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Mass spectrometric analysis of global histone modification profiles during xenopus laevis development
Mass spectrometric analysis of global histone modification profiles during xenopus laevis development
Vertebrate embryos are derived from a transitory pool of pluripotent embryonic cells. By the process of induction, these precursor cells are assigned to specific fates and differentiation programs. Histone post-translational modifications are thought to play a key role in the establishment and maintenance of stable gene expression patterns underlying these processes. While at gene level histone modifications are known to change during differentiation, very little is known about the quantitative fluctuations in bulk histone modifications during development. To investigate this issue histones isolated from four different developmental stages of Xenopus laevis were analysed by mass spectrometry. Initally, a variety of different protocols for histone extraction from Xenopus laevis embryos and stable cell lines was tested and evaluated. Since non of the available methods worked sufficiently, a new reliable and effective protocol for nuclei preparation and histone extraction was established. Using mass spectrometry, core histone modifications were unambiguously determined. The techniques for identification and quantification of histone modifications by tandem mass spectrometry were improved as well. In total, an average sequence coverage of 68% of modification sites for the four core histones was achived by tryptic digestion after covalent modification of lysine residues with propionic anhydride. Using both LC-MS/MS and MALDI-TOF mass spectrometry, a total number of 2 modifications of H2A and 3 modifications H2B, 39 modifications of H3 and 20 modifications of H4 were identified and quantified. During this developmental period, an increase in the unmodified states, and a shift from histone modifications associated with transcriptionally active to transcriptionally repressive histone marks, was observed. Furthermore, these naturally occurring histone modifications were compared to the histone modifications of murine ES cells, detecting large differences in the methylation patterns of lysines 27 and 36 of histone H3 between pluripotent cells from Xenopus blastulae and murine ES cells. By combining all detected modification transitions, their patterns could be clustered according to their embryonic origin, defining specific histone modification profiles for each developmental stage. These specific histone modification profiles indicated a stepwise maturation of the embryonic epigenome, which may be cause to the progressing restriction of cellular potency during development. This thesis has revealed major quantitative shifts for several histone modifications known to be involved in gene regulation and furthermore enabled the definition of stage specific histone modification profiles accompanying and potentially regulating the transition from pluripotent to determined cell states using an antibody-independent method.
Epigenetic, Histone Modification, Development, Xenopus laevis,
Schneider, Tobias
2012
Englisch
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Schneider, Tobias (2012): Mass spectrometric analysis of global histone modification profiles during xenopus laevis development. Dissertation, LMU München: Medizinische Fakultät
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Abstract

Vertebrate embryos are derived from a transitory pool of pluripotent embryonic cells. By the process of induction, these precursor cells are assigned to specific fates and differentiation programs. Histone post-translational modifications are thought to play a key role in the establishment and maintenance of stable gene expression patterns underlying these processes. While at gene level histone modifications are known to change during differentiation, very little is known about the quantitative fluctuations in bulk histone modifications during development. To investigate this issue histones isolated from four different developmental stages of Xenopus laevis were analysed by mass spectrometry. Initally, a variety of different protocols for histone extraction from Xenopus laevis embryos and stable cell lines was tested and evaluated. Since non of the available methods worked sufficiently, a new reliable and effective protocol for nuclei preparation and histone extraction was established. Using mass spectrometry, core histone modifications were unambiguously determined. The techniques for identification and quantification of histone modifications by tandem mass spectrometry were improved as well. In total, an average sequence coverage of 68% of modification sites for the four core histones was achived by tryptic digestion after covalent modification of lysine residues with propionic anhydride. Using both LC-MS/MS and MALDI-TOF mass spectrometry, a total number of 2 modifications of H2A and 3 modifications H2B, 39 modifications of H3 and 20 modifications of H4 were identified and quantified. During this developmental period, an increase in the unmodified states, and a shift from histone modifications associated with transcriptionally active to transcriptionally repressive histone marks, was observed. Furthermore, these naturally occurring histone modifications were compared to the histone modifications of murine ES cells, detecting large differences in the methylation patterns of lysines 27 and 36 of histone H3 between pluripotent cells from Xenopus blastulae and murine ES cells. By combining all detected modification transitions, their patterns could be clustered according to their embryonic origin, defining specific histone modification profiles for each developmental stage. These specific histone modification profiles indicated a stepwise maturation of the embryonic epigenome, which may be cause to the progressing restriction of cellular potency during development. This thesis has revealed major quantitative shifts for several histone modifications known to be involved in gene regulation and furthermore enabled the definition of stage specific histone modification profiles accompanying and potentially regulating the transition from pluripotent to determined cell states using an antibody-independent method.