Alexander S. Brodsky

Chromatin structure affects the accessibility of DNA to transcription, repair, and replication. Changes in chromatin structure occur during development, but less is known about changes during aging. We examined the state of chromatin structure and its effect on gene expression during aging in Drosophila at the whole genome and cellular level using whole-genome tiling microarrays of activation and repressive chromatin marks, whole-genome transcriptional microarrays and single-cell immunohistochemistry. We found dramatic reorganization of chromosomal regions with age. Mapping of H3K9me3 and HP1 signals to fly chromosomes reveals in young flies the expected high enrichment in the pericentric regions, the 4th chromosome, and islands of facultative heterochromatin dispersed throughout the genome. With age, there is a striking reduction in this enrichment resulting in a nearly equivalent level of H3K9me3 and HP1 in the pericentric regions, the 4th chromosome, facultative heterochromatin, and euchromatin. These extensive changes in repressive chromatin marks are associated with alterations in age-related gene expression. Large-scale changes in repressive marks with age are further substantiated by single-cell immunohistochemistry that shows changes in nuclear distribution of H3K9me3 and HP1 marks with age. Such epigenetic changes are expected to directly or indirectly impinge upon important cellular functions such as gene expression, DNA repair, and DNA replication. The combination of genome-wide approaches such as whole-genome chromatin immunoprecipitation and transcriptional studies in conjunction with single-cell immunohistochemistry as shown here provide a first step toward defining how changes in chromatin may contribute to the process of aging in metazoans.

RNA export from the nucleus is thought to be linked to proper processing and packaging into ribonucleoprotein protein complexes. A system to observe mRNA nuclear export in living yeast cells was developed by fusing the U1A RNA-binding protein to the green fluorescent protein to follow specific mRNAs with U1A hairpins engineered into them. RNAs encoding Rpl25, Pgk1, and Ssa4 were examined for the effects of 3' UTRs, introns, RNA processing factors, nucleoporins, and transport factors on their export. All accumulated in the nucleus in mutants affecting components of the nuclear export machinery and certain nucleoporins. However, under conditions of stress, PGK1 and RPL25 transcripts accumulate in the nucleus whereas SSA4 RNA is exported. Moreover, when export is blocked, only RNAs containing the ASH1 3' UTR accumulated in the nucleolus. Mutations in the splicing machinery selectively blocked export of only intron-containing RNAs. Mutations in RNA14, RNA15, and PAP1, which encode factors important for 3' processing, also blocked export of all RNAs, including SSA4, thereby linking export to the process of polyadenlyation. Taken together, these data graphically display the connections between mRNA processing and nuclear export.