Signalling to the nucleosome

Eukaryotic cells react to changes in their environment and signals from the outside by adjusting their gene expression program. Signals have to be 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 at histone H3. During the last years, H3-10 phosphorylation has been linked to the transcriptional activation of numerous mammalian genes. However, little is known how the phospho-S10 mark at histone H3 is recognized and how the signal is transmitted to the RNA Pol II machinery. In this project we intend to analyze the impact of S10 phosphorylation at histone H3 for gene regulation in mouse fibroblasts. For this purpose, we have recently established a model system that allows the induction of phosphorylation and acetylation at histone H3 thereby activating the expression of specific target genes. Using this system, we will examine the targeting of S10 phosphorylation at histone H3 to responsive promoters and the resulting changes in other modifications at the histone H3 tail. Furthermore, we will analyze the consequence of H3-S10 phosphorylation for initiation and elongation of transcription and the way in which the transient phosphorylation signal at target genes is turned off. To learn more about the signal transmission through phospho-S10 marks, we have recently initiated a screen for proteins that bind specifically to phosphorylated H3-S10. To our knowledge no phospho-S10 recognizing factors have been published up to now. We have isolated two nuclear factors, called here Phospho Histone Binding 1 and 2 (PHB1 and PHB2) that specifically bind to phosphorylated S10 at histone H3. Acetylation at lysine K14 increases the affinity of these proteins for the H3 tail. Importantly, PHB1 and PHB2 are recruited to target promoters that are associated with phosphorylated histone H3. We will characterize the role of these proteins during the transcriptional activation of target genes. Using the fibroblast model system, we will ask whether PHB1 and PHB2 can act as adapter proteins involved in the recruitment of chromatin remodeling machines, histone modifying enzymes or components of the basal transcription machinery. Our project intends to significantly contribute to our understanding of transcription control by a mechanism that integrates phosphorylation signals at the level of the nucleosome.

Eukaryotic cells react to changes in their environment and signals from the outside by adjusting their gene expression program. Signals have to be 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 at histone H3. Serine 10 phosphorylation facilitates the acetylation of the neighbouring lysine residues 9 and 14 at the H3 histone tail resulting in simultanous aceltylation and phosphorylation called phosphoacetylation. In this project, we have characterized the enzymes that control the phosphorylation of histone H3 in response to stress signals. Most importantly, we have identified two 14-3-3 proteins as the first factors that specifically detect and bind phosphorylated histone H3 thereby mediating the biolical read-out of stress signals to the cellular transcription machinery. In addition, we have successfully analyzed changes in histone H3 modification patterns and recruitment of histone-associated proteins such as 14-3-3 and HP1 upon activation of the signalling cascade by stress. It has become increasingly clear over the past decade that in addition to the genetic information provided by the DNA sequence, modifications of the packaging proteins are also involved in the control of gene expression. According to the histone code hypothesis, the combination of multiple chromatin modifications regulates the accessibility of specific target genes. In accordance with this idea, our results indicate that the cross-talk between methylation, acetylation and phosphorylation on histone H3 have a strong impact on the transcription of stress induced genes. Taken together, these data significantly contribute to the understanding of transcription control by a mechanism that integrates phosphorylation signals at the level of the nucleosome.