Expression profiling by high throughput sequencing
Summary
Alternative mRNA isoforms and long noncoding RNAs (lncRNA) make up a large fraction of the transcriptome and play key functions in cell-fate programming. These transcripts often initiate upstream of coding gene promoters from alternative transcription start sites (TSS) where they can regulate gene expression in cis through transcription-coupled chromatin alterations. How, when and where transcription of alternative cis-acting RNAs regulates local gene expression remains poorly understood. Here, we use a high-resolution quantitative approach to study alternative TSS and transcript end site (TES) usage during three different cell fate transitions in yeast: entry into gametogenesis, commitment to meiotic divisions and return to vegetative growth. We propose that an alternative transcriptome of mRNA isoforms and lncRNAs shapes local gene expression during cell fate transitions. Hence, changes in the types and proportions of different RNAs transcribed at a locus are important inputs for gene expression at distinct stages of development.
Overall design
5’ end RNA sequencing analysis of Saccharomyces cerevisiae SK1 strains. All strains used in this study harbor the IME1gene controlled by a copper inducible promoter (pCUP1), the NDT80 gene controlled by a galactose inducible promoter (pGAL) and a constitutively expressed Gal4 transcription factor fused to the hormone binding domain of the estrogen receptor (Gal4-ER). In addition, mutant strains include those with the following genotypes: set2, set3, set2 set3 and Spt16 with a C-terminal degron (SPT16-AID). 3 biological replicates of each condition were analysed for the sporulation and return to growth time courses. For the sporulation time course, cells were grown overnight in YPD medium, diluted, and shifted to sporulation media (SPO). IME1 was induced after 2h in Spo by adding 50 μM CuSO4. Degradation of Spt16-AID was induced at Spo 2h with 500 μM 3-indole-acetid acid (3-IAA, auxin). For the return to growth (RTG) time course, cells in meiotic prophase (Spo 6h) were washed and re-suspended in an equal volume of YPD. Details for library preparation are described below.
Less...
More...