High-mobility group (HMG) proteins are DNA binding proteins with low sequence specificity that were originally identified on the basis of their physical characteristics (1
). There are three functional classes of HMG protein: HMGA, HMGB, and HMGN. HMGB proteins have a molecular mass of ~25 kDa, and they contain HMG boxes that partially intercalate the DNA in the minor groove and cause a sharp bend. HMGB proteins have a preference for binding to distorted DNA, and they are believed to function as architectural proteins that stabilize multiprotein complexes on DNA. HMGB proteins are found in many eukaryotic species, and they are involved in a number of cellular processes, including transcription, replication, and DNA repair. However, little is known about the genomic association and biological functions of these proteins.
The yeast Saccharomyces cerevisiae
contains 10 HMG proteins, including the HMGB protein, Hmo1. Hmo1 has two HMG boxes, A and B. Box A has low affinity for DNA with some structural specificity, whereas box B has higher affinity for DNA with lower structural specificity (16
). Strains lacking Hmo1 show a decreased growth rate, higher rates of plasmid loss, and increased sensitivity of chromatin to micrococcal nuclease treatment (23
). Hmo1 interacts genetically and physically with FKBP12, a conserved prolyl isomerase (8
), and it also plays a role in mutagenesis control (2
). Hmo1 also plays a role in transcription of the rRNA (9
In S. cerevisiae
, the ribosome consists of the 5S, 5.8S, 18S, and 25S rRNAs as well as 137 ribosomal proteins (RPs). The 5S RNA is transcribed by RNA polymerase (Pol) III, the 5.8S, 18S, and 25S RNAs are transcribed as a single unit by Pol I, and the RP genes are transcribed by Pol II. Together this represents more than 70% of total cellular RNA and approximately 50% of all mRNA (43
). Pol I-directed transcription of rRNA requires the factors TATA binding protein (TBP), core factor (CF), and upstream activation factor (UAF), whereas an additional factor, upstream binding factor (UBF), is required for mammalian cells. UBF contains six HMG boxes and binds throughout the repeated rRNA gene locus (30
), and it shows remote sequence similarity to Hmo1 (9
Transcription of all these genes is regulated positively in response to growth stimuli and negatively in response to environmental stress, and coordinated regulation of these processes is critical for ribosome synthesis and cell growth (43
). During rapid growth, Fhl1 and Rap1 bound at RP promoters recruit Ifh1, which in turn activates transcription to maximal levels (25
). Following stress, Ifh1 dissociates from RP promoters and Fhl1 recruits the inhibitory factor Crf1, resulting in decreased transcription (25
). An additional factor, Sfp1, is also required for maximal transcription from RP promoters. Sfp1 associates with RP promoters specifically under conditions of rapid growth and is exported from the nucleus following stress (15
), but its precise role in RP transcription is unclear.
Here we use chromatin immunoprecipitation coupled with microarray analysis to identify the physiological targets of Hmo1 on a genome-wide level. Although Hmo1 exhibits minimal DNA binding specificity in vitro, we show that Hmo1 associates strongly and specifically with most RP promoters at an IFHL sequence motif. Our results strongly suggest that Hmo1 binds cooperatively with Rap1 and Fhl1 to RP promoters, whereas Hmo1 binding at the rRNA gene locus occurs independently of Rap1 and Fhl1. Surprisingly, loss of Hmo1 abolishes binding of Fhl1 and Ifh1 to RP promoters but does not significantly affect the level of transcriptional activity, suggesting that proteins other than Fhl1 and Ifh1 play an important role in RP transcription. Lastly, we show that Hmo1 is also required for both transcription and processing of the rRNA and, like mammalian UBF, associates throughout the rRNA gene locus. We propose that Hmo1 is involved in the coordination of rRNA and RP gene transcription.