Actively transcribed rRNA genes in S. cerevisiae are organized in a specialized chromatin associated with the high-mobility group protein Hmo1 and are largely devoid of histone molecules

Abstract

Synthesis of ribosomal RNAs (rRNAs) is the major transcriptional event in proliferating cells. In eukaryotes, ribosomal DNA (rDNA) is transcribed by RNA polymerase I from a multicopy locus coexisting in at least two different chromatin states. This heterogeneity of rDNA chromatin has been an obstacle to defining its molecular composition. We developed an approach to analyze differential protein association with each of the two rDNA chromatin states in vivo in the yeast Saccharomyces cerevisiae. We demonstrate that actively transcribed rRNA genes are largely devoid of histone molecules, but instead associate with the high-mobility group protein Hmo1.[Keywords: Chromatin; transcription; ribosomal DNA] Supplemental material is available at http://www.genesdev.org. Received December 11, 2007; revised version accepted February 29, 2008. In the yeast Saccharomyces cerevisiae (hereafter called yeast), >60% of total cellular transcripts are produced from the ribosomal DNA (rDNA) locus by a specific multiprotein enzyme, RNA Polymerase I (Pol I), and an assorted set of additional transcription factors (Reeder 1999;Moss and Stefanovsky 2002;Russell and Zomerdijk 2006). The basal Pol I transcription machinery has been largely defined and consists of two multiprotein complexes, the upstream activating factor (UAF) , and the core factor (CF) (Keys et al. 1994), as well as the TATA-box-binding protein (TBP) (Steffan et al. 1996).The template of RNA Pol I transcription, the yeast ribosomal RNA (rRNA) genes are located on the right arm of chromosome XII in a tandem array of 150-200 copies (Fig. 1), representing almost 10% of the yeast genome. Each of the repeats contains the Pol I-transcribed 35S rRNA gene (precusor of the 5.8S, 18S, and 25S rRNAs) and the RNA Polymerase III (Pol III)-transcribed 5S rRNA gene (Fig. 1). Three different regulatory DNA elements have been identified within the 35S rRNA gene sequence, two of which are located at the 5Ј end of the 35S rDNA, spanning only ∼170 base pairs (bp) (Musters et al. 1989;Kulkens et al. 1991): the upstream element (UE), which is the binding site for the UAF; and the core promoter (CP), where CF can be recruited (Fig. 1). The third element, called the enhancer (ENH), is located at the 3Ј end of the 35S transcription unit (Fig. 1) and was originally identified as exhibiting a stimulatory effect on Pol I transcription in experiments using episomal reporter systems Warner 1984, 1986). However, this sequence has been shown subsequently to be dispensable for RNA Pol I transcription in the chromosomal context (Wai et al. 2001).In each cell, Pol I-transcribed rDNA coexists in two different chromatin states (Conconi et al. 1989). In these studies, cells were treated with the DNA cross-linking drug psoralen, and it was shown that actively transcribed and transcriptionally inactive rRNA genes differed substantially in their degree of psoralen incorporation. The different migration behavior of the corresponding restriction fragments in agarose gel electrophoresis led to the terms s-ban...

Ribosome Biogenesis in the YeastSaccharomyces cerevisiae

“…The actively transcribed rRNA genes are devoid of histones and are instead covered with the Hmo1 protein (analogous to UBF1 in mammals). As expected, RNA polymerase I associates with the slowermoving band but not the faster-moving one by chromatin endogenous cleavage (ChEC) (Merz et al 2008). While biochemical analyses indicate that only about half of the repeats are active, single-transcript counting adds an interesting spin (Tan and van Oudenaarden 2010).…”

Distinguishing the Roles of Topoisomerases I and II in Relief of Transcription-Induced Torsional Stress in Yeast rRNA Genes

“…This length of DNA will have a relaxed linking number (Lk 0 ) of 642 (6,740/10.5). An inactive rRNA gene will have constrained negative supercoiling due to nucleosomal packaging, but the linking number will decrease upon activation and loss of nucleosomes (51), which constrain ϳ1 negative supercoil each (59). Assuming ϳ41 nucleosomes per gene (6,740 bp/ϳ165 bp/nucleosome ϭ ϳ41), this corresponds to a ⌬Lk of Ϫ41 and a superhelical density (, where ϭ ⌬Lk/Lk 0 ) of Ϫ0.06 for a nucleosome-free rRNA gene in control strains.…”

Nucleosome fragility is associated with future transcriptional response to developmental cues and stress inC. elegans

“…We next sought to test the relationship between fragile nucleosomes and trans-factors more explicitly. At extremely highly transcribed genes, such as heat-shock-responsive genes after induction, it has been proposed that RNA polymerase II (Pol II) molecules occupy the entire gene body (Schwabish and Struhl 2004;Merz et al 2008;Cole et al 2014). We hypothesized that fragility would increase at gene bodies after inducing high levels of transcription, as a result of nucleosome competition with transcribing Pol II.…”