We have devised a method for isolating virtually pure and comprehensive libraries of restriction fragments that contained replication initiation sites (bubbles) in vivo. We have now sequenced and mapped the bubble-containing fragments from GM06990, a near-normal EBV-transformed lymphoblastoid cell line, and have compared origin distributions with a comprehensive replication timing study recently published for this cell line. We find that early-firing origins, which represent~32% of all origins, overwhelmingly represent zones, associate only marginally with active transcription units, are localized within large domains of open chromatin, and are significantly associated with DNase I hypersensitivity. Origin ''density'' falls from early-to mid-S-phase, but rises again in late S-phase to levels only 17% lower than in early S-phase. Unexpectedly, late origin density calculated on the 1-Mb scale increases as a function of increasing chromatin compaction. Furthermore, the median efficiency of origins in late-replicating, heterochromatic domains is only 25% lower than in early-replicating euchromatic loci. Thus, the activation of early-and late-firing origins must be regulated by quintessentially different mechanisms. The aggregate data can be unified into a model in which initiation site selection is driven almost entirely by epigenetic factors that fashion both the long-range and local chromatin environments, with underlying DNA sequence and local transcriptional activity playing only minor roles. Importantly, the comprehensive origin map we have prepared for GM06990 overlaps moderately well with origin maps recently reported for the genomes of four different human cell lines based on the distributions of small nascent strands.

We have used a novel bubble-trapping procedure to construct nearly pure and comprehensive human origin libraries from early S-and log-phase HeLa cells, and from log-phase GM06990, a karyotypically normal lymphoblastoid cell line. When hybridized to ENCODE tiling arrays, these libraries illuminated 15.3%, 16.4%, and 21.8% of the genome in the ENCODE regions, respectively. Approximately half of the origin fragments cluster into zones, and their signals are generally higher than those of isolated fragments. Interestingly, initiation events are distributed about equally between genic and intergenic template sequences. While only 13.2% and 14.0% of genes within the ENCODE regions are actually transcribed in HeLa and GM06990 cells, 54.5% and 25.6% of zonal origin fragments overlap transcribed genes, most with activating chromatin marks in their promoters. Our data suggest that cell synchronization activates a significant number of inchoate origins. In addition, HeLa and GM06990 cells activate remarkably different origin populations. Finally, there is only moderate concordance between the log-phase HeLa bubble map and published maps of small nascent strands for this cell line.

BackgroundThe sirtuins are a conserved family of NAD+-dependent histone/protein deacetylases that regulate numerous cellular processes, including heterochromatin formation and transcription. Multiple sirtuins are encoded by each eukaryotic genome, raising the possibility of cooperativity or functional overlap. The scope and variety of chromatin binding sites of the sirtuins in any specific organism remain unclear.ResultsHere we utilize the ChIP-seq technique to identify and functionally characterize the genome-wide targets of the sirtuins, Sir2, Hst1 to Hst4, and the DNA binding partner of Hst1, Sum 1, in Saccharomyces cerevisiae. Unexpectedly, Sir2, Hst1 and Sum1, but not the other sirtuins, exhibit co-enrichment at several classes of chromatin targets. These include telomeric repeat clusters, tRNA genes, and surprisingly, the open reading frames (ORFs) of multiple highly expressed RNA polymerase II-transcribed genes that function in processes such as fermentation, glycolysis, and translation. Repression of these target genes during the diauxic shift is specifically dependent on Sir2/Hst1/Sum1 binding to the ORF and sufficiently high intracellular NAD+ concentrations. Sir2 recruitment to the ORFs is independent of the canonical SIR complex and surprisingly requires Sum1. The shared Sir2/Hst1/Sum1 targets also significantly overlap with condensin and cohesin binding sites, where Sir2, Hst1, and Sum1 were found to be important for condensin and cohesin deposition, suggesting a possible mechanistic link between metabolism and chromatin architecture during the diauxic shift.ConclusionsThis study demonstrates the existence of overlap in sirtuin function, and advances our understanding of conserved sirtuin-regulated functions, including the regulation of glycolytic gene expression and condensin loading.

Genes encoding thiamine biosynthesis enzymes in microorganisms are tightly regulated such that low environmental thiamine concentrations activate transcription and high concentrations are repressive. We have determined that multiple thiamine (THI) genes in Saccharomyces cerevisiae are also regulated by the intracellular NAD ؉ concentration via the NAD ؉ -dependent histone deacetylase (HDAC) Hst1 and, to a lesser extent, Sir2. Both of these HDACs associate with a distal region of the affected THI gene promoters that does not overlap with a previously defined enhancer region bound by the thiamine-responsive Thi2/Thi3/Pdc2 transcriptional activators. The specificity of histone H3 and/or H4 deacetylation carried out by Hst1 and Sir2 at the distal promoter region depends on the THI gene being tested. Hst1/Sir2-mediated repression of the THI genes occurs at the level of basal expression, thus representing the first set of transcription factors shown to actively repress this gene class. Importantly, lowering the NAD ؉ concentration and inhibiting the Hst1/Sum1 HDAC complex elevated the intracellular thiamine concentration due to increased thiamine biosynthesis and transport, implicating NAD ؉ in the control of thiamine homeostasis.

Yeast chronological lifespan (CLS) is extended by multiple genetic and environmental manipulations, including caloric restriction (CR). Understanding the common changes in molecular pathways induced by such manipulations could potentially reveal conserved longevity mechanisms. We therefore performed gene expression profiling on several long-lived yeast populations, including an ade4∆ mutant defective in de novo purine (AMP) biosynthesis, and a calorie restricted WT strain. CLS was also extended by isonicotinamide (INAM) or expired media derived from CR cultures. Comparisons between these diverse long-lived conditions revealed a common set of differentially regulated genes, several of which were potential longevity biomarkers. There was also enrichment for genes that function in CLS regulation, including a long-lived adenosine kinase mutant (ado1∆) that links CLS regulation to the methyl cycle and AMP. Genes co-regulated between the CR and ade4∆ conditions were dominated by GO terms related to metabolism of alternative carbon sources, consistent with chronological longevity requiring efficient acetate/acetic acid utilization. Alternatively, treating cells with isonicotinamide (INAM) or the expired CR media resulted in GO terms predominantly related to cell wall remodeling, consistent with improved stress resistance and protection against external insults like acetic acid. Acetic acid therefore has both beneficial and detrimental effects on CLS.