The VCSD project is in the write-up phase prior to submission to a journal. The intended journal is eLife.
VCSD aimed to utilize two-color, three-dimensional (3D) Stochastic Optical Reconstruction Microscopy (STORM) to visualize chromatin structure (DNA and associated histone proteins) within intact nuclei of human cells.
Therein, I have successfully identified a methodology enabling the co-imaging of both DNA and histone proteins using stochastic optical reconstruction microscopy (STORM) together with DNA-PAINT super-resolution methodologies. In this fashion, I have overcome the primary obstacle impeding my project for the past 3 years.
It is well established in the field of super-resolution microscopy that the best performing STORM dyes are Alexa 647 and Cy5. Previously using Alexa 647 to label histones, the Lakadamyali lab established that histones form clusters within the nucleus and that these clusters undergo reorganization in nuclei treated with trichostatin A (TSA), which causes histone hyperacetylation. I have found, firstly, that the histone structure within nuclei was unreliable when using dyes other than Alexa 647 including Cy3b, Alexa 568, Atto 488 and Alexa 750. STORM-dye reliability was determined by the ability of STORM data analysis to reveal the previously established histone reorganizations at the nanoscale. In lieu of STORM, I found that the DNA-PAINT methodology for protein labeling and super-resolution imaging successfully recovered the histone reorganization previously identified in the hyperacetylated nuclei. I have established that DNA can be imaged via direct-STORM (dSTORM) by first incorporating the artificial nucleotide EdC (5-Ethynyl-2′-deoxycytidine) into the cellular genome during DNA replication, which contains a click-chemistry compatible alkyne linker moiety. Upon performing copper-mediated click chemistry between the alkyne and Alexa 647 azide, the DNA becomes directly labeled with the optimal STORM-compatible dye. I then combined the DNA-PAINT methodology with traditional dSTORM to co-image histone proteins and DNA, respectively, in individual nuclei.
Super-resolution imaging of both DNA and histones in 3D, together, as described above is permitting novel quantification of chromatin structure with 20nm lateral and 50nm axial resolution. The Lakadamyali lab previously established that histones decompact in hyperacetylated nuclei to form smaller clusters. Now we visualize DNA decompaction and reveal hyperacetylated DNA to be globally less-dense, with quantification providing an area per localization metric to describe the different levels of compaction between heterochromatin in control nuclei and the chemically-induced euchromatin in TSA treated nuclei. Co-analysis of the DNA and histone images are revealing two-levels of DNA compaction in the area surrounding histone clusters. The first level occurs within a radius of 50nm around a histone cluster and is a regime where DNA density does not change upon hyperacetylation. The second level occurs within a range of 50-70nm from the center of a histone cluster wherein hyperacetylation causes a loss a DNA compaction. Thus, we are concluding that the DNA lying in a range of 50-70nm around a cluster is compacted by neighboring histone-histone interactions that are disrupted by the hyperacetylated state.
