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Myocardial injury leads to scar formation and pathological fibrosis that has a significant impact on the development and progression of cardiac disease. Increasing evidence suggests alteration in the chromatin landscape of cells can exacerbate the extracellular matrix deposition and enhance disease progression. Chromatin alterations and fibrosis mediate several cardiac cellular changes, including scar formation, DNA damage, collagen deposition, and increased TGFB expression which are all disease-driving mechanisms during heart failure. Targeting epigenetic dependent fibrosis pathways is thus a promising strategy for the prevention and treatment after myocardial injury. The polycomb complex protein Bmi1, an epigenetic regulator, is associated with numerous biological functions including mediating DNA damage, cellular fate, and proliferation. However, there is currently a lack of understanding on how Bmi1 mediated epigenetic modifications affect adult heart function after injury. It was previously determined that Bmi1 modulates the epigenetic landscape of cardiac stem cells that mediates various molecular processes during a stress condition. In the present study, using a Bmi1 global and fibroblast specific knockout model, cardiac function was assessed through echocardiography using adult mice following cardiac injury. The loss of Bmi1 caused a significant decrease in heart function after injury, which was associated with increased fibrosis and DNA damage. Specifically, we found that the adult cardiac fibroblasts, isolated from the Bmi1 knockout model, had increased expression of pro-fibrotic genes including TGFB, aSMA, and Collagen1a1. Through multiomic sequencing, we found significant changes in the pathological fibrotic signaling pathways of TGFB, specifically with SMAD3 chromatin accessibility with the loss of Bmi1 epigenetic regulation. Concluding, Bmi1 epigenetic regulation mediates repair during pathological challenge by regulating adult cardiac fibroblasts and pathological fibrosis after cardiac injury. / Biomedical Sciences

The Testis Specific Serine Kinases (Tssks) are a family of proteins that show testis and sperm-specific expression. Members of this family are most conserved among mammals, however there are homologs in vertebrates like birds and amphibians, chordates, and other invertebrates like insects and cnidarians. This specific expression suggests that these kinases are highly regulated. Analysis of murine and human Tssk1, Tssk2, and Tssk6 sequences show that these genes are comprised of one exon each, suggesting they are retrotransposons. The expression of these genes shows their importance, since many retrotransposons are silenced due to the foreign nature of the DNA, and knock-out mouse models have shown that these kinases are required for fertility. Understanding the properties of these kinases not only expands our scientific knowledge, but also lends itself to understanding fertility issues in men as well as being a contraceptive target. We looked at an epigenetic regulation factor, DNA methylation at CpG dinucleotides, to see if this caused the testis-specific gene expression we saw. Tssk2 and preliminary results from Tssk1 showed that there is no differential methylation at CpG dinucleotides or between tissues. Preliminary results for Tssk6 did show one site that may be differentially methylated, thus the tissue specific expression. We then started looking further into biochemically characterizing TSSK1 and TSSK2 to determine functionally relevant sites and new substrates. Understanding how these kinases function in sperm is relevant in our understanding in the fertility field and poses new targets for developing contraceptives.

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No / Significance: Epigenetic regulatory mechanisms are essential for epidermal homeostasis and contribute to the pathogenesis of many skin diseases, including skin cancer and psoriasis. However, while the epigenetic regulation of epidermal homeostasis is now becoming active area of research, the epigenetic mechanisms controlling the wound healing response remain relatively untouched. Recent Advances: Substantial progress achieved within the last two decades in understanding epigenetic mechanisms controlling gene expression allowed defining several levels, including covalent DNA and histone modifications, ATP-dependent and higher-order chromatin chromatin remodeling, as well as noncoding RNA- and microRNA-dependent regulation. Research pertained over the last few years suggests that epigenetic regulatory mechanisms play a pivotal role in the regulation of skin regeneration and control an execution of reparative gene expression programs in both skin epithelium and mesenchyme. Critical Issues: Epigenetic regulators appear to be inherently involved in the processes of skin repair, and are able to dynamically regulate keratinocyte proliferation, differentiation, and migration, together with influencing dermal regeneration and neoangiogenesis. This is achieved through a series of complex regulatory mechanisms that are able to both stimulate and repress gene activation to transiently alter cellular phenotype and behavior, and interact with growth factor activity. Future Directions: Understanding the molecular basis of epigenetic regulation is a priority as it represents potential therapeutic targets for the treatment of both acute and chronic skin conditions. Future research is, therefore, imperative to help distinguish epigenetic modulating drugs that can be used to improve wound healing.

No / The epidermal differentiation program is regulated at several levels including signaling pathways, lineage-specific transcription factors, and epigenetic regulators that establish well-coordinated process of terminal differentiation resulting in formation of the epidermal barrier. The epigenetic regulatory machinery operates at several levels including modulation of covalent DNA/histone modifications, as well as through higher-order chromatin remodeling to establish long-range topological interactions between the genes and their enhancer elements. Epigenetic regulators exhibit both activating and repressive effects on chromatin in keratinocytes (KCs): whereas some of them promote terminal differentiation, the others stimulate proliferation of progenitor cells, as well as inhibit premature activation of terminal differentiation-associated genes. Transcription factor-regulated and epigenetic mechanisms are highly connected, and the p63 transcription factor has an important role in the higher-order chromatin remodeling of the KC-specific gene loci via direct control of the genome organizer Satb1 and ATP-dependent chromatin remodeler Brg1. However, additional efforts are required to fully understand the complexity of interactions between distinct transcription factors and epigenetic regulators in the control of KC differentiation. Further understanding of these interactions and their alterations in different pathological skin conditions will help to progress toward the development of novel approaches for the treatment of skin disorders by targeting epigenetic regulators and modulating chromatin organization in KCs. / National Alopecia Areata Foundation; (R13AR067088-01) from the National Institute of Arthritis and Musculoskeletal and Skin Diseases; and the National Center for Advancing Translational Sciences

No / This volume highlights recent studies identifying epigenetic mechanisms as essential regulators of skin development, stem cell activity and regeneration. Chapters are contributed by leading experts and promote the skin as an accessible model system for studying mechanisms that control organ development and regeneration. The discussions contained throughout are of broad relevance to other areas of biology and medicine and can help inform the development of novel therapeutics for skin disorders as well as new approaches to skin regeneration that target the epigenome. Part of the highly successful Stem Cells and Regenerative Medicine series, Epigenetic Regulation of Skin Development and Regeneration uncovers the fundamental significance of epigenetic mechanisms in skin development and regeneration, and emphasizes the development of new therapies for a number of skin disorders, such as pathological conditions of epidermal differentiation, pigmentation and carcinogenesis. At least six categories of researchers will find this book essential, including stem cell, developmental, hair follicle or molecular biologists, and gerontologists or clinical dermatologists.

<p dir="ltr">Nitrogen (N) is a macronutrient required for plant growth and is a major constituent of nucleic acids and proteins that are essential for several life processes. Plant response to N has been well understood at a molecular level but little is known about the chromatin or epigenetic level regulation of N response. Uncovering the epigenetic level regulation essential for plant N signaling and response is essential to improving our molecular understanding of N use efficiency (NUE). To fill this knowledge gap, we first performed a meta-analysis intersecting the published transcriptomic study of N-responsive genes in <i>Arabidopsis thaliana </i>with EpiNet, an extensive epigenetic regulatory network previously constructed in our lab through machine learning approaches, to identify a list of 18 potential epigenetic regulators that are predicted to control N response in plants. Next, by adopting a reverse genetics approach, we aimed to validate the <i>in-silico</i> prediction of these essential epigenetic regulators. To do this, we grew T-DNA insertional mutants for the genes encoding these epigenetic regulators, along with wild-type controls, under high and low N conditions, and compared them in various physiological traits. Our results indicate that 8 out of 10 confirmed knock-down mutants do show altered N-responsive phenotypes in comparison to the wild type. One of the mutants, <i>ashr2-1, </i>which is mutated in a gene encoding a putative SET-domain containing group protein (SDG) of putative histone methyltransferase, displayed reduced growth of primary root compared to WT in response to N. We performed RNA-sequencing to identify the differentially expressed genes that are induced or repressed by ASHR2 in N treatments to gain further insight into the molecular underpinnings of the ASHR2-mdediated N response in roots<i>.</i> In summary, our study has revealed knowledge on important epigenetic regulators in plant N responses, which has the potential to be extended to crop species as novel targets for enhancing NUE.</p>

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25183号 / 医博第5069号 / 新制||医||1071(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 遊佐 宏介, 教授 小林 恭, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

It is well established that epigenetic modifications to the genome are crucial for the exquisite control of gene expression required for an organism to develop and differentiate. These modifications are maintained through mitotic rounds of cell division, but must be cleared and reset through meiosis in order for the cells of the early embryo to achieve totipotency. Although we know these mechanisms exist, the rules determining which modifications are established where on the genome and the genes involved in these processes remain poorly characterised. Much of what is known about epigenetic processes has come from studies in non-mammalian organisms, such as Drosophila. However, in our laboratory we have developed a mammalian system for identifying modifiers of epigenetic gene silencing. An ENU mutagenesis screen is being carried out using an inbred mouse line carrying a GFP transgene, with an erythroid-specific promoter, that is particularly sensitive to changes in epigenetic modifications. Currently, 14 mutant lines that display a heritable shift in GFP expression have been recovered. These have been termed Modifiers of Murine Metastable Epialleles (Mommes). When I began my PhD in 2005, we had not identified any of the mutations underlying the phenotypes observed. To confirm the efficacy of the screen, I have tested the effect of heterozygosity for null alleles of two known epigenetic modifiers, Dnmt3a and Dnmt3b, on expression of the GFP transgene. Heterozygosity for the Dnmt3b knockout allele does shift expression while heterozygosity for the Dnmt3a knockout allele does not. This highlights the limitations of the screen. With this particular screen we will only detect modifiers that are expressed during haematopoiesis in the bone marrow. I have also worked on MommeD5. MommeD5 is a semi-dominant, homozygous embryonic lethal mutation that acts as an enhancer of variegation. I have found that the MommeD5 allele carries a 7 bp deletion in the major histone deacetylase, Histone deacetylase 1 (Hdac1), and this significantly alters the C-terminus of the mutant protein. The finding of Hdac1 attests to the screen design. The MommeD5 homozygous mutants die at approximately the same time as the published knockout of Hdac1 and the heterozygous mutants show increased levels of Hdac2 and acetylated histone H3, as reported in Hdac1-deficient embryonic stem cells. In addition, I have studied the effect of heterozygosity for each of the mutations on the phenotype of the mouse. In general, heterozygous Momme mutants are viable and fertile, but show subtle abnormal phenotypes. However, in the case of MommeD5 none were observed and this may relate to the compensatory upregulation of other histone deacetylases. In the case of Dnmt3a and Dnmt3b a sex ratio distortion is seen in the colonies, with less males seen than expected. Also, Dnmt3a heterozygous mutant males that inherited the mutant allele from the dam are smaller and show an increased range of body weights compared to their wild-type male littermates. This may be an example of intangible variation, i.e. phenotypic variation observed in isogenic individuals raised in standardised environments. These results suggest that epigenetic mechanisms have a role in intangible variation, also known as developmental noise. Despite the fact that it is now acknowledged by many that stochastic events occur at the level of the cell, the idea that it can happen at the level of the whole organism is rarely considered.