Heterochromatin protein 1a functions for piRNA biogenesis predominantly from pericentric and telomeric regions in Drosophila

Abstract

In metazoan germline, Piwi-interacting RNAs (piRNAs) provide defence against transposons. Piwi-piRNA complex mediates transcriptional silencing of transposons in nucleus. Heterochromatin protein 1a (HP1a) has been proposed to function downstream of Piwi-piRNA complex in Drosophila. Here we show that HP1a germline knockdown (HP1a-GLKD) leads to a reduction in the total and Piwi-bound piRNAs mapping to clusters and transposons insertions, predominantly in the regions close to telomeres and centromeres, resulting in derepression of a limited number of transposons from these regions. In addition, HP1a-GLKD increases the splicing of transcripts arising from clusters in above regions, suggesting HP1a also functions upstream to piRNA processing. Evolutionarily old transposons enriched in the pericentric regions exhibit significant loss in piRNAs targeting these transposons upon HP1a-GLKD. Our study suggests that HP1a functions to repress transposons in a chromosomal compartmentalised manner.

Fig. 1

HP1a is required for the repression of selective transposons. a Representative images showing control and HP1a-GLKD fly ovaries stained for HP1a and HeT-A Gag proteins. Scale bars = 25 μm; n > 5. b Scatterplot showing the fold up-regulation of normalised RNA-seq reads mapping to canonical transposons in HP1a-GLKD compared to control ovaries. Telomeric transposons HeT-A, TART, and TAHRE are labelled in red. c Boxplot representing fold up-regulation in normalised RNA-seq reads mapping to telomeric and non-telomeric transposons in HP1a-GLKD vs. control. The middle line represents median. Box represents 25–75 percentile range called inter quartile range (IQR). Upper and lower whisker extend highest or lowest values till 1.5*IQR. Values above and below are outliers and plotted individually. d Ovaries with an HP1a mitotic clone (arrowhead) generated by FRT/FLP recombination stained for HP1a, GFP, and HeT-A Gag proteins. HP1a-null cells recapitulate show HeT-A upregulation. Scale bars = 25 μm; n = 3

Fig. 2

Loss of HP1a leads to the concomitant selective loss of piRNAs mapping to transposons. a, b Comparison of reduction in canonical transposon mapping piRNAs for a total cellular piRNAs in HP1a-GLKD[2] ovaries and b Piwi-bound piRNAs among two replicates of HP1a-GLKD[2] ovaries in comparison to control. Telomeric transposons, HeT-A, TART, and TAHRE, are labelled in red and correlation in piRNA loss among the replicates was estimated using Spearman’s rank correlation test. c Fold reduction in the total cellular piRNAs mapping to clusters on the chromosomal arms. Clusters close to the telomeres and centromeres are represented in red. d Fold reduction in the piRNAs uniquely mapping to transposon insertions, for different cytolocations. The piRNAs uniquely mapping to transposon insertions were consolidated for each cytolocation and the cytolocations having more than 1000 uniquely-mapping piRNAs in control were analysed. e Loess smoothening of the reduction in the 23–29-nt reads uniquely mapping to the transposon insertions in chromosome arms 2L and 2R. Fold reductions in those reads in HP1a-GLKD (HP1a-GLKD[2] and HP1a-GLKD[3] average), aub, ago3, and aub-ago3 ovaries compared with those in their respective controls are presented

Fig. 3

HP1a represses Het-A in a location-dependent manner a Schematic representation of the HeT-A/lacZ reporter construct. The HeT-A promoter is fused to the lacZ gene. b Schematic representation of the HeT-A/lacZ reporter insertion at the cytological position 66C (H18). c Representative confocal images of HP1a, LacZ, and HeT-A immunostaining in tej^48-5/CyO, tej^48-5, and HP1a-GLKD ovaries. All genotypes harbour the HeT-A/lazZ transgene (H18 line). Three independent experiments were performed. Scale bars = 25 μm. d Bar graph showing the percentage of ovarioles expressing LacZ in tej^48-5/CyO, tej^48-5, and HP1a-GLKD genotypes bearing the HeT-A/lacZ transgene (H18 and H111). LacZ positive and negative ovarioles are represented by black and grey, respectively. n represents the number of ovarioles counted (n ≥ 100, from three independent experiments). e Representative confocal images of LacZ and HP1a immunostaining in the mitotic clones lacking HP1a. Red arrows and white arrows denote the HP1a clones with LacZ and those without LacZ expression, respectively. Scale bars = 15 μm. Three biological replicates were examined. f Scatterplot showing the mean likelihood of HP1a enrichment on chromosomal arms. Cytolocations marking telomeres and centromeres are represented in red. g UCSC browser screenshot showing the changes in total piRNAs and HP1a enrichment, following the normalisation with reads in the IgG ChIP-seq library, at cluster 42AB (upper panel) and cluster 38C (lower panel) in the control and HP1a-GLKD ovaries

Fig. 4

HP1a is necessary for the proper splicing of piRNA precursors from selected clusters. a Amplification of regions from piRNA clusters at 42AB, 20F, and sox102F. Representative gel images of RT-PCRs show amplification and levels of the spliced and unspliced transcripts originating from clusters 42AB, 20F, and sox102F in HP1a-GLKD[2] ovaries. cuff-GLKD and krimp mutants were used as positive and negative controls, respectively. Three independent experiments were performed. b Spliced to unspliced transcript level ratio (SSR) calculated for the piRNA clusters 42AB, 20F, and sox102F. qRT-PCR was conducted with a primer set detecting either unspliced or spliced transcript. Three independent experiments were performed. The significance of difference in levels of spliced transcript between control and HP1a-GLKD[2] and between control and cuff-GLKD, ovaries was statistically tested using Mann-Whitney U test. n = 3, three independent biological replicates, error bars represent standard error for all replicates. p values: ^**represent p value <0.05 and ^*** represent p value <0.005. c Boxplot showing the changes in the average FPKM values of spliced transcripts mapping to the piRNA clusters in the HP1a-GLKD[2] ovaries vs. the controls. d The average FPKM values of spliced transcripts mapping to the piRNA clusters in cytolocations 1, 20, 21, 40, 41, 60, 61, 80, 810, 100, and 102 with that in the clusters in rest of genome, in control and HP1a-GLKD[2] ovaries. e Fold increase in the spliced transcript coming from major piRNA clusters in HP1a-GLKD ovaries. f Representative confocal images of Rhi immunostaining in the control and HP1a-GLKD ovaries (left panel) and comparison of Rhi foci sizes between the control and HP1a-GLKD[2] ovaries (right panel). Scale bar = 5 μm. Sixty egg-chambers from three biological replicates were analysed for the Rhi foci sizes. Statistical significance in differences of Rhi foci between the genotypes was examined using Kolmogorov-Smirnov test. For the boxplots in c–e, the middle line represents median. Box represents 25–75 percentile range called inter quartile range (IQR). Upper and lower whisker extend highest or lowest values till 1.5 * IQR. Values above and below are outliers and plotted individually

Fig. 5

HP1a is required for the biogenesis of piRNAs mapping to evolutionarily older transposons. a The percentage of the transposons insertions from major transposon families that showed at least three-fold loss in the uniquely-mapping piRNAs in the HP1a-GLKD ovaries. The average reduction between the 23~29-nt read levels in the HP1a-GLKD[2] and HP1a-GLKD[3] ovaries was plotted, and the insertions targeted by at least 20 unique piRNAs were considered. The number in red represents number of such insertions for each transposon family. b Boxplots showing the reduction in the piRNAs mapping to the evolutionarily fixed, common, and rare transposon insertions, based on their occurrence in population, in the HP1a-GLKD and ago3, aub, and aub-ago3 double mutants. The significance of higher reduction of piRNAs mapping to evolutionarily fixed transposon insertions in comparison with those mapping to common or rare insertions was examined using Mann-Whitney U-test. The middle line represents median. Box represents 25–75 percentile range called inter quartile range (IQR). Upper and lower whisker extend highest or lowest values till 1.5 * IQR. Values above and below are outliers and plotted individually