Silencing Lysine-Specific Histone Demethylase 1 (LSD1) Causes Increased HP1-Positive Chromatin, Stimulation of DNA Repair Processes, and Dysregulation of Proliferation by Chk1 Phosphorylation in Human Endothelial Cells

Abstract

The methylation of histone lysine residues modifies chromatin conformation and regulates the expression of genes implicated in cell metabolism. Lysine-specific demethylase 1 (LSD1) is a flavin-dependent monoamine oxidase that can demethylate mono- and dimethylated histone lysines 4 and 9 (H3K4 and H3K9). The removal of methyl groups from the lysine residues of histone and non-histone proteins was found to be an important regulatory factor of cell proliferation. However, its role has not been fully elucidated. In this study, we assessed LSD1-mediated cell cycle progression using a human endothelial cell model. The short hairpin RNA knockdown of LSD1 inhibits the G2/M phase of cell cycle progression by checkpoint kinase 1 (Chk1) phosphorylation (S137). We observed elevated DNA damage, which was consistent with the increased detection of double-strand breaks as well as purines and pyrimidines oxidation, which accompanied the activation of ATR/ATRIP signaling by H2AXS139 phosphorylation. The irreversible pharmacological inhibition of LSD1 by 2-phenylcyclopropylamine (2-PCPA) inactivated its enzymatic activity, causing significant changes in heterochromatin and euchromatin conformation assessed by chromatin assembly factor 1 subunit A (CAF1A) and heterochromatin protein 1 isoform α and γ (HP1α/γ) immunofluorescence analysis. We conclude that the knockdown of LSD1 in endothelial cells leads to increased HP1-positive chromatin, the stimulation of DNA repair processes, and the dysregulation of proliferation machinery.

The Role of Histone Lysine Methylation in the Response of Mammalian Cells to Ionizing Radiation

“…Once again, it is likely that one function of LSD1-mediated demethylation in this pathway is to facilitate a repressive chromatin environment near DSBs, which may silence transcription near these sites. Consistent with a role in DDR, knockdown of LSD1 results in moderate hypersensitivity to γ-irradiation, although, paradoxically, the efficiency of HR appears increased (Mosammaparast et al, 2013;Wojtala et al, 2019). To explain this ambivalent effect, it should be considered that LSD1 can also promote p53 demethylation, thereby inhibiting p53 function in DDR activation (Huang et al, 2007).…”

Kinetics of DNA Repair in Vicia faba Meristem Regeneration Following Replication Stress

“…There are few papers which report the analysis of the heterochromatin replication clusters’ shape as mathematical descriptors. The obtained data appear to be sufficient to draw the conclusion that the analysis of heterochromatin clusters, using classic photo analysis programs, turns out to be very helpful and seems to be universal in heterochromatin imaging (not only during replication), but can also be useful in the analysis of proteins related to heterochromatin, e.g., proteins from the HP1 family [ 50 , 51 ].…”

Hydroxyurea and Caffeine Impact pRb-like Protein-Dependent Chromatin Architecture Profiles in Interphase Cells of Vicia faba

“…Although HU induces ssDNA at the sites of stalled replication forks, V. faba cells do not seem to accumulate an excessive number of fragile sites even after 32h of treatment. Studies show that upon replication arrest, nucleosome components disengage from DNA [ 54 ], allowing the ssDNA-RPA complex to act as a landing site for—among others—ATR kinase [ 63 , 64 ] and the prevention of fork collapse. Strikingly, these results were also reported for human cells after acute HU treatment, but the same cells lacked the ability to restart replication forks after prolonged HU incubation [ 54 ].…”

“…Cell cycle analysis was performed using the RSLII flow cytometer and FlowJo 10.4.1. software (FlowJo LLC, Ashland, OR, United States), similarly to the procedure described in [ 64 ]. The forward and side scatter channels (FSC and SSC, respectively) were used for the identification of cells in different phases of the cell cycle.…”