Rap1-Mediated Chromatin And Gene Expression Changes At Senescence

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Doctor of Philosophy (PhD)
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Biochemistry & Molecular Biophysics
cellular senescence
pace of senescence
pioneer transcription factor
Cell Biology
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Song, Shufei

ABSTRACT RAP1-MEDIATED CHROMATIN AND GENE EXPRESSION CHANGES AT SENESCENCE The telomeric protein Rap1 has been extensively studied for its roles as a transcriptional activator and repressor. Indeed, in both yeast and mammals, Rap1 is known to bind throughout the genome to reorganize chromatin and regulate gene transcription. Previously, our lab published evidence that Rap1 plays important roles in cellular senescence. In telomerase-deficient S. cerevisiae, Rap1 relocalizes from telomeres and subtelomeres to new Rap1 target at senescence (NRTS). This leads to two types of histone loss: Rap1 lowers global histone levels by repressing histone gene transcription and it also results in local nucleosome displacement at the promoters of the activated NRTS. Here, I examine mechanisms of site-specific histone loss by presenting evidence that Rap1 can directly interact with histone tetramers H3/H4, and map this interaction to a three-amino-acid-patch within the DNA binding domain. Functional studies are performed in vivo using a mutant form of Rap1 with weakened histone interactions, and deficient promoter clearance as well as blunted gene activation is observed, indicating that direct Rap1-H3/H4 interactions are involved in nucleosome displacement. In addition, I explore histone chaperones and chromatin remodelers that may function as Rap1 co-activators at senescence, and found that the histone H3/H4 chaperone Asf1 is required for full Rap1-mediated nucleosome displacement and gene activation. The epigenetic mark H3K4me3 is similarly involved, though the exact details of how this crosstalk occur remain to be elucidated. Remarkably, blunting NRTS activation, at least in the cases of the Rap1 mutant and Asf1 deletion, do not affect the pace of senescence-related cell cycle arrest, suggesting that negative aspects of senescence-related gene expression changes can be uncoupled from the tumor-suppressive properties of cell senescence. Furthermore, as features of the yeast and mammalian Rap1 proteins are conserved, I also explore roles of hRAP1 in human fibroblast senescence. Genome-wide ChIP-seq in senescent IMR-90s and RNA-seq in fibroblasts overexpressing hRAP1 reveal important roles for hRAP1, including relocalization to and regulation of histone genes and senescence-related genes. Direct H3/H4 interactions are also conserved, though more mapping studies are needed to determine the exact interaction surface on mammalian RAP1. Together, these studies illuminate mechanisms of Rap1-mediated senescence changes.

F. Brad . Johnson
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