Archaeal nucleosomes

Abstract

Archaea contain histones that have primary sequences in common with eukaryal nucleosome core histones and a three-dimensional structure that is essentially only the histone fold. Here we report the results of experiments that document that archaeal histones compact DNA in vivo into structures similar to the structure formed by the histone (H3+H4)2 tetramer at the center of the eukaryal nucleosome. After formaldehyde cross-linking in vivo, these archaeal nucleosomes have been isolated from Methanobacterium thermoautotrophicum and Methanothermus fervidus, visualized by electron microscopy on plasmid and genomic DNAs, and shown by immunogold labeling, SDS/PAGE, and immunoblotting to contain archaeal histones, cross-linked into tetramers. Archaeal nucleosomes protect approximately 60 bp of DNA and multiples of approximately 60 bp from micrococcal nuclease digestion, and immunoprecipitation has demonstrated that most, but not all, M. fervidus genomic DNA sequences are associated in vivo with archaeal histones.

Figure 1

Isolation and EM of archaeal nucleosomes from M. thermoautotrophicum strain Marburg. (a) Electrophoretic separation of pME2001-containing complexes in sucrose gradient fractions from a lysate of formaldehyde-fixed protoplasts. (b) Immunoblot of the complexes in a, generated with anti-HMt antiserum. (c) EM of a pME2001–HMt complex (Bar = 200 nm). (d) pME2001–HMt–anti-HMt–IgG complex immunogold-labeled (black dots) with a protein A–gold bead conjugate. (e) EM of genomic DNA from a formaldehyde-fixed protoplast.

Figure 2

DNA and protein content of archaeal nucleosomes. (a) Electrophoretic separation of DNA fragments protected from MN digestion in nucleoprotein complexes isolated from formaldehyde-fixed M. fervidus. MN digestion was for 1, 2, 4, 6, 8, 10, 15, 30, and 45 min at 37°C. Control tracks contained size standards and undigested complexes (O). (b) Electrophoretic separation and silver staining of the proteins isolated from the complexes indicated in a that protected ≈60 bp fragments of DNA from MN digestion. Purification of these proteins involved incubations with β-agarase and DNase I, and the control tracks demonstrate the polypeptides present in these reagents. Small amounts of these polypeptides remained in the experimental material. An immunoblot of the purified proteins generated with anti-HMf antibodies is shown adjacent to the stained gel.

Figure 3

Quantitation of HMf. The immunoblot of a ¹²⁵I-labeled standard curve, adjacent to an aliquot of a M. fervidus lysate, is shown above a graphical quantitation of this result.

Figure 4

(A) Outline of the procedure used to isolate and immunoprecipitate histone–DNA complexes cross-linked in vivo (20). (B) Southern blots generated with probes specific for the M. fervidus genes listed (27, 28). The probes (short heavy lines) were designed to hybridize, as indicated, to similar-sized restriction fragments, ApoI (A), HincII (H), RsaI (R), and SspI (S), that spanned the sites of transcription (bent arrow) and translation initiation (thick bar). As illustrated, the ftr and mcr genes encode the enzymes that catalyze the second and seventh steps in CO₂ reduction to CH₄ (–29). At the time of formaldehyde fixation, the cells were actively synthesizing methane, and Northern blot analyses demonstrated that the ftr, mcr, hmfA, hmfB, 7S, and 16S transcripts were abundant. Aliquots (10% and 2%) of the total DNA present before the immunoprecipitation (see A) were electrophoresed in the tracks adjacent to the tracks that contained the DNA isolated from complexes immunoprecipitated (IP) by anti-HMf IgG (+ab) or by the control antiserum (−ab).