Biology subjects

Cawte, A. D.

Publications and source records attributed to Cawte, A. D..

5 recordsLinked to original sources

bioRxiv2026

A contact-mediated transfer mechanism directs in cis localization and spread of Xist RNA

X chromosome inactivation in mammals is driven by the long non-coding RNA Xist, which localizes and spreads across the chromosome from which it is transcribed. Although several factors required for Xist localization have been identified, the underlying mechanism remains unclear. Here, we combine Nuclear-BRITE, a method for visualizing and quantifying the dynamics of individual Xist RNA molecules, with acute depletion of key localization factors. We find that Xist molecules remain constrained throughout interphase, consistent with continuous association with chromatin. Building on this observation, we identify a contact-mediated transfer mechanism in which 3D chromosome dynamics promote handover of Xist molecules between chromosomal sites. We further show that the Xist-binding proteins SPEN and HNRNPU play distinct roles in this process by promoting Xist - Xist and Xist - chromatin interactions, respectively. Together, our findings define a mechanism for Xist RNA spreading and provide a framework for understanding its unique chromosome-wide localization.

molecular biology↗

bioRxiv2026

Chromatin structure of the inactive X chromosome revealed by in situ cryo-electron tomography

The intricate organisation of chromatin in eukaryotic cells plays a central role in regulating gene transcription and other DNA-templated processes. Prior studies using light microscopy, biochemistry, and genomics have implicated histone and DNA modifications, histone variants, chromosomal proteins, non-coding RNA, and intrinsic biophysical properties of chromatin in determining chromatin organisation across multiple scales. In this study, we apply an innovative approach, direct visualisation of native chromatin structure and its modulation by different factors using cryo-electron tomography, exploiting the inactive X chromosome (Xi) in differentiating mouse embryonic stem cells as a model system of facultative heterochromatin. We show that Xi chromatin undergoes progressive compaction across scales, from individual nucleosomes to chromatin domains. We observe an enrichment of histone H1-bound chromatosomes that are distributed to form the core of Xi chromatin domains. Moreover, we demonstrate that histone deacetylation demarcates chromatin domains. Specifically, perturbations leading to histone acetylation in the Xi dissolve discrete chromatin-domain boundaries, resulting in a homogeneous chromatin distribution with a dense and uniform packing of nucleosomes. Together, these findings illuminate the multiscale organisation of native facultative heterochromatin and the contributions of histone H1 and histone acetylation to this organisation. This study opens a new avenue for investigating how chromatin modification and structure relate to gene activity in near-native cells.

molecular biology↗

bioRxiv2026

Nuclear-BRITE: an enhanced MS2 system for live-cell imaging of nuclear RNAs

The MS2 system has been widely adopted for live-cell single molecule imaging of mRNA, but is of limited applicability for RNAs present in the cell nucleus due to excess nuclear-localised fluorescent capsid protein. Here we describe Nuclear-BRITE, an improved methodology that reduces nuclear background signal, enabling fast live-cell imaging of a wide range of endogenously tagged nuclear RNAs at single-molecule resolution without perturbing their abundance, localisation or function.

cell biology↗

bioRxiv2025

Selective interaction of the protein SMCHD1 with specific chromatin regions is governed by the loading factor LRIF1 and SMCHD1 ATPase activity

The chromosomal protein SMCHD1 is a GHKL ATPase that plays important roles in epigenetic silencing, including on the inactive X chromosome (Xi) and at the D4Z4 macrosatellite linked to regulation of DUX4 expression in the disorders facioscapulohumeral muscular dystrophy (FSHD2) and Bosma arrhinia micropthalmia syndrome (BAMS). In this study we use live-cell and single-molecule imaging approaches to investigate SMCHD1 interactions with chromatin and its function in epigenetic silencing. We show that chromatin binding of SMCHD1 genome-wide, including on the Xi, is critically dependent on the protein LRIF1 that mediates interaction with H3K9me2/3 modified nucleosomes. Using engineered mutations in the GHKL ATPase domain we show that ATP hydrolysis is required for selective enrichment of SMCHD1 at specific chromatin regions, which is critical for gene silencing on the Xi. A gain-of-function mutation, G137E, that occurs in BAMS patients, results in accelerated Xi recruitment and greater Xi chromosome compaction. Together, our findings advance mechanistic understanding of SMCHD1 function on the Xi and at other target sites in the genome.

molecular biology↗

bioRxiv2019

Live Cell Imaging of Single RNA Molecules with Fluorogenic Mango II Arrays

RNA molecules play vital roles in many cellular processes. Visualising their dynamics in live cells at single-molecule resolution is essential to elucidate their role in RNA metabolism. RNA aptamers, such as Spinach and Mango, have recently emerged as a powerful background-free technology for live-cell RNA imaging due to their fluorogenic properties upon ligand binding. Here, we report a novel array of Mango II aptamers for RNA imaging in live and fixed cells with high contrast and single-molecule sensitivity. Direct comparison of Mango II and MS2-tdMCP-mCherry dual-labelled mRNAs show marked improvements in signal to noise ratio using the fluorogenic Mango aptamers. Using both coding ({beta}-actin mRNA) and long non-coding (NEAT1) RNAs, we show that the Mango array does not affect cellular localisation. Additionally, we can track single mRNAs for extended time periods, likely due to fluorophore exchange. This property makes the arrays readily compatible with structured illumination super-resolution microscopy.

cell biology↗