RNA-DamID reveals cell-type-specific binding of roX RNAs at chromatin-entry sites

Abstract

Thousands of long noncoding RNAs (lncRNAs) have been identified in eukaryotic genomes, many of which are expressed in spatially and temporally restricted patterns. Nonetheless, the roles of the majority of these transcripts are still unknown. One of the mechanisms by which lncRNAs function is through the modulation of chromatin states. To assess the functions of lncRNAs, we developed RNA-DamID, a novel approach that detects lncRNA-genome interactions in a cell-type-specific manner in vivo with high sensitivity and accuracy. Identifying the cell-type-specific genome occupancy of lncRNAs is vital to understanding their mechanisms of action in development and disease. We used RNA-DamID to investigate targeting of the lncRNAs in the Drosophila dosage-compensation complex (DCC) and show that initial targeting is cell-type specific.

Figure 1. RNA-DamID accurately detects lncRNA-chromatin interactions in vivo.

(a) Schematic representation of RNA-DamID. A lncRNA of interest (red) tagged with 3xMS2 stem loops is co-expressed with a Dam-MCP fusion protein (blue and green, respectively) under the spatial and temporal control of GAL4 (purple). The Dam-MCP fusion is recruited to sites of lncRNA-chromatin interaction and methylates adenines within the sequence GATC. lncRNA binding sites are identified genome-wide by DamID. (b) A UAS-3xMS2-roX2 transgene was inserted on chromosome 3L. (c) Fold enrichment of roX2 RNA-DamID binding in whole male larvae, normalised to the negative control, reveals binding exclusively to the X chromosome (average of two biological replicates).

Figure 2. roX2 co-localises with the male-specific lethal complex.

(a) Peaks identified by roX2 RNA-DamID co-localise with roX2 ChIRP , Msl3 TaDa and H4K16ac ChIP . RNA-DamID scale represents log2 fold change of 3xMS2-roX2 and Dam-MCP compared to 3xMS2 and Dam-MCP (average of two biological replicates). Msl3 TaDa scale is a log2 fold change of Msl3-Dam fusion compared to Dam-alone. Chirp signal is log2 transformed. H4K16ac represents log2 fold change of H4K16ac ChIP over input. (b) Heat map of roX2 RNA-DamID, roX2 ChIRP, Msl3 and H4K16 ChIP signal plotted with over a 20kb window either side of roX2 ChIRP peaks. The majority of ChIRP X chromosome peaks, but not autosomal ChIRP peaks, show an enrichment of RNA-DamID, Msl3 TaDa and H4K16ac ChIP signal.

Figure 3. roX1 binding to CES is cell-type specific

(a) roX1 RNA-DamID signal from NSCs, salivary glands and whole larvae is enriched on the X chromosome (average of two biological replicates). Signal is plotted as fold enrichment over negative control. (b) Heat map of cell-type-specific binding to CES. Quantile-normalised log2 RNA-DamID is plotted over each CES (c) Example of cell-type-specific CES. log2 fold change of 3xMS2-roX2, Dam-MCP compared to 3xMS2, Dam-MCP.

Figure 4. roX2 targets a subset of chromatin entry sites in females

(a) roX2 is enriched on the X chromosome when expressed in female larvae. Data is plotted as fold enrichment over negative control. (b) roX2 occupancy is not enriched over most male binding sites. Data is plotted as the average log2 fold enrichment over the midpoint of each male peak. (c) Examples of female-bound chromatin entry sites. (d) Heatmap of roX2 occupancy on a subset of CES. Binding is abolished in msl-2 mutants . Data are represented as log2 transformed RNA-DamID signal. CES are k-means clustered according to roX2 signal in wild-type female larvae.