In Saccharomyces cerevisiae, the telomeres and rDNA repeats are repetitive silent chromatin domains that are tightly regulated to maintain silencing and genome stability. Disruption of the Cohibin complex, which maintains rDNA silencing and stability, also abrogates telomere localization and silencing. Cohibin-deficient cells have decreased Sir2 localization at telomeres, and restoring telomeric Sir2 concentrations rescues the telomeric defects observed in Cohibin-deficient cells. Genetic and molecular interactions suggest that Cohibin clusters telomeres to the nuclear envelope by binding inner nuclear membrane proteins. Futhermore, telomeric and rDNA sequences can form G-quadruplex structures. G-quadruplexes are non-canonical DNA structures that have been linked to processes affecting chromosome stability. Disruption of the G-quadruplex stabilizing protein Stm1, which also interacts with Cohibin, increases rDNA stability without affecting silent chromatin formation. In all, our findings have led to the discovery of new processes involved in the maintenance of repetitive silent chromatin domains that may be conserved across eukaryotes.

In Saccharomyces cerevisiae, the telomeres and rDNA repeats are repetitive silent chromatin domains that are tightly regulated to maintain silencing and genome stability. Disruption of the Cohibin complex, which maintains rDNA silencing and stability, also abrogates telomere localization and silencing. Cohibin-deficient cells have decreased Sir2 localization at telomeres, and restoring telomeric Sir2 concentrations rescues the telomeric defects observed in Cohibin-deficient cells. Genetic and molecular interactions suggest that Cohibin clusters telomeres to the nuclear envelope by binding inner nuclear membrane proteins. Futhermore, telomeric and rDNA sequences can form G-quadruplex structures. G-quadruplexes are non-canonical DNA structures that have been linked to processes affecting chromosome stability. Disruption of the G-quadruplex stabilizing protein Stm1, which also interacts with Cohibin, increases rDNA stability without affecting silent chromatin formation. In all, our findings have led to the discovery of new processes involved in the maintenance of repetitive silent chromatin domains that may be conserved across eukaryotes.

In eukaryotic cells, genomic DNA exists as chromatin in association with histone octamers called nucleosomes, and various other chromatin proteins. Chromatin structure varies along the chromosome and this influences the state of gene expression. Based on such variations in structure and gene expression, chromatin can be broadly classified into euchromatin (transcriptionally active) and heterochromatin (silent or transcriptionally repressed). In the budding yeast, Saccharomyces cerevisiae, there are four canonical transcriptionally silent regions, namely, the HMR, the HML (cryptic mating loci), the telomeres and the RDN1. Silencing at the HM loci and the telomeres is very well characterized. The repressive structure at the HMR spans around 3.5 Kb and extends between the two silencers E and I. It is well established that silencing in HMR is due to a specialized chromatin organization brought about by Orc1p, Rap1p, Abf1p and Sir proteins. Following recruitment, the Sir proteins spread along the DNA to form a repressive chromatin domain believed to arise from the deacetylation of amino-terminal tails of histones H3 and H4 by Sir2p (an NAD dependent deacetylase) and the interaction of Sir3p and Sir4p with the histones. The bi-directional spreading of silencing at HMR is restricted by barrier or boundary elements that flank the silencers. A tRNAThr gene in the right boundary of HMR acts as a strong barrier. Mutations in the promoter of this tRNA gene (tDNA) or in RNA polymerase III subunits/ transcription factors weaken the barrier activity of this tDNA. The barrier activity of this tDNA is also dependent on histone acetyltransferases like Sas2p and Gcn5p. Silencing in HML is uniformly high between the silencers E and I and falls sharply outside I. Recently, barriers to HML silencing have been discovered. A 0.71Kb sequence near E, which maps to the upstream activating sequence of YCL069W, acts as a robust barrier to spread of HML silencing. This is effectively the left boundary of silent HML. The right boundary maps to the promoter of CHA1 gene though silencing is believed to terminate at HML-I. An unusual form of silencing occurs at the RDN1, which contains 100-150 copies of tandemly repeated rRNA genes. Some RNA polymerase II transcribed genes integrated within the array are silenced by a Sir2p dependent mechanism whereas genes driven by RNA polymerases III and I are transcriptionally active. Though all the three forms of silencing (RDN1, HM and telomere) require Sir2p, RDN1 silencing differs from the others in its relative strength and factors responsible for repression. Several trans-acting factors required for RDN1 silencing are known. However, it is still unclear as to what limits the spread of RDN1 heterochromatin into neighbouring essential genes. RDN1 silencing spreads unidirectionally in its left hand side sequence. However, the zone of RDN1 heterochromatin does not engulf the essential gene, ACS2, which is present ~3 kb away from NTS1. This implies that there is a mechanism by which rDNA heterochromatin is contained. There could be several ways by which this is accomplished. Firstly, the cell could be critically maintaining the levels of Sir2p, the protein required for silencing at all the four silenced loci, such that silencing in the left flank of RDN1 does not spread beyond 300 bp of NTS1 (Buck et al, 2002). There is a ~2.5 kb gene free intervening sequence between NTS1 of the rDNA array and the Ty1 LTR, in which interval Sir2p level could fall below the threshold mark required for causing repression. In fact Buck et al. have demonstrated that Sir2p is bound to upto 1.5 kb from the NTS1 in the left flank but there is no accompanying silencing of the mURA3 reporter in these regions (1200L and 2000L), suggesting that the level of Sir2p at these sequences could be lower than the threshold required for initiation of silencing. Secondly, there could be cis-acting boundary elements or barriers as in the case of HMR, which prevents the propagation of RDN1 silencing. The third option is that termination of RNA polymerase I transcription at the terminator sites automatically halts the spread of rDNA silencing since Buck et al. have demonstrated that progression of rDNA heterochromatin is dependent on RNA pol I transcription. This however, does not seem to be the case as deletion of both the terminator sites within NTS1 does not lengthen the zone of silencing. Finally, there could be an euchromatin organizing center further from the array, which creates an “open” chromatin configuration required to confront the Sir2p mediated condensed chromatin. The balance of these two opposing activities, much like that at the telomeres, could set up a molecular boundary for containing rDNA silent chromatin. We have attempted to identify whether there are any sequences in the unique left flank of RDN1 that can act as a heterochromatin barrier. Towards that end we tested four overlapping fragments from NTS1 of RDN1 to the promoter of ACS2 for boundary activity in a quantitative mating assay. We have found that of all the four fragments tested, only a 0.427 kb tRNAGln-Ty1 LTR fragment, which is present 2.4 Kb from the NTS1 acts as a robust barrier in this assay. Further mapping revealed that the barrier activity of this sequence resides in the tRNAGln gene and that its activity is orientation-independent. tDNAs are transcribed by RNA polymerase III from internal promoters termed Box A and Box B. It has been shown for the HMR-tRNAThr that the transcriptional potential of the tDNA is crucial for its barrier function. Mutations in genes encoding various subunits of the RNA polymerase III complex, or transitions in the conserved bases within Box B known to disrupt transcription complex assembly and subsequent transcription, abrogate the barrier activity of HMR-tRNAThr. Similarly, loss of transcriptional ability of the tRNAAla in the centromere of S. pombe also abolishes its barrier activity, enforcing the fact that RNA polymerase III transcription is a decisive factor for a tDNA barrier. Contrary to the above observations, we report that barrier activity of tRNAGln is very negligibly dependent on RNA polymerase III mediated transcription. Mating assays done with the RNA pol III mutants and promoter point mutants, G18C and C55G in boxes A and B respectively, underline the fact that for this tDNA barrier, RNA pol III driven transcription is dispensable. We also show by RT-PCR analysis that in the C55G tRNAGln mutant there is loss of transcription as expected, whereas other wild type copies of tRNAGln are transcribed. Studies with another tDNA barrier, TRT2-tRNAThr, yielded similar results, again emphasizing the point that transcription through the tDNA, which leads to nucleosome displacement and therefore barrier activity, may not be applicable for all tDNA barriers. Acetylation of amino terminal tails of histones is known to influence the epigenetic state of chromatin. Addition of acetyl moiety to histones H3 and H4 initiates a cascade of events, which involves recruitment of a host of other chromatin modifiers to the target sequence, and ultimately culminates in the formation of an euchromatin-favouring environment. As reported for the HMR right boundary, we find that barrier activity of tRNAGln depends on two histone acetyl transferase complexes, SAS-I (comprised of Sas2p, Sas4p and Sas5p) and SAGA (contains Gcn5p HAT). Contrary to the HMR boundary, the barrier activity of tRNAGln is independent of two other nucleoplasmic HATs, NuA3 (Sas3p being the HAT) and NuA4 (Esa1p is the HAT). Barrier function of TRT2-tRNAThr also depends on HATs. Therefore it appears that requirement of HATs for boundary activity is a conserved theme, albeit with differential effects at different barrier sequences. We next attempted to determine the function of tRNAGln in its natural location on chromosome XII. As mentioned earlier, RDN1 silencing spreads upto ~0.3 kb in its left flanking sequence. However, Sir2p occupancy has been observed till 1.5 kb although there is no silencing of reporter genes observed beyond 0.3 kb of NTS1. This lead us to speculate that there could be a boundary sequence in the left flank that stops silencing, or a euchromatin-organizing element, which counters the propagation of silencing by a long-range effect. Since over expression of Sir2p extends the domain of silencing from 0.3 kb to 2.0 kb and the tRNAGln is present at 2.3 kb from NTS1, it was a good candidate for a heterochromatin barrier/ euchromatiniser. However, deletion of tRNAGln does not affect the zone of RDN1 silencing as is evident from our cell viability assays (which is a measure of the expression of the essential gene ACS2 situated further to the left of tRNAGln). Deletion of SAS2 and GCN5, factors that are required for barrier activity of tRNAGln in mating assays, also have no effect on the extent of spreading of RDN1 silencing in normal or Sir2p over expression conditions. Together, these observations imply that in situ, tRNAGln does not act as a barrier or an element with long-range euchromatin inducing properties. It still remains unclear as to what contains RDN1 silencing. It is possible that the cell critically monitors the level of Sir2p in order to maintain boundaries of silencing at the rDNA locus. Telomeres also nucleate the formation of silenced domain which spreads along the subtelomeric region upto ~ 2Kb. The key players in the formation of telomeric heterochromation are the Sir proteins, Sir2p, Sir3p and Sir4p, Rap1p, yKu complex and ORC. Protein-protein interactions between the telosome and the subtelomeric repeat bound silencing proteins create a domain of core heterochromatin that spreads in the adjacent sequences. While Sir2p deacetylates H4K16, Sir3p interacts with the hypoacetylated histone tails and helps in the spreading of the repressive chromatin structure. As a result telomere proximal genes are silent whereas the ones further away are expressed. There is a gradient of acetylation of histone H4, with the hypoacetylated histones at the telomeric ends and the hyperacetylated ones distant from the telomere. Recently it has been shown that this gradient is maintained by the concerted and antagonistic actions of Sir2p and Sas2p. In a sas2Δ strain Sir3p spreads to ~15 kb in the subtelomeric regions and there is increase in the levels of hypoacetylated histones. Though the molecular mechanism by which telomeric silencing is restrained is beginning to be understood, it remains unanswered whether there are any cis-acting sequences, capable of recruiting euchromatin-inducing factors such as Sas2p, near the telomeres. We have identified a RNA polymerase II driven gene, AAD3, in the subtelomeric region of chromosome III that has robust anti-silencing activity. Deletion mapping revealed that only 0.381 kb in the 5′ portion of the gene (excluding the promoter) is sufficient for barrier activity and that this property is orientation-independent (henceforth referred to as TEL-B). The barrier acivity of TEL-B depends strongly on Sas2p and Esa1p but not on Gcn5p and Sas3p, and is independent of cohesin. Previous investigations have shown that acetylation of H4K16 by Sas2p at subtelomeric regions of chromosome VI leads to deposition of HTZ1 in the nucleosome and its subsequent acetylation by Esa1p of NuA4. All these events together are required to contain the onslaught of telomeric core heterochromatin on neighbouring active regions. Since barrier activity of TEL-B depends on Sas2p and Esa1p, it is possible that TEL-B has the potential to act as a bona fide barrier in situ in its endogenous context. Our hypothesis is further cemented by the observation that there is a physical association between Sas2p, the molecule at the top of the entire cascade of events, with TEL-B by yeast one hybrid analysis. Further experiments will shed light on the role of this sequence in its natural location. In summary, I have identified and characterized two different barrier sequences in S. cerevisiae. Not many barriers are known in budding yeast and there is extensive ongoing research dedicated to understand the mechanism(s) of barrier function. In chapter I of my thesis I present a review of current literature regarding silencing barriers in yeast and other systems. In chapter II I have outlined a detailed characterization of a tDNA barrier element, tRNAGln, present near the silenced rDNA array on chromosome XII. My work addresses the various models for barrier activity and their applicability to the tRNAGln barrier. I have also attempted to understand the role of this tDNA in its natural location on the chromosome with respect to limitation of RDN1 silencing. In chapter III I have described an intensive study of a RNA polymerase II transcribed gene, AAD3, present near the right telomere of chromosome III, which acts as a robust barrier to silencing. I have attempted to answer which mechanism(s) is/are operational at this sequence so as to endow it with barrier potential. My studies with the two barrier elements highlight novel trans-acting factors required for barrier function, differential and selective requirements of certain factors for different barriers, and provide a mechanistic view of the boundary activity of these sequences.

In Saccharomyces cerevisiae, telomeric and HM silencing requires the histone deacetylase Sir2 and the chromatin binding proteins Sir3 and Sir4, which interact to form the SIR complex. Silent chromatin formation begins with a nucleation step, followed by spreading of Sir proteins along chromatin. Overexpression of Sir3 extends silent chromatin domains, however the role of Sir protein interactions within silent chromatin extensions remains unknown. Here, we generated the Sir3 mutant, Sir3-4A, which cannot interact with Sir4 but is capable of forming silent chromatin extensions when overexpressed. Within extended silent domains, Sir2 and Sir4 enrichments are similar whether Sir3 or Sir3-4A is overexpressed, suggesting that silent chromatin extensions require Sir4 but not the interaction between Sir3 and Sir4. Tethering Sir3-4A at an HMR silencer cannot nucleate silencing in the absence of Sir3, suggesting that in addition to Sir3 recruitment, the Sir3-Sir4 interaction has at least one other function during silent chromatin nucleation.