Ume6 transcription factor is part of a signaling cascade that regulates autophagy

Abstract

Autophagy has been implicated in a number of physiological processes important for human heath and disease. Autophagy involves the formation of a double-membrane cytosolic vesicle, an autophagosome. Central to the formation of the autophagosome is the ubiquitin-like protein autophagy-related (Atg)8 (microtubule-associated protein 1 light chain 3/LC3 in mammalian cells). Following autophagy induction, Atg8 shows the greatest change in expression of any of the proteins required for autophagy. The magnitude of autophagy is, in part, controlled by the amount of Atg8; thus, controlling Atg8 protein levels is one potential mechanism for modulating autophagy activity. We have identified a negative regulator of ATG8 transcription, Ume6, which acts along with a histone deacetylase complex including Sin3 and Rpd3 to regulate Atg8 levels; deletion of any of these components leads to an increase in Atg8 and a concomitant increase in autophagic activity. A similar regulatory mechanism is present in mammalian cells, indicating that this process is highly conserved.

Fig. 1.

(A) Diagram depicting the URS1 site in the ATG8 promoter. (B) The Ume6-Sin3-Rpd3 complex represses Atg8 expression. Wild-type (BY4742), ume6Δ, sin3Δ, and rpd3Δ yeast cells were grown in rich medium to midlog phase. Protein extracts from cells were prepared and subjected to immunoblotting with anti-Atg8 and anti-Pgk1 antiserum (the latter as a loading control).

Fig. 2.

Ume6 binds the ATG8 promoter and negatively regulates ATG8 transcription. (A) Expression of ATG8p-LacZ in a UME6 deletion strain. Wild-type and ume6Δ cells containing LacZ driven by the ATG8 promoter were grown to midlog phase and switched to nitrogen starvation medium (SD-N) for 2 h. β-galactosidase activity was measured from protein extracts. (B) Protein A–tagged Ume6 binds the ATG8 promoter. ChIP analysis was conducted on two regions of the ATG8 promoter: the URS1 region and a region −3 kb upstream of the ATG8 start codon (-3K), which was used as a negative control. The URS1 region in the INO1 promoter served as a positive control. The ChIP results were normalized to the input DNA and calibrated to the -3K PCR product, which was set to 1.0. Error bars represent the SD of at least three independent experiments.

Fig. 3.

Rim15 promotes Ume6 phosphorylation and functions as a positive regulator of Atg8 induction. (A) Rim15 is required for Ume6 phosphorylation in starvation conditions. Wild-type (WT, BY4742) and rim15Δ cells were grown in rich medium and starved in SD-N for up to 1 h. Cells were collected, and protein extracts were analyzed with anti-Ume6 and anti-Pgk1 (loading control) antisera. (B) Wild-type (WT, YZD005), rim15Δ (YZD006), and rim15Δ ume6Δ (YZD007) cells in a pep4Δ background were grown in rich medium and shifted to SD-N for starvation. Cells were collected at the indicated time points and subjected to immunoblotting with anti-Atg8 and anti-Pgk1 antisera.

Fig. 4.

Ume6 negatively regulates autophagy. (A) Autophagy as measured by the Pho8Δ60 assay is increased in ume6Δ cells. Wild-type (YCB193, SEY6210), atg1Δ (YCB194), and ume6Δ (YCB197) cells were grown in SMD medium and then starved for 0, 0.5, 1, 2, 3, and 4 h. The Pho8Δ60 activity was measured as described in Materials and Methods and normalized to the activity of the wild-type cells, which was set to 100%. Error bars indicate the SEM of three independent experiments. (B) Autophagosome size is increased in ume6Δ cells. Wild-type (FRY143, SEY6210) and ume6Δ (YCB234) strains with deletions of VPS4 and PEP4 to eliminate vesicles generated from the multivesicular body pathway and the breakdown of autophagic bodies, respectively, were grown in rich medium and starved in SD-N for 2 h. Samples were collected, prepared, and examined by TEM as described in Materials and Methods. The radius of each autophagosome was determined as described in Materials and Methods. The error represents the SEM for >400 autophagic bodies. (C) Representative TEM images of the cells in B. (Scale bars: 500 nm.)

Fig. 5.

SIN3A and SIN3B play redundant roles in regulating LC3 expression. (A) SIN3A- and SIN3B-targeted shRNA was prepared and used to generate viruses as described in Materials and Methods. The shRNA-expressing viruses were infected in combination into HeLa, HEK293T, and human fibroblast cells using scrambled DNA as a control (C). Cell lysates were analyzed by immunoblotting with the indicated antibodies. (B) SIN3A and SIN3B mRNA levels were monitored by quantitative PCR in shRNA-treated cells. The values for scrambled DNA were set to 1.0, and the other values were normalized. *P < 0.05; **P < 0.01. Error bars represent the SD.