Germ cells can be thought of as the guardians of the genome, utilizing various mechanisms to ensure their chromatin is structured to optimally maintain genome integrity and cell fate. The model organism C. elegans provides a means to investigate precisely how these chromatin structures are organized to achieve this. In a collaborative effort, research in the labs of Nobel Laureate Andrew Fire from Stanford University, USA, Sam Guoping Gu from Rutgers, the State University of New Jersey, USA, and James McGhee from the University of Calgary, Canada, reveals how endogenous nuclease activity in C. elegans oocytes shape a particularly unique structure in its chromatin. Guoping discusses their surprising results, published in a study in Epigenetics & Chromatin, and explains how this builds on what was previously known.
Why did you decide to study chromatin organization in C. elegans oocytes?
C. elegans has been a valuable system to study germline chromatin at various levels. For example, phenotypic analysis has been performed for a large number of genes with functions in germline chromatin, and optical analysis has revealed in a great detail the dynamic changes of chromatin structures during germline development. One crucial piece of information that was missing in this field is a high-resolution map of germline chromatin organization. We decided to start with C. elegans oocytes to accomplish this goal because we can get lots of pure samples.
What does your article set out to investigate and why?
A previous study by Andrew Fire and colleagues discovered that part of the C. elegans genome exhibits a strong bias toward a 10-nucleotide periodic occurrence of A(n)/T(n) sequences (Genetics. 2006 Jul;173(3):1259-73). In our most recent work, we decided to investigate whether DNA with this periodic signal is associated with a distinct chromatin structure in vivo. Studies from multiple species have shown that highly positioned nucleosomes tend to be associated with periodic DNA sequences. The periodic A(n)/T(n) signal in the C. elegans genome often continues over a long segment of DNA (>500 bp), which is unusual compared to the same signal found in other species. In addition, this periodic A(n)/T(n) signal is not randomly distributed in the C. elegans genome, but highly enriched in germline-expressed genes. We are very interested in identifying the molecular basis for the connection between periodic A(n)/T(n) signals and germline gene expression. It was clear to us from the start that having a high-resolution map of germline chromatin organization is essential to test various hypotheses that we had and this approach turned out to be quite productive in the end.
Could you describe what was surprising about your analysis?
There were a couple of surprises. The first one is the endogenous nuclease activity activated in the unfertilized C. elegans oocytes. As a result of this nuclease activity, the chromatin becomes fragmented. The second one is the large-scale chromatin organization in which long segments of DNA (>500 bp) are consistently organized on a surface that constrains accessibility to one helical face.
How do your findings build upon what was previously known about germ cell chromatin organization?
The germline has a remarkable ability to silence the part of the genomic DNA that is not needed for germline activities. Various silencing mechanisms have evolved to safeguard the genome integrity across species. In the mean time, genes that are essential for germline identity and early embryonic development must be actively transcribed at certain stages during germline development. How do these genes escape the global silencing activity in the germline? The specification of germline gene expression turns out to be highly complex. Promoters only play a very limited role in this case. Gene copy number and non-coding sequences such as introns and UTRs have been shown to regulate germline gene expression. Based on these previous studies, we were quite open to the possibility that the long-range periodic A(n)/T(n) sequences may play important roles in specifying germline gene expression.
What are the controversies surrounding your findings?
We initially used a standard assay to map nucleosome positioning by performing micrococcal nuclease digestion of the oocyte chromatin. In a control experiment, we were surprised to discover that unfertilized C. elegans oocytes eventually activate an endogenous nuclease activity, which fortuitously probes the chromatin structure in its native environment. However, the presence of the endogenous nuclease activity raised a valid question of whether the sample used in our study can faithfully indicate normal germline chromatin structures. To address this question, we performed a number of control analyses to demonstrate the oocyte chromatin used in our study still maintains several germline characteristics even though these cells have adopted a terminal fate.
What sort of impact will this work have on other fields?
Our work into long-range periodic chromatin organization provides a paradigm to study an uncharted area in which genetic information, DNA biophysical properties, chromatin structures, and germline biology are closely connected. Therefore our work has a broad impact on all of the related fields.
What’s next for your research?
Our current working hypothesis is that the long-range periodic A(n)/T(n) sequences in germline-expressed genes promote a highly constrained DNA packaging mechanism in the oocyte chromatin. This unique chromatin structure may represent a novel mechanism to specify germline gene expression. We plan to test various aspects of this model. For example, is this long-range chromatin organization a mechanism to resist the spread of heterochromatin? To what extend do periodic A(n)/T(n) sequences contribute to the observed long-range constraints in chromatin? What are the protein compositions of this type of chromatin structure? Does it require any special core histone or linker histone?
Your article was transferred to Epigenetics & Chromatin from a different publisher, could you comment on how you found the transfer process?
The mechanics of transfer were very helpful. I would certainly be open to engaging in such process in the future. By considering the previous reviews, Epigenetics & Chromatin were able to turn around a decision quickly. I just started my new lab when this work was submitted. So this quick turn around was tremendously helpful for me. Also, since different journals have different scopes and emphasis, it really makes a lot of sense to ’recycle’ the reviews from a previous submission process. I think this transfer process can drastically reduce the peer review effort for the scientific community.