UNCOVERING LNCRNA-CHROMATIN INTERACTIONS SHAPING HUMAN MONOCYTE TO MACROPHAGE DIFFERENTIATION

Mammalian genomes are pervasively transcribed, producing a large repertoire of coding and non-coding transcripts, that can associate with chromatin and contribute to the regulation of genome organization and gene expression. However, the functional roles of most chromatin-associated RNAs, particularly long non-coding RNAs (lncRNAs), are still being elucidated, with new functions continuously emerging as the filed advances. In recent years, several technologies have been developed to study RNA-chromatin interactions at a genome-wide scale, including RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq). Using a multi-omics approach, we aim to identify regulatory roles of chromatin-associated lncRNAs in a dynamic model of monocyte to macrophage differentiation, a key process in innate immunity. To achieve this, we generated a comprehensive transcript annotation of the system using a novel approach that combines long-reads sequencing with transcription start site mapping. This enabled the expansion of current annotations with a large repertoire of previously unannotated transcripts, many of which are highly cell-stage specific. We then mapped RNA-chromatin interactions genome-wide using RADICL-seq and integrated these data with transcriptional dynamics. We found that lncRNAs-chromatin interactions are dynamically reorganized during differentiation: lncRNAs upregulated during differentiation shift from predominantly local interactions in monocytes to more distal contacts in macrophages, whereas downregulated lncRNAs exhibit higher proportion of interactions in monocytes. The expression profiles of target genes largely paralleled those of their source lncRNAs in a stage specific manner suggesting that activated lncRNAs may reinforce macrophage differentiation, while repressed lncRNAs may contribute to maintaining the monocyte state. We further reconstructed gene regulatory networks integrating transcription factors and RNA-chromatin interactions to define the regulatory landscape and identify candidate lncRNAs for functional validation. Silencing of selected novel lncRNAs resulted in the enrichment of genes involved in phosphoinositide binding, a pathway relevant to macrophage specific functions. This work provides an improved transcript annotation and reveals RNA-chromatin interactions as an additional layer of regulation associated with lncRNAs during macrophage differentiation.