Understanding the cell cycle and cellular senescence is crucial in unraveling the mysteries of how cells divide and age. Cells grow, change and age while they fulfill their functions within our bodies, greatly in part due to the happenings of the cell cycle. A cell grows continuously and spends most of its time in interphase, which is subdivided into the G₁, S, G₂, and M phases. During these phases, the cell will replicate its DNA, duplicate its organelles, and eventually mitotically divide to produce an identical daughter cell. Throughout the cell cycle, there are certain checkpoints to ensure there are favorable internal and external conditions to the cell before committing to the extensive process of replication and division¹.

A cell may be delayed in a specialized resting state, known as G₀, if environmental factors are unfavorable. The amount of time spent in the G₀ rest phase depends on the cell type, function and resource availability and may be categorized as reversible or irreversible. A cell in a reversible state is said to be quiescent and may re-enter the cell cycle once activated. In an irreversible state the cell may either be differentiated or senescent. A differentiated cell would be a stem cell that has asymmetrically divided into a specialized cell that stays in a G₀ state while performing their functions indefinitely. Conversely, a senescent cell is permanently removed from the cell cycle and can no longer replicate and will remain in this state until the cell’s death². Considering this, cellular senescence is a subcategorization of the G₀ checkpoint in which the cell is removed from the cell cycle differing only in the state of permanence.

Cellular senescence is an occurrence defined by the cessation of cell division in response to a multitude of reasons, including telomere damage, oxidative stress, mitochondrial and DNA dysfunction^3,4. The depletion of stem cells and cellular senescence are part of the normal cell ageing process. Additionally, senescence serves as a cancer deterrent in that a cell that is expressing abnormal qualities is removed from the cell cycle at the G₀ checkpoint, is not allowed to duplicate and may undergo apoptosis⁴.

Shifting gears to DNA promoter regions, let’s explore how these regions influence gene expression in cells. A promoter region is defined as an upstream region of DNA that tells transcription factors where to bind and initiate the transcription of a certain gene to produce an RNA molecule⁵. A mutation or alteration in the sequence of a promoter region’s base pairs may lead to a cryptic promoter region. Cryptic promoters differ from regular DNA promoter regions in that they are hidden or unexpected sequences within the DNA that can mistakenly activate gene expression. On the other hand, regular DNA promoter regions are the usual sites where transcription factors bind to initiate the transcription of specific genes in a controlled and predictable manner⁶. Cryptic transcription differs from normal DNA transcription in that it involves the activation of genes by unregulated look-alike promoter sequences in the DNA, leading to gene expression in an abnormal or unintended manner that interferes with normal cellular processes⁷.

            The differences in transcription between proliferating/replicating cells and senescent cells are very interesting and were explored in the 2023 scientific paper, Spurious intragenic transcription is a feature of mammalian cellular senescence and tissue aging. In normal proliferating cells, the transcription start site begins right after the promoter region, where RNA polymerase binds. In this study it is demonstrated in figure 1a, that in senescent cells the percentage of RNA produced from the promoter region was around 3%. This is significantly less than in proliferating cells, with an RNA production from the promoter region at 13.5%. A greater percentage of RNA production in senescent cells was seen within the genetic sequence (also known a genic region) at 58.9% compared to proliferating cells with 45.3%⁸. This data demonstrates a clear difference in transcriptional activity between active proliferating cells and senescent cells, by means of the transcriptional start site.

            In addition to the change of transcription start sites in senescent cells, new sites also appeared in intron regions that were not present in proliferating cells. Introns are non-coding regions of DNA that are transcribed by RNA polymerase but are spliced out and not actively expressed in the final RNA product⁹. These peaks in transcriptional activity found in introns 8,6 and 4 of the genetic sequence, as seen in figures 1b-f, were not seen in the same introns of proliferating cells⁸. The appearance of new transcription start sites within introns of senescent cells is compelling, because this is not standard for proliferating cells. A transcriptional start site within an intron indicates that these are cryptic promoter regions.

            The aforementioned study also explored the differences in chromatin structure in relation to senescent cells. Chromatin is DNA compacted around histone proteins. The histone complex has two of each histone H2A, H2B, H3, and H4, that together form a histone octamer. There are tails off each histone, that are subject to modifications that make the complex either more or less transcriptionally active by altering how tightly the DNA is wound about the histone¹⁰. Adding an acetyl group to a histone tail is typically associated with active transcription, or a loosening of DNA and increase of chromatin accessibility. In the study, figure 2c depicts that when histone 3 of senescent cells are acetylated there is a major peak of transcriptional activity between -900 and 900 base pairs, where replicating cells had diminished activity comparatively. Figure 2d also demonstrates that senescent cells show more acetylation than replicative cells⁸. This finding shows that these alterations are transcriptionally activating within senescent cells, which had more activity within these transcription start site regions than replicating cells. This is another indicator of cryptic promoter regions.

            Continuing with the alterations of histone complexes, histone tails may also be methylated. Methylation of a histone tail can occur anywhere and typically add one to three methyl groups. Depending on where these alterations occur, single (mono-) methyl addition and three methyl additions (tri-) are associated with an increase in transcriptional activity¹¹. Figures 3 a and b of the study explore these changes between replicating and senescent cells in histone 3. The ratio of tri-methylated (me3) and mono-methylated (me1) regions determine if the region is a promoter or an enhancer. An enhancer region is a short region of DNA to which transcription factors bind and increase the likelihood that transcription of that gene will occur and control gene expression¹². In proliferating cells, the me1 to me3 ratio demonstrate that they normally behave as enhancers. While senescent cells showed a pattern of gaining me3 at the same sites while losing me1. This suggests that the sites were designated enhancers while actively replicating, and switch to a promoter state when the cell becomes senescent. It was also indicated in the study, that histone substitution played a role in enhancer to promoter conversions within senescent cells. This can be seen in figures 3f and g, where cryptic transcription start sites gained the histone H2A.Z, while there was a decrease in histone H3.3 in senescence⁸. The takeaway from this data indicates that replicating cells are normally enhancers, while senescent cell site undergo enhancer to promoter conversions.

References

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2. Wikipedia contributors. G0 phase 2024; https://en.wikipedia.org/wiki/G0_phase#:~:text=The%20G0%20phase%20describes,of%20as%20a%20resting%20phase.
3. Wikipedia contributors. Cellular senescence 2024; https://en.wikipedia.org/wiki/Cellular_senescence#cite_note-1.
4. McHugh, D., Gil, J. Senescence and aging: Causes, consequences, and therapeutic avenues. J Cell Biol. 2018; 217,1: 65-77
5. Segre, J. Promoter. 2024; National Human Genome Research Institute, https://www.genome.gov/genetics-glossary/Promoter#:~:text=Definition&text=A%20promoter%2C%20as%20related%20to,molecule%20(such%20as%20mRNA).
6. Pattenden, S., Gogol, M., Workman, J. Features of Cryptic Promoters and Their Varied Reliance on Bromodomain-Containing Factors. 2010; National Library of Medicine, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2944879/.
7. Shalchi, H. Cryptic transcription in mammalian stem cells linked to aging. 2021; Baylor College of Medicine, https://www.bcm.edu/news/cryptic-transcription-in-mammalian-stem-cells-linked-to-aging.
8. Sen, P., Donahue, G., Li, C., Egervari, G., Yang, N., Lan, Y., Robertson, N., Shah, P.P., Kerkhoven, E., Schultz, D.C., et al. (2023). Spurious intragenic transcription is a feature of mammalian cellular senescence and tissue aging. Nat Aging 3, 402-417. 10.1038/43587-023-00384-3.
9. Wikipedia contributors. Intron 2024; https://en.wikipedia.org/wiki/Intron
10. Annunziato, A. DNA Packaging: Nucleosomes and Chromatin. 2008; Nature Education, https://www.nature.com/scitable/topicpage/dna-packaging-nucleosomes-and-chromatin-310/
11. Neidhart M. DNA Methylation. Academic Press. 2016; 1: pages 1-8
12. Wikipedia contributors. Enhancer (genetics) 2024; https://en.wikipedia.org/wiki/Enhancer_(genetics)