The considerable length of DNA in eukaryotic genomes requires packaging into chromatin to fit inside the small dimensions of the cell nucleus. Histone H1 functions in the compaction of chromatin into higher order structures derived from the repeating 'beads on a string' nucleosome polymer. Modulation of H1 binding activity is thought to be an important step in the potentiation/depotentiation of chromatin structure for transcription. It is generally accepted that H1 binds less tightly than other histones to DNA in chromatin and can readily exchange in living cells. Fusion proteins of Histone H1 and green fluorescent protein (GFP) have been shown to associate with chromatin in an apparently identical fashion to native histone H1. This provides a means by which to study histone H1-chromatin interactions in living cells. Here we have used human cells with a stably integrated H1.1-GFP fusion protein to monitor histone H1 movement directly by fluorescence recovery after photobleaching in living cells. We find that exchange is rapid in both condensed and decondensed chromatin, occurs throughout the cell cycle, and does not require fibre-fibre interactions. Treatment with drugs that alter protein phosphorylation significantly reduces exchange rates. Our results show that histone H1 exchange in vivo is rapid, occurs through a soluble intermediate, and is modulated by the phosphorylation of a protein or proteins as yet to be determined.

Activation of the serine-threonine kinase p34cdc2 at an inappropriate time during the cell cycle leads to cell death that resembles apoptosis. Premature activation of p34cdc2 was shown to be required for apoptosis induced by a lymphocyte granule protease. The kinase was rapidly activated and tyrosine dephosphorylated at the initiation of apoptosis. DNA fragmentation and nuclear collapse could be prevented by blocking p34cdc2 activity with excess peptide substrate, or by inactivating p34cdc2 in a temperature-sensitive mutant. Premature p34cdc2 activation may be a general mechanism by which cells induced to undergo apoptosis initiate the disruption of the nucleus.

Histone acetylation plays an important role in regulating chromatin structure and thus gene expression. Here we describe the functional characterization of HDAC4, a human histone deacetylase whose C-terminal part displays significant sequence similarity to the deacetylase domain of yeast HDA1. HDAC4 is expressed in various adult human tissues, and its gene is located at chromosome band 2q37. HDAC4 possesses histone deacetylase activity intrinsic to its C-terminal domain. When tethered to a promoter, HDAC4 represses transcription through two independent repression domains, with repression domain 1 consisting of the N-terminal 208 residues and repression domain 2 containing the deacetylase domain. Through a small region located at its N-terminal domain, HDAC4 interacts with the MADS-box transcription factor MEF2C. Furthermore, HDAC4 and MEF2C individually upregulate but together downmodulate c-jun promoter activity. These results suggest that HDAC4 interacts with transcription factors such as MEF2C to negatively regulate gene expression.In eukaryotic cells, genetic information is packaged into chromatin, a highly organized DNA-protein complex which controls gene activities. A central question in studying eukaryotic gene regulation is how the generally repressive chromatin structure is regulated when necessary. In the past several years, three regulatory mechanisms have been recognized: DNA methylation, posttranslational modifications of histones, and ATP-dependent chromatin remodeling (53,55,57). The most extensively studied form of posttranslational modifications of histones is acetylation of ε-amino groups of lysine residues located at the flexible N-terminal tails of core histones (53, 55). The level of histone acetylation at a given region of chromatin correlates well with its transcriptional activity (39). Mechanistically, histone acetylation affects nucleosome stability and/or internucleosomal interaction (2, 29). The dynamic level of histone acetylation in vivo is maintained through opposing actions of histone acetyltransferases and deacetylases. Several known transcriptional coactivators possess intrinsic histone acetyltransferase activity (14,27,49,57).The first histone deacetylase, originally called HD1 (histone deacetylase 1) and later renamed HDAC1 (histone deacetylase 1), was cloned from mammalian cells (18, 50). HDAC1 was found to be highly homologous to the known yeast transcriptional coregulator RPD3 (50). Two HDAC1 homologs (HDAC2 and HDAC3) have been cloned from human cDNA libraries (10,58,59). Transcriptional repressors recruit RPD3 or HDAC1 to -3 to downregulate transcription (reviewed in references 41 and 56). The deacetylase activity of HDAC1 and RPD3 has been found to be important for transcriptional repression (18, 24), suggesting that histone deacetylation directly leads to transcriptional repression. Consistent with this contention, recruitment of RPD3 by the yeast repressor Ume6 leads to local histone deacetylation and formation of a highly localized domain of repressed chromatin in vivo...

We have used a combination of kinetic measurements and targeted mutations to show that the C-terminal domain is required for high-affinity binding of histone H1 to chromatin, and phosphorylations can disrupt binding by affecting the secondary structure of the C terminus. By measuring the fluorescence recovery after photobleaching profiles of green fluorescent protein-histone H1 proteins in living cells, we find that the deletion of the N terminus only modestly reduces binding affinity. Deletion of the C terminus, however, almost completely eliminates histone H1.1 binding. Specific mutations of the C-terminal domain identified Thr-152 and Ser-183 as novel regulatory switches that control the binding of histone H1.1 in vivo. It is remarkable that the single amino acid substitution of Thr-152 with glutamic acid was almost as effective as the truncation of the C terminus to amino acid 151 in destabilizing histone H1.1 binding in vivo. We found that modifications to the C terminus can affect histone H1 binding dramatically but have little or no influence on the charge distribution or the overall net charge of this domain. A comparison of individual point mutations and deletion mutants, when reviewed collectively, cannot be reconciled with simple charge-dependent mechanisms of C-terminal domain function of linker histones.Histone H1 is the fifth histone subtype and is not one of the histones that form the histone octamer of the nucleosome. Rather, histone H1 binds to the surface of the nucleosome and interacts with nucleosomal DNA at the entry and exit points (1). In doing so, histone H1 is critical in determining the higher-order folding states of chromatin. Because of this property, histone H1 has traditionally been considered a general repressor of transcription (3). Consistent with this hypothesis, histone H1 was found to be modestly depleted in transcriptionally active genes (4 -6). More recently, genetic studies have revealed contributions of H1 histones to the establishment of epigenetic silencing (7-10). In addition to a structural role, histone H1 also functions in gene-specific regulation. A large number of studies have demonstrated that H1 histones or specific variants are directly involved in the regulation of specific genes (3,(11)(12)(13)(14), consistent with the observation of differential gene expression when the sole histone H1 gene was knocked out in Tetrahymena thermophila (15).The structure of H1 histones is typically considered to consist of three separate domains (16). A short stretch of amino acids on the N terminus and a much larger stretch that comprises the C terminus show significant variability between individual subtypes as well as between species. The amino and carboxyl termini have diverged considerably throughout the evolution of metazoans (17). If we restrict the analysis to mammals, the C termini diverge between individual histone H1 variants, but the sequences of the individual C termini are well conserved between species. When histone H1 sequences are examined in a broader range of ...

YB-1 is a DNA/RNA-binding nucleocytoplasmic shuttling protein whose regulatory effect on many DNA-and RNAdependent events is determined by its localization in the cell. Distribution of YB-1 between the nucleus and the cytoplasm is known to be dependent on nuclear targeting and cytoplasmic retention signals located within the C-terminal portion of YB-1. Here, we report that YB-1 undergoes a specific proteolytic cleavage by the 20S proteasome, which splits off the C-terminal 105-amino-acidlong YB-1 fragment containing a cytoplasmic retention signal. Cleavage of YB-1 by the 20S proteasome in vitro appears to be ubiquitin-and ATP-independent, and is abolished by the association of YB-1 with messenger RNA. We also found that genotoxic stress triggers a proteasome-mediated cleavage of YB-1 in vivo and leads to accumulation of the truncated protein in nuclei of stressed cells. Endoproteolytic activity of the proteasome may therefore play an important role in regulating YB-1 functioning, especially under certain stress conditions.