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Genetics, Vol. 172, 963-974, February 2006, Copyright © 2006
doi:10.1534/genetics.105.045286
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* Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, Novosibirsk 630090, Russia and Howard Hughes Medical Institute, Harvard-Partners Center for Genetics and Genomics, Harvard Medical School, Boston, Massachusetts 02115
2 Corresponding author: Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, Lavrentjeva 10, Novosibirsk 630090, Russia.
E-mail: zhimulev{at}bionet.nsc.ru
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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On the polytene male X, the DCC is localized in 
300 discrete regions of decompacted chromatin, referred to as interbands (KELLEY et al. 1999). Still, the DCC-binding pattern is far from uniform, since many chromosomal regions were mapped and shown to be reproducibly devoid of DCC binding (DCC gaps) (BAKER et al. 1994; DEMAKOVA et al. 2003). In general, one of the fundamental questions of the dosage compensation mechanism is what are the factors that determine association of the functional complex with the whole set of its targets, on the one hand, and arrest it in distinct regions on the other hand. Of hundreds of DCC-binding sites along the male X chromosome 70 associate with an incomplete complex (LYMAN et al. 1997; GU et al. 1998; DEMAKOVA et al. 2003). These were proposed to be DNA targets serving as chromatin entry sites (CESs) and playing a key role both in recruitment of DCC to the X chromosome and in subsequent complex spreading to all additional sites (LYMAN et al. 1997; KELLEY et al. 1999; PARK et al. 2002). Further studies on DCC binding to the X under varying levels of MSL2 have led to the idea that this process is directed by a hierarchy of target sites displaying different affinities for the complex (DEMAKOVA et al. 2003). Nevertheless, this model accepts that, finally, upon achievement of high DCC titers, the CES might provoke complex spreading into adjacent low-affinity sites. Unfortunately, it still remains unknown what CESs are in molecular terms. Moreover, some authors suggest that CESs do not possess any special molecular properties except their high affinity to the complex (FAGEGALTIER and BAKER 2004).
It was shown earlier that roX RNAs, as well as histone acetyltransferase and ATPase activities of MOF and MLE, respectively, are needed for recruiting the DCC to multiple non-CES binding sites (GU et al. 2000; PARK et al. 2002). Recently, it was reported that the DCC-binding pattern on the male X reflects the distribution of genes, which are active in the tissue at this moment of development (SASS et al. 2003). Thus, the transcriptional activity of a locus could be determinative for DCC binding to most of its targets. The same idea has also been used to explain discontinuous DCC spreading patterns from autosomal roX transgenes (KELLEY et al. 1999; SASS et al. 2003).
To test this hypothesis and to further understand the requirements of additional non-CESs for DCC targeting, we compared the binding patterns of DCC and of PolIIo, the form of RNA polymerase II engaged in efficient transcription (WEEKS et al. 1993; KOMARNITSKY et al. 2000; CHENG and SHARP 2003). Productive transcription is characterized by the phosphorylation of the carboxy-terminal domain (CTD) at serine 5 and 2 in the largest subunit of PolII and the recruitment of a number of elongation and RNA-processing factors (HAMPSEY and REINBERG 2003; PALANCADE and BENSAUDE 2003). In this study, we make use of antibody H14, which recognizes PolII molecules carrying the phosphoserine 5 epitope. Since phosphorylation of serine 5 is required to initiate elongation complex assembly (KOMARNITSKY et al. 2000; PALANCADE and BENSAUDE 2003) and is found in vivo distributed across the entire transcription unit (SCHWARTZ et al. 2003), the H14 staining patterns mark entire active areas along the polytene male X.
We tracked visible changes of PolIIo and DCC distribution through larval development both on the male X and on an autosomal region with DCC spreading from a roX1 transgene. Temporal changes in the expression of distinct genes active in the salivary gland are accompanied by puffing of distinct chromosome regions in well-described patterns that mark the steps of development through larval and pupal stages (ASHBURNER et al. 1974; reviewed in ZHIMULEV 1999). Therefore, we were able to compare binding patterns of the proteins with the main visible developmental changes of gene expression.
We found that transcriptionally active areas can be revealed within all the DCC gaps, and in some cases these corresponded to the ecdysone-induced puffs. While the PolIIo appeared dynamic and predominantly associated with puffs, DCC was reproducibly absent from them and generally demonstrated surprising stability of binding patterns on the male X during larval developmental stages. Moreover, in the regions where both proteins are abundant, complete colocalization at the electron microscopy (EM) level appears not to be a rule. We also found that in the ectopic DCC spreading system, similarly to the wild-type male X, a number of transcriptionally active regions are reproducibly devoid of DCC binding. These data combined indicate that many active genes escape DCC binding, suggesting that the transcriptional activity could be necessary but not sufficient for DCC recruitment and stabilization at all its targets.
In this study, we also tested the expectation that, in addition to its proposed function in assembling roX RNAs into DCC (MELLER et al. 2000), the RNA-helicase MLE might direct the complex to active genes by associating with nascent transcripts (RICHTER et al. 1996; STUCKENHOLZ et al. 1999). Since both the data on its localization on polytene chromosomes and the interpretations of these data are still contradictory (KURODA et al. 1991; LEE et al. 1997; BHADRA et al. 1999; RUIZ et al. 2000), we set out to map all MLE targets in the nucleus. To this end, we separately investigated the distribution through larval development of MLE in both sexes in a range of wild-type and mutant backgrounds as well as upon heat-shock treatment. Our data indicate that, separately from the DCC, MLE associates dynamically with all puffs, thus resembling the PolIIo distribution. This type of puff stage-dependent binding is demonstrated by MLE on female X chromosomes, on autosomes of both sexes, and, surprisingly, in puffs developing within some DCC gaps on the male X chromosome—that is to say, whenever it functions independently of the other MSL proteins. Thus, it is tempting to assume that MLE might contribute to the general mechanisms of transcription regulation and RNA processing in addition to its still-unknown functions in dosage compensation.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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To generate males with extensive DCC spreading from the autosomal roX transgene, we utilized two stocks: y w roX1– roX2– cos4D; CyO, GM roX1/+ (PARK et al. 2002) and y w roX1ex6; GM roX1-
DHS-72D (BAI et al. 2004). The females of genotype y w roX1– roX2– cos4D; +/+ were crossed to the males from the second stock. The sons of the genotype y w roX1– roX2– cos4D; GM roX1-DHS-72D were used for cytological analysis.
All crosses to generate larvae for immunostaining were carried out at 18°. A laboratory stock of Drosophila simulans was used.
Staging of larvae:
Each developmental stage of third instar larvae or prepupae displays a specific and exclusively constant puffing pattern. To classify a puff stage (PS) correctly, we used a detailed schedule of puff changes in ontogenesis, which was established earlier (reviewed in ZHIMULEV 1999) and is routinely used for this purpose.
Immunofluorescent staining:
The immunostaining procedure was as in KURODA et al. (1991) and as in DEMAKOVA et al. 2003). Both primary affinity-purified rabbit anti-MSL2 and anti-MLE antibodies were used at a dilution of 1:100, and anti-MSL1 was used at 1:50 and detected with a 1:150 dilution of fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG secondary antibodies (Sigma, St. Louis). Primary affinity-purified goat anti-MLE antibodies were used at a 1:150 dilution and detected with a 1:400 dilution of FITC-conjugated rabbit anti-goat IgG secondary antibodies (Sigma). Primary affinity-purified goat anti-MSL3 antibodies were used at a 1:50 dilution and detected with a 1:500 dilution of Cy3-conjugated donkey anti-goat IgG secondary antibody (Rockland, Gilbertsville, PA). Primary monoclonal H14 mouse antibodies against the CTD of RNA polymerase II phosphorylated at Ser5 (Covance) were used at a dilution of 1:50 and detected with a 1:250 dilution of FITC-conjugated goat anti-mouse IgM secondary antibody (Sigma). For double-staining experiments, the antibodies raised in different hosts were incubated simultaneously overnight at 4° in a humidified chamber. After primary antibody incubation, slides were thoroughly washed in PBT and subsequently incubated with the secondary antibodies, first specific for one antigene and then specific for the other (2 hr each). Chromosomes were viewed using epifluorescent optics with the Olympus microscope (Japan) or with Axioscope 2 plus (Zeiss). Images were obtained and treated using the corresponding software: DPController 1.2.1.108 for Olympus and ISIS–CPD1E for Axioscope.
The descriptions of protein-binding patterns are based on the data obtained from at least five slides (100–120 nuclei in total) in each experiment.
Heat-shock treatment:
To obtain heat-shocked salivary glands, third instar larvae were collected in a polypropylene tube and submerged in a 37° water bath for 30 min. Squashes were made and immunostained as described above.
Electron microscopy double immunostaining:
For these experiments the transgenic w; msl3 [w+; H83M2-61]/TM6, Tb females were used, since their two X chromosomes in most of the regions demonstrate the DCC pattern resembling that of the wild-type males (DEMAKOVA et al. 2003) but display the morphology, which is more convenient for electron microscopy analysis (SEMESHIN et al. 2002). The immunostaining procedure was as in SEMESHIN et al. (2002) with minor modifications. Primary affinity-purified rabbit anti-MSL2 antibodies and primary monoclonal H14 mouse IgM antibodies against the phosphorylated at Ser5 CTD of RNA polymerase II (Covance) were used at a dilution of 1:50. For EM colocalization experiments, the antibodies were incubated simultaneously overnight at 4° in a humidified chamber. Then slides were washed four times for 5 min in PBT and then incubated at room temperature for 3 hr with a mixture of secondary antibodies: anti-mouse IgM FITC-conjugated (developed in goat) (1:250) and anti-rabbit IgG Gold (6 nm) conjugated (developed in donkey) (1:30) (Jackson ImmunoResearch, West Grove, PA) antibodies. After incubation with secondary antibodies, the slides were washed with double-distilled water not fewer than six times (3 min each), and then chromosomes were treated with Silver Enhancement reagent (Boehringer Mannheim, Indianapolis) for 20 min to increase the size of gold particles. Thereafter, slides were washed six times (3 min each) with double-distilled water and incubated with anti-goat IgG Gold (18 nm) conjugated antibodies (developed in donkey) (1:30) (Jackson ImmunoResearch) for 3 hr at room temperature, thoroughly washed in PBT and double-distilled water, dehydrated in a graded ethanol series (20, 35, 50, and 70%) for 5 min in each, and left overnight in a 1.5% solution of uranyl acetate in 70% ethanol for staining. Further procedures of dehydration and embedding in epoxy resin have been described elsewhere (SEMESHIN et al. 1998). Ultra-thin sections were examined in a JEM-100C electron microscope at 80 kV.
To localize chromosome regions, we referred to the revised cytological maps of polytene chromosomes of C. B. Bridges (represented in LINDSLEY and ZIMM 1992).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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PS5 (FURIA et al. 1993) manifests as a puff. While the DCC is reproducibly absent from this region during the whole third instar, the PolIIo pattern appears dynamic and varies from intensive diffuse labeling of the puff material at PS1-2 to two weak sites at PS11 (Figure 2, A–D). It should be noted that Sgs4 is known to be dosage compensated (BREEN and LUCCHESI 1986; KAISER et al. 1986; CHIANG and KURNIT 2003). In addition to the prominent puffs, there are several dozens of so-called "small puffs" on the X chromosome, which do not form any significant swelling of the polytene chromosome in the corresponding region (BELYAEVA et al. 1974). For example, such a small puff is described to form in the region 5A1 at PS11, where the two adjacent bands split. PolIIo diffusely paints the puff material; however, no specific MSL binding sites are observed in this region throughout the larval stages, and a typical DCC gap is formed (Figure 2, E–I). A number of other regions on the X chromosome that undergo puff formation during the third instar stage, such as 2C1-2 (Figure 3A), 9CD, and 16B (data not shown), also appear completely devoid of DCC.
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In general, in contrast to the PolIIo distribution, the DCC pattern looks stable over the developmental stages analyzed (region 3A-D in Figure 2, B and C). Some of the faint DCC-binding sites bordering some gaps, as exemplified by the regions 3C7-11 or 6EF, may appear undetectable in some nuclei of a single individual.
Discontinuous spreading of the DCC from a roX1 transgene does not fully reflect localization of active genes in the flanking autosomal region:
We asked whether DCC chooses active genes when it spreads locally from autosomal roX transgenes. To generate the most efficient DCC spreading in all nuclei, we tested male larvae, which carry the GM roX1-DHS-72D transgene in a roX1–roX2– double-mutant background (PARK et al. 2002; BAI et al. 2004). We found that a large number of good Po1IIo-binding sites, including some of ecdysone-induced puffs, do not associate with the DCC (Figure 4, A and C). The DCC spreading patterns are well known to be variable from nucleus to nucleus, although we failed to confirm this variability to be PS dependent (Figure 4, B and D). It should be noted that in the region 63BC the DCC binds one site, which was found to associate often with the complex in wild-type males (DEMAKOVA et al. 2003) and never binds to neighboring sites regardless of their active transcriptional status (Figure 4, A–D). Thus, similarly to the male X, the patterns of ectopic DCC binding do not directly reflect the localization of active transcriptional domains in autosomes carrying a GM roX1-DHS-72D transgene.
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Upon closer examination of the well-stretched chromosomes, we managed to reveal also a number of chromosomal regions, in which the signals tend to show only partial overlap or to border each other (Figures 1 and 2, A–D). To determine precisely the relative localization of DCC and PolIIo within distinct small chromosomal regions, we carried out EM double immunostaining. Figure 5 shows data obtained for the 6A-D and the 8E-10A regions. Earlier it was shown that MSL2 tends to demonstrate unipolar labeling of narrow band/interband borders along the whole male X (SEMESHIN et al. 2002). Here we found that this specific localization of MSL2 looks different from PolIIo binding to decompacted interbands (Figure 5, C–F). The protein labels look partly overlapping in some regions (Figure 5, D–F) but localize apart in others (Figure 5C). Thus, EM data also indicate that relative localization of PolIIo and MSL2 within a distinct interband may be very close but still not identical.
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Although MLE is the first DCC subunit that has been immunolocalized on the polytene chromosomes, it is the only one of the MSLs whose detailed localization still remains unclear. MLE was first reported to bind predominantly the male X chromosome and additionally to demonstrate numerous faint sites of association on male autosomes and all female chromosomes, these autosomal targets being uncharacterized (KURODA et al. 1991). Later, BHADRA et al. (1999), using the same MLE-specific antibody, restricted male MLE binding exclusively to the X chromosome. At the same time, in females, they reported msl-dependent MLE binding to all chromosomes, and this finding catalyzed an idea about the existence of a reduced MSL complex in females that might contribute to an inverse dosage effect (BHADRA et al. 1999). Finally, recently it was shown that in addition to labeling the X chromosome, MLE binds only a few autosomal sites in males, similar to other MSLs (RUIZ et al. 2000).
To gain further insight into possible roles of MLE in transcription regulation, we performed precise mapping of MLE-binding sites in both sexes at different points of larval development. First, we noted that MLE autosomal distribution looked identical in both sexes. As demonstrated in Figure 6, A and B, the RNA-helicase MLE binds essentially to all the developmental puffs typical for the PS analyzed and to many interbands. The observation that MLE binds puffs also is true for the female X chromosome (Figures 3F and 6B). Diffuse PS-dependent binding sites on female X look quite different when compared with the reproducible banded pattern in males. Also, we found that several regions (18F, 19A, 22B, two sites in 29C, 30A, 50C, and 86C) reproducibly demonstrate intensive MLE staining, some of them appearing PS specific (Figure 6, A and B). To illustrate, the strong MLE-binding site in the region 30A can be detected only at PS1, whereas the one in the region 29C appears exclusively at the PS8-9. Thus, when associating with the DCC, the MLE distribution pattern on the male X is highly reproducible, whereas its MSL-independent chromosomal binding appears to be essentially a function of the transcriptional status of the region, resembling the PolIIo pattern. Additionally, we observed punctate MLE staining of the chromocenter with the intensity of staining varying from nucleus to nucleus (Figure 6, A, B, and D), as it was shown earlier for the species from the Obscura group (BONE and KURODA 1996). Under the staining conditions used, no MLE-binding sites could be detected in mle1/mle1 females (data not shown).
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MLE associates with heat-shock-induced puffs and apparently shows increased affinity to the 
-element, which produces the noncoding heat-shock RNA:
Upon brief exposure to heat shock, the MLE protein can be found in many heat-shock-induced puffs. In particular, we observed a very robust permanent signal in the region 87C. Strong labeling could also be seen in the puff regions 93D and 95D, but never in the region 87A (Figure 7, A and B). The two adjacent puffs 87A and 87C are known to develop due to the activity of a cluster of hsp70 genes (ISH-HOROWICZ et al. 1979). However, in contrast to the 87A region, the 87C region contains not only the hsp70 cluster, but also the -element, coding for the hshRNA with unknown functions (SHARMA and LAKHOTIA 1995). Since we never observed any MLE-binding site in region 87C in both sexes under non-heat-shock conditions, we speculate that this particular hshRNA, which belongs to the same class of RNA molecules as the roX RNAs, might be the target for MLE. To test the idea, we utilized D. simulans, a sibling species for D. melanogaster, which lacks the sequences homologous to the -element in this region (LIS et al. 1981). Consistent with this proposal, when staining the polytene chromosomes from D. simulans heat-shocked larvae with MLE-specific antibodies, we failed to detect labeling of the 87C region (Figure 7C). It should be noted that -element RNA sequence, when compared to that of the roX1 or roX2, produced no significant homologies (http://www.ncbi.nlm.nih.gov/blast/).
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Thus, independently of other MSL proteins, MLE interacts with the most prominent transcriptionally active regions of chromosomes and likely shows increased affinity to the transcripts of certain genes.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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35) that recruit the DCC and accumulate locally at high levels, which in turn results in association of the complex with numerous sites of low affinity (KELLEY et al. 1999; KAGEYAMA et al. 2001). Nevertheless, the modern view of the problem assumes that there might be many more DNA sequences required both for the initial recruitment/assembly of DCC (CES) (DEMAKOVA et al. 2003) and for the association of a functional complex with additional sites (non-CES) (FAGEGALTIER and BAKER 2004; GILFILLAN et al. 2004; OH et al. 2004). In contrast to these postulated DNA sequences, most sites on the X, which are targets for functional DCC, are thought to mark genes actively transcribed in a given tissue and time of development (KELLEY et al. 1999; SASS et al. 2003). This idea implies that DCC mediates transcription enhancement via direct involvement in transcription regulation of each active gene. In this article, we made an effort to test further this model by precisely investigating the relative localization of DCC and PolIIo along the male X in the course of larval development. Previously it was described that in vivo PolIIo and various elongation factors, as well as the H3.3 histone variant, dynamically associate with active genes, accompany their expression, and look colocalized in Drosophila polytene chromosomes. This overlap is most obvious as diffuse labeling of developmental and heat-shock-induced puffs (WEEKS et al. 1993; KAPLAN et al. 2000; GERBER et al. 2001; SAUNDERS et al. 2003; SCHWARTZ et al. 2003; SCHWARTZ and AHMAD 2005). Intriguingly, we found that DCC demonstrates striking stability in both the number and intensity of binding sites along the X throughout larval development. Moreover, despite being targets for MLE, the sites of the most intensive gene expression both on the male X and within an autosomal DCC-spreading area appear to not be targets for DCC at all. We also demonstrated all the DCC gaps that comprise active genes. Additionally, DCC skips over a number of transcriptionally active regions when it inappropriately spreads in cis from an autosomal roX1 transgene. We therefore suggest that active transcriptional status of the chromosomal region or association with MLE is not sufficient for DCC targeting and that, if DCC binds to actively transcribed regions, it does so very selectively.
Our findings on MLE localization on the polytene chromosomes might reflect dual functioning of MLE on the male X. In addition to being a subunit of DCC, MLE probably accomplishes some unrelated functions, which are neither X nor sex specific. On the basis of the observed MLE association with a large number of sites of active transcription in both sexes, we believe that this RNA-helicase might be important not only for splicing certain genes, as was shown for the gene para (REENAN et al. 2000), but also for playing some general role in the transcription process. In support of this idea, the mammalian MLE homolog, RHA, was demonstrated to contribute to various steps of transcription—from initiation to processing of nascent transcripts (ZHANG and GROSSE 2004). Assuming that MLE apparently is able to bridge DCC with transcriptionally active regions, this might occur only if some additional requirements for DCC binding are realized.
Earlier, it was reported that a partial MSL complex lacking MSL2 protein is present in normal female nuclei. Accordingly, mutations in various msl genes except mof disassociate all MSLs from the chromosomes in females (BHADRA et al. 1999). Nevertheless, our data on MLE distribution in polytene chromosomes of females homozygous for msl1 or msl3 null alleles indicate that even if MSLs form a partial complex in females, MLE binds the chromatin in an MSL-independent manner.
Our findings raise questions as to what are the reasons for exclusion of DCC from some active X-linked regions and whether dosage compensation does take place there. One can speculate that highly active chromatin in puffing regions turns into a poor substrate for the DCC due to drastic changes in packaging, possibly, up to nucleosome removal (ORPHANIDES and REINBERG 2000; KIREEVA et al. 2002). However, a cluster of CESs bound by DCC is detected in the puffed 2B region throughout larval development. Moreover, strong transcription induced in EP transposons on the male X sometimes results in ectopic DCC recruitment, suggesting that the complex is able to recognize very active chromatin (SASS et al. 2003).
Revealing active genes within each of the cytologically extensive DCC gaps provides yet another puzzle. On the one hand, in the neo X chromosome of D. miranda, the blocks of chromatin escaping dosage compensation do alternate with other blocks that are dosage compensated and therefore bind DCC (BONE and KURODA 1996; MARIN and BAKER 1998). However, no data indicate that such clustering takes place in D. melanogaster (GHOSH et al. 1992). If X-linked genes actually possess still unknown features needed for DCC targeting (FAGEGALTIER and BAKER 2004), then DCC gaps might reflect evolutionary incompleteness of this process in D. melanogaster. It should be noted that, in contrast, 70 autosomal regions are competent to recruit functional DCC in wild-type males (DEMAKOVA et al. 2003). Alternatively, the active genes located within the DCC gaps might serve as targets for the complex but cannot realize this ability probably due to the chromatin environment.
Whether the genes within puffs and DCC gaps undergo dosage compensation remains to be answered. It seems plausible to suggest that transcription upregulation could be not so essential for a subset of highly expressed genes. Nevertheless, whatever the reasons for highly expressed loci to escape association with DCC, these genes are probably dosage compensated, which was shown at least for Sgs4 and the Broad-Complex (BREEN and LUCCHESI 1986; KAISER et al. 1986; CHIANG and KURNIT 2003). If many active genes lack DCC-binding sites in the immediate vicinity, they might achieve dosage compensation by a yet unknown pathway. It is very possible that upregulation of active genes within DCC gaps and puffing regions might be achieved, at least to some extent, via DCC-mediated establishment of a more open chromatin structure of the whole male X, suggesting that DCC affects transcription indirectly. Site-specific localization of H4Ac16 probably initiates a cascade of molecular remodeling events resulting in diffuse appearance of the whole male X chromosome (BONE et al. 1994). Generally, such a chromatin state would facilitate the access of various transcription and replication factors. Accordingly, in females having ectopic dosage compensation induced, the DCC gap corresponding to the intercalary heterochromatin region on the polytene X demonstrated a greater extent of both polytenization and replication than in the wild type. This clearly correlated with the higher local concentrations of the DCC in neighboring areas (ALEKSEYENKO et al. 2002). Thus, despite the fact that the DCC-mediated site-specific histone acetylation pattern correlates with an increase in transcription of the underlying sequences (AKHTAR et al. 2000; HENRY et al. 2001; SMITH et al. 2001), we believe it would be more accurate to suggest that there is no common scenario of dosage compensation for all the X-linked genes. Also, the DCC pattern appears essentially permanent and displays only negligible variations, both in the course of larval development (our data) and in different tissues (SASS et al. 2003), which might point to the contribution of yet unidentified epigenetic factors in the establishment and maintenance of DCC binding. For example, the transcriptional activity of X-linked genes could govern DCC settling on the male X in early embryogenesis, and this pattern might be subsequently reproduced epigenetically. Hence, this scenario would imply high stability of the DCC pattern at least for the housekeeping genes, rather than dramatic changes in DCC distribution resulting from fine-tuned transcriptional programs further in development.
Future molecular studies of the dosage compensation status of active genes mapping to the DCC gaps could determine whether dosage compensation can also utilize some unknown mechanisms other than site-specific acetylation of H4 at lysine 16 leading to site-specific transcription enhancement. Alternatively, there may be many more X-linked genes whose expression does not require dosage compensation than was expected to date. Regardless, the important question remains how functional DCC recognizes its targets among the active genes on the male X chromosome.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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