Biology subjects

Chen, R.-W.

Publications and source records attributed to Chen, R.-W..

5 recordsLinked to original sources

bioRxiv2026

Fusion Oncoprotein EWSR1::FLI1 Invades Nucleosomes at Consensus ETS Motifs and GGAA Microsatellites

Ewing sarcoma is an aggressive bone malignancy occurring in children, adolescents, and young adults. Most cases are caused by expression of the fusion oncoprotein EWSR1::FLI1, which contains the low complexity domain (LCD) of EWSR1 and the DNA-binding domain (DBD) of FLI1. Previous genomic studies indicate EWSR1::FLI1 accesses GGAA microsatellites in chromatin to function as a potent transcriptional regulator. Due to the technical challenges of purifying full-length EWSR1::FLI1, mechanistic studies biochemically characterizing its pioneer activities have been lacking. Here, we purified both full-length EWSR1::FLI1 and truncated DBD constructs to conduct biochemical and fluorescence-based experiments investigating interactions with different motifs in free DNA and nucleosomes. Both truncated and full-length EWSR1::FLI1 show efficient target binding in nucleosomes, and that the fourth alpha-helix in the FLI1 DBD enhances nucleosome-binding efficiency. Surprisingly, we also observe differences in both free DNA binding affinity and sequence preference between truncated and full-length proteins, though these changes are not apparent in nucleosome-binding assays. These findings reveal that EWSR1::FLI1 possesses a key pioneer factor property, efficiently targeting its binding site within nucleosomes, and that full-length EWSR1::FLI1 binding shifts to preferentially target GGAA repeats, even on motifs that bind a single EWSR1::FLI1 protein.

biochemistry↗

bioRxiv2026

In-Situ ssDNA Isolation from dsDNA Sources as a Streamlined Pathway to DNA Origami Assembly and Testing

Scaffolded DNA origami has become a valuable nanoscale tool for applications in biomedical and physical sciences. Critical to leveraging the modular and programmable properties of DNA origami nanodevices is access to the scaffold strand, a long single-stranded DNA (ssDNA) of precise length and sequence, which is folded into a compact shape via piecewise base-pairing with many staple strands, short ssDNA oligonucleotides. Current methods to produce and manipulate long ssDNA scaffolds can be costly, time-consuming, and cumbersome. In contrast, methods to produce and manipulate the sequence of double-stranded DNA (dsDNA) are efficient and scalable. Here, we present a method for the rapid isolation of target ssDNA sequences from a variety of dsDNA sources using oligonucleotides as blocking strands that bind continuously to the undesired strand, thereby releasing the target scaffold strand. We report successful ssDNA isolation from linear and supercoiled dsDNAs of various sequences and lengths, ranging from 769 to 15,101 nucleotides. In addition to isolating ssDNA, we demonstrated this approach enables folding of DNA origami directly from dsDNA templates using both blocking and staple strands in a single-pot thermally controlled reaction. Furthermore, we explore multi-scaffold and gene-encoding DNA origami structures, expanding the framework for application-based designs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/709872v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@f7ada7org.highwire.dtl.DTLVardef@a9bab8org.highwire.dtl.DTLVardef@121b695org.highwire.dtl.DTLVardef@5a1dea_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Cohesin Facilitates Nucleosome Invasion by Transcription Factors

Nucleosomes present a major barrier to transcription factor (TF) binding. However, a subgroup of TFs known as pioneer factors (PFs) can recognize motifs covered by nucleosomes and initiate chromatin opening. PFs also bind nucleosomal substrates with high affinity in vitro, which may facilitate their nucleosome invasion in vivo. Here, we show that LexA, a bacterial TF with poor nucleosome binding in vitro, can rapidly invade into a well-positioned nucleosome from a motif positioned at the dyad when expressed ectopically in yeast. This striking contrast between LexA binding in vitro and in vivo raises the possibility that TFs can exploit nucleosome dynamics in vivo to access occluded sites. Surprisingly, we find that LexA-mediated chromatin opening can occur in the absence of DNA replication, chromatin remodeling, histone turnover, and a few histone chaperones. Instead, nucleosome invasion by LexA and a native PF, Cbf1, is promoted by the cohesin complex, illustrating intriguing a connection between cohesin and TF binding. Together, our results demonstrate that even non-pioneer TFs like LexA can bind and displace nucleosomes in vivo, through a process facilitated by cohesin.

molecular biology↗

bioRxiv2025

Free energy spectroscopy reveals the mechanistic landscape of chromatin compaction

Eukaryotic genomic DNA is repeatedly wrapped into nucleosome spools: the basic building block of chromatin. This organization regulates the physical accessibility of the genome to gene transcription, replication, and repair regulatory factors. Chromatin compaction is controlled by multivalent weak interactions, resulting in a complicated conformational landscape that remains challenging to characterize. This work reports a method for characterizing chromatin compaction, Free Energy Spectroscopy (FES), which is based on DNA nanotechnology and transmission electron microscopy. This method experimentally determines the chromatin compaction free energy landscape in terms of end-to-end distance and nucleosome stacking interactions. By deconvolving the free energy landscapes of partially and fully compact tetranucleosomes, FES revealed three separate mechanisms by which linker histones reshape the compaction energetics to condense chromatin. This study establishes FES as a method with the potential to help answer a broad range of mechanistic questions about genome and epigenome function.

biophysics↗

bioRxiv2025

Native nucleosome-positioning elements for the investigation of nucleosome repositioning

Nucleosome repositioning is essential for establishing nucleosome-depleted regions (NDRs) to initiate transcription. This process has been extensively studied using structural, biochemical, and single-molecule approaches, which require homogenously positioned nucleosomes. This is often achieved using the Widom 601 sequence, a highly efficient nucleosome positioning element (NPE) selected for its unusually strong binding to the H3-H4 histone tetramer. Due to the artificial nature of 601, native NPEs are needed to explore the role of DNA sequence in nucleosome repositioning. Here, we characterize the position distributions and nucleosome formation free energy for a set of yeast native nucleosomes (YNNs) from Saccharomyces cerevisiae. We show these native NPEs can be used in biochemical studies of nucleosome repositioning by transcription factors (TFs) and the chromatin remodeler Chd1. TFs could directly reposition a fraction of nucleosomes containing native NPEs, but not 601-containing nucleosomes. In contrast, partial unwrapping was similar for 601 and native NPE sequences, and the rate of ATP-dependent remodeling by Chd1 was within the range of the fast and slow directions of the 601 nucleosomes. This set of native NPEs provides an alternative to the 601 NPE that can be used for probing the repositioning of nucleosomes that contain native DNA sequences.

molecular biology↗