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Proximity labeling identifies a repertoire of site-specific R-loop modulators

ABSTRACT: R-loops are three stranded nucleic acid structures that accumulate on chromatin in neurological diseases and cancers and that contribute to genome instability. Using a proximity-dependent labeling system, we identified distinct classes of proteins that regulate R-loops in vivo through different mechanisms. We show that ATRX suppresses R-loops by interacting with RNAs and preventing R-loop formation. Our proteomics screen also discovered an unexpected enrichment for proteins containing zinc fingers and homeodomains at R-loops. One of the most consistently enriched proteins at R-loops was activity-dependent neuroprotective protein (ADNP), which is frequently mutated in ASD and causal in ADNP syndrome. We find that ADNP is necessary to suppress R-loops in vivo at its genomic targets and that it resolves R-loops in vitro. Furthermore, deletion of the homeodomain severely diminishes R-loop resolution activity in vitro, results in R-loop accumulation at ADNP targets and compromises neuronal differentiation. Notably, patient derived human induced pluripotent stem cells that contain an ADNP syndrome-causing mutation exhibit R-loop and CTCF accumulation at ADNP targets. Our findings point to a specific role for ADNP-mediated R-loop resolution in physiological and pathological neuronal function and, more broadly, to a previously unappreciated role for zinc finger and homeodomain proteins in R-loop regulation, with important implications for developmental disorders and cancers.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Kavitha Sarma

PROVIDER: MSV000087568 | MassIVE | Thu Jun 03 14:01:00 BST 2021

SECONDARY ACCESSION(S): PXD026473

REPOSITORIES: MassIVE

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Project description:Abnormal accumulation of aggregated proteins and sustained microglial activation are important contributors of neurodegenerative process in neurological diseases. Recent studies have shown that aggregation-prone proteins, such as a-synuclein, the protein implicated in Parkinson’s disease (PD), are released from neuronal cells and thus present in the extracellular fluid, pointing to the possible paracrine effects of these proteins on microglial immune responses. However, the mechanism underlying the disease-associated microglial activation and the role of neuronal proteins in this process remain unknown. Here, we show that extracellular a-synuclein released from neuronal cells is an endogenous ligand of toll-like receptor 2 (TLR2) and activates microglia, which in turn induces neurodegeneration. Interaction between neuron-released a-synuclein and TLR2 and subsequent activation of the TLR2 signaling were demonstrated comprehensively by using computational modeling of signaling network and by the experimental validation in TLR2-deficient microglia both in vitro and in vivo. In contrast to the neuron-released a-synuclein, recombinant a-synuclein proteins, including monomer, oligomer, fibril, or nitrated forms, were not able to interact or activate TLR2, suggesting that neuronal cells have a mechanism of enriching specific forms of a-synuclein capable of activating TLR2 during the process of releasing this protein. Taken together, the results suggest that both neuron-released extracellular a-synuclein and TLR2 might be novel therapeutic targets for modifying neuroinflammation in PD and related neurodegenerative diseases. We collected culture media from differentiated SH-SY5Y cells overexpressing either human a-synuclein (alpha-SCM) or beta-galactosidase (LZCM) and treat these media to primary rat microglia at the concentration of a-synuclein of 1.1M. Transcriptome analyses with microglial cells treated with either aSCM or LZCM at two different time points, 6 h and 24 h.

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Proximity labeling identifies a repertoire of site-specific R-loop modulators

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