In this study we showed that the histone demethylase LSD1 plays an important role in neural stem cell proliferation. Either inhibition of LSD1 activity or knockdown of LSD1 expression led to dramatically reduced neural stem cell proliferation. We also identified TLX as a critical effector of LSD1 function in neural stem cells. This idea was supported by several pieces of evidence. First, LSD1 is recruited to the promoters of TLX target genes p21 and pten by TLX to repress their expression. Second, knockdown of TLX expression diminished LSD1 siRNA-mediated inhibition of neural stem cell proliferation significantly. Furthermore, treatment of adult mice with the LSD1 siRNA or the LSD1 inhibitors pargyline and tranylcypromine led to dramatically reduced neural progenitor proliferation in the hippocampal dentate gyri of wild-type mouse brains; however, the inhibitor treatment had almost no effect on cell proliferation in TLX−/− mouse brains.
The data presented here demonstrated that the function of TLX is modulated by LSD1 in neural stem cells. The association of LSD1 with TLX in neural stem cells led to transcriptional repression of TLX target gene expression. Both LSD1 inhibitor treatment and siRNA knockdown resulted in an increase in H3K4 methylation levels on TLX target gene promoters and a concomitant induction of TLX target gene expression, suggesting that LSD1 mediates transcriptional repression of TLX target genes via histone demethylation. These results mirrored the observation in retinoblastoma cells, in which LSD1 was found to be present in the TLX immunocomplex to modulate its activity (26
LSD1 has been shown to catalyze demethylation of mono- and dimethyl H3K4 through a flavin adenine dinucleotide (FAD)-dependent oxidative reaction (16
). Treatment with both LSD1 inhibitors and siRNA led to increased monomethyl and dimethyl H3K4 levels on p21 and pten promoters, providing strong evidence that LSD1 participates in the demethylation of TLX target gene promoters. Because LSD1 is unable to remove trimethyl H3K4, no induction of trimethyl H3K4 levels was detected on the p21 promoter in LSD1 siRNA-treated cells, as expected. No change in TLX binding was detected in LSD1 siRNA-treated cells, although a slight decrease of TLX binding was seen in LSD1 inhibitor-treated cells, which may be due to an indirect effect.
To determine the role of LSD1 in neural stem cells, we treated neural stem cells with the LSD1 inhibitors pargyline and tranylcypromine, which resulted in significant inhibition of cell proliferation. However, chemical inhibition may have pleiotropic effects. We therefore knocked down LSD1 expression using its sequence-specific siRNA. Both induction of TLX target gene expression and reduced neural stem cell proliferation were observed upon LSD1 knockdown, similar to the effect of LSD1 inhibitor treatment.
We demonstrated that TLX recruits both LSD1 and HDAC5 to its target genes in neural stem cells. Knockdown of either LSD1 or HDAC5 led to induction of p21 and pten gene expression and inhibition of neural stem cell proliferation. When LSD1 and HDAC5 were knocked down simultaneously, a more dramatic effect on both induction of TLX target gene expression and inhibition of neural stem cell proliferation was detected, compared to individual siRNA treatment. Similarly, when TLX was knocked down, substantial induction of p21 and pten gene expression and inhibition of neural stem cell proliferation were observed. These results suggest that TLX recruits both LSD1 and HDAC5, presumably in a corepressor complex, to mediate its cellular function.
In summary, this study has uncovered a novel role for LSD1 in neural stem cell proliferation. The findings presented here revealed a mechanism by which recruitment of the histone demethylase LSD1 and the histone deacetylase HDAC5 enables TLX to mediate transcriptional repression in neural stem cells. The partnership of TLX with LSD1 and HDAC5 links transcriptional repression and epigenetic modulation at TLX target genes to neural stem cell proliferation. Stem cells provide great hope for the treatment of a variety of human diseases that lack efficacious therapies to date. We show here that LSD1 inhibitors, such as pargyline and tranylcypromine, control the demethylase activity of LSD1, thereby regulating TLX signaling. Considering the essential role of TLX in neural stem cell proliferation and self-renewal, specific modulation of LSD1 activity may become a promising therapeutic tool for the treatment of neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases.