京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21023号 / 医科博第84号 / 新制||医科||6(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 斎藤 通紀, 教授 萩原 正敏, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Lysine-specific Demethylase 1B (KDM1B) is a chromatin regulator which functions as a histone eraser through the removal of the post-translational modifications mono and dimethylation of histone 3 on lysine 4 (H3K4me1/2). This process is enhanced by the formation of a complex with Nuclear Protein Glyoxylate Reductase (NPAC). NPAC resolves the sequestration of the nucleosome histone tail to allow robust demethylation of H3K4me2 by KDM1B, during transcriptional elongation by RNA polymerase 2 (RNAP II). KDM1B is involved in many crucial processes during development. Its physiological functions include the establishment of maternal genomic imprints, reset of the epigenome during somatic cell reprogramming, and regulation of brown adipogenic differentiation. In light of this, the role of KDM1B in human embryonic stem cells (hESCs) is examined through CRISPR/Cas9-editing to further dissect its biological functions during embryogenesis. CRISPR-induced knockouts of KDM1B exhibited similar cell proliferation rate and expression of OCT4 and NANOG pluripotency markers to wildtype cells. Furthermore, KDM1B-/- clones were able to maintain their pluripotency potential by differentiating to all germ layers in teratoma and embryoid body formation assays. In addition, RNA-seq of KDM1B-/- clones showed enrichment of mesoderm lineage-related gene ontology (GO) terms in the downregulated differentially expressed genes. Thus, KDM1B is believed to be dispensable during the pluripotent stage of the cell but proved fundamental during later stages of development.

Transplanted embryonic stem (ES) cells following myocardial infarction (MI) contribute to limited cardiac repair and regeneration with improved function. Therefore novel strategies are still needed to enhance the efficacy by which ES cells differentiate into cardiac cell types and inhibit adverse remodeling in the infarcted myocardium. Our studies evaluate whether genetic manipulation of transplanted ES cells employing miR- 1, a pro-cardiac microRNA, and TIMP-1, an anti-apoptotic and anti-fibrotic protein, will enhance cardiac myocyte differentiation, inhibit native cardiac apoptosis, and reduce fibrosis in the infarcted myocardium. Furthermore, we assess levels of associated pro- (caspase-3, PTEN) and anti-(Akt) apoptotic proteins as well as a pro-fibrotic protein (MMP-9) in the post-MI and cell transplanted heart. microRNAs (miRs) have emerged as critical regulators of various physiological processes including development, differentiation, metabolism, and death. Indeed, miR- 1 plays an integral role in early cardiac development in Drosophila and mice as well as mediates differentiation of cardiac myocytes in vitro. To that end, we generated ES cells overexpressing miR-1 (miR-1-ES cells), transplanted them into the infarcted myocardium, and evaluated their impact on cardiac myocyte differentiation, myocardial repair, and left ventricular dysfunction post-MI. We provide evidence demonstrating enhanced cardiac myocyte commitment of transplanted miR-1-ES cells in the mouse infarcted heart as compared to ES cell and culture media transplanted hearts. Assessment of apoptosis revealed overexpression of miR-1 in transplanted ES cells protected host myocardium from MI-induced apoptosis through activation of p-Akt and inhibition of caspase-3, PTEN, and superoxide anion production. A significant reduction iv in interstitial and vascular fibrosis was quantified in miR-1-ES and ES cell transplanted groups compared with control MI. However, no statistical significance between miR-1- ES cell and ES cell groups was observed. Finally mice receiving miR-1-ES cell transplantation post-MI had significantly improved heart function compared with respective controls. Our data suggests miR-1 drives cardiac myocyte differentiation from transplanted ES cells and inhibits apoptosis post-MI ultimately giving rise to enhanced cardiac repair, regeneration, and function. Next, we assessed the role of miR-1-ES cells in a chronic model of MI as research has shown that apoptosis occurs not only hours but months following ischemia. 4 weeks following transplantation into the infarcted myocardium, we provide evidence demonstrating reduced cardiac apoptosis in miR-1-ES cell transplanted hearts compared to respective controls. Moreover, we show significant elevation of p-Akt levels and diminished PTEN levels in hearts transplanted with miR-1-ES cells as determined by enzyme-linked immunoassays. Finally, using echocardiography, we reveal mice receiving miR-1-ES cell transplantation post-MI had significantly improved cardiac function compared with animals transplanted with ES cell and culture media. Our data suggests that miR-1, when overexpressed in transplanted ES cells, has the capacity to inhibit apoptosis long term while attenuating contractility loss. In addition to enhancing cardiac-specific donor cell differentiation, improving the efficacy by which stem cells promote cell survival and repair in the host myocardium is imperative in the pursuit of refining and optimizing stem cell therapy. To that end, we overexpressed TIMP-1, an endogenous inhibitor of apoptosis and fibrosis, in ES cells (TIMP-1-ES cells), transplanted them into infarcted myocardium, and evaluated their v impact on adverse cardiac remodeling. Immunofluorescence, TUNEL staining, caspase-3 activity, ELISAs, histology, and echocardiography were used to assess apoptosis, fibrosis, and heart function. Hearts transplanted with TIMP-1-ES cells demonstrated a reduction in apoptosis as well as an increase in p-Akt activity compared with ES cells or culture media controls. Interstitial and vascular fibrosis was significantly decreased in the TIMP-1-ES cell group compared to controls. Furthermore, MMP-9, a key pro-fibrotic protein, was significantly reduced following TIMP-1-ES cell transplantation. Echocardiography data showed fractional shortening and ejection fraction were significantly improved in the TIMP-1-ES cell group compared with respective controls. Our data suggest that transplanted ES cells overexpressing TIMP- 1 attenuate adverse myocardial remodeling and improve cardiac function compared with ES cells. Overall, our data suggest that genetic manipulation of ES cells following transplantation in the infarcted heart enhances cardiac myocyte differentiation, inhibits apoptosis and fibrosis as well as improves cardiac function.

Long undecoded transcript isoforms (LUTIs) are a class of non-canonical mRNAs that repress gene expression by a combined mechanism of transcriptional and translational interference. Although this mechanism has been shown to be widespread in yeast, its prevalence in mammals has not been established. Using human embryonic stem cells (hESCs) differentiated into endoderm, mesoderm, and ectoderm lineages and further differentiation into polyhormonal cells, cardiomyocytes, and motor neurons, respectively, we obtained a comprehensive dataset through mRNA-seq, ribosome profiling, and quantitative mass spectrometry measurements. Our analysis revealed that LUTI-based regulation is context-dependent, with a total of 271 genes identified in ectoderm to motor neuron differentiation, 69 genes in mesoderm to cardiomyocyte differentiation, and 99 genes in endoderm to polyhormonal cell differentiation. Translational repression of LUTI candidates was found to be primarily dependent on upstream open reading frames (uORFs), while LUTI-based transcriptional repression displayed variability. This study enhances our understanding of gene expression and regulation during mammalian development and highlights the potential significance of LUTI-based regulation in the development of specific cell types or tissues. The findings lay the groundwork for further exploration into the role of LUTI- based regulation in other mammalian developmental programs and its potential implications for therapeutic targets in developmental disorders and diseases.

Tse, Chi Lok. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 116-131). / Abstracts in English and Chinese. / Declaration --- p.i / Abstract --- p.iii / Abstract in Chinese --- p.v / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Figures --- p.X / List of Tables --- p.xii / List of Abbreviations --- p.xiii / Chapter CHAPTER 1 --- General Introduction / Chapter 1.1 --- Regenerative Medicine --- p.1 / Chapter 1.2 --- Embryonic Stem Cells and Reprogramming --- p.3 / Chapter 1.3 --- Transdifferentiation --- p.6 / Chapter 1.4 --- The Cerebellum --- p.7 / Chapter 1.4.1 --- Functions of the cerebellum --- p.7 / Chapter 1.4.2 --- Structure and organization of the cerebellum --- p.8 / Chapter 1.4.3 --- Principle cellular components in the cerebellum --- p.12 / Chapter 1.4.3.1 --- Purkinje cells --- p.12 / Chapter 1.4.3.2 --- Granule cells --- p.12 / Chapter 1.4.3.3 --- Mossy fibres --- p.13 / Chapter 1.4.3.4 --- Climbing fibres --- p.13 / Chapter 1.4.3.5 --- Deep cerebellar nuclei --- p.13 / Chapter 1.4.3.6 --- Other cerebellar neurons --- p.14 / Chapter 1.4.3.7 --- Neuroglia of the cerebellum --- p.16 / Chapter 1.4.4 --- Circuitry of the cerebellum --- p.17 / Chapter 1.5 --- Development of the Cerebellum --- p.21 / Chapter 1.5.1 --- Anatomical changes during cerebellar development --- p.21 / Chapter 1.5.2 --- Molecular control of cerebellar development --- p.25 / Chapter 1.5.2.1 --- Specification of the cerebellar region --- p.25 / Chapter 1.5.2.2 --- Neurogenesis from the ventricular zone --- p.26 / Chapter 1.5.2.3 --- Neurogenesis from rhombic lip --- p.29 / Chapter 1.6 --- Scope of the Thesis --- p.33 / Chapter CHAPTER 2 --- Materials and General Methods / Chapter 2.1 --- Materials for Molecular Biological Work --- p.35 / Chapter 2.1.1 --- Enzymes --- p.35 / Chapter 2.1.2 --- Chemicals and others --- p.35 / Chapter 2.1.3 --- Plasmid vectors and plasmid --- p.36 / Chapter 2.1.4 --- Solutions and media --- p.36 / Chapter 2.2 --- Materials for Tissue/Cell Culture --- p.38 / Chapter 2.2.1 --- Chemicals --- p.38 / Chapter 2.2.2 --- Culture media and solutions --- p.38 / Chapter 2.2.3 --- Culture cells --- p.39 / Chapter 2.3 --- Animals --- p.40 / Chapter 2.4 --- Materials for Immunocytochemistry --- p.40 / Chapter 2.5 --- Oligonucleotide Primers --- p.41 / Chapter 2.6 --- RNA Extraction --- p.44 / Chapter 2.7 --- Generation of cDNA from mRNA --- p.44 / Chapter 2.8 --- Preparation of Recombinant Plasmid DNA --- p.45 / Chapter 2.8.1 --- Small scale preparation of DNA --- p.45 / Chapter 2.8.2 --- QLAGEN plasmid midiprep kit method --- p.46 / Chapter 2.9 --- Preparation of Specific DNA Fragment from Agarose Gel --- p.46 / Chapter 2.10 --- Subcloning of DNA Fragments --- p.47 / Chapter 2.10.1 --- Preparation of cloning vectors --- p.47 / Chapter 2.10.2 --- Subcloning of DNA fragment --- p.48 / Chapter 2.10.3 --- Transformation of DNA into competent cells --- p.48 / Chapter 2.11 --- Preparation of Competent Cells --- p.48 / Chapter CHAPTER 3 --- Generation and Characterization of Induced Neurons / Chapter 3.1 --- Introduction --- p.50 / Chapter 3.2 --- Experimental Procedures --- p.51 / Chapter 3.2.1 --- Construction of expression vector --- p.51 / Chapter 3.2.1.1 --- Preparation of insert DNA --- p.51 / Chapter 3.2.1.2 --- Construction of entry vector --- p.52 / Chapter 3.2.1.3 --- Construction of destination vector --- p.52 / Chapter 3.2.1.4 --- Construction of expression vector --- p.52 / Chapter 3.2.2 --- Generation of induced neural cells --- p.57 / Chapter 3.2.2.1 --- Culture of mouse embryonic fibroblasts (MEF) --- p.57 / Chapter 3.2.2.2 --- Production of expression vector containing retroviruses --- p.57 / Chapter 3.2.2.3 --- Transfection and induction of neural fate of MEF --- p.57 / Chapter 3.2.3 --- Immunocytochemcial analysis --- p.58 / Chapter 3.2.4 --- Efficiency calculation --- p.59 / Chapter 3.3 --- Results --- p.62 / Chapter 3.3.1 --- A screen for cerebellar Purkinje and granule cell fate-inducing factors --- p.62 / Chapter 3.3.2 --- Characterization of the induced neurons --- p.67 / Chapter 3.3.2.1 --- Granule cell induction --- p.67 / Chapter 3.3.2.2 --- Purkinje cell induction --- p.71 / Chapter 3.4 --- Discussion --- p.102 / Chapter 3.4.1 --- Roles of inducing factors in Purkinje cells and granule cells development --- p.102 / Chapter 3.4.2 --- Mechanism of neural transdifferentiation --- p.107 / Chapter CHAPTER 4 --- Future Directions / Chapter 4.1 --- Complete Induction of Purkinje Cell Fate --- p.111 / Chapter 4.2 --- Induced Neurons of Different Subtypes --- p.112 / Chapter 4.3 --- Mechanism of Transdifferentiation --- p.114 / Chapter 4.4 --- Transdifferentiation and Regenerative Medicine --- p.114 / Bibliography --- p.116

Wong, Chun Kit. / "September 2010." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 104-118). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Acknowledgements --- p.ii / Contents --- p.iii / Declaration --- p.vi / Abstract --- p.vii / 摘要 --- p.x / Abbreviations --- p.xi / List of Figures --- p.xiii / Chapter CHAPTER ONE: --- INTRODUCTION / Chapter 1.1 --- Embryonic Stem Cells (ESCs) / Chapter 1.1.1 --- Characteristics of ESC --- p.1 / Chapter 1.1.2 --- Therapeuticotential of ESCs --- p.2 / Chapter 1.2 --- 17β-estradiol (E2) / Chapter 1.2.1 --- Genomic Actions of E2 --- p.3 / Chapter 1.2.2 --- Non-genomic Actions of E2 --- p.5 / Chapter 1.2.3 --- hysiological Roles of E2 on Early Mammalian Development --- p.9 / Chapter 1.2.4 --- E2 and Cell Proliferation --- p.10 / Chapter 1.3 --- Ca2+ homeostasis / Chapter 1.3.1 --- Overview --- p.11 / Chapter 1.3.2 --- Ca2+ Signaling in mESCs --- p.14 / Chapter 1.4 --- Store-operated Ca2+ Entry (SOCE) / Chapter 1.4.1 --- Overview --- p.15 / Chapter 1.4.2 --- Store Depletion --- p.15 / Chapter 1.4.3 --- Activation of SOCE --- p.16 / Chapter 1.5 --- Molecular Identities of Store-operated Ca2+ Channels (SOCCs) on plasma Membrane / Chapter 1.5.1 --- TRPC Channels --- p.17 / Chapter 1.5.2 --- ORAI Channels --- p.18 / Chapter 1.5.3 --- Regulation of SOCCs at Different Levels --- p.18 / Chapter 1.5.4 --- Regulation of SOCE --- p.19 / Chapter 1.6 --- Nuclear Factor of Activated T-cells (NFAT) / Chapter 1.6.1 --- Overview --- p.20 / Chapter 1.6.2 --- Mechanisms of Action --- p.21 / Chapter 1.6.3 --- Functions --- p.22 / Chapter 1.7 --- Aims of the Study --- p.23 / Chapter CHAPTER TWO: --- MATERIALS AND METHODS / Chapter 2.1 --- Maintenance of mESCs --- p.24 / Chapter 2.2 --- Cell proliferation Assay and Viability Test --- p.24 / Chapter 2.3 --- "RNAreparation, Reverse Transcription (RT) and Quantitative Polymerase Chain Reaction (qPCR)" --- p.25 / Chapter 2.4 --- Totalrotein Extraction --- p.27 / Chapter 2.5 --- Measurement of protein Concentration --- p.27 / Chapter 2.6 --- De-phosphorylation Assay --- p.28 / Chapter 2.7 --- Western Blot --- p.28 / Chapter 2.8 --- Ca2+ Measurement by Confocal Microscopy --- p.30 / Chapter 2.9 --- Ca2+ Measurement by Flow Cytometry --- p.31 / Chapter 2.10 --- siRNA Transfection --- p.31 / Chapter 2.11 --- DNAlasmid Transfection --- p.32 / Chapter 2.12 --- Molecular and Fluorescence Imaging --- p.33 / Chapter 2.13 --- Statistical Analysis --- p.34 / Chapter 2.14 --- Primers used in the Study (Table 1:Primers List) --- p.34 / Chapter 2.15 --- Drugs used in the Study (Table 2: Drugs List) --- p.36 / Chapter 2.16 --- Antibodies used in the Study (Table 3: Antibodies List) --- p.37 / Chapter CHAPTER THREE: --- RESULTS / Chapter 3.1 --- Expression of SOCE in mESCs --- p.38 / Chapter 3.2 --- SOCC Blockers Attenuated mESCroliferation --- p.43 / Chapter 3.3 --- E2 Increased mESCroliferation --- p.48 / Chapter 3.4 --- E2 Increased Intracellular Ca2+ ([Ca2+]i) Level in mESCs --- p.48 / Chapter 3.5 --- E2 Increased the Amplitude of SOCE --- p.51 / Chapter 3.6 --- Increase in mESC proliferation and SOCE Caused by E2 Could be Reversed by SOCC Blocker --- p.51 / Chapter 3.7 --- Relative Expression of SOCC Candidates at mRNA Level Under the Treatment of E2 --- p.56 / Chapter 3.8 --- E2 Down-regulated the Expression of ORAI3 --- p.56 / Chapter 3.9 --- Knockdown of ORAI3 in mESCs --- p.61 / Chapter 3.10 --- Identification of NFATc3 Specific Bands --- p.63 / Chapter 3.11 --- E2 Increased the phosphorylation of NFATc3 --- p.67 / Chapter 3.12 --- Effects of 2-APB on NFATc3 phosphorylation Status --- p.67 / Chapter 3.13 --- Identification of NFATc4 Specific Bands ？ --- p.72 / Chapter 3.14 --- E2 Increased the Translocation of GFP-NFATc4 From the Cytoplasm to the Nucleus and This Effect Could be Reversed by 2-APB --- p.80 / Chapter 3.15 --- CsA Reversed E2-induced Increase in proliferation --- p.82 / Chapter CHAPTER FOUR: --- DISCUSSION / Chapter 4.1 --- Expression of SOCE in mESCs --- p.84 / Chapter 4.2 --- proliferation of mESCs Depends on SOCE --- p.85 / Chapter 4.3 --- E2 Acts an Extrinsic Factor for Stimulatingroliferation of mESCs Via SOCE --- p.87 / Chapter 4.4 --- roposed Mechanism to Show an Increment of SOCE Can be Due to a Down-regulation of ORAI3 --- p.89 / Chapter 4.5 --- Experiments Aiming to Knockdown ORAI3 --- p.92 / Chapter 4.6 --- roposed Mechanism to Show an Increment of SOCE by Other SOCC Candidates Rather than ORAI3 --- p.93 / Chapter 4.7 --- Activation of NFATc3 and NFATc4 by E2 in mESCs --- p.94 / Chapter 4.8 --- possible Downstream Targets of NFAT Responsible for E2-induced mESCs proliferation --- p.96 / Chapter CHAPTER FIVE: --- FUTUREERSPECTIVES --- p.98 / Chapter CHAPTER SIX: --- CONCLUSION --- p.100 / REFERENCES --- p.104

Precise control of stem cell fate is a fundamental issue in the use of human embryonic stem (hES) cells in the context of cell therapy We examined three ways in which the microenvironment can be controlled to alter hES cell behavior, providing insight into the best conditions for maintenance of pluripotency and neural differentiation in developmental and therapeutic studies. We first examined the effects of polydimethylsiloxane (PDMS) growth surfaces on hES cell survival and maintenance of pluripotency. Lightly cured, untreated PDMS was shown to be a poor growth surface for hES cells. Some of the adverse effects caused by PDMS could be mitigated with increased curing or UV treatment of the surface, but neither modification provided a growth surface that supported pluripotent hES cells as well as polystyrene. This work provides a basis for further optimizing PDMS for hES cell culture, moving towards the use of microdevices in establishing precise control over stem cell fate. The second study explored the use of an easily constructed diffusion-based device to grow hES cells in culture on a defined, physiologic oxygen (O₂) gradient. We observed greater hES cell survival and higher levels of pluripotency markers in the lower O₂ regions of the gradient. The greatest benefit was observed at O₂ levels below 5%, narrowing the potential optimal range of O₂ for the maintenance of pluripotent hES cells. Finally, we developed a small molecule-mediated adherent and feeder-free neural differentiation protocol that reduced the cost and time scale for in vitro differentiation of neural precursors and functional neurons from human pluripotent cells. hES cell-derived neural precursors transplanted into a murine model of focal ischemic stroke survived, improved neurogenesis, and differentiated into neurons. Transplant also led to a more consistent and measurable sensory recovery after stroke as compared to untransplanted controls. This protocol represents a potentially translatable method for the generation of CNS progenitors from human pluripotent stem cells.

Stem and progenitor cells are an attractive cell source for the treatment of degenerative diseases due to their potential to differentiate into multiple cell types and provide large cell yields. Thus far, however, clinical applications have been limited due to inefficient differentiation into desired cell types with sufficient yields for adequate tissue repair and regeneration. The ability to spontaneously aggregate in suspension makes embryonic stem cells (ESCs) amenable to large-scale culture techniques for the production of large yields of differentiating cell spheroids (termed embryoid bodies or EBs); however, the introduction of hydrodynamic conditions may alter differentiation profiles within EBs and should be methodically examined. The work presented here employs a novel, laboratory-scale hydrodynamic culture model to systematically interrogate the effects of ESC culture hydrodynamics on cardiomyocyte differentiation through the modulation of a developmentally-relevant signaling pathway. The fluidic environment was defined using computational fluid dynamic modeling, and the effects of hydrodynamic conditions on EB formation, morphology and structure were assessed. Additionally, EB differentiation was examined through gene and protein expression, and indicated that hydrodynamic conditions modulate differentiation patterns, particularly cardiogenic lineage development. This work illustrates that mixing conditions can modulate common signaling pathways active in ESC differentiation and suggests that differentiation may be regulated via bioprocessing parameters and bioreactor design.

Current therapeutic strategies to stimulate endogenous angiogenic processes within injured tissue areas are typically based on introducing exogenous pro-angiogenic molecules or cell populations. Stem cell transplantation for angiogenic therapy aims to deliver populations of cells that secrete angiogenic factors and/or engraft in the new branching vasculature within the damaged tissue. Utilizing stem or progenitor cells has been shown to induce a rather robust angiogenic response despite minimal repopulation of the host vasculature, suggesting that stem cells may provide paracrine factors that transiently induce endogenous angiogenesis of tissues undergoing regeneration. Early differentiating embryonic stem cell (ESC) aggregates, referred to as embryoid bodies (EBs), can undergo vasculogenic differentiation, and also produce extracellular matrix and growth factors that induce proliferation, differentiation, and tissue morphogenesis. Taken together, the ESC extracellular environment may be an effective means by which to manipulate cell behavior. Thus, the objective of this project was to harness morphogens derived from ESCs undergoing differentiation and analyze their bioactive potential. To examine the expression of extracellular factors within EBs, gene expression arrays in conjunction with a variety of analytical tools were utilized to gain an understanding of the importance of extracellular factors in ESC differentiation. Furthermore, the soluble fraction of secreted factors contained within EB-conditioned media was compared to the matrix-associated factors produced by EBs, which led to the development of novel ESC-derived matrices via mechanical acellularization methods. Acellular embryonic stem cell-derived matrices demonstrated the retention of bioactive factors that impacted aspects of angiogenesis. In conclusion, extracellular factors were modulated in response to the progression of EB differentiation and can further be harnessed via acellularization techniques, in order to deliver bioactive ESC-secreted factors in a cell-free manner.

H1 linker histone facilitates the formation of higher order chromatin structure and is essential for mammalian development. Mice have 11 H1 variants which are differentially regulated and conserved in human. Previous research indicates that H1 regulates the expression of specific genes in mouse embryonic stem cells (ESCs). However, whether individual variants have distinct functions and how H1 participates in gene regulation remain elusive. An investigation of the precise localization of individual H1 variants in vivo would facilitate the elucidation of mechanisms underlying chromatin compaction regulated gene expression, while it has been extremely difficult due to the lacking of specific antibodies toward H1 variants. In this dissertation, I have generated a knock-in system in ESCs and shown that the N-terminally tagged H1 proteins are functionally interchangeable to their endogenous counterparts in vivo. H1d and H1c are depleted from GC- and gene-rich regions and active promoters, inversely correlated with H3K4me3, but positively correlated with H3K9me3 and associated with characteristic sequence features. Surprisingly, both H1d and H1c are significantly enriched at major satellites, which display increased nucleosome spacing compared with bulk chromatin. While also depleted at active promoters and enriched at major satellites, overexpressed H10 displays differential binding patterns in specific repetitive sequences compared with H1d and H1c. Depletion of H1c, H1d ,and H1e causes pericentric chromocenter clustering and de-repression of major satellites. Collectively, these results integrate the localization of an understudied type of chromatin proteins, namely the H1 variants, into the epigenome map of mouse ESCs, and demonstrate significant changes at pericentric heterochromatin upon depletion of this epigenetic mark.