Effect of LEM domain protein depletion on the expression of the <i>60D1</i> gene-cluster.

<p>Bars show increased expression of the cluster genes in <i>S2</i> cells determined with RT-qPCR after treatment with the mixture of <i>dMAN1</i>, <i>Bocksbeutel</i>, and <i>Otefin</i> dsRNAs. The control <i>LacZ</i> dsRNA-treated cells served as the reference. Gene symbols are shown on the X-axis; the <i>60D1</i> cluster is boxed. n = 6; error bars show SEM; *, p≤0.05 for comparison of individual transcript levels between <i>LacZ</i> RNAi and target RNAi; ††, p≤0.01 for comparison between the <i>60D1</i> cluster and control housekeeping genes. Inserts show the knockdown efficiency of the RNAi at the RNA levels.</p

Role of Histone Deacetylases in Gene Regulation at Nuclear Lamina

<div><p>Theoretical models suggest that gene silencing at the nuclear periphery may involve “closing” of chromatin by transcriptional repressors, such as histone deacetylases (HDACs). Here we provide experimental evidence confirming these predictions. Histone acetylation, chromatin compactness, and gene repression in lamina-interacting multigenic chromatin domains were analyzed in Drosophila <em>S2</em> cells in which B-type lamin, diverse HDACs, and lamina-associated proteins were downregulated by dsRNA. Lamin depletion resulted in decreased compactness of the repressed multigenic domain associated with its detachment from the lamina and enhanced histone acetylation. Our data reveal the major role for HDAC1 in mediating deacetylation, chromatin compaction, and gene silencing in the multigenic domain, and an auxiliary role for HDAC3 that is required for retention of the domain at the lamina. These findings demonstrate the manifold and central involvement of class I HDACs in regulation of lamina-associated genes, illuminating a mechanism by which these enzymes can orchestrate normal and pathological development.</p> </div

Effect of B-type Lamin depletion on chromatin compactness and histone acetylation.

<p>(<b>A</b>) Increase in general sensitivity to DNase I upon dsRNA-induced depletion of <i>LamDm_o</i>. Permeabilized cells were treated with DNase I and DNA damage was quantified by qPCR and normalized to the amplicons located at 37–39 kb (outside the <i>60D1</i> cluster), as shown for the <i>LamDm_o</i>-depleted cells in comparison to control <i>LacZ</i> dsRNA-treated cells. Horizontal axis shows positions of amplicons relative to the testis-specific <i>60D1</i> gene cluster outlined with a box, and its genes highlighted in black. (<b>B</b>) Increase in histone acetylation along the <i>60D1</i> cluster in <i>LamDm_o</i> dsRNA-treated cells as compared to control <i>LacZ</i> dsRNA-treated cells. Acetylation of histones H3 (left panel) and H4 (right panel) was detected by ChIP assay. Horizontal axis is same as in (A). n = 3 to 6; error bars show SEM; *, p≤0.05; **, p≤0.01 for comparisons to the control. Inserts show the knockdown efficiency of the dsRNA at the RNA levels.</p

Effects of Class I HDAC depletion on histone acetylation and chromatin compactness.

<p>(<b>A</b>) ChIP assay shows increased acetylation of histones H3 (left panel) and H4 (right panel) in cells treated with <i>HDAC1</i> dsRNA and <i>HDAC3</i> dsRNAs as compared to the <i>LacZ</i> dsRNA-treated control cells. n = 4; error bars represent SEM. (<b>B</b>) Decreased chromatin compactness revealed by the general sensitivity to DNase I assay in <i>HDAC1</i> dsRNA-treated cells as compared to the control <i>LacZ</i> dsRNA treatment. Gene positions are shown below the X-axis with the <i>60D1</i> cluster framed. n = 2 to 4; error bars show SEM. *, p≤0.05; **, p≤0.01; ***, p≤0.001 for comparisons to the control. Inserts show the knockdown efficiency of the RNAi at the RNA levels.</p

Effect of HDAC depletion on retention of the 60D1 locus at the nuclear periphery.

<p>(<b>A</b>) Position of the locus was determined by FISH (red) and the nuclear envelope visualized with immunostaining for <i>LamDm_o</i> (green). Figure shows representative nuclei of cells treated with control <i>LacZ</i> dsRNA or a mixture of <i>HDAC1</i> and <i>HDAC3</i> dsRNAs. (<b>B</b>) Bars show the proportion of nuclei with FISH signals ≤0.4 µm apart from the nuclear envelope. dsRNAs used for depletion are indicated below the X-axis. <i>LacZ</i>, n = 256, 3 independent experiments; <i>HDAC1</i>, n = 199, 2 independent experiments; <i>HDAC3</i>, n = 201, 2 independent experiments; <i>HDAC1</i>+<i>HDAC3</i>, n = 208, 2 independent experiments; <i>LamDm_o</i>, n = 89, 2 independent experiments. Error bars show SEM. *, p≤0.05 for comparisons to <i>LacZ</i> control.</p

Effect of Class I HDAC depletion on expression of the <i>60D1</i> gene cluster.

<p>(<b>A</b>) Treatment of cells with <i>HDAC1</i> dsRNA results in increased transcript levels for the testis-specific cluster. Bars show changes in transcript levels detected with RT-qPCR in cells treated with <i>HDAC1</i> dsRNA or <i>HDAC3</i> dsRNA, as compared to the <i>LacZ</i> dsRNA-treated control cells. (<b>B</b>) Increased changes in transcript levels upon treatment of the cells with the mixture of <i>HDAC1</i> and <i>HDAC3</i> dsRNAs. Gene symbols are shown on the X-axis; the <i>60D1</i> gene-cluster is framed. <i>Rp49</i> and <i>Actin5C</i> are housekeeping genes used as controls. <i>Rpl9</i> served as a template for loading reference. n = 6 to 9; error bars show SEM; **, p≤0.01; ***, p≤0.001 for comparison of individual transcript levels between <i>LacZ</i> RNAi and target RNAi; †††, p≤0.001 for comparison between the <i>60D1</i> cluster and control housekeeping genes. Inserts show the knockdown efficiency of the RNAi at the RNA levels.</p