Extended Archaeal Histone-Based Chromatin Structure Regulates Global Gene Expression in Thermococcus kodakarensis

Abstract

Histone proteins compact and organize DNA resulting in a dynamic chromatin architecture impacting DNA accessibility and ultimately gene expression. Eukaryotic chromatin landscapes are structured through histone protein variants, epigenetic marks, the activities of chromatin-remodeling complexes, and post-translational modification of histone proteins. In most Archaea, histone-based chromatin structure is dominated by the helical polymerization of histone proteins wrapping DNA into a repetitive and closely gyred configuration. The formation of the archaeal-histone chromatin-superhelix is a regulatory force of adaptive gene expression and is likely critical for regulation of gene expression in all histone-encoding Archaea. Single amino acid substitutions in archaeal histones that block formation of tightly packed chromatin structures have profound effects on cellular fitness, but the underlying gene expression changes resultant from an altered chromatin landscape have not been resolved. Using the model organism Thermococcus kodakarensis, we genetically alter the chromatin landscape and quantify the resultant changes in gene expression, including unanticipated and significant impacts on provirus transcription. Global transcriptome changes resultant from varying chromatin landscapes reveal the regulatory importance of higher-order histone-based chromatin architectures in regulating archaeal gene expression.

Keywords: RNA-seq; Thermococcus; archaea; chromatin; histone; transcriptome.

FIGURE 1

A single wild-type histone protein is sufficient for normal DNA protection in T. kodakarensis. (A) Diagrammatic representation of wildtype chromatin modeled from the archaeal histone-based chromatin crystal structure: 9 polymerized histone B dimers (pale green) wrapped by DNA (gray) adapted from Mattiroli et al. (2017). The central glycine in the AGA motif at the Loop1–Loop1 interface is colored in red. A Logo-plot highlights the conservation of this motif. Histone dimers may be heterogeneously composed. (B) DNA fragments resulting from micrococcal nuclease (MNase) digested chromatin demonstrate the state of chromatin structure in TS600, TS622, and TS620. Chromatin purified from TS600 (TK1413^WT:histone A/ΔTK2289:histone B) resists MNase digestion over time, resulting in a laddered DNA banding pattern. Prominent 60 and 90 bp bands in addition to higher molecular weight bands (increasing 30 bp increments up to ∼300 bp) represent varying levels of histone dimerization and MNase protection. Chromatin purified from TS622 (TK1413^G17D:histone A/TK2289^WT:histone B) exhibits an identical protection pattern to TS600 despite encoding a variant (G17D) histone A. This suggests a single WT histone is sufficient for normal chromatin structure formation. Chromatin purified from TS620 (TK1413^G17D:histone A/ΔTK2289:histone B) exhibits a markedly different protection pattern from TS600 and TS622. The presence of only a variant (G17D) histone A results in a loss of DNAs protected above 90 bp, demonstrating the disruption of the L1–L1 interface interferes with continued histone dimer polymerization. (C) Diagrammatic representation of the potential chromatin structures in TS600, TS622, and TS620.

FIGURE 2

Altering 3-dimensional chromatin structure dramatically alters gene expression in T. kodakarensis. (A) In TS620 (compared to TS600) 100 protein-coding genes were downregulated while 134 protein-coding genes were upregulated. In TS622 (compared to TS600), 49 protein-coding genes were downregulated while 18 protein-coding genes were upregulated. (B) In TS620 (TK1413^G17D:histone A/ΔTK2289:histone B) differential RNA-sequencing, represented in an MA plot, revealed a number of genes are significantly upregulated and downregulated when compared to TS600 (TK1413^WT:histone A/ΔTK2289:histone B). Green transcripts are significantly (≥2 fold change) enriched in TS620 when compared to TS600. Red transcripts are significantly depleted (≥2 fold change) in TS620 when compared to TS600. Notably, in TS620, a number of single stranded binding proteins (TK1959-1961: replication protein A subunits Rpa32, Rpa14, and Rpa41) are significantly enriched in TS620. TK1413^G17D was found to be enriched in TS620 when compared to TK1413^WT in TS600. In TS620 transcripts involved in cell motility and cell signaling (TK0038-TK0049: archaeal Fla operon and archaeal che operon (TK0629-TK0639) were significantly depleted. Additionally, a portion (TK0394-TK0410) of viral region 2 (TK0381-TK0421) was significantly depleted. (C) In TS622 (TK1413^G17D:histone A/TK2289^WT:histone B) far fewer genes were upregulated when compared to TS600. Downregulated genes related in cell motility and environmental signal sensing revealed similar trends to TS620 (che and fla operons).

FIGURE 3

Disruption of 3-dimensional chromatin structure results in genome instability in T. kodakarensis. (A) A circos plot comparing TS620 to TS600. The outermost black circle represents genomic position. The outer coverage plot (blue) represents Fragments Per Kilobase of transcript per Million mapped reads (FPKM) for TS620. The inner coverage plot (purple) represents FPKM for TS600. Notably, nearly zero reads mapped to TKVR2 in TS620 (highlighted in yellow). Red lines represent fragments enriched in TS600 while green lines represent fragments enriched in TS620. (B) A loci diagram of the annotated T. kodakarensis viral region 2 (TKVR2: TK0381-TK0421) that highlights the observed region of excision (∼TK0389 – ∼TK0412) superimposed over a genome alignment plot derived from PacBio long read sequencing of TS620.