The covalent modifications to the histone 2A, 2B, 3, and 4 N-terminal tails that affect gene expression have been deemed the “Histone Code.” Mis-regulation of these signalling pathways is of great interest as are important in human disease. A variety of peptides containing post-translationally modified histone 3 and 4 sequences were read using a supramolecular sensor array approach, where two or three sensors gave a unique response for each analyte when compared to others. These sequences were chosen to determine what type of modifications could be read (phosphorylation, acetylation, methylation) and if this type of array would be suitable for reading analytes on which antibodies—the leading technology—typically perform poorly. It was found that three sensors, which operate in neutral aqueous solution, were able to discriminate 16 different histone analytes. Additionally, it was shown that this array could report simultaneously on both concentration and the identities of histone analytes. / Graduate

No description available.

<p>The accurate and timely replication of eukaryotic DNA during S-phase is of critical importance for the cell and for the inheritance of genetic information. Missteps in the replication program can activate cell cycle checkpoints or, worse, trigger the genomic instability and aneuploidy associated with diseases such as cancer. Eukaryotic DNA replication initiates asynchronously from hundreds to tens of thousands of replication origins spread across the genome. The origins are acted upon independently, but patterns emerge in the form of large-scale replication timing domains. Each of these origins must be localized, and the activation time determined by a system of signals that, though they have yet to be fully understood, are not dependent on the primary DNA sequence. This regulation of DNA replication has been shown to be extremely plastic, changing to fit the needs of cells in development or effected by replication stress. </p><p>We have investigated the role of chromatin in specifying the eukaryotic DNA replication program. Chromatin elements, including histone variants, histone modifications and nucleosome positioning, are an attractive candidate for DNA replication control, as they are not specified fully by sequence, and they can be modified to fit the unique needs of a cell without altering the DNA template. The origin recognition complex (ORC) specifies replication origin location by binding the DNA of origins. The <italic>S. cerevisiae</italic> ORC recognizes the ARS (autonomously replicating sequence) consensus sequence (ACS), but only a subset of potential genomic sites are bound, suggesting other chromosomal features influence ORC binding. Using high-throughput sequencing to map ORC binding and nucleosome positioning, we show that yeast origins are characterized by an asymmetric pattern of positioned nucleosomes flanking the ACS. The origin sequences are sufficient to maintain a nucleosome-free origin; however, ORC is required for the precise positioning of nucleosomes flanking the origin. These findings identify local nucleosomes as an important determinant for origin selection and function. Next, we describe the <italic>D. melanogaster</italic> replication program in the context of the chromatin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium. We find that while the cell lines exhibit similar replication programs, there are numerous cell line-specific differences that correlate with changes in the chromatin architecture. We identify chromatin features that are associated with replication timing, early origin usage, and ORC binding. Primary sequence, activating chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute in an additive manner to specify ORC-binding sites. We also generate accurate and predictive models from the chromatin data to describe origin usage and strength between cell lines. Multiple activating chromatin modifications contribute to the function and relative strength of replication origins, suggesting that the chromatin environment does not regulate origins of replication as a simple binary switch, but rather acts as a tunable rheostat to regulate replication initiation events. </p><p>Taken together our data and analyses imply that the chromatin contains sufficient information to direct the DNA replication program.</p> / Dissertation

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.

No description available.

Myeloid-derived suppressor cells (MDSCs) prolong sepsis by promoting immunosuppression. We reported that sepsis MDSC development requires long non-coding RNA Hotairm1 interactions with S100A9. Using a mouse model that simulates the immunobiology of sepsis, we find that histone demethylase KDM6A promotes Hotairm1 transcription by demethylating transcription repression H3K27me3 histone mark. We show that chemical targeting of KDM6A by GSK-J4 represses Hotairm1 transcription, which coincides with decreases in transcription activation H3K4me3 histone mark and transcription factor PU.1 binding to the Hotairm1 promoter. We further show that immunosuppressive IL-10 cytokine promotes KDM6A binding at the Hotairm1 promoter. IL-10 knockdown repletes H3K27me3 and reduces Hotairm1 transcription. GSK-J4 treatment also relocalizes nuclear S100A9 protein to the cytosol. To support translation to human sepsis, we demonstrate that inhibiting H3K27me3 demethylation by KDM6A ex vivo in MDSCs from patients with protracted sepsis decreases Hotairm1 transcription. These findings suggest that epigenetic targeting of MDSCs in human sepsis might resolve post-sepsis immunosuppression and improve sepsis survival.