The ribosome is a highly complex and abundant protein-RNA machinery responsible for translating mRNA into protein. It is largely composed of specialized RNA known as ribosomal RNA (rRNA) as well as dozens of distinct proteins. Since protein synthesis is such a crucial process for growth, one would expect rapidly growing cells to have a massive demand for ribosomes. Indeed, genes encoding rRNA constitute the most abundant genes in the eukaryotic genome. These genes reside in tandem repetitive clusters known as the ribosomal DNA (rDNA) locus, in some cases totaling hundreds of copies. In the budding yeast Saccharomyces cerevisiae, 150 – 200 tandem repeats can be found in the rDNA locus on chromosome 12; within each repeat are a 35S rRNA gene and inter-genic spacers (IGS), which contain regulatory elements. Due to their repetitive structure and highly active transcription, the rDNA locus has to be tightly regulated to achieve a balance in size and replication efficiency. For these complex, DNA-dependent processes to occur, chromatin has to be structurally permissive. The most widely characterized chromatin-modifying complexes can be classified into two major groups, based on their modes of action. ATP-dependent complexes use the energy of ATP hydrolysis to locally disrupt or alter the association of histones with DNA, while histone acetyltransferase (HAT) and histone deacetylase (HDAC) complexes determine the level of acetylation of amino-terminal domains of nucleosomal histones to regulate transcription. Of these, little is known about how ATP-dependent chromatin remodeling factors dynamically regulate chromatin structure at the S.cerevisiae rDNA locus.
Dr. Toshio Tsukiyama and members of his laboratory in the Basic Sciences Division studied the nature of chromatin regulation by ATP-dependent remodeling factors at the yeast rDNA locus, and published their results in a recent issue of Genetics. Led by graduate student Sam Cutler, the authors focused on two known chromatin remodeling factors: lsw2 and lno80, and performed chromatin immune-precipitation followed by deep sequencing (ChIP-seq) to show that these factors are targeted to the rDNA locus. Using a series of mutant strains, they further showed that lsw2 and lno80 affected nucleosome occupancy over the 35S rRNA gene, and can increase the fraction of active rDNA repeats. These data led them to wonder if the transcription of the 35S rRNA gene would be regulated by these two factors, but to their surprise, lsw2 and lno80 do not affect overall levels of 35S rRNA transcription. However, the authors found that lsw2 and lno80 indeed affected the position of nucleosomes within the rDNA inter-genic spacer, a known function of chromatin remodeling factors.
The maintenance of ~150 – 200 rDNA repeats within the locus is subject to the balance between the demand for rRNA transcription, genome stability and limitation of replication resources. To determine if lsw2 and lno80 play a role in rDNA copy number, the authors turned to strains that had shortened rDNA arrays (20 repeats instead of 150). The rDNA copy number increases through the action of the Fob1 protein, which is required for this process; Fob1 is absent in the low rDNA copy number strains, but can be introduced in the form of an expression plasmid. The rDNA copy number is tracked over time in the presence or absence of lsw2 and lno80 mutants. Their results show most strikingly that lsw2 and lno80 facilitated the increase of rDNA copy number in the array when Fob1 is expressed.
Sam Cutler explains the impact of this work: “This study shows for the first time that specific ATP-dependent chromatin remodeling factors, Isw2 and Ino80, are targeted to the budding yeast ribosomal DNA (rDNA) locus and that loss of these factors affects both local chromatin structure and critical biological processes of the rDNA. In addition, this work contains the first demonstration of chromatin remodeling-mediated changes in chromatin structure that coincide with changes in replication origin efficiency at a totally natural origin.”
Cutler S, Lee LJ, Tsukiyama T. 2018. Chromatin Remodeling Factors Isw2 and Ino80 Regulate Chromatin, Replication, and Copy Number of the Saccharomyces cerevisiae Ribosomal DNA Locus. Genetics. Dec 1, 210 (4): 1543-1556
Funding was provided by the National Science Foundation and the National Institutes of Health.