Hungry for Sex: Differential Roles for Ustilago maydisb Locus Components in Haploid Cells vis à vis Nutritional Availability.

Mating-types allow single-celled eukaryotic organisms to distinguish self from non-self in preparation for sexual reproduction. The components of mating-type loci provide initial self/non-self-recognition through pheromone and receptor interactions that control early cell fusion events. However, they may also provide a second level of scrutiny that requires differences in alleles leading to production of a transcription factor required for successful downstream developmental pathways after initial cell fusion. Interestingly, the protein subunits of these transcription factors have not been thoroughly examined for their roles, if any, in the haploid cells themselves. In Ustilago maydis, the causative agent of galls in maize plants, the b locus, encoding bEast (bE) and bWest (bW), components of the eventual requisite transcription factor, has been extensively studied for its role in formation of the stable dikaryon after mating and subsequent pathogenic program. Little is known, however, about any roles for bE or bW in haploid cells. Since mating in fungi is often induced under conditions of nitrogen starvation, we have explored connections between the b locus and the nitrogen-sensing and response pathways in U. maydis. We previously identified a connection in haploid cells between the b locus and Ump2, the high-affinity transceptor, a protein that both transports ammonium and triggers filamentous growth as a response to nitrogen starvation. Deletion of the entire b locus abrogates the filamentous response to low ammonium, a phenotype that is rescued by overexpression of Ump2. Here we further investigated the individual roles of bE and bW in haploid cells. We show that bE and bW are expressed differentially in haploid cells starved for ammonium. Their respective deletion elicits different effects on transcription of mating and pathogenic-related genes and, importantly, on the degree of pathogenic development in host plants. This is the first demonstration of a role for these mating locus components on haploid development and the first to demonstrate a connection to the ammonium transceptors.

Regulation of miR-29b-1/a transcription and identification of target mRNAs in CHO-K1 cells.

miR-29b and miR-29a transcript levels were reported to increase in exponentially growing CHO-K1 cells. Here, we examine the regulation of miR-29b-1/a in CHO-K1 cells. We observed that 4-hydroxytamoxifen (4-OHT) increased pri-miR-29b-1 and pri-miR-29a transcription in CHO-K1 cells by activating endogenous estrogen receptor α (ERα). DICER, an established, bona fide target of miR-29b-1/a, was shown to be regulated by 4-OHT in CHO-K1 cells. We showed that miR-29b-1 and miR-29a serve a repressive role in cell proliferation, migration, invasion, and colony formation in CHO-K1 cells. To identify other targets of miR-29b-1 and miR-29a, RNA sequencing was performed by transfecting cells with anti-miR-29a, which inhibits both miR-29a and miR-29b-1, pre-miR-29b-1, and/or pre-miR-29a. In silico network analysis in MetaCore™ identified common and unique putative gene targets of miR-29b-1 and miR-29a. Pathway analysis of identified putative miR-29 targets were related to cell adhesion, cytoskeletal remodeling, and development. Further inquiry revealed regulation of pathways mediating responses to growth factor stimulus and cell cycle regulation.

Tamoxifen differentially regulates miR-29b-1 and miR-29a expression depending on endocrine-sensitivity in breast cancer cells.

Endocrine-resistance develops in ∼40% of breast cancer patients after tamoxifen (TAM) therapy. Although microRNAs are dysregulated in breast cancer, their contribution to endocrine-resistance is not yet understood. Previous microarray analysis identified miR-29a and miR-29b-1 as repressed by TAM in MCF-7 endocrine-sensitive breast cancer cells but stimulated by TAM in LY2 endocrine-resistant breast cancer cells. Here we examined the mechanism for the differential regulation of these miRs by TAM in MCF-7 versus TAM-resistant LY2 and LCC9 breast cancer cells and the functional role of these microRNAs in these cells. Knockdown studies revealed that ERα is responsible for TAM regulation of miR-29b-1/a transcription. We also demonstrated that transient overexpression of miR-29b-1/a decreased MCF-7, LCC9, and LY2 proliferation and inhibited LY2 cell migration and colony formation but did not sensitize LCC9 or LY2 cells to TAM. Furthermore, TAM reduced DICER1 mRNA and protein in LY2 cells, a known target of miR-29. Supporting this observation, anti-miR-29b-1 or anti-miR-29a inhibited the suppression of DICER by 4-OHT. These results suggest miR-29b-1/a has tumor suppressor activity in TAM-resistant cells and does not appear to play a role in mediating TAM resistance.