Biology subjects

Lei, M.

Publications and source records attributed to Lei, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Integrated transcriptome and lineage analyses reveal novel catecholaminergic cardiomyocytes contributing to the cardiac conduction system in murine heart

Cardiac conduction system (CCS) morphogenesis is essential for correct heart function yet is incompletely understood. Here we established the transcriptional landscape of cell types populating the developing heart by integrating single-cell RNA sequencing and spatial enhanced resolution omics-sequencing (Stereo-seq). Stereo-seq provided a spatiotemporal transcriptomic cell fate map of the murine heart with a panoramic field of view and in situ cellular resolution of the CCS. This led to the identification of a previously unrecognized cardiomyocyte population expressing dopamine beta-hydroxylase (Dbh+-CMs), which is closely associated with the CCS in transcriptomic analyses. To confirm this finding, genetic fate mapping by using DbhCre/Rosa26-tdTomato reporter mouse line was performed with Stereo-seq, RNAscope, and immunohistology. We revealed that Dbh+-derived CMs first emerged in the sinus venosus at E12.5, then populated the atrial and ventricular CCS components at E14.5, with increasing abundance towards perinatal stages. Further tracing by using DbhCFP reporter and DbhCreERT/Rosa26-tdTomato inducible reporter, we confirmed that Dbh+-CMs are mostly abundant in the AVN and ventricular CCS and this persists in the adult heart. By using DbhCre/Rosa26-tdTomato/Cx40-eGFP compound reporter line, we validated a clear co-localization of tdTomato and eGFP signals in both left and right ventricular Purkinje fibre networks. Finally, electrophysiological optogenetic study using cell-type specific Channelrhodopsin2 (ChR2) expression further elucidated that Dbh+-derived CMs form a functional part of the ventricular CCS and display similar photostimulation-induced electrophysiological characteristics to Cx40CreERT/ChR2-tdTomato CCS components. Thus, by utilizing advanced transcriptomic, mouse genetic, and optogenetic functional analyses, our study provides new insights into mammalian CCS development and heterogeneity by revealing novel Dbh+-CMs. HighlightsO_LIStereo-seq provided a spatiotemporal transcriptomic cell fate map of the murine heart with a panoramic field of view and in situ cellular resolution of the CCS. C_LIO_LIEstablished the transcriptional landscape of cell types populating the developing murine heart. C_LIO_LIRevealed previously unreported catecholaminergic cardiomyocyte populations contributing to the developing and mature murine cardiac conduction system. C_LI

cell biology↗

bioRxiv2022

Functional Integration of 3D-Printed Cerebral Cortical Tissue into a Brain Lesion

Engineering human tissue with diverse cell types and desired cellular architectures and functions is a considerable challenge. The cerebral cortex, which has a layered cellular architecture composed of layer-specific neurons organised into vertical columns, delivers higher cognition through intricately wired neural circuits. However, current tissue engineering approaches cannot produce such structures. Here, we use a droplet printing technique to fabricate tissues comprising simplified cerebral cortical columns. Human induced pluripotent stem cells (hiPSCs) were differentiated into upper- and deep-layer neural progenitors, which were then printed to form cerebral cortical tissues with a two-layer organization. The tissues showed layer-specific biomarker expression and developed an integrated network of processes. Implantation of the printed cortical tissues into mouse brain explants resulted in substantial implant-host integration across the tissue boundaries as demonstrated by the projection of processes, the migration of neurons and the appearance of correlated Ca2+ signals. The approach we have developed might be used for the evaluation of drugs and nutrients that promote tissue integration. Importantly, our approach might be applied in personalised implantation treatments that restore the cellular structure and function of a damaged brain by using 3D tissues derived from a patients own iPSCs.

bioengineering↗

bioRxiv2022

Generation of cardiomyocytes from human induced pluripotent stem cells resembling atrial cells with ability to respond to adrenoceptor agonists

Cardiovascular disease is the leading cause of global mortality and morbidity. Cardiac dysrhythmias contribute significantly to this disease burden. Atrial fibrillation (AF) is the most common chronic dysrhythmia. Human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-AMs) present an exciting new model for AF but currently fail to reach maturity and so are limited in translational potential currently. We report a new approach using a combination of Gremlin 2 and retinoic acid treatment of human iPSCs for generating cardiomyocytes resembling atrial cells. More than 40% of myocytes generated by this approach showed rod-shaped morphology, expression of cardiomyocyte proteins (including RyR2 receptors, a-actinin-2, F-actin) and typically a striated appearance, all of which were broadly similar to the characteristics of adult atrial myocytes. Isolated myocytes were electrically quiescent until stimulated to fire action potentials with an atrial myocyte profile and an amplitude of approximately 100 mV, arising from a resting potential of approximately -70 mV. Single-cell RNA sequence (scRNASeq) analysis showed a high level of expression of several atrial specific transcripts including NPPA, MYL7, HOXA3, SLN, KCNJ4, KCNJ5 and KCNA5. Amplitudes of calcium transients recorded from spontaneously beating cultures were increased by the stimulation of -adrenoceptors (activated by phenylephrine and blocked by prazosin) or {beta}-adrenoceptors (activated by isoproterenol and blocked by CGP20712A). Thus, our new method provides an efficient approach for differentiating human atrial myocytes with mature characteristics from hiPSCs. This preparation will be very useful for studying signalling pathways in human atrial myocytes, and provides a valuable model for investigating atrial fibrillation and drug discovery.

bioengineering↗

bioRxiv2022

CD169-mediated restrictive SARS-CoV-2 infection of macrophages induces pro-inflammatory responses

Exacerbated and persistent innate immune response marked by pro-inflammatory cytokine expression is thought to be a major driver of chronic COVID-19 pathology. Although macrophages are not the primary target cells of SARS-CoV-2 infection in humans, viral RNA and antigens in activated monocytes and macrophages have been detected in post-mortem samples, and dysfunctional monocytes and macrophages have been hypothesized to contribute to a protracted hyper-inflammatory state in COVID-19 patients. In this study, we demonstrate that CD169, a myeloid cell specific I-type lectin, facilitated ACE2-independent SARS-CoV-2 fusion and entry in macrophages. CD169- mediated SARS-CoV-2 entry in macrophages resulted in expression of viral genomic and sub-genomic (sg) RNAs with minimal viral protein expression and no infectious viral particle release, suggesting a post-entry restriction of the SARS-CoV-2 replication cycle. Intriguingly this post-entry replication block was alleviated by exogenous ACE2 expression in macrophages. Restricted expression of viral gRNA and sgRNA in CD169+ macrophages elicited a pro-inflammatory cytokine expression (TNF, IL-6 and IL-1{beta}) in a RIG-I, MDA-5 and MAVS-dependent manner, which was suppressed by remdesivir pre- treatment. These findings suggest that de novo expression of SARS-CoV-2 RNA in macrophages contributes to the pro-inflammatory cytokine signature and that blocking CD169-mediated ACE2 independent infection and subsequent activation of macrophages by viral RNA might alleviate COVID-19-associated hyperinflammatory response. Author SummaryOver-exuberant production of pro-inflammatory cytokine expression by macrophages has been hypothesized to contribute to severity of COVID-19 disease. Molecular mechanisms that contribute to macrophage-intrinsic immune activation during SARS- CoV-2 infection are not fully understood. Here we show that CD169, a macrophage- specific sialic-acid binding lectin, facilitates abortive SARS-CoV-2 infection of macrophages that results in innate immune sensing of viral replication intermediates and production of proinflammatory responses. We identify an ACE2-independent, CD169- mediated endosomal viral entry mechanism that results in cytoplasmic delivery of viral capsids and initiation of virus replication, but absence of infectious viral production. Restricted viral replication in CD169+ macrophages and detection of viral genomic and sub-genomic RNAs by cytoplasmic RIG-I-like receptor family members, RIG-I and MDA5, and initiation of downstream signaling via the adaptor protein MAVS, was required for innate immune activation. These studies uncover mechanisms important for initiation of innate immune sensing of SARS-CoV-2 infection in macrophages, persistent activation of which might contribute to severe COVID-19 pathophysiology.

immunology↗

bioRxiv2021

Flow Cytometric Evaluation of Yeast-Bacterial Cell-Cell Interactions

Synthetic cell-cell interaction systems can be useful for understanding multicellular communities or for screening binding molecules. We adapt a previously characterized set of synthetic cognate nanobody-antigen pairs to a yeast-bacteria coincubation format and use flow cytometry to evaluate cell-cell interactions mediated by binding between surface-displayed molecules. We further use fluorescence-activated cell sorting (FACS) to enrich for a specific yeast-displayed nanobody within a mixed yeast-display population. Finally, we demonstrate that this system supports characterization of a therapeutically relevant nanobody-antigen interaction: a previously discovered nanobody that binds to the intimin protein expressed on the surface of enterohemorrhagic E. coli. Overall, our findings indicate that the yeast-bacteria format supports efficient evaluation of ligand-target interactions. With further development, this format may facilitate systematic characterization and high throughput discovery of bacterial surface-binding molecules.

synthetic biology↗

bioRxiv2021

Comprehensive analysis of RNA-seq and whole genome sequencing data reveals no evidence for SARS-CoV-2 integrating into host genome

SARS-CoV-2, as the causation of severe epidemic of COVID-19, is one kind of positive single-stranded RNA virus with high transmissibility. However, whether or not SARS-CoV-2 can integrate into host genome needs thorough investigation. Here, we performed both RNA sequencing (RNA-seq) and whole genome sequencing on SARS-CoV-2 infected human and monkey cells, and investigated the presence of host-virus chimeric events. Through RNA-seq, we did detect the chimeric host-virus reads in the infected cells. But further analysis using mixed libraries of infected cells and uninfected zebrafish embryos demonstrated that these reads are falsely generated during library construction. In support, whole genome sequencing also didnt identify the existence of chimeric reads in their corresponding regions. Therefore, the evidence for SARS-CoV-2s integration into host genome is lacking. One-Sentence SummarySARS-CoV-2 does not integrate into host genome through whole genome sequencing.

genomics↗

bioRxiv2021

A H3K9me2-Binding Protein AGDP3 Limits DNA Methylation and Transcriptional Gene Silencing in Arabidopsis

DNA methylation is critical for tuning gene expression to prevent potentially deleterious gene-silencing. The Arabidopsis DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1) initiates active DNA demethylation and is required for the prevention of DNA hypermethylation at thousands of genomic loci. However, the mechanism recruiting ROS1 to specific loci is not well understood. Here, we report the discovery of Arabidopsis AGENET Domain Containing Protein 3 (AGDP3) as a cellular factor required for ROS1-mediated DNA demethylation, and targets ROS1 to specific loci. We found that AGDP3 could bind to the H3K9me2 mark by its AGD12 cassette. The crystal structure of the AGDP3 AGD12 in complex with an H3K9me2 peptide reveals the molecular basis for the specific recognition, that the dimethylated H3K9 and unmodified H3K4 are specifically anchored into two different surface pockets. Interestingly, a histidine residue located in the methylysine binding aromatic cage enables AGDP3 pH-dependent H3K9me2 binding capacity. Considering the intracellular pH correlates with the histone acetylation status, our results provide the molecular mechanism for the regulation of ROS1 DNA demethylase by the gene silencing H3K9me2 mark and the potential crosstalk with active histone acetylation mark.

plant biology↗

bioRxiv2021

Pharmacological targeting of a PWWP domain demonstrates cooperative control of NSD2 localization

NSD2 is the primary enzyme responsible for the dimethylation of lysine 36 of histone 3 (H3K36me2), a mark associated with active gene transcription and intergenic DNA methylation. In addition to a methyltransferase domain, NSD2 harbors two PWWP and five PHD domains believed to serve as chromatin reading modules, but their exact function in the regulation of NSD2 activity remains underexplored. Here we report a first-in-class chemical probe targeting the N-terminal PWWP (PWWP1) domain of NSD2. UNC6934 binds potently (Kd of 91 {+/-} 8 nM) to PWWP1, antagonizes its interaction with nucleosomal H3K36me2, and selectively engages endogenous NSD2 in cells. Crystal structures show that UNC6934 occupies the canonical H3K36me2-binding pocket of PWWP1 which is juxtaposed to the DNA-binding surface. In cells, UNC6934 induces accumulation of endogenous NSD2 in the nucleolus, phenocopying the localization defects of NSD2 protein isoforms lacking PWWP1 as a result of translocations prevalent in multiple myeloma. Mutation of other NSD2 chromatin reader domains also increases NSD2 nucleolar localization, and enhances the effect of UNC6934. Finally we identified two C-terminal nucleolar localization sequences in NSD2 that appear to drive nucleolar accumulation when one or more chromatin reader domains are disabled. These data support a model in which NSD2 chromatin engagement is achieved in a cooperative manner and subcellular localization is controlled by multiple competitive structural determinants. This chemical probe and the accompanying negative control, UNC7145, will be useful tools in defining NSD2 biology.

cell biology↗

bioRxiv2020

Mechanistic insights into ventricular arrhythmogenesis of hydroxychloroquine and azithromycin for the treatment of COVID-19

Background: Recent reports on the use of hydroxychloroquine (HCQ) alone, or combined with azithromycin (AZM) in the management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have raised cardiac safety concerns. Currently, there is limited mechanistic data evaluating cardiac safety with HCQ and AZM therapy. Methods: Using comprehensive In Vitro ProArrhythmia Assay (CiPA) Schema IC50 paradigms, we examined the cardiac electrophysiological effects of HCQ and HCQ/AZM. Molecular modelling explored HCQ and AZM binding properties to hERG. Langendorff-perfused guinea-pig hearts were electrically and optically mapped by multi-electrode array and voltage (RH237) and Ca2+ (Rhod-2 AM) dyes. Human action potential and ion current reconstructions were performed in silico. Results: HCQ blocked IKr and IK1 with IC50 concentrations (10+-0.6 and 34+-5.0 microM) within the therapeutic range observed clinically. HCQ also blocked INa and ICaL but at higher IC50, whilst Ito and IKs were unaffected. Contrastingly, AZM produced minor inhibition of INa, ICaL, IKs, and IKr,, with no effect on IK1 and Ito. HCQ + AZM combined inhibited IKr and IK1 with IC50s of 7.7 +- 0.8 microM and 30.4 +- 3.0 microM, but spared INa, ICaL and Ito,. Molecular modelling confirmed potential HCQ binding to hERG. Cardiac mapping and ECG studies in isolated hearts demonstrated that HCQ slowed heart rate and ventricular conduction with associated prolongation of PR, QRS and QT intervals. Optical mapping demonstrated, and prolonged, more heterogeneous, action potential durations and intracellular Ca2+ transients. These effects were accentuated with combined HCQ+AZM treatment, which elicited electrical alternans, re-entrant circuits and wave breaks. Reconstruction in a human in-silico model demonstrated that this is attributable to the integrated action of HCQ and AZM reducing IKr, IKs and IK1. Conclusions: These data provide an electrophysiological basis for recent FDA guidelines cautioning against combined HCQ/AZM administration for the treatment of Covid-19 on the grounds of potential cardiac safety. We would strongly recommend monitoring of electrocardiographic QT interval with the use of this combination of medications.Competing Interest StatementThe authors have declared no competing interest.

pharmacology and toxicology↗

bioRxiv2020

Latent defense response to non-pathogenic microbial factors impairs plant-rhizobacteria mutualism

Unlike pathogens that trigger plant defense responses, commensal or beneficial microbes are compatible with plants and do not elicit a defense response. An assumption underlying the compatibility is that plants are inert in mounting a defense response to non-pathogenic microbial factors. However, the mechanisms underlying this inertness in defense are unknown. Here a forward genetic screen led to the isolation of an Arabidopsis mutant displaying a new type of immunity which we named as latent defense response (LDR) to a beneficial rhizobacterium. The mutant, known as gp1 for Growth-Promotion 1, is impaired in rhizobacteria-induced plant growth-promotion due to disrupted oleic acid homeostasis and consequent activation of defense responses. Several bacterial volatile compounds trigger LDR in gp1 but not wild type plants. GP1 dysfunction strongly represses colonization of the beneficial rhizobacterium and alters root-associated microbiota. Our findings reveal a hidden layer of plant defense, LDR, which is suppressed by GP1 to allow mutualistic association between plants and beneficial rhizobacteria. One Sentence SummaryA hidden layer of host immunity against non-pathogenic microbes leads to plant incompatibility with beneficial rhizobacteria.

plant biology↗

bioRxiv2019

Ultra-low input single tube linked-read library method enables short-read NGS systems to generate highly accurate and economical long-range sequencing information for de novo genome assembly and haplotype phasing

Long-range sequencing information is required for haplotype phasing, de novo assembly and structural variation detection. Current long-read sequencing technologies can provide valuable long-range information but at a high cost with low accuracy and high DNA input requirement. We have developed a single-tube Transposase Enzyme Linked Long-read Sequencing (TELL-Seq) technology, which enables a low-cost, high-accuracy and high-throughput short-read next generation sequencer to routinely generate over 100 Kb long-range sequencing information with as little as 0.1 ng input material. In a PCR tube, millions of clonally barcoded beads are used to uniquely barcode long DNA molecules in an open bulk reaction without dilution and compartmentation. The barcode linked reads are used to successfully assemble genomes ranging from microbes to human. These linked-reads also generate mega-base-long phased blocks and provide a cost-effective tool for detecting structural variants in a genome, which are important to identify compound heterozygosity in recessive Mendelian diseases and discover genetic drivers and diagnostic biomarkers in cancers.

genomics↗

bioRxiv2019

Reporter system architecture affects measurements of noncanonical amino acid incorporation efficiency and fidelity

The ability to genetically encode noncanonical amino acids (ncAAs) within proteins supports a growing number of applications ranging from fundamental biological studies to enhancing the properties of biological therapeutics. Currently, our quantitative understanding of ncAA incorporation systems is confounded by the diverse set of characterization and analysis approaches used to quantify ncAA incorporation events. While several effective reporter systems support such measurements, it is not clear how quantitative results from different reporters relate to one another, or which details influence measurements most strongly. Here, we evaluate the quantitative performance of single-fluorescent protein reporters, dual-fluorescent protein reporters, and cell surface displayed protein reporters of ncAA insertion in response to the TAG (amber) codon in yeast. While different reporters support varying levels of apparent readthough efficiencies, flow cytometry-based evaluations with dual reporters yielded measurements exhibiting consistent quantitative trends and precision across all evaluated conditions. Further investigations of dual-fluorescent protein reporter architecture revealed that quantitative outputs are influenced by stop codon location and N-and C-terminal fluorescent protein identity. Both dual-fluorescent protein reporters and a \"drop-in\" version of yeast display support quantification of ncAA incorporation in several single-gene knockout strains, revealing strains that enhance ncAA incorporation efficiency without compromising fidelity. Our studies reveal critical details regarding reporter system performance in yeast and how to effectively deploy such reporters. These findings have substantial implications for how to engineer ncAA incorporation systems--and protein translation apparatuses--to better accommodate alternative genetic codes for expanding the chemical diversity of biosynthesized proteins.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC=\"FIGDIR/small/737197v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (18K):\norg.highwire.dtl.DTLVardef@342d13org.highwire.dtl.DTLVardef@17b6ab6org.highwire.dtl.DTLVardef@19aed21org.highwire.dtl.DTLVardef@4648b8_HPS_FORMAT_FIGEXP M_FIG C_FIG Design, System, Application ParagraphOn earth, the genetic code provides nearly invariant instructions for generating the proteins present in all organisms using 20 primary amino acid building blocks. Scientists and engineers have long recognized the potential power of altering the genetic code to introduce amino acids that enhance the chemical versatility of proteins. Proteins containing such \"noncanonical amino acids\" (ncAAs) can be used to elucidate basic biological phenomena, discover new therapeutics, or engineer new materials. However, tools for measuring ncAA incorporation during protein translation (reporters) exhibit highly variable properties, severely limiting our ability to engineer improved ncAA incorporation systems. In this work, we sought to understand what properties of these reporters affect measurements of ncAA incorporation events. Using a series of ncAA incorporation systems in yeast, we evaluated reporter architecture, measurement techniques, and alternative data analysis methods. We identified key factors contributing to quantification of ncAA incorporation in all of these categories and demonstrated the immediate utility of our approach in identifying genomic knockouts that enhance ncAA incorporation efficiency. Our findings have important implications for how to evolve cells to better accommodate alternative genetic codes.

synthetic biology↗

bioRxiv2019

Super-resolution imaging reveals changes in Escherichia coli SSB localization in response to DNA damage.

The E. coli single stranded DNA binding protein (SSB) is essential to viability. It plays key roles in DNA metabolism where it binds to nascent single strands of DNA and to target proteins known as the SSB interactome. There are >2,000 tetramers of SSB per cell with perhaps 100-150 associated with genome at any one time, either at DNA replication forks or at sites of DNA repair. The remaining 1,900 tetramers could constantly diffuse throughout the cytosol or be associated with the inner membrane as observed for other DNA metabolic enzymes such as DnaA and RecA. To visualize SSB directly and to ascertain spatiotemporal changes in tetramer localization in response to DNA damage, SSB-GFP chimeras were visualized using a novel, super-resolution microscope optimized for visualization of prokaryotic cells. Results show that in the absence of DNA damage, SSB localizes to a small number of foci and the excess protein is observed associated with the inner membrane where it binds to the major phospholipids. Within five minutes following DNA damage, the vast majority of SSB disengages from the membrane and is found almost exclusively in the cell interior. Here, it is observed in a large number of foci, in discreet structures or, in diffuse form spread over the genome, thereby enabling repair events. In the process, it may also deliver interactome partners such as RecG or PriA to sites where their repair functions are required.

cell biology↗

bioRxiv2019

Environmental chemical diethylhexyl phthalate alters intestinal microbiota community structure and metabolite profile in mice

Exposure to environmental chemicals during windows of development is a potentially contributing factor in gut microbiota dysbiosis, and linked to chronic diseases and developmental disorders. We used a community-level model of microbiota metabolism to investigate the effects of diethylhexyl phthalate (DEHP), a ubiquitous plasticizer implicated in neurodevelopmental disorders, on the composition and metabolite outputs of gut microbiota in young mice. Administration of DEHP by oral gavage increased the abundance of Lachnoclostridum, while decreasing Akkermansia, Odoribacter, and Clostridium sensu stricto. Addition of DEHP to in vitro cultured cecal microbiota increased the abundance of Alistipes, Paenibacillus, and Lachnoclostridium. Untargeted metabolomics showed that DEHP broadly altered the metabolite profile in the culture. Notably, DEHP enhanced the production of p-cresol, while inhibiting butyrate synthesis. Metabolic model-guided correlation analysis indicated that the likely sources of p-cresol are Clostridium species. Our results suggest that DEHP can directly modify the microbiota to affect production of bacterial metabolites linked with neurodevelopmental disorders.\n\nImportanceSeveral previous studies have pointed to environmental chemical exposure during windows of development as a contributing factor in neurodevelopmental disorders, and correlated these disorders with microbiota dysbiosis, little is known about how the chemicals specifically alter the microbiota to interfere with development. The findings reported in this paper unambiguously establish that a pollutant linked with neurodevelopmental disorders can directly modify the microbiota to promote the production of a potentially toxic metabolite (p-cresol) that has also been correlated with neurodevelopmental disorders. Further, we use a novel modeling strategy to identify the responsible enzymes and bacterial sources of this metabolite. To the best of our knowledge, the present study is the first to characterize the functional consequence of phthalate exposure on a developed microbiota. Our results suggest that specific bacterial pathways could be developed as diagnostic and therapeutic targets against health risks posed by ingestion of environmental chemicals.

microbiology↗

bioRxiv2019

Plasticity at the DNA recognition site of the MeCP2 mCG-binding domain

MeCP2 is an abundant protein, involved in transcriptional repression by binding to CG and non-CG methylated DNA. However, MeCP2 might also function as a transcription activator as MeCP2 is found bound to sparsely methylated promoters of actively expressed genes. Furthermore, Attachment Region Binding Protein (ARBP), the chicken ortholog of MeCP2, has been reported to bind to Matrix/scaffold attachment regions (MARs/SARs) DNA with an unmethylated 5-CAC/GTG-3 consensus sequence. In this study, we investigated how MeCP2 recognizes unmethylated 5-CAC/GTG-3 motif containing DNA by binding and structural studies. We found that MeCP2-MBD binds to MARs DNA with a comparable binding affinity to mCG DNA, and the MeCP2-CAC/GTG complex structure revealed that MeCP2 residues R111 and R133 form base-specific interactions with the GTG motif. For comparison, we also determined crystal structures of the MeCP2-MBD bound to mCG and mCAC/GTG DNA, respectively. Together, these crystal structures illustrate the adaptability of the MeCP2-MBD toward the GTG motif as well as the mCG DNA, and also provide structural basis of a biological role of MeCP2 as a transcription activator and its disease implications in Rett syndrome.

biochemistry↗

bioRxiv2018

Crystal structure of the MBD domain of MBD3 in complex with methylated CG DNA

MBD3 is a core subunit of the Mi-2/NuRD complex, and has been previously reported to lack methyl-CpG binding ability. However, recent reports show that MBD3 recognizes both mCG and hmCG DNA with a preference for hmCG, and is required for the normal expression of hmCG marked genes in ES cells. Nevertheless, it is not clear how MBD3 recognizes the methylated DNA. In this study, we carried out structural analysis coupled with isothermal titration calorimetry (ITC) binding assay and mutagenesis studies to address the structural basis for the mCG DNA binding ability of the MBD3 MBD domain. We found that the MBD3 MBD domain prefers binding mCG over hmCG through the conserved arginine fingers, and this MBD domain as well as other mCG binding MBD domains can recognize the mCG duplex without orientation selectivity. Furthermore, we found that the tyrosine-to-phenylalanine substitution at Phe34 of MBD3 is responsible for its weaker mCG DNA binding ability compared to other mCG binding MBD domains. In summary, our study demonstrates that the MBD3 MBD domain is a mCG binder, and also illustrates its binding mechanism to the methylated CG DNA.

biochemistry↗

bioRxiv2018

Structural and histone binding studies of the chromo barrel domain of TIP60

TIP60 consists of an N-terminal chromo barrel domain (TIP60-CB) and a C-terminal acetyltransferase domain and acetylates histone and non-histone proteins within diverse cellular processes. Whereas the TIP60-CB is thought to recognize histone tails, molecular details of this interaction remain unclear. Here we attempted a quantitative analysis of the interaction between the TIP60-CB and histone peptides, but did not observe any binding through either fluorescence polarization or isothermal titration calorimetry. We solved a crystal structure of the TIP60-CB alone. Analysis of the crystal structure demonstrates a putative peptide binding site that may be occluded by the basic side chain of a residue in a unique {beta} hairpin between the two N-terminal strands of the {beta} barrel.

biochemistry↗

bioRxiv2018

Crystal structure of chromo barrel domain of RBBP1

RBBP1 is a retinoblastoma protein (pRb) binding protein acting as a repressor of gene transcription. RBBP1 is a multidomain protein including a chromo barrel domain, and its chromo barrel domain has been reported to recognize histone H4K20me3 weakly, and this binding is enhanced by the simultaneous binding of DNA. However, the molecular basis of this DNA-mediated histone binding by the chromo barrel domain of RBBP1 is unclear. Here we attempted to co-crystallize the chromo barrel domain of RBBP1 with either a histone H4K20me3 peptide alone or with both a histone H4K20me3 peptide and DNA, but only solved the peptide/DNA unbound crystal structure. Our structural analysis indicates that RBBP1 could interact with histone H4K20me3 similar to other histone binding chromo barrel domains, and the surface charge representation analysis of the chromo barrel domain of RBBP1 suggests that the chromo barrel domain of RBBP1 does not have a typical DNA binding surface, indicating that it might not bind to DNA. Consistently, our ITC assays also showed that DNA does not significantly enhance the histone binding ability of the chromo barrel domain of RBBP1.

biochemistry↗