The role of Piwi in Drosophila germline nuclei

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Germline bi-directional and uni-directional piRNA clusters have a distinct chromatin structure.

In order to understand the chromatin status of piRNA clusters we performed ChIP-seq for a variety of active and repressive histone marks34 together with the HP1a protein in Drosophila melanogaster ovaries. The profiling of these marks along bi-directional piRNA clusters (Fig. 1), expressed in the germline, shows an evident depletion for active marks, such as H3K36me1, H3K36me3, and also H3K4me2/3, whereas a strong enrichment for repressive marks is observed, as seen for HP1a, H3K9me2, and more specifically H3K9me3 and Rhino.
To quantitatively compare the chromatin of piRNA clusters with other genomic regions, we defined enrichment of H3K9me3 (repressive) and H3K36me3 (active) chromatin marks (defined as a ratio of depth-normalized read number in ChIP to input) for prominent piRNA clusters in the genome, compared to their immediate genomic environment (Fig. 2). All bidirectional clusters, including the most prominent cluster at region 42AB, show a significantly higher H3K9me3 mark enrichment and lower H3K36me3 mark enrichment than their neighboring regions. The only uni-directional cluster expressed in the germline, X upstream, in contrary shows a totally opposite profile. These results indicate that high level of H3K9me3 mark is an important characteristic of piRNA clusters.

piRNA cluster promoters are necessary for piRNA expression

Along with chromatin marks, we also ChIPed for the RNA polymerase II phosphorylated on the serine 5 of its CTD domain to identify putative promoter regions of every piRNA cluster (Fig. 1,2). Doing so, we were able to find distinct RNA pol II signals around bidirectional clusters, and a unique signal upstream of uni-stranded clusters X upstream and Flamenco.
In order to determine if the regions corresponding to the RNA Pol II ChIP signal are really promoting transcription of piRNA clusters, we performed two types of experiment: first, we tested the ability of putative promoter cluster sequences to drive transcription by cloning them in front of a LacZ reporter gene and transfecting resulting constructs into embryonic S2 culture cells. The transcription output was assessed by measuring the level of LacZ expression in the cells (Fig. 5). Each putative promoter tested was able to drive expression of LacZ, indicating that those regions are indeed promoters, and do not need the presence of an active piRNA pathway to perform their function.

Cluster promoter motifs, and effect on transcription

Once we identified promoter sequences, we decided to align together those of major bidirectional piRNA clusters (5 peaks). We quickly noticed a 150-200 bp region that is highly similar in every promoter sequence. Inside this homologous region, we can identify several motifs that have already been described previously for promoters: a TATA box, an Initiation motif (Inr), and a CCAAT-box like35,36 motif (Fig.7). These motifs all seem to be relatively well conserved between different Drosophila species (see UCSC Genome Browser). In addition to these known motifs, we can also find another one that appear three times in that same region: G[G,T,C]C[A,C]C[A,T]C[C,T]. It is an eight nucleotides motif that was only identified previously in large-scale computational studies of Drosophila gene promoters. It was shown to be present mostly at genes expressed in Drosophila ovaries37,38.

Activity of transgenic piRNA clusters correlates with the level of H3K9me3 mark and with the presence of trans-generationally inherited piRNAs

To identify the features that discriminate piRNA-generating regions from other genomic loci, we searched for examples when the same locus is active in piRNA generation in one Drosophila strain but inactive in another. Two D. melanogaster strains, T1 and BX2, contain an identical number of tandem repeats of the P-lacZ transgene inserted in the same locus in the middle of chromosome arm 2R, a genomic position that does not give rise to piRNAs in other strains42,43. In the T1 strain the transgene gives rise to abundant piRNAs in germ cells, and these piRNAs are able to silence expression of another lacZ transgene in trans. In contrast, no piRNAs are generated from the same transgene in the BX2 strain40 (Fig. 1A). As the sequence of the T1 and the BX2 transgenes is identical, the reason for their differential ability to generate piRNAs is unknown. Recent studies revealed that two chromatin factors, SetDB1 (a methyltransferase responsible for installation of the H3K9me3 mark) and the HP1 homologue Rhino, are required for piRNA biogenesis32,33. Based on this, we hypothesized that a difference in the chromatin state of the T1 and the BX2 transgenes explains their differential ability to produce piRNAs. To test this we profiled the histone 3 lysine 9 trimethylation (H3K9me3) mark on the transgene sequences in ovaries of flies from both strains using ChIP-qPCR. The H3K9me3 mark was enriched by ~4 fold over the transgene in the ‘active’ T1 strain compared to the ‘inactive’ BX2 strain (Fig. 1B). To rule out the possibility that the differences in H3K9me3 mark between T1 and BX2 are caused by changes in nucleosome occupancy, we performed total H3 ChIP that showed no difference between the two strains (Fig. S1). This result indicates that the ability of a genomic locus to generate piRNAs indeed correlates with its chromatin structure and in particular with a high level of the H3K9me3 mark.
In Drosophila, piRNAs expressed in germ cells during oogenesis are deposited into the embryo and are important for transposon silencing in the next generation28. It was previously found that the BX2 locus, which is deficient in piRNA production, can be converted into an active locus (designated as BX2*) by exposure to maternally inherited cytoplasm, carrying piRNAs homologous to the lacZ transgene40 (Fig. 1A). To determine whether activation of the BX2 locus caused by trans-generationally inherited piRNAs was coupled with a change in the chromatin state of the locus, we analyzed this region for the presence of the H3K9me3 mark before and after conversion. Conversion of BX2 to BX2* was accompanied by an increase in the H3K9me3 mark on the transgene (Fig. 1C). The level of the H3K9me3 mark on the activated BX2* transgene was similar to the signal observed on the locus in the T1 strain. Therefore, exposure of an inactive locus to homologous piRNAs inherited from the previous generation leads to installment of the H3K9me3 mark and conversion of the locus to an active piRNA cluster.
The ability of the T1 and the activated BX2* loci to generate piRNAs was reported to be stable over many generations if the locus was inherited from the mother40 (Fig. 1A). However, the transgenic loci lose their capacity to generate piRNAs after paternal transmission, suggesting that the presence of piRNAs is necessary to maintain the locus in an active state. We measured the enrichment of the H3K9me3 mark on the T1 transgene after maternal and paternal transmission. The paternal transmission reduced the level of the H3K9me3 mark ~2 fold, although levels were slightly higher than those on the non-activated BX2 locus (Fig. 1D). Thus, lack of trans-generationally inherited piRNAs homologous to the transgene correlates with decreased levels of the H3K9me3 mark.

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Table of contents :

Introduction
Chapter 1 – The role of Piwi in Drosophila germline nuclei
Chapter 2 – piRNA cluster promoter and chromatin
Chapter 3 – Selection of piRNA cluster precursors
Part 1 – Drosophila melanogaster
Part 2 – Drosophila virilis
Discussion
Materials & Methods
References
Annexes – Supplementary Tables
Annexes – Supplementary Figures

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