Biology subjects

Brockdorff, N.

Publications and source records attributed to Brockdorff, N..

15 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↗

bioRxiv2026

A key role for chromosome compartment interactions in directing Xist RNA localisation

X-chromosome inactivation, the mechanism for dosage compensation in mammals, is orchestrated by the long non-coding RNA Xist which localises across the X chromosome in cis. The basis for in cis localisation remains poorly understood. To investigate in situ Xist localisation, we established MCPH1-deficient cell models that retain compacted interphase chromosomes, enabling visualisation of individualised chromosome territories. We find that Xist RNA is directed to sites around the periphery of compacted chromosome territories, and moreover that these sites correlate closely with A1-sub-compartments on the X chromosome. We further show that peripheral positioning of A1-sub-compartments occurs on all chromosomes and is a hallmark of early prophase. A key role for compartment interactions in Xist localisation is further supported by analysis of MCPH1-deficient models where Xist is overexpressed (from the X chromosome or an autosomal Xist transgene), and following depletion of HNRNPU, a key factor that anchors Xist RNA to chromosome territories.

genetics↗

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↗

bioRxiv2025

N6-methyladenosine and the NEXT complex direct Xist RNA turnover and X inactivation dynamics

X chromosome inactivation (XCI) in mammals is orchestrated by the non-coding RNA Xist which together with specific interacting proteins, functions in cis to silence an entire X chromosome. Defined sites on Xist RNA carry the N6-methyladenosine (m6A) modification, and perturbation of the m6A writer complex has been found to abrogate Xist-mediated gene-silencing. However, the relative contribution of m6A and its mechanism of action remain unclear. Here we investigate the role of m6A in XCI by applying rapid degron-mediated depletion of METTL3, the catalytic subunit of the m6A writer complex, an approach that minimises indirect effects due to transcriptome-wide depletion of m6A. We find that acute loss of METTL3/m6A accelerates Xist-mediated gene silencing, and that this correlates with increased levels and stability of Xist transcripts. We show that Xist RNA turnover is mediated by the nuclear exosome targeting (NEXT) complex but is independent of the principal nuclear m6A reader protein YTHDC1. Our findings demonstrate that the primary function of m6A on Xist RNA is to promote Xist RNA turnover which in turn regulates XCI dynamics.

biochemistry↗

bioRxiv2024

PWWP-ADD and N-terminal domains of DNMT3B1 confer specificity for developmentally regulated CpG island methylation

CpG methylation in mammalian genomes is established by the closely related de novo DNA methyltransferases DNMT3A and DNMT3B. Whilst both enzymes contribute to pervasive genome-wide CpG methylation, DNMT3B has a unique role in developmentally regulated CpG island (CGI) methylation both on the inactive X chromosome and at other sites in the genome. The mechanistic basis for this specificity is poorly understood. Here we have developed an in vitro embryonic stem cell model system to dissect critical determinants of DNMT3B specificity. Our model faithfully recapitulates developmentally regulated CGI methylation and additionally provides novel insights into CpG methylation at cis-regulatory elements. Using genetic complementation, we show that DNMT3B specificity is attributable solely to the catalytic isoform DNMT3B1. Domain swap experiments demonstrate an important role both for the PWWP-ADD chromatin binding and unstructured N-terminal domains. Together, these findings advance our mechanistic understanding of the unique roles of DNMT enzymes in establishing CpG methylation in development.

genetics↗

bioRxiv2023

YY1 binding is a gene-intrinsic barrier to Xist-mediated gene silencing

X chromosome inactivation (XCI) in mammals is mediated by Xist RNA which functions in cis to silence genes on a single X chromosome in XX female cells, thereby equalising levels of X-linked gene expression relative to XY males. XCI progresses over a period of several days, with some X-linked genes silencing faster than others. Chromosomal location of a gene is an important determinant of silencing rate, but uncharacterised gene-intrinsic features also mediate resistance or susceptibility to silencing. In this study, we integrate time-course data of gene silencing and decreasing inactive X (Xi) chromatin accessibility in mouse embryonic stem cell lines with an inducible Xist allele (iXist-ChrX mESCs). Our analysis reveals that motifs bound by the transcription factor YY1 are associated with persistently accessible regulatory elements, including many promoters and enhancers of slow-silencing genes. We further show that YY1 is evicted relatively slowly from target sites on Xi, and that silencing of X-linked genes is increased upon YY1 degradation. Together our results indicate that YY1 acts as barrier to Xist-mediated silencing that is removed only at late stages of the XCI process.

genomics↗

bioRxiv2021

Xist-mediated silencing requires additive functions of SPEN and Polycomb together with differentiation-dependent recruitment of SmcHD1

X chromosome inactivation (XCI) is mediated by the non-coding RNA Xist which directs chromatin modification and gene silencing in cis. The RNA binding protein SPEN and associated corepressors have a central role in Xist-mediated gene silencing. Other silencing factors, notably the Polycomb system, have been reported to function downstream of SPEN. In recent work we found that SPEN has an additional role in correct localisation of Xist RNA in cis, indicating that its contribution to chromatin-mediated gene silencing needs to be reappraised. Making use of a SPEN separation-of-function mutation we show that SPEN and Polycomb pathways in fact function in parallel to establish gene silencing. Additionally, we find that differentiation-dependent recruitment of the chromosomal protein SmcHD1 is required for silencing many X-linked genes. Our results provide important insights into the mechanism of X inactivation and the coordination of chromatin-based gene regulation with cellular differentiation and development.

molecular biology↗

bioRxiv2020

Time-resolved structured illumination microscopy reveals key principles of Xist RNA spreading

Xist RNA directs the process of X-chromosome inactivation in mammals by spreading in cis along the chromosome from which it is transcribed and recruiting chromatin modifiers to silence gene transcription. To elucidate mechanisms of Xist RNA cis-confinement, we established a sequential dual color labeling, super-resolution imaging approach to trace individual Xist RNA molecules over time, enabling us to define fundamental parameters of spreading. We demonstrate a feedback mechanism linking Xist RNA synthesis and degradation, and an unexpected physical coupling between preceding and newly synthesized Xist RNA molecules. Additionally, we show that the protein SPEN, a key factor for Xist-mediated gene-silencing, has a distinct function in Xist RNA localization, stability and in coupling behavior. Our results provide important insights towards understanding the unique dynamic properties of Xist RNA. One Sentence SummaryVisualizing Xist RNA dynamics in single cells during X chromosome inactivation

developmental biology↗

bioRxiv2020

A polyglutamine domain is required for de novo CIZ1 assembly formation at the inactive X chromosome

CIP1-interacting zinc finger protein 1 (CIZ1) forms large assemblies at the inactive X chromosome (Xi) in female fibroblasts in an Xist lncRNA-dependent manner. Here we address the requirements for assembly formation, and show that CIZ1 interacts directly with Xist via two independent domains in its N- and C-terminus. Interaction with Xist repeat E, assembly at Xi in cells, and the complexity of self-assemblies formed in vitro, are all modulated by alternatively-spliced exons that include two glutamine-rich prion-like domains (PLD1 and PLD2), both conditionally excluded from the N-terminal domain. Exclusion of PLD1 alone is sufficient to abrogate de novo establishment of new CIZ1 assemblies and Xi territories enriched for H3K27me3 in CIZ1-null fibroblasts. Together the data suggest that PLD1-driven CIZ1 assemblies form at Xi, are nucleated by interaction with Xist and amplified by multivalent interaction with RNA, so implicating a polyglutamine tract in the maintenance of epigenetic state.

cell biology↗

bioRxiv2020

Acute depletion of METTL3 identifies a role for N6-methyladenosine in alternative intron/exon inclusion in the nascent transcriptome

RNA N6-methyladenosine (m6A) modification plays important roles in multiple aspects of RNA regulation. m6A is installed co-transcriptionally by the METTL3/14 complex, but its direct roles in RNA processing remain unclear. Here we investigate the presence of m6A in nascent RNA of mouse embryonic stem cells (mESCs). We find that around 10% m6A peaks are in introns, often close to 5-splice sites. RNA m6A peaks significantly overlap with RBM15 RNA binding sites and the histone modification H3K36me3. Interestingly, acute dTAG depletion of METTL3 reveals that inclusion of m6A-bearing alternative introns/exons in the nascent transcriptome is disrupted. For terminal or variable-length exons, m6A peaks are generally located upstream of a repressed 5-splice site, and downstream of an enhanced 5-splice site. Intriguingly, genes with the most immediate effects on splicing include several components of the m6A pathway, suggesting an autoregulatory function. Our findings demonstrate a direct crosstalk between m6A machinery and the regulation of RNA processing.

genetics↗

bioRxiv2018

Selective roles of vertebrate PCF11 in premature and full-length transcript termination

The pervasive nature of RNA polymerase II (Pol II) transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation (CPA) factor PCF11, which directly binds to the Pol II C-terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11-mediated termination is essential for vertebrate development. A range of genomic analyses, including: mNET-seq, 3 mRNA-seq, chromatin RNA-seq and ChIP-seq, reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome-wide. PCF11 binds preferentially between closely spaced genes, where it prevents transcriptional interference and downstream gene silencing. Notably, PCF11 is sub-stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto-regulatory mechanism involving premature termination of its own transcript, and are important for normal development. Both in human cell culture and during zebrafish development, PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination.

molecular biology↗

bioRxiv2018

Systematic Allelic Analysis Defines the Interplay of Key Pathways in X Chromosome Inactivation

Xist RNA, the master regulator of X chromosome inactivation, acts in cis to induce chromosome silencing through the stepwise recruitment of factors that modify underlying chromatin structure. Whilst considerable progress has been made towards defining key silencing factors and the elements to which they bind, their relative contribution to silencing different genes, and their relationship with one another is poorly understood. Here we describe a systematic analysis of Xist-mediated allelic silencing in ES cell-based models. We show that Spen, recruited through the Xist A-repeat, plays a central role, being critical for silencing of all except a subset of weakly expressed genes. Polycomb, recruited through the Xist B/C-repeat, also plays a key role, favouring silencing of genes with pre-existing H3K27me3 chromatin. LBR and the Rbm15/ m6A-methyltransferase complex, previously proposed to have a central role, make at most a minor contribution to gene silencing. We integrate our findings in a comprehensive model for Xist-mediated chromosome silencing.

genetics↗

bioRxiv2018

The non-canonical SMC protein SmcHD1 antagonises TAD formation on the inactive X chromosome

The inactive X chromosome (Xi) in female mammals adopts an atypical higher-order chromatin structure, manifested as a global loss of local topologically associated domains (TADs), and formation of two mega-domains. In this study we demonstrate that the non-canonical SMC family protein, SmcHD1, which is important for gene silencing on Xi, contributes to this unique chromosome architecture. Specifically, allelic mapping of the transcriptome and epigenome in SmcHD1 null cells revealed the appearance of sub-megabase domains defined by gene activation, CpG hypermethylation and depletion of Polycomb-mediated H3K27me3. These domains, which correlate with sites of SmcHD1 enrichment on Xi in wild-type cells, additionally adopt features of active X chromosome higher-order chromosome architecture, including partial restoration of TAD boundaries. Xi chromosome architecture changes also occurred in an acute SmcHD1 knockout model, but in this case, independent of Xi gene de-repression. We conclude that SmcHD1 is a key factor in antagonising TAD formation on Xi.

molecular biology↗