Chromatin Regulation of Planarian Stem Cells

Date of Award

Fall 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular, Cellular, and Developmental Biology

First Advisor

van Wolfswinkel, Josien

Abstract

Stem cells are vital for multicellular organisms both during embryonic development and through adulthood. During embryogenesis, organisms grow from a pluripotent zygote and eventually develop all of the cell types needed to form their tissues. In adulthood, stem cells are still needed for replacing aged cells to maintain tissue homeostasis, as well as for repairing damaged tissues. The differentiation from a stem cell to a specialized cell type is a highly complex process that needs to be tightly coordinated to perform its critical function throughout development, and errors in differentiation can lead to severe consequences ranging from birth defects to cancer. The gene expression changes that drive these crucial cell state transitions are guided by changes in chromatin state, including chromatin accessibility, epigenetic modifications, and the 3D organization of chromatin within the nucleus. A more complete knowledge of the regulation of stem cells and differentiation will enable future advances in the fields of regenerative medicine and stem cell-based therapies. Many highly regenerative organisms maintain adult pluripotent stem cells throughout their life, but how the long-term maintenance of pluripotency is accomplished is unclear. Current insights in the transcriptional regulation of pluripotent cells have been based on embryonic stem cells, which remain in a pluripotent state for only a short time. Adult pluripotent stem cells may need to employ a different system of genome organization to maintain their pluripotency over a longer timeframe. To decipher the regulatory logic of adult pluripotent stem cells, we analyzed the chromatin organization of stem cell genes in the planarian Schmidtea mediterranea. Planaria are a powerful model for the study of stem cells, as about 20% of their cells are adult pluripotent cells, called neoblasts. This large population of neoblasts allows planaria to regenerate any missing tissues and maintain their tissues indefinitely. We hypothesized that planarian neoblasts may employ a specialized system of genome organization in order to maintain their pluripotent state over time. In order to understand how planaria regulate their neoblasts at the level of chromatin, we examined their euchromatin and heterochromatin organization, 3D genome organization, and the promoter organization and regulation of neoblast genes. We used ATAC-seq, CUT&Tag, and Hi-C to examine chromatin accessibility, histone modifications, and 3D chromatin organization in planaria. We found that planaria have some expected characteristics of euchromatin and heterochromatin, such as H3K4me3 and H3K27ac at gene promoters, H3K4me1 and H3K27ac at putative enhancers, and H3K9me2 and H3K9me3 over transposable elements (TEs). However, when analyzing chromatin states, there did not seem to be a strong distinction between euchromatic and heterochromatic states. When looking at 3D chromatin organization, the A and B compartments also did not seem to directly correspond to euchromatin and heterochromatin, as chromatin accessibility, euchromatic histone modifications, and heterochromatic histone modifications were all fairly evenly distributed between both compartments. The structure of the planarian genome, which is very dense with TEs interspersed with genes, may require gene regulation to occur on a more local scale. In order to understand neoblast gene regulation, we examined the organization of their promoters. We found that neoblast gene promoters have a chromatin state distinct from that of tissue-specific genes and instead resemble constitutive gene promoters in their accessibility, H3K4me3 signal, and nucleosome positioning. Tissue-specific gene promoters had detectable transcription factor binding sites and the predicted transcription factors were required for tissue-specific gene expression. The promoters of both constitutive and neoblast genes lacked such binding sites, and instead they contained homopolymeric AT stretches, which broadly decrease nucleosome binding affinity, pointing toward regulation by chromatin remodelers. Chromatin remodelers ISWI and SNF2 were enriched in the neoblasts and specifically regulated the expression of neoblast genes, as the knockdown of either remodeler by RNAi caused a decrease in expression of neoblast genes while constitutive genes remained unaffected. The loss of either of these remodelers also impaired the ability of the neoblasts to expand after irradiation. Remarkably, a large fraction of the neoblast genes also had a distinct region of H3K9me3 upstream of their promoters. It has previously been suggested that HP1 plays a key role in stem cell function, and is required for the transcription of stem cell genes. We hypothesize that HP1 may directly enhance transcription of neoblast genes through interactions with the promoter-associated H3K9me3 at these genes. Together, these findings contribute to our understanding of the mechanisms of long-term maintenance of pluripotency, which could be useful for future applications involving stem cells.

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