Signalling to the nucleosome: Targets and detectors

Eukaryotic cells adjust their gene expression program in response to changes in their environment and signals from the outside. Extracellular signals are transmitted from the cell surface to the nucleus, where transcription factors regulate gene expression with the help of associated chromatin modifying enzymes. Activation of stress and mitogen activated kinase cascades ultimately leads to specific marks at nucleosomes through phosphorylation of serine 10 on histone H3. During the last years, H3S10 phosphorylation has been linked as part of the nucleosomal response to the transcriptional activation of numerous mammalian genes. In addition, phosphorylation of H3S28 was shown to correlate with gene activation in response to different stimuli. However, little is known about the mechanisms by which the phosphorylated histone H3 is recognized and how the signals are conveyed to the RNA Pol II machinery. We and others have recently identified two members of the 14-3-3 family, 14-3-3 epsilon and zeta, as H3S10ph binding modules. Interestingly, additional acetylation of the neighboring lysine residues K9 or K14 significantly increases the affinity of 14-3-3 for S10 phosphorylated histone H3 suggesting that 14-3-3 recognizes multiple modifications on the H3 tail. In addition, 14-3-3 strongly binds to the H3S28ph mark in in vitro binding assays. In response to extracellular signals, 14-3-3 is recruited to the promoters of specific target genes and in knockdown experiments we have demonstrated that the presence of 14-3-3 is crucial for gene activation of these genes. During the last years, we have created valuable tools such as 14-3-3 and histone modification specific antibodies and stable cell lines expressing TAP-tagged 14-3-3 and myc-tagged histone H3 isoforms. Using these tools, we intend to analyze the impact of histone H3 phosphorylation and 14-3-3 recruitment on gene regulation in mouse fibroblasts. By TAP-tag affinity purification of associated factors, knockdown studies and ChIP assays, we will unravel the molecular function of 14-3-3 in chromatin-dependent regulation of gene expression. 14-3-3 binds to both phosphorylated serine 10 and serine 28 albeit with different affinity. Published data indicate that the two modifications target different pools of nucleosomes. We have established a system, in which histone H3 phosphorylation can be triggered by activation of stress-induced MAP kinase cascades and have identified target genes by gene expression profiling. By ChIP sequencing, we will examine genome-wide histone H3 phosphorylation at serine 10 and serine 28 in response to stress and correlate these data with the identified target genes. To understand the regulatory mechanisms we will examine genome-wide recruitment of 14-3-3 and RNA polymerase II and the effect of MAP kinase signaling on H3 acetylation and transcription factors by quantitative ChIP analysis. Our project will significantly contribute to our understanding of transcription control by a mechanism that integrates phosphorylation signals at the level of the nucleosome. In addition to core histones, several other proteins involved in signal transduction, apoptosis and tumor suppression show multiple modifications that are regulated in a well-orchestrated manner and define the biological function of the target protein. Thus, the crosstalk of multiple modifications on histone H3 might be a paradigm for a more general protein modification code and the outcome of this study will therefore be of general interest for the scientific community.

In this project we have drawn a full picture of the stress-dependent transcriptional response in mammalian cells. In our cells external signals are transmitted via signal cascades from the cell surface to the nucleus mediating changes in the gene expression program. This allows for rapid adaptations of vital cellular functions to changes in internal and external conditions. In response to stress histones, the proteins which pack our genetic material become modified by phosphorylation. Applying state-of-the-art technology we have identified all the genes that are marked by histone phosphorylation. We could show that this modification results in reduced packaging of these genes and thereby allowing for stress-induced activation. The marking of histones by phosphorylation is part of the histone code and triggers the induction of a specific response to external signals. This work is of high relevance for our understanding of the impact of histone phosphorylation and contributes to the deciphering of the histone code.