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Molecular and Cellular Biology, April 2009, p. 2023-2031, Vol. 29, No. 8
0270-7306/09/$08.00+0     doi:10.1128/MCB.01448-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Two Classes of Dosage Compensation Complex Binding Elements along Caenorhabditis elegans X Chromosomes {triangledown} ,{dagger}

Timothy A. Blauwkamp{ddagger} and Gyorgyi Csankovszki*

Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109-1048

Received 15 September 2008/ Returned for modification 21 October 2008/ Accepted 23 January 2009


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ABSTRACT
 
Dosage compensation equalizes X-linked gene products between the sexes. In Caenorhabditis elegans, the dosage compensation complex (DCC) binds both X chromosomes in XX animals and halves the transcription from each. The DCC is recruited to the X chromosomes by a number of loci, rex sites, and is thought to spread from these sites by an unknown mechanism to cover the rest of the chromosome. Here we describe a novel class of DCC-binding elements that we propose serve as "way stations" for DCC binding and spreading. Both rex sites and way stations comprise strong foci of DCC binding on the native X chromosome. However, rex sites maintain their ability to bind large amounts of DCC even on X duplications detached from the native X, while way stations do not. These results suggest that two distinct classes of DCC-binding elements facilitate recruitment and spreading of the DCC along the X chromosome.


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INTRODUCTION
 
In animals with chromosomally determined sex, including mammals, flies, and worms, the process of dosage compensation equalizes transcription of X-linked genes in males (XY or XO) and females (XX), despite their different gene doses (23). Female mammals accomplish this by inactivating most of the genes on one of the two X chromosomes (36), Drosophila males upregulate most genes on the single male X chromosome twofold (26, 34), and Caenorhabditis elegans hermaphrodites (XX) halve transcription from each X chromosome (27). Although the details of the dosage compensation mechanisms differ between species, all require chromosome-specific targeting of molecular complexes, and all regulate transcription over large domains. While dosage compensation is one of the best studied models for domain-specific transcriptional regulation, other examples of domain-specific regulation include clusters of imprinted genes (9), coregulation of 20 to 80 gene clusters throughout the human genome (13), coregulation of HOX gene clusters in several organisms (7), and coordinate regulation of the entire fourth chromosome in Drosophila (17, 31). The mechanism by which domain-specific regulatory complexes are recruited to specific regions and function over large distances remains an intriguing question.

In C. elegans, the dosage compensation complex (DCC) binds along the entire length of both X chromosomes in XX animals (hermaphrodites) and halves transcription from each (27). The SDC-2, SDC-3, and DPY-30 proteins recruit the condensin-like subcomplex composed of MIX-1, DPY-27, DPY-26, DPY-28, and CAPG-1, as well as the SDC-1 and DPY-21 proteins, to the X chromosome (reviewed in reference 27). The condensin-like subcomplex shares the MIX-1 subunit with the C. elegans mitotic condensin complex, which functions in chromosome condensation and segregation. However, the two complexes have separate functions, and mutations in the gene encoding the DCC-specific subunit dpy-27 do not affect mitosis or meiosis (22, 27).

Despite the observed DCC binding along the entire length of both hermaphrodite X chromosomes in wild-type animals, some large duplicated X-chromosome fragments maintain their ability to bind the DCC, while some do not (4). The current model for chromosome-wide regulation is that the DCC is recruited to specific sites on the X chromosome and subsequently spreads from these sites to cover the rest of the X chromosome. Two independent studies recently identified similar DNA sequences implicated in recruiting the DCC to the X chromosome. In one study, confocal microscopy was used to detect DCC binding to transgenic arrays containing progressively smaller DNA fragments of the X chromosome, leading to the identification of two DNA motifs that were sufficient to recruit the DCC when clustered, the so-called A and B motifs (25). Another study used a genome-wide chromatin immunoprecipitation (ChIP)-on-chip approach to identify sites of DCC binding on the X chromosomes (10). Antibodies to SDC-3 and DPY-27 were both used to detect DCC binding and both analyses yielded similar results. Their study confirmed DCC binding along the entire length of the X chromosome but showed that binding levels were highly variable across the X. Approximately 1,500 "peaks" were observed to bind significantly more DCC than the rest of the X chromosome. Among the peaks, approximately 50 were observed to bind especially high levels of DCC and were referred to as "foci" (10). DNA sequence analysis of the foci identified an over-represented 10-bp DNA sequence similar to the A and B motifs, leading to the hypothesis that foci are DNA sequence-based DCC recruitment centers. However, neither the A and B motifs nor the motif derived by Ercan et al. (10) are specific to the X chromosome. In addition, only 42% of DCC foci actually contain one or more of the motifs. Although these motifs are likely to play a role in binding the DCC (25), they are neither necessary nor sufficient for the formation of DCC peaks on the native hermaphrodite X chromosome.

Here we explore the mechanism of DCC recruitment and spreading by using ChIP and fluorescence microscopy to test the hypothesis that foci are sites of DCC recruitment. We found that some foci can recruit the DCC to genomic duplications, while other foci are not sufficient to recruit the DCC to genomic duplications. The ability to recruit the DCC to genomic duplications was not correlated with the presence or absence of strong DCC-binding motifs, the amount of DCC bound to the native X foci, or the local genomic architecture (upstream, downstream, or within genes). Our data suggest that the X chromosome contains at least two classes of strong DCC-binding elements: those that recruit the DCC (rex sites) and others that facilitate spreading of the DCC by acting as way stations along the X chromosome.


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MATERIALS AND METHODS
 
C. elegans strains. Animals were maintained on NG agar plates with Escherichia coli (OP50) as a food source, using standard methods. The following four strains were used in these experiments: (i) N2 (Bristol), wild type; (ii) SP0262, mnDp1(X;V)/+; mnDf1 X; (iii) RW2551, stDp2(X;II); stDf5 X; and (iv) SP0957, dpy-8(e130) stDf1 X; mnDp30(X;f).

ChIP. Embryos were harvested from synchronous cultures of gravid adults grown on egg plates with E. coli (HB101) by using standard bleaching protocols. Portions (100 to 500 µl) of embryos were fixed in 40 ml of 2% formaldehyde for 30 min, followed by two washes with M9 and one wash with homogenization buffer (50 mM HEPES-KOH [pH 7.6], 1 mM EDTA, 140 mM KCl, 0.5% NP-40, 10% glycerol, and fresh protease inhibitors). Embryos in homogenization buffer were sonicated to an average DNA fragment length of 100 to 1,000 bp. Embryonic sonicate was cleared by centrifugation, and the protein concentration was determined by a Bio-Rad assay (Bio-Rad).

For each ChIP experiment, 100 µg of protein in 300 µl of homogenization buffer was precleared with 50 µl of ChIP-ready protein A beads (50% slurry of protein A-agarose beads supplemented with 400 µg of sonicated salmon sperm DNA/ml and 500 µg of bovine serum albumin/ml; Upstate) at 4°C for 30 min, followed by incubation with 27 µg of affinity-purified rabbit DPY-27 antibody (raised against N-terminal peptide QPFKRRALTSDDDRPYADTDSMPEVDLDVDRRR) at 4°C for 2 h. Immunocomplexes were isolated by incubation with 50 µl of ChIP-ready protein A beads at 4°C for 30 min, followed by four washes of the beads with low-salt buffer (100 mM KCl, 50 mM HEPES-KOH [pH 7.6], 1 mM EDTA, and 0.05% NP-40), two washes with high-salt buffer (1 M KCl, 50 mM HEPES-KOH [pH 7.6], 1 mM EDTA, and 0.05% NP-40), and two washes with Tris-EDTA. Immunocomplexes were eluted with two washes with 0.1 M NaHCO3-1% sodium dodecyl sulfate at room temperature for 15 min. Immunoprecipitated DNA was isolated by reversing cross-links with 100 mM NaCl at 65°C for 5 to 12 h, followed by protein digestion with 20 µg of proteinase K at 45°C for 1 h, phenol-chloroform-isoamyl alcohol extraction, and ethanol precipitation.

Real-time PCR analysis. Immunoprecipitated DNA was measured by using real-time PCRs, carried out in standard PCR buffer supplemented with 25% dimethyl sulfoxide, SYBR green, 10 mM fluorescein, and Platinum Taq (Invitrogen) in an iCycler IQ real-time PCR detection system (Bio-Rad). Primers sequences are listed in Table S1 in the supplemental material.

In Fig. 3B, 4B, and 5B, the values plotted on the y axis were calculated as follows. The percentage of input DNA pulled down by DPY-27 antibodies in the mutant strain (mutant %IP) was divided by the percentage of input DNA pulled down in the wild-type strain (wild-type %IP). This ratio was calculated for all 13 foci and is arbitrarily designated the relative IP. The average relative IP at foci not affected by the chromosomal rearrangement was set to 1.0, and data from the affected foci were adjusted proportionately: in other words, (mutant %IP)/(wild-type %IP) = relative IP. Also, the relative IP/the average relative IP = the normalized relative IP. The normalized relative IP is plotted on the y axes in Fig. 3B, 4B, and 5B.


Figure 3
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  		FIG. 3. Foci within the stDp2 duplication do not bind the DCC when removed from the endogenous X chromosome. The role of chromosomal context in recruiting the DCC to foci was examined by comparing DCC binding in wild-type (N2) embryos to embryos derived from a strain homozygous for the stDf5 X-chromosome deficiency and the stDp2 X-chromosome duplication (attached to chromosome II) (RW2551), as shown in the diagram. (A) The relative input levels from RW2551 embryos confirm that most foci are present only on the X, that Dpy-27_17 and Dpy-27_19 are present on the X and on the stDp2 duplication, and that Dpy-27_18 is present only on the stDp2 duplication. (B) The percentage of input DNA immunoprecipitated by DPY-27 ChIP in RW2551 embryos was compared to the percentage of input DNA immunoprecipitated in wild-type (N2) embryos. The results are plotted as the ratio of these values. The average value for unaffected peaks was set to 1.0 (arbitrary units) to account for differences in cross-linking efficiency from experiment to experiment. In all cases, the ratio at Dpy-27_17 and Dpy-27_19 was ca. 50% the average, and the ratio at Dpy-27_18 was 15 to 20% of the average. (C) DPY-27 binding to foci was examined in intestinal nuclei of wild-type (N2) and RW2252 hermaphrodite worms. FISH probes Dpy-27_17, Dpy-27_18, and Dpy-27_19 (listed on the right) marked the location of foci, and DPY-27 specific antibodies were used to analyze DCC binding. Whereas in wild-type worms the FISH probes only marked locations within the DPY-27-bound territory (arrows), in RW2551 the foci on the X chromosome were within the DPY-27-bound territory (arrows), and foci on the duplication were not (arrowhead). The FISH probe is shown in red, the anti-DPY-27 is indicated in green, and DAPI is indicated in blue.


Figure 4
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  		FIG. 4. Foci within the mnDp30 duplication do not bind the DCC when removed from the endogenous X chromosome. (A) Relative input levels from embryos homozygous for the stDf1 X-chromosome deficiency and also containing mnDp30 X-chromosome free duplication (SP957) confirm that Dpy-27_16 is only present on the mnDp30 duplication in these embryos. Dpy-27_15 and Dpy-27_17 appear to be present on both the native X chromosome and the mnDp30 free duplication, but the relatively low contribution of mnDp30-derived sequences to total copy number makes it difficult to assess the affects of chromosomal context at these foci. (B) The percentage of input DNA immunoprecipitated by DPY-27 ChIP in SP957 embryos was compared to the percentage of input DNA immunoprecipitated in wild-type (N2) embryos. The results are plotted as the ratio of these values. The average value for unaffected peaks was set to 1.0 (arbitrary units) to account for differences in cross-linking efficiency from experiment to experiment. In all cases, the ratio at Dpy-27_16 was ca. 20% of the average. (C) DPY-27 binding to focus Dpy-27_16 was examined in the intestinal nuclei of wild-type (N2) and SP957 hermaphrodite worms. Whereas in wild-type worms the Dpy-27_16 sequences are found on the two X chromosomes within the DPY-27-bound territory (arrow), in SP957 the Dpy-27_16 is located only on the duplication and is not bound by DPY-27 (arrowhead). The FISH probe is shown in red, anti-DPY-27 is indicated in green, and DAPI is indicated in blue.


Figure 5
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  		FIG. 5. DCC binding to some foci is not affected by chromosomal context. (A) Relative input levels from a strain homozygous for the mnDf1 X-chromosome deficiency and heterozygous for the mnDp1 X-chromosome duplication (attached to chromosome V) confirm that Dpy-27_34 to Dpy-27_40 foci are present only on the mnDp1 duplication and at half the copy number of the other foci. (B) The percentage of input DNA immunoprecipitated by DPY-27 ChIP in mnDp1/+; mnDf1 embryos was compared to the percentage of input DNA immunoprecipitated in wild-type embryos. The results are plotted as the ratio of these values. The average value for unaffected peaks was set to 1.0 (arbitrary units) to account for differences in cross-linking efficiency from experiment to experiment. In all cases, the DCC bound equally well to foci on the native X and on the mnDp1 duplication.

Fluorescence microscopy. Combined fluorescence in situ hybridization (FISH) and immunofluorescence (IF) analyses were performed as described previously (4). To generate FISH probes, the following yeast artificial chromosomes (YACs) were labeled by using a Prime-a-Gene kit (Promega) and Cy3-dCTP (Amersham): YAC Y72A10 was used to indicate the location of Dpy-27_16, Y49B2 was used to indicate the location of Dpy-27_17, Y46B8 was used to indicate the location of Dpy-27_18, and Y44D2 was used to indicate the location of Dpy-27_19. YAC DNA was purified by using pulsed-field gel electrophoresis and then amplified using degenerate oligonucleotide-primed PCR prior to labeling (1). IF was performed using the same antibody as used for ChIP, diluted 1:100.


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RESULTS AND DISCUSSION
 
We performed ChIP of the DCC subunit DPY-27 and analyzed the enriched DNA fragments by real-time PCR. To confirm that our antibody and protocols could reliably distinguish the level of DCC binding at foci, peaks, and other X-linked loci from background, we first examined DPY-27 binding at rex-1 and rex-2, two foci previously shown to recruit the DCC to extrachromosomal arrays (25). DPY-27 bound to rex-1 and rex-2 very efficiently and at higher levels than it bound to three representative "peaks" or the five sites representative of "basal" binding to the X chromosome (Fig. 1). Importantly, even the basal levels of DPY-27 bound to the X between peaks were >2-fold higher than the binding to autosomal sequences (not predicted to bind the DCC). We interpret the levels of binding at these autosomal sequences as the background since the binding was similar to the level observed in experiments lacking antibody. We conclude that our protocols reliably distinguish DCC binding at foci, peaks, and X-linked sites with basal levels of binding from background and therefore present a suitable system in which to explore the mechanism of DCC recruitment and spreading along the X chromosome.


Figure 1
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  		FIG. 1. DCC binding at foci, peaks, basal X loci, and autosomes. Chromatin was immunoprecipitated from wild-type embryos with DPY-27 antibodies and quantified by using real-time PCR. Immunoprecipitated DNA is expressed as the percentage of input. Foci (rex-1 and rex-2), peaks (DPY-27_628, DPY-27_637, and DPY-27_704, and basal X sequences (five sequences not identified as foci or peaks by Ercan et al. [10]) all bind significantly more DPY-27 than autosomal sequences (myo-1 coding and myo-1 promoter sequences). No antibody control ChIP experiments (averaged level of Foci sequences detected in ChIP experiment lacking antibody) are similar to autosomal ChIP levels, suggesting that both result from nonspecific immunoprecipitation of DNA.

We tested the hypothesis that foci are sites of DCC recruitment by measuring the ability of genomic duplications containing foci to recruit the DCC when removed from the endogenous X chromosome and present as free or autosome-attached duplications. It has been demonstrated before that multimerizing DNA sequences can change their ability to recruit the complex (14, 16). Therefore, we limited our analysis to chromosomal rearrangements to test the ability of single-copy DNA sequences containing foci to recruit the DCC, as opposed to the hundreds or thousands of copies of DNA sequences in extrachromosomal arrays. We focused our analysis on 13 of the 35 foci previously shown to bind DPY-27, 12 of which also bind SDC-3 (10). To avoid confusion, we use the focus numbering system of Ercan et al. (10). Throughout the present study, DCC binding is defined as the fraction of input DNA pulled down by DPY-27 ChIP. Input DNA is measured individually for each focus in all experiments. We found that DCC binding to individual foci in the wild-type strain was highly variable, but in all cases was significantly higher than the level of binding across the rest of the X chromosome (Fig. 2).


Figure 2
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  		FIG. 2. DCC binding to foci on the wild-type X chromosome is highly variable in amplitude and distributed along the entire length of X-chromosome. DCC binding to 13 sites identified as foci on wild-type X chromosomes was measured using ChIP with DPY-27 antibodies. (A) Input levels for all 13 foci were highly similar in wild-type (N2) embryos. (B) Immunoprecipitated DNA is expressed as the percentage of input for each of the foci. The level of DCC binding to wild-type X is variable across the chromosome.

We next measured how well foci bind DPY-27 when removed from the X chromosome. stDp2 is a duplication of ~4 Mb of DNA near the middle of the X chromosome and is attached to chromosome II (WormBase [http://www.wormbase.org], release WS193). It includes three foci (Dpy-27_17, Dpy-27_18, and Dpy-27_19). In order to discern DCC binding at X-derived sequences on the duplication from binding to native X sequences, we used a strain that also contains the stDf5 deletion, which lacks a portion of the X chromosome harboring the Dpy-27_18 focus (Fig. 3A). Real-time PCR analysis of genomic DNA confirms that this strain contains only two copies of Dpy-27_18 (both on stDp2), four copies each of foci Dpy-27_17 and Dpy-27_19 (two copies on the native X and two on stDp2), and two copies of all other foci, which are not affected by the duplication or deletion (Fig. 3A).

We compared DCC binding at 13 foci in the stDp2; stDf5 strain to binding in wild-type worms. The results are displayed as the ratio of DCC binding in stDp2; stDf5 relative to binding in wild type for each individual focus. Although foci not included in the duplication and deficiency were unaffected, we found that Dpy-27_18 was severely deficient in binding the DCC when removed from the native X chromosome, binding <20% of the wild-type levels (Fig. 3B). We also observed a 50% reduction in DCC binding at the other foci on this duplication, Dpy-27_17 and Dpy-27_19. Both of these foci are present in four copies per cell. Since the stDp2 duplication overall does not bind large amounts of the DCC (4), it is unlikely that the duplication titrates the DCC away from the X chromosome. Therefore, we interpret the 50% reduction in binding as severely compromised binding to the two copies of these foci present on the duplication, with normal binding to the two copies on the native X chromosome.

To confirm this interpretation, we performed in situ analysis of DPY-27 binding to individual foci using fluorescence microscopy (Fig. 3C). In an earlier study, prior to the discovery that some DPY-27 binding foci map to stDp2, we reported that DCC binding to this duplication cannot be detected by IF (4). To analyze binding at individual foci, we used FISH probes prepared from individual YAC clones that map to the location of the foci analyzed. FISH was followed by IF analysis of DPY-27 binding. To facilitate visualization, we analyzed polyploid intestinal nuclei, as was done earlier (4). Due to the polyploid nature of the cells, loci appear as clusters of dots. In wild-type worms, probes corresponding to foci Dpy-27_17, Dpy-27_18, and Dpy-27_19, all marked two loci in the nucleus, both within the large territory of the X chromosome bound by DPY-27. In contrast, in the duplication strain, probes Dpy-27_17 and Dpy-27_19 marked loci both inside and outside the DPY-27-bound territory, corresponding to foci on the X chromosome and on the duplication, respectively. Probe Dpy-27_18 only marked a location outside the DPY-27 territory, a finding consistent with this focus being present on the duplication but not on the X chromosome. In all three cases, foci on the duplication appeared severely compromised in binding DPY-27 (Fig. 3C).

We confirmed the observation above, that not all sites of strong DCC binding on the X could recruit DCC when detached from the X chromosome, by analyzing another duplication-deletion combination (Fig. 4). The mnDp30 free duplication includes three foci: Dpy-27_15, Dpy-27_16, and Dpy-27_17. We analyzed DCC binding to these foci in a strain that also contained the mnDf1 deficiency that removes a portion of the X harboring Dpy-27_16. Free duplications are frequently inherited at a lower efficiency than attached duplications, and real-time PCR measurement of genomic DNA levels indicate that the mnDp30 duplication (as detected by Dpy-27_16 input levels) is present at approximately only one copy for every five copies of the endogenous X chromosome (Fig. 4A). Genetic mapping of the mnDp30 and stDf1 boundaries indicate that the Dpy-27_15 and Dpy-27_17 foci are present on both the native X chromosome and the mnDp30 free duplication, whereas Dpy-27_16 is present only on the mnDp30 duplication (WormBase). Consistently, we observe higher than average input levels for Dpy-27_15 and Dpy-17 and lower than average input levels of Dpy-27_16 in the mnDp30; stDf1 strain. Importantly, DPY-27 ChIP showed that DCC binding to the Dpy-27_16 foci on mnDp30 was also compromised when removed from the native X chromosome, similar to the foci on the stDp2 duplication (Fig. 4B). We confirmed that binding to Dpy-27_16 is severely compromised on the duplication using FISH and IF (Fig. 4C). A probe that detects focus Dpy-27_16 marked two loci in wild-type cells, both within the DPY-27-bound territory. In contrast, in the duplication strain, the probe detected a single location, outside the DPY-27 bound territory, confirming reduced or absent binding of DPY-27 to this focus in the duplication strain.

Not all foci bind the DCC less well when removed from the X chromosome. Several foci near the right end of the X chromosome are present on the mnDp1 duplication attached to chromosome V. We analyzed DCC binding to these foci in a strain that was also homozygous for mnDf1, a deletion that removes the portion of X duplicated in mnDp1 (Fig. 5A). Only animals heterozygous for the mnDp1 duplication are viable, and real-time PCR measurement of input DNA levels showed the right end of the X chromosome was present at half the level of the rest of the X chromosome in the mnDp1/+; mnDf1 strain, as expected (Fig. 5A). Unlike binding to foci on the mnDp30 and stD2 duplications, the DCC binds foci on the mnDp1 duplication just as efficiently as it binds these sequences on the native X chromosome in wild-type cells. These results are consistent with earlier studies showing strong DPY-27 binding to mnDp1 by IF (4). These analyses clearly indicate that two classes of foci exist—those that can recruit the DCC when detached from the X chromosome (rex sites) and those that only bind high levels of the DCC when attached to the endogenous X chromosome.

We refer to foci that bind high levels of the DCC only on the intact X chromosome as "way stations," since they appear to store large amounts of DCC on the native X but do not recruit large quantities of the complex de novo. According to our model (Fig. 6), high levels of DCC binding are observed at way stations only when the complex can spread in cis from rex sites. If a duplication does not contain a rex site, DCC binding is severely reduced at all way stations. Based on the analyses described above, we conclude that the Dpy-27_16, Dpy-27_17, Dpy-27_18, and Dpy-27_19 foci are all way stations and that the mnDp1 duplication must contain at least one rex site.


Figure 6
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  		FIG. 6. Summary and model for DCC recruitment and spreading. Our data suggest two classes of strong DCC-binding elements exist on the X chromosome: sites that must be attached to the native X chromosome to bind high levels of DCC (way stations) and sites that bind high levels of DCC regardless of chromosomal context (rex sites). ChIP experiments indicate that the four previously identified sites of strong DCC binding between 6.29 and 9.46 Mb on the physical map function as way stations, while at least one of the sites of strong DCC binding between 14.81 Mb and the right end of the X chromosome must function as a rex site. We propose that the DCC is initially recruited to rex sites and that way stations aid in the spreading of dosage compensation away from these sites of DCC recruitment to encompass the entire X chromosome. It is currently unclear what distinguishes a way station from a rex site molecularly, although differences in the DNA sequence to which the DCC binds, local chromatin modifications, and/or nearby genetic elements may all play a role.

Although we have only identified four way stations, we can use this information to test several hypotheses. Previous studies identified a DNA sequence motif within DPY-27 foci and rex sites, but only 42% of foci actually contain the consensus motif (10, 25). This observation led us to the hypothesis that true rex sites contain the motif and way stations do not. However, our current data do not support this hypothesis, since all four way stations contain at least one binding motif (Matrixscan, P ≤ 10–5), and both the Dpy-27_16 and the Dpy-27_18 foci contain at least one near-perfect binding motif (Matrixscan, P ≤ 10–6), making them the fifth and seventh highest in terms of quality motifs present in the foci (10). We also tested the hypothesis that foci that bind the most DCC on the X will be true recruiting elements. As shown in Fig. 2, however, the Dpy-27_16 and Dpy-27_18 foci bind more DCC than any of the other peaks we examined, making it unlikely that amplitude of DCC binding has a significant correlation with foci that act as recruiting elements or as way stations. Finally, we tested the hypothesis that the strong DCC binding observed at way stations was a secondary consequence of converging transcription at these foci. Foci are over-represented at sites of converging transcription (10), a finding consistent with data showing that yeast RNA polymerases can push the related cohesin complex along the DNA (15, 21). However, the genetic architecture surrounding Dpy-27_16 to Dpy-27_19 is inconsistent with this hypothesis, since Dpy-27_16 is inside the mom-1 transcription unit, Dpy-27_17 is downstream of two genes, Dpy-27_18 is upstream of two genes, and Dpy-27_19 is downstream of one gene and upstream of another. Analysis of larger number of way stations will be needed to conclusively prove, or rule out, these and other emerging hypotheses.

It is informative to compare our model presented above to models of binding and spreading of the dosage compensation machinery in other systems. In mammals, X chromosome inactivation is initiated from a single locus on the chromosome, the X-inactivation center (XIC/Xic). The XIST gene, encoding a functional RNA essential for the initiation of X inactivation, is located in this region (reviewed in reference 29). As X inactivation is initiated, XIST/Xist is transcribed locally from the XIC/Xic, and the RNA subsequently spreads bidirectionally to cover the entire chromosome (3). When a portion of the X not containing the XIC/Xic region is translocated to an autosome, this chromosomal region will not be bound by Xist and will not be subjected to dosage compensation. However, when the X-inactivation center is moved adjacent to autosomal sequences (either on X-autosomal translocations, or Xic transgenes inserted on autosomes), variable and incomplete spreading into the autosomal region has been observed (8, 20, 30, 32, 33, 35). These types of observations led to the "way station" hypothesis, originally proposed by Gartler and Riggs (12). According to this hypothesis, booster elements along the X chromosome facilitate spreading of the inactive state from the X-inactivation center. A repetitive DNA element enriched on the X chromosome would be an ideal candidate to serve as way stations. LINE-1 (L1) elements, comprising one such repeat, were subsequently proposed to function as way stations (24). Although mammals and worms differ in the number of dosage compensation initiation sites (one for mammals, multiple for worms), the DCC way stations identified herein may serve a role analogous to the proposed mammalian booster elements in facilitating the spread of dosage compensation from a limited number of initiation sites.

Flies also use multiple classes of cis-acting sequences to achieve the same goal: the binding of DCC to an entire chromosome. According to one of the models, the X chromosome contains multiple classes of DCC-binding sites (as in worms); these sites differ not so much in their ability to recruit the complex, but in their affinities for the DCC (as opposed to our proposed model in worms) (5, 6, 11, 14). High-affinity sites are thought to create a high local concentration of DCC to facilitate binding to nearby lower-affinity sites (5, 14), which bind the DCC in a transcription-dependent manner (18, 19). A recent study (2) proposed an alternative model based on the discovery of a sequence motif found in the highest peaks of DCC binding. When these sequences are inserted into autosomes, they direct local spreading into neighboring chromatin (2). The authors of that study propose a two-step model of DCC binding to X: sequence-dependent recruitment, followed by sequence-independent local spreading.

Based on the fly model and on our data, it is possible to postulate an alternative mechanism for DCC binding in worms, in which different classes of DNA elements with a range of affinities for the DCC bind the complex. According to this model, low-affinity sites (our proposed way stations) would only bind the DCC when located nearby a high-affinity site. Although we cannot conclusively rule out this alternative model, we favor the way station model for the following reasons. First, in Drosophila male cells, DCC binding patterns on the X portion of X-A translocations are identical to the pattern observed on the native X chromosome, indicating that a large chromosomal translocation provide a "chromosomal context" similar to the native X chromosome (11, 28). This is clearly different from our results, where X-chromosome duplications of several megabases in size differ from the X chromosome in DCC-binding ability. Second, in flies, sites with the ability to bind the most amount of DCC correspond to sites with recruitment ability (2). Again, this result contrasts with our findings in worms, where regions containing near-perfect consensus motifs and corresponding to the highest peaks of DCC binding do not recruit the complex de novo. To further refine the worm model, it will be necessary to analyze DCC binding to a large number of sites in different contexts and with different concentrations of DCC available.

Binding of the dosage compensation machinery to X chromosomes serves as a paradigm for many models of domain-specific transcriptional regulation. Our work shows that in the worm at least two different classes of DCC-binding elements play a role recruiting and spreading of a complex that exerts domain-specific gene regulation. It is currently unclear what distinguishes a way station from a recruiting element on an X site. The fact that similar consensus DCC-binding motifs were identified in two independent analyses strongly suggests a role for this DNA sequence, but this is clearly not the whole story (10, 25). Other factors such as histone modifications, subnuclear localization, and other functional DNA sequences may also play important roles in DCC binding and spreading. A more thorough analysis of which sites of strong DCC binding on the X can recruit when detached and which ones act as way stations would help identify additional factors that influence DCC recruitment and spreading. Our work raises new questions about the mechanism by which protein complexes bind and spread to cover large chromosomal domains and suggests that the recruitment and spreading steps of widespread transcriptional regulation may involve distinct sets of cis-acting sites.


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ACKNOWLEDGMENTS
 
We thank Sevinc Ercan and members of the Csankovszki lab for critical reading of the manuscript.

The strains used in this study were provided by the C. elegans Genetics Center, which is funded by the NIH National Center for Research resources. This study was supported by a National Institutes of Health grant R01 GM079533 to G.C. and a Biological Sciences Scholars Program grant at the University of Michigan to G.C.


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FOOTNOTES
 
* Corresponding author. Mailing address: University of Michigan, MCDB, 830 N. University Ave., Ann Arbor, MI 48109-1048. Phone: (734) 764-3412. Fax: (734) 647-0884. E-mail: gyorgyi{at}umich.edu Back

{triangledown} Published ahead of print on 2 February 2009. Back

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/. Back

{ddagger} Present address: Stanford University Medical Center, 279 Campus Dr., Stanford, CA 94305-5323. Back


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Molecular and Cellular Biology, April 2009, p. 2023-2031, Vol. 29, No. 8
0270-7306/09/$08.00+0     doi:10.1128/MCB.01448-08
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