UC Berkeley

Transcriptional silencing in the budding yeast Saccharomyces cerevisiae occurs at the cryptic mating-type loci HML and HMR on chromosome III and at all 32 telomeres. Transcriptional silencing occurs through the formation of a repressive chromatin structure, featuring nucleosome compaction and removal of active chromatin marks. These features are the result of the activity of the Silent Information Regulator (SIR) complex, made up of Sir2, Sir3, and Sir4. Sir2, founding member of the wide spread class of protein deacetylases known as sirtuins, deacetylates histone tails, whereas Sir3 and Sir4 serve structural roles, binding to histones and compacting chromatin.

The formation of silent chromatin by the SIR complex conceptually involves two steps: recruitment and spreading. Recruitment, also referred to as nucleation, occurs at DNA sequence elements called silencers that are present at telomeres and flank HML and HMR. The E and I silencers that flank both HML and HMR contain different combinations of binding sites for the Origin Recognition Complex (ORC) and the general transcription factors Rap1 and Abf1. Silencers at telomeres are less well characterized, but include an array of Rap1 binding sites in telomere repeats and possibly ORC/Abf1 sites further into the chromosome. Fully silent chromatin displays SIR complex binding beyond recruitment sites for multiple kilobases. The difference in SIR complex occupancy between nucleation and full silencing occurs through an ill-defined process referred to as ‘spreading’.

To date, most studies on spreading of the SIR complex have focused on telomeres, sometimes only one, and relied on low-resolution ChIP-PCR, inducible systems that resulted in massive overexpression of Sir3, or both. These studies defined to a limited resolution positions of SIR complex binding and occupancy, and provided a foundation for genome-wide studies with higher temporal and spatial resolution. My work further characterized and distinguished the two processes of recruitment and spread at telomeres as well as HML and HMR. I accomplished this by developing a new method for tracing the history and trajectory of SIR complex binding: measuring DNA methylation by long-read nanopore sequencing of DNA from cells expressing a fusion protein between Sir3 and an N6-methyladenosine methyltransferase, M.EcoGII.

The fusion protein Sir3-M.EcoGII strongly and specifically methylated HML, HMR, and telomeres and was able to detect transient or low-affinity Sir3-chromatin interactions better than ChIP-seq. This new method allowed me to characterize the occupancy of a SIR3 allele encoding a protein deficient in binding to nucleosomes, sir3-bah∆. The sir3-bah∆-M.EcoGII fusion protein methylated DNA only at recruitment sites, clearly providing evidence that recruitment and spread of Sir3 were separable processes and that the interaction between Sir3 and nucleosomes was not required for recruitment but was required for spread. I also tested prior claims that overexpression of SIR3 results in binding of the protein at even longer distances from recruitment sites. The overexpression of SIR3-M.ECOGII, with few exceptions, did not extend regions of DNA methylation.

I also defined the dynamics of Sir3 spreading during silencing establishment and how its occupancy related to transcriptional silencing of HML and HMR. A fusion between sir3-8–a temperature sensitive allele of SIR3–and M.ECOGII allowed for regulated induction of DNA methylation without straying above the endogenous level of SIR3 expression. Over the course of about one cell cycle, methylation appeared only at the E and I silencers and the promoters of HML and HMR, demonstrating recruitment. Despite a lack of Sir3 occupancy between these recruitment sites, repression of transcription occurred early in the time course, suggesting that the early stages of silencing did not require Sir3 occupancy across the entire locus.

Once silent chromatin is established, it must be faithfully inherited through the disruptive process of DNA replication. Certain point mutations in PCNA (POL30), the processivity clamp for DNA polymerase at replication forks, result in loss of transcriptional silencing at HML and HMR. I used classical genetics to study three of these alleles, pol30-6, pol30-8, and pol30-79, in more detail. All three alleles disrupted silencing only in actively-cycling cells, and the disruption in silencing was only transient, suggesting that the inheritance of silent chromatin through cell division was not as robust as in wild-type cells. All three alleles of POL30 destabilized silencing through disrupting the function of histone chaperones, highlighting the importance of histone trafficking at the replication fork for the stability of transcriptional silencing.