Published online 25 January 2011 Nucleic Acids Research, 2011, Vol. 39, No. 10 4023–4034 doi:10.1093/nar/gkq1286 Yeast H2A.Z, FACT complex and RSC regulate transcription of tRNA gene through differential dynamics of flanking nucleosomes Sahasransu Mahapatra, Pooran S. Dewari, Anubhav Bhardwaj and Purnima Bhargava* Centre for Cellular and Molecular Biology (Council of Scientific and Industrial Research), Uppal Road, Hyderabad 500007, India Received November 12, 2010; Accepted November 30, 2010 ABSTRACT FACT complex is involved in elongation and ensures fidelity in the initiation step of transcription by RNA polymerase (pol) II. Histone variant H2A.Z is found in nucleosomes at the 50-end of many genes. We report here H2A.Z-chaperone activity of the yeast FACT complex on the short, nucleosome-free, non-coding, pol III-transcribed yeast tRNA genes. On a prototype gene, yeast SUP4, chromatin re- modeler RSC and FACT regulate its transcription through novel mechanisms, wherein the two gene- flanking nucleosomes containing H2A.Z, play differ- ent roles. Nhp6, which ensures transcription fidelity and helps load yFACT onto the gene flanking nucleosomes, has inhibitory role. RSC maintains a nucleosome abutting the gene terminator down- stream, which results in reduced transcription rate in active state while H2A.Z probably helps RSC in keeping the gene nucleosome-free and serves as stress-sensor. All these factors maintain an epigen- etic state which allows the gene to return quickly from repressed to active state and tones down the expression from the active SUP4 gene, required probably to maintain the balance in cellular tRNA pool. INTRODUCTION Eukaryotic genome is organized into transcriptionally inactive, condensed heterochromatin and transcriptionally active euchromatin in different epigenetic states. Chromatin formation, which results in repressive effect on elongation, can suppress non-speciﬁc initiations from the transcribed regions as well. A complex, facilitates chromatin transcription (FACT) with two subunits Spt16 and SSRP1 in humans, can counter this nucleosom- al obstacle during elongation and even ensure the ﬁdelity of transcription initiation by RNA polymerase (pol) II (1– 3). FACT travels with pol II and removes/reinstates the H2A–H2B dimer from the nucleosome after passage of the pol II during elongation (4,5). Yeast FACT consists of three proteins: Spt16, Pob3 and a small, HMG box-containing, non-histone protein Nhp6 (6). Cells with deletion of both the copies of Nhp6 are viable (7), while Spt16/Pob3 are essential for cell viability (8), suggesting their functions are independent of each other. Nucleosomal arrangement on pol II-transcribed genes follows a general pattern, wherein a nucleosome-free region (NFR) is ﬂanked by two positioned nucleosomes (9). Several studies suggest that tDNAs are generally devoid of nucleosomes (9–11) and hence, refractory to chromatin mediated repression. Out of approximately 280 targets of pol III in the Saccharomyces cerevisiae genome (12,13), 275 genes code for different tRNAs. The most abundant ATP-dependent chromatin remodeler of yeast, RSC which interacts with all the three RNA polymerases, is found on most of the pol III-transcribed genes in yeast (14). Genome-wide localization studies of the chromatin modifying complexes also suggest that chromatin may be having an important regulatory role in expression of these genes (14–16). Recent studies have revealed that histone H2A variant H2A.Z (coded by Htz1 in S. cerevisiae) localizes to hundreds of repressed or basal, preferably TATA-less promoters of pol II (9). It acts as boundary mark for pol II-transcribed genes and is found around 200 and +200 bp from the tran- scription start site (TSS) of the tRNA genes (9). However, the role of H2A.Z in pol III transcription is largely an unexplored area. Earlier studies from our lab had shown that in vivo chromatin structure of a yeast pol III-transcribed gene, SNR6 is remodelled by RSC in *To whom correspondence should be addressed. Tel: +91 40 27192603; Fax: +91 40 27160591; Email: purnima@ccmb.res.in Present address: Sahasransu Mahapatra, Room No. 2300, La Jolla Institute for Allergy and Immunology, 9420, Athena Circle, CA92037, USA. The Author(s) 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.