Final Report Summary - NUCLEARACTIN (Actin as the Master Organizer of Nuclear Structure and Function)
In addition to its essential functions within the cytoskeleton, actin also localizes to the cell nucleus, where has been linked to many important nuclear processes from gene expression to maintenance of genomic integrity. However, both the molecular mechanisms and the in vivo relevance of nuclear actin have remained unclear. The NuclearActin project has made significant breakthroughs on both of these aspects. One major issue that has prevented mechanistic studies on nuclear actin has been the lack of well-characterized binding partners for actin in this cellular compartment. To tackle this problem, we have utilized two complementary mass-spectrometry based methods to reveal both stable and dynamic interactions for nuclear actin. Our analysis identifies already established binding partners for actin, such as components of chromatin remodeling complexes, and provides new insights into known actin-regulated processes, such as transcription. In addition, we identify completely novel binding partners and functions for actin in the nucleus. We show that actin binds directly to the histone acetyl transferase (HAT) KAT14, and regulates its HAT activity both in vitro and in cells. We also demonstrate that actin interacts with the spliceosome and is required for alternative splicing in cells. We further analyze the genome-wide binding pattern of actin on chromatin by using chromatin immunoprecipitation followed by deep sequencing (ChIP-seq), which demonstrates, for the first time, that actin interacts with essentially all transcribed genes. Actin is found, together with RNA polymerase II, near transcription start sites of most genes, but also on the gene bodies of highly expressed genes. This result agrees with our mass spectrometry data, which identified both components of the pre-initiation complex and transcription elongation factors as part of the nuclear actin interactome. Finally, we take advantage of the nucleo-cytoplasmic shuttling properties of actin to study the in vivo roles of actin in the fruit fly Drosophila melanogaster. Our results show that adequate nuclear actin levels are required for Drosophila oogenesis by regulating transcription of the egg shell genes. This study thus demonstrates that actin is required for transcription during a developmental process. Taken together, the NuclearActin project has brought novel mechanistic and functional information on how actin operates in the nucleus to regulate gene expression. Especially significant are the findings that actin binds to all transcribed genes, interacts with proteins required for gene transcription and that actin has a functional role in gene transcription also in vivo. In addition, this study has also opened completely new avenues for this research field by identifying novel binding partners and functions for nuclear actin, thus enabling further mechanistic studies.