Understanding the mechanisms of adjacent gene coregulation

The requirements for ordered cell growth and cell division depend, to a large extent, upon the varied metabolic pathways involved in producing new proteins. At the heart of these pathways is the ribosome: the large, complex, rRNA and protein containing machine that efficiently translates the nucleotide code of mRNAs into the amino acid code of the proteins. Evidence for the important role that ribosome metabolism plays with regards to controlled cell growth and division – beyond the essential requirement for translation – has come from a number of eukaryotic systems. For example, the c-myc oncoprotein functions to regulate ribosome biogenesis, and in turn, it is regulated itself by the ribosomal protein RPL11 (reviewed in Dai and Lu, 2008). In Drosophila, c-myc has been shown to control ribosome biogenesis, in part, by binding to the promoters of ribosome biogenesis genes (Grewal, 2005). In humans, the cancer predisposing diseases Diamond-Blackfan anemia, and Myelodyplastic Syndrome are associated with mutations in the small ribosomal subunit proteins RPS19, RPS17, and RPS24 respectively. Therefore, regulated cell growth and division does not simply depend upon the need for translation, it depends upon a delicate balance whereby cells coordinately regulate the hundreds of genes and proteins that participate in the various stages of ribosome biogenesis and activity.

The impetus for our research stems from our discovery that there is a large, evolutionarily conserved, metabolically important set of transcriptionally co-regulated genes that function in the rRNA and ribosome biosynthesis (RRB) pathway in budding yeast (Fig. 1). Containing some 200 members that function in various levels of the rRNA and ribosome biogenesis pathways, this regulon is one of the largest sets of co-regulated genes in yeast. Our computational analysis of the RRB gene sequences identified the conserved PAC and RRPE promoter motifs, and we subsequently demonstrated that they play important regulatory roles in vivo. Additionally, we discovered that an unusually high fraction (28/188 or 15%) of the RRB genes exist on the chromosomes as immediately adjacent gene pairs (in all orientations: tandem, divergent and convergent). This adjacent gene pairing – although previously not recognized – is extensive, and is present in the RRB and ribosomal protein (RP) regulons across widely divergent yeasts.

Figure. 1 Ribosome biogenesis requires the coordinated regulation of three extensive gene networks, including 137 cytoplasmic RP genes, 150 rRNA genes and some 200 RRB genes.

By using a genetic approach, we demonstrated that the PAC and RRPE promoter motifs from one gene of a convergent RRB gene pair are important for the regulated expression of the neighboring RRB gene who’s promoter lies in the opposite orientation, and over 3.5 kb away. This discovery of adjacent gene co-regulation is novel, and suggests that one way that cells control the regulated expression of sets of genes involved in a common biochemical pathway (i.e. ribosome biogenesis) is related to their immediate physical adjacency. Since significant RP gene pairing can be recognized in higher eukaryotes as well, understanding the mechanisms whereby adjacent gene co-regulation is achieved will be an important advance in understanding how cells coordinately regulate large sets of genes under varying conditions.

Through bioinformatics approaches, we have found that the members of the RRB regulon are highly enriched for two promoter motifs (known as PAC and RRPE). Since these short consensus sequences are ideally positioned upstream of the majority of the RRB genes, we are testing the hypothesis that they are important for regulating RRB gene expression levels in response to changing cellular conditions. We are currently taking a genetic approach to define the roles that RRPE and PAC play in modulating RRB gene expression, both when situated upstream of single or pairs of RRB genes. We are also interested in identifying the factors that bind to the PAC and RRPE motifs, and to characterize how their activities may contribute to altered RRB gene expression levels in response to varying cellular conditions (i.e. heat shock, osmotic shock etc.).

Current lab members: Anand Soorneedi, Jeff Arace, Jun Huang, Tadeshi Kamitaki