Post discussion, questions, or comments about the Week 3 course material here.
Question for Brownell et al:
At the end of the section Tetrahymena is Homologous to Yeast Gcn5p, the authors say that Gcn5p "migrates anomolously in SDS gels". Why would that happen?
Question for Taunton et al:
I noticed that they related RbAp48, the protein that binds to the retinoblastoma gene product, to histone deacetylase. They also found that inhibition of the deacetylase arrests the cell cycle. Could any of the research be useful for cancer treatment?
Comments from the second paper (Taunton et al)
There are three classes of HDAC proteins with class I being homologous to Rpd3 and containing 4 members (H. Santos-Rosa, C. Caldas/ European Journal of Cancer 41 (2005) 2381-2402). Why did they identified only one protein? Maybe some of the others were among the six bands that initialy appeared?
They predicted that RbAp48 is an adaptor subunit targeting HDAC to chromatin domains; it is found in the HATB complex in the cyoplasm, with CAF-1 in the hucleus, participating in nuclesome assembly and with HDAC. What is its precise role in all these processes?
Also, can we have a more in depth discussion about the various HAT and HDAC complexes, their specific functions, etc.?
Taunton et al paper:
Why did RbAp48 not co-precipitate with the recombinant HD 1 protein? Did this mean that the HD 1 was not targeted effectively? What would the effect of this be on the cells?
Brownell et al paper:
It is suggested that a conserved bromodomain is a source of HAT A specificity. Presumably the genes that need transcribing change during the lifetime of the cell (e.g. during differentiation, apoptosis etc) – how is this accommodated? Is the bromodomain modified in any way?
How does the charcoal precipitation assay work? Has any subsequent work regarding "cell cycle checkpoints may exist that monitor histone acetylation" been done?
I don't quite understand why this paper was important. It had already been known that histone acetylation was important for transcription. The main finding seems to be of this conserved bromodomain, but it seemed like the authors only speculated/theorized on its function. Did they really present any experimental evidence that this domain is indeed what directs HAT A to specific sites? What data did Marcus et al, 1994 and Georgakopoulos et al, 1995 show?
Comments from the first paper (Brownell et al)
The authors show that p55 and Gcn5p both contain bromodomain; and that Hat1p doesn't possess a bromodomain. They speculate that the bromodomain tethers HAT A to other factors at specific chromosomal sites. This is remarkably accurate - later research has shown that bromodomains bind to acetylated lysine residues; helping regulate transciptional remodelling and transciptional activation (Zeng, L., Zhou, M.M. (2002)Bromodomain: an acetyl lysine binding domain. FEBS Letters. Vol 213, 124-128)
Question about the second paper (Taunton et al.)
On p. 410, they say "RPD3 has yet to be implicated in silencing at telomeres or at the mating loci." Presumably this work has been done since 1996. Do you know what the outcomes were?
Taunton et al.:
As an organic chemistry enthusiast, I loved that they showed how trapoxin looks like an acetylated lysine side chain to the histone deacetylase enzyme; and you can see how the reactive and nucleophilic epoxide ring of trapoxin binds to and destroys the HD1 enzyme as soon as it attempts to deacetylate the trapoxin. I found it very interesting and counter-intuitive that trapoxin and trichostatin increase acetylation, and thus increase transcription, but because they selectively stimulate transcription they actually depress the cell cycle. At the end of the paper, the authors allude to the fact that they don't know what mechanism causes this paradoxical cell cycle arrest.
Brownell et al.:
Gcn5p (and tetrahymena p55) from this paper and RbAp48 from the Taunton paper both help to target an enzyme to certain histones. How do they do this? Do these proteins actually bind to specific sequences of DNA? Do they interact with other gene regulatory proteins (it seems that Gcn5p might work this way)? What is a bromodomain? If Gcn5p is only an "adaptor protein," then why does it have a domain that is similar to an aminotransferase?