Epigenetic regulation of DNA replication studied by super resolution microscopy

Master's Thesis , 2014 , 98 Pages , Grade: 1.0 (A+)

Biology - Genetics / Gene Technology

DNA replication is a fundamental biological process responsible for accurate duplication of genetic information necessary for its faithful inheritance to the two daughter cells. Despite much effort, the underlying mechanisms controlling this process are not fully understood. In order to accommodate very large and complex genomes, replication dynamics in eukaryotes evolved to become controlled by major epigenetic mechanisms. Moreover, the spatio-temporal organization of S-phase progression changes throughout cell differentiation and development. The study of genome duplication has been largely hindered by the lack of appropriate monitoring techniques, and any comprehensive understanding ultimately requires quantitative approach.
In this master’s thesis, we analyzed replication patterns in mouse somatic and embryonic stem cells (mESCs) with newly developed three-dimensional structured illumination microscopy (3D-SIM) to register the progression of S-phase in more detail than previously described. We successfully established an automated workflow to produce reliable and reproducible replication foci (RF) counts in C2C12 cells from 3DSIM data and TANGO (Tools for Analysis of Nuclear Genome Organization). Such an approach has not been described before, and could be used to evaluate further cell types and schemes.
We observed significant differences in replication timing and progression between somatic (C2C12, C127) and mESCs (HI5). In this report we show that in mESCs S-phase lasts significantly longer (15 h), with a ‘leaky’ chromocenter replication profile compared to somatic cells. Furthermore, differentiated HI5 female mESCs into epiblast-like cells (EpiLCs) exhibit inactive X chromosome and differential replication timing of Xi within two distinct EpiLC populations, and a much shorter S-phase (10 h).
As a final aim of this work, we interfered with specific histone modifications with inhibitors and knockout cell lines. Inhibition of EZH2 methyltransferase resulted in global reduction of H3k27me3 levels in both somatic and mESCs, however replication dynamics were not affected. In contrast to somatic cells, viability of mESCs in presence of inhibitor was greatly reduced, suggesting a more important role of H3K27me3 in mESCs. Suv39H1/H2 double knockout mESCs had no observable effect on replication dynamics or proliferation. Moreover, differentiation of these cells into EpiLCs resulted in a distinct S-phase progression, with replication resembling HI5 EpiLCs.

Inhaltsverzeichnis (Table of Contents)

• Abstract
• Zusammenfassung
• List of figures
• Abbreviations
• 1. Introduction
  • 1.1 DNA replication is a basic biological process required by all living organisms
    • 1.1.1 Mammalian DNA is organized into a higher-order chromatin structure…
    • 1.1.2 Origins of replication and visualization of distinct replication patterns
    • 1.1.3 DNA replication patterns are formed by replication foci.......
    • 1.1.4 Chromatin structure can be directly influenced by DNA methylation
    • 1.1.5 Histones exhibit a multitude of post-translational modifications that define chromatin structure .....
    • 1.1.6 Histone modifications are brought about by specific histone modifying enzymes..
    • 1.1.7 Disruption of histone modifying enzymes affects replication dynamics..........\n
  • 1.2 Breaking the resolution limit with super resolution microscopy.
    • 1.2.1 Principles and applications of 3D structured illumination microscopy..\n
    • 1.2.2 Utilization of 3DSIM data for analysis of nuclear genome organization\n
  • 1.3 Aims of this study...\n
• 2. Materials...........
  • 2.1 Cell lines
  • 2.2 Antibodies.
  • 2.3 Consumables........
  • 2.4 Equipment.
  • 2.5 EdU and BrdU detection reagents.......
  • 2.6 Reagents and chemicals
  • 2.7 Buffers ...........\n
  • 2.8 Somatic and stem cell medium
  • 2.9 Fluorescent in situ hybridization primers.\n
  • 2.10 Software\n
• 3. Methods
  • 3.1 Cell Culturing
    • 3.1.1 C2C12 and C127 propagation
    • 3.1.2 Seeding C2C12 and C127 cells for immunostaining
    • 3.1.3 Freezing and thawing of C2C12 and C127 cells
    • 3.1.4 Embryonic stem cells propagation
    • 3.1.5 Seeding embryonic stem cells for immunostaining..\n
    • 3.1.6 Pre-coating of flasks and coverslips for embryonic stem cells.
    • 3.1.7 Freezing and thawing of embryonic stem cells..\n
    • 3.1.8 Embryonic stem cell differentiation....\n
  • 3.2 Combined pulse-chase-pulse and immunostaining protocol........
    • 3.2.1 EdU click chemistry and pulse-chase-pulse experiments ......
    • 3.2.2 Immunostaining for pulse-chase-pulse experiments….......\n
  • 3.3 Transfection of embryonic stem cells for live-cell-imaging.....\n
  • 3.4 Fluorescence in situ hybridization (FISH) of somatic and embryonic stem cells ..........
    • 3.4.1 DNA FISH probe preparation for major and minor satellites and LINES-1.
    • 3.4.2 DNA FISH probe preparation for telomeric repeats.......
    • 3.4.3 Nick translation for amplified PCR products for DNA FISH probes......
    • 3.4.4 DNA FISH probe preparation from nick translated products ....
    • 3.4.5 DNA FISH probe hybridization protocol........
    • 3.4.6 DNA FISH probe post-hybridization protocol...\n
    • 3.4.7 FISH treatment combined with EdU pulse ………………
    • 3.4.8 Xist RNA FISH probe preparation .......
    • 3.4.9 Pre-treatment and fixation of cells for Xist RNA FISH
    • 3.4.10 Xist RNA FISH probe hybridization and detection .........
  • 3.5 3D-SIM to wide-field deconvolution protocol\n
  • 3.6 Microscopy and image acquisition\n
• 4. Results....……………….\n
  • 4.1 DNA replication patterns in somatic mouse cells......
    • 4.1.1 DNA replication patterns in C2C12 cells
    • 4.1.2 DNA replication patterns in C127 cells.........\n
  • 4.2 Fluorescent in situ hybridization (FISH) of repetitive sequences in C2C12 cells..........
  • 4.3 Histone modification patterns in C2C12 cells\n
  • 4.4 Quantification of replication foci in C2C12 cells..\n
    • 4.4.1 Effect of laser intensity on count of RF.
    • 4.4.2 Comparison of RF between 3D-SIM and WFD data......\n
  • 4.5 Analysis of EZH2 inhibitor effect on replication and proliferation of C2C12 cells\n
  • 4.6 DNA replication patterns in mouse embryonic stem cells.......
    • 4.6.1 DNA replication patterns in HI5 female mESCs
    • 4.6.2 Live-cell S-phase progression in HI5 female mESCs.
    • 4.6.3 FISH hybridization of major satellite repeats in HI5 mESCs..\n
  • 4.7 H3K27me3 distribution in HI5 mESCs and the effect of EZH2 inhibitor.
  • 4.8 Differentiation of HI5 mESCs into EpiLCs.
    • 4.8.1 DNA replication patterns in HI5 EpiLCs
    • 4.8.2 Inactive X chromosome replication analysis in EpiLCs.........\n
  • 4.9 Hybridization of Xist RNA probe in somatic, stem, and EpiLCs.
  • 4.10 Effect of EZH2 inhibition during differentiation of HI5 mESCs into epiblasts
  • 4.11 Comparison of DNA replication patterns in undifferentiated and differentiated Suv39H1/2 knockout male mESCs\n
• 5. Discussion ......
  • 5.1 Replication patterns in C2C12 and C127 cells with 3D-SIM.
  • 5.2 Combination of fluorescent in situ hybridization of specific sequences and 3D-SIM ....
  • 5.3 RF counts in C2C12 cells with TANGO and 3D-SIM..\n
  • 5.4 Differences in replication dynamics in somatic and mESCs.
  • 5.5 Effect of interference with histone modifications on S-phase progression ............\n
• References
• Acknowledgements...\n

Zielsetzung und Themenschwerpunkte (Objectives and Key Themes)

This master's thesis aims to analyze replication patterns in mouse somatic and embryonic stem cells (mESCs) using three-dimensional structured illumination microscopy (3D-SIM) to gain a more detailed understanding of S-phase progression.

• Replication patterns and dynamics in somatic and embryonic stem cells
• The role of epigenetic mechanisms in regulating DNA replication
• The impact of histone modifications on replication dynamics
• The application of super-resolution microscopy for studying genome organization
• Analysis of replication foci and their relationship to chromatin structure

Zusammenfassung der Kapitel (Chapter Summaries)

• Chapter 1: Introduction This chapter provides a comprehensive overview of DNA replication, chromatin structure, and the role of epigenetic mechanisms in regulating this process. It also introduces the principles and applications of super-resolution microscopy, specifically 3D-SIM, and outlines the aims of the study.
• Chapter 2: Materials This chapter details the materials and methods used in the study, including cell lines, antibodies, reagents, equipment, and software.
• Chapter 3: Methods This chapter describes the experimental protocols employed, including cell culturing, immunostaining, fluorescence in situ hybridization (FISH), and 3D-SIM imaging.
• Chapter 4: Results This chapter presents the findings of the study, analyzing replication patterns in somatic and embryonic stem cells, including the effects of histone modifications and inhibitors on replication dynamics.
• Chapter 5: Discussion This chapter discusses the implications of the findings, drawing conclusions about the role of epigenetic mechanisms in regulating DNA replication and the potential of super-resolution microscopy for studying genome organization.

Schlüsselwörter (Keywords)

This master's thesis focuses on the epigenetic regulation of DNA replication using super-resolution microscopy. Key terms include DNA replication, chromatin structure, replication foci, histone modifications, 3D-SIM, embryonic stem cells, somatic cells, and epigenetic regulation.

• College: LMU Munich (Department Biologie II)
• Grade: 1.0 (A+)
• Author: Alexei Budco (Author)
• Publication Year: 2014
• Pages: 98
• Catalog Number: V302366
• ISBN (eBook): 9783668010598
• ISBN (Book): 9783668010604
• Language: English
• Tags: Genetics, Epigenetics, Cell biology, Microscopy, DNA, RNA, Live cell imaging, Stem cells, Somatic cells, super-resolution, 3DSIM, knockouts, histone modifications, inactive x, TANGO, DNA replication, S-phase