Analysis of Developmental Epistasis by Chromatin Immunoprecipitation in Xenopus laevis

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.