Rbmx Regulates YTHDC1 Nuclear Condensates to Promote Nascent Transcription in Acute Myeloid Leukemia

Genetic mutations and altered expression of transcription factors and chromatin modulators are critical drivers for myeloid leukemogenesis. We and others previously found that the RNA binding protein and mRNA methylation associated factor RBMX/L1 is upregulated in AML and essential for myeloid leukemia development through control of nascent transcription of key chromatin regulators (Prieto, C., Nguyen, T.T.D., et al., Nat Cancer 2021). However, how this regulatory axis is controlled is not known.

We postulated that discovery of RBMX interactors and associated programs could elucidate this mechanism. To identify such programs, we first performed RBMX co-immunoprecipitation followed by mass spectrometry. We found that YTHDC1 a nuclear RNA methylation (m6A) reader was enriched. Immunofluorescence coupled with high resolution microscopy revealed 80% of endogenous YTHDC1 to co-localize with RBMX while 20% of RBMX co-localized with YTHDC1. We previously reported that YTHDC1 binds m6A modified transcripts and forms a distinct class of liquid-like nuclear condensates required for myeloid leukemogenesis (Cheng, Y., et al., Cancer Cell, 2021). We found that RBMX depletion increased liquid-like properties of YTHDC1 condensates while ectopic expression reduced their liquid-like properties, number, and size, suggesting that RBMX alters the biophysical properties and molecular function of YTHDC1.

To determine whether this RBMX-YTHDC1 interaction results in co-regulation of shared targets, we first compared YTHDC1's mRNA binding targets mapped by iCLIP and RBMX's binding targets mapped by PARCLIP. We found YTHDC1 to bind 67% of RBMX transcript targets (4,613 transcripts; p < 0.001). Moreover, de-novo motif analysis indicated the two proteins bind a similar motif matching to the known m6A DRACH sequence (p < 10^-104). Crucially, our re-analysis of published YTHDC1 ChIP-seq and RBMX CUT&Tag datasets along with the CLIP data above revealed 1,345 sites (p < 0.001) to be bound at both the DNA and RNA level with 45% of the binding within the promoter region. Notably, these sites were enriched for histone modifications associated with active transcription such as H3K4me3 and H3K27ac and depleted for H3K9me3 associated with repressed transcription.

Transcriptome profiling of YTHDC1 deficient MOLM13 cells enriched for similar gene expression signatures obtained from the top 300 upregulated (NES = 2.96; p < 0.05) and downregulated genes (NES = -3.44; p < 0.05) following RBMX depletion. Remarkably, metabolic labeling of nascent transcripts with ethylene uridine coupled with qPCR in THP1 and MOLM13 cells revealed a severe impairment in nascent transcription of our previously identified RBMX targets CBX5, CBS, XBP1 and PABPC4 with a corresponding reduction in their protein levels following YTHDC1 depletion.

To measure the most proximal effects of RBMX depletion, we fused RBMX to an FKBP12^F36V degron tag. Treatment of cells carrying this fusion with dTAG^v-1 led to rapid and yet reversible depletion of RBMX within 4 hours. RBMX depletion severely impaired proliferation, induced apoptosis (9-fold increase in Annexin V/7AAD cells; p < 0.005) and increased myeloid differentiation (8-fold increase in CD11b⁺ CD13⁺ cells; p < 0.005) within 3 days, which could be partially rescued by overexpressing YTHDC1. RNA-sequencing revealed subsets of genes that undergo both rapid (4hrs) and gradual differential expression (24hrs). To examine the mechanism underlying the differential gene expression, we combined transcriptome profiling, chromatin accessibility by ATAC-seq and epigenomic profiling by ChIP-seq. We found that RBMX depletion led to a potent alteration in chromatin accessibility within 4 hours with 4,405 sites gaining accessibility and 3,745 losing accessibility and a reduction of H3K4me3 and H3K27ac at promoters within 24 hours. Most importantly, re-analyzing published RNA Pol II S5P and S2P ChIP-seq datasets revealed that RBMX depletion reduces transcription start site and transcription elongation associated Pol II, respectively, at key shared RBMX and YTHDC1 targets. Thus, we propose, that RBMX controls nascent transcription by creating more fixed YTHDC1 condensates that sequester Pol II at joint DNA and RNA bound sites. Collectively, our results uncover an unorthodox RNA regulatory paradigm of transcriptional control by RBPs in myeloid leukemia and may provide new therapeutic strategies.

Kharas:858 Therapeutics, Inc: Other: Equity; Professional Services and Activities; Astra Zeneca: Other: Professional Services and Activities; Transition Bio, Inc.: Other: Professional Services and Activities.