Search

Set1 is the catalytic subunit and the central component of the evolutionarily conserved Set1 complex (Set1C) that methylates histone 3 lysine 4 (H3K4). Here we have determined protein/protein interactions within the complex and related the substructure to function. The loss of individual Set1C subunits differentially affects Set1 stability, complex integrity, global H3K4 methylation, and distribution of H3K4 methylation along active genes. The complex requires Set1, Swd1, and Swd3 for integrity, and Set1 amount is greatly reduced in the absence of the Swd1-Swd3 heterodimer. Bre2 and Sdc1 also form a heteromeric subunit, which requires the SET domain for interaction with the complex, and Sdc1 strongly interacts with itself. Inactivation of either Bre2 or Sdc1 has very similar effects. Neither is required for complex integrity, and their removal results in an increase of H3K4 mono- and dimethylation and a severe decrease of trimethylation at the 5' end of active coding regions but a decrease of H3K4 dimethylation at the 3' end of coding regions. Cells lacking Spp1 have a reduced amount of Set1 and retain a fraction of trimethylated H3K4, whereas cells lacking Shg1 show slightly elevated levels of both di- and trimethylation. Set1C associates with both serine 5- and serine 2-phosphorylated forms of polymerase II, indicating that the association persists to the 3' end of transcribed genes. Taken together, our results suggest that Set1C subunits stimulate Set1 catalytic activity all along active genes. more...

Hat1 is the catalytic subunit of the only type B histone acetyltransferase known (HAT-B). The enzyme specifically acetylates lysine 12, and to a lesser extent lysine 5, of free, non-chromatin-bound histone H4. The complex is usually isolated with cytosolic fractions and is thought to be involved in chromatin assembly. The Saccharomyces cerevisiae HAT-B complex also contains Hat2, a protein stimulating Hat1 catalytic activity. We have now identified by two-hybrid experiments Hif1 as both a Hat1- and a histone H4-interacting protein. These interactions were dependent on HAT2, indicating a mediating role for Hat2. Biochemical fractionation and co-immunoprecipitation assays demonstrated that Hif1 is a component of a yeast heterotrimeric HAT-B complex, in which Hat2 bridges Hat1 and Hif1 proteins. In contrast to Hat2, this novel subunit does not appear to regulate Hat1 enzymatic activity. Nevertheless, similarly to Hat1, Hif1 influences telomeric silencing. In a localization analysis by immunofluorescence microscopy on yeast strains expressing tagged versions of Hat1, Hat2, and Hif1, we have found that all three HAT-B proteins are mainly localized in the nucleus. Thus, we propose that the distinction between A- and B-type enzymes should henceforth be based on their capacity to acetylate histones bound to nucleosomes and not on their location within the cell. Finally, by Western blotting assays, we have not detected differences in the in vivo acetylation of H4 lysine 12 (acK12H4) between wild-type and hat1Delta, hat2Delta, or hif1Delta mutant strains, suggesting that the level of HAT-B-dependent acK12H4 may be very low under normal growth conditions. more...

We have analyzed the histone acetyltransferase enzymes obtained from a series of yeast hat1, hat2, and gcn5 single mutants and hat1,hat2 and hat1,gcn5 double mutants. Extracts prepared from both hat1 and hat2 mutant strains specifically lack the following two histone acetyltransferase activities: the well known cytoplasmic type B enzyme and a free histone H4-specific histone acetyltransferase located in the nucleus. The catalytic subunits of both cytoplasmic and nuclear enzymes have identical molecular masses (42 kDa), the same as that of HAT1. However, the cytoplasmic complex has a molecular mass (150 kDa) greater than that of the nuclear complex (110 kDa). The possible functions of HAT1 and HAT2 in the yeast nucleus are discussed. In addition, we have detected a yeast histone acetyltransferase not previously described, designated HAT-A4. This enzyme is located in the nucleus and is able to acetylate free and nucleosome-bound histones H3 and H4. Finally, we show that the hat1, gcn5 double mutant is viable and does not exhibit a new phenotype, thus suggesting the existence of several histone acetyltransferases with overlapping functions. more...

We have assayed several methods to quantitatively recover yeast histone acetyltransferases in an attempt to study the multiplicity of enzymatic activities. Two methods, namely (NH4)2SO4 precipitation and salt dissociation of chromatin in 0.5 M NaCl, yielded convenient preparations of total histone acetyltransferases. DEAE-Sepharose chromatography of the crude extracts resulted in the separation of three peaks of activity when total yeast histones were used as substrate. However, the scanning of the enzymatic activity toward individual histones along the chromatography, achieved by determining the specific activity of the individual histones after incubating whole histones and [14C]acetyl-CoA with the chromatographic fractions, yielded four peaks. The first two peaks showed specificity toward H2B and H3, respectively. Although they partially overlapped, rechromatography on cation exchangers allowed us to resolve the two activities, and several criteria were used to prove that they correspond to different enzyme molecules. The last two peaks were H4-specific, but the present data suggest that one of the activities is chromatin-bound, whereas the other surely corresponds to the cytoplasmic B-form of the enzyme. The enzyme specific for yeast H2B acetylates chicken erythrocyte H2A, rather than H2B. The detected multiplicity of yeast histone acetyltransferases may correspond to the multiplicity of roles proposed for histone acetylation. more...