The development of an organism from the fertilized zygote to a
multicellular organism is a unidirectional process. It occurs in a spatially and
temporally tightly controlled fashion. To understand how the genetic information
is interpreted and how the cellular identity is inherited, are major challenges
towards the understanding of developmental processes. Epigenetic marks like
histone modifications, changes of the protein composition binding to DNA or the
remodeling of nucleosomes have been shown to be important for the
establishment of tissue-specific transcription profiles.
Chromatin immunoprecipitation (ChIP) is a method to investigate the
association of proteins to specific genomic loci. In this study, I have established
two protocols for ChIP analyses of Xenopus laevis embryos: the In Situ ChIP and
the Douncer ChIP. In addition, I have generated several antibodies in
collaboration with Dr. Elisabeth Kremmer (GSF München) for ChIP analyses,
which were directed against the muscle determination factor MyoD and the
Wnt/β-catenin signaling components Lef/Tcf transcription factors Lef1 and Tcf1.
While optimizing of the ChIP protocols, I have analyzed successfully
the binding of various transcription factors, chromatin remodeling enzymes and
histone modifications on genomic loci of key developmental regulators. With the
In Situ ChIP, I have shown that the serum response factor SRF interacts
predominantly with the actively transcribed myoD gene. Together with other data,
this result helps to define a specific role of SRF protein in the stable maintenance
of myoD transcription, which is essential for proper muscle differentiation.
With the Douncer ChIP protocol, a time course study has been
performed in order to understand, when and which histone modification marks
appear during muscle cell determination and differentiation on the myoD locus.
The temporal and spatial distribution of the analyzed histone modification marks
was correlated for the most part with the expected patterns. Furthermore, I have
demonstrated that direct binding of the chromatin remodeler CHD4/Mi2-β to the
5' part of the sip1 gene in gastrula stage embryos. This interaction represents a
crucial regulatory module, which determines the position along the
animal-vegetal axis of the embryo, where the border between the mesodermal
and neuroectodermal germ layer will be formed. These examples represent on of
the very few successful ChIP applications for the endogenous proteins in young
Xenopus embryos, and I hope that my protocols will turn out useful for future
investigations of regulatory interactions in this vertebrate model organism.