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.