DNA Wrapping Research Articles

Nucleosome placement and polymer mechanics explain genomic contacts on 100kbp scales.

The 3d organization of the genome - in particular, which two regions of DNA are in contact with each other - plays a role in regulating gene expression. Several factors influence genome 3d organization. Nucleosomes (where ~ 100 basepairs of DNA wrap around histone proteins) also bend, twist and compactify chromosomal DNA, altering its polymer mechanics. How much does the positioning of nucleosomes between gene loci influence contacts between those gene loci? And, to what extent is polymer mechanics responsible for this? To address this question, we combine a stochastic polymer mechanics model of chromosomal DNA including twists and wrapping induced by nucleosomes with two data-driven pipelines. The first estimates nucleosome positioning from ATACseq data in regions of high accessibility. Most of the genome is low-accessibility, so we combine this with a novel image analysis method that estimates the distribution of nucleosome spacing from electron microscopy data. There are no free parameters in the biophysical model. We apply this method to IL6, IL15, CXCL9, and CXCL10, inflammatory marker genes in macrophages, before and after immune stimulation, and compare the predictions with contacts measured by conformation capture experiments (4C-seq). We find that within a 500 kilo-basepairs genomic region, polymer mechanics with nucleosomes can explain 71% of close contacts. These results suggest that, while genome contacts on 100kbp-scales are multifactorial, they may be amenable to mechanistic, physical explanation. Our work also highlights the role of nucleosomes, not just at the loci of interest, but between them, and not just the total number of nucleosomes, but their specific placement. The method generalizes to other genes, and can be used to address whether a contact is under active regulation by the cell (e.g., a macrophage during inflammatory stimulation). Importantly, our findings suggest that gene function may have evolved through selective pressures that co-opted contact-mediated regulatory mechanisms reliant largely on polymer mechanics.

Impact of Surfactant to DNA Exchange on Carbon Nanotube Emission for Biosensing Applications

Synthesis methods of single walled carbon nanotubes (SWCNTs) result in mixtures of different chiralites. Chirality affects SWCNT diameter as well electronic and optical properties. For use in optical sensors, mono-chirality SWCNTs offer better signal compared to chirality mixtures. A common chirality separation method is aqueous two-phase extraction, where solutions of surfactants are used to partition SWCNTs based on chirality. The process results in a mono-chiral solution of SWCNTs wrapped in surfactant, however for sensing applications SWCNTs need to be exchanged from this surfactant wrapping to single stranded DNA wrapping. Concerns have been raised about residual surfactant remaining on the SWCNT surface after the exchange process and the effect this would have on the sensing capabilities of the SWCNT. To investigate this, we compared the emission spectra of covalently modified SWCNTs sonicated directly in DNA to that of SWCNTs exchanged from surfactant into DNA. We observed that emission from exchanged SWCNTs was red shifted compared to direct DNA sonicated SWCNTs, however exchanged SWCNTs had similar environmental responsivity to direct DNA sonicated SWCNTs.

Partial wrapping of single-stranded DNA by replication protein A and modulation through phosphorylation.

Single-stranded DNA (ssDNA) intermediates which emerge during DNA metabolic processes are shielded by replication protein A (RPA). RPA binds to ssDNA and acts as a gatekeeper to direct the ssDNA towards downstream DNA metabolic pathways with exceptional specificity. Understanding the mechanistic basis for such RPA-dependent functional specificity requires knowledge of the structural conformation of ssDNA when RPA-bound. Previous studies suggested a stretching of ssDNA by RPA. However, structural investigations uncovered a partial wrapping of ssDNA around RPA. Therefore, to reconcile the models, in this study, we measured the end-to-end distances of free ssDNA and RPA-ssDNA complexes using single-molecule FRET and double electron-electron resonance (DEER) spectroscopy and found only a small systematic increase in the end-to-end distance of ssDNA upon RPA binding. This change does not align with a linear stretching model but rather supports partial wrapping of ssDNA around the contour of DNA binding domains of RPA. Furthermore, we reveal how phosphorylation at the key Ser-384 site in the RPA70 subunit provides access to the wrapped ssDNA by remodeling the DNA-binding domains. These findings establish a precise structural model for RPA-bound ssDNA, providing valuable insights into how RPA facilitates the remodeling of ssDNA for subsequent downstream processes.

Nanoscale Structure, Interactions, and Dynamics of Centromere Nucleosomes.

Centromeres are specific segments of chromosomes comprising two types of nucleosomes: canonical nucleosomes containing an octamer of H2A, H2B, H3, and H4 histones and CENP-A nucleosomes in which H3 is replaced with its analogue CENP-A. This modification leads to a difference in DNA wrapping (∼121 bp), considerably less than 147 bp in canonical nucleosomes. We used atomic force microscopy (AFM) and high-speed AFM (HS-AFM) to characterize nanoscale features and dynamics for both types of nucleosomes. For both nucleosomes, spontaneous asymmetric unwrapping of DNA was observed, and this process occurs via a transient state with ∼100 bp DNA wrapped around the core, followed by a rapid dissociation of DNA. Additionally, HS-AFM revealed higher stability of CENP-A nucleosomes compared with H3 nucleosomes in which dissociation of the histone core occurs prior to the nucleosome dissociation. These results help elucidate the differences between these nucleosomes and the potential biological necessity for CENP-A nucleosomes.

Rapid, DNA-induced interface swapping by DNA gyrase

DNA gyrase, a ubiquitous bacterial enzyme, is a type IIA topoisomerase formed by heterotetramerisation of 2 GyrA subunits and 2 GyrB subunits, to form the active complex. DNA gyrase can loop DNA around the C-terminal domains (CTDs) of GyrA and pass one DNA duplex through a transient double-strand break (DSB) established in another duplex. This results in the conversion from a positive (+1) to a negative (–1) supercoil, thereby introducing negative supercoiling into the bacterial genome by steps of 2, an activity essential for DNA replication and transcription. The strong protein interface in the GyrA dimer must be broken to allow passage of the transported DNA segment and it is generally assumed that the interface is usually stable and only opens when DNA is transported, to prevent the introduction of deleterious DSBs in the genome. In this paper, we show that DNA gyrase can exchange its DNA-cleaving interfaces between two active heterotetramers. This so-called interface ‘swapping’ (IS) can occur within a few minutes in solution. We also show that bending of DNA by gyrase is essential for cleavage but not for DNA binding per se and favors IS. Interface swapping is also favored by DNA wrapping and an excess of GyrB. We suggest that proximity, promoted by GyrB oligomerization and binding and wrapping along a length of DNA, between two heterotetramers favors rapid interface swapping. This swapping does not require ATP, occurs in the presence of fluoroquinolones, and raises the possibility of non-homologous recombination solely through gyrase activity. The ability of gyrase to undergo interface swapping explains how gyrase heterodimers, containing a single active-site tyrosine, can carry out double-strand passage reactions and therefore suggests an alternative explanation to the recently proposed ‘swivelling’ mechanism for DNA gyrase (Gubaev et al., 2016).

Rapid, DNA-induced interface swapping by DNA gyrase.

DNA gyrase, a ubiquitous bacterial enzyme, is a type IIA topoisomerase formed by heterotetramerisation of 2 GyrA subunits and 2 GyrB subunits, to form the active complex. DNA gyrase can loop DNA around the C-terminal domains (CTDs) of GyrA and pass one DNA duplex through a transient double-strand break (DSB) established in another duplex. This results in the conversion from a positive (+1) to a negative (-1) supercoil, thereby introducing negative supercoiling into the bacterial genome by steps of 2, an activity essential for DNA replication and transcription. The strong protein interface in the GyrA dimer must be broken to allow passage of the transported DNA segment and it is generally assumed that the interface is usually stable and only opens when DNA is transported, to prevent the introduction of deleterious DSBs in the genome. In this paper, we show that DNA gyrase can exchange its DNA-cleaving interfaces between two active heterotetramers. This so-called interface 'swapping' (IS) can occur within a few minutes in solution. We also show that bending of DNA by gyrase is essential for cleavage but not for DNA binding per se and favors IS. Interface swapping is also favored by DNA wrapping and an excess of GyrB. We suggest that proximity, promoted by GyrB oligomerization and binding and wrapping along a length of DNA, between two heterotetramers favors rapid interface swapping. This swapping does not require ATP, occurs in the presence of fluoroquinolones, and raises the possibility of non-homologous recombination solely through gyrase activity. The ability of gyrase to undergo interface swapping explains how gyrase heterodimers, containing a single active-site tyrosine, can carry out double-strand passage reactions and therefore suggests an alternative explanation to the recently proposed 'swivelling' mechanism for DNA gyrase (Gubaev et al., 2016).

Molecular motor in a box: Attractors, repellers, and ratchets in chromatin

The spatiotemporal organization of eukaryotic DNA is controlled by an intricate combination of passive (thermally activated) and active (ATP-consuming) processes. Based on recent experimental insights into the mechanochemical cycle of chromatin remodelers, molecular machines that actively control nucleosome positions, we introduce a model to study the competition between active and passive mechanisms at the most basic layer of DNA packaging, the wrapping of DNA into nucleosomes. Depending on the level of remodeler activity, the positions of nucleosomes are controlled by either the bending or the stretching energy of the wrapped DNA involved. Since these energies are highly sequence dependent, DNA guides its own packaging. However, since this dependence differs for the two deformation modes, active processes can drive nucleosomes from their equilibrium positions. Furthermore, for repetitive DNA sequences, such as telomeres, we find thermal ratchets that propel nucleosomes in a preferred direction. Published by the American Physical Society 2024

Abstract 3505: Classification of cancer subtypes by cfDNA fragmentomics analysis

Abstract Introduction Fragmentation patterns of cell-free DNA (cfDNA) from whole genome sequencing offer insights into nucleosome occupancy, providing a tool to infer epigenomic and transcriptomic information. The nucleosome complex, the primary protector of cfDNA, is reflected in the size distribution of cfDNA fragments, with a mode fragment size of 167 bp corresponding to the wrapping of DNA around a single nucleosome. Nucleosome occupancy patterns at transcription factor binding sites (TFBS) exhibit differences between circulating tumor DNA in cancer patients and normal plasma controls, as well as among different tumor subtypes. Therefore, cfDNA fragmentomics can be employed for cancer subtyping analysis. Methods We utilized the Griffin framework to classify tumor subtypes based on nucleosome profiling of cancer-specific TFBS and tumor subtype-specific chromatin accessibility regions from low-pass whole genome sequencing data of cfDNA. For prostate cancer subtyping, AR and ASCL1 binding sites were used to distinguish between androgen receptor-dependent prostate cancer (ARPC) and neuroendocrine prostate cancer (NEPC). ER and ERBB2 were employed for breast cancer subtyping to differentiate between ER-positive and ER-negative tumor subtypes. Results Nucleosome profiling patterns at AR binding sites (ARBS) were compared among over 500 prostate cancer plasma samples and 42 normal plasma backgrounds. The ARBS nucleosome profiling abnormality score (ARBS score) was quantified by comparing its GC-corrected fragment central coverage with the normal background using a standard Z-score.Majority of prostate cancer samples with a high ARBS score could be classified as ARPC. For 12 samples with high tumor fraction but low ARBS score and no AR copy number amplification, we found that 8 had a high ASCL1 nucleosome profiling abnormality score. All these samples also exhibited RB1 copy number loss, suggesting that these 8 samples likely belong to NEPC. Nucleosome profiling of 171 breast cancer plasma samples at breast cancer-specific ER binding sites revealed that majority of samples had a high ERBS abnormality score, indicating likely ER-positive breast cancer. Conclusion Our results demonstrate that nucleosome profiling of tumor cfDNA exhibits distinct patterns across different tumor types. Integrating nucleosome profiling at cancer-specific transcription factor binding sites can facilitate cancer subtyping, highlighting its potential clinical utility for patient stratification. Citation Format: Lisha Zhu, Chao Dai, Shuang Gan, Shidong Jia, Pan Du. Classification of cancer subtypes by cfDNA fragmentomics analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3505.

Archaeal histone-based chromatin structures regulate transcription elongation rates

Many archaea encode and express histone proteins to compact their genomes. Archaeal and eukaryotic histones share a near-identical fold that permits DNA wrapping through select histone-DNA contacts to generate chromatin-structures that must be traversed by RNA polymerase (RNAP) to generate transcripts. As archaeal histones can spontaneously assemble with a single histone isoform, single-histone chromatin variants provide an idealized platform to detail the impacts of distinct histone-DNA contacts on transcription efficiencies and to detail the role of the conserved cleavage stimulatory factor, Transcription Factor S (TFS), in assisting RNAP through chromatin landscapes. We demonstrate that substitution of histone residues that modify histone-DNA contacts or the three-dimensional chromatin structure result in radically altered transcription elongation rates and pausing patterns. Chromatin-barriers slow and pause RNAP, providing regulatory potential. The modest impacts of TFS on elongation rates through chromatin landscapes is correlated with TFS-dispensability from the archaeon Thermococcus kodakarensis. Our results detail the importance of distinct chromatin structures for archaeal gene expression and provide a unique perspective on the evolution of, and regulatory strategies imposed by, eukaryotic chromatin.

Detection of new pioneer transcription factors as cell-type-specific nucleosome binders.

Wrapping of DNA into nucleosomes restricts accessibility to DNA and may affect the recognition of binding motifs by transcription factors. A certain class of transcription factors, the pioneer transcription factors, can specifically recognize their DNA binding sites on nucleosomes, initiate local chromatin opening, and facilitate the binding of co-factors in a cell-type-specific manner. For the majority of human pioneer transcription factors, the locations of their binding sites, mechanisms of binding, and regulation remain unknown. We have developed a computational method to predict the cell-type-specific ability of transcription factors to bind nucleosomes by integrating ChIP-seq, MNase-seq, and DNase-seq data with details of nucleosome structure. We have demonstrated the ability of our approach in discriminating pioneer from canonical transcription factors and predicted new potential pioneer transcription factors in H1, K562, HepG2, and HeLa-S3 cell lines. Last, we systematically analyzed the interaction modes between various pioneer transcription factors and detected several clusters of distinctive binding sites on nucleosomal DNA.

Detection of new pioneer transcription factors as cell-type-specific nucleosome binders

Wrapping of DNA into nucleosomes restricts accessibility to DNA and may affect the recognition of binding motifs by transcription factors. A certain class of transcription factors, the pioneer transcription factors, can specifically recognize their DNA binding sites on nucleosomes, initiate local chromatin opening, and facilitate the binding of co-factors in a cell-type-specific manner. For the majority of human pioneer transcription factors, the locations of their binding sites, mechanisms of binding, and regulation remain unknown. We have developed a computational method to predict the cell-type-specific ability of transcription factors to bind nucleosomes by integrating ChIP-seq, MNase-seq, and DNase-seq data with details of nucleosome structure. We have demonstrated the ability of our approach in discriminating pioneer from canonical transcription factors and predicted new potential pioneer transcription factors in H1, K562, HepG2, and HeLa-S3 cell lines. Last, we systematically analyzed the interaction modes between various pioneer transcription factors and detected several clusters of distinctive binding sites on nucleosomal DNA.

Prediction of Mycotoxin Response of DNA-Wrapped Nanotube Sensor with Machine Learning

DNA-wrapped single-walled carbon nanotubes (SWCNTs) have demonstrated great versatility in their use as optical sensors. SWCNTs emit a near-infrared fluorescence that is responsive to even the slightest changes in the nanotube environment, enabling sensors that can respond to single-molecule fluctuations within the vicinity of their surfaces. The fluorescence response and surface interactions of these sensors are determined by the DNA wrapping sequence. However, the lack of information on the relationship between the DNA sequence and its effect on the SWCNT fluorescence remains a bottleneck for designing sequences that are specific to analytes of interest. We have recently demonstrated the use of directed volution to control the fluorescence response of SWCNTs through DNA design. Iterative cycles of DNA mutation, screening, and selection allowed us to evolve sequences that yield DNA-wrapped SWCNT sensors with a desired fluorescence response to mycotoxins.In this work, we apply the screening results of the DNA libraries used in this approach to train machine learning (ML) algorithms. Artificial neural network (ANN) and support vector machine (SVM) methods were used to predict the response of ssDNA-SWCNT sensors to a specific mycotoxin. The reliability of these models was further assessed through cross-validation. The ANN and SVM models with cross-validation were able to accurately classify the various DNA sequences as yielding either a high or low fluorescence response with an accuracy of 73 and 81%, respectively. The models were further used to predict the performance of alternative DNA sequences outside the initial training dataset, using the Hierarchy and k-means++ clustering methods to evaluate the similarity and dissimilarity of each DNA sequence. Compared to the SVM model, the ANN model showed an improved ability to predict high responses for dissimilar DNA sequences. We further applied a combinatorial approach based on SVM and ANN models to design new DNA sequences for improving sensor performance. The success of this approach was validated experimentally, demonstrating the rational design of improved sensors with 95% prediction accuracy.The application of ML algorithms to directed evolution libraries of DNA thus allows one to accurately map the performances of these sensors within a particular sequence space. The computational success of this mapping provides a framework for replacing current empirical approaches with the rational design of DNA sequences for SWCNT sensing. Keywords: Nano Biosensor, DNA-SWCNT, Mycotoxin, Machine learning, Directed evolution method.

Prediction of Mycotoxin Response of DNA-Wrapped Nanotube Sensor with Machine Learning

DNA-wrapped single-walled carbon nanotubes (SWCNTs) have demonstrated great versatility in their use as optical sensors. SWCNTs emit a near-infrared fluorescence that is responsive to even the slightest changes in the nanotube environment, enabling sensors that can respond to single-molecule fluctuations within the vicinity of their surfaces. The fluorescence response and surface interactions of these sensors are determined by the DNA wrapping sequence. However, the lack of information on the relationship between the DNA sequence and its effect on the SWCNT fluorescence remains a bottleneck for designing sequences that are specific to analytes of interest. We have recently demonstrated the use of directed evolution to control the fluorescence response of SWCNTs through DNA design. Iterative cycles of DNA mutation, screening, and selection allowed us to evolve sequences that yield DNA-wrapped SWCNT sensors with a desired fluorescence response to mycotoxins.In this work, we apply the screening results of the DNA libraries used in this approach to train machine learning (ML) algorithms. Artificial neural network (ANN) and support vector machine (SVM) methods were used to predict the response of ssDNA-SWCNT sensors to a specific mycotoxin. The reliability of these models was further assessed through cross-validation. The ANN and SVM models with cross-validation were able to accurately classify the various DNA sequences as yielding either a high or low fluorescence response with an accuracy of 73 and 81%, respectively. The models were further used to predict the performance of alternative DNA sequences outside the initial training dataset, using the Hierarchy and k-means ++ clustering methods to evaluate the similarity and dissimilarity of each DNA sequence. Compared to the SVM model, the ANN model showed an improved ability to predict high responses for dissimilar DNA sequences. We further applied a combinatorial approach based on SVM and ANN models to design new DNA sequences for improving sensor performance. The success of this approach was validated experimentally, demonstrating the rational design of improved sensors with 95% prediction accuracy.The application of ML algorithms to directed evolution libraries of DNA thus allows one to accurately map the performances of these sensors within a particular sequence space. The computational success of this mapping provides a framework for replacing current empirical approaches with the rational design of DNA sequences for SWCNT sensing. Keywords: Nano Biosensor, DNA-SWCNT, Mycotoxin, Machine learning, Directed evolution method

What DNA Sequence Strongly Binds to Carbon Nanotube? High-Throughput Study

Noncovalent functionalization of single-walled carbon nanotubes (SWCNT) with amphiphilic polymers has significantly advanced the colloidal SWCNT efficacy for biomedical optical sensors. Single-strand DNA (ssDNA), as an amphiphilic biocompatible polymer, has been employed to fabricate ssDNA-wrapped SWCNT complexes, however the correlation between DNA sequence and wrapping affinity is not fully studied. In this research, we evolved and screened high-affinity ssDNA sequences from ~1015 ssDNA (random 30-mer), and analyzed the influence of DNA sequence on the wrapping affinity. Wrapping affinity of specific ssDNA sequence was quantitatively measured by the rate constant when ssDNA at the surface was exchanged to sodium cholate. The wrapping affinity relies on the base composition and k-mer motif. Also, we build a machine learning model to identify the high affinity DNA sequence with >85% accuracy. These results indicate the presence of “sequence motif(s)” to control the wrapping affinity and chemical conformation of ssDNA molecule on the SWCNT surface, which can be useful for developing DNA-assisted carbon nanosensors.

Structural insights into histone binding and nucleosome assembly by chromatin assembly factor-1.

Chromatin inheritance entails de novo nucleosome assembly after DNA replication by chromatin assembly factor-1 (CAF-1). Yet direct knowledge about CAF-1's histone binding mode and nucleosome assembly process is lacking. In this work, we report the crystal structure of human CAF-1 in the absence of histones and the cryo-electron microscopy structure of CAF-1 in complex with histones H3 and H4. One histone H3-H4 heterodimer is bound by one CAF-1 complex mainly through the p60 subunit and the acidic domain of the p150 subunit. We also observed a dimeric CAF-1-H3-H4 supercomplex in which two H3-H4 heterodimers are poised for tetramer assembly and discovered that CAF-1 facilitates right-handed DNA wrapping of H3-H4 tetramers. These findings signify the involvement of DNA in H3-H4 tetramer formation and suggest a right-handed nucleosome precursor in chromatin replication.

Arginine anchor points govern H3 tail dynamics.

Chromatin is dynamically reorganized spatially and temporally, and the post-translational modification of histones is a key component of this regulation. The basic subunit of chromatin is the nucleosome core particle, consisting of two copies each of the histones H2A, H2B, H3, and H4 around which ∼147 base pairs of DNA wrap. The intrinsically disordered histone termini, or tails, protrude from the core and are heavily post-translationally modified. Previous studies have shown that the histone tails exist in dynamic ensembles of DNA-bound states within the nucleosome. Histone tail interactions with DNA are involved in nucleosome conformation and chromatin organization. Charge-modulating histone post-translational modifications (PTMs) are poised to perturb the dynamic interactions between histone tails and DNA. Arginine side chains form favorable interactions with DNA and are sites of charge-modulating PTMs such as citrullination. Our current focus is on the H3 tail, the longest histone tail. Four arginine residues are relatively evenly spaced along the H3 tail sequence, suggesting multivalent interactions with DNA poised for regulation by PTMs. In this study, we use NMR nuclear spin relaxation experiments to investigate the contribution of arginine residues to H3 tail dynamics within the nucleosome core particle. By neutralizing arginine via mutation to glutamine, we begin to work towards a comprehensive understanding of the contribution of individual residues to H3 tail dynamics. We find that neutralization of arginine residues results in increased regional mobility of the H3 tails, with implications for understanding the direct effects of arginine citrullination. Altogether, these studies support a role for dynamics within the histone language and emphasize the importance of charge-modulating histone PTMs in regulating chromatin dynamics, starting at the level of the basic subunit of chromatin.

Composite of DNA-Stabilized Multiwalled Carbon Nanotube and Nematic Liquid Crystal for Display Performance Optimization

We have prepared composites of a nematic liquid crystal (NLC) mixture to improve display performance by doping multiwalled carbon nanotubes (MWCNTs) and single-stranded-DNA-wrapped MWCNTs. Polarized optical microscopy images indicated the uniformity of the composite. Infrared absorption spectra showed there existed an interaction between MWCNTs and 4-pentyl-4′-cyanobiphenyl (5CB, a practical solvent-type monomer NLC) due to π–π stacking and charge transfer, indicating the anchoring of 5CB molecules on the MWCNT surface. The electro-optical property proved the incorporation of MWCNTs and DNA-wrapped MWCNTs reduced the threshold voltage and response time, thus following the changed splay elastic constant and rotational viscosity coefficient of LC, reflecting that DNA can improve the stability of MWCNTs in 5CB. Moreover, dielectric measurement showed the enhanced value to dielectric anisotropy and shift in relaxation frequency, including the changes of dielectric permittivity and loss, revealing the electrically “inertness” MWCNTs at high frequencies and the DNA-induced broadened frequency range of low dielectric loss. Then electrical conductivity tests further proved the cooperative orientation of MWCNTs with 5CB under the applied voltage and the contribution of MWCNTs’ high conducting along the long axis. This study revealed that MWCNT doping could improve the physical properties of NLCs, and DNA wrapping could pave a promising way toward stabilized MWCNT/NLC composites for display performance optimization.

Application of mPEG-CS-cRGD/Bmi-1RNAi-PTX nanoparticles in suppression of laryngeal cancer by targeting cancer stem cells

Although surgery-based comprehensive therapy is becoming the main approach to treat laryngeal cancer, recurrence, metastasis, radiotherapy resistance and chemotherapy tolerance are still the main causes of death in patients. Targeted inhibition of laryngeal cancer stem cells has been considered as the consensus to cure laryngeal cancer. Our previous study has confirmed proto-oncogene Bmi-1 as a key regulator for self-renewal of laryngeal cancer stem cells. Targeted knockdown of Bmi-1 gene effectively inhibited the self-renewal and differentiation of laryngeal cancer stem cells, leading to the promoted sensitivity to chemotherapy including paclitaxel. However, due to off-target effects and quick degradation of the naked Bmi-1-RNAi small RNA oligo by nuclease in body fluids, it is urgently needed to develop a tumor-targeted delivery system with a protective shell. In this study, we designed and synthesized cRGD peptide-modified chitosan-polyethylene glycol slow-release nanoparticles (mPEG-CS-cRGD/Bmi-1RNAi-PTX) containing Bmi-1RNAi siRNA oligo and paclitaxel, which showed spherical in shape, 200 nm diameter in size, low cytotoxicity, strong DNA wrapping, resistance to nuclease degradation and high transfection efficiency to cells. Functional analysis indicated significant suppression of cell proliferation and migration and induction of apoptosis by the nanocomplex in laryngeal cancer cells in vitro. By application to the mouse model with laryngeal cancer, the nanocomplex inhibited tumor growth significantly in vivo. In addition, cRGD peptide, paclitaxel and Bmi-1 siRNA in the nanoparticles showed synergistic effects to suppress laryngeal cancer stem cells. In conclusion, this study not only developed a laryngeal tumor-targeted chemotherapeutic system, but also demonstrated a Bmi-1 RNAi-based chemotherapeutic strategy to inhibit cancer stem cells, having strong potential to treat laryngeal cancer patients suffering therapy resistance and/or tumor recurrence.

Single molecule evidence for linker histone H1 DNA sliding and nucleosomal bypassing regulated by the C-terminal disordered domain.

Linker histone H1 proteins are amongst the most ubiquitous chromatin-binding proteins in eukaryotic cells. H1 binds to nucleosomes via interactions of the winged helix domain (WHD) with both the nucleosomal DNA at the dyad and the linker DNA that extends out from the nucleosome. The C-terminal domain (CTD) of H1 is a key domain for linker histone function, which includes regulating nucleosomal DNA wrapping, chromatin compaction, and transcription factor occupancy. Currently, dynamics of the DNA binding interactions between different H1 subtypes in the context of chromatin is poorly understood. Using an optical trap single molecule strategy combined with confocal microscopy, we investigated the DNA binding interactions between two different subtypes of human H1, H1.0 and H1.2, and nucleosomal DNA. We determined the impact of mechanical force on H1 binding interactions with linker and nucleosomal DNA, the role of the H1 CTD in regulating DNA binding interactions, and the regulation of linker histone H1 interactions with DNA by the linker histone chaperone protein Nap1. Our findings reveal the importance of the H1 CTD on how linker histones load onto DNA and find their nucleosome targets. These findings can help advance the biophysical and structural understanding of the mechanism behind how linker histone H1 targets nucleosomes to regulate chromatin compaction and transcription.

Thermodynamics of E. coli SSB phase separation. Effects of monovalent salt concentration and type and intrinsically disordered linker modifications.

E. coli single strand (ss) DNA binding protein (SSB) is an essential protein that binds ssDNA intermediates formed during genome maintenance. Depending on solution conditions SSB can bind to ssDNA in different binding modes characterized by different extent of DNA wrapping around the SSB tetramer and different cooperativities. We and others showed recently that SSB can undergo salt dependent phase separation (PS) which is facilitated by increases in concentration of the physiological salt, KGlu, while inhibited by [KCl] and/or deletion of the intrinsically disordered linker ( IDL) within the SSB C-terminal tail. These same factors influence non-nearest neighbor (NNN) cooperative binding of SSB to long ssDNA resulting in collapse of the nucleoprotein complexes. Here we report a thermodynamic study of EcSSB PS over a broad range of [KCl] and [KGlu] and find that PS is driven by favorable enthalpy with unfavorable entropy changes. While the effects of [KGlu] and [KCl] are similar at low salt concentrations, dramatic differences are observed at [salt] above 0.1 M. At low [salt] PS is driven mostly by electrostatic (coulombic) interactions, whereas at high [salt] Hofmeister effects dominate involving weak preferential interactions relative to water. We also have examined the effects on PS of amino acid sequence and composition of the IDL. The results are discussed in relation with SSB function in E. coli (supported by NIH GM136632 to TML and GM118100 to MTR).