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Strickfaden, H.

Publications and source records attributed to Strickfaden, H..

7 recordsLinked to original sources
bioRxiv2025

MeCP2 NID interaction with RNA: Implications for Rett Syndrome-Relevant Protein Regulation

Mutations in the MECP2 gene cause the progressive neurodevelopmental disorder Rett syndrome. Pathogenic missense mutation hotspots exist in the proteins Methyl DNA binding Domain (MBD), and the NCoR Interaction Domain (NID), indicating these regions as critical for MeCP2 function. The NID binds to a co-repressor complex allowing transcriptional repression at target genes. A putative RNA Binding Domain (RBD) was identified that overlaps with the NID, yet the role that RNA interaction plays in MeCP2 function remains underexplored. Using cell-based and in vitro molecular assays, we validated RNA interaction at the NID/RBD of MeCP2 both to a dsRNA probe in vitro and to the lncRNA NEAT1_2 in cells. As expected, this region did not appear to affect MeCP2-chromatin interactions; however, we found that RNA-RBD interaction precludes MeCP2-NCoR binding in cells. Taken together, we find that RNA interaction at this non-canonical RNA binding domain regulates important MeCP2-protein interactions and therefore may be a key part of the pathophysiology of Rett syndrome.

molecular biology↗
bioRxiv2024

Space-time dynamics of genome replication studied with super-resolved microscopy

Genome replication requires duplication of the complete set of DNA sequences together with nucleosomes and epigenetic signatures. Notwithstanding profound knowledge on mechanistic details of DNA replication, major problems of genome replication have remained unresolved. In this perspective article, we consider the accessibility of replication machines to all DNA sequences in due course, the maintenance of functionally important positional and structural features of chromatid domains during replication, and the rapid transition of CTs into prophase chromosomes with two chromatids. We illustrate this problem with EdU pulse-labeling (10 min) and chase experiments (80 min) performed with mouse myeloblast cells. Following light optical serial sectioning of nuclei with 3D structured illumination microscopy (SIM), seven DNA intensity classes were distinguished as proxies for increasing DNA compaction. In nuclei of cells fixed immediately after the pulse-label, we observed a relative under-representation of EdU-labeled DNA in low DNA density classes, representing the active nuclear compartment (ANC), and an over-representation in high density classes representing the inactive nuclear compartment (INC). Cells fixed after the chase revealed an even more pronounced shift to high DNA intensity classes. This finding contrasts with previous studies of the transcriptional topography demonstrating an under-representation of epigenetic signatures for active chromatin and RNAPII in high DNA intensity classes and their over-representation in low density classes. We discuss these findings in the light of current models viewing CDs either as structural chromatin frameworks or as phase-separated droplets, as well as methodological limitations that currently prevent an integration of this contrasting evidence for the spatial nuclear topography of replication and transcription into a common framework of the dynamic nuclear architecture.

cell biology↗
bioRxiv2022

True-to-scale DNA-density maps correlate with major accessibility differences between active and inactive chromatin

Chromatin compaction differences may have a strong impact on accessibility of individual macromolecules and macromolecular assemblies to their DNA target sites. Estimates based on fluorescence microscopy with conventional resolution, however, suggested only modest compaction differences ([~]2-10x) between active and inactive nuclear compartments (ANC and INC). Here, we present maps of nuclear landscapes with true-to-scale DNA-densities, ranging from <5 Mbp/{micro}m3 to >300 Mbp/{micro}m3. Maps were generated from individual human and mouse cell nuclei with single-molecule localization microscopy at [~]20 nm lateral and [~]100 nm axial resolution and supplemented by electron spectroscopic imaging. Microinjection of fluorescent nanobeads with sizes corresponding to macromolecular assemblies for transcription and replication into nuclei of living cells, demonstrated their localization and movements within the ANC and exclusion from the INC.

cell biology↗
bioRxiv2020

Condensed chromatin behaves like a solid on the mesoscale in vitro and in living cells

The association of nuclear DNA with histones to form chromatin is essential to the temporal and spatial control of eukaryotic genomes. In this study, we examined the physical state of chromatin in vitro and in vivo. Our in vitro studies demonstrate that MgCl2-dependent self-association of native chromatin fragments or reconstituted nucleosomal arrays produced supramolecular condensates whose constituents are physically constrained and solid-like. Liquid chromatin condensates could be generated in vitro, but only using non-physiological conditions. By measuring DNA mobility within heterochromatin and euchromatin in living cells, we show that chromatin also exhibits solid-like behavior in vivo. Representative heterochromatin proteins, however, displayed liquid-like behavior and coalesced around a solid chromatin scaffold. Remarkably, both euchromatin and heterochromatin showed solid-like behavior even when transmission electron microscopy revealed limited interactions between chromatin fibers. Our results therefore argue that chromatin is not liquid but exists in a solid-like material state whose properties are tuned by fiber-fiber interactions.

biochemistry↗
bioRxiv2019

KMT5C displays robust retention and liquid-like behavior in phase separated heterochromatin

The pericentromere exists as a distinct chromatin compartment that is thought to form by a process of phase separation. This reflects the ability of the heterochromatin protein CBX5 (aka HP1) to form liquid condensates that encapsulate pericentromeres.1,2 In general, phase separation compartmentalizes specific activities within the cell, but unlike membrane-bound organelles, their contents rapidly exchange with their surroundings.3 Here, we describe a novel state for the lysine methyltransferase KMT5C where it diffuses within condensates of pericentromeric heterochromatin but undergoes strikingly limited nucleoplasmic exchange, revealing a barrier to exit similar to that of biological membranes. This liquid-like behavior maps to a discrete protein segment with a small number of conserved sequence features and containing separable determinants for localization and retention that cooperate to confer strict spatial control. Accordingly, loss of KMT5C retention led to aberrant spreading of its catalytic product (H4K20me3) throughout the nucleus. We further found that KMT5C retention was reversible in response to chromatin state, which differed markedly for CBX5 and the methyl-CpG binding protein MeCP2, revealing considerable plasticity in the control of these phase separated assemblies. Our results establish that KMT5C represents a precedent in the biological phase separation4 continuum that confers robust spatial constraint of a protein and its catalytic activity without progression to a gel or solid.

cell biology↗
bioRxiv2019

A charge-dependent phase transition determines interphase chromatin organization

An emerging principle of cellular compartmentalization is that liquid unmixing results in formation of compartments by phase separation. We used electron spectroscopic Imaging (ESI), a transmission electron microscopy technology, to distinguish chromatin and nucleoplasmic phases of mammalian cell lines and their responses towards different environmental changes. We tested the hypothesis that charge-dependent phase separation mediated by the histone N-termini could explain the organization of chromatin. 3D images of nuclear chromatin with electron spectroscopic imaging (ESI) demonstrates that the amount of chromatin proximal to the interchromatin compartment (IC) differs between cell types, reflecting major differences in chromatin organization. These differences were lost when cells were treated overnight with a histone deacetylase inhibitor. We show that drastic, reversible changes in chromatin mixing or unmixing with the nucleoplasm/interchromatin space can be induced by modulating osmolarity of the medium or acetylation status of the chromatin. In vitro phase separation experiments demonstrated that chromatin separated from solution through a phase transition towards a more solid chromatin state.

cell biology↗