Epigenetic control of planarian stem cell potency limits stem activity and accurately defines differentiation programs

Planarian flatworms are gaining popularity in regenerative medicine research due to the fact that they have unparalleled regeneration capacity. Their tissue recovery abilities are dependent on a pool of adult stem cells (neoblasts). Studies in the recent years have shown that epigenetic mechanisms have an important role in neoblasts’ self-renewal and differentiation properties. This thesis focuses on the study of trithorax-related genes and their function in neoblast regulation. Despite the fact that mammalian trithorax-related genes Mll3 and Mll4 are among the most frequently mutated genes in cancer, trithorax-related genes are the least well-studies members of the trithorax gene group (TrxG) of histone modifiers. The current study traced the evolutionary history of trithorax-related genes and concluded that they have undergone a number of independent gene fission events across phyla. In planarians, there are three partial orthologue of the mammalian Mll3 and Mll4 genes – Smed-LPT (corresponding to the N-terminus of Mll3/4), Smed-trr-1 and Smed-trr-2 (both corresponding to the C-terminus of Mll3/4). The three planarian trithorax-related genes are expressed in stem cells and control neoblast differentiation down certain lineages (brain, gut, eyes, pharynx, epidermis). Down-regulation of Smed-LPT results in hyperproliferation of stem cells, leading to tumour-like outgrowth formation. It was shown that trithorax-related genes’ function in stem cell regulation correlates with histone modification changes, specifically alterations in H3K4me1, H3K4me3 and H3K27me3. Future studies will focus on examining this correlation further via Next-Generation sequencing techniques

Conservation of epigenetic regulation by the MLL3/4 tumour suppressor in planarian pluripotent stem cells

Currently, little is known about the evolution of epigenetic regulation in animal stem cells. Here we demonstrate, using the planarian stem cell system to investigate the role of the COMPASS family of MLL3/4 histone methyltransferases that their function as tumor suppressors in mammalian stem cells is conserved over a long evolutionary distance. To investigate the potential conservation of a genome-wide epigenetic regulatory program in animal stem cells, we assess the effects of Mll3/4 loss of function by performing RNA-seq and ChIP-seq on the G2/M planarian stem cell population, part of which contributes to the formation of outgrowths. We find many oncogenes and tumor suppressors among the affected genes that are likely candidates for mediating MLL3/4 tumor suppression function. Our work demonstrates conservation of an important epigenetic regulatory program in animals and highlights the utility of the planarian model system for studying epigenetic regulation

Epigenetic control of planarian stem cell potency limits stem activity and accurately defines differentiation programs

Planarian flatworms are gaining popularity in regenerative medicine research due to the fact that they have unparalleled regeneration capacity. Their tissue recovery abilities are dependent on a pool of adult stem cells (neoblasts). Studies in the recent years have shown that epigenetic mechanisms have an important role in neoblasts’ self-renewal and differentiation properties. This thesis focuses on the study of trithorax-related genes and their function in neoblast regulation. Despite the fact that mammalian trithorax-related genes Mll3 and Mll4 are among the most frequently mutated genes in cancer, trithorax-related genes are the least well-studies members of the trithorax gene group (TrxG) of histone modifiers. The current study traced the evolutionary history of trithorax-related genes and concluded that they have undergone a number of independent gene fission events across phyla. In planarians, there are three partial orthologue of the mammalian Mll3 and Mll4 genes – Smed-LPT (corresponding to the N-terminus of Mll3/4), Smed-trr-1 and Smed-trr-2 (both corresponding to the C-terminus of Mll3/4). The three planarian trithorax-related genes are expressed in stem cells and control neoblast differentiation down certain lineages (brain, gut, eyes, pharynx, epidermis). Down-regulation of Smed-LPT results in hyperproliferation of stem cells, leading to tumour-like outgrowth formation. It was shown that trithorax-related genes’ function in stem cell regulation correlates with histone modification changes, specifically alterations in H3K4me1, H3K4me3 and H3K27me3. Future studies will focus on examining this correlation further via Next-Generation sequencing techniques

Antitumor Properties of Epitope-Specific Engineered Vaccine in Murine Model of Melanoma

Finding new effective compounds of natural origin for composing anti-tumor vaccines is one of the main goals of antitumor research. Promising anti-cancer agents are the gastropodan hemocyanins–multimeric copper-containing glycoproteins used so far for therapy of different tumors. The properties of hemocyanins isolated from the marine snail Rapana thomasiana (RtH) and the terrestrial snail Helix aspersa (HaH) upon their use as carrier-proteins in conjugated vaccines, containing ganglioside mimotope GD3P4 peptide, were studied in the developed murine melanoma model. Murine melanoma cell line B16F10 was used for solid tumor establishment in C57BL/6 mice using various schemes of therapy. Protein engineering, flow cytometry, and cytotoxicity assays were also performed. The administration of the protein-engineered vaccines RtH-GD3P4 or HaH-GD3P4 under the three different regimens of therapy in the B16F10 murine melanoma model suppressed tumor growth, decreased tumor incidence, and prolonged the survival of treated animals. The immunization of experimental mice induced an infiltration of immunocompetent cells into the tumors and generated cytotoxic tumor-specific T cells in the spleen. The treatment also generates significantly higher levels of tumor-infiltrated M1 macrophages, compared to untreated tumor-bearing control mice. This study demonstrated a promising approach for cancer therapy having potential applications for cancer vaccine research

The CCR4-NOT Complex Mediates Deadenylation and Degradation of Stem Cell mRNAs and Promotes Planarian Stem Cell Differentiation

<div><p>Post-transcriptional regulatory mechanisms are of fundamental importance to form robust genetic networks, but their roles in stem cell pluripotency remain poorly understood. Here, we use freshwater planarians as a model system to investigate this and uncover a role for CCR4-NOT mediated deadenylation of mRNAs in stem cell differentiation. Planarian adult stem cells, the so-called neoblasts, drive the almost unlimited regenerative capabilities of planarians and allow their ongoing homeostatic tissue turnover. While many genes have been demonstrated to be required for these processes, currently almost no mechanistic insight is available into their regulation. We show that knockdown of planarian Not1, the CCR4-NOT deadenylating complex scaffolding subunit, abrogates regeneration and normal homeostasis. This abrogation is primarily due to severe impairment of their differentiation potential. We describe a stem cell specific increase in the mRNA levels of key neoblast genes after <i>Smed-not1</i> knock down, consistent with a role of the CCR4-NOT complex in degradation of neoblast mRNAs upon the onset of differentiation. We also observe a stem cell specific increase in the frequency of longer poly(A) tails in these same mRNAs, showing that stem cells after <i>Smed-not1</i> knock down fail to differentiate as they accumulate populations of transcripts with longer poly(A) tails. As other transcripts are unaffected our data hint at a targeted regulation of these key stem cell mRNAs by post-transcriptional regulators such as RNA-binding proteins or microRNAs. Together, our results show that the CCR4-NOT complex is crucial for stem cell differentiation and controls stem cell-specific degradation of mRNAs, thus providing clear mechanistic insight into this aspect of neoblast biology.</p></div

Increased level of transcripts and increased frequency of long poly(A) tails are restricted to neoblast-containing cell populations.

<p>(A–B) Quantification of the level of expression by qRT-PCR of the neoblast markers <i>Smedwi-1</i>, <i>Smedtud-1</i>, <i>Smed-vasa-1</i> and <i>Smed-pcna</i> (A) and <i>Smed-not1</i> (B) in FACS sorted populations X1, X2 and Xins in <i>control(RNAi)</i> (c) and <i>Smed-not1(RNAi)</i> (n) animals 10 and 15 days after RNAi, normalized expression and relative to respective X1 <i>control(RNAi)</i> samples. (A) <i>Smedwi-1</i>, <i>Smedtud-1</i>, <i>Smed-vasa-1</i> and <i>Smed-pcna</i> transcripts accumulate progressively after 10 and 15 days of RNAi in X1 and X2 cells, the two fractions that contain neoblasts to different extents, but this accumulation is not observed in Xins cells, which contain differentiated cells exclusively, including CNS cells. (B) <i>Smed-not1</i> is significantly depleted across all three cell fractions, showing that the absence of accumulation and increased frequency of long poly(A) tails of neoblast mRNAs that are expressed also in CNS is not due to absence of effective gene knock down in differentiated cells. Error bars represent standard deviation and asterisks represent statistical significance in A–B (C–E) PAT assays reflecting the distribution of mRNA poly(A) tail lengths for the neoblast and CNS expressed mRNAs <i>Smedtud-1</i> and <i>Smed-vasa-1</i> (C), the neoblast specific mRNAs <i>Smed-pcna</i> and <i>Smedwi-1</i> (D) and the housekeeping and tissue specific mRNAs <i>Smed-mhc</i> and <i>Smed-ef-2</i> (E) in FACS sorted populations X1, X2 and Xins from <i>control(RNAi)</i> (c) and <i>Smed-not1(RNAi)</i> (n) animals 10 and 15 days after RNAi. Size markers used are represented in blue, the theoretical length of the amplicon corresponding to the deadenylated mRNA species given the primers used in each assay is given in green. (C). The marked differences in poly(A) tail length distribution detected for the neoblast and CNS mRNAs <i>Smed-vasa-1</i> and <i>Smedtud-1</i> are only detected in X1 and X2 but not in Xins FACS sorted populations, showing that the fractions of these mRNA populations localised in the CNS show no differences after <i>Smed-not1</i> knock down. (D) The marked differences in poly(A) tail length distribution detected for the neoblast specific mRNAs <i>Smed-pcna</i> and <i>Smedwi-1</i> are only detected in X1 and X2 but the mRNAs are not detected in Xins FACS sorted populations. (E) No differences in poly(A) tail length distribution are detected for the tissue specific mRNA <i>Smed-mhc</i>, and only slight differences are detected in X1 and X2 but not in Xins fractions for the housekeeping mRNA <i>Smed-ef-2</i>.</p

Increased levels of neoblast transcripts and their increased frequency of long poly(A) tails are irradiation sensitive.

<p>(A) WMISH of the neoblast marker <i>Smedtud-1</i> in <i>control(RNAi)</i> animals (left panels) and <i>Smed-not1(RNAi)</i> animals (right panels) non irradiated (top panels) and irradiated 24 hours (bottom panels) prior to data collection time points 10 and 15 days after RNAi. A consistent qualitative difference is observed in non irradiated animals, however, no qualitative differences are observed in irradiated animals, showing that <i>Smed-not1(RNAi)</i> animals do not overexpress ectopically <i>Smedtud-1</i>. Anterior is to the left. Scale bars: 500 µm. (B) Quantification of the level of expression by qRT-PCR of the neoblast markers <i>Smedtud-1</i>, <i>Smed-vasa-1</i> and <i>Smed-pcna</i> in <i>control(RNAi)</i> (c) and <i>Smed-not1(RNAi)</i> (n) animals non-irradiated and irradiated 24 hours prior to data collection time points 10 and 15 days after RNAi, normalized expression and relative to respective <i>control(RNAi)</i> samples. Error bars represent standard deviation, asterisks represent statistical significance. <i>Smedtud-1</i>, <i>Smed-vasa-1</i> and <i>Smed-pcna</i> transcripts accumulate progressively after 10 and 15 days of RNAi, but this accumulation is eliminated 24 hours post irradiation, with <i>Smed-not1(RNAi)</i> irradiated animals showing levels similar to <i>control(RNAi)</i> irradiated animals. (C–F) PAT assays reflecting the distribution of mRNA poly(A) tail lengths for the neoblast specific mRNAs <i>Smedwi-1</i> and <i>Smed-pcna</i> (C), the neoblast and CNS expressed mRNAs <i>Smed-vasa-1</i> and <i>Smedtud-1</i> (D), the progeny specific mRNA <i>Smed-nb.21.11e</i> and <i>Smed-agat-1</i> (E) and the housekeeping and tissue specific mRNAs <i>Smed-ef-2</i>, <i>Smed-eif-3</i>, <i>Smed-mhc</i> and a spiked-in control in <i>control(RNAi)</i> (c) and <i>Smed-not1(RNAi)</i> (n) animals non irradiated and irradiated 24 hours prior to data collection time points 15 days after RNAi. Size markers used are represented in blue, the theoretical length of the amplicon corresponding to the deadenylated mRNA species given the primers used in each assay is given in green. Neoblast specific markers are not detected after irradiation (C). The marked differences in poly(A) tail length distribution detected for the neoblast and CNS mRNAs <i>Smed-vasa-1</i> and <i>Smedtud-1</i> are eliminated by irradiation, showing that the fractions of these mRNA populations localised in the CNS show no differences after <i>Smed-not1</i> knock down (D). The differences in poly(A) tail length distribution detected for the progeny specific mRNAs <i>Smed-nb.21.11e</i> and <i>Smed-agat-1</i> are not affected by irradiation, as these cells are not eliminated after 24 hours of irradiation (E). No differences are detected for <i>Smed-ef-2</i>, <i>Smed-eif-3</i>, <i>Smed-mhc</i> and a spike-in control RNA.</p

<i>Smed-not1(RNAi)</i> animals maintain mitotic neoblasts.

<p>(A) Quantification of mitosis by counting of h3p-positive cells in whole mount immunohistochemistry on <i>control(RNAi)</i> and <i>Smed-not1(RNAi)</i> animals 5, 10, 15 and 20 days after RNAi (N = 7 per time point). No significant differences are detected. Representative <i>control(RNAi)</i> (B) and different <i>Smed-not1(RNAi)</i> worms (C–E) 20 days after RNAi, immunostained with the mitotic marker h3p (h3p, green) and counterstained with phospho-tyrosine (p-tyr, red) in order to show head regression defects. <i>Smed-not1(RNAi)</i> animals still display detectable mitotic cells, even as head regression defects occur. The number of mitotic cells detected is smaller in the animals with the most severe head regression defects (D–E). Anterior is to the left. Scale bars: 500 µm. (F) Quantification of FACS sorted X1 cells in <i>control(RNAi)</i> and <i>Smed-not1(RNAi)</i> animals 10 and 15 days after RNAi, and wild type irradiated animals. While no significant differences are observed 10 days after knock down, <i>Smed-not1(RNAi)</i> animals show a reduced but significant decrease in percentage of X1 cells. Error bars represent standard deviation and asterisks represent statistical significance. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004003#pgen.1004003.s004" target="_blank">Figure S4</a>.</p