Painting of fourth and chromosome-wide regulation of the 4th chromosome in Drosophila melanogaster
2007; Springer Nature; Volume: 26; Issue: 9 Linguagem: Inglês
10.1038/sj.emboj.7601604
ISSN1460-2075
AutoresAnna‐Mia Johansson, Per Stenberg, Carolina Bernhardsson, Jan Larsson,
Tópico(s)Invertebrate Immune Response Mechanisms
ResumoArticle22 February 2007free access Painting of fourth and chromosome-wide regulation of the 4th chromosome in Drosophila melanogaster Anna-Mia Johansson Anna-Mia Johansson UCMP, Umeå University, Umeå, Sweden Search for more papers by this author Per Stenberg Per Stenberg UCMP, Umeå University, Umeå, Sweden Search for more papers by this author Carolina Bernhardsson Carolina Bernhardsson UCMP, Umeå University, Umeå, Sweden Search for more papers by this author Jan Larsson Corresponding Author Jan Larsson UCMP, Umeå University, Umeå, Sweden Search for more papers by this author Anna-Mia Johansson Anna-Mia Johansson UCMP, Umeå University, Umeå, Sweden Search for more papers by this author Per Stenberg Per Stenberg UCMP, Umeå University, Umeå, Sweden Search for more papers by this author Carolina Bernhardsson Carolina Bernhardsson UCMP, Umeå University, Umeå, Sweden Search for more papers by this author Jan Larsson Corresponding Author Jan Larsson UCMP, Umeå University, Umeå, Sweden Search for more papers by this author Author Information Anna-Mia Johansson1,‡, Per Stenberg1,‡, Carolina Bernhardsson1 and Jan Larsson 1 1UCMP, Umeå University, Umeå, Sweden ‡These authors contributed equally to this work *Corresponding author. UCMP, Umeå University, SE-901 87 Umeå, Sweden. Tel.: +46 090 7856 785; Fax: +46 090 7780 07; E-mail: [email protected] The EMBO Journal (2007)26:2307-2316https://doi.org/10.1038/sj.emboj.7601604 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Drosophila melanogaster 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. Here we show 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, we show that POF binding is dependent on heterochromatin and that POF and HP1 bind interdependently to the 4th chromosome. We propose a balancing mechanism involving POF and HP1 that provides a feedback system for fine-tuning expression status of genes on the 4th chromosome. Introduction The dosage compensation system of Drosophila melanogaster is a well-known example of whole-chromosome regulation. Dosage compensation is the mechanism used to equalize the transcriptional activities of the two X chromosomes in the homogametic sex with that of the single X chromosome in the heterogametic sex. Dosage compensation strategies vary widely between species. In female mammals, one X chromosome is inactivated and forms the Barr body, whereas in Caenorhabditis elegans, transcription from both X chromosomes in hermaphrodites is downregulated. In D. melanogaster, dosage compensation is achieved by doubling transcription of the single male X chromosome (reviewed in Baker et al, 1994; Lucchesi et al, 2005; Larsson and Meller, 2006). In D. melanogaster, two non-coding RNAs, roX1 and roX2, have been shown to be essential components of the dosage compensation system. Together with the five MSL (male-specific lethal) proteins, roX1 and roX2 'paint' the dosage compensated male X chromosome. The MSL complex mediates acetylation of H4 at lysine 16 on the male X chromosome, which in part explains the subsequent hypertranscription (reviewed in Akhtar, 2003; Larsson and Meller, 2006). A number of recent reports have suggested that components generally associated with heterochromatin are important for dosage compensation. A knock-down of Su(var)2-5 (HP1, heterochromatin-associated protein 1) was shown to result in significantly more lethality in males than in females (Liu et al, 2005). A low amount of HP1 along the entire male X chromosome has been found in genome-wide mapping of HP1 using the DamID technique (de Wit et al, 2005). The Su(var)3-7 protein is another component associated with pericentric heterochromatin. Mutation in Su(var)3-7 causes sex-biased lethality and bloating of the male X chromosome. This bloating is dependent on a functional dosage compensation complex (Spierer et al, 2005). Mild overexpression of Su(var)3-7 causes hypercompaction of the male X chromosome (Delattre et al, 2004). These results suggest that Su(var)3-7 is also involved in dosage compensation. All the above-mentioned mechanisms for regulating entire chromosomes affect the expression of sex chromosomes, but in Drosophila, an additional example of whole-chromosome targeting is provided by the specific binding of POF to the 4th chromosome (Larsson et al, 2001). This suggests that chromosome-specific regulation may be a more general process. POF is a 495-aa protein with a predicted RNA-binding domain (RRM1) in its central part (Larsson et al, 2001) that binds throughout the entire euchromatic portion of the 4th chromosome. The association with the 4th chromosome appears to nucleate in the basal region of this chromosome and spread in cis or trans to coat the length of the chromosome (Larsson et al, 2001). In several species within the genus Drosophila, for example D. virilis and D. pseudoobscura, POF binds specifically to the F-element (the chromosome corresponding to chromosome 4 in D. melanogaster) (Larsson et al, 2004), suggesting that there is a functional association between POF and chromatin. POF also shows a strong association with the MSL dosage compensation complex. In D. busckii, POF binds specifically to the male X chromosome and colocalizes with H4K16Ac. In D. ananassae and D. malerkotliana, POF again binds specifically to the male X chromosome and colocalizes with the MSL complex protein MSL3 (Larsson et al, 2004). These results support the notion that the 4th chromosome in D. melanogaster has a strong relationship with the X chromosome. Indeed it has been argued that the 4th chromosome originates from the X (reviewed in Hochman, 1976; Larsson and Meller, 2006; Riddle and Elgin, 2006). The 4th chromosome is in many ways an atypical chromosome. The sequenced part of the chromosome is 1.28-Mb long and corresponds roughly to the banded region seen in polytene chromosomes, that is cytogenetic bands 101E–102F. The remaining 3–4 Mb proximal heterochromatic part of the chromosome consists of simple satellite repeats (Locke and McDermid, 1993). The sequenced and banded part of the chromosome includes 92 genes leading to a gene density similar to that of the major chromosome arms. However, this banded part of the chromosome shares properties typical of heterochromatin. Under normal conditions, chromosome 4 does not undergo meiotic recombination (Hochman, 1976; Sandler and Szauter, 1978; Ashburner et al, 2005) and is late replicating (Barigozzi et al, 1966). The banded region appears as a mosaic of unique sequences interspersed with repetitive DNA with a high content of transposable elements (Barigozzi et al, 1966; Miklos et al, 1988; Pimpinelli et al, 1995; Locke et al, 1999a, 1999b; Kaminker et al, 2002; Stenberg et al, 2005). The heterochromatin protein HP1 is abundantly present on the 4th chromosome as are certain histone modifications used to identify heterochromatin, for example, methylated H3K9 (Eissenberg et al, 1992; Czermin et al, 2002; Schotta et al, 2002). Reporter genes inserted in this chromosome often display a partially silenced, variegated expression (Wallrath and Elgin, 1995; Wallrath et al, 1996). Therefore, how expression of the genes on this highly heterochromatic chromosome is controlled remains an intriguing question. Here we present evidence that POF and HP1 are involved in regulation of the entire 4th chromosome. Pof is not essential for survival of flies with a diplo-4th karyotype, but is necessary for survival of haplo-4th individuals. We show that genes along the entire length of chromosome 4 are downregulated in the absence of Pof and upregulated in the absence of HP1, and that POF binds preferentially within genes. This binding is dependent on heterochromatin, and HP1 and POF colocalize at cytological level and their binding is interdependent. Results Characterization of the genomic Pof locus In our previous study of Pof, we induced and analyzed short deletions in the promoter regions of the Pof gene. These deletions, which include PofD31A and PofD2, result in no detectable Pof transcription, cause female sterility and are homozygous lethal. The lethality could be rescued by a transgenic construct P[w+ Pof] (Figure 1A; Larsson et al, 2004). On the basis of these findings, we proposed that the sterility and lethality were caused by disruption of the Pof gene. However, in later releases of the genome annotation, a novel gene was predicted in the promoter region of Pof, namely CG33228 (Figure 1A). As this predicted gene would be affected by PofD31A and PofD2 deletions and would be included in the transgenic construct P[w+ Pof], we decided to characterize further CG33228. To verify the existence and extent of the CG33228 gene, we performed 3′RACE and 5′RACE experiments using RNA templates from embryos, third instar larvae and adult females and males. We found CG33228 transcript in all these stages and one single 3′ end and one 5′ end were verified (Figure 1A). We cloned the gene using RT–PCR and confirmed its exon/intron structure. In contrast to the predicted gene structure, we found the 5′ end upstream of the transcription start point of Pof and a different exon/intron organization (Figure 1A). We could not detect a transcript with the first exon as predicted in the genome annotation (results not shown). Two P-element insertions, CB-6312-3 and l(2)SH0542, are located within the ORF of CG33228 (Figure 1A). Both these insertions cause recessive lethality, and complementation analysis shows that all trans-heterozygous combinations of PofD31A, PofD2, CB-6312-3 and l(2)SH0542 are lethal. The lethality of these trans-heterozygotes is fully rescued by the P[w+ Pof] transgene, indicating that the lethality lies within the Pof genomic region. These findings indicated that lethality could be because of disruption of CG33228 rather than Pof. To test this possibility, we induced new deletions in the Pof gene, selectively searching for deletions in the transcribed region. Thirteen unique deletions were isolated by imprecise excisions of the EP element in line with EP(2)2285. All of these deletions were homozygous-viable including PofD140, which uncovers the entire transcribed gene. PofD140 (null mutation), PofD119 and PofD114 (probable null mutations) were used for further experiments. Staining of polytene chromosomes from PofD119 homozygotes or PofD119 in combination with PofD2, CB-6312-3 and l(2)SH0542 confirmed that PofD119 and PofD2 do not produce any detectable POF in salivary gland cells, whereas POF expression is unaffected in CB-6312-3 and l(2)SH0542 (Figure 1B). RT–PCR analysis further confirmed that PofD119 and PofD2 have no detectable Pof transcript, whereas CB-6312-3 and l(2)SH0542 produce roughly normal amounts of Pof transcript. The level of transcription from the neighboring gene CG4806 is not affected by these mutations (Figure 1C). We conclude that the lethality and the female sterility caused by PofD2 and PofD31A are the result of disruption of the CG33228 gene and that Pof is not required for viability. The CG33228 gene encodes a novel protein of 215 amino acids with no clear similarity to any protein or domain of known function. Figure 1.Map of the Pof gene region. (A) The exon–intron structure of the Pof and CG33228 genes are shown below the genomic DNA line. CG4806 and Mmp1 genes extend outside the region shown. Pof is transcribed from left to right, all other genes shown are transcribed in the opposite direction. The confirmed exon–intron structure of CG33228 is shown in yellow and the predicted gene structure from the genome annotation (release 4) is shown in gray. The positions of the P-element insertions in CB-6312-3 and l(2)SH0542 lines are indicated (CB and SH). The deletions PofD31A and PofD2 have been previously described (Larsson et al, 2004). The extent of induced deletions and the transgenic construct P[w+Pof] are shown. (B) Localization of POF on polytene chromosomes from PofD119/PofD119, PofD2/PofD119, CB-6312-3/PofD119 and l(2)SH0542/PofD119 third instar larvae salivary gland cells. DNA is stained with DAPI (blue) and POF is recognized by an anti-POF antibody (yellow). (C) RT–PCR analysis on wild-type, PofD119/ PofD119, PofD2/ PofD119, CB-6312-3/PofD119 and l(2)SH0542/PofD119female poly(A)+ RNA. The primer pairs amplify Pof (top panel), CG4806 (middle) and CG33228 (bottom). Download figure Download PowerPoint Pof is essential for survival of haplo-4 flies What are the consequences of lacking a functional Pof gene? It has been proposed that the 4th chromosome also possesses a dosage compensation function, which allows haplo-4th individuals to survive (Hochman, 1976). Crossing males with a compound 4th chromosome (C(4)RM) to females with a normal diploid set of chromosomes will give two classes of offspring, one with three 4th chromosomes and one with a single 4th chromosome. Crosses between PofD140/PofD140; ci57g/ci57 g females and PofD140/CyO; C(4)RM svspa-pol/0 males showed that no haplo-4th PofD140/PofD140 individuals survived (Figure 2A). Other allelic combinations, that is PofD31A/PofD114, PofD119/PofD114 and PofD2/PofD119, also caused haplo-4th-specific lethality (Figure 2B–D). The haplo-4th lethality can be rescued by a single copy of the P[w+ Pof] transgene (Figure 2B). Reciprocal crosses gave similar results (not shown). The trans-heterozygotes with genotypes CB-6312-3/PofD140 and l(2)SH0542/PofD140 did not cause haplo-4th lethality (Figure 2E and F), excluding an influence of CG33228 on haplo-4th lethality. Although most of the PofD140/PofD140; ci57g/0 offspring died as embryos or in early larval stages, a small proportion reached the pupae stage (Supplementary Figure 1). We conclude that Pof is essential for survival of haplo-4th individuals. Figure 2.Mutations in Pof cause haplo-4th lethality. The y-axis shows the number of hatched adult flies. The x-axis shows the offspring genotype and the number of 4th chromosomes in roman numerals. Females are shown in red and males in blue (A, C–F). In (B), flies with the P[w+Pof] rescue construct are shown in yellow and their siblings lacking the transgenic construct are shown in light blue. (A) Offspring from PofD140/PofD140; ci57g/ci57 g females × PofD140/CyO; C(4)RM svspa-pol/0 males. No PofD119/PofD119; ci57g/0 (haplo-4th) offspring survive. (B) The haplo-4th lethality is rescued by a transgenic P[w+Pof] construct. The PofD119/PofD114 allelic combination (C) and the PofD2/PofD119 allelic combination (D) causes haplo-4th lethality. In CB-6312-3/PofD140 (E) and l(2)SH0542/PofD140 trans-heterozygotes (F) the haplo-4th individuals survive. The haplo-4th individuals are delayed by 2–4 days and have lower viability than their triplo-4th siblings. This caused the haplo-4th classes to be smaller than theoretically expected in all experiments. Download figure Download PowerPoint Pof mutations cause decreased levels of chromosome 4 gene expression The haplo-4th lethality in Pof mutants shows that Pof is essential for survival of haplo-4th individuals. However, under normal conditions flies are diploid for the 4th chromosome. We therefore tested if Pof mutations have a general effect on chromosome 4 gene expression. We prepared four biological replicates of mRNA from PofD2/PofD2 first instar larvae and compared them to three biological wild-type replicates. An Affymetrix microarray analysis showed that the lack of Pof caused a low but significant reduction of mRNA from a majority of the genes on the 4th chromosome (Supplementary Figure 2A). Although the reduction is too small to be significant at the level of individual genes, the overall reduction on chromosome 4 compared with any of the other chromosome arms is significant (Supplementary Figure 2B and C). However, PofD2/PofD2 larvae were selected from heterozygous parents with maternal POF present and the PofD2 allele also affects the neighboring gene CG33228. We, therefore, performed an additional microarray and compared three biological replicates of mRNA from PofD119/PofD119 first instar larvae with three new biological wild-type replicates. The results confirm and strengthen the conclusion that lack of Pof causes a significant reduction of gene expression from the 4th chromosome (Figure 3A and B; Supplementary Figure 2D). As chromosome 4 only represents 105 expression units on the microarray, we also performed a simulation to verify our results. One-hundred and five genes were randomly selected from the other chromosome arms and the mean reduction was compared with chromosome 4 mean reduction. After 100 million simulations no group with a reduction equal to or stronger than the 4th chromosome was found. Figure 3.Levels of mRNA from genes located on the 4th chromosome are decreased in a Pof mutant background and increased upon HP1 RNAi when compared with wild type. (A, C) Microarray analysis showing the median level of mRNA from genes on the 4th chromosome in PofD119/PofD119 mutant (A) and HP1 RNAi (C) as a function of the median levels in controls (log2 scale). Genes for which there were reduced amounts of mRNA (below dotted line) are marked with filled circles and genes for which there were increased amounts with open circles. (B, D) Mean change in gene expression for each chromosome arm, PofD119 – wild-type expression (B) and HP1 RNAi – control expression (D) (log2 scale). Squares indicate the mean value and whiskers indicate 95% confidence interval. (E) The microarray results plotted with respect to the position of the genes along the 4th chromosome (annotation release 4.3). The widths of the columns indicate the length of corresponding genes. Expression change (log2 scale) after HP1 RNAi is shown in blue and expression change in PofD119 mutants in red. Tick marks on the scale bar (middle) indicate 100 kb and genes transcribed from left to right are shown above the line and genes transcribed in opposite direction below. Download figure Download PowerPoint The 4th chromosome is known to be highly heterochromatic and targeted also by the heterochromatin-associated protein HP1. We therefore decided to test also HP1 for general effects on chromosome 4 gene expression. We reanalyzed the microarray data from Liu et al (2005) to see if HP1 RNAi causes chromosome 4-specific expression changes. RNAi-mediated knock-down of HP1 caused a significant general increase in gene expression on chromosome 4 genes (Figure 3C and D; Supplementary Figure 2E). After 100 million simulations (as above), the frequency of gene groups with an increase equal to or stronger than the 4th chromosome was 8.6 × 10−6. Both Pof and HP1 microarray data were reanalyzed after removal of low expressed genes. The results were consistent (results not shown). We next tested if the reduction in gene expression in PofD119 or the increase in gene expression after HP1 RNAi is more pronounced at certain parts of the 4th chromosome, by plotting the relative change against the chromosomal position (Figure 3E). Genes that changed in gene expression are spread along the length of the chromosome. This result suggested a negative correlation between the change caused by PofD119 and HP1 RNAi. Indeed, when tested, the correlation was significant (P<<0.001) with an r-value of −0.60 (Supplementary Figure 2F). This means that removing Pof gives an inverse but proportional effect compared with HP1 RNAi at an individual gene level. We chose nine genes with different expression levels for confirmatory tests using real-time PCR from RNA templates. Four new biological replicates were prepared from both PofD119/PofD119 and wild-type first instar larvae. All nine genes showed decreased mRNA levels in PofD119/PofD119 mutants when compared with wild-type (Figure 4A). Figure 4.POF regulates and binds within chromosome 4 genes. (A) Comparison of the mean levels of mRNA from nine genes by real-time RT–PCR in wild type (dark gray) and PofD119/PofD119 mutants (light gray). mRNA levels were normalized against actin gene mRNA in each replicate and 100% was set as the mean value of expression in wild type. Error bars represent the standard deviations of four biological replicates. The y-axis shows the relative amount of transcript and the x-axis shows the gene name with an estimate of the relative amount of mRNA (expression level) as a percentage of actin mRNA below. (B) The binding of POF to six chromosome 4 genes and two genes from chromosome 2R (CG4016 and Xbp1) were analyzed using ChIP. The y-axis indicates enrichment plotted as percent of input. Mean POF enrichments (gray bars) and MOCK (black bars) with standard deviations from three independent chromatin preparations are shown. The binding of POF to three additional genes CG31998, region 220 kb (C), MED26, 800 kb (D) and Zyx102EF, 1080 kb (E) were analyzed using ChIP. The extent of the genes is shown below the x-axis. Download figure Download PowerPoint POF binds preferentially within genes The above results indicate that POF is necessary to assist transcription of genes on chromosome 4. We, therefore, decided to examine in more detail the distribution of POF on the chromosome in Schneider 2 cells by chromatin immunoprecipitation (ChIP), followed by real-time PCR. To get a rough idea of POF binding, we analyzed approximately half of the sequenced chromosome at 10 kb resolution (Supplementary Figure 3). The results suggest that POF is highly enriched on this part of the chromosome, with two regions showing less enrichment (230–320 and 730–790 kb regions). During the course of this study, high-resolution ChIP results were published showing that the MSL proteins bind within genes on the X chromosome (Alekseyenko et al, 2006; Gilfillan et al, 2006; Legube et al, 2006). As the 10 kb spacing was not suitable to draw conclusion on binding of POF at the gene level, we chose three well isolated genes with POF binding according to ChIP results for more detailed mapping: CG31998, MED26 and Zyx102EF. We analyzed these three genes at approximately 2 kb resolution using ChIP and the results are shown in Figure 4C–E. In these three genes POF clearly binds within the transcribed region rather than outside the genes. In addition, the amount of transcript from these three genes is decreased in a Pof mutant background (Figure 4A). We also tested the remaining six genes in Figure 4A for POF binding by ChIP. The results show that all chromosome 4 genes tested except CG1732 represent targets for POF binding. It should be noted that CG1732 shows the smallest reduction in gene expression in PofD119 mutants in Figure 4A. Binding of POF to the 4th chromosome depends on heterochromatin To identify POF requirements for binding to the 4th chromosome, we immunostained polytene chromosomes from larval strains with translocated 4th chromosomes. Six different T(1;4)s with karyotype pXd4; p4dX; 4 were stained to detect POF distal-4th chromosome (d4) binding and scored as no binding, single-band binding, partial binding (a few bands) and entire d4 binding (Supplementary Figure 4). In two of these translocations, we detected nuclei with single-band binding (T(1;4)wm5 and T(1;4)wm258−18) or partial binding (T(1;4)wm258−18). We decided to study these two translocations in more detail. By growing the larvae at different temperatures it became clear that at 18°C a majority of the nuclei in T(1;4)wm5 showed no binding on d4, although the normal 4th chromosome was still decorated with POF as usual. However, a fraction of the nuclei showed single-band binding (Figure 5 and Supplementary Figure 4). In contrast, in T(1;4)wm258−18, which includes more of the centromere proximal heterochromatin, the majority of the nuclei showed single-band binding and a fraction showed partial binding. If these larvae were grown at 25°C, no binding was detected in T(1;4)wm5, the fraction of single-band binding in T(1;4)wm258−18 was decreased and no partial binding was found. Because an increase in temperature is known to reduce the compaction of heterochromatin, we decided to also analyze chromosomes from T(1;4)/0 males, that is males lacking the Y chromosome. It is also known that partial or total removal of the Y chromosome increases the compaction of heterochromatin (Gowen and Gay, 1934; Spofford, 1976; Dimitri and Pisano, 1989). Removal of the Y chromosome caused a similar effect to lowering the temperature and, strikingly, in T(1;4)wm258−18/0 males grown at 18°C, a fraction of the d4 showed staining of the entire polytenized region (Figure 5). We conclude that the binding of POF depends on the amount and compaction of nearby heterochromatin. It should be stressed that POF is not detected within the centromere proximal heterochromatic region of the 4th chromosome. Whenever a single-band is seen in nuclei it is located at 101F–102B2. Figure 5.POF localization on translocated chromosomes. T(1;4) males stained with DAPI (blue) anti-POF (red) are shown as merged images. Original images are shown in Supplementary Figure 4. Schematic illustrations of the two translocations are shown on top (not to scale). The proximal X region is labeled pX and the distal 4 portion translocated to pX is labeled d4. The vertical black lines indicate the translocation breakpoints and are shown by white lines in the images. 3C indicates the cytological position of the X chromosome breakpoint, and h60 and h61 correspond to the heterochromatic regions 60 and 61. The temperature for growing the larvae is shown to the left of the images and the presence or absence of the Y chromosome is indicated below the images. The T(1;4)wm258−18/0 males grown at 18°C (bottom right) is a composite image in which half the POF-stained d4 has been collated on the DAPI-stained 4th chromosome. Download figure Download PowerPoint To analyze further the dependence on heterochromatin for POF binding to the 4th chromosome, we analyzed POF binding in H2Av, HP1, Su(var)3-7, Su(var)3-9 and Su(UR) larvae (mutants in which heterochromatin formation is affected). The results are shown in Figure 6A. On the 4th chromosomes from H2Av810 and HP104/HP105, mutant larvae the staining of POF was dramatically decreased although not completely absent. Upon increasing the exposure time, weak staining with the normal distribution of POF on the 4th chromosome was seen (results not shown). No effect was seen in mutants of Su(var)3-7, Su(var)3-9 or Su(UR). We then tested the possible interdependence of binding by POF and HP1 or H2Av by staining chromosomes from PofD119/PofD119 mutant larvae with antibodies raised against H2Av and HP1 (Figure 6B). A PofD119 mutant background has no effect on H2Av staining, but markedly reduces the binding of HP1 to the 4th chromosome. Pericentric heterochromatic regions show no detectable difference in HP1 binding between PofD119 mutants and wild type (Figure 6B). The reduction of HP1 binding to the 4th chromosome in PofD119/PofD119 mutant background is less dramatic than the reduction of POF binding in HP104/HP105 mutants. Notably, immunostaining also showed that POF and HP1 colocalized close to perfect at the level of cytology within the 4th chromosome (Figure 6C). We conclude that POF is dependent on heterochromatin for binding to the 4th chromosome and that POF and HP1 binding to the 4th chromosome is interdependent. Figure 6.Association of POF and HP1 with the 4th chromosome is interdependent. (A) Polytene chromosomes were prepared from wild-type, H2Av810/H2Av810, HP104/HP105, Su(var)3-7R2a8/ Su(var)3-7R2a8, Su(var)3-9evo/Su(var)3-906 and Su(UR)/Su(UR) third instar larvae and stained with DAPI (blue) and POF (red). The staining of POF is dramatically reduced in H2Av810 and HP1 mutant backgrounds. (B) Polytene chromosomes from wild type and PofD119/PofD119 mutants were stained with antibodies raised against H2Av (left panel) and against HP1 (right panel). Two nuclei from each genotype are shown with HP1 staining. Note the reduction of HP1 staining on the 4th chromosome but not in the chromocenter in PofD119/PofD119 mutants compared with wild type. (C) Localization of POF and HP1 on chromosome 4. The combined POF/HP1 image shows that POF colocalizes perfectly with HP1 on the 4th chromosome. Download figure Download PowerPoint Discussion Chromosome-specific regulation has long been known as a mechanism used to maintain correct expression levels of sex chromosomes, that is, dosage compensation. Discovery of the binding of POF to the 4th chromosome in, for example, D. melanogaster and to the male X chromosome in some related species suggests that the 4th chromosome is also regulated by a chromosome-specific system. Further, haplo-4th individuals of D. melanogaster can survive, and two hypotheses have been proposed to explain their survival: (i) a dosage compensation mechanism may compensate for the lack of one 4th chromosome (Hochman, 1976) or (ii) lo
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