Global regulation of gene expression in stem cells and regeneration

Rapid developments in genomics and transcriptomics fields have made it possible to ask new questions as well as solve various old problems in biology that were not achievable previously. Novel techniques such as RNA sequencing and Hi-C became available at the time I started my PhD. Therefore, in order to study regeneration in salamanders and genome-wide regulatory interactions in mouse embryonic stem cells, my first goals were to make use of these techniques. Regeneration in salamanders has not been fully understood despite being studied for a few centuries. One of the reasons was the scarcity of genomic data. We mainly solved this problem by providing a high-quality transcriptome of red spotted newt, using latest tools (Paper I). Combining Hi-C with promoter capture probes increased the resolution for finding regulatory interactions, mainly promoter-enhancer (distal element). One of the surprising discoveries was enhancer-enhancer interactions, which was actually due to imperfect promoter capture efficiency. Our method, HiCap (Paper II), had a highest resolution for locating enhancers, yet had a modest improvement over assigning enhancers to their closest gene. Further analysis of regulatory networks showed a strong connectivity of enhancers and promoters individually than promoter-enhancers together. My last two projects involved studying gene regulation at a single cell level. The role of small RNAs in gene regulation in individual cells was not studied at that time. Aiming to shed a light on this, we developed a single-cell method for small RNAs, where I performed all the computational analysis (Paper III). This novel method, Small-seq, mainly revealed that microRNAs could be used to cluster different cell types. Since almost all of the available singlecell methods quantify polyadenylated RNAs (mainly mRNAs), Small-seq showed that one can get equally good clustering of cells using an order of magnitude less number of genes (about 200 microRNAs in human embryonic stem cells compared to a few thousand mRNAs). By making use of the newt transcriptome from Paper I, we aim to decipher the cellular composition of blastema – a small bud of cell mass formed on the amputation surface of regenerating newt limb. Adult newt limbs, upon amputation, undergo a precisely controlled “magic” of regenerating fully functional copy of its original limb. Newt cells are shown to dedifferentiate back to progenitor-like cellular state, populate and differentiate back to necessary cell types. The extend of this dedifferentiation and which cells contribute and how much is unknown. In paper IV, we have studied limb regeneration in newt and identified 8 cell types in blastema, where one cell type has significantly enriched for transposable elements, DNA fragments that are able to change their genomic positions, and has been shown to play a critical role in stem cell pluripotency, disease and development. Overall, this thesis covers studies of gene regulation in regeneration and several types of stem cells, both at an individual cell level as well as using millions of cells, by applying latest experimental and computational methods

Additional file 1: of Genome-wide mapping of promoter-anchored interactions with close to single-enhancer resolution

Supplementary tables. (ZIP 20649 kb

Reading and editing the Pleurodeles waltl genome reveals novel features of tetrapod regeneration

Salamanders exhibit an extraordinary ability among vertebrates to regenerate complex body parts. However, scarce genomic resources have limited our understanding of regeneration in adult salamanders. Here, we present the ~20 Gb genome and transcriptome of the Iberian ribbed newt Pleurodeles waltl, a tractable species suitable for laboratory research. We find that embryonic stem cell-specific miRNAs mir-93b and mir-427/430/302, as well as Harbinger DNA transposons carrying the Myb-like proto-oncogene have expanded dramatically in the Pleurodeles waltl genome and are co-expressed during limb regeneration. Moreover, we find that a family of salamander methyltransferases is expressed specifically in adult appendages. Using CRISPR/Cas9 technology to perturb transcription factors, we demonstrate that, unlike the axolotl, Pax3 is present and necessary for development and that contrary to mammals, muscle regeneration is normal without functional Pax7 gene. Our data provide a foundation for comparative genomic studies that generate models for the uneven distribution of regenerative capacities among vertebrates