Stem cells are unique cells with the ability to self-renew and differentiate into multiple cell types. Neural stem cells are a subset of undifferentiated precursors that retain the ability to proliferate, self-renew, and have the capacity to differentiate into both neuronal and glial lineages1
. Although the functional properties of neural stem cells have been studied extensively, understanding the molecular mechanisms underlying neural stem cell self-renewal and differentiation remains one of the most important tasks in stem cell biology.
Accumulating evidence indicates that both transcriptional and post-transcriptional regulations are important mechanisms for regulating genes that are essential for neural stem cell self-renewal and neurogenesis. MicroRNAs (miRNAs) are a recently identified large family of 20–22 nucleotide non-coding RNAs that are involved in numerous cellular processes, including development, proliferation, and differentiation2,3
. In the past decade, a network of regulatory circuitries operating at the posttranscriptional level has been uncovered with the finding of a class of miRNA regulators. These molecules play pivotal roles by mediating mRNA stability and translational inhibition of specific target genes3
. The function of miRNAs has been correlated with many complex cellular functions2
. While some miRNAs are ubiquitously expressed, others exhibit developmental stage or tissue-specific expression4
. The study of tissue-specific miRNAs will provide important clues for identifying new regulatory circuitries involved in the control of cell-specific differentiation. Recently, several brain-specific miRNAs have been identified. Among these is miR-137, which has been shown to promote neuronal differentiation of adult subventricular neural stem cells and to inhibit neuronal maturation in adult hippocampal neurons5,6
. However, the role of miR-137 in embryonic brains is still unknown and the upstream regulators of miR-137 in neural stem cells remain to be identified.
TLX (NR2E1) is an orphan nuclear receptor that is expressed in vertebrate forebrains7
. Mature Tlx
knockout mice have significantly reduced cerebral hemispheres and exhibit increased aggressiveness and progressively violent behavior8–11
. We have shown that TLX is an essential regulator of self-renewal in adult neural stem cell9
. TLX maintains adult neural stem cells in an undifferentiated and self-renewable state, in part through transcriptional repression of its downstream target genes, such as the cyclin-dependent kinase inhibitor p21 and the tumor suppressor pten, by complexing with histone deacetylases and the lysine-specific histone demethylase LSD112,13
. In addition to modulation of TLX activity by histone modifying enzymes, the expression of TLX has been shown to be regulated by miRNAs, including miR-9 and let-7b14,15
. The TLX-positive neural stem cells in the hippocampal dentate gyrus play an important role in spatial learning and memory16
, whereas the TLX-expressing cells in the subventricular zone of adult brains were shown to be the slowly-dividing neural stem cells17–19
. Recently, we demonstrated that TLX activates the canonical Wnt/β-catenin pathway to stimulate adult neural stem cell proliferation and self-renewal19
. In addition to its function in adult brains, TLX also plays an important role in neural development by regulating cell cycle progression in neural stem cells of the developing brain20–22
. TLX is therefore a key regulator of neural stem cells in both embryonic and adult brains, although aspects of its downstream events have yet to be uncovered.
Epigenetic mechanisms, such as histone modifications, have been shown to play significant roles in the regulation of stem cell proliferation and differentiation23
. Histone modifications, such as acetylation, phosphorylation, and methylation, are switches that alter chromatin structure to form a binding platform for downstream “effector” proteins to allow transcriptional activation or repression24
. The recent discovery of a large number of histone demethylases indicates that demethylases play a central role in the regulation of histone methylation dynamics25–28
. The first lysyl demethylase identified is lysine-specific demethylase 1 (LSD1), which demethylates H3K4 or H3K9 in a reaction employing flavin as a cofactor. LSD1 is limited to mono- or dimethylated substrates26
. We have previously shown that the histone demethylase LSD1 is expressed in neural stem cells and plays an important role in neural stem cell proliferation via modulating TLX signaling13
. LSD1 is recruited to the promoters of TLX downstream target genes to repress their expression, consequently promoting neural stem cell proliferation. However, the upstream events that regulate LSD1 expression remain largely unknown.
In this study, we identified miR-137 as a new target of TLX and a novel upstream regulator of LSD1. We demonstrate that TLX represses miR-137 expression in neural stem cells by binding to the 5′ and 3′ sequences of miR-137. Remarkably, one of the targets of miR-137 is the TLX transcriptional co-repressor, LSD1, which is shown to be recruited to the genomic region of miR-137 by TLX to inhibit miR-137 expression. miR-137 inhibits LSD1 expression to regulate neural stem cell proliferation and differentiation. Increased expression of miR-137 led to reduced mouse neural stem cell proliferation and accelerated neural differentiation. In utero electroporation of miR-137 into neural stem cells in the ventricular zone of embryonic mouse brains triggered premature differentiation and outward migration of the transfected cells. Moreover, co-electroporation of an LSD1 expression vector lacking the endogenous 3′ UTR rescued the premature differentiation induced by miR-137 overexpression. Our findings point to the regulatory loop formed between miR-137 and TLX/LSD1 as a critical player in the molecular circuitry controlling neural stem cell proliferation and differentiation during neural development.