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Buffering and proteolysis are induced by segmental monosomy in Drosophila melanogaster.

Lundberg LE, Figueiredo ML, Stenberg P, Larsson J - Nucleic Acids Res. (2012)

Bottom Line: We have examined the expression effects of seven heterozygous chromosomal deficiencies, both singly and in all pairwise combinations, in Drosophila melanogaster.Furthermore, long genes are significantly more highly buffered than short genes and gene length appears to be the primary determinant of the buffering degree.Furthermore, the results show that in deficiency heterozygotes the expression of genes involved in proteolysis is enhanced and negatively correlates with the degree of buffering.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Umeå University, SE-90187 Umeå, Sweden.

ABSTRACT
Variation in the number of individual chromosomes (chromosomal aneuploidy) or chromosome segments (segmental aneuploidy) is associated with developmental abnormalities and reduced fitness in all species examined; it is the leading cause of miscarriages and mental retardation and a hallmark of cancer. However, despite their documented importance in disease, the effects of aneuploidies on the transcriptome remain largely unknown. We have examined the expression effects of seven heterozygous chromosomal deficiencies, both singly and in all pairwise combinations, in Drosophila melanogaster. The results show that genes in one copy are buffered, i.e. expressed more strongly than the expected 50% of wild-type level, the buffering is general and not influenced by other monosomic regions. Furthermore, long genes are significantly more highly buffered than short genes and gene length appears to be the primary determinant of the buffering degree. For short genes the degree of buffering depends on expression level and expression pattern. Furthermore, the results show that in deficiency heterozygotes the expression of genes involved in proteolysis is enhanced and negatively correlates with the degree of buffering. Thus, enhanced proteolysis appears to be a general response to aneuploidy.

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Related in: MedlinePlus

Segmental monosomic regions are buffered. (A) Schematic illustration of the seven Drosophila deficiency regions included in the study. (B) Heatmap table showing the total number of uncovered genes (number of Affymetrix probe sets), total number of uncovered expressed genes (i.e. probe sets with a wild-type expression level >6) and the total length of the deficiency regions. (C) Absolute expression level (in log2 scale) after RMA normalization of all genes uncovered by a deficiency. The black line represents the median expression of six wild-type replicates (diploid gene copy number) sorted by expression levels. The grey line represents expression of the deficiency strains (monosomic gene copy number). The deficiency data were plotted as running averages of 50 genes. Genes with a wild-type expression level <6 or >12 were excluded from further analysis (grey boxes). (D) Standard deviation (in log2 scale) of the mean calculated from the six wild-type replicates for all expressed genes (after removing <6; >12), sorted in descending order. A total of 259 genes with SD>1 are defined as unstably expressed and were thus excluded from further analysis (black line). (E) Expression ratios (log2 scale) of 1000 expressed genes along chromosome 2, showing a representative dataset containing two adjacent deficiency regions (in black). (F) The mean expression ratio (log2 scale) for each deletion (diamonds indicate mean values for all genes from the two or three single replicates). Whiskers indicate 95% CIs. Black lines represent 0 in log2 scale or 100% of wild-type expression level and dashed lines represent −1 in log2 scale or 50% of wild-type expression level (i.e. the expected expression level in the absence of a buffering effect).
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gks245-F1: Segmental monosomic regions are buffered. (A) Schematic illustration of the seven Drosophila deficiency regions included in the study. (B) Heatmap table showing the total number of uncovered genes (number of Affymetrix probe sets), total number of uncovered expressed genes (i.e. probe sets with a wild-type expression level >6) and the total length of the deficiency regions. (C) Absolute expression level (in log2 scale) after RMA normalization of all genes uncovered by a deficiency. The black line represents the median expression of six wild-type replicates (diploid gene copy number) sorted by expression levels. The grey line represents expression of the deficiency strains (monosomic gene copy number). The deficiency data were plotted as running averages of 50 genes. Genes with a wild-type expression level <6 or >12 were excluded from further analysis (grey boxes). (D) Standard deviation (in log2 scale) of the mean calculated from the six wild-type replicates for all expressed genes (after removing <6; >12), sorted in descending order. A total of 259 genes with SD>1 are defined as unstably expressed and were thus excluded from further analysis (black line). (E) Expression ratios (log2 scale) of 1000 expressed genes along chromosome 2, showing a representative dataset containing two adjacent deficiency regions (in black). (F) The mean expression ratio (log2 scale) for each deletion (diamonds indicate mean values for all genes from the two or three single replicates). Whiskers indicate 95% CIs. Black lines represent 0 in log2 scale or 100% of wild-type expression level and dashed lines represent −1 in log2 scale or 50% of wild-type expression level (i.e. the expected expression level in the absence of a buffering effect).

Mentions: To study the buffering of genes in monosomic condition we chose seven segmental deficiencies (Df) differing in length, number of uncovered genes and number of uncovered expressed genes (Figure 1A and B). To explore their effects we outcrossed strains, each heterozygously harbouring one of the deficiencies, to wild-type (to make single, Df/+, deletion strains) and created all combinations of pairwise crosses (Supplementary Table S1). We then analysed adult female expression levels in the wild-type (six biological replicates) and each Df/+ strain, both single-deletion (2–3 biological replicates) and all non-lethal pairwise combinations (single replicates) by Affymetrix microarray analysis. To minimize the effect of genetic background we used isogenic DrosDel strains in all our crosses (29,30). Two of the 21 Df combinations were lethal. Since genes with non- or sub-detectable expression and genes with very high expression (which tend to reach saturation on the arrays) will behave as fully compensated in expression microarray studies, we plotted transcript levels in each Df/+ against wild-type transcript levels for Affymetrix probe sets (hereafter called genes) within the affected regions. The resulting plot showed that that transcript levels of genes with an expression level <6 (log2-scale) and >12 did not differ between wild-type and any Df/+ (Figure 1C). This implies that genes with transcript levels below or above these thresholds are either fully buffered or their transcript levels cannot be reliably measured. These genes were therefore removed from all further analysis. We have previously noticed that expression levels of a subset of genes recorded in all microarray analyses are highly variable due to technical reasons and/or the developmental stage analysed. We therefore used our six wild-type replicates to identify genes with highly variable expression and excluded all genes from further analysis for which the standard deviation of expression level for the wild-type replicates exceeded 1 in log2 scale (corresponding to a ±2-fold variation in expression) (Figure 1D). In summary, out of the 18 769 genes (probe sets) on the arrays we removed 52% due to low expression, 2.6% due to very high expression and 1.4% due to high variability between replicates. All further analysis was performed on the 8255 remaining genes, of which 320 were uncovered by a deficiency. The expression values of these 8255 genes correlates very well between the wild-type replicates (average rs = 0.93, for all pairwise comparisons).Figure 5.


Buffering and proteolysis are induced by segmental monosomy in Drosophila melanogaster.

Lundberg LE, Figueiredo ML, Stenberg P, Larsson J - Nucleic Acids Res. (2012)

Segmental monosomic regions are buffered. (A) Schematic illustration of the seven Drosophila deficiency regions included in the study. (B) Heatmap table showing the total number of uncovered genes (number of Affymetrix probe sets), total number of uncovered expressed genes (i.e. probe sets with a wild-type expression level >6) and the total length of the deficiency regions. (C) Absolute expression level (in log2 scale) after RMA normalization of all genes uncovered by a deficiency. The black line represents the median expression of six wild-type replicates (diploid gene copy number) sorted by expression levels. The grey line represents expression of the deficiency strains (monosomic gene copy number). The deficiency data were plotted as running averages of 50 genes. Genes with a wild-type expression level <6 or >12 were excluded from further analysis (grey boxes). (D) Standard deviation (in log2 scale) of the mean calculated from the six wild-type replicates for all expressed genes (after removing <6; >12), sorted in descending order. A total of 259 genes with SD>1 are defined as unstably expressed and were thus excluded from further analysis (black line). (E) Expression ratios (log2 scale) of 1000 expressed genes along chromosome 2, showing a representative dataset containing two adjacent deficiency regions (in black). (F) The mean expression ratio (log2 scale) for each deletion (diamonds indicate mean values for all genes from the two or three single replicates). Whiskers indicate 95% CIs. Black lines represent 0 in log2 scale or 100% of wild-type expression level and dashed lines represent −1 in log2 scale or 50% of wild-type expression level (i.e. the expected expression level in the absence of a buffering effect).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3401434&req=5

gks245-F1: Segmental monosomic regions are buffered. (A) Schematic illustration of the seven Drosophila deficiency regions included in the study. (B) Heatmap table showing the total number of uncovered genes (number of Affymetrix probe sets), total number of uncovered expressed genes (i.e. probe sets with a wild-type expression level >6) and the total length of the deficiency regions. (C) Absolute expression level (in log2 scale) after RMA normalization of all genes uncovered by a deficiency. The black line represents the median expression of six wild-type replicates (diploid gene copy number) sorted by expression levels. The grey line represents expression of the deficiency strains (monosomic gene copy number). The deficiency data were plotted as running averages of 50 genes. Genes with a wild-type expression level <6 or >12 were excluded from further analysis (grey boxes). (D) Standard deviation (in log2 scale) of the mean calculated from the six wild-type replicates for all expressed genes (after removing <6; >12), sorted in descending order. A total of 259 genes with SD>1 are defined as unstably expressed and were thus excluded from further analysis (black line). (E) Expression ratios (log2 scale) of 1000 expressed genes along chromosome 2, showing a representative dataset containing two adjacent deficiency regions (in black). (F) The mean expression ratio (log2 scale) for each deletion (diamonds indicate mean values for all genes from the two or three single replicates). Whiskers indicate 95% CIs. Black lines represent 0 in log2 scale or 100% of wild-type expression level and dashed lines represent −1 in log2 scale or 50% of wild-type expression level (i.e. the expected expression level in the absence of a buffering effect).
Mentions: To study the buffering of genes in monosomic condition we chose seven segmental deficiencies (Df) differing in length, number of uncovered genes and number of uncovered expressed genes (Figure 1A and B). To explore their effects we outcrossed strains, each heterozygously harbouring one of the deficiencies, to wild-type (to make single, Df/+, deletion strains) and created all combinations of pairwise crosses (Supplementary Table S1). We then analysed adult female expression levels in the wild-type (six biological replicates) and each Df/+ strain, both single-deletion (2–3 biological replicates) and all non-lethal pairwise combinations (single replicates) by Affymetrix microarray analysis. To minimize the effect of genetic background we used isogenic DrosDel strains in all our crosses (29,30). Two of the 21 Df combinations were lethal. Since genes with non- or sub-detectable expression and genes with very high expression (which tend to reach saturation on the arrays) will behave as fully compensated in expression microarray studies, we plotted transcript levels in each Df/+ against wild-type transcript levels for Affymetrix probe sets (hereafter called genes) within the affected regions. The resulting plot showed that that transcript levels of genes with an expression level <6 (log2-scale) and >12 did not differ between wild-type and any Df/+ (Figure 1C). This implies that genes with transcript levels below or above these thresholds are either fully buffered or their transcript levels cannot be reliably measured. These genes were therefore removed from all further analysis. We have previously noticed that expression levels of a subset of genes recorded in all microarray analyses are highly variable due to technical reasons and/or the developmental stage analysed. We therefore used our six wild-type replicates to identify genes with highly variable expression and excluded all genes from further analysis for which the standard deviation of expression level for the wild-type replicates exceeded 1 in log2 scale (corresponding to a ±2-fold variation in expression) (Figure 1D). In summary, out of the 18 769 genes (probe sets) on the arrays we removed 52% due to low expression, 2.6% due to very high expression and 1.4% due to high variability between replicates. All further analysis was performed on the 8255 remaining genes, of which 320 were uncovered by a deficiency. The expression values of these 8255 genes correlates very well between the wild-type replicates (average rs = 0.93, for all pairwise comparisons).Figure 5.

Bottom Line: We have examined the expression effects of seven heterozygous chromosomal deficiencies, both singly and in all pairwise combinations, in Drosophila melanogaster.Furthermore, long genes are significantly more highly buffered than short genes and gene length appears to be the primary determinant of the buffering degree.Furthermore, the results show that in deficiency heterozygotes the expression of genes involved in proteolysis is enhanced and negatively correlates with the degree of buffering.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Umeå University, SE-90187 Umeå, Sweden.

ABSTRACT
Variation in the number of individual chromosomes (chromosomal aneuploidy) or chromosome segments (segmental aneuploidy) is associated with developmental abnormalities and reduced fitness in all species examined; it is the leading cause of miscarriages and mental retardation and a hallmark of cancer. However, despite their documented importance in disease, the effects of aneuploidies on the transcriptome remain largely unknown. We have examined the expression effects of seven heterozygous chromosomal deficiencies, both singly and in all pairwise combinations, in Drosophila melanogaster. The results show that genes in one copy are buffered, i.e. expressed more strongly than the expected 50% of wild-type level, the buffering is general and not influenced by other monosomic regions. Furthermore, long genes are significantly more highly buffered than short genes and gene length appears to be the primary determinant of the buffering degree. For short genes the degree of buffering depends on expression level and expression pattern. Furthermore, the results show that in deficiency heterozygotes the expression of genes involved in proteolysis is enhanced and negatively correlates with the degree of buffering. Thus, enhanced proteolysis appears to be a general response to aneuploidy.

Show MeSH
Related in: MedlinePlus