The eukaryotic nucleosome is the fundamental unit of chromatin, comprising a protein octamer that wraps ∼147 bp of DNA and has essential roles in DNA compaction, replication and gene expression. Nucleosomes and chromatin have historically been considered to be unique to eukaryotes, yet studies of select archaea have identified homologs of histone proteins that assemble into tetrameric nucleosomes. Here we report the first archaeal genome-wide nucleosome occupancy map, as observed in the halophile Haloferax volcanii. Nucleosome occupancy was compared with gene expression by compiling a comprehensive transcriptome of Hfx. volcanii. We found that archaeal transcripts possess hallmarks of eukaryotic chromatin structure: nucleosome-depleted regions at transcriptional start sites and conserved −1 and +1 promoter nucleosomes. Our observations demonstrate that histones and chromatin architecture evolved before the divergence of Archaea and Eukarya, suggesting that the fundamental role of chromatin in the regulation of gene expression is ancient.DOI: http://dx.doi.org/10.7554/eLife.00078.001

No abstract

Systematic analysis of gene overexpression phenotypes provides an insight into gene function, enzyme targets, and biological pathways. Here, we describe a novel functional genomics platform that enables a highly parallel and systematic assessment of overexpression phenotypes in pooled cultures. First, we constructed a genome-level collection of ~5100 yeast barcoder strains, each of which carries a unique barcode, enabling pooled fitness assays with a barcode microarray or sequencing readout. Second, we constructed a yeast open reading frame (ORF) galactose-induced overexpression array by generating a genome-wide set of yeast transformants, each of which carries an individual plasmid-born and sequence-verified ORF derived from the Saccharomyces cerevisiae full-length EXpression-ready (FLEX) collection. We combined these collections genetically using synthetic genetic array methodology, generating ~5100 strains, each of which is barcoded and overexpresses a specific ORF, a set we termed “barFLEX.” Additional synthetic genetic array allows the barFLEX collection to be moved into different genetic backgrounds. As a proof-of-principle, we describe the properties of the barFLEX overexpression collection and its application in synthetic dosage lethality studies under different environmental conditions.

Histones are the primary protein component of chromatin, the mixture of DNA and proteins that packages the genetic material in eukaryotes. Large amounts of histones are required during the S phase of the cell cycle when genome replication occurs. However, ectopic expression of histones during other cell cycle phases is toxic; thus, histone expression is restricted to the S phase and is tightly regulated at multiple levels, including transcriptional, post-transcriptional, translational, and post-translational. In this review, we discuss mechanisms of regulation of histone gene expression with emphasis on the transcriptional regulation of the replication-dependent histone genes in the model yeast Saccharomyces cerevisiae.

Bacteria of the genusT he genus Shigella comprises 4 species, including Shigella flexneri, which is comprised of 14 different serotypes and subserotypes (2). S. flexneri strain M90T Sm is commonly used in the laboratory to understand the molecular basis of shigellosis (1,5,9).Two amplification-free libraries were constructed from 5 g of M90T Sm genomic DNA each, and the genome was sequenced using a 2-by-110-base paired-end strategy on the Illumina HiSeq2000 platform. Libraries were constructed using the NEBNext DNA sample prep master mix set 1 (NEB; part number E6040L) and barcoded in an overnight ligation. Libraries were size selected from 450 bp to 500 bp, quantified by quantitative PCR (qPCR) (Kapa BioSystems), pooled, requantified, and clustered using cBot on a single lane with TruSeq PE cluster kit v2 chemistry. A total of 129,937,458 single-end reads were filtered by demultiplexing using two indexes (AGTCGATC/CAGTCTGA) and the signal purity filter. The resulting 73,325,436 reads were trimmed to 54 bp, the point at which the mean quality score dropped below 30 (Illumina 1.5ϩ phred quality score), using the Fastx toolkit (version 0.0.13) (http://hannonlab.cshl.edu/fastx_toolkit/index .html). Half of the preprocessed reads (34,231,825 reads) were mapped onto the chromosomal sequence of S. flexneri serotype 5b strain 8401 (GenBank accession no. NC_008258) (6) using the Burrows-Wheeler aligner (BWA) (version 0.5.9) (3). A total of 28,876,862 reads (84.3%) were successfully mapped to a depth of 340ϫ. Single nucleotide polymorphism (SNP) calls and consensus sequence generation were performed using the pileup function in SAMtools (version 0.1.16) (4). The integrated genomics viewer (IGV) (version 1.5) (7) was used to examine regions of low coverage (0ϫ to 160ϫ) and/or low quality. A total of 39 positions within the single-copy region of the genome were verified by Sanger sequencing. The remaining 119 gaps were within multicopy regions, such as insertion sequences (IS), ribosomal DNAs, and the tufA gene. The GϩC content was calculated using Artemis (version 13.0) (8).The sequence of the S. flexneri M90T Sm genome covers 99.9% of the 4,574,284-bp chromosome of S. flexneri 8401 (6). A total of 1,433 SNPs and 176 indels were identified. About one-third of the SNPs (543 sites) cause nonsynonymous substitutions in S. flexneri M90T Sm. Twenty-one indels in M90T Sm cause frameshifts, and 9 indels correct 9 frame-shifted genes in S. flexneri 8401. A tandem repeat of tRNAs (tRNA-Lys and tRNA-Val) and one hypothetical protein that both do not exist in S. flexneri 8401 were found in M90T Sm. The M90T Sm genome contains 3,676 open reading frames (ORFs) (excluding IS ORFs), 99 tRNA genes, and 22 rRNA genes. The sequence has an average GϩC content of 50.9% in its entirety with a GϩC content of 52% within coding genes.The S. flexneri M90T Sm genome will enhance ongoing wholegenome comparisons across all S. flexneri serotypes for a fuller understanding of the evolution of S. flexneri and provides accurate sequence information need...