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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - UNICODE (Evolution and Impact of Heterochromatin on a Young Drosophila Y chromosome)

Teaser

Summary

This project addresses three basic questions that concern the evolution of sex chromosomes, and its impact on the rest of the genome: 1) how does the Y chromosome become a genetic junk full of selfish repetitive elements? 2) How does the cell tame such male-specific expansion of selfish elements with small RNAs? 3) How does this arms-race between small RNAs and repeat elements drive the rest of the genome to evolve? Y chromosomes of most species only exist for the function of determining the maleness, with its genetic composition enriched for repetitive elements (sometimes can comprise over 90% of the entire Y chromosome sequence) but very few functional genes. This has been puzzling biologists for long, as the counterpart of Y chromosome, the X chromosome is fully functional and contains thousands of genes, which suggests the ancestor of X and Y chromosomes is a pair of ordinary autosomes. The highly degenerated Y chromosomes have been so widely observed in both plant and animal species, there must be some general evolutionary and molecular driving forces that are yet to be uncovered. The difficulty of tackling this question comes from the fact that most Y chromosomes have undergone at least tens of millions of years of evolution, for example, the human Y chromosome is 170 million years old. They have already become too degenerated with few evolutionary traces left to study. In this project, we are taking advantage of a very young Y chromosome system (termed â€˜neo-Yâ€™) aged only 1.5 million years of Drosophila miranda. It formed through fusion of an autosome to the ancestral Y chromosome, thus acquiring the same inheritance pattern as the Y chromosome. My previous studies showed that although most genes still exist on this young Y chromosome, it already has shown clear signatures of degeneration: over two thirds of the Y-linked genes have accumulated mutations that disrupt their protein structure, and a similar proportion of Y-linked genes have reduced their expression levels relative to their X-linked counterparts. And the sequence of this neo-Y has become much more repetitive than the neo-X. We are going to use the cutting-edge technologies of the third-generation sequencing and ChIP-seq to uncover 1) how this young Drosophila Y chromosome has shift its entire gene regulatory landscape from euchromatin to heterochromatin, the latter of which usually only exists in highly repetitive centromere and telomere regions of the genome. The underlying mechanisms have profound implications beyond the Y chromosome evolution alone, as most eukaryotic genomes are organised as intertwined euchromatin and heterochromatin regions, yet their dynamic transitions are unclear for the mechanisms. 2) The second objective involves identifying the responsive changes of small RNA pathways, driven by such expansion of repetitive elements on the Y chromosome, i.e., specifically in males. Small RNA (specifically â€˜piRNAsâ€™ and â€˜siRNAsâ€™) pathways are several ancient pathways that specifically silence the repeat elementsâ€™ activity to guard the integrity of the genome. I will test the hypothesis that the Y chromosome evolution has driven the adaptive changes of small RNA pathway, through birth of new small RNA genes, and/or upregulation of existing small RNA genes. This objective concerns the interaction between the Y chromosome vs. the rest of the genome, i.e., the evolution of Y chromosome is not an isolated event, would rather drive the rest genome to change. 3) The last objective aims to functionally validate the small RNA genes identified from 2). Paradoxically, previous studies showed that small RNAs are mostly deposited by the mother into the embryos, which cannot silence repeat elements from the Y chromosome if there are male-specific sequence changes. Therefore, the evolution of Y chromosome may ultimately drive the female genome to change, so that those repeat elements can be silenced. Overall, the results of these research objectives

Work performed

From August, 2016 unitil March, 2019, I have established a group comprising one postdoc scholar and four Ph.D. students in the Department of Molecular Evolution and Development at the University of Vienna. During this first part of the project, we have several achievements: 1) We have finished collecting the major body of the experimental data that are planned for this project. This includes massive ChIP-seq datasets of 11 histone modifications spanning 10 different tissues and stages. We have also finished collecting DNA-seq data of the focused species, including 200-fold PacBio sequencing, 200-fold 10x linked reads sequencing, and 100-fold Hi-C chromatin conformation data. We have used these data and performed the preliminary assembly of the genome. Finally, we have produced antibodies targeting the key genes that we plan to study in this project, including Rhino, Piwi, Ago2, Aub, and Ago3. We have verified these antibodiesâ€™ function by immunostaining. 2) We have published a research article on the peer-reviewed journal Molecular Biology and Evolution, which has been later highlighted by F1000 third-party recommendation website. We have also been invited to write a review paper in 2018 on the topic of â€˜evolution of heterochromatinâ€™ by the journal The Annals of the New York Academy of Sciences. The research paper looks into the dynamic change of chromatin configuration of genes by their age. We found for a new gene copy recently borned in the genome, it takes time for it to gradually acquire active histone modifications and evolve important functions of housekeeping genes. This is directly related to one of the project goals of looking into how chromatin evolves. 3) I have been invited to give several seminar and conference talks at Ludwig Maximilian University of Munich, Uppsala University at Sweden, and annual Drosophila conference of 2017 and annual Evolution Meeting at Marseilles etc. I also have co-organized the 1st Asian Evolution conference in 2018, and hosted a session of â€˜Evolution of Sexâ€™. These work greatly helped to disseminate our project.

Final results

We plan to finish all the experimental data collection before the summer of 2019. At the same time, we have already started the bioinformatic analyses including genome assembly, ChIP-seq data processing in Drosophila pseudoobscura (whose genome is already available). We expect to finish determining the chromatin state of D. pseudoobscura also before the summer of 2019, and also finish annotating the genomes of D. miranda for genes and repetitive elements as well. Then before the end of 2019, we expect to submit two research papers, one is about the chromatin evolution of neo-Y chromosome in D. miranda. We will focus on describing the genome assembly of the neo-Y chromosome, by comparing the chromatin state between D. miranda and D. pseudoobscura, we will also be able to elucidate the mechanisms of heterochromatin formation on the neo-Y. Another research paper will be about evolution of dosage compensation between the two species. We recently found, among the collected data, that Drosophila testis also has dosage compensation. This is quite unexpected given previous results reported a lack of expression of key protein complex (MSL complex) of dosage compensation process within Drosophila testis. We will test a hypothesis that the evolution of dosage compensation on the X chromosome may accelerate the degeneration of Y chromosome.