Speaker: Kirti Prakash
Department: Institute of BMolecular Biology, Mainz, Heidelberg University Heidelberg. Carnergie institution for science, Baltimore usa, current affiliation institute paris
Subject: Structurally distinct periodic and dynamic chromatin domains revealed by super-resolution fluorescence microscopy
Location: Bionanoscience Department, TU Delft
Author: Mirte Golverdingen
A large spectrum of chromatin domains between DNA and the chromosome territory level remains unexplored. We do understand the morphology of the DNA helix since the 1950’s and a decade ago Bolzer et al. showed the morphology of intra-chromatic departments of chromatins (see Figure 1). However, the morphology and behavior of the region between single DNA morphology and intra-chromatic departments is still fairly unknown. Genomic methods have already found a large spectrum of chromatin domains between the DNA and the chromosomal territory level, however their regulation is not known yet. To learn more of the regulation of gene expression, we have to obtain a better understanding of the unknown region.
Figure 1: False color representation of all CTs visible in this mid-section after classification with the program goldFISH.
Adapted from: Bolzer, A., et al. (2005). Three-dimensional maps of all chromosomes in human male fibroblast nuclei and prometaphase rosettes. PLoS biology, 3(5), e157.
Kirti Prakash studied these intermediate chromatin features by using single-molecule localization methods (SMLM). Classical microscopy methods, such as conventional light and electronic microscopy, are not able to identify intermediate chromatin features because of specificity and a poor resolution. Therefore, nanoscale imaging is required to describe chromatin complexity.
SMLM is the best method to study chromatin organization because it has the best resolution in a small area. From all SMLM methods, PALM/STROM has the best resolution and distance in xyz area. When asked if the time resolution had not to be taken into account, Prakash mentioned that time resolution is not important for the research he is doing. Moreover, he mentioned that the PALM/STORM microscope set-up technique is very simple, because no much alignment is needed.
However, the preparation of the buffers and fluorophores is very complicated, because the combination of buffer and fluorophores need to have a high photon expression number, prevents photo-bleaching and it has to blink for a long time. The search to a combination that has all three characteristics is time consuming and hard. Therefore, I think that PALM/STORM is not that better than other SMLM methods as Prakash suggested in his talk.
Dyes as Hoechst and DAPI undergo phot-conversion when binding to the minor DNA groove, therefore they are a good tool of chromatin studies for SMLM. The red shift of the fluorophore makes that you can exclusively excite the dyes that are bound to the DNA. This makes that the dyes do not bleach and that there are still a lot of signals. This high labeling density is required to study unknown structures.
According to Prakash, this method of direct DNA staining is better than previous methods based on antibody staining o histone proteins. This is because you can have a 5 times higher labeling density. However, can we really compare the DNA-staining with histone labeling? Histones behave different from DNA itself, so are you not comparing apples with oranges?
By using the technique Prakash was able to study the hierarchical structure of the chromatin in a single experiment. He made 2D images of a 3D domain, so different in z-direction could not be measured. He showed that there were clusters of chromatin in the cell. However, is this really a fair conclusion if you have no idea of the clustering of chromatin in the z-direction.
By studying the clusters visualized, Prakash could research the chromatin complex morphology. He observed ring and rod like chromatin domains and displacement of active histone modification during stress. In mammalian cells, the chromatin complexes rearrange after stress. So, according to Prakash SMLM contributes to describe the spectrum of unknown chromatin domains between the nucleosome and chromosome level.
Prakash, however, did not convince me of the value of SMLM to the study of unknown chromatin domains. He compared the results of Histone labeling with his DNA staining assay, which was, I think, not fair. Moreover, his visualization of the chromatin complexes was in 2D without a verification that this 2D representation was valid. I think that a more validated study to chromatin complexes with this assay would have a better contribution to research on these complexes.