The Roles of the Catalytic and Noncatalytic Activities of Rpd3L and Rpd3S in the Regulation of Gene Transcription in Yeast

<div><p>In budding yeasts, the histone deacetylase Rpd3 resides in two different complexes called Rpd3L (large) and Rpd3S (small) that exert opposing effects on the transcription of meiosis-specific genes. By introducing mutations that disrupt the integrity and function of either Rpd3L or Rpd3S, we show here that Rpd3 function is determined by its association with either of these complexes. Specifically, the catalytic activity of Rpd3S activates the transcription of the two major positive regulators of meiosis, <i>IME1</i> and <i>IME2</i>, under all growth conditions and activates the transcription of <i>NDT80</i> only during vegetative growth. In contrast, the effects of Rpd3L depends on nutrients; it represses or activates transcription in the presence or absence of a nitrogen source, respectively. Further, we show that transcriptional activation does not correlate with histone H4 deacetylation, suggesting an effect on a nonhistone protein. Comparison of <i>rpd3</i>-null and catalytic-site point mutants revealed an inhibitory activity that is independent of either the catalytic activity of Rpd3 or the integrity of Rpd3L and Rpd3S. </p> </div

Model for the regulation of meiosis-specific genes by Rpd3.

<p>Blue rectangle: Core components; Purple rectangle: components specific to Rpd3L; Green rectangles components specific to Rpd3S. The components whose function was examined in this research are marked. Growth: Transcription under vegetative growth conditions with acetate as the sole carbon source. Meiosis: nitrogen depletion in the presence of acetate as the sole carbon source.</p

Diploids with <i>DEP1/RCO1</i> or <i>SDS3/RCO1</i> deletions arrest in meiosis before nuclear division.

<p>Isogenic wt (Y1631, closed squares), <i>dep1∆/dep1∆</i> (Y1894, open circles), and <i>rco1∆/rco1∆ </i><i>sds3∆/sds3∆</i> (Y1883, open triangle) diploids were shifted to meiotic conditions (SPM medium). Samples were taken at the indicated times for FACS analysis to calculate the percentage of cells with 4C DNA content and to count the percentage of cells with more than 2 nuclei (DAPI stain).</p

Experimental Proof for the Role of Nonlinear Photoionization in Plasmonic Phototherapy

Targeting individual cells within a heterogeneous tissue is a key challenge in cancer therapy, encouraging new approaches for cancer treatment that complement the shortcomings of conventional therapies. The highly localized interactions triggered by focused laser beams promise great potential for targeting single cells or small cell clusters; however, most laser-tissue interactions often involve macroscopic processes that may harm healthy nearby tissue and reduce specificity. Specific targeting of living cells using femtosecond pulses and nanoparticles has been demonstrated promising for various potential therapeutic applications including drug delivery via optoporation, drug release, and selective cell death. Here, using an intense resonant femtosecond pulse and cell-specific gold nanorods, we show that at certain irradiation parameters cell death is triggered by nonlinear plasmonic photoionization and not by thermally driven processes. The experimental results are supported by a physical model for the pulse-particle-medium interactions. A good correlation is found between the calculated total number and energy of the generated free electrons and the observed cell death, suggesting that femtosecond photoionization plays the dominant role in cell death

Repression of transcription by Rpd3 requires Rpd3L and Rpd3S.

<p>Cells carrying the <i>UAS</i>_<i>GAL1</i><i>-</i>UAS_<i>HIS4</i><i>-his4-lacZ</i> reporter gene were grown in SD medium to a density of 10⁷ cells/ml. The activity of β-galactosidase (Miller units) in cells expressing Gal4(dbd)-Rpd3 is relative to the level in the control cells (c) expressing only Gal4(dbd). Diploid strains used were as follows: wild-type (Y1884), <i>rco1</i>Δ/<i>rco1</i>Δ (Y1814), <i>sds3</i>Δ/<i>sds3</i>Δ (Y1845), <i>dep1</i>Δ/<i>dep1</i>Δ (Y1894), <i>ume6</i>Δ/<i>ume6</i>Δ (Y1388), and <i>rco1</i>Δ/<i>rco1</i>Δ <i>sds3</i>Δ/<i>sds3</i>Δ (Y1883). These strains carried either <i>pADH1-gal4</i>(dbd)<i>-</i>RPD3 (YEp2593) or <i>pADH1-gal4</i>(dbd) (YEp2149) on a 2-µ vector. Proteins were extracted from at least three independent transformants. </p

The kinetics of Rpd3 binding, histone H4 acetylation, and transcription of meiosis-specific genes.

<p><i>MAT</i>a/<i>MAT</i>α <i>RPD3-13xmyc</i>/<i>RPD3-13xmyc</i> (Y1767) cells were shifted to meiotic conditions (SPM media) for the times indicated and subjected to ChIP analysis to determine Rpd3 binding (white column) and acetylated H4 (gray column). Sequences of the <i>IME2</i> (A), and <i>NDT80</i> promoters (B), or the <i>TEL1</i> locus were amplified using qPCR. Enrichment values represent the ratio between the relative levels of PCR amplicons recovered from the specific versus the non-specific probe, then the bound fraction was divided by input. Samples were taken simultaneously to isolate RNA for qPCR analysis (black line with triangles).</p

Possible molecular mechanisms of transcriptional activation by Rpd3.

<p>A. Deletion of <i>SUM1</i> did not suppress <i>rpd3</i>Δ. Isogenic wild-type (Y1631, closed black squares), <i>rpd3</i>Δ/<i>rpd3</i>Δ (Y1888, empty black squares, dashed line) and <i>rpd3</i>Δ/<i>rpd3</i>Δ <i>sum1</i>Δ<i>/sum1</i>Δ (Y1914, gray circles, dashed lines). B. Ectopic transcription of <i>NDT80</i> partially suppressed the effect of <i>rpd3</i>Δ on the transcription of <i>SPS1</i> and nuclear division. Isogenic <i>NDT80</i>/<i>NDT80</i> (Y1631, black squares), <i>IME2p-6xHA-NDT80/IME2p-6xHA-NDT80</i> (Y1763, gray triangles), <i>rpd3</i>Δ/<i>rpd3</i>Δ (Y1537, empty gray squares, dashed gray lines) and <i>rpd3</i>Δ/<i>rpd3</i>Δ <i>IME2p-6xHA-NDT80/IME2p-6xHA-NDT80</i> (Y1870, empty gray triangle, dashed gray lines) cells were shifted to meiotic conditions (SPM medium), and at the indicated times, samples were taken for RNA extraction and DAPI staining to determine the percentage of cells with more than 1 nucleus. <i>NDT80</i> expression was measured using q-RT PCR. Levels of expression are relative to that of <i>ACT1</i>. The results of a representative experiment are shown. Similar results were obtained from three independent experiments.</p