Epigenetic regulation of planarian stem cells by the SET1/MLL family of histone methyltransferases

San Diego State University
Epigenetics: official journal of the DNA Methylation Society (Impact Factor: 4.78). 12/2012; 8(1). DOI: 10.4161/epi.23211
Source: PubMed


Chromatin regulation is a fundamental mechanism underlying stem cell pluripotency, differentiation, and the establishment of cell type-specific gene expression profiles. To examine the role of chromatin regulation in stem cells in vivo, we study regeneration in the freshwater planarian Schmidtea mediterranea. These animals possess a high concentration of pluripotent stem cells, which are capable of restoring any damaged or lost tissues after injury or amputation. Here, we identify the S. mediterranea homologs of the SET1/MLL family of histone methyltransferases and COMPASS and COMPASS-like complex proteins and investigate their role in stem cell function during regeneration. We identified six S. mediterranea homologs of the SET1/MLL family (set1, mll1/2, trr-1, trr-2, mll5-1 and mll5-2), characterized their patterns of expression in the animal, and examined their function by RNAi. All members of this family are expressed in the stem cell population and differentiated tissues. We show that set1, mll1/2, trr-1, and mll5-2 are required for regeneration and that set1, trr-1 and mll5-2 play roles in the regulation of mitosis. Most notably, knockdown of the planarian set1 homolog leads to stem cell depletion. A subset of planarian homologs of COMPASS and COMPASS-like complex proteins are also expressed in stem cells and implicated in regeneration, but the knockdown phenotypes suggest that some complex members also function in other aspects of planarian biology. This work characterizes the function of the SET1/MLL family in the context of planarian regeneration and provides insight into the role of these enzymes in adult stem cell regulation in vivo.

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    • "Planarians were killed in ice cold 2% hydrochloric acid for 5 min and fixed in Carnoy's solution (6:3:1 ethanol, chloroform, and glacial acetic acid) for 2 h at 4 °C then incubated in 100% methanol for 1 h at 4 °C and bleached overnight in 6% hydrogen peroxide diluted in methanol. Animals were washed out of methanol into PBSTx and blocked in 1% BSA diluted in PBSTx for 2 h prior to anti- phosphohistone-H3 (Ser10) (1:1000, Cell Signaling) (Hendzel et al., 1997; Hubert et al., 2013) staining. For anti-3G9 staining, animals were processed as previously described (Forsthoefel et al., 2012). "
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    ABSTRACT: E3 ubiquitin ligases constitute a large family of enzymes that modify specific proteins by covalently attaching ubiquitin polypeptides. This post-translational modification can serve to regulate protein function or longevity. In spite of their importance in cell physiology, the biological roles of most ubiquitin ligases remain poorly understood. Here, we analyzed the function of the HECT domain family of E3 ubiquitin ligases in stem cell biology and tissue regeneration in planarians. Using bioinformatic searches, we identified 17 HECT E3 genes that are expressed in the Schmidtea mediterranea genome. Whole-mount in situ hybridization experiments showed that HECT genes were expressed in diverse tissues and most were expressed in the stem cell population (neoblasts) or in their progeny. To investigate the function of all HECT E3 ligases, we inhibited their expression using RNA interference (RNAi) and determined that orthologs of huwe1, wwp1, and trip12 had roles in tissue regeneration. We show that huwe1 RNAi knockdown led to a significant expansion of the neoblast population and death by lysis. Further, our experiments showed that wwp1 was necessary for both neoblast and intestinal tissue homeostasis as well as uncovered an unexpected role of trip12 in posterior tissue specification. Taken together, our data provide insights into the roles of HECT E3 ligases in tissue regeneration and demonstrate that planarians will be a useful model to evaluate the functions of E3 ubiquitin ligases in stem cell regulation. Copyright © 2015. Published by Elsevier Inc.
    Developmental Biology 05/2015; 404(2). DOI:10.1016/j.ydbio.2015.04.021 · 3.55 Impact Factor
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    • "Lineage-restricted intestinal stem cells on the other hand were found to undergo little change in overall DNA methylation upon differentiation (Kaaij et al. 2013), suggesting that their chromatin was already mostly in the ''closed'' differentiated state. In planarians, chromatin remodeling is thought to play a role during differentiation of stem cell progeny (Scimone et al. 2010; Hubert et al. 2013; Jaber-Hijazi et al. 2013), but it is currently unclear whether differentiation coincides with a global increase in heterochromatin, and whether neoblast chromatin is in an ''open'' state. Speculating further along these lines, it is possible that the role of PIWI proteins in the stabilization of the stem cell genome extends beyond the control of repetitive sequences, and includes the maintenance of this ''open'' chromatin structure. "
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    ABSTRACT: PIWI proteins are well known for their roles in the animal germline. They are essential for germline development and maintenance, and together with their binding partners, the piRNAs, they mediate transposon silencing. More recently, PIWI proteins have also been identified in somatic stem cells in diverse animals. The expression of PIWI proteins in these cells could be related to the ability of such cells to contribute to the germline. However, evaluation of stem cell systems across many different animal phyla suggests that PIWI proteins have an ancestral role in somatic stem cells, irrespective of their contribution to the germ cell lineage. Moreover, the data currently available reveal a possible correlation between the differentiation potential of a cell and its PIWI levels.
    Integrative and Comparative Biology 06/2014; 54(4). DOI:10.1093/icb/icu084 · 2.93 Impact Factor
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    ABSTRACT: Planarian adult stem cells (pASCs) or neoblasts represent an ideal system to study the evolution of stem cells and pluripotency as they underpin an unrivaled capacity for regeneration. We wish to understand the control of differentiation and pluripotency in pASCs and to understand how conserved, convergent or divergent these mechanisms are across the Bilateria. Here we show the planarian methyl-CpG Binding Domain 2/3 (mbd2/3) gene is required for pASC differentiation during regeneration and tissue homeostasis. The genome does not have detectable levels of 5-methylcytosine (5(m)C) and we find no role for a potential DNA methylase. We conclude that MBD proteins may have had an ancient role in broadly controlling animal stem cell pluripotency, but that DNA methylation is not involved in planarian stem cell differentiation.
    Developmental Biology 09/2013; 384(1). DOI:10.1016/j.ydbio.2013.09.020 · 3.55 Impact Factor
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