Watt, A. | bioRxiv Science

bioRxiv2026

Benchmarking normalisation methods for differential binding analysis in CUT&RUN

CUT&RUN (Cleavage Under Targets and Release Using Nuclease) is an increasingly popular method for profiling protein interactions (transcription factors, histone modifications, etc) with DNA across the whole genome. When performing differential binding analysis of CUT&RUN data to identify genomic regions where interaction profiles vary between conditions, data normalisation is essential for accurate biological interpretations. Despite this, there are no clear guidelines on the optimal normalisation method for CUT&RUN datasets. Here, we examine five normalisation approaches (spike-in, library size, background, reads-in-peak and greenlist) and highlight that different methods can result in widely discrepant interpretations of the data. We test these normalisation methods by simulating a variety of plausible differential binding scenarios as well as an in-house generated dataset. We determined that normalisation by either (i) library size or (ii) background to be the most robust. Importantly, we find spike-in normalisation to be the least reliable method. Our findings inform the use of normalisation methods for CUT&RUN data and should thus facilitate reproducible and robust analysis.

bioinformatics↗

bioRxiv2022

Local axonal translation sustains synapse-type-specific neurotransmission

Dendritic protein synthesis is a well-known regulator of neurotransmission in the central nervous system. For example, local on-demand protein synthesis in dendrites is required for hippocampal long-term1,2 and homeostatic plasticity3. Although axonal protein synthesis is a key regulator in neural circuit assembly during early development4-6, it is unclear if local translation in mature axons plays a functional role in neurotransmission of the mammalian central nervous system. Here we show that local translation in axons regulates pyramidal cell neurotransmission in primary visual cortex microcircuits in a synapse-type-specific manner7,8. Using paired recordings to manipulate pre- and postsynaptic neurons independently, we found that presynaptic translation sustains neurotransmission and regulates short-term plasticity. This was mediated via mTOR and cap-dependent translation, with presynaptic NMDA receptors acting as an up-stream trigger. By isolating the axon from the soma with laser microsurgery, we unveiled that axonal translation was required to sustain neurotransmission. With live endogenous RNA imaging, we found that RNA granules stably docked in individual presynaptic compartments, suggesting bouton-specific regulation. In agreement, presynaptic translation influenced pyramidal cell synapses to neighbouring pyramidal cells, but not to inhibitory Martinotti cells. Our study establishes local protein synthesis in mature axons as a fundamental regulatory principle that governs information transfer at central synapses.

neuroscience↗