The large fraction of heterochromatin in Drosophila neurons is bound by both B-type lamin and HP1a

CONCLUSIONS: In various differentiated Drosophila cell types, we discovered the existence of peripheral heterochromatin, similar to that observed in mammals. Our findings support the model that peripheral heterochromatin matures enhancing the repression of unwanted genes as cells terminally differentiate.BACKGROUND: In most mammalian cell lines, chromatin located at the nuclear periphery is represented by condensed heterochromatin, as evidenced by microscopy observations and DamID mapping of lamina-associated domains (LADs) enriched in dimethylated Lys9 of histone H3 (H3K9me2). However, in Kc167 cell culture, the only Drosophilla cell type where LADs have previously been mapped, they are neither H3K9me2-enriched nor overlapped with the domains of heterochromatin protein 1a (HP1a).RESULTS: Here, using cell type-specific DamID we mapped genome-wide LADs, HP1a and Polycomb (Pc) domains from the central brain, Repo-positive glia, Elav-positive neurons and the fat body of Drosophila third instar larvae. Strikingly, contrary to Kc167 cells of embryonic origin, in neurons and, to a lesser extent, in glia and the fat body, HP1a domains appear to overlap strongly with LADs in both the chromosome arms and pericentromeric regions. Accordingly, centromeres reside closer to the nuclear lamina in neurons than in Kc167 cells. As expected, active gene promoters are mostly not present in LADs, HP1a and Pc domains. These domains are occupied by silent or weakly expressed genes with genes residing in the HP1a-bound LADs expressed at the lowest level

Alterations in Synthesis and Repair of DNA during the Development of Loach Misgurnus fossilis

Using a modified radiolabeled primer extension method (we named this modification misGvA—“misincorporation of G versus A”) we have investigated the DNA synthesis and repair at early and late stages of development of loach Misgurnus fossilis. The misincorporation activity of DNA polymerase iota (Pol ι) in wild-type loach could not be detected by this method at any stage of loach development. In transgenic loach overexpressing human Pol ι we have shown that the bypassing of DNA synthesis arrest after incorporation of mismatched nucleotide by Pol ι (the T-stop) was not associated with this enzyme. Non-transgenic loach larvae are virtually lacking the capacity for error correction of DNA duplex containing a mismatched nucleotide. Such repair activity develops only in the adult fish. It appears that the initial stages of development are characterized by more intensive DNA synthesis, while in terminal stages the repair activities become more prominent. The misGvA approach clearly indicates substantial changes in the DNA synthesis intensity, although the role of particular replicative and repair DNA polymerases in this process requires further study

Transcription of <i>HOX</i> Genes Is Significantly Increased during Neuronal Differentiation of iPSCs Derived from Patients with Parkinson’s Disease

Parkinson’s disease (PD) is the most serious movement disorder, but the actual cause of this disease is still unknown. Induced pluripotent stem cell-derived neural cultures from PD patients carry the potential for experimental modeling of underlying molecular events. We analyzed the RNA-seq data of iPSC-derived neural precursor cells (NPCs) and terminally differentiated neurons (TDNs) from healthy donors (HD) and PD patients with mutations in PARK2 published previously. The high level of transcription of HOX family protein-coding genes and lncRNA transcribed from the HOX clusters was revealed in the neural cultures from PD patients, while in HD NPCs and TDNs, the majority of these genes were not expressed or slightly transcribed. The results of this analysis were generally confirmed by qPCR. The HOX paralogs in the 3′ clusters were activated more strongly than the genes of the 5′ cluster. The abnormal activation of the HOX gene program upon neuronal differentiation in the cells of PD patients raises the possibility that the abnormal expression of these key regulators of neuronal development impacts PD pathology. Further research is needed to investigate this hypothesis

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 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

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

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

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