Biology subjects

Zhong, Z.

Publications and source records attributed to Zhong, Z..

At least 91 records · Page 5Linked to original sources

bioRxiv2022

Improving disease resistance in plants by editing the epigenome

Pathogens rely on expression of host susceptibility (S) genes to promote infection and disease. As DNA methylation is an epigenetic modification that affects gene expression, blocking access to S genes through targeted methylation could increase disease resistance. Xanthomonas phaseoli pv. manihotis, the causal agent of cassava bacterial blight (CBB), uses transcription activator-like20 (TAL20) to induce expression of the S gene MeSWEET10a. In this work, we direct methylation to the TAL20 effector binding element within the MeSWEET10a promoter using a synthetic zinc-finger DNA binding domain fused to a component of the RNA-directed DNA methylation pathway. We demonstrate that this methylation prevents TAL20 binding, blocks transcriptional activation of MeSWEET10a in vivo and that these plants display decreased CBB symptoms while maintaining normal growth and development. This work therefore presents an epigenome editing approach useful for crop improvement.

plant biology↗

bioRxiv2021

Correlational networking guides the discovery of cryptic proteases for lanthipeptide maturation

Bacterial natural product biosynthetic genes, canonically clustered, have been increasingly found to rely on cryptic enzymes encoded elsewhere in the genome for completion of biosynthesis. The study and application of lanthipeptides are frequently hindered by cryptic proteases required for final maturation. Here, we establish a global correlation network bridging the gap between lanthipeptide precursors and cryptic proteases. Applying our analysis to 161,954 bacterial genomes, we establish 5,209 correlations between precursors and cryptic proteases, with 91 prioritized. We use network predictions and co-expression analysis to reveal a previously missing protease for the maturation of class I lanthipeptide paenilan. We further discover widely distributed bacterial M16B metallopeptidases of previously unclear biological function as a new family of lanthipeptide proteases. We show the involvement of a pair of bifunctional M16B proteases in the production of novel class III lanthipeptides with high substrate specificity. Together, these results demonstrate the strength of our correlational networking approach to the discovery of cryptic lanthipeptide proteases and potentially other missing enzymes for natural products biosynthesis. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biochemistry↗

bioRxiv2021

The tetrameric assembly of 2-aminomuconic acid dehydrogenase is a functional requirement of cofactor NAD+ binding

The bacterium Pseudomonas sp. AP-3 is able to use the environmental pollutant 2-aminophenol as its sole source of carbon, nitrogen, and energy. Eight genes (amnA, B, C, D, E, F, G, and H) encoding 2-aminophenol metabolizing enzymes are clustered into a single operon. 2-aminomuconic 6-semialdehyde dehydrogenase (AmnC), a member of the aldehyde dehydrogenase (ALDH) superfamily, is responsible for oxidizing 2-aminomuconic 6-semialdehyde to 2-aminomuconate. In contrast to many other members of the ALDH superfamily, the structural basis of the catalytic activity of AmnC remains elusive. Here, we present the crystal structure of AmnC, which displays a homotetrameric quaternary assembly that is directly involved in its enzymatic activity. The tetrameric state of AmnC in solution was also presented using small-angle X-ray scattering. The tetramerization of AmnC is mediated by the assembly of a protruding hydrophobic beta-strand motif and residues V121 and S123 located in the NAD+-binding domain of each subunit. Dimeric mutants of AmnC dramatically lose NAD+ binding affinity and enzyme activity, indicating that tetrameric assembly of AmnC is required for oxidizing the unstable metabolic intermediate 2-aminomuconic 6-semialdehyde to 2-aminomuconic acid in the 2-aminophenol metabolism pathway.

microbiology↗

bioRxiv2021

Structural insight into molecular inhibitory mechanism of InsP6 on African Swine Fever Virus mRNA-decapping enzyme g5Rp

Removal of 5' cap on cellular mRNAs by the African Swine Fever Virus (ASFV) decapping enzyme g5R protein (g5Rp) is beneficial to viral gene expression during the early stages of infection. As the only nucleoside diphosphate linked moiety X (Nudix) decapping enzyme encoded in the ASFV genome, g5Rp works in both the degradation of cellular mRNA and hydrolyzation of the diphosphoinositol polyphosphates. Here, we report the structures of dimeric g5Rp and its complex with inositol hexakisphosphate (InsP6). The two g5Rp protomers interact head-to-head to form a dimer, and the dimeric interface is formed by extensive polar and nonpolar interactions. Each protomer composed a unique N-terminal helical domain and C-terminal classic Nudix domain. As a mRNA decapping enzyme, we identified key residues, including K8, K94, K95, K98, K175, R221, and K243 located on the substrate RNA binding interfaces of g5Rp, are important to RNA binding and decapping enzyme activity. Furthermore, we identified that the g5Rp-mediated mRNA decapping was inhibited by the InsP6. The g5Rp-InsP6 complex structure showed that the InsP6 molecules occupy the same regions that primarily mediate g5Rp-RNA interaction, elucidating the roles of InsP6 in the regulation of the viral decapping activity of g5Rp in mRNA degradation. Collectively, these results provide the structural basis of interaction between RNA and g5Rp and highlight the inhibitory mechanism of InsP6 on mRNA decapping by g5Rp.

biochemistry↗

bioRxiv2021

Single-molecule mechanical unfolding kinetics of unmodified Saccharomyces cerevisiae tRNAPhe: a hint to the tRNA chaperone-tRNA interaction mechanism

The biological activity of tRNA is closely related to its mechanical folding properties. Although previous studies focused on the folding and unfolding mechanism of tRNA, its kinetics are largely unknown. In this study, combining optical tweezers and molecule dynamics simulations, we characterized the mechanical folding and unfolding processes of a single unmodified Saccharomyces cerevisiae tRNAphe. We identified the intermediates and pathways for tRNA mechanical folding and unfolding in the presence of Mg2+, discovering that the folding/unfolding kinetics of D stem-loop and T stem-loop but not the anti-codon stem-loop significantly affected by their upstream and downstream structures. The cooperative unfolding of the tRNA in the presence of Mg2+ lead to a large hysteresis between the folding and unfolding pathway, and such hysteresis and unfolding cooperativity are significantly reduced by lowering the Mg2+ concentration or mutating the nucleotides forming the elbow structure. Moreover, both steered molecular dynamics simulation and optical tweezers experiment results support that, formation of tertiary interactions in the elbow region increases energy barriers of the mechanical unfolding pathway, including those in between intermediates, and determines the overall unfolding cooperativity. Our studies may shed light on the detailed tRNA chaperone mechanism of TruB and TrmA.

biophysics↗

bioRxiv2021

Direct sulphate-TOR signalling controls transcriptional reprogramming for shoot apex activation in Arabidopsis

Photosynthetic plants play a primary role for the global sulphur cycle in the earth ecosystems by reduction of inorganic sulphate from the soil to organic sulphur-containing compounds. How plants sense and transduce the sulphate availability in soil to mediate their growth remains largely unclear. The target of rapamycin (TOR) kinase is an evolutionarily conserved master regulator of nutrient sensing and metabolic signalling to control cell proliferation and growth in all eukaryotes. Here, we discovered that inorganic sulphate exhibits higher potency than organic cysteine and glutathione for activation of TOR and cell proliferation in the leaf primordium to promote true leaf development in Arabidopsis. Chemical genetic analyses further revealed that this sulphate activation of TOR is independent of the sulphate-assimilation process and glucose-energy signalling. Significantly, tissue specific transcriptome analyses uncovered previously unknown sulphate-orchestrating genes involved in DNA replication, cell proliferation, autophagy and various secondary metabolism pathways, which are completely depending on TOR signalling. Systematic comparison between the sulphate- and glucose-TOR controlled transcriptome further revealed that, as the central growth integrator, TOR kinase can sense different upstream nutrient signals to control both shared and unique transcriptome networks, therefore, precisely modulate plant proliferation, growth and stress responses.

plant biology↗

bioRxiv2020

Sterilizing Immunity against SARS-CoV-2 Infection in Mice by a Single-Shot and Modified Imidazoquinoline TLR7/8 Agonist-Adjuvanted Recombinant Spike Protein Vaccine

The search for vaccines that protect from severe morbidity and mortality as a result of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19) is a race against the clock and the virus. Several vaccine candidates are currently being tested in the clinic. Inactivated virus and recombinant protein vaccines can be safe options but may require adjuvants to induce robust immune responses efficiently. In this work we describe the use of a novel amphiphilic imidazoquinoline (IMDQ-PEG-CHOL) TLR7/8 adjuvant, consisting of an imidazoquinoline conjugated to the chain end of a cholesterol-poly(ethylene glycol) macromolecular amphiphile). This amphiphile is water soluble and exhibits massive translocation to lymph nodes upon local administration, likely through binding to albumin. IMDQ-PEG-CHOL is used to induce a protective immune response against SARS-CoV-2 after single vaccination with trimeric recombinant SARS-CoV-2 spike protein in the BALB/c mouse model. Inclusion of amphiphilic IMDQ-PEG-CHOL in the SARS-CoV-2 spike vaccine formulation resulted in enhanced immune cell recruitment and activation in the draining lymph node. IMDQ-PEG-CHOL has a better safety profile compared to native soluble IMDQ as the former induces a more localized immune response upon local injection, preventing systemic inflammation. Moreover, IMDQ-PEG-CHOL adjuvanted vaccine induced enhanced ELISA and in vitro microneutralization titers, and a more balanced IgG2a/IgG1 response. To correlate vaccine responses with control of virus replication in vivo, vaccinated mice were challenged with SARS-CoV-2 virus after being sensitized by intranasal adenovirus-mediated expression of the human angiotensin converting enzyme 2 (ACE2) gene. Animals vaccinated with trimeric recombinant spike protein vaccine without adjuvant had lung virus titers comparable to non-vaccinated control mice, whereas animals vaccinated with IMDQ-PEG-CHOL-adjuvanted vaccine controlled viral replication and infectious viruses could not be recovered from their lungs at day 4 post infection. In order to test whether IMDQ-PEG-CHOL could also be used to adjuvant vaccines currently licensed for use in humans, proof of concept was also provided by using the same IMDQ-PEG-CHOL to adjuvant human quadrivalent inactivated influenza virus split vaccine, which resulted in enhanced hemagglutination inhibition titers and a more balanced IgG2a/IgG1 antibody response. Enhanced influenza vaccine responses correlated with better virus control when mice were given a lethal influenza virus challenge. Our results underscore the potential use of IMDQ-PEG-CHOL as an adjuvant to achieve protection after single immunization with recombinant protein and inactivated vaccines against respiratory viruses, such as SARS-CoV-2 and influenza viruses.

microbiology↗

bioRxiv2020

Blocking of EGFR Signaling is a Latent Strategy for the Improvement of Prognosis of HPV Induced Cancer

Human papillomavirus (HPV) is a dsDNA virus and its high-risk subtypes increase cancer risks. Yet, the mechanism of HPV infection and pathogenesis still remain unclear. Therefore, understanding the molecular mechanisms, and the pathogenesis of HPV are crucial in the prevention of HPV related cancers. In this study, we analyzed cervix squamous cell carcinoma (CESC) and head and neck carcinoma (HNSC) combined data to investigate various HPV induced cancer common feature. We showed that epidermal growth factor receptor (EGFR) was downregulated in HPV positive (HPV+) cancer, and that HPV+ cancer patients exhibited better prognosis than HPV negative (HPV-) cancer patients. Our study also showed that TP53 mutation rate is lower in HPV+ cancer than in HPV- cancer and that TP53 can be modulated by HPV E7 protein. However, there was no significant difference in the expression of wildtype TP53 in both groups. Subsequently, we constructed HPV-human interaction network and found that EGFR is a critical factor. From the network, we also noticed that EGFR is regulated by HPV E7 protein and hsa-miR-944. Moreover, while phosphorylated EGFR is associated with a worse prognosis, EGFR total express level is not significantly correlated with prognosis. This indicates that EGFR activation will induce a worse outcome in HPV+ cancer patients. Further enrichment analysis showed that EGFR downstream pathway and cancer relative pathway are diversely activated in HPV+ cancer and HPV- cancer. In summary, HPV E7 protein downregulates EGFR that downregulates phosphorylated EGFR and inhibit EGFR related pathways which in turn and consequently induce better prognosis. ImportanceAlthough HPV infection has been studied in various cancer types, there are only limited studies that have focused on the common effect of HPV related cancer. Consequently, this study focused on CESC and HNSC, two cancer types with high HPV infection proportion in cohort, thereby, intending to dig out the common effects and mechanisms of HPV+ cancers. Unlike some virus-human interaction prediction studies, the P-HIPSter database provides virus-human protein interaction based on protein structure prediction. Through this data, our interaction network was able to uncover previously unnoticed protein interactions. Our finding revealed that HPV infection caused various gene expression differences, and a great amount of which interact with EGFR, a cancer related gene. Therefore, since EGFR is associated with HPV+ cancer patients survival, some FDA proved EGFR inhibitors would be potential anti-HPV drugs.

microbiology↗

bioRxiv2020

Corticosteroids and cellulose purification improve respectively the in vivo translation and vaccination efficacy of self-amplifying mRNAs

Synthetic mRNAs are an appealing therapeutic platform with multiple biomedical applications ranging from protein replacement therapy to vaccination. In comparison to conventional mRNA, synthetic self-amplifying mRNAs (sa-mRNAs) are gaining increased interest due to their higher and longer-lasting expression. However, sa-mRNAs also elicit an innate immune response, which may complicate the clinical translation of this platform. Approaches to reduce the innate immunity of sa-mRNAs have not been studied in detail. In this work we investigated the effect of several innate immune inhibitors and a novel cellulose-based mRNA purification approach on the type I interferon (IFN) response, translation and vaccination efficacy of our formerly developed sa-mRNA vaccine against Zika virus. Among the investigated inhibitors, we found that topical application of clobetasol at the sa-mRNA injection site was the most efficient in suppressing the type I IFN response and increasing the translation of sa-mRNA. However, clobetasol prevented the formation of antibodies against sa-mRNA encoded antigens and should therefore be avoided in a vaccination context. Residual dsRNA by-products of the in vitro transcription reaction are known inducers of immediate type I IFN responses. We additionally demonstrate drastic reduction of these dsRNA by-products upon cellulose-based purification, consequently reducing the innate immune response and improving sa-mRNA vaccination efficacy.

immunology↗

bioRxiv2020

Cytoplasmic CPSF6 regulates HIV-1 capsid trafficking and infection in a cyclophilin A-dependent manner

Human immunodeficiency virus type 1 (HIV-1) capsid binds host proteins during infection, including cleavage and polyadenylation specificity factor 6 (CPSF6) and cyclophilin A (CypA). We observe that HIV-1 infection induces higher-order CPSF6 formation and capsid-CPSF6 complexes co-traffic on microtubules. CPSF6-capsid complex trafficking is impacted by capsid alterations that reduce CPSF6 binding or by excess cytoplasmic CPSF6 expression, both of which are associated with decreased HIV-1 infection. Higher-order CPSF6 complexes bind and disrupt HIV-1 capsid assemblies in vitro. Disruption of HIV-1 capsid binding to CypA leads to increased CPSF6 binding and altered capsid trafficking, resulting in reduced infectivity. Our data reveal an interplay between CPSF6 and CypA that is important for cytoplasmic capsid trafficking and HIV-1 infection. We propose that CypA prevents HIV-1 capsid from prematurely engaging cytoplasmic CPSF6 and that differences in CypA cellular localization and innate immunity may explain cell-specific variations in HIV-1 capsid trafficking and uncoating. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/136697v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@646829org.highwire.dtl.DTLVardef@1f25cf9org.highwire.dtl.DTLVardef@b5e696org.highwire.dtl.DTLVardef@3593a7_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2020

Room-temperature-storable PCR Mixes for SARS-CoV-2 Detection

A novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) emerged in late 2019, causing an outbreak of pneumonia [coronavirus disease 2019 (COVID-19)] in Wuhan, China, which then rapidly spread globally. Although the use of ready-made reaction mixes can enable more rapid PCR-based diagnosis of COVID-19, the need to transport and store these mixes at low temperatures presents challenges to already overburdened logistics networks. Here, we present an optimized freeze-drying procedure that allows SARS-CoV-2 PCR mixes to be transported and stored at ambient temperatures, without loss of activity. Additive-supplemented PCR mixes were freeze-dried. The residual moisture of the freeze-dried PCR mixes was measured by Karl-Fischer titration. We found that freeze-dried PCR mixes with [~]1.2% residual moisture are optimal for storage, transport, and reconstitution. The sensitivity, specificity, and repeatability of the freeze-dried reagents were similar to those of freshly prepared, wet reagents. The freeze-dried mixes retained activity at room temperature (18[~]25{degrees}C) for 28 days, and for 14 and 10 days when stored at 37{degrees}C and 56{degrees}C, respectively. The uptake of this approach will ease logistical challenges faced by transport networks and make more cold storage space available at diagnosis and hospital laboratories. This method can also be applied to the generation of freeze-dried PCR mixes for the detection of other pathogens.

microbiology↗

bioRxiv2020

Automated continuous evolution of proteins in vivo

We present automated continuous evolution (ACE), a platform for the hands-free directed evolution of biomolecules. ACE pairs OrthoRep, a genetic system for continuous targeted mutagenesis of user-selected genes in vivo, with eVOLVER, a scalable and automated continuous culture device for precise, multi-parameter regulation of growth conditions. By implementing real-time feedback-controlled tuning of selection stringency with eVOLVER, genes of interest encoded on OrthoRep autonomously traversed multi-mutation adaptive pathways to reach desired functions, including drug resistance and improved enzyme activity. The durability, scalability, and speed of biomolecular evolution with ACE should be broadly applicable to protein engineering as well as prospective studies on how selection parameters and schedules shape adaptation.

synthetic biology↗

bioRxiv2020

Genetic architecture of oxidative stress tolerance in the fungal wheat pathogen Zymoseptoria tritici

Reactive oxygen species are toxic byproducts of aerobic respiration produced during cell growth. They also are an important component of plant defenses to inhibit microbial pathogens. Tolerance to oxidative stress contributes to viability and pathogenicity of plant pathogens. However, the complex molecular network of oxidative stress responses hinders identification of the genes contributing to variation in this trait. Variation in genes affecting responses to oxidative stress is likely to affect the evolutionary potential of pathogen tolerance to host defences. Here, we employed a forward genetic approach to investigate the genetic architecture of oxidative stress tolerance in the fungal wheat pathogen Zymoseptoria tritici. By performing quantitative trait locus (QTL) mapping in two crosses, we identified several genomic regions associated with tolerance to oxidative stress, including a QTL having a large effect on growth under oxidative stress. We found evidence for a significant trade-off between growth under non-stressful conditions and growth inhibition under oxidative stress. We identified a large QTL associated with this trade-off and with growth under non-stressful conditions, suggesting that differences in fungal growth could result in different sensitivities to oxidative stress. Our results suggest that genes related to fungal growth could also contribute to variation in oxidative stress tolerance among fungal strains.

genetics↗

bioRxiv2020

Wnt-regulated lncRNA discovery enhanced by in vivo identification and CRISPRi functional validation

BackgroundWnt signaling is an evolutionarily conserved developmental pathway that is frequently hyperactivated in cancer. While multiple protein-coding genes regulated by Wnt signaling are known, the functional lncRNAs regulated by Wnt signaling have not been systematically characterized. ResultsWe comprehensively mapped lncRNAs from an orthotopic Wnt-addicted pancreatic cancer model, identifying 3,633 lncRNAs, of which 1,503 were regulated by Wnt signaling. We found lncRNAs were much more sensitive to changes in Wnt signaling in xenografts than in cultured cells. To functionally validate Wnt-regulated lncRNAs, we performed CRISPRi screens to assess their role in cancer cell proliferation. Consistent with previous genome-wide lncRNA CRISPRi screens, around 1% (13/1,503) of the Wnt-regulated lncRNAs could modify cancer cell growth in vitro. This included CCAT1 and LINC00263, previously reported to regulate cancer growth. Using an in vivo CRISPRi screen, we doubled the discovery rate, identifying twice as many Wnt-regulated lncRNAs (25/1,503) that had a functional effect on cancer cell growth. ConclusionsOur study demonstrates the value of studying lncRNA functions in vivo, provides a valuable resource of lncRNAs regulated by Wnt signaling and establishes a framework for systematic discovery of functional lncRNAs.

genomics↗

bioRxiv2020

Antibiotics-induced alterations in gut microbiol composition leads to blood-brain barrier permeability increase in rhesus monkeys

Blood-brain barrier (BBB) contributes to maintenance of brain homeostasis. Gut microbiome composition affected BBB development and expression of tight junction proteins in rodents. However, we still do not know if theres any direct effect of gut microbiol composition on BBB permeability and function in normal adult animals. In current study, we determined temporal and spatial change of BBB permeability in rhesus monkeys receiving either oral or intravenous amoxicillin-clavulanic acid (AC), by monitoring CSF/serum albumin ratio (AR) and the volume transfer constant (Ktrans). We showed that oral, but not intravenous AC led to a significant alteration in gut microbiol composition and increase of BBB permeability in all monkeys, especially in thalamus area. Acetic acid and propionic acid might play a pivotal role in this newly found communication between gut and central nervous system. Antibiotics-induced gut microbiol composition change, especially the decreasing of acetic and propionic acid producing phyla and genera, leads to increase of BBB permeability, which may contribute to a variety of neurological and psychological diseases.

neuroscience↗

bioRxiv2019

Visualizing in vitro-in vivo correlation of miktoarm copolymer nanomicelles in cancer cellular uptake and trafficking

In this study, a clear correlation between the in vitro and in vivo cellular uptake and trafficking was discovered by delivering miktoarm copolymer nanomicelles (MCNs) to cancer cells and tumor tissues. To monitor this process, two different FRET pairs, DiO and DiI, DiD and DiR, were loaded into MCNs to monitor the Forster resonance energy transfer (FRET) efficiency. The change in FRET efficiency in vitro and in vivo demonstrated a similar sequence of events for the transport of MCNs: hyperbranched block PCL inserted into cytomembrane, while the loaded hydrophobic fluorescence probes were released and followed by time-dependent intracellular clustering within endocytic vesicles. Additionally, uptake of loaded fluorescence probes with successively increasing ratios of copolymers suggested that with the increase of mass ratio of copolymer to fluorescence probes, cellular uptake of probes significantly decreased. This result was also consistent with the uptake behavior in cancer tissues. Collectively, the interaction between MCNs and cellular membrane dictated the uptake and trafficking of core-loaded hydrophobic probes. This concept paves a new way to analyze in vitro-in vivo correlation of other nanocarriers for endocytosis mechanism studies as well as further novel copolymers design in biomedical applications.

pharmacology and toxicology↗

bioRxiv2019

N-Acetyl Cysteine Alleviates Coxsackievirus B-Induced Myocarditis by Suppressing caspase-1

Viral myocarditis caused by Coxsackievirus B (CVB) infection is a severe inflammatory disease of the myocardium, which may develop to cardiomyopathy and heart failure. No effective medicine is available to treat CVB infection. Here we evaluated the anti-CVB effect of N-acetyl cysteine (NAC), a widely used antioxidant. NAC significantly alleviated myocarditis and improved the overall condition of CVB type 3 (CVB3)-infected mice. Importantly, NAC treatment suppressed viral replication in both myocardium and cell culture. We show that NAC inhibited CVB3 replication when it was applied at the early stage of CVB3 infection. NACs antiviral mechanism, while independent of its antioxidant property, relies on its inhibition on caspase-1 activation, since the knockdown of caspase-1 blocked CVB3 replication. Moreover, NAC promotes procaspase-1 degradation via ubiquitin proteasome system, which may further contribute to the inhibited activity of caspase-1. NAC also inhibits the activity of viral proteases. Taken together, this study shows that NAC exerts potent anti-CVB effect by inhibiting caspase-1 and viral proteases. This study suggests that NAC can be a safe therapeutic option for CVB-induced myocarditis.

microbiology↗

bioRxiv2019

Arabidopsis SWR1-associated protein methyl-CpG-binding domain 9 is required for histone H2A.Z deposition.

Deposition of the histone variant H2A.Z by the SWI2/SNF2-Related 1 chromatin remodeling complex (SWR1-C) is important for gene regulation in eukaryotes, but the composition of the Arabidopsis SWR1-C has not been thoroughly characterized. Here identify interacting partners of a conserved Arabidopsis SWR1 subunit, ACTIN-RELATED PROTEIN 6 (ARP6). We isolated nine predicted components, and identified additional interactors implicated in histone acetylation and chromatin biology. One of the novel interacting partners, methyl-CpG-binding domain 9 (MBD9), also strongly interacted with the Imitation SWItch (ISWI) chromatin remodeling complex. MBD9 was required for deposition of H2A.Z at a distinct subset of ARP6-dependent loci. MBD9 was preferentially bound to nucleosome-depleted regions at the 5 ends of genes containing high levels of activating histone marks. These data suggest that MBD9 is a SWR1-C interacting protein required for H2A.Z deposition at a subset of actively transcribing genes.

molecular biology↗