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Dosage analysis of Z chromosome genes using microarray in silkworm, Bombyx mori
Insect Biochemistry and Molecular Biology 39 (2009) 315–321
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Dosage analysis of Z chromosome genes using microarray in silkworm, Bombyx mori Xingfu Zha a, Qingyou Xia a, b, *, Jun Duan a, b, Chunyun Wang a, Ningjia He a, Zhonghuai Xiang a a b
The Key Sericultural Laboratory of Agricultural Ministry, College of Biotechnology, Southwest University, Beibei, Chongqing 400715, China The Institute of Agronomy and Life Sciences, Chongqing University, Chongqing 400030, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 November 2007 Received in revised form 20 October 2008 Accepted 4 December 2008
In many organisms, dosage compensation is needed to equalize sex-chromosome gene expression in males and females. Several genes on silkworm Z chromosome were previously detected to show a higher expression level in males and lacked dosage compensation. Whether silkworm lacks global dosage compensation still remains poorly known. Here, we analyzed male:female (M:F) ratios of expression of chromosome-wide Z-linked genes in the silkworm using microarray data. The expression levels of genes on Z chromosome in each tissue were significantly higher in males compared to females, which indicates no global dosage compensation in silkworm. Interestingly, we also found some genes with no bias (M:F ratio: 0.8–1.2) on the Z chromosome. Comparison of male-biased (M:F ratio more than 1.5) and unbiased genes indicated that the two sets of the genes have functional differences. Analysis of gene expression by sex showed that M:F ratios were, to some extent, associated with their expression levels. These results provide useful clues to further understanding roles of dosage of Z chromosome and some Z-linked sexual differences in silkworms. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Bombyx mori Dosage compensation Microarray Z chromosome Genome
1. Introduction Sex chromosomes show striking difference between sexes in many eukaryotes, such as humans, Drosophila melanogaster and Caenorhabditis elegans. Male-heterogametic species carry a pair of X chromosomes in female and an XY pair in male. Female-heterogametic species have two Z chromosomes in male and a ZW in female. The X and Z chromosomes can carry many genes, but the Y and W chromosomes are often composed mainly of transposable elements and possess few functional genes (Abe et al., 2005; Venter et al., 2001; Adams et al., 2000; International Chicken Genome Sequencing Consortium, 2004). Females and males have a different genomic dosage of sex-chromosome genes. To balance the dosage difference, some species use different molecular mechanisms. Mammals randomly inactivate one of X chromosomes in females and increase the expression of the single active X genes in both sexes to be on par with that of the autosomal genes (Nguyen and Disteche, 2006). D. melanogaster increases transcription from the single X in males (Baker et al., 1994; Meller and Kuroda, 2002). In C. elegans, hermaphrodites have a pair of sex chromosomes (XX);
the males have only one sex chromosome (X0). Hermaphrodites (XX) reduce the level of transcripts from each of their two X chromosomes by half to equal the expression from the single male X (Meyer and Casson, 1986). The silkworm, Bombyx mori, is a female-heterogametic insect. Suzuki et al. (1998) first reported a Z-linked gene, named as T15.180a, showed twice as much mRNA transcript from this in males than in females (Suzuki et al., 1998). A second Z-linked gene, Bmkettin, had the same expression pattern as T15.180a in that it lacked dosage compensation (Suzuki et al., 1999). Recently, expression levels of 13 Z-chromosome-linked genes around the Bmkettin locus were detected, and most of them were shown to express more abundant mRNA in males than in females (Koike et al., 2003). In order to determine whether chromosome-wide Z-linked genes in the silkworm are dosage compensated, we tested the mRNA level of Z-chromosome genes between sexes of the silkworm using microarray. 2. Materials and methods 2.1. Animals, tissues and microarray
* Corresponding author. Key Sericultural Laboratory of Agricultural Ministry, Southwest University, Tiansheng Road, Beibei, Chongqing 400715, China. Tel.: þ86 23 68250099; fax: þ86 023 68251128. E-mail address: [email protected] (Q. Xia). 0965-1748/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2008.12.003
Gene-expression profiles of silkworm tissues/organs using oligonucleotide microarrays were obtained from our previous study (Xia et al., 2007). Briefly, microarrays were designed based on the draft silkworm genome sequence database, and included probe
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sequences for over 18,000 silkworm genes. The silkworm strain Dazao used in this study was provided by the Silkworm Genetic Resource Center of Southwest University, China. In order to investigate gene-expression differences by sex, we prepared male and female samples at the same time. In this research, gene-expression profiles were surveyed in five tissues/organs including anterior/ median silk gland (A/MSG), gonads, integument, malpighian tubules and head from silkworm larvae on day 3 of the fifth instar. Biological replicates were done 12 times in A/MSG, 12 times in gonads, 4 times in integument, 8 times malpighian tubules and 4 times in head, respectively. 2.2. Identification of probes located on Z chromosome A genome-wide microarray with 70-mer oligonucleotide probes used in this study was designed by using the predicted genes from the draft sequence of the silkworm genome and the expressed sequence tags (Xia et al., 2004, 2007). Recently, the complete sequence of the silkworm genome has been finished and newly predicted genes from the complete sequence have also been done (Unpublished). We identified lists of the candidate Z-chromosome probes using BLAST. Z-chromosome genome sequences were used to search the database of the sequences of the probes using BLASTN. Since probes are 70-mer oligonucleotides, all BLAST matches with 100% identity and >35-bp length shows E values from 5E33 to 1E11. Thus, we used BLASTN with a relaxed threshold E < 1e11. To exclude the hitted probes from all other chromosomes, we did the second BLAST search. High-score probes with 99% identity were BLASTNed in the complete sequences of all chromosomes to identify the best hit. If the best hit was the same site of Z chromosome, the probe was confirmed to locate on Z chromosome. Then, the probes were searched to match newly predicted genes from the complete Z-chromosome sequence. If the probes were searched to the same gene, probe IDs were combined to decrease gene redundancy. 2.3. Expression data analysis We analyzed the male:female gene-expression ratio on five silkworm tissues/organs as mentioned above. We scanned each array with a confocal LuxScan scanner and evaluated the raw intensity value based on the approach of median of pixel intensity at each channel subtract background using LuxScan 3.0 software. Raw data for silkworm genes were background-subtracted before calculating M:F ratios. Weak spots with signal intensities below 400 units and uneven signal were filtered out. To determine which genes showed a sex difference in expression, hybridization to each spot was normalized relative to the mean hybridization of four autosomal house-keeping genes (those encoding proteasome beta subunit, eIF-3 subunit 4, eIF-3A subunit 5, and eIF 4A), and the M:F ratio of gene expression was calculated for each gene and tissue . Considering that global gene-expression changes may exist across different silkworm tissues, we applied a linear normalization
method to normalize individual channel data instead of the prevalent LOWESS normalization method for dual-channel microarrays. Three categories were arbitrarily developed in this study: (1) M:F ratio 1.5, a ratio of greater than or equal to 1.5 used as a cut-off indicating higher expression in males, which supports no dosage compensation (Craig et al., 2004); (2) M:F ratio between 1.2 and 0.8, indicating relatively equal expression in males and females; and (3) M:F ratio <0.5, indicating higher expression in females. To compare expression levels of Z genes in males and those in females, OneSample T Test was used to compare the mean score of M:F ratios of Z genes to the population mean of 1. The distribution of M:F ratios on the Z chromosome was analyzed by computing the running average of M:F ratios with a mean length of 30 genes.
2.4. Annotation of gene function We annotated genes systematically using the following bioinformatics tools. The annotations include homologous searches using BLASTX and functional classifications using Gene Ontologies (GO) (Ashburner et al., 2000). Gene sequences were BLASTXed to nr database in GenBank. GO annotations were assigned using the program Blast2GO (Conesa et al., 2005). After BLASTX, the default settings were used to assign GO terms to gene sequences. From these annotations, analysis was made using 2nd level GO terms based on biological process, molecular function, and cellular component (Ye et al., 2006).
3. Results 3.1. Expression of silkworm genes on Z chromosome By using Z-chromosome sequences as described above, on microarrays, we identified 697 probes on the Z chromosome. The lists of these probes and their signal intensity after normalization are showed in Table S1. The total of 697 probes represented 579 genes on silkworm Z chromosome owing to some probes recognizing the same gene. We calculated male:female (M:F) geneexpression ratio change in each tissue as shown in Table S2. Microarray detection revealed that active genes are most in the gonads and least in A/MSG, and that majority (65–90%) of genes exhibit M:F ratio higher than 1 in these tissues/organs (Table 1). In addition, the expression ratios of 55% genes are greater than 2 in the gonads while only 3% do in head. To determine whether genes on silkworm Z chromosome in general are expressed at a significantly higher levels in males, we performed T test on expression data. The results were shown in Fig. 1. In each of five samples, the mean of the log2 M:F is significantly greater than zero (T test: p ¼ 4.8e28 for integument, p ¼ 4.1e28 for head, p ¼ 7.5e7 for malpighian tubules, p ¼ 0.004 for A/MSG and p ¼ 5.7e32 for gonads), which clearly demonstrates Z-linked genes are expressed at significantly higher levels in males compared to females, indicating silkworm lacks global dosage compensation.
Table 1 Distribution of genes that are differentially expressed between males and females in integument, head, malpighian tubule, A/MSG and gonad. Tissue/organ
Active genes
Integument Head Malpighian tubule A/MSG Gonad
180 186 155 132 314
Genes with M:F ratio (%) >1.0
>1.2
>1.5
>2.0
162 158 115 86 246
138 128 93 57 222
74 48 56 28 204
20 5 26 10 172
(90%) (85%) (74%) (65%) (78%)
(77%) (69%) (60%) (43%) (71%)
(41%) (26%) (36%) (21%) (65%)
(11%) (3%) (17%) (8%) (55%)
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Fig. 1. T test analysis of male:female (M:F) ratios of gene expression for Z-linked genes. The vertical red line is centered at an M:F ratio of 1 (log2 ratio of 0). A/MSG represents anterior and median silk gland. y-axis denotes the frequency of genes expressing within a certain bin. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.2. Functional analysis of Z-linked genes sorted by M:F ratios Most Z-linked genes in the silkworm showed higher expression level in males than in females. However, we also found some Z-linked genes showed no male-biased expression. Some genes with somewhat lower expression in males than in females were also found. To compare the sets of different genes by sex, we
classified Z-linked genes into three categories by M:F ratios: malebiased genes (M:F 1.5), female-biased genes (M:F < 0.5) and genes with no bias (M:F 0.8–1.2). In somatic tissues, there are 94 male-biased genes, 57 unbiased genes, and 6 female-biased genes (Table S3). Among them, 8 male-biased genes, 8 unbiased genes and 1 female-biased gene showed no similarity to genes reported in nr database, and these genes probably are species-specific. For
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male-biased genes, its male-biased expression probably reflected some silkworm sex-limited characters. For example, the gene, BGIBMGA000498, encoded circadian locomoter output cycles kaput protein, a central component of circadian clock. Male-biased expression of the circadian clock gene may explain some sexual differences in adult behavior of silkworms. To further understand gene function, we carried out GO annotation. Thirty-two malebiased genes and 16 unbiased genes had GO functional classifications while female-biased genes lacked them. Comparing GO function of the male-biased genes to the unbiased ones, results indicated that the male-biased genes were enriched for binding activity, catalytic activity, transport, transcriptional regulation, molecular transduction, antioxidant activity, enzyme regulation and motor function (Fig. 2). In contrast, the unbiased genes did not include any enzyme regulator activity and motor function genes. Genes encoding structural proteins were part of the unbiased gene category. These results probably reflected functional differences between two sets of genes. Interestingly, six female-biased genes were identified on Z chromosome (Table S3). One of these, BGIBMGA000639, encoded a homolog of iroquois-class homeodomain protein irx, which has been confirmed to express at a higher level in female mice than in males in a previous study (Jorgensen and Gao, 2005). These results suggested that six femalebiased genes might play a role in female-specific differences, thus expressed at a higher level in female, although they are located on Z chromosome. In the gonads, great changes in M:F ratios were shown (Table S2). Surprisingly, there were more than 50 genes with ratios in double digits, including 4 genes with ratios greater than 100. It was a possible reason that the gonads including testes and ovaries are so different morphologically by sex, and they contain different cell types, so they expressed quite different sets of genes. We carried out functional annotation for these genes (Table S4). Interestingly, we found a few genes coding testis-specific proteins, such as BGIBMGA002056 and BGIBMGA000508. The former coded a protein of outer dense fiber of sperm tails 2 isoform 1, and the latter was similar to sperm associated antigen 17. 3.3. Distribution of M:F ratios of gene expression In order to learn whether genes showing male-biased and unbiased expression are concentrated in specific regions of the Z
chromosome, we mapped M:F ratio by gene position along the Z chromosome (Fig. 3A). The map indicated that genes with high and low M:F ratios were found across the entire Z chromosome. We computed a running average of M:F ratios on the Z chromosome (Fig. 3B–F) and observed probably similar trend of M:F ratios on Z chromosome in the integument and head. To further verify whether the trend exists, we carried out Independent-Samples T Test using M:F ratio data in Table S2. Results showed that M:F value of gene expression in integument is significantly different from that in head (T test: p < 0.0004), which does not support the opinion about similar trend. To compare the levels of expression of genes on the Z chromosome in each sex to M:F ratios, we calculated median expression of M:F ratios. Fig. 4 illustrates that genes with high (1.5) M:F ratios have generally lower expression than the genes that are equally expressed (M:F ratio near 1) in both sexes. In other words, malebiased genes showed low expression, but unbiased genes had higher expression levels. The results suggested expression difference of Z-chromosome genes by sex in silkworm was also associated with their expression level to some extent.
3.4. Verification of expression differences of Z-linked genes by sex Our analysis of expression differences of Z-linked genes by sex was based strictly on microarray data. To verify that expression differences of Z-linked genes by sex did not result from an artifact of microarray, we compared previously reported expression data of Z-linked genes with differential expression by sex obtained using different techniques. Among 579 Z-linked genes in this study, 10 genes were previously detected with higher expression level in males than in females using real time RT-PCR (Koike et al., 2003); 8 of these genes showed significantly higher than 1:1 expression ratio using microarray data (Table S5). The results suggested that our analysis of expression differences of Z-linked genes by sex using microarray was reproducible. The remaining 2 genes, named as Bmhepa and Bmmiple, showed no significant deviation from 1:1 in this study. Koike et al. detected mRNA expression levels of the genes in thoracic segments from newly emerged moths, whereas we used five tissues/organs of 5 fifth-instar larvae as experimental materials. This is a possible reason for different results of two
Fig. 2. GO categories of male-biased and unbiased genes on silkworm Z chromosome. Thirty-two male-biased genes and 16 unbiased genes have GO functional classifications. All genes are assigned a gene ontology. The y-axis denotes the percentage of male-biased genes, or unbiased genes, with a GO classification to 32 male-biased genes or 16 unbiased genes. Along the x-axis, these are three independent gene ontologies. The male-biased genes are with M:F ratio more than 1.5, and the unbiased genes with M:F ratio of 0.8–1.2.
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Fig. 3. Region distribution of M:F ratios on Z chromosome. (A) Individual M:F ratios in the integument, graphed by gene position on the Z chromosome. Two genes (BGIBMGA002087 and BGIBMGA000725) are the outliers with respect to M/F ratio. The former with M:F expression ratio 5.315 codes macrophage migration inhibitory factor in Bombyx mori, and the latter with the ratio 4.010 codes ATP-binding cassette transporter. (B–F) The running average of 30 M:F ratios is plotted at the median gene position, for integument, head, malpighian tubule, A/MSG (anterior and median silk gland), and gonad.
experiments. Of course, we trend to carry out further real time RT-PCR and northern blot experiments to validate results presented in the study since all expression level differences from microarrays are putative and often do not correlate with real time PCR results (Morey et al., 2006). 4. Discussion In the silkworm, female is the heterogametic (ZW) sex and the male is homogametic (ZZ). In this study, we have analyzed the expression level of genes on Z chromosome in the two sexes, and found male:female ratios of genes significantly higher in males than in females, indicating that silkworm lacks chromosome-wide dosage compensation. The mean of M:F ratios in each tissue was 1.25 in A/MSG, 1.34 in head, 1.53 in malpighian tubules, 1.54 in integument and 11.15 in gonads (Table S2). Aside from gonads, other tissues showed the means of M:F ratios less than 2. The results concur with the previous reports, which suggested that, in the absence of dosage compensation, the fold-change in expressed
gene dose is often considerably less than the difference in gene copy number in the genome (Birchler et al., 2002, 2005; Itoh et al., 2007). Thus, we would expect a distribution of M:F ratios with a mean less than 2, as observed here. At the level of transcription no ‘‘twofold increase’’ rule seems to exist. Several genes were previously reported in silkworm to show expression differences between males and females, such as vitellogenin, beta-tubulin, Bmdsx, BmDmc1, tektin and BmAha1 (Yano et al., 1994; Mita et al., 1995; Ohbayashi et al., 2001; Kusakabe et al., 2001; Ota et al., 2002; Miyagawa et al., 2005), which play a role in oogenesis, spermatogenesis and sex determination. These genes are not present on Z chromosome (data not shown). They show sex-difference expression principally due to regulation of sex determination and difference pathways. The reason of male-biased expression pattern of Z-chromosome genes is different from them. It has previously been reported that dosage compensation was ineffective in some other ZW species such as zebra finch and chicken also (Itoh et al., 2007), and that some dosage compensated genes occurred in specific regions of chicken Z chromosome
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Fig. 4. Correlation of M:F ratios with male and female expression levels. For each somatic tissue, all genes showing expression from the Z chromosome were grouped into bins according to M:F ratios. Bin width is 0.2. Median values of expression levels of genes in each bin are calculated. Values are plotted at mid-point of bin. A small number of genes, with M:F ratios outside of the range shown, are included in the most extreme bins.
(Melamed and Arnold, 2007). Another microarray analysis indicated that chickens have no global dosage compensation (Ellegren et al., 2007). In silkworm, we found no sets of genes with M:F ratios of 0.8–1.2 clustered on specific region of silkworm Z chromosome in any tissue. Interestingly, genes on Z chromosome with high (>1.5) M:F ratios showed generally lower expression, a situation reverse from birds (Melamed and Arnold, 2007). It seems that the mechanisms of dosage of Z gene expression in silkworms and birds are different although they both lack chromosome-wide dosage compensation. Silkworms clearly differ from D. melanogaster in smaller sexual difference of X gene expression achieved owing to dosage compensation. In D. melanogaster, at least eight genes (msl-1, msl-2, msl-3, mle, mof, roX1, roX2, JIL-1) are required for dosage compensation. Five proteins coded by msl-1, msl-2, msl-3, mle and mof, together with roX1 and roX2, compose a large complex, which specifically targets hundreds of sites on the male X chromosome to activate transcription of the X-linked genes only in males. However, only homologs of msl-3, mle and mof were found in silkworms (Xia et al., 2004; Mita et al., 2004). It is believed that mammalian orthologs of MOF, MLE, and MSL-3 compose a complex for transcriptional regulation other than dosage compensation (Marin, 2003), which suggests that D. melanogaster recently utilized these proteins to regulate sex-linked genes transcriptionally. These results might indicate different mechanisms of silkworm regulated dosage of Z-chromosome genes. In butterflies (Heliconius), activity of 6-phosphogluconate dehydrogenase (6PGD) in males is approximately twice that in females (Johnson and Turner, 1979). As the results referred in Naisbit’s paper, phenotype analysis on Z-linked triose-phosphate isomerase suggested no dosage compensation in butterflies (Naisbit et al., 2002). Both of the results from butterflies and silkworms revealed that lepidoptera lack no dosage compensation of sex-linked genes. Dosage tolerance and the evolutionary pressure for sex-chromosome genes are certainly related to gene function (Straub and Becker, 2007). Differences of enzyme levels and morphological characters by sex in butterflies indicate that lepidoptera does not evolve any mechanisms for post-transcriptional regulation of Z-chromosome gene dosage. Double Z-linked genes bring too much more gene expression in males than in females in lepidoptera. This may reflect why there is a relationship between expression levels
and M:F ratios. Our analysis of Z gene functions showed two sets of Z genes with different M:F ratios involved in different molecular functions. Genes with M:F ratios higher than 1.5 were involved in regulatory activity and motor functions, whereas genes with ratios in the lower range of 0.8–1.2 did not fall in these categories. These male-biased Z genes are more likely to have evolved for a role in controlling sexual differentiation in silkworms. Our results provide valuable information for further understanding the roles of gene dosage on Z chromosome for sexual differences during silkworm development. Acknowledgments This work was supported by research grants from the National Basic Research Program of China (No. 2005CB121000), the National Hi-Tech Research and Development Program of China (No. 2006AA10A118), and the National Natural Science Foundation of China (No. 30800804 and No. 30330460). Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version, at doi:10.1016/j.ibmb.2008.12.003. References Abe, H., Mita, K., Yasukochi, Y., Oshiki, T., Shimada, T., 2005. Retrotransposable elements on the W chromosome of the silkworm, Bombyx mori. Cytogenet. Genome Res. 110 (1–4), 144–151. Adams, M.D., Celniker, S.E., Holt, R.A., Evans, C.A., et al., 2000. The genome sequence of Drosophila melanogaster. Science 287 (5461), 2185–2195. Ashburner, M., Ball, C.A., Blake, J.A., Botstein, D., Butler, H., Cherry, J.M., Davis, A.P., Dolinski, K., Dwight, S.S., Eppig, J.T., Harris, M.A., Hill, D.P., Issel-Tarver, L., Kasarskis, A., Lewis, S., Matese, J.C., Richardson, J.E., Ringwald, M., Rubin, G.M., Sherlock, G., 2000. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 25 (1), 25–29. Baker, B.S., Gorman, M., Marin, I., 1994. Dosage compensation in Drosophila. Annu. Rev. Genet. 28, 491–521. Birchler, J.A., Bhadra, U., Bhadra, M.P., Auger, D.L., 2002. Dosage-dependent gene regulation in multicellular eukaryotes: implications for dosage compensation, aneuploid syndromes, and quantitative traits. Dev. Biol. 234, 275–288. Birchler, J.A., Riddle, N.C., Auger, D.L., Veitia, R.A., 2005. Dosage balance in gene regulation: biological implications. Trends Genet. 21, 219–226.
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Dosage analysis of Z chromosome genes using microarray in silkworm, Bombyx mori Xingfu Zha a, Qingyou Xia a, b, *, Jun Duan a, b, Chunyun Wang a, Ningjia He a, Zhonghuai Xiang a a b
The Key Sericultural Laboratory of Agricultural Ministry, College of Biotechnology, Southwest University, Beibei, Chongqing 400715, China The Institute of Agronomy and Life Sciences, Chongqing University, Chongqing 400030, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 November 2007 Received in revised form 20 October 2008 Accepted 4 December 2008
In many organisms, dosage compensation is needed to equalize sex-chromosome gene expression in males and females. Several genes on silkworm Z chromosome were previously detected to show a higher expression level in males and lacked dosage compensation. Whether silkworm lacks global dosage compensation still remains poorly known. Here, we analyzed male:female (M:F) ratios of expression of chromosome-wide Z-linked genes in the silkworm using microarray data. The expression levels of genes on Z chromosome in each tissue were significantly higher in males compared to females, which indicates no global dosage compensation in silkworm. Interestingly, we also found some genes with no bias (M:F ratio: 0.8–1.2) on the Z chromosome. Comparison of male-biased (M:F ratio more than 1.5) and unbiased genes indicated that the two sets of the genes have functional differences. Analysis of gene expression by sex showed that M:F ratios were, to some extent, associated with their expression levels. These results provide useful clues to further understanding roles of dosage of Z chromosome and some Z-linked sexual differences in silkworms. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Bombyx mori Dosage compensation Microarray Z chromosome Genome
1. Introduction Sex chromosomes show striking difference between sexes in many eukaryotes, such as humans, Drosophila melanogaster and Caenorhabditis elegans. Male-heterogametic species carry a pair of X chromosomes in female and an XY pair in male. Female-heterogametic species have two Z chromosomes in male and a ZW in female. The X and Z chromosomes can carry many genes, but the Y and W chromosomes are often composed mainly of transposable elements and possess few functional genes (Abe et al., 2005; Venter et al., 2001; Adams et al., 2000; International Chicken Genome Sequencing Consortium, 2004). Females and males have a different genomic dosage of sex-chromosome genes. To balance the dosage difference, some species use different molecular mechanisms. Mammals randomly inactivate one of X chromosomes in females and increase the expression of the single active X genes in both sexes to be on par with that of the autosomal genes (Nguyen and Disteche, 2006). D. melanogaster increases transcription from the single X in males (Baker et al., 1994; Meller and Kuroda, 2002). In C. elegans, hermaphrodites have a pair of sex chromosomes (XX);
the males have only one sex chromosome (X0). Hermaphrodites (XX) reduce the level of transcripts from each of their two X chromosomes by half to equal the expression from the single male X (Meyer and Casson, 1986). The silkworm, Bombyx mori, is a female-heterogametic insect. Suzuki et al. (1998) first reported a Z-linked gene, named as T15.180a, showed twice as much mRNA transcript from this in males than in females (Suzuki et al., 1998). A second Z-linked gene, Bmkettin, had the same expression pattern as T15.180a in that it lacked dosage compensation (Suzuki et al., 1999). Recently, expression levels of 13 Z-chromosome-linked genes around the Bmkettin locus were detected, and most of them were shown to express more abundant mRNA in males than in females (Koike et al., 2003). In order to determine whether chromosome-wide Z-linked genes in the silkworm are dosage compensated, we tested the mRNA level of Z-chromosome genes between sexes of the silkworm using microarray. 2. Materials and methods 2.1. Animals, tissues and microarray
* Corresponding author. Key Sericultural Laboratory of Agricultural Ministry, Southwest University, Tiansheng Road, Beibei, Chongqing 400715, China. Tel.: þ86 23 68250099; fax: þ86 023 68251128. E-mail address: [email protected] (Q. Xia). 0965-1748/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2008.12.003
Gene-expression profiles of silkworm tissues/organs using oligonucleotide microarrays were obtained from our previous study (Xia et al., 2007). Briefly, microarrays were designed based on the draft silkworm genome sequence database, and included probe
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sequences for over 18,000 silkworm genes. The silkworm strain Dazao used in this study was provided by the Silkworm Genetic Resource Center of Southwest University, China. In order to investigate gene-expression differences by sex, we prepared male and female samples at the same time. In this research, gene-expression profiles were surveyed in five tissues/organs including anterior/ median silk gland (A/MSG), gonads, integument, malpighian tubules and head from silkworm larvae on day 3 of the fifth instar. Biological replicates were done 12 times in A/MSG, 12 times in gonads, 4 times in integument, 8 times malpighian tubules and 4 times in head, respectively. 2.2. Identification of probes located on Z chromosome A genome-wide microarray with 70-mer oligonucleotide probes used in this study was designed by using the predicted genes from the draft sequence of the silkworm genome and the expressed sequence tags (Xia et al., 2004, 2007). Recently, the complete sequence of the silkworm genome has been finished and newly predicted genes from the complete sequence have also been done (Unpublished). We identified lists of the candidate Z-chromosome probes using BLAST. Z-chromosome genome sequences were used to search the database of the sequences of the probes using BLASTN. Since probes are 70-mer oligonucleotides, all BLAST matches with 100% identity and >35-bp length shows E values from 5E33 to 1E11. Thus, we used BLASTN with a relaxed threshold E < 1e11. To exclude the hitted probes from all other chromosomes, we did the second BLAST search. High-score probes with 99% identity were BLASTNed in the complete sequences of all chromosomes to identify the best hit. If the best hit was the same site of Z chromosome, the probe was confirmed to locate on Z chromosome. Then, the probes were searched to match newly predicted genes from the complete Z-chromosome sequence. If the probes were searched to the same gene, probe IDs were combined to decrease gene redundancy. 2.3. Expression data analysis We analyzed the male:female gene-expression ratio on five silkworm tissues/organs as mentioned above. We scanned each array with a confocal LuxScan scanner and evaluated the raw intensity value based on the approach of median of pixel intensity at each channel subtract background using LuxScan 3.0 software. Raw data for silkworm genes were background-subtracted before calculating M:F ratios. Weak spots with signal intensities below 400 units and uneven signal were filtered out. To determine which genes showed a sex difference in expression, hybridization to each spot was normalized relative to the mean hybridization of four autosomal house-keeping genes (those encoding proteasome beta subunit, eIF-3 subunit 4, eIF-3A subunit 5, and eIF 4A), and the M:F ratio of gene expression was calculated for each gene and tissue . Considering that global gene-expression changes may exist across different silkworm tissues, we applied a linear normalization
method to normalize individual channel data instead of the prevalent LOWESS normalization method for dual-channel microarrays. Three categories were arbitrarily developed in this study: (1) M:F ratio 1.5, a ratio of greater than or equal to 1.5 used as a cut-off indicating higher expression in males, which supports no dosage compensation (Craig et al., 2004); (2) M:F ratio between 1.2 and 0.8, indicating relatively equal expression in males and females; and (3) M:F ratio <0.5, indicating higher expression in females. To compare expression levels of Z genes in males and those in females, OneSample T Test was used to compare the mean score of M:F ratios of Z genes to the population mean of 1. The distribution of M:F ratios on the Z chromosome was analyzed by computing the running average of M:F ratios with a mean length of 30 genes.
2.4. Annotation of gene function We annotated genes systematically using the following bioinformatics tools. The annotations include homologous searches using BLASTX and functional classifications using Gene Ontologies (GO) (Ashburner et al., 2000). Gene sequences were BLASTXed to nr database in GenBank. GO annotations were assigned using the program Blast2GO (Conesa et al., 2005). After BLASTX, the default settings were used to assign GO terms to gene sequences. From these annotations, analysis was made using 2nd level GO terms based on biological process, molecular function, and cellular component (Ye et al., 2006).
3. Results 3.1. Expression of silkworm genes on Z chromosome By using Z-chromosome sequences as described above, on microarrays, we identified 697 probes on the Z chromosome. The lists of these probes and their signal intensity after normalization are showed in Table S1. The total of 697 probes represented 579 genes on silkworm Z chromosome owing to some probes recognizing the same gene. We calculated male:female (M:F) geneexpression ratio change in each tissue as shown in Table S2. Microarray detection revealed that active genes are most in the gonads and least in A/MSG, and that majority (65–90%) of genes exhibit M:F ratio higher than 1 in these tissues/organs (Table 1). In addition, the expression ratios of 55% genes are greater than 2 in the gonads while only 3% do in head. To determine whether genes on silkworm Z chromosome in general are expressed at a significantly higher levels in males, we performed T test on expression data. The results were shown in Fig. 1. In each of five samples, the mean of the log2 M:F is significantly greater than zero (T test: p ¼ 4.8e28 for integument, p ¼ 4.1e28 for head, p ¼ 7.5e7 for malpighian tubules, p ¼ 0.004 for A/MSG and p ¼ 5.7e32 for gonads), which clearly demonstrates Z-linked genes are expressed at significantly higher levels in males compared to females, indicating silkworm lacks global dosage compensation.
Table 1 Distribution of genes that are differentially expressed between males and females in integument, head, malpighian tubule, A/MSG and gonad. Tissue/organ
Active genes
Integument Head Malpighian tubule A/MSG Gonad
180 186 155 132 314
Genes with M:F ratio (%) >1.0
>1.2
>1.5
>2.0
162 158 115 86 246
138 128 93 57 222
74 48 56 28 204
20 5 26 10 172
(90%) (85%) (74%) (65%) (78%)
(77%) (69%) (60%) (43%) (71%)
(41%) (26%) (36%) (21%) (65%)
(11%) (3%) (17%) (8%) (55%)
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Fig. 1. T test analysis of male:female (M:F) ratios of gene expression for Z-linked genes. The vertical red line is centered at an M:F ratio of 1 (log2 ratio of 0). A/MSG represents anterior and median silk gland. y-axis denotes the frequency of genes expressing within a certain bin. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.2. Functional analysis of Z-linked genes sorted by M:F ratios Most Z-linked genes in the silkworm showed higher expression level in males than in females. However, we also found some Z-linked genes showed no male-biased expression. Some genes with somewhat lower expression in males than in females were also found. To compare the sets of different genes by sex, we
classified Z-linked genes into three categories by M:F ratios: malebiased genes (M:F 1.5), female-biased genes (M:F < 0.5) and genes with no bias (M:F 0.8–1.2). In somatic tissues, there are 94 male-biased genes, 57 unbiased genes, and 6 female-biased genes (Table S3). Among them, 8 male-biased genes, 8 unbiased genes and 1 female-biased gene showed no similarity to genes reported in nr database, and these genes probably are species-specific. For
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male-biased genes, its male-biased expression probably reflected some silkworm sex-limited characters. For example, the gene, BGIBMGA000498, encoded circadian locomoter output cycles kaput protein, a central component of circadian clock. Male-biased expression of the circadian clock gene may explain some sexual differences in adult behavior of silkworms. To further understand gene function, we carried out GO annotation. Thirty-two malebiased genes and 16 unbiased genes had GO functional classifications while female-biased genes lacked them. Comparing GO function of the male-biased genes to the unbiased ones, results indicated that the male-biased genes were enriched for binding activity, catalytic activity, transport, transcriptional regulation, molecular transduction, antioxidant activity, enzyme regulation and motor function (Fig. 2). In contrast, the unbiased genes did not include any enzyme regulator activity and motor function genes. Genes encoding structural proteins were part of the unbiased gene category. These results probably reflected functional differences between two sets of genes. Interestingly, six female-biased genes were identified on Z chromosome (Table S3). One of these, BGIBMGA000639, encoded a homolog of iroquois-class homeodomain protein irx, which has been confirmed to express at a higher level in female mice than in males in a previous study (Jorgensen and Gao, 2005). These results suggested that six femalebiased genes might play a role in female-specific differences, thus expressed at a higher level in female, although they are located on Z chromosome. In the gonads, great changes in M:F ratios were shown (Table S2). Surprisingly, there were more than 50 genes with ratios in double digits, including 4 genes with ratios greater than 100. It was a possible reason that the gonads including testes and ovaries are so different morphologically by sex, and they contain different cell types, so they expressed quite different sets of genes. We carried out functional annotation for these genes (Table S4). Interestingly, we found a few genes coding testis-specific proteins, such as BGIBMGA002056 and BGIBMGA000508. The former coded a protein of outer dense fiber of sperm tails 2 isoform 1, and the latter was similar to sperm associated antigen 17. 3.3. Distribution of M:F ratios of gene expression In order to learn whether genes showing male-biased and unbiased expression are concentrated in specific regions of the Z
chromosome, we mapped M:F ratio by gene position along the Z chromosome (Fig. 3A). The map indicated that genes with high and low M:F ratios were found across the entire Z chromosome. We computed a running average of M:F ratios on the Z chromosome (Fig. 3B–F) and observed probably similar trend of M:F ratios on Z chromosome in the integument and head. To further verify whether the trend exists, we carried out Independent-Samples T Test using M:F ratio data in Table S2. Results showed that M:F value of gene expression in integument is significantly different from that in head (T test: p < 0.0004), which does not support the opinion about similar trend. To compare the levels of expression of genes on the Z chromosome in each sex to M:F ratios, we calculated median expression of M:F ratios. Fig. 4 illustrates that genes with high (1.5) M:F ratios have generally lower expression than the genes that are equally expressed (M:F ratio near 1) in both sexes. In other words, malebiased genes showed low expression, but unbiased genes had higher expression levels. The results suggested expression difference of Z-chromosome genes by sex in silkworm was also associated with their expression level to some extent.
3.4. Verification of expression differences of Z-linked genes by sex Our analysis of expression differences of Z-linked genes by sex was based strictly on microarray data. To verify that expression differences of Z-linked genes by sex did not result from an artifact of microarray, we compared previously reported expression data of Z-linked genes with differential expression by sex obtained using different techniques. Among 579 Z-linked genes in this study, 10 genes were previously detected with higher expression level in males than in females using real time RT-PCR (Koike et al., 2003); 8 of these genes showed significantly higher than 1:1 expression ratio using microarray data (Table S5). The results suggested that our analysis of expression differences of Z-linked genes by sex using microarray was reproducible. The remaining 2 genes, named as Bmhepa and Bmmiple, showed no significant deviation from 1:1 in this study. Koike et al. detected mRNA expression levels of the genes in thoracic segments from newly emerged moths, whereas we used five tissues/organs of 5 fifth-instar larvae as experimental materials. This is a possible reason for different results of two
Fig. 2. GO categories of male-biased and unbiased genes on silkworm Z chromosome. Thirty-two male-biased genes and 16 unbiased genes have GO functional classifications. All genes are assigned a gene ontology. The y-axis denotes the percentage of male-biased genes, or unbiased genes, with a GO classification to 32 male-biased genes or 16 unbiased genes. Along the x-axis, these are three independent gene ontologies. The male-biased genes are with M:F ratio more than 1.5, and the unbiased genes with M:F ratio of 0.8–1.2.
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Fig. 3. Region distribution of M:F ratios on Z chromosome. (A) Individual M:F ratios in the integument, graphed by gene position on the Z chromosome. Two genes (BGIBMGA002087 and BGIBMGA000725) are the outliers with respect to M/F ratio. The former with M:F expression ratio 5.315 codes macrophage migration inhibitory factor in Bombyx mori, and the latter with the ratio 4.010 codes ATP-binding cassette transporter. (B–F) The running average of 30 M:F ratios is plotted at the median gene position, for integument, head, malpighian tubule, A/MSG (anterior and median silk gland), and gonad.
experiments. Of course, we trend to carry out further real time RT-PCR and northern blot experiments to validate results presented in the study since all expression level differences from microarrays are putative and often do not correlate with real time PCR results (Morey et al., 2006). 4. Discussion In the silkworm, female is the heterogametic (ZW) sex and the male is homogametic (ZZ). In this study, we have analyzed the expression level of genes on Z chromosome in the two sexes, and found male:female ratios of genes significantly higher in males than in females, indicating that silkworm lacks chromosome-wide dosage compensation. The mean of M:F ratios in each tissue was 1.25 in A/MSG, 1.34 in head, 1.53 in malpighian tubules, 1.54 in integument and 11.15 in gonads (Table S2). Aside from gonads, other tissues showed the means of M:F ratios less than 2. The results concur with the previous reports, which suggested that, in the absence of dosage compensation, the fold-change in expressed
gene dose is often considerably less than the difference in gene copy number in the genome (Birchler et al., 2002, 2005; Itoh et al., 2007). Thus, we would expect a distribution of M:F ratios with a mean less than 2, as observed here. At the level of transcription no ‘‘twofold increase’’ rule seems to exist. Several genes were previously reported in silkworm to show expression differences between males and females, such as vitellogenin, beta-tubulin, Bmdsx, BmDmc1, tektin and BmAha1 (Yano et al., 1994; Mita et al., 1995; Ohbayashi et al., 2001; Kusakabe et al., 2001; Ota et al., 2002; Miyagawa et al., 2005), which play a role in oogenesis, spermatogenesis and sex determination. These genes are not present on Z chromosome (data not shown). They show sex-difference expression principally due to regulation of sex determination and difference pathways. The reason of male-biased expression pattern of Z-chromosome genes is different from them. It has previously been reported that dosage compensation was ineffective in some other ZW species such as zebra finch and chicken also (Itoh et al., 2007), and that some dosage compensated genes occurred in specific regions of chicken Z chromosome
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Fig. 4. Correlation of M:F ratios with male and female expression levels. For each somatic tissue, all genes showing expression from the Z chromosome were grouped into bins according to M:F ratios. Bin width is 0.2. Median values of expression levels of genes in each bin are calculated. Values are plotted at mid-point of bin. A small number of genes, with M:F ratios outside of the range shown, are included in the most extreme bins.
(Melamed and Arnold, 2007). Another microarray analysis indicated that chickens have no global dosage compensation (Ellegren et al., 2007). In silkworm, we found no sets of genes with M:F ratios of 0.8–1.2 clustered on specific region of silkworm Z chromosome in any tissue. Interestingly, genes on Z chromosome with high (>1.5) M:F ratios showed generally lower expression, a situation reverse from birds (Melamed and Arnold, 2007). It seems that the mechanisms of dosage of Z gene expression in silkworms and birds are different although they both lack chromosome-wide dosage compensation. Silkworms clearly differ from D. melanogaster in smaller sexual difference of X gene expression achieved owing to dosage compensation. In D. melanogaster, at least eight genes (msl-1, msl-2, msl-3, mle, mof, roX1, roX2, JIL-1) are required for dosage compensation. Five proteins coded by msl-1, msl-2, msl-3, mle and mof, together with roX1 and roX2, compose a large complex, which specifically targets hundreds of sites on the male X chromosome to activate transcription of the X-linked genes only in males. However, only homologs of msl-3, mle and mof were found in silkworms (Xia et al., 2004; Mita et al., 2004). It is believed that mammalian orthologs of MOF, MLE, and MSL-3 compose a complex for transcriptional regulation other than dosage compensation (Marin, 2003), which suggests that D. melanogaster recently utilized these proteins to regulate sex-linked genes transcriptionally. These results might indicate different mechanisms of silkworm regulated dosage of Z-chromosome genes. In butterflies (Heliconius), activity of 6-phosphogluconate dehydrogenase (6PGD) in males is approximately twice that in females (Johnson and Turner, 1979). As the results referred in Naisbit’s paper, phenotype analysis on Z-linked triose-phosphate isomerase suggested no dosage compensation in butterflies (Naisbit et al., 2002). Both of the results from butterflies and silkworms revealed that lepidoptera lack no dosage compensation of sex-linked genes. Dosage tolerance and the evolutionary pressure for sex-chromosome genes are certainly related to gene function (Straub and Becker, 2007). Differences of enzyme levels and morphological characters by sex in butterflies indicate that lepidoptera does not evolve any mechanisms for post-transcriptional regulation of Z-chromosome gene dosage. Double Z-linked genes bring too much more gene expression in males than in females in lepidoptera. This may reflect why there is a relationship between expression levels
and M:F ratios. Our analysis of Z gene functions showed two sets of Z genes with different M:F ratios involved in different molecular functions. Genes with M:F ratios higher than 1.5 were involved in regulatory activity and motor functions, whereas genes with ratios in the lower range of 0.8–1.2 did not fall in these categories. These male-biased Z genes are more likely to have evolved for a role in controlling sexual differentiation in silkworms. Our results provide valuable information for further understanding the roles of gene dosage on Z chromosome for sexual differences during silkworm development. Acknowledgments This work was supported by research grants from the National Basic Research Program of China (No. 2005CB121000), the National Hi-Tech Research and Development Program of China (No. 2006AA10A118), and the National Natural Science Foundation of China (No. 30800804 and No. 30330460). Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version, at doi:10.1016/j.ibmb.2008.12.003. References Abe, H., Mita, K., Yasukochi, Y., Oshiki, T., Shimada, T., 2005. Retrotransposable elements on the W chromosome of the silkworm, Bombyx mori. Cytogenet. Genome Res. 110 (1–4), 144–151. Adams, M.D., Celniker, S.E., Holt, R.A., Evans, C.A., et al., 2000. The genome sequence of Drosophila melanogaster. Science 287 (5461), 2185–2195. Ashburner, M., Ball, C.A., Blake, J.A., Botstein, D., Butler, H., Cherry, J.M., Davis, A.P., Dolinski, K., Dwight, S.S., Eppig, J.T., Harris, M.A., Hill, D.P., Issel-Tarver, L., Kasarskis, A., Lewis, S., Matese, J.C., Richardson, J.E., Ringwald, M., Rubin, G.M., Sherlock, G., 2000. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 25 (1), 25–29. Baker, B.S., Gorman, M., Marin, I., 1994. Dosage compensation in Drosophila. Annu. Rev. Genet. 28, 491–521. Birchler, J.A., Bhadra, U., Bhadra, M.P., Auger, D.L., 2002. Dosage-dependent gene regulation in multicellular eukaryotes: implications for dosage compensation, aneuploid syndromes, and quantitative traits. Dev. Biol. 234, 275–288. Birchler, J.A., Riddle, N.C., Auger, D.L., Veitia, R.A., 2005. Dosage balance in gene regulation: biological implications. Trends Genet. 21, 219–226.
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