MOESM1 of Blue light induces a neuroprotective gene expression program in Drosophila photoreceptors

Additional file 1: Fig. S1. The blue light treatment conditions used for RNA-seq analysis do not induce retinal degeneration. Fig. S2. Affinity-enrichment of photoreceptor nuclear RNA from day one dark-treated flies. Fig. S3. Newly-eclosed flies do not show any unique blue light-induced gene expression changes. Fig. S4. Promoter motifs enriched at blue light-regulated genes. Fig. S5. Distribution of promoter motifs in blue light-regulated genes

MOESM2 of Blue light induces a neuroprotective gene expression program in Drosophila photoreceptors

Additional file 2: Table 1. Significantly differentially expressed genes identified under each comparison

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Additional file 3: Table 2. GO term analysis of differentially regulated genes

MOESM4 of Blue light induces a neuroprotective gene expression program in Drosophila photoreceptors

Additional file 4: Table 3. Transcription factors matches for all motifs identified for blue light-regulated genes

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Additional file 5: Table 4. Fly stocks used in this study

Timely Activation of Budding Yeast APC^Cdh1 Involves Degradation of Its Inhibitor, Acm1, by an Unconventional Proteolytic Mechanism

<div><p>Regulated proteolysis mediated by the ubiquitin proteasome system is a fundamental and essential feature of the eukaryotic cell division cycle. Most proteins with cell cycle-regulated stability are targeted for degradation by one of two related ubiquitin ligases, the Skp1-cullin-F box protein (SCF) complex or the anaphase-promoting complex (APC). Here we describe an unconventional cell cycle-regulated proteolytic mechanism that acts on the Acm1 protein, an inhibitor of the APC activator Cdh1 in budding yeast. Although Acm1 can be recognized as a substrate by the Cdc20-activated APC (APC^Cdc20) in anaphase, APC^Cdc20 is neither necessary nor sufficient for complete Acm1 degradation at the end of mitosis. An APC-independent, but 26S proteasome-dependent, mechanism is sufficient for complete Acm1 clearance from late mitotic and G1 cells. Surprisingly, this mechanism appears distinct from the canonical ubiquitin targeting pathway, exhibiting several features of ubiquitin-independent proteasomal degradation. For example, Acm1 degradation in G1 requires neither lysine residues in Acm1 nor assembly of polyubiquitin chains. Acm1 was stabilized though by conditional inactivation of the ubiquitin activating enzyme Uba1, implying some requirement for the ubiquitin pathway, either direct or indirect. We identified an amino terminal predicted disordered region in Acm1 that contributes to its proteolysis in G1. Although ubiquitin-independent proteasome substrates have been described, Acm1 appears unique in that its sensitivity to this mechanism is strictly cell cycle-regulated via cyclin-dependent kinase (Cdk) phosphorylation. As a result, Acm1 expression is limited to the cell cycle window in which Cdk is active. We provide evidence that failure to eliminate Acm1 impairs activation of APC^Cdh1 at mitotic exit, justifying its strict regulation by cell cycle-dependent transcription and proteolytic mechanisms. Importantly, our results reveal that strict cell-cycle expression profiles can be established independent of proteolysis mediated by the APC and SCF enzymes.</p></div

Acm1 degradation requires both the 20S core particle and the 19S regulatory complex of the 26S proteasome.

<p>A) Strain YKA407 carrying plasmid pHLP298 expressing 3HA-Acm1^5A from the <i>GAL1</i> promoter was treated first with galactose to induce 3HA-Acm1^5A expression, second with 50 µM MG-132 or a mock treatment, and third with glucose and cycloheximide to terminate expression (Time = 0). The level of 3HA-Acm1^5A was then monitored over time by immunoblotting with an HA antibody. G6PD is a loading control. B) and C) The same experiment described in panel A was performed with wild-type (YWO0607 for B, MHY753 for C) or the indicated temperature-sensitive proteasome mutant strains (YWO0612 for B, MHY754 for C) carrying plasmids expressing either 3HA-Acm1^5A, 3HA-Acm1, or Fin1-3HA from the <i>GAL1</i> promoter. Instead of MG-132 treatment, cultures were shifted to 37°C prior to terminating protein expression. For 3HA-Acm1, and Fin1-3HA, cells were arrested first in G1. D) The same strains from panels B and C were grown to exponential phase and shifted to the restrictive temperature to compare the steady-state level of endogenous Acm1 by immunoblotting with an anti-Acm1 antibody. G6PD was used as a loading control.</p

APC^Cdc20 is neither necessary not sufficient for complete Acm1 degradation at mitotic exit.

<p>A) <i>dbf2-2</i> cells expressing endogenous chromosomally tagged Clb5-3HA and Pds1-9Myc were released from a G1 arrest at 37°C and the levels of Clb5, Pds1, and Acm1 monitored by immunoblotting over the indicated time period. G6PD is a loading control. Numbers under each lane were obtained by fluorescence microscopic analysis of at least 100 cells at that timepoint stained with DAPI and scored for the presence of 2 segregated DNA masses indicative of the <i>dbf2-2</i> late anaphase arrest point. cyc, asynchronous cycling cultures. B) Protein levels were quantified from the immunoblots in panel A. The abundance of each protein was plotted as a percentage of its maximal expression level. C) Extracts from synchronized cultures of yBRT135 (Wild-type) and mutant strain yBRT159 lacking several subunits of APC (<i>apc2Δ apc11Δ cdc20Δ cdh1Δ pds1Δ clb5Δ SIC1^10x</i>) were generously provided by David Toczyski <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103517#pone.0103517-Thornton1" target="_blank">[17]</a>, and were probed for Acm1, Clb2, and the loading control G6PD by immunoblotting. The budding index under each lane, taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103517#pone.0103517-Thornton1" target="_blank">[17]</a>, is used as an indicator of cell cycle progression.</p

Proteolysis of an Acm1-ProtA fusion protein in G1 requires the N-terminal 52 amino acids of Acm1.

<p>A) YKA247 cells transformed with <i>P_GAL1</i> constructs expressing <i>ACM1</i>, <i>acm1^NΔ42</i>, <i>acm1^NΔ52</i>, <i>acm1^NΔ60</i>, <i>acm1^NΔ72</i> or <i>acm1^NΔ80</i> fused to the ZZ domain of Protein A were arrested in G1 and protein stability assayed over time by immunoblotting with anti-Protein A antibody. B) The same assay as panel A with cells expressing Acm1 amino acids 1–52 fused to Protein A. C) The stability of Acm1 and Acm1^NΔ52 without the Protein A fusion were compared using the same assay as in panel A, but with anti-Acm1 antibody for immunoblot detection. G6PD is a loading control. G6PD loading controls were performed for all blots in panels A and B as well (not shown).</p

Acm1^NΔ52 is still cleared at mitotic exit, but constitutive expression impairs growth of <i>sic1Δ</i> cells.

<p>A) YKA859 cells carrying either pHLP117 (for expression of 3HA-Acm1) or pHLP505 (for expression of Acm1^NΔ52-ProtA) were arrested at metaphase by methionine repression of <i>P_MET3-CDC20</i> and then released in the absence of methionine. Cells were collected at regular intervals and analyzed by immunoblotting using antibodies against Acm1, Clb2, or G6PD (loading control). B) Quantitation of chemiluminescent immunoblots from panel A. Data are the average of 4 independent experiments. C) Growth of <i>sic1Δ</i> cells transformed with either an empty vector, pHLP361 (<i>P_ADH</i>-<i>Acm1-ProtA</i>) or pHLP363 (<i>P_ADH</i>-<i>acm1^NΔ52-ProtA</i>) were compared using a plate reader to measure absorbance at 600 nm. Data are the average of three independent experiments with standard deviation error bars. D) Serial 10-fold dilutions of the strains from panel C as well as isogenic wild-type and <i>cdh1Δ</i> strains were spotted and grown on selective agar plates.</p