bernard et al., 2001;
Nonaka et al., 2002
;
Partridge et al., 2002
).
Moreover, RNAi-mediated silencing of a non-centromeric gene in S.
pombe causes the recruitment of heterochromatin proteins and cohesin to
the silenced gene (Schramke and Allshire,
2003
).
Mechanisms for the effects of cohesin on gene expression
We favor the idea that cohesin inhibits enhancer-promoter communication in
cut. This idea originates from the allele-specific effects of
Nipped-B mutations on cut expression. The known cut
regulators required for activation by the wing margin enhancer, including
scalloped, vestigial, mastermind, Chip, Nipped-A and
l(2)41Af, all display different cut allele specificities
than Nipped-B (Morcillo et al.,
1996; Morcillo et al.,
1997
; Rollins et al.,
1999
). In contrast to these factors, Nipped-B is most limiting
when enhancer-promoter communication is partially compromised by a weak
gypsy insulator, suggesting that Nipped-B facilitates long-range
communication (Rollins et al.,
1999
).
Binding of cohesin to multiple sites between the wing margin enhancer and the cut promoter is consistent with the hypothesis that Nipped-B facilitates enhancer-promoter communication by regulating the binding of cohesin to chromosomes. To explain how Nipped-B aids activation, we theorize that Nipped-B can remove cohesin from chromosomes. In a simple model, Nipped-B facilitates the cohesin-binding equilibrium. When Nipped-B is partially reduced but not abolished, it takes longer to achieve equilibrium, but the extent of cohesin binding is not altered and there is little effect on sister chromatid cohesion. The reduced cohesin on-off rates, however, would diminish the opportunities for gene activation that require cohesin removal or repositioning.
We do not know how cohesin inhibits long-range activation, but we can
envision mechanisms for various long-range activation models. For example,
cohesin could inhibit the folding or looping of the chromosome that is
required to bring the enhancer into contact with the promoter. Alternatively,
cohesin could block a Chip-mediated spread of protein binding between the
enhancer and promoter in linking models for long-range activation
(Dorsett, 1999;
Bulger and Groudine, 1999
), or
block the transfer or tracking of RNA polymerase from the enhancer to the
promoter, as appears to occur in the chicken beta-globin gene locus (Zhou and
Dean, 2004).
Effects of Pds5 on cohesin function
We found that Drosophila Pds5 is required for sister chromatid
cohesion, consistent with studies on fungal Pds5
(Hartman et al., 2000;
Panizza et al., 2000
;
Tanaka et al., 2001
). To
explain the effects of the pds5e6 mutation on cohesin
chromosome binding and cut expression, we propose that it produces a
mutant protein that blocks cohesin binding, or causes cohesin to be released
from chromosomes. This agrees with observations on vertebrate and S.
pombe Pds5 suggesting that Pds5 has both positive and negative effects on
cohesion, possibly by regulating the association of cohesin with chromosomes
(Losada et al., 2005
;
Tanaka et al., 2001
).
Vertebrates contain two Pds5 isoforms that associate with chromosomal
cohesin (Losada et al., 2005;
Rankin et al., 2005
;
Sumara et al., 2000
).
Reduction of Pds5 partially decreases sister chromatid cohesion
(Losada et al., 2005
).
Consistent with our finding that the pds5e3 null mutation
does not reduce the binding of cohesin to polytene chromosomes, and with
previous work on S. cerevisiae and S. pombe Pds5
(Hartman et al., 2000
;
Stead et al., 2003
;
Tanaka et al., 2001
),
Xenopus Pds5 is not required for the binding of cohesin to
chromosomes (Losada et al.,
2005
). One report suggested that S. cerevisiae Pds5 is
required for the association of cohesin with chromosomes
(Panizza et al., 2000
), but it
is possible that this discrepancy might be caused by differences in the mutant
alleles, similar to the differences we find between pds5e3
and pds5e6.
Depletion of Pds5 from Xenopus extracts increases the amount of
cohesin associated with chromatin (Losada
et al., 2005). A similar increase in cohesin binding could explain
the slight decrease in cut expression caused by the
pds5e3 null allele. Consistent with the idea that
wild-type Pds5 partially reduces cohesin binding, deletion of S. pombe
pds5 partially suppresses a temperature-sensitive mutation in
mis4, which encodes the homolog of the Nipped-B and Scc2
cohesin-loading factors (Tanaka et al.,
2001
).
Because wild-type Pds5 appears to partially reduce the binding of cohesin
to chromosomes, we speculate that the pds5e6 mutation
increases this activity, which may be related to the cohesin-loading function
of Nipped-B/Scc2. Scc2 interacts with cohesin, and is thought to open the
cohesin ring (Arumugam et al.,
2003). In synchronized yeast cells, cohesin loads at Scc2-binding
sites and translocates away (Lengronne et
al., 2004
). Like Nipped-B/Scc2, Pds5 contains several HEAT repeats
(Neuwald and Hirano, 2000
),
and thus might also open the cohesin ring during DNA replication to allow it
to encompass both sister chromatids. It could play a similar role in the snap
model (Milutinovich and Koshland,
2003
), in which cohesin complexes bound to the two sisters
interlock to hold the sisters together. If the pds5e6
mutant protein interacts with cohesin non-productively, it could block access
to Nipped-B and prevent loading. Alternatively, when the mutant Pds5 attempts
to establish cohesion, it might fail, releasing cohesin from the chromosome.
Wild-type Pds5 might partially reduce cohesin binding by competing with
Nipped-B for cohesin, or by occasionally failing to establish cohesion.
Implications for Cornelia de Lange syndrome
The effects of cohesin on cut expression are likely pertinent to
the etiology of Cornelia de Lange syndrome (CdLS) (reviewed by
Strachan, 2005). CdLS is
caused by mutations in the Nipped-B-Like (NIPBL) human
homolog of Nipped-B (Krantz et
al., 2004
; Tonkin et al.,
2004
). Most missense mutations that cause CdLS affect residues
conserved in Nipped-B (Borck et al.,
2005
; Gillis et al.,
2004
; Krantz et al.,
2004
; Miyake et al.,
2005
; Tonkin et al.,
2004
). CdLS is characterized by several physical and mental
deficits, including slow growth, mental retardation, and upper limb,
gastroesophageal and cardiac deformities
(de Lange, 1933
;
Ireland et al., 1993
;
Jackson et al., 1993
).
Heterozygous loss-of-function NIPBL mutations cause CdLS, and thus
the developmental changes likely reflect gene expression effects similar to
those caused by heterozygous Nipped-B mutations
(Rollins et al., 1999
). At
least some birth defects in CdLS, such as limb truncations and cardiac
abnormalities, could be caused by changes in expression of the known homeotic
genes. The observations presented here indicate that cohesin likely plays a
role in CdLS by inhibiting the long-range gene control of homeotic genes.
The possibility that some developmental changes in CdLS reflect reduced
sister chromatid cohesion cannot be ruled out. A recent study found evidence
for cohesion deficits in 41% of CdLS patients compared with in 9% of controls
(Kaur et al., 2005). Also, the
autosomal recessive Roberts syndrome has some similarities to CdLS, and is
caused by mutations in a human homolog of the Eco1/Eso1/Deco cohesion factor
(Vega et al., 2005
). Cells
from Roberts patients display defects in sister chromatid cohesion. Homozygous
Drosophila deco1 mutants appear to affect cohesin binding
only at centromeric regions (Williams et
al., 2003
), and, as described above, we did not see dominant
effects of deco mutations on cut gene expression, leading us
to favor the idea that most CdLS developmental deficits reflect changes in
gene expression instead of in sister chromatid cohesion.
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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/132/21/4743/DC1
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