Revealing long noncoding RNA architecture and functions using domain-specific chromatin isolation by RNA purification

Abstract

Little is known about the functional domain architecture of long noncoding RNAs (lncRNAs) because of a relative paucity of suitable methods to analyze RNA function at a domain level. Here we describe domain-specific chromatin isolation by RNA purification (dChIRP), a scalable technique to dissect pairwise RNA-RNA, RNA-protein and RNA-chromatin interactions at the level of individual RNA domains in living cells. dChIRP of roX1, a lncRNA essential for Drosophila melanogaster X-chromosome dosage compensation, reveals a 'three-fingered hand' ribonucleoprotein topology. Each RNA finger binds chromatin and the male-specific lethal (MSL) protein complex and can individually rescue male lethality in roX-null flies, thus defining a minimal RNA domain for chromosome-wide dosage compensation. dChIRP improves the RNA genomic localization signal by >20-fold relative to previous techniques, and these binding sites are correlated with chromosome conformation data, indicating that most roX-bound loci cluster in a nuclear territory. These results suggest dChIRP can reveal lncRNA architecture and function with high precision and sensitivity.

Figure 1

dChIRP uses antisense oligos to purify specific RNA domains and associated RNAs, proteins, and chromatin. (a) dChIRP oligo design strategy. Biotinylated antisense oligos are designed to tile specific regions of the target RNA. (b) dChIRP workflow. To prepare chromatin, whole cells are cross-linked to preserve protein-nucleic acid interactions. Sonication is used to solubilize the nuclear fraction and shear nucleic acids. Next, the chromatin is subdivided into equal samples. OPs are added to each sample, which hybridize to the targeted RNA fragments. The biotinylated oligos, RNA targets, and cross-linked biomolecules are then purified on magnetic streptavidin beads, and unbound material is washed away. (c) RNA-, protein-, and DNA-sensitive modalities of dChIRP. RNA, protein, and DNA fractions are extracted from each dChIRP sample. Intra- or inter- molecular RNA-RNA, RNA-protein, and RNA-DNA interactions may be measured by RT-qPCR, immunoblotting, and qPCR or sequencing, respectively.

Figure 2

dChIRP RNA co-recovery reveals roX1’s topological architecture. (a) Schematic representation of known roX1 domain interactions with MLE protein and dChIRP OP design strategy. MLE directly contacts the three D domains (D1, D2, and D3). The three intervening U domains (U1, U2, and U3) exhibit minimal binding. Six OPs were designed to target and recover each domain. (b) roX1 dChIRP specifically enriches for roX1 RNA. roX1 RNA is >1000-fold enriched over the abundant GAPDH mRNA in roX1 dChIRP samples. LacZ ChIRP does not enrich for roX1 over GAPDH. Average of technical triplicates +s.d. shown. (c, d) roX1 RNA recovery by dChIRP. To confirm that roX1 dChIRP successfully recovers the targeted RNA domain, the RNA fraction of each dChIRP sample was analyzed by RT-qPCR, using primers within each domain of roX1. Within each sample, roX1 domain recovery was quantified against input and normalized to total roX1 RNA recovery (% roX1 RNA recovery). As expected, each OP best enriches for the target roX1 domain (b, red diagonal). Whereas domains D1, D2, and D3 were recovered independently, domains U1, U2, and U3 were co-recovered. To demonstrate that the co-recovery of domains U1, U2, and U3 is cross-linking-dependent, fixed chromatin was thermally de-cross-linked and dChIRP was performed (c). Each of the domains of roX1 is independently recovered. (e) Schematic representation of roX1 intramolecular topology. Domains U1, U2, and U3 are topologically proximal to one another, forming the core “palm” of roX1. Domains D1, D2, and D3 extend as “fingers” and are distant from one another and the intervening U domains.

Figure 3

roX1 D domains interact with the MSL complex and chromatin on the X. (a) Schematic representation of roX1 interactions. The three D domains (D1, D2, and D3) directly contact MLE by iCLIP. The three intervening U domains do not contact MLE, but are topologically associated (gray dotted lines). (b) dChIRP-Western blot confirms known MLE-bound domains of roX1. The protein fraction from each roX1 dChIRP sample was analyzed by immunoblotting against MLE, MSL3, CLAMP, and Actin. roX1 domains D1, D2, and D3 efficiently recovered MLE and MSL3 proteins. D3 recovered more protein than D2, and D2 recovered more than D1. Domains U1, U2, and U3 recovered minimal or undetectable MLE and MSL3. Only D3 recovered CLAMP appreciably, albeit very weakly. LacZ ChIRP recovered no detectable protein. Actin was not detected in any sample. (c) The three D domains of roX1 are associated with chromatin at dosage compensated loci on the X chromosome. DNA fractions from each roX1 dChIRP sample were analyzed by qPCR and normalized to input. Five genomic loci were investigated: three MSL-bound X-linked loci (dlg1, suv4-20, u2af50), one locus from an autosome (gstd2), and an unbound X-linked locus (ovo). dChIRP of domains D1, D2, and D3 significantly enrich for X-bound loci relative to control loci (*P-value < 0.01, t-test). Domains D2 and D3 recover significantly more X-bound DNA than D1 or the three U domains. LacZ ChIRP fails to recover substantial DNA from any locus. Average of technical triplicates +s.d. shown.

Figure 4

dChIRP boosts signal-to-noise ratio relative to traditional ChIRP-sequencing. (a) Genomic tracks of dChIRP-seq results at representative X-linked locus. Sequencing tracks from roX1 dChIRP (U1, D2, D3) and traditional ChIRP (roX1, roX2). roX1 peaks (gray highlight) align with peaks from roX2, MSL3 ChIP, and CLAMP ChIP^,. (b) Comparison of signal from the X and noise on autosomes. Signal was calculated at 457 peaks on the X and 457 random loci on autosomes. Box-plot represents 1st, 2nd, and 3rd quartiles; whiskers denote 5th and 95th percentiles. Signal-to-noise ratio (X peak mean to autosome mean) is indicated above each sample. (c) Average peak diagram of 457 peaks on X. roX1 dChIRP produces higher signal and more focal peaks than traditional ChIRP. The MRE GA-repeat motif is significantly enriched within peaks (P = 5.2e-526, MEME) and is located specifically at peak summits (P = 5.3e-182, CentriMo). (d, e) Correlation between ChIRP-sequencing experiments. (d) roX1-D3 dChIRP and roX2 ChIRP signal are highly correlated (r=0.9619), especially on the X chromosome (red). (e) roX1-D2 and -D3 dChIRP are very highly correlated (r=0.9912). roX1 D2, D3, and roX2 co-occupy the same loci on the X.

Figure 5

CES cluster together in a dosage compensation territory of the nucleus. (a, b) Correlation between (a) roX2 occupancy by ChIRP and roX2 proximity by Hi-C, and (b) roX1-D3 occupancy by ChIRP and roX1 proximity by Hi-C. roX2 RNA occupancy is correlated with roX2 proximity (r=0.5332); roX1 RNA occupancy is not correlated with roX1 proximity (r=-0.0255). roX1 457 peaks (magenta) are clustered at sites of high roX RNA occupancy. 400kb around the roX gene loci were excluded (gray mask) for correlation calculation (Pearson’s r), so as to exclude signal from direct ChIRP oligo-DNA recovery and one-dimensionally proximal chromosome sites. (c) Gene set enrichment analysis (GSEA) of roX occupied genes. Genes that are occupied by roX RNAs are significantly more likely to be proximal to the roX2 locus (FDR<0.001). (d) Instances of the MRE motif that are more proximal to the roX2 locus are significantly more likely to be bound by CLAMP and co-occupied by roX RNAs. P-value<0.001 by Kolmogorov-Smirnov test. (e) Model of X chromosome conformation. The roX2 locus and CES are clustered in a dosage compensation (DC) territory. The roX1 locus lies outside of the DC territory.

Figure 6

roX1’s D domains are independent, functional RNA subunits. (a) Transgene designs. Transgenic constructs of full-length roX1 and the six individual domains were cloned, chromosomally integrated, and expressed under the tubulin-GAL4 promoter in roX-null flies. (b) Rescue of male lethality by roX1 transgenes. Transgenic males surviving to adulthood were counted and normalized to females. Only the D domains rescued males appreciably. Rescue by D3 is not significantly different from that of full-length roX1 (t-test, P-value=0.20). Average of three separate crosses +s.d. shown (on average, n=800). roX transgene expression was quantified and normalized to endogenous roX1 expression in wild-type males, represented as relative fold (transgene/endogenous) ±s.d. (c, d) Integrated interaction map of the dosage compensation complex with chromatin. (c) roX1 RNA is topologically organized such that the three U domains form a core palm and each of the D domains extends independently as a finger. Each D domain finger directly binds to proteins of the MSL complex, with domain D3 having the highest affinity and D1 the weakest. (d) CLAMP binds the GAGA motif at X-linked CES, and associates with MLE. MLE binds to stem-loops on roX1, which tethers MLE to the core MSL complex. MOF of the MSL complex recognizes and acetylates H4K16 of adjacent nucleosomes.