During replication, nucleosomes are disrupted immediately upstream of the replication fork and re-form downstream of the fork (reviewed in ^{ref. 3}). Furthermore, parental H3/H4 tetramers and H2A/H2B dimers are re-deposited at random on either daughter strand. Finally the gaps in the nucleosomal arrays on the daughter strands are filled in with newly synthesized, specifically acetylated H3/H4 and H2A/H2B dimers.⁴ The Hat1p complex acetylates newly synthesized histone H4 on K5 and K12,⁵ while the Rtt109 complex acetylates newly synthesized histone H3 on K9 and K56.⁶ This entire process of histone deposition during replication utilizes various histone chaperones such as Nap1, Asf1 and CAF1 which co-ordinate deposition with the replication machinery.^7,8

Studies on gene activation in yeast have identified some of the factors involved in the stable maintenance of genes in an active state. Transcription of the GAL genes occurs only in the presence of galactose following glucose depletion.¹³ For example, the GAL1 gene is maintained in a repressed state by the Mig1 and Gal80 repressors. While Mig1 binds the promoter of GAL1, Gal80 directly binds the transcriptional activator, Gal4 and prevents it from activating the GAL1 gene. Activation involves the release of Mig1 from the GAL1 promoter, the upregulation of Gal4 as well its release from Gal80 that is mediated by Gal3. The Gal1 protein is a close homolog of Gal3 and can substitute for Gal3 in dissociating Gal4 from Gal80. While the de novo induction of the GAL genes is a slow process, re-induction of the genes following a period of quiescence is extremely rapid demonstrating that the cell maintains a memory of the gene activity state for several generations.^14,15 This begs the obvious question of which factors mediate transcriptional memory. A series of experiments showed that the memory is directly related to the levels of Gal1 protein.¹⁶ As long as the cells have a reservoir of Gal1 in the cytoplasm, upon reactivation, the elevated levels of Gal1 protein can rapidly aid in the dissociation of Gal80 from Gal4 allowing Gal4 to recruit various chromatin remodelers and modifiers to activate the gene. Detailed molecular analyses of this process demonstrated that the active gene localizes to nuclear pores via interactions with pore protein Mlp1 and remains localized during quiescence and this localization is necessary for transcriptional memory. Rapid reinduction correlates with the persistence of the activator Gal4 at the promoter of the gene, and also requires the chromatin remodeler Swi/Snf, the histone variant Htz1p (which localizes at the promoters of genes) and the general transcription factor TFIIB.^14,15,17,18 These data suggest that while transcriptional memory is dependent upon the levels of the Gal1 protein, memory is critically dependent upon the ability of the promoter bound transcription factors to rapidly recruit cofactors and remodel the chromatin structure at the transcription start site enabling rapid reinitiation of transcription and these results are consistent with a large body of literature demonstrating that the peripheral localization of genes promotes efficient induction of transcription.¹⁹

The establishment of silencing requires passage through the G₂/M phase of the cell cycle but does not require DNA replication.²¹ While the replication-independent S-phase event necessary to establish the silent state is not fully clear, the Sir proteins are unable to stably bind to chromatin during the G₂ phase of the cell cycle and full silencing is only achieved in telophase concomitant with the degradation of cohesin subunits.²¹

While establishment of silencing requires passage through S-M, the silenced state once established is also disrupted in each cell cycle and must be faithfully re-established in every cell cycle following replication. The chromatin state after replication mimics euchromatin and therefore re-formation of the silenced chromatin is likely an active process. The proper deposition of nucleosomes during replication, removal of the acetyl marks by histones deacetylases and the even spacing of nucleosomes during chromatin maturation are all necessary for efficient inheritance of the silent state.^22,23

Most importantly, the inheritance of the silenced state also requires the silencers. Deletion of the silencer in cells progressing through the cell cycle leads to de-repression of the silent domain within a single generation,²⁴ indicating that an intact silencer is required for the efficient inheritance of the repressed chromatin structures following replication. The silencer appears to provide the genomic memory that promotes the reformation of the silent chromatin in the progeny.²⁵ Furthermore, mutations in the silencer bound proteins indicate redundancies in the silencer. Mutations in any one silencer bound protein leads to an increase in the probability of loss of silencing^26,27 presumably because the stability of the protein complex at the silencer is weakened and the probability of inheriting the repressed state is thus reduced. Thus silencers can be considered as elements that efficiently ensure epigenetic memory.

In addition to the silencers, it is likely that the Sir proteins are important for the efficient inheritance of the repressed state. SUM1-1 is a dominant mutation of the local repressor Sum1.²¹SUM1-1 is recruited to the silenced HMR domain by the silencer bound proteins and represses genes even in the absence of the Sir proteins.²¹ However, unlike Sir mediated repression, the SUM1-1 mediated repressed state is not stably inherited²⁸ suggesting that the silencers are not sufficient for inheritance and the Sir proteins either directly or indirectly also play a role.

Outside of the silenced HMR domain in S. cerevisiae is an actively transcribed tRNA^Thr gene. This gene acts as a chromatin insulator and restricts the spread of silencing.³² Insulation involves a direct competition between heterochromatin and the tRNA gene mediated chromatin remodeling/modification.³³ Detailed molecular analyses of tRNA mediated insulation have identified some unexpected links between replication coupled chromatin assembly, transcription factors that bind the tRNA gene and chromatin remodelers that are recruited to and required for the activation of tRNA genes.³⁴ The tRNA gene lacks nucleosomes and numerous proteins are required for the efficient formation and maintenance of the specialized chromatin structure at the tRNA^Thr insulator. Mutants in DNA polymerase ε (which is associated with leading strand DNA synthesis), the histone acetylase Rtt109 (which acetylates free histones prior to their deposition into chromatin) and the chromatin remodeler Rsc (which is recruited to tRNA genes and evicts nucleosomes) allow the tRNA specific transcription factors (TFIIIB and TFIIIC) to bind the gene. Most interestingly, the tRNA bound transcription factors and the acetylase Rtt109 are also required for the binding/recruitment of Rsc to the tRNA suggesting a self-reinforcing mechanism between the transcription factors and Rsc in potentiating this gene. Clustering of tRNA genes in the nucleus is a phenomenon that is conserved from yeast to humans. The clustering could aid in the process of inheritance of the gene state, providing a local environment rich in RNA pol III transcription factors and cofactors.