Effects Of Dicer Knockdown On Chromatin Structure And The Related Cellular Function In Human Cells

Dicer is a ribonuclease III-like enzyme that plays a key role in the biogenesis of miRNAs and siRNAs. In addition, Dicer is essential for the regulation of chromatin structure and function, DNA replication timing, genome stability and cellular senescence. Targeted knockout of Dicer leads to the inability to maintain the stem cell population during early mouse development, aberrant T cell differentiation, meiosis arrest of mouse oocytes, dilated cardiomyopathy, poor proliferation of primordial germ cells and spermatogonia, aberrant B cell development, glomerular disease, degeneration of the mouse retina, and neurological disorders.It has been reported that RNAi-dependent chromatin silencing in vertebrates is not restricted to the centromeres. To address whether RNAi machinery could regulate the chromatin structure of imprinted genes, we knocked down Dicer in HEK293 cells, and found that the expression of PHLDA2, one of the several genes in the imprinted gene domain of 11p15.5, was specifically upregulated. This was accompanied by a shift towards more activated chromatin at PHLDA2 locus as indicated by change in H3K9 acetylation, however, the methylation state at this locus was not affected. Furthermore, we found that PHLDA2 was downregulated in growth-arrested HEK293 cells induced by either serum deprivation or contact inhibition. This suggests that PHLDA2 upregulation might be a direct result of Dicer knockdown-induced chromatin decomdensation rather than the consequence of growth arrest induced by Dicer knockdown.Chromatin structure plays pivotal roles in maintaining genome stability. Loss of Dicer2, a key enzyme in the RNAi pathway, not only results in decondensation of heterochromatin but also leads to accumulation of extrachromosomal circular (ecc) repeated DNAs. Ligase IV, an essential regulator of nonhomologous end joining, and perhaps other DNA damage repair machinery, participate in ecc DNA formation, this suggests that, in addition to increased accessibility of DNA repair and recombination proteins to repeated DNA caused by heterochromatin decondensation, activation of the DNA damage response may also contribute to the formation of ecc DNA in Dicer2 mutant cells. DNA replication timing is tightly regulated and correlates with chromatin state, and the timing of satellite DNA replication is misregulated in Dicer -deficient embryonic stem cells. Stalled and collapsed replication forks elicit the DNA damage response. In addition, loss of Dicer may activate transposons, which in turn leads to DNA damage. These observations collectively suggest that DNA damage response may be elicited in Dicer -deficient cells. To test this hypothesis, we knocked down Dicer in HEK293 cells and human hepatoma HepG2 cells. DNA damage was measured by immunostaining for the phosphorylated form of histone H2AX (γ-H2AX), a widely used marker for DSBs, and the replication protein A 70 (RPA70), a protein involved in DNA replication, recombination, and repair, that becomes phosphorylated and forms intranuclear foci upon exposure of cells to DNA damage, our results indicated that a much higher percentage of Dicer knockdown cells also displayed intense RPA foci andγ-H2AX foci. Consistent with the RPA andγ-H2AX staining results, an increase in checkpoint kinase 1 (Chk1) phosphorylation on S345, an event associated with DNA damage, was found in Dicer knockdown cells. As a more direct assessment of DNA damage, comet assay revealed that knockdown of Dicer resulted in accumulation of DNA breaks, as indicated by formation of a comet-like tail after single-cell gel electrophoresis. As a consequence of DNA damage, the DNA damage-induced genes, including growth arrest– and DNA damage– inducible geneα(GADD45A), growth arrest– and DNA damage– inducible geneβ(GADD45B), p21, B cell translocation gene 3 (BTG3), activating transcription factor 3 (ATF3), and early growth response 1 (EGR1) were up-regulated in Dicer knockdown cells.The DNA damage pathway regulates innate immune system ligands for the NKG2D receptor, and human NKG2D ligands are up-regulated by genotoxic stress and stalled DNA replication, conditions known to activate a major DNA damage checkpoint pathway. To test whether Dicer knockdown-induced DNA damage could activate the expression of NKG2D ligands, we checked the expression of major histocompatibility complex class I– related molecules A and B (MICA and MICB), ULBP1, -2, and -3 using quantitative RT-PCR and flow cytometry. Our results indicated that MICA and MICB were upregulated in Dicer knockdown cells, while the expression of ULBP1, -2, and -3 was not affected by Dicer knockdown. Up-regulation of MICA and MICB by Dicer knockdown is prevented by pharmacologic or genetic inhibition of DNA damage pathway components, including ataxia telangiectasia mutated (ATM) kinase, ATM- and Rad3-related kinase, or checkpoint kinase 1, indicating that up-regulation of MICA and MICB is the result of DNA damage response activation caused by Dicer knockdown. As a consequence of MICA and MICB upregulation, Dicer knockdown sensitized HEK293T cells and HepG2 cells to the lysis of NKL, a cell line derived from an aggressive form of human natural killer (NK) cell leukemia.To further confirm that chromatin decondensation causes DNA damage and induces the expression of MICA and MICB, we treated HEK293T cells and HepG2 cells with 5-Aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor. DNA methylation is essential for the heterochromatin condensation, 5-aza-dC treatment induces global DNA demethylation and hence heterochromatin decondensation. Our results demonstrated that 5-aza-dC treatment leads DNA damage and upregulation of MICA and MICB.Depletion of Dicer was found to impair the migration of endothelial cells, and fibronectin-1 (FN1) was upregulated in Dicer knockdown endothelial cells. EGR1 binds to the fibronectin-1 promoter and induces the expression of fibronectin-1. We found that EGR1 was upregulated in Dicer knockdown cells. Therefore, we hypothesized that Dicer knockdown may induce fibronectin-1 expression via an EGR1-dependant mechanism. To test this hypothesis, we knocked-down Dicer expression in HEK293T cells, and found that decreased Dicer expression induced the expression of fibronectin-1, and that the upregulation of fibronectin-1 in Dicer knockdown cells was caused by EGR1. Knockdown of Dicer sensitized cells to apoptosis induced by fibronectin-1 knockdown. Furthermore, we found that knockdown of Dicer impairs the migratory capacity of HEK293T cells.We proposed the following model: Dicer is essential for heterochromatin formation, loss of Dicer leads to chromatin decondensation, which in turn disrupts DNA replication timing and induces DNA damage; chromatin decondensation may also activate transponsable elements, in addition, decreased Dicer expression may stabilize the dsRNAs transcribed from transposable elements and hence enhance transposition, activation of transposition leads to DNA damage. DNA damage induces the expression of EGR1, which in turn activates the transcription of fibronectin-1, upregulation of fibronectin-1 impair cell migratory capacity. In addition, Dicer knockdown-induced DNA damage leads to the upregulation of MICA and MICB, and Dicer knockdown cells may be eliminated by immune cells.