Sub-nuclear distribution and mobility of nuclear proteins involved in histone acetylation and pre-mRNA splicing

The mitotic relationship between levels of highly acetylated chromatin, chromatin condensation, and HAT/HDAC organization was examined. HATs and HDACs were found to dissociate from chromosomes along with a loss of highly acetylated histones in condensed chromatin in mitosis. We demonstrate that, rather than being enzymatically inactivated, HAT and HDAC activities are decreased in mitosis because the enzymes are sequestered to a non-chromatin domain. Highly acetylated histone species reappear coincident with the reassociation of HATS and HDACs in late telophase/early interphase and before reinitiation of transcription. We propose that HATS and HDACs are spatially regulated through the cell cycle and that this regulation influences which chromatin domains are available for acetylation and deacetylation.; We examined the movement of a splicing factor, ASF, green fluorescent fusion protein (ASF:GFP) using timelapse microscopy and the technique fluorescence recovery after photobleaching (FRAP). We found that ASF:GFP moves significantly slower than free diffusion when it is associated with speckles and, surprisingly, also when it is dispersed in the nucleoplasm. The mobility of ASF is consistent with frequent but transient interactions with relatively immobile nuclear binding sites. This mobility is slightly increased in the presence of transcription inhibitors and the ASF molecules further enrich in speckles. We propose that the nonrandom organization of splicing factors reflects spatial differences in the concentration of relatively immobile binding sites.; Through a careful analysis of HDAC4 expression we found that HDAC4-containing MAD bodies are not a consistent component of the interphase nucleus. By comparing MAD bodies to PML bodies we found that the assembly, maintenance and distribution of PML bodies is regulated.; We investigated the involvement of chromatin condensation in establishing mitotic transcription repression, by analyzing transcriptional activity in mitotic cells infected with herpes simplex virus type 1. We found that virus-infected cells enter mitosis and that transcription is repressed on viral genes that are maintained in a nucleosome-free and non-condensed state and on cellular genes that are condensed into mitotic chromosomes. We also examined the structure of hyperacetylated mitotic chromosomes, and found that hyperacetylated chromatin has a minimal influence on the formation and structure of the mitotic chromosomal conformation.