One of the reviews of the back-to-back submission to Biophysical Journal of Contributions of Linker Histones and Histone H3 to Chromatin Structure: SFM Studies on Trypsinized Fibers and Linker Histone Tails and N-tails of Histone H3 Are Redundant: SFM Studies of Reconstituted Fibers to Biophysical Journal Comments from Reviewer No. 1. These two manuscripts have been submitted for joint "back-to-back" publication by the authors as they are closely related. I will therefore submit a single review applicable to both manuscripts. The studies described in these manuscripts represents a collaboration between two very well known laboratories: one in the field of chromatin structure; and one in applications of scanning force microscope(SFM), a structural tool in biology. The purpose of the work is to use scanning force microscopy in one of its more advanced applications to deduce important structural information about the three-dimensional structure of the extended chromatin fiber. Since this fiber is the extended or putatively unfolded form of the condensed or "30 nm" fiber typically thought to be associated with transcriptionally inactive chromatin, there is good reason to believe that this extended form is relevant during biological processes such as transcription that occur in the milieu of chromatin structure in actual cell nuclei. In particular, this work examines the contributions of the linker histone (H1/5) structural domains and the N-terminal tails of histone H3 to the extended fiber conformation. In the first paper, SFM imaging is conducted on fibers which have been subjected to progressive proteolysis by trypsin. This enzyme progressively cleaves the basic tails of linker and H3 histones. Because the cleavages of these two histones partially overlap, the second paper provides similar imaging on fibers reconstituted with both intact and the globular domain only of linker histone H5 onto linker histone-stripped fibers that contained either normal core histone octamers or octamers in which the basic tails of core histories H3 had been removed. Thus, the second set of results provide both new information and important controls for the first set. In both papers, the limitations of SFM imaging is supplemented with mathematical modeling in which the critical determinants of the extended fiber are the lengths of linker DNA and the entry-exit angles between successive linkers. The principal conclusion of the first paper is that fiber extension is primarily determined by the linker and H3 core histone basic tails. The second paper enables the effects of these two to be sorted out and the results demonstrate that they are evidently redundant in their effects upon fiber structure. Both manuscripts are very well written and clearly describe both the problem and its antecedent history and provide a novel and, at present, unique approach to understanding key questions that govern the adaptive roles of chromatin structure to dynamic biological processes. Thus, the question of chromatin fiber structure is one of utmost importance in understanding phenomena such as transcription, but it must be admitted that these and related questions have been argued for many years by people in the chromatin structure field. I suspect that the present papers will be somewhat controversial. This is especially so since the SFM imaging as amplified by molecular modeling clearly show less regularity in fiber structure than predicted by the models widely discussed in the past. Nevertheless, it seems to me that they provide a novel approach that leads to important conclusions about chromatin fiber structure that must be taken very seriously. Further, the conclusions drawn seem to flow inexorably from the experimental results reported. I have read both papers carefully and repeatedly, and can find no significant weaknesses in the experiments other that the basic limitation in resolution of the SFM technique itself. (One only wishes that the images could have been obtained under solution, at higher resolution and under conditions leading to less stress on the fibers form the scanning stylus itself; this would place far less burden upon associated molecular modeling.) Nevertheless, I think that these papers make a very important contribution to the chromatin structure field and need to be made available to the full scientific community. Biophysical Journal is an appropriate place to publish them.
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