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 … Show more
“…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).…”
Section: Measuring Active Vs Inactive Rdna Repeatsmentioning
Ribosomes are highly conserved ribonucleoprotein nanomachines that translate information in the genome to create the proteome in all cells. In yeast these complex particles contain four RNAs (.5400 nucleotides) and 79 different proteins. During the past 25 years, studies in yeast have led the way to understanding how these molecules are assembled into ribosomes in vivo. Assembly begins with transcription of ribosomal RNA in the nucleolus, where the RNA then undergoes complex pathways of folding, coupled with nucleotide modification, removal of spacer sequences, and binding to ribosomal proteins. More than 200 assembly factors and 76 small nucleolar RNAs transiently associate with assembling ribosomes, to enable their accurate and efficient construction. Following export of preribosomes from the nucleus to the cytoplasm, they undergo final stages of maturation before entering the pool of functioning ribosomes. Elaborate mechanisms exist to monitor the formation of correct structural and functional neighborhoods within ribosomes and to destroy preribosomes that fail to assemble properly. Studies of yeast ribosome biogenesis provide useful models for ribosomopathies, diseases in humans that result from failure to properly assemble ribosomes.
TABLE OF CONTENTS
Abstract 643Introduction 644
“…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).…”
Section: Measuring Active Vs Inactive Rdna Repeatsmentioning
Ribosomes are highly conserved ribonucleoprotein nanomachines that translate information in the genome to create the proteome in all cells. In yeast these complex particles contain four RNAs (.5400 nucleotides) and 79 different proteins. During the past 25 years, studies in yeast have led the way to understanding how these molecules are assembled into ribosomes in vivo. Assembly begins with transcription of ribosomal RNA in the nucleolus, where the RNA then undergoes complex pathways of folding, coupled with nucleotide modification, removal of spacer sequences, and binding to ribosomal proteins. More than 200 assembly factors and 76 small nucleolar RNAs transiently associate with assembling ribosomes, to enable their accurate and efficient construction. Following export of preribosomes from the nucleus to the cytoplasm, they undergo final stages of maturation before entering the pool of functioning ribosomes. Elaborate mechanisms exist to monitor the formation of correct structural and functional neighborhoods within ribosomes and to destroy preribosomes that fail to assemble properly. Studies of yeast ribosome biogenesis provide useful models for ribosomopathies, diseases in humans that result from failure to properly assemble ribosomes.
TABLE OF CONTENTS
Abstract 643Introduction 644
“…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.…”
To better understand the role of topoisomerase activity in relieving transcription-induced supercoiling, yeast genes encoding rRNA were visualized in cells deficient for either or both of the two major topoisomerases. In the absence of both topoisomerase I (Top1) and topoisomerase II (Top2) activity, processivity was severely impaired and polymerases were unable to transcribe through the 6.7-kb gene. Loss of Top1 resulted in increased negative superhelical density (two to six times the normal value) in a significant subset of rRNA genes, as manifested by regions of DNA template melting. The observed DNA bubbles were not R-loops and did not block polymerase movement, since genes with DNA template melting showed no evidence of slowed elongation. Inactivation of Top2, however, resulted in characteristic signs of slowed elongation in rRNA genes, suggesting that Top2 alleviates transcription-induced positive supercoiling. Together, the data indicate that torsion in front of and behind transcribing polymerase I has different consequences and different resolution. Positive torsion in front of the polymerase induces supercoiling (writhe) and is largely resolved by Top2. Negative torsion behind the polymerase induces DNA strand separation and is largely resolved by Top1.Eukaryotic cells have two major topoisomerases that are capable of efficiently relaxing torsionally stressed DNA: topoisomerase I (Top1) and topoisomerase II (Top2) (75). They are both abundant nuclear proteins with roles in many DNA activities, and since they both can relax positive and negative torsion, they can substitute for each other in most situations (11,28,29,35,62). In spite of this partial functional redundancy, they control DNA topology by very different mechanisms (65). Top1 (a type IB topoisomerase) makes transient single-strand breaks in torsionally stressed DNA (recognizing the torque in such DNA), followed by controlled rotation of the nicked strand and resealing of the DNA in a more relaxed state (38). Top2 (a type IIA topoisomerase) recognizes juxtaposed DNA helices (as in supercoiled DNA) and passes one DNA helix through the other by making a transient doublestrand break in one of the helices (61, 65
“…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.…”
Section: Nucleosome Fragility Increases Throughout Heat-shock Genes Umentioning
Nucleosomes have structural and regulatory functions in all eukaryotic DNA-templated processes. The position of nucleosomes on DNA and the stability of the underlying histone-DNA interactions affect the access of regulatory proteins to DNA. Both stability and position are regulated through DNA sequence, histone post-translational modifications, histone variants, chromatin remodelers, and transcription factors. Here, we explored the functional implications of nucleosome properties on gene expression and development in Caenorhabditis elegans embryos. We performed a time-course of micrococcal nuclease (MNase) digestion and measured the relative sensitivity or resistance of nucleosomes throughout the genome. Fragile nucleosomes were defined by nucleosomal DNA fragments that were recovered preferentially in early MNase-digestion time points. Nucleosome fragility was strongly and positively correlated with the AT content of the underlying DNA sequence. There was no correlation between promoter nucleosome fragility and the levels of histone modifications or histone variants. Genes with fragile nucleosomes in their promoters tended to be lowly expressed and expressed in a contextspecific way, operating in neuronal response, the immune system, and stress response. In addition to DNA-encoded nucleosome fragility, we also found fragile nucleosomes at locations where we expected to find destabilized nucleosomes, for example, at transcription factor binding sites where nucleosomes compete with DNA-binding factors. Our data suggest that in C. elegans promoters, nucleosome fragility is in large part DNA-encoded and that it poises genes for future context-specific activation in response to environmental stress and developmental cues.[Supplemental material is available for this article.]The fundamental unit of eukaryotic chromatin is the nucleosome, which consists of 147 bp of DNA wrapped around an octamer of histone proteins (Luger et al. 1997). Nucleosomes have important structural and regulatory functions in organizing the genome and restricting access of regulatory factors to the DNA sequence (Henikoff 2008). As such, the interactions between nucleosomes and DNA strongly influence the regulation of gene expression by determining DNA accessibility for transcription factors (TFs) and RNA polymerase. In addition to regulated nucleosome assembly and disassembly through the action of histone chaperones and chromatin remodelers, nucleosome stability is influenced by histone modifications, histone variants, DNA features encoded in cis, and competition with DNA-binding factors in trans . A complete picture of the mechanisms governing nucleosome stability is fundamental to understanding how gene expression is dynamically regulated.Nucleosome stability has been studied in vitro using sensitivity to enzymatic digestion or salt concentration (Bloom and Anderson 1978;Burton et al. 1978;Li et al. 1993;Polach and Widom 1995;Wu and Travers 2004;Jin and Felsenfeld 2007). Genome-wide adaptations of these methods have been used to identify nuc...
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