An HMG Protein, Hmo1, Associates with Promoters of Many Ribosomal Protein Genes and throughout the rRNA Gene Locus in Saccharomyces cerevisiae

ABSTRACT HMG proteins are architectural proteins that bind to DNA with low sequence specificity, but little is known about their genomic location and biological functions. Saccharomyces cerevisiae encodes 10 HMG proteins, including Hmo1, which is important for maximal transcription of rRNA. Here we use chromatin immunoprecipitation coupled with microarray analysis to determine the genome-wide association of Hmo1. Unexpectedly, Hmo1 binds strongly to the promoters of most ribosomal protein (RP) genes and to a number of other specific genomic locations. Hmo1 binding to RP promoters requires Rap1 and (to a lesser extent) Fhl1, proteins that also associate with RP promoters. Hmo1, like Fhl1 and Ifh1, typically associates with an IFHL motif in RP promoters, but deletion of the IFHL motif has a very modest effect on Hmo1 binding. Surprisingly, loss of Hmo1 abolishes binding of Fhl1 and Ifh1 to RP promoters but does not significantly affect the level of transcriptional activity. These results suggest that Hmo1 is required for the assembly of transcription factor complexes containing Fhl1 and Ifh1 at RP promoters and that proteins other than Fhl1 and Ifh1 also play an important role in RP transcription. Lastly, like mammalian UBF, Hmo1 associates at many locations throughout the rRNA gene locus, and it is important for processing of rRNA in addition to its role in rRNA transcription. We speculate that Hmo1 has a role in coordinating the transcription of rRNA and RP genes.

Transcription in the yeast rRNA gene locus: distribution of the active gene copies and chromatin structure of their flanking regulatory sequences.

In growing yeast cells, about half of the 150 tandemly repeated rRNA genes are transcriptionally active and devoid of nucleosomes. By using the intercalating drug psoralen as a tool to mark accessible sites along chromatin DNA in vivo, we found that the active rRNA gene copies are rather randomly distributed along the ribosomal rRNA gene locus. Moreover, results from the analysis of a single, tagged transcription unit in the tandem array are not consistent with the presence of a specific subset of active genes that is stably maintained throughout cell divisions. In the rRNA intergenic spacers of yeast cells, an enhancer is located at the 3' end of each transcription unit, 2 kb upstream of the next promoter. Analysis of the chromatin structure along the tandem array revealed a structural link between transcription units and adjacent, 3' flanking enhancer sequences: each transcriptionally active gene is flanked by a nonnucleosomal enhancer, whereas inactive, nucleosome-packed gene copies are followed by enhancers regularly packaged in nucleosomes. From the fact that nucleosome-free enhancers were also detected in an RNA polymerase I mutant strain, we interpret these open chromatin structures as being the result of specific protein-DNA interactions that can occur before the onset of transcription. In contrast, in this mutant strain, all of the rRNA coding sequences are packaged in nucleosomal arrays. This finding indicates that the establishment of the open chromatin conformation on the activated gene copies requires elongating RNA polymerase I molecules advancing through the template.