Summary
The pluripotent state of embryonic stem cells (ESCs) is maintained by a unique gene expression program, which is characterized by an elevated transcription across the genome and by unique chromatin modification signatures. These characteristics are lost upon lineage commitment of ESCs. A precise and detailed understanding of the molecular basis for this is important to exploit the full potential of ESCs for clinical applications. Here we propose a systematic proteomic analysis of the composition of the SAGA chromatin modifying and TFIID transcription regulatory complexes and their interacting proteins in pluripotent ESCs and upon their differentiation into neural cells. For this a BAC transgene tagging approach will be combined with a highly-efficient affinity purification protocol based on streptavidin capture (see also T4.2) and with quantitative mass spectrometry methods. To understand the interplay between different histone modifications we will employ a peptide affinity assay focusing on modifications which co-occur on promoters and enhancers in ESCs. This will identify the molecular basis of the interplay between different ‘readers’ of the histone code. Together, these two experimental approaches will bring novel mechanistic insights into regulation of transcription and of chromatin in pluripotent ESCs and in neural differentiation pathways.