Learn about histone acetylation.
In 1964 Vincent Allfrey discovered that histones can be found in both acetylated and unacetylated forms. The acetylated forms have acetyl groups added to the amino groups on lysine side chains. Allfrey also discovered that acetylation correlates with gene activity. Unacetylated histones, added to DNA, tend to repress transcription, while acetylated histones are weaker repressors of transcription. These results therefore imply that there are enzymes in nuclei that acetylate and deacetylate histones, and therefore influence gene activity.
James Brownell and David Allis in 1996 succeeded in identifying and purifying a histone acetyltransferase (HAT). HAT is an enzyme that transfers acetyl groups from a donor (acetyl-CoA) to core histones. In order to isolate this enzyme Brownell and Allis started with Tetrahymena (ciliated protozoan) cells because this organism has histones that are heavily acetylated, which implies that these cells contain relatively high concentrations of HAT. They prepared extracts from macronuclei (the large Tetrahymena nuclei that contain the active genes) and subjected them to gel electrophoresis in an SDS gel impregnated with histones. HAT activity was detected by soaking the gel in a solution of acetyl-CoA with a radioactive label in the acetyl group. If the gel contained a band with HAT activity, the HAT would transfer labeled acetyl groups from acetyl-CoA to the histones. This would create a labeled band of acetylated histones in the gel at the position of the HATactivity. To detect the labeled histones, they washed away the unreacted acetyl-CoA, then subjected the gel to flurography.
This initial identification of HAT activity was further followed by Allis and colleagues using a molecular cloning technique to learn more about p55 and its gene. First they purified the HAT activity further , using standard biochemical techniques. Once they purified the HAT activity essentially to homogeneity, they isolated enough of it to obtain a partial amino acid sequence. Using this sequence they designed a set of degenerate oligonucleotides that coded for parts of the amino acid sequence and therefore hybridized to the macronuclear genomic DNA (or to cellular RNA). Using these oligonucleotides as primers, they performed PCR, then cloned the PCR products. They obtained the base sequences of some of the cloned PCR products and checked them to verify that the internal parts also coded for known HAT amino acid sequences. None of the PCR clones contained complete cDNAs, so these workers extended them in both the 5' and 3' directions, using the RACE procedure or Rapid Amplification of cDNA ends. (Refer to RACE diagram for further explanation).
Allis and colleagues used a modified version of this 5' RACE procedure in which they cyclized the cDNA so they could use internal primers for both forward and reverse PCR priming. This trick allowed them to avoid tailing the 3' end of the first strand cDNA with terminal transferase. Finally, they obtained a cDNA clone that encoded the full 421 amino acid p55 protein.
The amino acid sequence inferred from the base sequence of the p55 cDNA was very similar to the amino acid sequence of a yeast protein called Gcn5p. Gcn5p has been identified as a mediator of acidic transcription activators such as Gcn4p, so the amino acid sequence similarity suggested that both the p55 and Gcn5p are HATs that are involved in gene activation. To verify that Gcn5p has HAT activity, Allis and colleagues expressed its gene in E. coli, then subjected it and p55 to the SDS-PAGE activity gel assay. Both proteins showed clear HAT activity. Thus, at least one HAT (Gcn5p) has both HAT and transcription co-activator activities. It appears to play a direct role in gene activation by acetylating histones.
p55 and Gcn5p are type A HATs (HAT As) that exist in the nucleus and are apparently involved in gene regulation. They acetylate core histones on their lysine-rich N-terminal tails. These are dustunct from type B HATs (HAT Bs) that are found in the cytoplasm and acetylate newly synthesized histones H3 and H4 so they can be assembled properly into nucleosomes. The acetyl groups added by HAT Bs are later removed in the nucleus by histone deacetylases. All known HAT As, including p55 and Gcn5p, contain a bromodomain, while all known HAT Bs lack a bromodomain. Since HAT As are involved in acetylating histones in chromatin, but HAT Bs are not, the bromodomain may help the HAT As bind to chromatin.
See the DNA Molecule in 3-Dimensions