Transcriptional activation in the context of repression mediated by archaeal histones

Abstract

Many archaea (including all the methanogens, nearly all euryarchaeotes, and some crenarchaeotes) use histones as components of the chromatin that compacts their genomes. The archaeal histones are homo- and heterodimers that pair on DNA to form tetrasomes (as the eukaryotic histones H3 and H4 do). The resulting DNA packaging is known to interfere with assembly of the archaeal transcription apparatus at promoters; the ability of transcriptional activation to function in repressive archaeal chromatin has not yet been explored in vitro. Using four of the Methanocaldococcus jannaschii (Mja) histones, we have examined activation of the model Mja rb2 transcription unit by the Mja transcriptional activator Ptr2 in this simplified-chromatin context. Using hydroxyl radical footprinting, we find that the Ptr2-specific rb2 upstream activating site is a preferred histone-localizing site that nucleates histone: DNA-binding radiating from the rb2 promoter. Nevertheless, Ptr2 competes effectively with histones for access to the rb2 promoter and most potently activates transcription in vitro at histone concentrations that extensively coat DNA and essentially silence basal transcription.

Fig. 1.

Repression of transcription by the Mja histones. (A) The relaxed pTerm plasmid template was incubated at 65 °C for 20 min with TBP, TFB, and RNAP, in the absence (Lanes 1, 8, 15, and 22), or with increasing concentrations of individual histones (specified above the panel and measured in terms of the monomer as specified in SI Text), and transcribed for 15 min at the same temperature. The transcript terminating at t_mcrA/M and the DNA recovery marker (RM) are shown. (B) residual transcription, data from A, quantified.

Fig. 2.

Full activation of transcription in the context of repressive HMjA1. Transcription factors TBP and TFB were incubated with relaxed pTerm DNA at 65 °C for 20 min in the absence (Lanes 1–7) or presence (Lanes 8–14) of 100 nM (monomer) HMjA1 and in the absence (Lanes 7–8) or presence of the indicated (monomer) concentrations of Ptr2 before addition of RNAP for a single round of transcription at the same temperature. The transcript terminating at t_mcrA/M and the RM are shown.

Fig. 3.

Partial activation at high HMjA4 concentration. pTerm DNA was incubated at 65 °C for 15 min in the absence (Lanes 1–7) or presence (Lanes 8–14) of 1.5 μM (monomer) HMjA4. The transcription machinery was then added without (Lanes 1 and 8) or with the indicated concentrations of Ptr2 for multiple rounds of transcription at the same temperature.

Fig. 4.

Activation of transcription in the presence of HMjA3. (A) pTerm DNA was incubated at 65 °C for 20 min in the absence (Lanes 1–4), or with increasing concentrations of HMjA3 (specified in terms of the monomer, above the panel), in the absence of His₆-Ptr2 (Lanes 1, 5, 9, and 13), or with increasing concentrations of His₆-Ptr2 (specified in terms of the monomer, above each lane) and transcribed for 15 min at the same temperature. The transcript terminating at t_mcrA/M and the DNA RM are indicated by arrowheads. Fold-activations are shown below each lane. (B) Levels of transcription; data from A, quantified.

Fig. 5.

Promoter occupancy by Ptr2 and histones. A 460 bp DNA probe encompassing positions -302 to +158 relative to the start site of transcription of the rb2_con promoter, 5^′-end labeled on the top (nontranscribed) strand (A) or the bottom (transcribed/template) strand (B), was generated by PCR using plasmid pTerm as template. This probe was incubated at 65 °C for 20 min in the absence of HMjA1 (Lane 1 of each panel) or with increasing concentrations of HMjA1 (indicated above each panel), and subjected to hydroxyl radical (•OH) cleavage for 30 s at the same temperature. In lanes 6 to 9, HMjA1 binding to DNA is analyzed in reaction mixtures also containing 100 nM His₆-Ptr2. In each panel, lane 10 shows His₆-Ptr2 binding to DNA in the absence of histone. The TATA box and the two Ptr2 consensus binding sites are identified at the left of each panel. Also shown are the untreated DNA probe (P) as well as the A + G chemical sequencing ladder. The bullets point to the shift of protection at the Site 2 dyad reflecting histone displacement by Ptr2. (C) Aligned density profiles of •OH footprints shown in panel A; profiles in black and red correspond to lanes 1 and 2, respectively. The horizontal bars indicate the two consensus Ptr2 binding sites, with the arrows pointing to their central base pairs, which are the most protected by Ptr2 against •OH cleavage.