Archaeal nucleosome positioning in vivo and in vitro is directed by primary sequence motifs

Abstract

BackgroundHistone wrapping of DNA into nucleosomes almost certainly evolved in the Archaea, and predates Eukaryotes. In Eukaryotes, nucleosome positioning plays a central role in regulating gene expression and is directed by primary sequence motifs that together form a nucleosome positioning code. The experiments reported were undertaken to determine if archaeal histone assembly conforms to the nucleosome positioning code.ResultsEukaryotic nucleosome positioning is favored and directed by phased helical repeats of AA/TT/AT/TA and CC/GG/CG/GC dinucleotides, and disfavored by longer AT-rich oligonucleotides. Deep sequencing of genomic DNA protected from micrococcal nuclease digestion by assembly into archaeal nucleosomes has established that archaeal nucleosome assembly is also directed and positioned by these sequence motifs, both in vivo in Methanothermobacter thermautotrophicus and Thermococcus kodakarensis and in vitro in reaction mixtures containing only one purified archaeal histone and genomic DNA. Archaeal nucleosomes assembled at the same locations in vivo and in vitro, with much reduced assembly immediately upstream of open reading frames and throughout the ribosomal rDNA operons. Providing further support for a common positioning code, archaeal histones assembled into nucleosomes on eukaryotic DNA and eukaryotic histones into nucleosomes on archaeal DNA at the same locations. T. kodakarensis has two histones, designated HTkA and HTkB, and strains with either but not both histones deleted grow normally but do exhibit transcriptome differences. Comparisons of the archaeal nucleosome profiles in the intergenic regions immediately upstream of genes that exhibited increased or decreased transcription in the absence of HTkA or HTkB revealed substantial differences but no consistent pattern of changes that would correlate directly with archaeal nucleosome positioning inhibiting or stimulating transcription.ConclusionsThe results obtained establish that an archaeal histone and a genome sequence together are sufficient to determine where archaeal nucleosomes preferentially assemble and where they avoid assembly. We confirm that the same nucleosome positioning code operates in Archaea as in Eukaryotes and presumably therefore evolved with the histone-fold mechanism of DNA binding and compaction early in the archaeal lineage, before the divergence of Eukaryotes.

Transcriptional Repressor TrmBL2 from Thermococcus kodakarensis Forms Filamentous Nucleoprotein Structures and Competes with Histones for DNA Binding in a Salt- and DNA Supercoiling-dependent Manner

“…Identical tetra-and hexamer nucleotide sequences analysis also leads to very similar conclusions, indicating robustness of these results (data not shown). Observed sequence selectivities of TrmBL2 and histones are consistent with previous experimental studies showing that archaeal nucleosomes tend to bind to G/C-rich genome regions and avoid A/T-rich transcription initiation and termination sequences (45) and that TrmBL2 protein functions as a global expression regulator of many T. kodakarensis genes by binding to some A/T-rich promoter regions (20).…”

“…It has been reported that T. kodakarensis histones form multimeric structures primarily in the form of tetramers binding on average to 60 bp of DNA (16,45), which is 12 times larger than a nucleotide pentamer. Therefore, 1.6 k B T energy difference per a nucleotide pentamer results in a large binding energy gap of ϳ19.2 k B T between the most and least probable histone positions on the genomic DNA.…”

Transcription Regulation in Archaea

“…The binding preferences and genomic locations of stable euryarchaeal histone proteins interactions have been mapped, and it has been shown that regions directly upstream from the start codon are nucleosome depleted on a global scale (164,165). The presence of histones bound at the promoter has been correlated with a decrease in total transcription in vitro (177), and it was suggested that both steric and torsional effects limited binding of basal transcription factors to the DNA (177).…”

“…The best-described complexes are homo-or hetero-histone tetramers, homologous to the H3/H4 tetramer in eukaryotes, that associate with ϳ60 bp of double-stranded DNA. Archaeal histones share similar biases with eukaryotic nucleosomes for flexible DNA sequences and are, in general, absent from the core promoters of archaeal genes (164,165). Archaeal histone proteins share the same core fold as eukaryotic histones but lack the extensions from this fold (i.e., tails) that are highly modified and essential for proper nucleosome dynamics in eukaryotes (166).…”

“…These studies contrast the view of histone proteins as general organizational factors with the global influence on gene expression and minimally suggest that the archaeal chromatin of some species is dependent on the activities of many nucleoid-associated proteins. When histone-encoding genes have been deleted, or have been attempted to be deleted from other species, more global disruption of gene expression has been noted (161,164,165,167,(171)(172)(173)(174)(175)(176). Some species are reliant on at least one histone protein, and it is unclear at this point whether the noted global changes in gene expression seen in deletion strains stem from reorganization or disorganization of the archaeal genomes or the primary, secondary, and tertiary effects of localized disruptions that lead to additional differences in regulation at remote sites.…”