Expression analysis identifies metabolism genes as altered in <i>HP1b</i> mutants.

GO terms significantly enriched (top) or depleted (bottom three terms) are reported along with the relevant p-values. The bar graphs show the observed (grey) and expected (black) number of genes associated with each GO term. GO terms show are top level terms from GO SLIM. For the results for all GO SLIM terms, see S2 Table. For the results from the basic GO term analysis see S3 (upregulated genes) and S4 (downregulated genes) Tables.</p

<i>HP1b</i>^<i>16</i> and <i>HP1b</i> ^<i>86</i> are null alleles.

A. Top—diagram showing the genomic region near the HP1b locus on the X chromosome. The P element mobilized to generate the HP1b alleles is indicated by a triangle above the genes shown in blue above the position on the X chromosome (bp). Black arrows mark the primers used to screen for deletions. Bottom—close-up of HP1b with the three deletion alleles shown below. All three deletions remove the HP1b start codon. B. RT-PCR analysis shows that full-length HP1b mRNA is transcribed in wildtype OR embryos, but not in HP1b16 or HP1b86 homozygous embryos. CG7346 and APC4 are transcribed in all samples, as is the Rpl32 positive control. RT: reverse transcriptase. C. No HP1B protein is detected in HP1b16 and HP1b86 homozygous larvae (left panel; *); Actin (right panel; **) serves as loading control.</p

<i>HP1b</i> is an enhancer of variegation.

A. If mutant HP1b alleles are introduced into stocks carrying hsp70-white reporter genes, the red eye color (reflecting white expression) is decreased compared to the yw control in male flies. Representative eye pictures are shown from males for two of the reporter insertions, 39C-3 located in the pericentric region of chromosome 2, and 118E-10 located in the pericentric region of chromosome 4. B. Quantitative eye pigment assays for males from the five reporter insertions assayed. All comparisons of mutant eye pigment values to the yw control are statistically significant (p480 measuring eye pigment. Results from the yw control are in red, from HP1b16 in blue, and from HP1b86 in green. Box plots: black bar—median; box—+25% and -25% quartiles; whiskers—maximum and minimum; circles—outliers; n = 6–10. C. Stubble variegation assays indicate that HP1b is an enhancer of variegation, as the presence of the HP1b mutant alleles significantly increases the number of wildtype bristles compared to the yw control (pyw control are in red, from HP1b16 in blue, and from HP1b86 in green. Box plots: black bar—median; box—+25% and -25% quartiles; whiskers—maximum and minimum; circles—outliers. For data from females, see S1 Fig.</p

Mutations in <i>HP1b</i> reduce female fertility.

A. The average offspring number for individual females of yw (red; n = 73), HP1b16 (blue; n = 80), and HP1b86 (green; n = 82) is shown (Y-axis). Error bars: SEM. The average number of offspring in both HP1b16 and HP1b86 females is significantly reduced compared to yw females (t test, pB. The average offspring number for individual males of yw (red; n = 80), HP1b16 (blue; n = 73), and HP1b86 (green; n = 76) is shown (Y-axis). Error bars: SEM. The average offspring number of HP1b16 and HP1b86 males does not differ from that of yw males (t test, not significant).</p

Loss of HP1B increases survival during starvation and oxidative stress, but not during heat stress.

A. HP1b mutant animals survive significantly longer during starvation condition than yw control animals (p = 1.022e-13 and p = 7.188e-06 for HP1b16 and HP1b86, respectively, Kruskal-Wallis rank sum test). B. HP1b mutant animals survive significantly longer after exposure to the oxidizer paraquat than yw control animals (p = 3.057e-13 and p HP1b16 and HP1b86, respectively, Kruskal-Wallis rank sum test). C. HP1b mutant animals do not show improved survival during heat stress conditions (37°C) compared to yw control animals. In contrast, they show lower survival (p = 0.04158 and p = 1.874e-09, for HP1b16 and HP1b86, respectively, Kruskal-Wallis rank sum test).</p

HP1B is a euchromatic Drosophila HP1 homolog with links to metabolism

Heterochromatin Protein 1 (HP1) proteins are an important family of chromosomal proteins conserved among all major eukaryotic lineages. While HP1 proteins are best known for their role in heterochromatin, many HP1 proteins function in euchromatin as well. As a group, HP1 proteins carry out diverse functions, playing roles in the regulation of gene expression, genome stability, chromatin structure, and DNA repair. While the heterochromatic HP1 proteins are well studied, our knowledge of HP1 proteins with euchromatic distribution is lagging behind. We have created the first mutations in HP1B, a Drosophila HP1 protein with euchromatic function, and the Drosophila homolog most closely related to mammalian HP1α, HP1β, and HP1γ. We find that HP1B is a non-essential protein in Drosophila, with mutations affecting fertility and animal activity levels. In addition, animals lacking HP1B show altered food intake and higher body fat levels. Gene expression analysis of animals lacking HP1B demonstrates that genes with functions in various metabolic processes are affected primarily by HP1B loss. Our findings suggest that there is a link between the chromatin protein HP1B and the regulation of metabolism.</div

Loss of HP1B impacts survival.

Survival curves differ significantly between the HP1b mutant strains and the yw control strain (p = 0.0483; log rank test). X-axis: Time to death in days. Y-axis: Survival probability. Data shown are combined from three trials, each with 100 animals per genotype/sex. For separate survival curves for males and females, see S4 Fig.</p

Loss of HP1B reduces food intake, alters body fat levels, and leads to oxidative stress resistance due to altered feeding behavior.

A. Food intake measured using a CAFÉ assay (Y-axis, decrease in food levels over an 8hr time period as measured by drop in capillary meniscus in mm) reveals lower food intake in HP1b mutants (HP1b16: p = 0.0003155; HP1b86: p = 0.0095041; Tukey’s multiple comparisons of means; n = 8 per sex and genotype). Data from one representative trial, both sexes combined are shown. For results from the additional trials and separated by sex, see S1 Table. B. Percent body fat (Y-axis) was measured by QMR in animals of different ages (X-axis). Body fat levels are increased in mutant animals early in life but decrease at a rapid rate after midlife (30–40 days). Red: yw; blue: HP1b16; green: HP1b86. Error bars: SD. N = 4 per sex and genotype. C. After paraquat concentration is adjusted to take into account differences in feeding behavior between HP1b mutants (blue, green) and the yw control strain (red), HP1b mutants no longer exhibit increased resistance to oxidative stress. X-axis: Time to death in hours. Y-axis: Survival probability. For separate survival curves for males and females, see S5 Fig.</p

Animals lacking HP1B show decreased levels of activity.

A. A significantly higher fraction of third instar larvae lacking HP1B (HP1b16 and HP1b86) failed to move in a 60 second observation period compared to the yw control (pB. Adult animals lacking HP1B show significantly lower activity levels than animals of the yw control genotype (p<0.001 for both comparisons; Tukey’s HSD). Y-axis: activity level as measured by recorded beam crossings in an activity monitor for 10 flies within a 2hr assay period. Black diamond and bar: mean +/- SD. N = 50 per genotype.</p

Krebs cycle metabolites and mitochondrial complex III activity are impacted by loss of HP1B.

A&B. Assay of Krebs cycle metabolites by mass spectroscopy reveals that citrate (A) and malate (B) levels are lower in HP1b mutants. X-axis: Genotypes. Y-axis: Metabolite levels in μg/ml. n = 4 per sex and genotype. C. Mitochondrial function assays indicate that Complex III activity is reduced in HP1b mutant animals (males). X-axis: Genotypes. Y-axis: enzyme activity in mmol/mg/min. n = 5 per genotype.</p