Our cells are usually frantically working to sustain life. Part of this work includes dividing, a process that creates more cells to support our growth when we are children or to replace old, worn-out cells when we are adults. Stem cells in our body receive cues from their environment to tell them when to divide or warn them when there are not enough resources to support division. When the body has enough of one type of cell or stem cells face stress, they can enter a sleepy state known as quiescence. During quiescence, cells are still alive and performing some of their biological functions, but they are not actively dividing. To make sure quiescent cell do not divide, the arrangement of the proteins and DNA, or chromatin, in the cell becomes more compact. Because DNA contains all the instructions for cell division, its compaction makes these instructions inaccessible. This allows cells to remain quiescent until they receive instructions to divide or until their stressors go away. Proper quiescence entry and exit ensures that cells stay healthy for as long as possible, and defects in this process can lead to diseases like cancer. Much work has already been done exploring the chromatin changes that accompany quiescence, but the precise mechanisms underlying these changes are still largely undefined.
To further investigate these mechanisms, Drs. Christine Cucinotta and Toshio Tsukiyama in the Basic Sciences Division at Fred Hutch analyzed how ribosomal DNA (rDNA) structure changes during quiescence. rDNA is necessary to synthesize the protein-making machines in cells, and there are dozens of rDNA repeat genes clustered together on the same chromosome. These genes are essential, and any aberrations or deletions could have dramatic consequences for cells. Defining how these repeats are organized during normal cell cycling and quiescence is crucial to understanding how quiescent cells preserve their genomes.
To start disentangling these structures, co-author Rachel Dell used super-resolution microscopy techniques to take pictures of the rDNA during the cell cycle and quiescence. Super-resolution microscopy lets researchers take pictures of cell structures in near-native states, making it an ideal tool to look for large-scale remodeling of DNA organization. The group found that rDNA forms distinct condensed-loop structures during quiescence. This implies that the DNA is less accessible to proteins that would normally transcribe it. To confirm these results, the group also analyzed Micro-C data. Micro-C lets researchers analyze which DNA regions are in contact with one another. Cucinotta found that rDNA was in contact with more distant regions of the genome during quiescence when compared to active cell cycling phases. Because these regions are typically far away, this implies that there is more compaction around the rDNA during quiescence. This novel DNA structure and compaction data raises questions about how the cells unravel their chromatin after exiting quiescence. “I don’t know, at this point, which…dots on the loop will become reactivated or stay repressed coming out of quiescence,” says Cucinotta, lead author of the study. Understanding how cells unravel their rDNA when they “wake up” from quiescence could lead to new tools to ensure this process happens correctly with our stem cells throughout life.
After discovering these large-scale chromatin changes during quiescence, the group wanted to know what mechanism underlies the changes. The RENT complex is known to compact rDNA. To see if the RENT complex could be responsible for rDNA silencing during quiescence, they investigated Sir2, one of the main members of the RENT complex. They found that Sir2 is indeed bound to rDNA during quiescence. When they deleted the SIR2 gene, they found that the cells were unable to form the condensed-loop chromatin structures previously observed in quiescent rDNA. Instead, their chromatin collapsed into a small dot structure, highlighting the importance of Sir2 to chromatin structure formation during quiescence.
SIR2 deletion also resulted in reduced long-term survival of the cells and delayed exit from quiescence. Despite these striking phenotypes, how Sir2 impacted DNA accessibility to proteins was unclear. The group found that in cells lacking SIR2, RNA polymerase II was mislocalized to the rDNA genes throughout quiescence and into the beginning stages of the cell cycle. Taken together, these results underscore the importance of Sir2 in cell cycling, chromatin structure, and transcriptional regulation.
This work advances the field’s understanding of large-scale chromatin remodeling events during the cell cycle. By unveiling how these basic biological processes are regulated, Cucinotta and Tsukiyama hope to define a framework for how chromatin is regulated by the cell cycle.
This work was supported by grants from the National Institutes of Health.
Fred Hutch/University of Washington/Seattle Children's Cancer Consortium Member Dr. Toshio Tsukiyama contributed to this work.
Cucinotta C, Dell R, Alavattam K, Tsukiyama T. 2024. Sir2 is required for the quiescence-specific condensed three-dimensional chromatin structure of rDNA. BioRxiv. 2024.12.12.628092.
Kelsey Woodruff is a PhD candidate in the Termini Lab at Fred Hutch Cancer Center. She studies how acute myeloid leukemia cells remodel the sugars on their membranes to reprogram cancer cell signaling. Originally from Indiana, she holds a bachelor's degree in Biochemistry from Ball State University. Outside of lab, you can find her crocheting and enjoying the Seattle summers.
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