Affinity-purified polyclonal antibodies recognizing the most highly acetylated forms of histones H3 and H4 were used in immunoprecipitation assays with chromatin fragments derived from 15-day chicken embryo erythrocytes by micrococcal nuclease digestion. The distribution of hyperacetylated H4 and H3 was mapped at the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the tissue-specific gene, carbonic anhydrase (CA). H3 and H4 acetylation was found targeted to the CpG island region at the 5 end of both these genes, falling off in the downstream direction. In contrast, at the ␤ A -globin gene, both H3 and H4 are highly acetylated throughout the gene and at the downstream enhancer, with a maximum at the promoter. Low level acetylation was observed at the 5 end of the inactive ovalbumin gene. Run-on assays to measure ongoing transcription showed that the GAPDH and CA genes are transcribed at a much lower rate than the adult ␤ Aglobin gene. The extensive high level acetylation at the ␤ A -globin gene correlates most simply with its high rate of transcription. The targeted acetylation of histones H3 and H4 at the GAPDH and CA genes is consistent with a role in transcriptional initiation and implies that transcriptional elongation does not necessarily require hyperacetylation.

Acetylation of histones H4 and H3 targeted to promoters/enhancers is linked to the activation of transcription, whereas widespread, long range acetylation of the same histones has been linked to the requirement for open chromatin at transcriptionally active loci and regions of V(D)J recombination. Using affinity-purified polyclonal antibodies to tetra/tri-acetylated histone H2B in chromatin immunoprecipitation (ChIP) assays with mononucleosomes from 15-day chicken embryo erythrocytes, a high resolution distribution of H2B acetylation has been determined and compared with that of H4 and H3 at the same genes/loci. At the ␤-globin locus, the H2B acetylation is high throughout and in general mirrors that of H3 and H4, consistent with the observation of co-precipitation of hyperacetylated H4 together with the hyperacetylated H2B. In contrast, at the weakly expressed genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Gas41 (housekeeping) and carbonic anhydrase (tissue specific), very little or no hyperacetylated H2B was found despite the presence of acetylated H4 and H3 at their promoters and proximal transcribed sequences. At the inactive lysozyme and ovalbumin genes essentially no acetylation of H2B, H3, or H4 was observed. Acetylation of H2B appears to be principally a feature of only the most actively transcribed genes/loci.

Mitotically active cells guard against genetic instability and increase their survival following DNA damage both by direct DNA repair mechanisms and by delaying progression through the cell cycle. A checkpoint mechanism operates in eukaryotic cells to ensure that mitosis is not initiated if S-phase or DNA repair during G2 is incomplete. Recently a new family of PI-3 kinase-related proteins have been identified which play an important role in the DNA-damage responsive checkpoints. ATR is a member of this family and is a large >250kDa protein containing a carboxyterminus kinase domain related to phosphatidylinositol-3 kinase. ATR is a human homologue of rad3MECl in yeast and is highly related to'the ATM gene product in human cells. The molecular mechanism controlling the activation of ATR following DNA replication arrest and its signalling to downstream elements are unknown. We have expressed full-length wild type and kinasedead mutant human ATR proteins in HEK293 cells and purified them to near homogeneity. In this poster we present data showing that ATR is a protein kinase. We have identified several substrates for this protein and have determined sites of phosphorylation. Importantly, we have uncovered a potential mechanism for the activation of ATR and data will be presented describing both the activation and the substrate specificity determinants for this kinase.