NEUROSPECIFIC LSD1 SPLICING ISOFORM LINKS EPIGENETICS TO MAMMALIAN BRAIN PHYSIOLOGY

LSD1, the first identified Lysine specific demethylase that removes methyl groups from mono- or di-methylated Histone 3 Lys4 (H3K4), has a mammalian-restricted neuronal isoform (LSD1-E8a), generated by the alternative inclusion of the 12-bp neurospecific exon E8a. LSD1 general function is to inhibit the expression of neuronal genes in non-neuronal cells, but LSD1-E8a isoform is characterized by a less gene repressing action. Indeed, the 4 aa coded by the exon E8a, which form a protruding loop in the LSD1 catalytic domain, contain a Threonine residue that can be phosphorylated and that is required to induce neuronal maturation, neurite outgrowth and the abrogation of LSD1-8a repressive activity. Using a Minigene reporter assay, we demonstrate that the exon E8a is surrounded by a highly conserved 800-bp intronic region that is sufficient per se to regulate exon E8a alternative splicing, containing at least in part the necessary cis-acting elements. Among them there are the binding sites for NOVA1 and nPTB, that we found to be exon E8a positive trans-acting splicing regulators. Exon E8a, indeed, is very tightly modulated and it is present only in neuronal brain tissues and not in cell line. Along with positive trans-acting factors, we identified an exon E8a complementary/inverted sequence with a very strong negative regulatory effect on exon E8a inclusion. Here we show that this sequence acts by forming a double strand pre-mRNA pairing in which exon E8a is masked. Indeed, the deletion of the 12 core nucleotides of such complementary region promotes a strong exon E8a inclusion, allowing binding of trans-acting factors that recognize single-strand cis-acting motifs. Furthermore, downstream of exon E8a we identified a new 77-bp human-restricted LSD1 alternative exon, that we called “exon E8b”. Its inclusion in mature transcripts is regulated by FOX1 and occurs in many different tissues, although these transcripts are present at a very low level. This is probably due to the fact that exon E8b, by introducing a premature STOP codon inside LSD1 mature transcripts, causes their degradation by the cellular Non-sense mRNA Mediated Decay (NMD) pathway. Exon E8b very low endogenous expression level makes it difficult to study its functional role. At the moment we can only say that its inclusion into LSD1 transcripts could be a tool at cells disposal to finely tune LSD1 RNA amount, providing a new human-restricted LSD1 level of regulation. Since the epigenetic function of the neurospecific LSD1 isoform has not been completely elucidated in-vivo, we generated a knock-out mouse model by replacing the sole LSD1 exon E8a with the Neomycin resistance cassette, flanked by two loxP sites. LSD1-E8a knockout animals are fertile, survive embryogenesis and show no histological differences as well as no obvious developmental defects. Interestingly, specific behavioral differences were detected in the exon E8a-deficient mice in response to a pharmacologically induced epileptic treatment, where they display a longer time of latency and a reduced number of seizures, in addition to a more restrained expression-burst of the two immediate early gene Egr-1 and c-Fos. From the immunohistological point of view, significative differences were observed in Calbindin, V-GLUT2 and Sox-2 expression and distribution, although the characterization is not completed yet. Our results indicate that, in vivo, neuronal LSD1 isoform is not strictly required for normal development but it may became relevant to later functional events inside the Central Nervous System.