2019) Identification and epigenetic analysis of divergent long non-coding RNAs in multilineage differentiation of human Neural Progenitor Cells, RNA Biology, 16:1, 13-24, ABSTRACT Long non-coding RNAs have emerged as an important regulatory layer in biological systems. Of the various types of lncRNAs, one class (designated as divergent RNAs/XH), which is in head-to-head overlap with the coding genes, has emerged as a critical biotype that regulates development and cellular differentiation. This work aimed to analyze previously published data on differential expression, epigenetic and network analysis in order to demonstrate the association of divergent lncRNAs, a specific biotype with the differentiation of human neural progenitor cells (hNPCs). We have analyzed various available RNAseq databases that address the neuronal and astrocytic differentiation of hNPCs and identified differentially expressed lncRNAs (DELs) during cell-fate determination. Key DELs identified from the databases were experimentally verified by us in our in-vitro hNPC differentiation system. We also analyzed the change in promoter activity using ChIP-seq datasets of the histone markers H3K4me3 (activation) and H3K27me3 (inactivation) of these DELs. Additionally, we explored the change in the euchromatinization state of DELs (by analyzing DNase-seq data) during lineage-specific differentiation of hNPCs and performed their network analysis. We were able to identify differences between neuronal and astrocytic differentiation of hNPCs at the level of divergent DELs epigenetic markers, DNAase hypersensitive sites and gene expression network. Divergent lncRNAs are more involved in neuronal rather than astrocytic differentiation, while the sense downstream lncRNA biotype appears to be more involved in astrocytic differentiation. By studying the lncRNA involvement of distinct biotypes, we have been able to indicate the preferential role of a particular biotype during lineage-specific differentiation. ARTICLE HISTORY

lncRNA genes can be genic or "intergenic". "Genic" RNAs can be further divided into six biotypes. Through genomewide analysis of a publicly available data set on corticogenesis, we found that the divergent lncRNA (XH) biotype, comprising the lncRNA and the coding gene being in opposite directions in a head-to-head manner, was most prominent during neural commitment. Within this biotype, a coding gene/divergent RNA pair of the BASP1 gene and the uncharacterized RNA loc285696 (hitherto referred as BASP1-AS1) formed a major HUB gene during neuronal differentiation. Experimental validation during the in vitro differentiation of human neural progenitor cells (hNPCs) showed that BASP1-AS1 regulates the expression of its adjacent coding gene, BASP1. Both transcripts increased sharply on the first day of neuronal differentiation of hNPCs, to fall steadily thereafter, reaching very low levels in differentiated neurons. BASP1-AS1 RNA and the BASP1 gene formed a molecular complex that also included the transcription factor TCF12. TCF12 is coded by the DYX1 locus, associated with inherited dyslexia and neurodevelopmental defects. Knockdown of BASP1-AS1, BASP1, or TCF12 impaired the neuronal differentiation of hNPCs, as seen by reduction in DCX and TUJ1-positive cells and by reduced neurite length. There was also increased cell proliferation. A common set of critical genes was affected by the three molecules in the complex. Our study thus identified the role of the XH biotype and a novel mediator of neuronal differentiation-the complex of BASP1-AS1, BASP1, and TCF12. It also linked a neuronal differentiation pathway to inherited dyslexia.

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