Post discussion, questions, or comments about the course material here.
Questions about the first paper:
I don't really understand why ATV genes are sensitive to digestion. The paper says the globin genes are active, and therefore more have an altered subunit structure, which is more susceptible to pancreatic DNAse digestion. I don't understand how this fits with less active RNA tumour virus genes also being digested.
Question about the second paper:
In figure 4C, the NPCs show a very clear line on the gel electrophoresis, which is not seen on the ESCs. This is present both before and after digestion with micrococcal nuclease. What does this line represent? It suggests some undigestible element of the nucleus, present in NPCs, and not in ESCs. I'd investigate this further.
1) For the Weintraub paper, I was wondering if today we have images of the
histone conformations they were speculating about at that time.
2) For the Meshorer one, I was curious about the leukemia inhibitory factor
(LIF) and why the cell differentiates when you deplete it.
What exactly are they doing with the acid precipitations/what do the acid-soluble fractions contain? (e.g. in pg. 849 middle column, pg. 850 middle column, figure 2 legend, pg. 853 middle column).
Having a hard time fully understanding the x-axis of their graphs... Cot is [DNA]*(time digested)?? Concentration of which DNA? I don't know how to predict what the curves should look like with those units.
What is FISH?
The HirA-/- data are the opposite of what I would expect. Since lack of HirA makes it harder to assemble complete nucleosomes ("reduced incorporation of core histones H3 and H3.3"), then it would seem that HirA-/- cells would be less able to form heterochromatin, and therefore prefer to stay more ESC-like rather than differentiating quickly. The authors' argument is that since H3 and H3.3 cannot be incorporated as well, they are more of them floating around; but in the end wouldn't you still need HirA function to use those extra H3/H3.3s?
Questions relating to Paper 1 (Chromosomal subunits in active genes have an
-It says that mature adult RBCs that don't synthesize RNA are also sensitive to
the nuclease - does this suggest that the original structure is not reinstated?
Could this be due to the lack of hyperdynamic chromatin proteins shown in paper
2? Does it also suggest that it isn't the structure of DNA that is manipulated
to silence the gene? In which case what is used?
- Why does staph nuclease not normally show preferential digestion - is it
because even with the more open conformation of active genes the enzyme is
still to bulky(?) to access them?
- A possible control to check that the preferential digestion is due to
the structural conformation would be to digest the histones with a protease,
then subsequently add the nuclease - would expect 100% digestion?
Questions relating to Paper 2 (Hyperdynamic etc etc)
- Possible follow up questions - What are the interactions of the hyperdynamic
chromatin proteins; what is the signal that instigates their actions in the
remodeling process? Is there anything that could reinstate the hyperdynamic
nature and would this cause the cell to revert back to pluripotency?
For "Chromosomal subunits in active genes have an altered conformation", I'm
confused by the passage on page 849, in the middle column, where it describes
the DNA being 'nibbled'. If the DNA is being digested (and presumably
differently in different cells, since different cells have different active
genes), doesn't that mean that then each type of cell has a different set of
DNA? I thought all the cells in an organism had the same set of DNA... Do
they just start off with the same set, and then they're modified?
For "Hyperdynamic plasticity of chromatin proteins in pluripotent embryonic stem
cells", I was confused by the discussion in the summary about chromatin binding
proteins. Are these proteins that simply bind to the chromatin, or are they
proteins that bind to multiple, pieces of chromatin and thus bind the chromatin
together, affecting the shape?
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?
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?
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?
Lachner et al:
Presumably the authors of this paper are short of time. It would have been nice if they'd replicated some of the studies shown in the second paper; including perhaps a loss of function analysis in a model organism.
Wysocka et al:
Figure 5D shows an increase in the levels of dimethylated K4 in HOXA9, when WDR5 is knocked down, using WDR5 siRNA. The authors say this data is reproducible; but it doesn't seem to be explained by their model.
Wysocka et al.
I think the increase in DiMeK4 in HOXA9 P2 can be explained by the decrease in trimethylation, e.g. less DiMeK4 is converted to TriMeK4, that's why the levels go up; which fits the model; moreover they see the same in HOXC8 P4; but in HOXC8 P3 the levels go down which can not be explained at all, I think. Maybe if they had more WDR5 targets the results would me more consistent. They say there might be differences in the dynamic of regulation between the two Hox genes anyway
Lachner et al.
In the WDR5 paper they do ChIP and gene expression analysis. Maybe they might have done it for the HP1 paper too, if, of course it was possible to do in 2001 (which I don't know). Also, is there any reason for using (Myc)3-tagging other than just not having specific anibodies against the proteins of interest?
Lachner et al.
What does it mean to "mock-infect" the fibroblasts during the experiment to look for nuclear localization of SUV39H1 proteins?
Wysocka el at.
When the authors look for defects from knockdown of xWDR5, they use use embryos derived from eggs of multiple frogs so that there will not be phenotypic differences from different genetic backgrounds. How does using eggs from many frogs do this? It seems like they would want to use embryos from the same frog instead.
Wysocka et al
They suggest that their data provides evidence that WDR5 acts as a "sensor" protein - in what way? It does not seem to show how the timing of WDR5 binding and subsequent activation is related to signals that indicate expression would be appropriate (although it does show that WDR5 would be a good target for manipulating/ regulating expression)
Lachner et al
This paper discusses what features of HP1 are required for it to bind the H3 lys 9 N terminus. However the experiments only show the characteristics needed in vitro - I think they should have done the pull down assay with nucleosomes. Also, what is the significance of understanding the binding? Why would dimerization be important and do these results actually show that it is necessary?
Wysocha et al
I found their suggestion that WDR5 specifically recognizes the H3 tail methylated at K4 a bit confusing, and do not understand how they made that leap from the analysis of Ash2 and RbBP5.
Lachner et al
I really enjoyed how they used the GFP analysis to better show that HP1 localization is dependent on Suv39h activity and not Suv39h proteins, and the result of 80-90% was so high. I would however, have liked to hear more about how Lys9 in H3 acts like a molecular switch, because I think that is one of the strongest suggestions in their paper.