Histone octamer trans-transfer: a signature mechanism of ATP-dependent chromatin remodelling unravelled in wheat nuclear extract

Abstract

Background and scope: In eukaryotes, chromatin remodelling complexes are shown to be responsible for nucleosome mobility, leading to increased accessibility of DNA for DNA binding proteins. Although the existence of such complexes in plants has been surmised mainly at the genetic level from bioinformatics studies and analysis of mutants, the biochemical existence of such complexes has remained unexplored.

Methods: Histone H1-depleted donor chromatin was prepared by micrococcal nuclease digestion of wheat nuclei and fractionation by exclusion chromatography. Nuclear extract was partially purified by cellulose phosphate ion exchange chromatography. Histone octamer trans-transfer activity was analysed using the synthetic nucleosome positioning sequence in the absence and presence of ATP and its analogues. ATPase activity was measured as (32)Pi released using liquid scintillation counting.

Key results: ATP-dependent histone octamer trans-transfer activity, partially purified from wheat nuclei using cellulose phosphate, showed ATP-dependent octamer displacement in trans from the H1-depleted native donor chromatin of wheat to the labelled synthetic nucleosome positioning sequence. It also showed nucleosome-dependent ATPase activity. Substitution of ATP by ATP analogues, namely ATPγS, AMP-PNP and ADP abolished the octamer trans-transfer, indicating the requirement of ATP hydrolysis for this activity.

Conclusions: ATP-dependent histone octamer transfer in trans is a recognized activity of chromatin remodelling complexes required for chromatin structure dynamics in non-plant species. Our results suggested that wheat nuclei also possess a typical chromatin remodelling activity, similar to that in other eukaryotes. This is the first report on chromatin remodelling activity in vitro from plants.

Fig. 1.

The functions and the characteristic domain architecture of the ATPase subunit of chromatin remodelling families from animal systems: chromatin remodellers from yeast and animal systems are classified into four families based on split ATPase domains and the characteristic flanking domains unique for each class. The callout boxes represent the functions that have been assigned to each family of the remodeller by the biochemical and genetic approaches from animal systems and yeast. The bottom panel indicates the domain architecture of each of these families that classifies them into the specific class of the remodeller as described by Clapier and Cairns (2009). Domain detail are described at www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml

Fig. 2.

The putative functions of the members of the chromatin remodelling families in plant systems: the callout boxes indicate the functions that have been detected mainly by genetic approaches in Arabidopsis as described by Hsieh and Fischer (2005). The table at the bottom indicates the classification of remodellers belonging to the SNF2 superfamily ATPase protein sequences from www.chromdb.org with chromdb i.d. for Arabidopsis, maize and rice on the basis of the domains annotated in the Conserved Domain Database (CDD) www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml. The sequences [42 from Arabidopsis, 47 from maize, 42 and 40 from rice (japonica and indica) and 14 from wheat], which were annotated as the SNF2 super family, were used for analysis.

Fig. 3.

Profile of DNA isolated from the soluble chromatin: DNA was extracted from fractionated nucleosomes obtained from chromatin derived from MNase-treated wheat nuclei and analysed on 1 % agarose gel. The lane numbers (25–39) indicate fraction numbers. Lane M: 100-bp DNA ladder. Chromatin from fractions 29 and 30 (boxed) was pooled, concentrated and used as ‘donor chromatin’ for the remodelling assay.

Fig. 4.

Nucleosome reconstitution in vitro: mononucleosomes were assembled in vitro both non-enzymatically and enzymatically as described in the Material and methods. Lane 1, mobility shift of NPS due to binding of histone octamer by the salt jump method. Lane 2, mobility shift of NPS due to ATP-dependent displacement of histone octamer in trans; Lane 3, free NPS.

Fig. 5.

(A) Schematic representation of histone octamer trans-transfer activity: the cartoon shows that the labelled NPS assembles into a mononucleosome in the presence of the chromatin remodeler and ATP, resulting in reduction in its mobility on PAGE. In the absence of ATP and/or remodeller the trans-transfer would not occur and hence there will be no reduction in the mobility of the labelled NPS. (B) Histone octamer trans-transfer activity of the elutes from cellulose phosphate: lane 1, activity eluted with SSB-0·2; lane 2, SSB-0·35; lane 3, SSB-0·5; lane 4, SSB-0·75; lane 5, free NPS.

Fig. 6.

Time course of chromatin remodelling activity: lanes 1–4, 15, 30, 45 and 60 min incubation, respectively, of the assay mixture. Lane 5, free NPS.

Fig. 7.

Chromatin remodelling activity in the presence of ATP and ATP analogues: lane 1, free NPS; lane 2, with ATP; lane 3, without ATP; lane 4, with ATPγS; lane 5, with AMP-PNP; lane 6, with ADP as mentioned at the bottom of each lane.

Fig. 8.

ATPase assay of the chromatin remodelling complex: the x-axis depicts the assay conditions and y-axis shows the concentration of ³²Pi released from hydrolysis of ATP in terms of counts (cpm). Nucl., donor chromatin as oligo-nucleosomal DNA; CRC, chromatin remodelling complexes.