InteractiveFly: GeneBrief

Painting of fourth: Biological Overview | References

BIOLOGICAL OVERVIEW

In Drosophila, two chromosome-wide compensatory systems have been characterized: the dosage compensation system that acts on the male X chromosome and the chromosome-specific regulation of genes located on the heterochromatic fourth chromosome. Dosage compensation in Drosophila is accomplished by hypertranscription of the single male X chromosome mediated by the male-specific lethal (MSL) complex. The mechanism of this compensation is suggested to involve enhanced transcriptional elongation mediated by the MSL complex, while the mechanism of compensation mediated by the painting of fourth (POF) protein on the fourth chromosome has remained elusive. This study shows that POF binds to nascent RNA, and this binding is associated with increased transcription output from chromosome 4. Genes located in heterochromatic regions spend less time in transition from the site of transcription to the nuclear envelope. These results provide useful insights into the means by which genes in heterochromatic regions can overcome the repressive influence of their hostile environment (Johansson, 2012).

Aneuploidy of entire chromosomes and chromosome segments is an important evolutionary driving force that increases variation but is accompanied by problems associated with changes in gene dosage and genomic instability. The evolution of buffering systems that compensate for dosage differences must therefore allow for a balance between allowing genomic variability and avoiding genomic instability. Buffering systems of this kind have been described in Drosophila; in haploid conditions, they cause the transcription output to increase by a factor of approximately 1.4. However, the evolution of heteromorphic sex chromosomes, such as the X and Y chromosome pair in flies and mammals, is accompanied by an expression problem that requires more extensive compensation. Since most genes on the X chromosome should be expressed at the same levels in males and females, dosage compensation mechanisms coevolve as the X-Y chromosome pair is formed. Notably, while the ancient homology between the mammalian X and Y is clear, the evolutionary origin of the Drosophila Y is more complicated. The evolution of dosage compensation mechanisms is attributable to evolutionary pressures that act at all levels of expression to compensate for the losses of functional gene copies. Two dosage compensation systems have been studied in Drosophila: the male-specific lethal (MSL) complex, which targets and upregulates the male X chromosome, and the painting of fourth (POF) protein, which stimulates the expression of the fourth chromosome in Drosophila melanogaster but is believed to have originated from a dosage-compensating mechanism (Johansson, 2007a; Larsson, 2001; Larsson, 2006; Larsson, 2004). The MSL complex and POF coexist; they are on different chromosomes in, e.g., D. melanogaster, but are colocalized on the same chromosome in, e.g., Drosophila ananassae. Their coexistence suggests that they probably act on different levels of gene regulation (Johansson, 2012).

The MSL complex is a ribonucleoprotein complex consisting of five male-specific lethal proteins (MSL1, MSL2, MSL3, MLE, and MOF) and two noncoding RNAs, roX1 and roX2. Because expression of MSL2 is sex restricted, the complex is formed only in males and specifically targets the male X chromosome. A model of its action has been proposed: MOF (a histone acetyltransferase) acetylates H4K16, and this modification leads to decompaction of the chromatin and hypertranscription of the male X chromosome genes. The prevailing idea is that the MSL complex stimulates transcriptional elongation. This idea is supported by genome-wide mappings showing that expression of the MSL complex and the associated H4K16 acetylation are both enhanced within gene bodies with a bias to their 3' end. A recent study (Larschen, 2011) confirmed that the MSL complex enhances transcription by facilitating the progression of RNP2 across the active X chromosomal genes (Johansson, 2012).

Less is known about the regulatory level at which POF acts. POF is a 55-kDa protein containing an RNA recognition motif (RRM). Like the MSL complex, POF binds within the bodies of expressed genes. However, in D. melanogaster, POF specifically targets the fourth chromosome in both males and females. The targeting of POF to the fourth chromosome is associated with a chromosome-specific increase in transcript levels that primarily affects differentially expressed genes (Johansson, 2007a; Stenberg, 2009). Flies can survive without POF or missing one copy of the fourth. However, haplo-fourth animals die if they also lack POF. Importantly, the expression of nonubiquitously expressed genes on the fourth chromosome has been shown to be compensated by POF in haplo-4 flies; suppressing or eliminating this compensation causes haplo-fourth lethality (Johansson, 2012).

The fourth chromosome of D. melanogaster has several unique characteristics. It is the smallest chromosome in the Drosophila genome, with an approximate size of 5 Mb. Of these 5 Mb, 3 to 4 Mb consists exclusively of simple AT-rich satellite repeats and does not contain any known genes. The sequenced part of chromosome 4 is only 1.3 Mb and represents the banded, polytenized, and gene-rich portion corresponding to cytological sections 101E to 102F. In principle, the entire fourth chromosome can be considered heterochromatic; more specifically, it consists of the HP1 enriched 'green chromatin' as defined by van Steensel and coworkers (Filion, 2010). This means that the fourth chromosome is enriched in the heterochromatin protein HP1 and in specific histone modification markers of heterochromatin, e.g., methylated H3K9. In keeping with its heterochromatic nature, the polytenized part of chromosome 4 contains large blocks of repeated sequences and transposable elements that are interspersed with the genes. Transgenes inserted on the fourth chromosome often show partially silenced, variegated expression. In fact, the structure and sequence composition of the fourth chromosome, with its scattered repetitive elements, is more reminiscent of the organization of mammalian chromosomes than that of the other D. melanogaster autosomes. It appears as though the genes located on the fourth chromosome have adapted to function in this repressive milieu (Johansson, 2012).

To study the fundamental compensatory processes acting on the fourth chromosome, RNA immunoprecipitation (RIP) experiments followed by tiling array analysis (RIP-chip) and transcript profiling experiments were performed. This study shows that POF associates with the newly transcribed RNA produced from the fourth chromosome. The data indicate that POF binds to the spliced form of the transcript and that this binding is associated with an increase in the amount of chromosome 4 transcripts. It was also shown that transcripts encoded by the fourth chromosome or pericentromeric heterochromatin have a shorter transition time from the site of transcription to the nuclear envelope (Johansson, 2012).

In Drosophila, two chromosome-wide compensatory systems have been characterized: the MSL complex, which targets and stimulates the expression of the X chromosome in males, and POF, which targets and stimulates the expression of the fourth chromosome (Larsson, 2006; Stenberg, 2011). It has been hypothesized that the MSL complex stimulates expression by facilitating transcriptional elongation, and this hypothesis was recently confirmed experimentally (Larschan, 2011). This study has used RNA immunoprecipitation and transcriptome profiling techniques to further clarify the mechanism by which POF stimulates gene expression (Johansson, 2012).

Previous studies have shown that POF binds to active genes on the fourth chromosome (Johansson, 2011). Since previously reported ChIP-chip results were obtained using cross-linked extracts, they could not be used to determine whether POF associates directly with the chromatin or binds via interactions with other components and was frozen in place by the cross-linking. In the work reported in this study, a relationship was observed between the binding of the POF protein to the chromosome and that of the RNA polymerase. This connection with transcription, together with the fact that POF possesses an RNA binding domain, suggested that POF binds to RNA rather than directly to chromatin. To test this hypothesis, a genome-wide POF RIP-chip experiment was performed. The POF-RIPs verified the association of POF with chromosome 4 transcripts (Johansson, 2012).

Two components of the MSL complex (MSL2 and MOF) were also investigated, both as controls and to determine whether the MSL complex also binds to transcripts from the chromosome it regulates, i.e., the male X chromosome. In contrast to the observed association of POF with transcripts from the fourth chromosome, no unambiguous evidence was found to support any binding of MSL2 or MOF to X-linked transcripts. However, at this point, it cannot be excluded that other components of the MSL complex, especially the more loosely bound MLE, may have a general affinity for transcripts of the X chromosome. A slight reduction was also noticed in the number of transcripts from the fourth chromosome in both the MSL2-RIPs and the MOF-RIPs, suggesting that the general RNA affinity of the MSL complex is less pronounced for the fourth chromosome than for the other chromosomes. This relative reduction in the number of chromosome 4 transcripts associated with the MSL complex might reflect the binding of POF to these transcripts, which could block their association with other RNA binding proteins (Johansson, 2012).

The POF-RIP results, together with the previously reported strong link between POF and the fourth chromosome, suggested that POF binds to nascent RNAs, while these RNAs are still bound to the RNP2. The most parsimonious explanation of the high similarity between the ChIP-chip and RIP-chip profiles for POF is that POF associates with nascent RNA and the ChIP profile is indicative of chromatin linked via RNP2. However, at this point it cannot be excluded that POF in addition to binding nascent RNAs also associates with fully processed mature mRNAs en route to the cytoplasm. It also remains possible that POF, in addition to binding nascent RNA, also associates with chromatin via interactions with other proteins (for instance, HP1). No evidence of physical association between POF and HP1 has so far been reported, but POF and Setdb1 (the HKMT responsible for H3K9me on the fourth chromosome) have been shown to interact in vitro (Tzeng, 2007). Thus, POF may be an element of an adaptor system linking histone marks to nascent RNA via HP1 and Setdb1, in a fashion similar to MRG15 and PTB. The fact that POF binding to chromatin is RNase resistant may be explained by a stabilization of POF via interaction with a chromatin-associated factor like HP1 or Setdb1. The RNase resistance may also be caused by inaccessibility, i.e., the nascent RNA associated with POF is not accessible to the RNase (Johansson, 2012).

POF was observed to bind to RNA from other chromosomes, indicating that it possesses a general affinity for RNA. This association with transcripts other than those from the fourth chromosome is more pronounced in the native samples, suggesting that it occurs during sample preparation as an equilibrium reaction rather than accurately reflecting in vivo associations. It is speculated that during the preparation of the native samples, some POF molecules are released from their normal target sites and become free to associate with any transcript in the nucleoplasm. In contrast, in the cross-linked samples, the in vivo POF binding is 'frozen' before any sample treatment steps are performed. Consequently, POF will only be observed to bind to chromosome 4 transcripts, and the apparent enrichment of RNAs encoded from other chromosomes is lost (Johansson, 2012).

Splicing is commonly regarded as a process that takes place after transcription. However, cotranscriptional splicing was visualized in Drosophila more than 25 years ago (Osheim, 1995). More recent studies have revealed that cotranscriptional splicing is more common than was previously believed and also that splicing can begin as a cotranscriptional event and continue posttranscriptionally (Brody, 2011; Neugebauer, 2002). Therefore, the strong exon bias reported in this study is compatible with POF binding to nascent RNAs. Cotranscriptional splicing is believed to depend on cooperation between exon recognition and the speed of transcription by RNP2. Further, it has been shown that the density of nucleosomes is higher within exons; they may thus function as 'speed bumps' to slow down the RNP2 elongation rate. Since POF is connected to nascent RNAs, this reduction in the speed of transcription over exons would explain the exon bias observed at the chromatin level in ChIP-chip experiments (Johansson, 2012).

Given that POF presumably binds to nascent RNA via its RRM1 domain, it is interesting to consider hypothetical mechanisms by which POF might regulate the expression of genes on chromosome 4. The binding of POF to nascent RNA may directly or indirectly (i.e., via chromatin structure modifications) stimulate transcription. There are several examples of interactions between chromatin-associated proteins recognizing histone modification marks and proteins that bind nascent RNA (Johansson, 2012).

To explore the potential difference in engaged RNP2 distribution on chromosome 4 compared to other autosomes, the previously published GRO-seq data (that maps the position, amount, and orientation of transcriptionally engaged polymerases genome wide) for Drosophila S2 cells was used. That study shows that the male X chromosome has a higher elongation density index (EdI) than the autosomes, which is interpreted as an enhanced transcription elongation. In contrast, comparing the fourth chromosome to all other autosomes, a significantly decreased EdI was found, consistent with a less efficient transcription elongation. The fourth chromosome also shows a decreased pausing index (PI). This is, on the other hand, in line with an increased transcription output from chromosome 4 genes. It is tempting to speculate that the heterochromatic nature of the fourth chromosome, with HP1 enriched over gene bodies of active genes, causes the decreased EdI. Considering that the fourth chromosome is expressed at levels equal to (or slightly higher than) those of the other chromosomes, this elongation disadvantage may be counteracted by a decreased RNP2 pausing. Since POF is bound to in principle all active chromosome 4 genes, it remains elusive whether POF is connected to the observed decrease in chromosome 4 PI (Johansson, 2012).

It is also possible that the binding of POF to nascent RNA has posttranscriptional effects. There are at least three possible posttranscriptional scenarios we must consider: splicing, protection, and transport. If splicing were the main function of POF targeting, we would expect a difference in the transcriptome profiles between Pof mutants and the wild type. However, the transcriptome profiles of Pof mutants are very similar to those of the wild type, and there is no evidence of an increased rate of incorrect splicing or more frequent use of introns in the Pof mutant. The only striking difference between the transcriptome profile of the Pof mutant and that of the wild type is the reduction in the amount of processed transcripts from chromosome 4. The same reduction is observed whether we look at the 5' end, 3' end, or middle part of the genes and is thus less consistent with the hypothesis that chromosome 4 transcripts are more prone to degradation in Pof mutants. This demonstrates that POF has a positive effect on the amount of chromosome 4 transcripts which is not caused by improved splicing efficiency (Johansson, 2012).

Analysis of the ratio of input RNA levels from WCFA (whole cells) to input RNA levels from FA (nuclei) revealed that the relative amounts of chromosome 4 transcripts and transcripts from genes in the pericentromeric region are higher in the cytoplasm than in the nucleoplasm, in relation to transcripts from the other chromosomes. Notably, although export rates per se have not been measured, the results are consistent with pericentromeric and chromosome 4 transcripts being more efficiently exported. Chromatin is highly organized within the nucleus: euchromatic blocks are preferentially located in the center, while heterochromatic regions, such as the fourth chromosome and pericentromeric heterochromatin, tend to localize closer to the nuclear rim. Whether the nuclear periphery is a repressive or permissive environment for gene expression has been debated. The transition time for the export of transcribed mRNAs from the site of synthesis to the nuclear pore would be minimized for chromosomal regions close to the nuclear pore. The gene-gating hypothesis postulates that nucleoporins associate with active genes and facilitate the export of the corresponding mRNAs. Components of the nuclear pore complex (nucleoporins) have been reported to interact with transcriptionally active genes. It has been shown in a mammalian system that transport of an mRNA from the site of transcription to the nuclear pore occurs within a time frame of 5 to 40 min. In the same study, no pileup of mRNAs at the nuclear pore was found, and export through the pore was rapid (0.5 s). Thus, a closer proximity to the nuclear pore may increase transcription output, especially in rapidly dividing cells, since reducing the transition time would allow a more rapid initiation of protein synthesis after cell division. It was observed that the whole cell/nuclei ratio for transcripts produced from the fourth chromosome and the pericentromeric heterochromatin was increased compared to the transcript ratio of the entire genome. It should be stressed that although these genes are located in seemingly repressive environments, both the number of expressed genes and gene expression are comparable to euchromatic chromosome regions. It may be that genes located in these heterochromatic regions benefit from their relative proximity to the nuclear pore, which would facilitate the export of transcribed mRNA. This may in fact be one reason why genes located in pericentric heterochromatin, such as light and rolled, are repressed by rearrangements that move them into euchromatic surroundings (Johansson, 2012).

It is tempting to speculate that the evolution of more efficient logistics for the export of transcripts from the fourth chromosome was driven by the need to facilitate the expression of its genes. This study has also shown that the quantity of chromosome 4 transcripts is reduced in Pof mutants, and the data indicate that this decrease is not caused by splicing defects or increased degradation. It is not yet known whether the binding of POF to nascent RNAs increases the efficiency of transcription or whether it facilitates their efficient export. However, it should be stressed that transcription levels are probably influenced by a number of stimulatory and repressive influences and that during the course of evolution these factors become increasingly interdependent (Johansson, 2012).

HP1a, Su(var)3-9, SETDB1 and POF stimulate or repress gene expression depending on genomic position, gene length and expression pattern in Drosophila melanogaster

Heterochromatin protein 1a (HP1a) is a chromatin-associated protein important for the formation and maintenance of heterochromatin. In Drosophila, the two histone methyltransferases SETDB1 and Su(var)3-9 mediate H3K9 methylation marks that initiates the establishment and spreading of HP1a-enriched chromatin. Although HP1a is generally regarded as a factor that represses gene transcription, several reports have linked HP1a binding to active genes, and in some cases, it has been shown to stimulate transcriptional activity. To clarify the function of HP1a in transcription regulation and its association with Su(var)3-9, SETDB1 and the chromosome 4-specific protein POF, genome-wide expression studies were performed and the results were combined with available binding data in Drosophila melanogaster. The results suggest that HP1a, SETDB1 and Su(var)3-9 repress genes on chromosome 4, where non-ubiquitously expressed genes are preferentially targeted, and stimulate genes in pericentromeric regions. Further, it was shown that on chromosome 4, Su(var)3-9, SETDB1 and HP1a target the same genes. In addition, it was found that transposons are repressed by HP1a and Su(var)3-9 and that the binding level and expression effects of HP1a are affected by gene length. The results indicate that genes have adapted to be properly expressed in their local chromatin environment (Lundberg, 2013a).

Heterochromatin protein 1 is a protein that has been well-studied in many model organisms, including Schizosaccharomyces pombe, mouse and D. melanogaster. Although D. melanogaster HP1a is best known for its role in heterochromatin formation and silencing, several reports have linked HP1a to regulation of transcriptional activity of heterochromatic and some euchromatic genes. This study asked if these conflicting results could partly be explained by a region-specific function of HP1a and the proteins involved in HP1a binding, i.e. SETDB1, Su(var)3-9 and POF. Based on polytene chromosome staining, it was clear that POF, HP1a and SETDB1 overlapped on chromosome 4 but not on the pericentromeric section or on the most distal part of the tip, which was only bound by HP1a. These POF and SETDB1 unbound regions also correspond to regions that are independent of SETDB1 for maintaining a proper H3K9me2 and me3 pattern. In line with previous studies, it was found that Su(var)3-9 binds to chromosome 4 when considering expressed genes, and more interestingly, this binding to active genes is, on average, stronger than the binding of Su(var)3-9 to active genes in the pericentromeric regions, although loss of Su(var)3-9 had minor effects on the methylation pattern of chromosome 4. The putative function of Su(var)3-9 on chromosome 4 therefore remains elusive (Lundberg, 2013a).

In addition to the persistent binding of POF to chromosome 4, it is interesting to note the presence of occasional binding to region 2L:31. It is known that POF binds to HP1a binding sites where HP1a binding is dependent on SETDB1, and since it has been previously shown that binding of HP1a in region 2L:31 is dependent on SETDB1, this could partially explain the sporadic binding of POF in this region. Region 2L:31 displayed similar properties to other euchromatic regions that are unbound by SETDB1 and HP1a. Thus, the reason for the targeting of this particular region remains to be explained (Lundberg, 2013a).

HP1a has long been known for its repressive function. It was initially identified as a dominant suppressor of position-effect variegation and was named Su(var)205, and it has been reported that HP1a represses gene expression on chromosome 4. However, several studies have reported an activating function of HP1a. The current study suggests that these conflicting reports can at least be partly explained by the observation that HP1a has different functions in different regions; chromosome 4 genes are, on average, repressed, whereas pericentromeric genes are stimulated. It is therefore believed that it is important to look at different groups of genes when studying the effects of HP1a. Otherwise, these opposing effects may cancel each other out on a genome-wide level (Lundberg, 2013a).

Nevertheless, the conflicting results cannot be fully explained by the current findings. For example, another study found that transcription was reduced in an RNAi-mediated HP1a knock-down, in contrast to the current results. Therefore, technical differences between experiments should also be considered; in the current study, the possibility of a maternal contribution of HP1a cannot be excluded, as mutants were studied in first-instar larvae from heterozygous mothers, and it is thus likely that we have a reduction in HP1a levels rather than complete removal. It has been shown that maternal HP1a contributes to ~20% of the HP1a protein found in heterozygous mutant first-instar larvae. Previously studies have shown that the average level of gene expression of chromosome 4 is comparable with, or even higher than, that of genes on other chromosomes. At least to some extent, this is a consequence of POF-mediated stimulation of gene expression output, which counteracts the repressing nature of the 4th chromosome (Johansson, 2007a; Stenberg, 2009; Johansson, 2012). It is hypothesized that due to POF and other factors, genes on the 4th chromosome have evolved to be functional in this repressive chromatin environment. A decrease in HP1a is mainly expected to cause a reduction of the low affinity binding of HP1a in the gene body, and consequently a de-repression of gene expression. However, prolonged loss or very strong depletion of HP1a will most probably have dramatic effects on the overall structure of chromosome 4 chromatin, and thus lead to a dysfunctional chromatin structure with decreased gene expression. This implies that the genes have adapted to be properly expressed in the local chromatin environment (Lundberg, 2013a).

Previous studies have shown that POF is involved in stimulating expression of active genes on chromosome 4. The observed effects in the Pof mutant on genes in the pericentromeric regions and region 2L:31 are most likely explained by indirect effects when HP1a is being redistributed from chromosome 4 to other binding sites, as binding of HP1a to chromosome 4 is dependent on the presence of POF (Riddle, 2012; Figueiredo, 2012; Johansson, 2007a). The increased transcriptional output of chromosome 4 genes in the Setdb1 mutant is likely due to loss of the repressive methylation marks, which in turn will reduce HP1a binding. Although it is known that HP1a binding to promoters is independent of methylation marks, it is possible that HP1a binding remains in promoters, where it exerts an activating function (Lundberg, 2013a).

The increased chromosome 4 expression observed in the Su(var)3-9 mutant is surprising but could be explained by indirect effects; it is speculated that when Su(var)3-9 is lost from the pericentromeric regions, SETDB1 is redirected from chromosome 4 to sustain normal H3K9 methylation in the pericentromeric regions, thus decreasing HP1a binding to chromosome 4. This could explain why both the Setdb1 and the Su(var)3-9 mutants give such similar up-regulating effects on chromosome 4 expression. An alternative explanation for this effect is that the observed binding of Su(var)3-9 to chromosome 4 has a yet-unknown repressing function independent of the HKMT function of Su(var)3-9 (Lundberg, 2013a).

The HP1a Pof double-mutant displayed weak non-significant up-regulation of chromosome 4, with marginally larger error bars than for the HP1a mutant, which supports the suggested balancing mechanism of chromosome 4, where HP1a and POF fine-tune the transcriptional output; in the absence of both components, the overall expression will not change but individual genes will start losing proper transcriptional control (Lundberg, 2013a).

Although previous studies have indicated that SETDB1 and Su(var)3-9 have separate main targets, the data show that the majority of genes that are up- or down-regulated in Su(var)3-9 mutants are correspondingly up- or down-regulated in Setdb1 mutants. These results suggest that a redundancy exists between these two proteins, in which both proteins, to some extent, have the ability to be redirected to other locations when needed, as we know that Su(var)3-9 has a chromosome 4 binding capacity. Alternatively, the HP1a system might affect a number of genetic networks so that even if different regions are affected by Su(var)3-9 and Setdb1, the same genetic networks may be indirectly affected. Because Su(var)3-9 affects larger regions than Setdb1, it is likely that more HP1a will be released and redirected to other regions in the Su(var)3-9 mutant than in a Setdb1 mutant, thus causing repression of genes normally unbound by HP1a. This would explain why more genes are down-regulated in the Su(var)3-9 mutant compared with the Setdb1 mutant (Lundberg, 2013a).

These results provide strong support for the suggested model in which transposons are repressed by HP1 proteins, as shown for HP1a, the HP1 homolog Rhino and also Su(var)3-9. In contrast, neither SETDB1 nor POF had any effects on transposon expression. Because SETDB1 is known to have a role in repression of chromosome 4, one could speculate that SETDB1 has a greater influence on repression of transposons located specifically on chromosome 4 than in other parts of the genome. However, due to the repetitive nature of the transposons and the methods used in this study, it was not possible to distinguish effects for transposons in specific regions (Lundberg, 2013a).

The observation that chromosome 4 displays a stronger effect on non-ubiquitously expressed genes (NUEGs), both in terms of down-regulation in the Pof mutant and up-regulation in the HP1a mutant, is supported by previous findings on chromosome 4. One potential explanation for this is that NUEGs have evolved to respond to a regulatory mechanism, whereas UEGs are more robust in expression. Although weak, it is noteworthy that the effect of the HP1a mutant in the pericentromeric regions (decreased gene expression) was slightly stronger for UEGs than NUEGs; this is in line with the relatively strong binding peak found in promoters compared with the gene body in pericentromeric UEGs, as it has been proposed that HP1a in the promoter has a stimulating effect and HP1a in the gene body of chromosome 4 genes has an inhibiting effect. Note that the number of NUEGs exceeds the number of UEGs on a whole-genome level and on chromosome 4, whereas in pericentromeric regions, the UEGs are over-represented (Lundberg, 2013a).

In summary, these data support a model in which HP1a binding to promoters in general has a positive function for transcriptional output, whereas HP1a binding in gene bodies has a negative function. If binding in the gene body is relatively large compared with binding to the promoter, the negative function will dominate. In contrast, if the binding to the promoter is stronger than the gene body, the stimulating effect will be larger, albeit not dominating. A reduction in HP1a levels will initially affect the low-affinity gene binding and sequentially, the loss of HP1a will also affect the promoter binding (Lundberg, 2013a).

The average binding level of HP1a is constant irrespective of gene length (the HP1a binding per length unit is constant), implying that longer genes have more HP1a molecules bound in total. This finding, in combination with the suggestion that the repressive effect of HP1a is mainly observed in the gene body, could explain the greater de-repression of longer genes. Stronger binding of HP1a to long genes has also been suggested in previous studies. In addition, some chromatin marks mostly associated with active chromatin have been shown to bind differently to different gene lengths, suggesting that gene length affects the level of association with chromatin marks. Furthermore, there are indications that HP1 proteins are involved with transcription machinery; the mammalian HP1 isoform gamma and H3K9me3 regulate transcriptional activation by associating with the RNA polymerase II (RNP2), and HP1 can interact with and guide the recruitment of the histone chaperone complex FACT to active genes, which facilitates RNP2 transcription elongation. This, along with the current findings, suggests a mechanism in which HP1a is involved in transcriptional elongation. It is speculated that HP1a slows down the progression of the RNP2 through the length of the gene body. HP1a binding mechanisms could also be connected with RNA interactions, as HP1a has been shown to directly interact with RNA transcripts and heterogeneous nuclear ribonucleoproteins, and HP1a association to centric regions in Drosophila and mice is sensitive to RNase treatment (Lundberg, 2013a).

Interestingly, it was discovered that a group of non-annotated short genes (<0.5 kb) were repressed by HP1a, even though the HP1a binding levels appeared to be low (which might be explained by technical aspects in determining the binding levels). The lack of annotation and lower wild-type expression level suggest that this group consists of many short genes encoding ncRNAs (Lundberg, 2013a).

In the pericentromeric regions of the genome, an interesting connection was observed between the binding and stimulating effects of HP1a and the position of the gene; the closer the gene is located to the centromeric chromatin, the more strongly HP1a binds and stimulates it (Lundberg, 2013a).

In conclusion, it was found that HP1a has opposite functions in different genomic regions, repressing expression on chromosome 4 and stimulating expression in pericentromeric regions. Furthermore, the targets of Su(var)3-9 and SETDB1 are considerably more redundant than previously reported, and the overlap between HP1a, Su(var)3-9 and SETDB1 on chromosome 4 genes is extensive. It is however important to note that the different effects caused by HP1a, SETDB1, Su(var)3-9 and POF could all be interrelated to create a balanced genome. Therefore, it is hard to distinguish the separate effects caused by the different proteins (Lundberg, 2013a).

Targeting of Painting of fourth to roX1 and roX2 proximal sites suggests evolutionary links between dosage compensation and the regulation of the fourth chromosome in Drosophila melanogaster

In Drosophila melanogaster, two chromosome-specific targeting and regulatory systems have been described. The male-specific lethal (MSL) complex supports dosage compensation by stimulating gene expression from the male X-chromosome, and the protein Painting of fourth (POF) specifically targets and stimulates expression from the heterochromatic 4^th chromosome. The targeting sites of both systems are well characterized, but the principles underlying the targeting mechanisms have remained elusive. This study presents an original observation, namely that POF specifically targets two loci on the X-chromosome, PoX1 and PoX2 (POF-on-X). PoX1 and PoX2 are located close to the roX1 and roX2 genes, which encode noncoding RNAs important for the correct targeting and spreading of the MSL-complex. The targeting of POF to PoX1 and PoX2 is largely dependent on roX expression, and a high-affinity target region was identified that ectopically recruits POF. The results presented support a model linking the MSL-complex to POF and dosage compensation to regulation of heterochromatin (Lundberg, 2013b).

High-affinity sites have been characterized for the MSL-complex and there are several published examples of short regions, including the roX1 and roX2 loci, that are capable of recruiting this complex when presented as transgenes. In contrast, until now no high-affinity sites for POF targeting have been identified. Translocated 4^th chromosomes will not be targeted by POF, unless the proximal heterochromatic region is present and under conditions that favor heterochromatin formation (Johansson, 2007a). The characterization of POF targeting to PoX1 and PoX2 in females thus provides a unique opportunity to study the targeting of POF to a nonheterochromatic target and to further understanding of the evolution of these two targeting systems (Lundberg, 2013b).

Considering the evolutionary relationship between POF and the MSL-complex (reviewed by Stenberg, 2011), it was intriguing to find POF targeting to two distinct regions on the X-chromosome, i.e., X:3E and X:10E-F. The apparent spreading of POF targeting in these two regions (resembling the spreading of the MSL-complex when it is targeted to roX transgenes) and the close location of these regions to roX1 and roX2 suggested a link with the MSL-complex and dosage compensation. It has been hypothesized that POF originated as a dosage compensation system, since POF targets the male X-chromosome in, for example, D. busckii and D. ananassae and in those species POF colocalizes with H4K16ac and the MSL-complex, respectively (Larsson, 2004; Stenberg. 2011). However, the targeting of POF to endogenous PoX1 and PoX2 in D. melanogaster is restricted to females. This sex-specific targeting is not caused by sex-specific expression of the targeted genes, since comparable expression levels of >RE64691 as well as SelG and CG1840 are consistently found in male and female salivary glands (Lundberg, 2013b).

Not only are the two targeted loci, PoX1 and PoX2, located in close proximity to roX1 and roX2, the targeting is also largely dependent on roX, because losses of roX1 alone or of roX1 and roX2 cause a clear decrease in the frequency of PoX targeting. Importantly, in all roX mutant conditions tested, complete loss of POF binding to the PoX sites was never found. Therefore, roX is not absolutely required for PoX targeting but rather it enhances or stabilizes the interaction. The dependence of targeting on roX is not caused by the close proximity of the PoX loci to the corresponding roX loci, because in the duplications tested the PoX1 and PoX2 are located on another chromosome, i.e., chromosome arm 3L, and the roX genes are not included in the duplicated region. Despite this, two of the duplications show targeting by POF, comparable to that to the endogenous loci. Furthermore, targeting to these transgenic regions was found to be largely dependent on roX, which indicates that roX can act in trans to enhance or stabilize POF targeting. The most parsimonious model to explain these observations is that it is the roX ncRNA species that enhance or stabilize targeting of POF to these non-chromosome 4 targets. This model is supported by the fact that roX2 overexpression seems to further increase the frequency of targeting. However, it should be stressed that endogenous roX expression in females is reported to be at very low levels or absent. In females, roX1 RNA has been reported in early embryos but it appears to be lost midway through embryogenesis, whereas in males expression is maintained through adulthood. roX2 RNA first appears a few hours after roX1, but only in male embryos (Lundberg, 2013b).

No high-affinity sites for POF targeting have previously been identified (Johansson, 2007a). It therefore came as a surprise that a short (6-kb) region from PoX2 functions as a strong ectopic target for POF in both males and females. The nonsex-specific targeting of POF to the P[w⁺ SelG CG1840] transgene, in contrast to Dp(1;3)DC246 and endogenous PoX, may be explained by a competition of targeting between POF and the MSL-complex in males. This competition will be more pronounced at the endogenous PoX sites and the duplications as these are also targets for the MSL-complex in males. This finding is supported by the fact that on polytene chromosomes, Dp(1;3)DC246 is targeted by MSL-complex in males while the P[w⁺ SelG CG1840] transgene is not targeted. A competition in targeting is also supported by the reduction frequency of PoX1 and PoX2 targeting by POF observed in females expressing a partial MSL-complex, i.e. w; P[w⁺ hsp83:msl2] msl3 females. It is important to note that the targeting of POF to the P[w⁺ SelG CG1840] transgene was not caused by genomic location of this transgene since the same attP docking site (3L:65B2) was used as for the duplications of the PoX1 and PoX2 loci. The lack of targeting of POF to translocated parts of the 4^th chromosome and the strong targeting to the PoX2 transgene suggest that the PoX regions may be POF targets that are functionally separable from the 4^th chromosome genes. Since both Setdb1 and HP1a are detected on the transgene, it appears likely that POF recruitment leads to, or is connected with, the formation of GREEN (HP1a and H3K9me enriched) chromatin structure (Filion, 2010) (Lundberg, 2013b).

The targeting of POF to the 4^th chromosome depends on its well-characterized heterochromatic nature and on the presence of HP1a and Setdb1. It is therefore important to note that links between the MSL-complex, roX1 and roX2 and heterochromatic regions have been reported previously, though they remain to be understood. It is known that in roX1 roX2 mutant males, the MSL-complex is still detected on the X-chromosome, albeit at a reduced number of sites, but binding is also found in the chromocenter and at a few reproducible sites on the 4^th chromosome. In contrast to the X-chromosome, where the MSL-complex is believed to stimulate gene expression, loss of roX RNA reduces expression from genes located in the chromocenter and on the 4^th chromosome (Deng, 2009). It has been suggested that roX RNAs participate in two distinct regulatory systems, the dosage compensation system and a system for the modulation of heterochromatin (Deng, 2009). Although the mechanism by which roX RNAs enhance binding of POF to PoX loci remains elusive, the observation supports a model linking dosage compensation to modulation of heterochromatin. Additional factors supporting a model linking heterochromatin to dosage compensation are the proposed binding of HP1a to the male X-chromosome (de Wit, 2005) and the fact that a reduction in the histone H3S10 kinase JIL-1 results in the spreading of heterochromatic markers (such as H3K9me2 and HP1a) along the chromosome arms, with the most marked increase taking place on the X-chromosomes. The JIL1 kinase, which is believed to counteract heterochromatin formation, is highly enriched on the male X-chromosome and is reported to be loosely attached to the MSL-complex). It is noteworthy that POF, which targets genes in a heterochromatic environment, i.e., on the 4^th chromosome, has an intrinsic ability to target the male X-chromosome, as seen in, e.g., D. ananassae, and the targeting to X-chromosome sites reported in this study is dependent on roX RNAs. At the same time the MSL-complex, which binds to and stimulates expression of genes on the male X-chromosome, has an intrinsic ability to target heterochromatin as seen in the roX1 roX2 mutant background. The link between these two systems is intriguing and promises to increase understanding of balanced gene expression (Lundberg, 2013b).

High-affinity targeting to the PoX1 and PoX2 loci therefore provides a novel system for further studies on targeting mechanisms involved in chromosome-wide gene regulation, the evolutionary relationship between POF and dosage compensation and the evolution of balanced gene expression, and the results favor a model involving not only the X-chromosome but also balance to heterochromatin (Lundberg, 2013b).

POF and HP1 bind expressed exons, suggesting a balancing mechanism for gene regulation

Two specific chromosome-targeting and gene regulatory systems are present in Drosophila melanogaster. The male X chromosome is targeted by the male-specific lethal complex believed to mediate the 2-fold up-regulation of the X-linked genes, and the highly heterochromatic fourth chromosome is specifically targeted by the Painting of Fourth (POF) protein, which, together with heterochromatin protein 1 (HP1), modulates the expression level of genes on the fourth chromosome. This study used chromatin immunoprecipitation and tiling microarray analysis to map POF and HP1 on the fourth chromosome in S2 cells and salivary glands at high resolution. The enrichment profiles were complemented by transcript profiles to examine the link between binding and transcripts. The results show that POF specifically binds to genes, with a strong preference for exons, and the HP1 binding profile is a mirror image of POF, although HP1 displays an additional 'peak' in the promoter regions of bound genes. HP1 binding within genes is much higher than the basal HP1 enrichment on Chromosome 4. The results suggest a balancing mechanism for the regulation of the fourth chromosome where POF and HP1 competitively bind at increasing levels with increased transcriptional activity. In addition, the results contradict the idea that transposable elements are a major nucleation site for HP1 on the fourth chromosome (Johansson, 2007b).

Previously studies have shown that POF and HP1 colocalize at the resolution given by polytene chromosome and that the same set of genes is regulated by these two proteins (Johansson, 2007a). This study shows that POF and HP1 binding colocalize at the gene level, which also provides insights into the modes of action for this regulatory system. The bias in within-gene binding towards exons shown by POF was also observed for HP1. HP1, like POF, preferentially binds exons but, in contrast to POF, HP1 shows a high basal binding to the fourth chromosome and a peak in binding associated with promoters for most targeted genes. The specificity of HP1 to certain promoters at the individual target gene level has previously been reported in mammals. According to the current data the promoter peak of HP1 is a more general characteristic of HP1-bound genes on the fourth chromosome and is related to transcription. It has been proposed that the presence of H3K9me at promoters is connected to gene repression, but that H3K9me within the genes is associated with gene activity. If the HP1 profile is assumed to be linked to the presence of H3K9me this implies that a combination of these two binding profiles is linked to the transcription of genes on the fourth chromosome (Johansson, 2007b).

The classical view is that HP1 is associated with gene repression. However, a number of recent reports have linked HP1 to gene activation, based on the enrichment of H3K9me and HP1 on active genes. It is well known that HP1 is enriched in pericentric regions and that genes in those regions are, therefore, connected to high HP1 levels. It has been shown that mutation in HP1 causes a reduction in the expression of a number of heterochromatin-located genes in Drosophila e.g., light and rolled, supporting the idea that some genes depend on their heterochromatic surroundings for correct expression. However, it has also been demonstrated that HP1 is associated with the transcribed regions of active genes located in euchromatic regions. In addition, HP1 has been shown to associate with developmental and heatshock-induced puffs of the polytene chromosome, which is indicative of intense gene activity. It has been shown that H3K9 methylation occurs in the transcribed region of active genes in mammalian chromatin and, in fact, increases during activation of transcription. In that case HP1 was found to be associated with the transcribed genes of several mammalian cell lines and also in primary cells. However, it is important to note that, except for the heterochromatin genes light and rolled,, it is not clear whether the binding of HP1 is associated with facilitated transcription. The current results indicate not only that HP1 binds to active genes on the fourth chromosome, but also that the genes on the fourth chromosome are up-regulated upon loss of HP1. Thus, although the genes on the fourth chromosome are bound in response to gene activity, HP1 still causes repression. It may be that, for example, heat-shock induced genes attract HP1 as a modulator that represses uncontrolled gene expression. In contrast to HP1, the loss of POF leads to a general decrease in gene expression from the fourth chromosome. The strong correlation with respect to binding between HP1 and POF and their correlation with transcription support the balancing model (Johansson, 2007a): POF stimulates and HP1 represses gene expression and the interdependent binding of these two proteins fine tunes the expression output from the fourth chromosome. It should be noted that the correlation between HP1 and POF seems to be linear, suggesting that highly expressed genes have the same POF/HP1 ratio as genes with weak expression, although they bind higher amounts of both proteins. A balancing mechanism may act as a buffering system in which the dual recruitment of a repressing and a stimulating factor makes the transcription efficiency more stable and less sensitive to fluctuations. Balancing mechanisms may be more general. For example, this may explain the proposed binding of HP1 to the male X chromosome. The facilitated transcription of X chromosomal genes by acetylation of H4K16 may need to be tempered by a repressing factor to reach the expected 2-fold increase. This repressing function might be supported by HP1, Su(var)3-7, or other unknown factors not yet linked to dosage compensation (Johansson, 2007b).

The targeting of POF to the fourth chromosome shows similarities to the targeting of the MSL complex to the male X chromosome. The striking similarity between POF and the dosage-compensating MSL complex in evolutionary terms, their function as chromosome-wide regulators, and their binding profiles, as presented as presented in this study, supports a common origin. For the MSL complex, expressed genes are the main targets. In addition, in fly embryos, S2 cells, and in a cell line derived from larval imaginal discs (Clone8 cells), MSL binding is also associated with expressed genes, but does not correlate with level of expression. It has been demonstrated that, to a large extent, MSL binding is stable throughout development and that the binding reflects the expression levels in young embryos (4–5 h). It is hypothesized that a similar strong correlation between levels of transcript and binding as well as cell type differences as seen for POF, might be true also for MSL if studied at higher resolution. A correlation between binding levels and levels of transcription would be in line with the expected 2-fold increase of gene expression independendent of expression levels (Johansson, 2007b).

It has been shown that transgenes inserted on the fourth chromosome are often partially silenced and that the localization of these variegated insertions in some regions of chromosome 4 is correlated to their distance from the transposable element 1360 (Sun, 2004). This, along with the fact that the 1360 element can contribute to the silencing of an adjacent reporter when close to pericentric heterochromatin, suggests that 1360 elements may serve as HP1 recruitment signals. Further support for this hypothesis is provided by the suggestion that repeat flanked genes are more likely to bind HP1. The current results show that HP1 binds Chromosome 4 genes, but no indications were found of transposable elements such as 1360 acting as nucleation sites for HP1. It should be stressed that these results do not contradict the reported correlation between transgenic silencing and distance from 1360 elements. It is possible that 1360 elements under certain conditions serve as nucleation sites for heterochromatin formation and spread, but that this does not involve HP1. It is also possible that 1360 elements act as initial nucleation sites for HP1, which are not maintained in the two cell types analyzed. Furthermore, silenced transgene insertions on Chromosome 4 are linked to regions with relatively high binding of HP1, but these regions are typically expressed. This implies that the transcriptional consequences of high HP1 levels differ between inserted transgenes and endogenous Chromosome 4 genes. It is speculated that POF is needed for expression of these Chromosome 4 genes and that inserted transgenes will be repressed by HP1 but will fail to recruit POF (Johansson, 2007b).

Painting of fourth and chromosome-wide regulation of the 4th chromosome in Drosophila melanogaster

Drosophila exhibits two expression-regulating systems that target whole, specific chromosomes: the dosage compensation system whereby the male-specific lethal complex doubles transcription of genes on the male X-chromosome and the chromosome 4-specific protein Painting of fourth, POF. POF is the first example of an autosome-specific protein and its presence raises the question of the universality of chromosome-specific regulation. This study shows that POF and heterochromatin protein 1 (HP1) are involved in the global regulation of the 4th chromosome. Contrary to previous conclusions, Pof is not essential for survival of diplo-4th karyotype flies. However, Pof is essential for survival of haplo-4th individuals and expression of chromosome 4 genes in diplo-4th individuals is decreased in the absence of Pof. Mapping of POF using chromatin immunoprecipitation suggested that it binds within genes. Furthermore, this study shows that POF binding is dependent on heterochromatin and that POF and HP1 bind interdependently to the 4th chromosome. A balancing mechanism involving POF and HP1 is proposed that provides a feedback system for fine-tuning expression status of genes on the 4th chromosome (Johnsson, 2007a).

Painting of fourth in genus Drosophila suggests autosome-specific gene regulation

Painting of fourth (POF) is a chromosome-specific protein in Drosophila and represents the first example of an autosome-specific protein. POF binds to chromosome 4 in Drosophila melanogaster, initiating at the proximal region, followed by a spreading dependent on chromosome 4-specific sequences or structures. Chromosome-specific gene regulation is known thus far only as a mechanism to equalize the transcriptional activity of the single male X chromosome with that of the two female X chromosomes. In Drosophila, a complex including the male-specific lethal proteins, 'paints' the male X chromosome, mediating its hypertranscription, explained to some extent by the acetylation of lysine 16 on histone H4. This study shows that Pof is essential for viability in both sexes and for female fertility. POF binding to an autosome, the F element, is conserved in genus Drosophila, indicating functional conservation of the autosome specificity. In three of nine studied species, POF binds to the male X chromosome. When bound to the male X, it also colocalizes with the dosage compensation protein male-specific lethal 3, suggesting a relationship to dosage compensation. The chromosome specificity is determined at the species level and not by the amino acid sequence. It is argued that POF is involved in a chromosome-specific regulatory function (Larsson, 2004).

Painting of fourth, a chromosome-specific protein in Drosophila

Chromosome-specific gene regulation is known thus far only as a mechanism to equalize the transcriptional activity of the single male X chromosome with that of the two female X chromosomes. In Drosophila melanogaster, a complex including the five Male-Specific Lethal (MSL) proteins, 'paints' the male X chromosome, mediating its hypertranscription. With the molecular cloning of Painting of fourth (Pof), this study describes a previously uncharacterized gene encoding a chromosome-specific protein in Drosophila. Unlike the MSL proteins, POF paints an autosome, the fourth chromosome of Drosophila melanogaster. Chromosome translocation analysis shows that the binding depends on an initiation site in the proximal region of chromosome 4 and spreads in cis to involve the entire chromosome. The spreading depends on sequences or structures specific to chromosome 4 and cannot extend to parts of other chromosomes translocated to the fourth. Spreading can also occur in trans to a paired homologue that lacks the initiation region. In the related species Drosophila busckii, POF paints the entire X chromosome exclusively in males, suggesting relationships between the fourth chromosome and the X and between POF complexes and dosage-compensation complexes (Larsson, 2001).

Search PubMed for articles about Drosophila Painting of fourth

Brody, Y., Neufeld, N., Bieberstein, N., Causse, S. Z., Bohnlein, E. M., Neugebauer, K. M., Darzacq, X. and Shav-Tal, Y. (2011). The in vivo kinetics of RNA polymerase II elongation during co-transcriptional splicing. PLoS Biol 9: e1000573. PubMed ID: 21264352

Deng, X. and Meller, V. H. (2009). Molecularly severe roX1 mutations contribute to dosage compensation in Drosophila. Genesis 47: 49-54. PubMed ID: 19101984

de Wit, E., Greil, F. and van Steensel, B. (2005). Genome-wide HP1 binding in Drosophila: developmental plasticity and genomic targeting signals. Genome Res 15: 1265-1273. PubMed ID: 16109969

Figueiredo, M. L., Philip, P., Stenberg, P. and Larsson, J. (2012). HP1a recruitment to promoters is independent of H3K9 methylation in Drosophila melanogaster. PLoS Genet 8: e1003061. PubMed ID: 23166515

Filion, G. J., van Bemmel, J. G., Braunschweig, U., Talhout, W., Kind, J., Ward, L. D., Brugman, W., de Castro, I. J., Kerkhoven, R. M., Bussemaker, H. J. and van Steensel, B. (2010). Systematic protein location mapping reveals five principal chromatin types in Drosophila cells. Cell 143: 212-224. PubMed ID: 20888037

Johansson, A. M., Stenberg, P., Bernhardsson, C. and Larsson, J. (2007a). Painting of fourth and chromosome-wide regulation of the 4th chromosome in Drosophila melanogaster. EMBO J 26: 2307-2316. PubMed ID: 17318176

Johansson, A. M., Stenberg, P., Pettersson, F. and Larsson, J. (2007b). POF and HP1 bind expressed exons, suggesting a balancing mechanism for gene regulation. PLoS Genet 3: e209. PubMed ID: 18020713

Johansson, A. M., Allgardsson, A., Stenberg, P. and Larsson, J. (2011). msl2 mRNA is bound by free nuclear MSL complex in Drosophila melanogaster. Nucleic Acids Res 39: 6428-6439. PubMed ID: 21551218

Johansson, A. M., Stenberg, P., Allgardsson, A. and Larsson, J. (2012). POF regulates the expression of genes on the fourth chromosome in Drosophila melanogaster by binding to nascent RNA. Mol Cell Biol 32: 2121-2134. PubMed ID: 22473994

Larschan, E., Bishop, E. P., Kharchenko, P. V., Core, L. J., Lis, J. T., Park, P. J. and Kuroda, M. I. (2011). X chromosome dosage compensation via enhanced transcriptional elongation in Drosophila. Nature 471: 115-118. PubMed ID: 21368835

Larsson, J., Chen, J. D., Rasheva, V., Rasmuson-Lestander, A. and Pirrotta, V. (2001). Painting of fourth, a chromosome-specific protein in Drosophila. Proc Natl Acad Sci U S A 98: 6273-6278. PubMed ID: 11353870

Larsson, J., Svensson, M. J., Stenberg, P. and Makitalo, M. (2004). Painting of fourth in genus Drosophila suggests autosome-specific gene regulation. Proc Natl Acad Sci U S A 101: 9728-9733. PubMed ID: 15210994

Larsson, J. and Meller, V. H. (2006). Dosage compensation, the origin and the afterlife of sex chromosomes. Chromosome Res 14: 417-431. PubMed ID: 16821137

Lundberg, L. E., Stenberg, P. and Larsson, J. (2013a). HP1a, Su(var)3-9, SETDB1 and POF stimulate or repress gene expression depending on genomic position, gene length and expression pattern in Drosophila melanogaster. Nucleic Acids Res 41: 4481-4494. PubMed ID: 23476027

Lundberg, L. E., Kim, M., Johansson, A. M., Faucillion, M. L., Josupeit, R. and Larsson, J. (2013b). Targeting of Painting of fourth to roX1 and roX2 proximal sites suggests evolutionary links between dosage compensation and the regulation of the fourth chromosome in Drosophila melanogaster. G3 (Bethesda) 3: 1325-1334. PubMed ID: 23733888

Neugebauer, K. M. (2002). On the importance of being co-transcriptional. J Cell Sci 115: 3865-3871. PubMed ID: 12244124

Osheim, Y. N., Miller, O. L., Jr. and Beyer, A. L. (1985). RNP particles at splice junction sequences on Drosophila chorion transcripts. Cell 43: 143-151. PubMed ID: 3935315

Riddle, N. C., Jung, Y. L., Gu, T., Alekseyenko, A. A., Asker, D., Gui, H., Kharchenko, P. V., Minoda, A., Plachetka, A., Schwartz, Y. B., Tolstorukov, M. Y., Kuroda, M. I., Pirrotta, V., Karpen, G. H., Park, P. J. and Elgin, S. C. (2012). Enrichment of HP1a on Drosophila chromosome 4 genes creates an alternate chromatin structure critical for regulation in this heterochromatic domain. PLoS Genet 8: e1002954. PubMed ID: 23028361

Stenberg, P., Lundberg, L. E., Johansson, A. M., Ryden, P., Svensson, M. J. and Larsson, J. (2009). Buffering of segmental and chromosomal aneuploidies in Drosophila melanogaster. PLoS Genet 5: e1000465. PubMed ID: 19412336

Stenberg, P. and Larsson, J. (2011). Buffering and the evolution of chromosome-wide gene regulation. Chromosoma 120: 213-225. PubMed ID: 21505791

Sun, F. L., Haynes, K., Simpson, C. L., Lee, S. D., Collins, L., Wuller, J., Eissenberg, J. C. and Elgin, S. C. (2004). cis-Acting determinants of heterochromatin formation on Drosophila melanogaster chromosome four. Mol Cell Biol 24: 8210-8220. PubMed ID: 15340080

Tzeng, T. Y., Lee, C. H., Chan, L. W. and Shen, C. K. (2007). Epigenetic regulation of the Drosophila chromosome 4 by the histone H3K9 methyltransferase dSETDB1. Proc Natl Acad Sci U S A 104: 12691-12696. PubMed ID: 17652514

date revised: 21 August, 2013

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