States of G0 in development, cancer and aging

Our research projects explore four related questions about the molecular control of the cell cycle machinery during the decision to enter or maintain quiescence or G0. Each of these projects illuminates a new aspect of cell cycle control and capitalizes on our unique model system-based approach, complemented by powerful new assays and tools. Ultimately this knowledge will allow us to generate tools for manipulating and controlling cellular quiescence, which become improperly regulated in both cancer and aging.

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  1. Modulators of the proliferation-quiescence decision

The PP2A phosphatase complex is a well-known tumor suppressor, yet its molecular targets that prevent tumorigenesis remain unknown. We uncovered a new role for PP2A in controlling the process of cell cycle exit upon terminal differentiation, which may underlie its critical function in tumor suppression. Loss of PP2A function leads to extra cell divisions in Drosophila (fruit fly) wings and eyes, at a time in development when all the cells are normally quiescent.  The PP2A complex has over 200 known targets, where target specificity is determined by which regulatory subunit is bound in the complex. By systematically testing each regulatory subunit in Drosophila, we discovered that the B56 subunit (termed WDB in Drosophila) is the regulatory subunit required for this function of PP2A at cell cycle exit. We found that PP2A-B56 dephosphorylates a residue on the T-Loop of Cdk2, required for high Cdk2 activity (a mechanism also conserved in mammalian cells), and showed that PP2A/B56 can inhibit Cdk2 activity at the end of the cell cycle to promote proper cell cycle exit (Sun and Buttitta, 2015).

We have recently extended our studies to mammalian cell lines, using single-cell tracking approaches with new fluorescent in vivo cell cycle sensors to monitor and quantify the proliferation-quiescence decision in real-time, under a variety of conditions.  Our preliminary results indicate that PP2A activity contributes to this decision.

The proliferation-quiescence decision is a critical issue for the prognosis of cancers with high rates of recurrence, such as breast and prostate cancer. In these cancers it is thought that a small population of malignant cells can somehow remain quiescent and lie dormant for months or even years, evading chemotherapy, only to later ‘seed’ a more aggressive, recurrent cancer. In collaboration with Russell Taichman’s group at University of Michigan School of Dentistry, we have generated prostate cancer cell lines with fluorescent in vivo cell cycle sensors to monitor G0, and examine how a signaling molecule associated with tumor dormancy, Gas6, promotes cell cycle arrest in culture (Jung et al., 2016).

  1. Silencing cell cycle genes – chromatin changes during G0

The cell cycle has an intimate, reciprocal relationship with the organization of DNA and its associated proteins, collectively termed chromatin, in the nucleus. Both the nuclear envelope and the chromatin must undergo robust disassembly and re-assembly each cell cycle during DNA replication and mitosis. Chromatin binding proteins and chromatin modifications influence the expression of critical cell cycle regulators and control the accessibility of DNA for replication and DNA damage repair. Thus the cell cycle influences chromatin, and chromatin in turn affects the expression of genes that are critical for the cell cycle, as discussed in our review (Ma et al., 2015).

Most studies of cell cycle gene expression use actively cycling cells or cells that can readily re-enter the cell cycle. A more relevant question for most cells though, is how to synchronously and robustly silence cell cycle genes when cells enter or maintain a prolonged G0 state that is stable over an organism’s lifetime, such as in terminally differentiated muscle or neurons. We hypothesized that the G0 state must somehow alter the ability of transcription factors to access certain cell cycle genes to turn them on. Transcription factors bind to specific areas of the DNA known as regulatory elements to turn on or off transcription of genes at the right places and times. Because the DNA in multicellular organisms is packaged with histone proteins and folded into units called nucleosomes, when a gene is turned on, the regulatory element must be accessible for the transcription factor to bind. If a regulatory region is occupied by nucleosomes, the region is inaccessible and the nucleosomes will need to be moved for activation, creating an additional barrier to gene expression. This would be a potent way to silence the cell cycle transcriptional oscillator and an effective anti-cancer mechanism, as it would block accidental re-activation of cell cycle genes in cells that should be quiescent.

Through collaboration with Daniel McKay at UNC Chapel Hill, we performed a technique called FAIRE-seq on Drosophila wings during specific developmental timepoints as the cells transition naturally from a proliferating state to a stable, long-term G0 state. Using this approach in parallel with RNA-seq to measure gene expression, we identified thousands of changes in active regulatory element accessibility across the genome as wing cells enter and maintain quiescence. These data are providing a wealth of information about developmental changes in gene regulatory networks as cells mature and differentiate in the Drosophila wing. Most relevant to the cell cycle, we found that regulatory sequences for just two critical cell cycle genes become permanently inaccessible when cells maintain a prolonged G0 state. These two genes, Cyclin E, and cdc25c, are essential cell cycle regulators that are normally activated by the transcriptional oscillator in proliferating cells. Our results suggest these regulators become dominantly silenced by nucleosome occupancy at regulatory elements to block their re-activation in late G0 (Fig.3). (Ma et al., PLOS Biology 2019).

Since regulatory elements controlling the expression of CyclinE and cdc25c become inaccessible when cells are in G0, we hypothesize that molecular complex(es) direct nucleosomes to occupy these sequences after cell cycle exit. In a genetic screen we identified two such complexes that are required for proper quiescence, the Brahma complex and the NuRD complex. We are now examining whether these complexes are responsible for the changes in accessibility we observe at CyclinE and cdc25c when cells enter G0. Consistent with this idea, NuRD localizes to certain regulatory regions of the cdc25c gene that become inaccessible (Fig.3). Most importantly, we want to determine how and why these complexes become targeted to these specific DNA regions during G0, but not during normal proliferation.

  1. Developmental signals that promote quiescence

For most tissues in Drosophila, the switch from a proliferative to a quiescent state occurs during metamorphosis, which is driven by pulses of the fly steroid hormone ecdysone. Ecdysone signals through a nuclear hormone receptor signaling pathway, which is most closely related to the retinoic acid signaling pathway in vertebrates. Importantly, retinoic acid signaling also triggers developmental changes, cell differentiation and even cell cycle exit in certain human cell types, but it is unclear how hormonal signaling influences the cell cycle to promote quiescence in specific cell types.

Ecdysone exposure causes cell cycle arrest in Drosophila cells in culture, although the mechanism of arrest and its relationship to cell cycle exit in vivo has also been a long-standing mystery. We found that ecdysone induces the expression of a transcription factor called Broad, which in turn binds to the regulatory sequences of cdc25c to inhibit its expression. Since cdc25c is essential to promote mitotic Cyclin/cdk activity and cell proliferation, its repression by Broad inhibits the cell cycle in culture, as well as during metamorphosis when Drosophila wing cells enter into a non-proliferating state (Fig.4).

Using our data on DNA accessibility, we have been able to reconstruct the timing of accessibility for the binding of Broad to repress cell cycle genes in the Drosophila wing, as well as other hormone-induced transcription factors (Guo et al., Biology Open 2016). We are now interested in investigating how the multiple pulses of hormones during metamorphosis contribute to increasing the stability of the cell cycle arrest and how this is coordinated with the program of wing terminal differentiation.

Developmental regulation of the cell cycle in the Drosophila Accessory Gland (prostate):

More recently we have extended this project to examine cell cycle regulation in the adult male Drosophila accessory gland, which performs functions analogous to the human prostate. Cells in the accessory gland are polyploid and bi-nucleate, as a result of sequential special types of cell cycles with incomplete cytokinesis and mitoses. These are often called “variant” cell cycles to distinguish them from the more common “canonical” cell cycles that result in full mitosis and cytokinesis and lead to formation of two separate cells.

We recently found that cells in the adult accessory gland undergo additional variant cell cycles in the adult that are highly regulated under normal physiological conditions and that can be induced as a response to tissue damage (Box et al., Biorxiv). We are interested in understanding how hormonal signals that change with age, or in response to damage, affect the cell cycle in the fly prostate.

  1. Cell cycle exit becomes compromised in the aged brain

What happens in non-dividing tissues that accumulate cell damage or loss when stem cells are not available or have been depleted during aging? In examples such as skin, liver and cornea, cells that are normally non-dividing and quiescent can fuse or re-enter the cell cycle and begin replicating their DNA in response to cell loss. This process leads to a larger than normal number of chromosome copies, a state termed hyperploidy, which is also associated with cancer.

Abnormal cell cycle re-entry by neurons in the brain may underlie age-related cognitive decline and neurodegeneration. Patients with neurodegeneration exhibit hyperploid neurons, and gene expression analysis of aged brains has shown aberrant cell cycle gene reactivation in organisms ranging from flies to humans. We recently discovered that under normal physiological conditions the aging adult fly brain exhibits age-associated cell cycle re-entry, making it an excellent model system to study this process. Our work thus far indicates that cell cycle re-entry occurs in multiple cell types, increases with age and can cause neurodegenerative phenotypes. We are now working to address questions such as: What causes cell cycle re-entry in the aging brain? How does cell cycle re-entry impact neurodegeneration? How does manipulation of re-entry impact age-associated decline?

We have developed genetic tools to force cell cycle re-entry of quiescent neurons and glia in the developing and adult Drosophila brain. Forcing re-entry leads to defects suggestive of neurodegeneration, and we are now in a position to observe what happens to neurons and glia that re-enter the cell cycle. We also use simple behavioral assays to examine the consequences of re-entry in specific cell types.