Biology subjects

Chen, R.

Publications and source records attributed to Chen, R..

At least 271 records · Page 15Linked to original sources

bioRxiv2022

Mapping whole-brain projections of anatomically defined prefrontal neurons using combined 3D convolution networks

To understand how the brain produces behavior, we must elucidate the relationships between neuronal connectivity and function. The medial prefrontal cortex (mPFC) is critical for complex functions including decision-making and mood. mPFC projection neurons collateralize extensively, but the relationships between mPFC neuronal activity and brain-wide connectivity are poorly understood. We performed whole-brain connectivity mapping and fiber photometry to better understand the mPFC circuits that control threat avoidance. Using tissue clearing and light sheet fluorescence microscopy we mapped the brain-wide axon collaterals of populations of mPFC neurons that project to nucleus accumbens (NAc), ventral tegmental area (VTA), or contralateral mPFC (cmPFC) in mice. We present DeepTraCE, for quantifying bulk-labeled axonal projections in images of cleared tissue, and DeepCOUNT, for quantifying cell bodies. Anatomical maps produced with DeepTraCE aligned with known axonal projection patterns and revealed class-specific topographic projections within regions. During threat avoidance, cmPFC and NAc-projectors encoded conditioned stimuli, but only when action was required to avoid threats. mPFC-VTA neurons encoded learned but not innate avoidance behaviors. Together our results present new and optimized approaches for quantitative whole-brain analysis and indicate that anatomically-defined classes of mPFC neurons have specialized roles in threat avoidance.

neuroscience↗

bioRxiv2022

BET inhibition sensitizes immunologically-cold Rb-deficient prostate cancer to immune checkpoint blockade

PurposeNon-T cell-inflamed immunologically "cold" tumor microenvironments (TME) are associated with poor responsiveness to immune checkpoint blockade (ICB), and can be sculpted by tumor cell genomics. Here we evaluated how Retinoblastoma (Rb) tumor suppressor loss of function (LOF), one of the most frequent alterations in human cancer and associated with lineage plasticity, poor prognosis and therapeutic outcomes, alters the TME, and whether therapeutic strategies targeting the molecular consequences of Rb loss enhance ICB efficacy. Experimental DesignWe performed bioinformatics analysis to elucidate the impact of endogenous Rb LOF on the immune TME in human primary and metastatic tumors. Next, we utilized isogenic murine models of Rb-deficient prostate cancer (PC) for in vitro and in vivo mechanistic studies to examine how Rb loss and bromodomain and extraterminal (BET) domain inhibition (BETi) reprograms the immune landscape, and evaluated in vivo therapeutic efficacy of BETi, singly and in combination with ICB and androgen deprivation therapy. ResultsRb loss was enriched in non-T cell-inflamed tumors, and Rb-deficient murine tumors demonstrated decreased immune infiltration in vivo. The BETi JQ1 increased immune infiltration into the TME through enhanced tumor cell STING/NF-{kappa}B activation and type I interferon (IFN) signaling within tumor cells, resulting in differential macrophage and T cell-mediated tumor growth inhibition and sensitization of Rb-deficient PC to ICB. ConclusionsBETi can reprogram the immunologically cold Rb-deficient TME via STING/NF-{kappa}B/IFN signaling to sensitize Rb-deficient PC to ICB. These data provide the mechanistic rationale to test combinations of BETi and ICB in clinical trials of Rb-deficient PC. STATEMENT OF TRANSLATIONAL RELEVANCERb LOF is one of the most common genomic alterations in human cancer, occurring in approximately 1/3 of advanced malignancies, Furthermore, loss of Rb correlates with enhanced aggressiveness and poor therapeutic outcomes. In this study, we demonstrate that loss of Rb is also associated with an immunosuppressive tumor microenvironment and lack of responsiveness to immune checkpoint blockade (ICB). As a strategy to overcome Rb LOF induced immunosuppression, we have demonstrated that BETi treatment drives STING/NF-{kappa}B signaling and type I interferon production within tumor cells, resulting in immune-mediated tumor control in Rb-deficient PC, which is accentuated by the combination with ICB and ADT. These findings provide a roadmap for maximizing the clinical translation of BET inhibitors into the clinic to treat aggressive-variant Rb-deficient PC.

cancer biology↗

bioRxiv2022

XY sex determination in a cnidarian

Sex determination occurs across animal species, but most of our knowledge about the mechanisms of sex determination comes from only a handful of bilaterian taxa. This limits our ability to infer the evolutionary history of sex determination within animals. In this study, we generated a linkage map of the genome of the colonial cnidarian Hydractinia symbiolongicarpus and used this map to determine that this species has an XX/XY sex determination system. We delineate the pseudoautosomal and non-recombining regions of the Y chromosome and show that the latter encodes a number of genes with male gonad-specific expression. These findings establish Hydractinia as a tractable non-bilaterian model system for the study of sex determination. Significance StatementWhat determines whether an animal is male or female? The answer depends on the species. Some rely on signals from their environment, while others take cues from their genome. Most of what we understand about sex determination comes from traditional model organisms (such as mice, flies, and worms) or groups of well-studied vertebrates and insects. Studying sex determination in other animals, especially those from phyla that diverged early in animal evolution, would allow us to better understand how sex has evolved across the animal kingdom and could reveal pathways present in the eumetazoan ancestor >600 million years ago. In this study, we show that the cnidarian Hydractinia symbiolongicarpus has XY sex determination, establishing this species as a model system for sex determination in an understudied group of animals.

genetics↗

bioRxiv2022

Chimeric mRNA based COVID-19 vaccine elicits potent neutralizing antibodies and protection against Omicron and Delta

The emerging SARS-CoV-2 variants of concern (VOCs) exhibit enhanced transmission and immune escape, reducing the efficacy and effectiveness of the two FDA-approved mRNA vaccines. Here, we explored various strategies to develop novel mRNAs vaccines to achieve safer and wider coverage of VOCs. Firstly, we constructed a cohort of mRNAs that feature a furin cleavage mutation in the spike (S) protein of predominant VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2). Not present in the mRNA vaccines currently in use, the mutation abolished the cleavage between the S1 and S2 subunits, potentially enhancing the safety profile of the immunogen. Secondly, we systematically evaluated the induction of neutralizing antibodies (nAb) in vaccinated mice, and discovered that individual VOC mRNAs elicited strong neutralizing activity in a VOC-specific manner. Thirdly, the IgG produced in mice immunized with Beta-Furin and Washington (WA)-Furin mRNAs showed potent cross-reactivity with other VOCs, which was further corroborated by challenging vaccinated mice with the live virus of VOCs. However, neither WA-Furin nor Beta-Furin mRNA elicited strong neutralizing activity against the Omicron variant. Hence, we further developed an Omicron-specific mRNA vaccine that restored protection against the original and the sublineages of Omicron variant. Finally, to broaden the protection spectrum of the new Omicron mRNA vaccine, we tested the concept of bivalent immunogen. Instead of just fusing two RBDs head-to-tail, we for the first time constructed an mRNA-based chimeric immunogen by introducing the RBD of Delta variant into the entire S antigen of Omicron. The resultant chimeric mRNA was capable of inducing potent and broadly acting nAb against Omicron (both BA.1 and BA.2) and Delta, which paves the way to develop new vaccine candidate to target emerging variants in the future.

immunology↗

bioRxiv2022

Deciphering the developmental patterns of white matter tracts in a diffusion MRI based fetal brain atlas

White matter of the fetal brain undergoes rapid development to form the early structural connections. Diffusion MRI has shown to be a useful tool to depict the fetal brain white matter in utero, and many studies have observed increasing fractional anisotropy and decreasing mean diffusivity in the fetal brain during the second-to-third trimester, whereas others reported non-monotonic changes. Unbiased diffusion MRI atlases of the fetal brain are important for characterizing developmental trajectories of white matter and providing normative references for in-utero diagnosis of prenatal abnormalities. To date, the sole fetal brain diffusion MRI atlas was collected from a Caucasian/mixed population, and was constructed based on the diffusion tensor model with limited spatial resolution. In this work, we proposed a fiber orientation distribution (FOD) based pipeline for the generation of fetal brain diffusion MRI atlases, which showed better registration accuracy than diffusion tensor-based pipeline. Based on the FOD pipeline, we constructed the first Chinese fetal brain diffusion MRI atlas using 89 normal fetal diffusion MRI scans at GA between 24 and 38 weeks. Complex non-monotonic trends of tensor- and FOD-derived microstructural parameters in eight white matter tracts were observed, which jointly pointed to different phases of microstructural development. Specifically, we speculated that the turning point of the diffusivity trajectory may correspond to the starting point of pre-myelination, based on which, the developmental order of white matter tracts can be mapped and the order was in agreement with order of myelination from histological studies. The normative atlas also provided a reference for detection of abnormal white matter development, such as that in congenital heart disease. Therefore, the high-order fetal brain diffusion MRI atlas established in this study depicted the spatiotemporal pattern of early white matter development, and findings may help decipher the distinct microstructural events in utero.

neuroscience↗

bioRxiv2022

Super-resolution analyzing spatial organization of lysosomes with an organic fluorescent probe

Lysosomes are multifunctional organelles involved in macromolecule degradation, nutrient sensing and autophagy. Live imaging has revealed lysosome subpopulations with dynamics and characteristic cellular localization. An as-yet unanswered question is whether lysosomes are spatially organized to coordinate and integrate their functions. Combined with super-resolution microscopy, we designed a small organic fluorescent probe, TPAE, that targeted lysosomes with a large Stokes shift. When we analyzed the spatial organization of lysosomes against mitochondria in different cell lines with this probe, we discovered different distance distribution patterns between lysosomes and mitochondria during increased autophagy flux. By using SLC25A46 mutation fibroblasts derived from patients containing highly fused mitochondria with low oxidative phosphorylation, we concluded that unhealthy mitochondria redistributed the subcellular localization of lysosomes, which implies a strong connection between mitochondria and lysosomes.

biophysics↗

bioRxiv2022

The interactome of Cryptococcus neoformans Rmt5 reveals multiple regulatory points in fungal cell biology and pathogenesis

Protein arginine methylation is a key post-translational modification in eukaryotes that modulates core cellular processes, including translation, morphology, transcription, and RNA fate. However, this has not been explored in Cryptococcus neoformans, a human-pathogenic basidiomycetous encapsulated fungus. We characterized the five protein arginine methyltransferases in C. neoformans and highlight Rmt5 as critical regulator of cryptococcal morphology and virulence. An rmt5{Delta} mutant was defective in thermotolerance, had a remodeled cell wall, and exhibited enhanced growth in an elevated carbon dioxide atmosphere and in chemically induced hypoxia. We revealed that Rmt5 interacts with post-transcriptional gene regulators, such as RNA-binding proteins and translation factors. Further investigation of the rmt5{Delta} mutant showed that Rmt5 is critical for the homeostasis of eIF2 and its phosphorylation state following 3-amino-1,2,4-triazole-induced ribosome stalling. RNA sequencing of one rmt5{Delta} clone revealed stable chromosome 9 aneuploidy that was ameliorated by complementation but did not impact the rmt5{Delta} phenotype. As a result of these diverse interactions and functions, loss of RMT5 enhanced phagocytosis by murine macrophages and attenuated disease progression in mice. Taken together, our findings link arginine methylation to critical cryptococcal cellular processes that impact pathogenesis, including post-transcriptional gene regulation by RNA-binding proteins. SignificanceThe fungal pathogen Cryptococcus neoformans is a huge threat for people living with immune deficits, especially HIV/AIDS. Its virulence potential is dependent on virulence factors, stress adaptation, and thermotolerance. Post-transcriptional gene regulation is important for these pathogenic processes, but the mechanisms that govern post-transcriptional regulator function are unexplored. Protein arginine methylation is a major modification of post-transcriptional regulators that has not been investigated in pathogenic fungi. Here we investigated the role of arginine methylation by arginine methyltransferases on the biology and virulence of C. neoformans. Phenotypic characterization of deletion mutants revealed pleiotropic functions for RMTs in this pathogen. Further investigation of the Rmt5 interactome using proximity-dependent biotinylation revealed interactions with RNA binding proteins and translation factors, thereby impacting virulence-associated processes.

microbiology↗

bioRxiv2022

An antibody targeting the N-terminal domain of SARS-CoV-2 disrupts the spike trimer

The protective human antibody response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus focuses on the spike (S) protein which decorates the virion surface and mediates cell binding and entry. Most SARS-CoV-2 protective antibodies target the receptor- binding domain or a single dominant epitope ( supersite) on the N terminal domain (NTD). Here, using the single B cell technology LIBRA-seq, we isolated a large panel of NTD-reactive and SARS-CoV-2 neutralizing antibodies from an individual who had recovered from COVID-19. We found that neutralizing antibodies to the NTD supersite commonly are encoded by the IGHV1-24 gene, forming a genetic cluster that represents a public B cell clonotype. However, we also discovered a rare human antibody, COV2-3434, that recognizes a site of vulnerability on the SARS-CoV-2 S protein in the trimer interface and possesses a distinct class of functional activity. COV2-3434 disrupted the integrity of S protein trimers, inhibited cell-to-cell spread of virus in culture, and conferred protection in human ACE2 transgenic mice against SARS-CoV-2 challenge. This study provides insight about antibody targeting of the S protein trimer interface region, suggesting this region may be a site of virus vulnerability.

immunology↗

bioRxiv2022

TCF7L2 acts as a molecular switch in midbrain to control mammal vocalization through a transcriptional repression mechanism

Vocalization is an essential medium for sexual and social signaling in birds and mammals. Periaqueductal gray (PAG) a conserved midbrain structure is believed to be responsible for innate vocalizations, but its molecular regulation remains largely unknown. Here, through a mouse forward genetic screening we identified one of the key Wnt/ {beta}-catenin effectors TCF7L2/TCF4 controls ultrasonic vocalization (USV) production and syllable complexity during maternal deprivation and sexual encounter. Expression of TCF7L2 in PAG excitatory neurons is necessary for the complex trait, while TCF7L2 loss reduces neuronal gene expressions and synaptic transmission in PAG. TCF7L2-mediated vocal {beta}-catenin-binding domain but dependent of its DNA binding ability. Patient mutations associated with severe speech delay disrupt the transcriptional repression effect of TCF7L2, while mice carrying those mutations display severe USV impairments. Therefore, we conclude that TCF7L2 orchestrates gene expression in midbrain to control vocal production through a transcriptional repression mechanism.

neuroscience↗

bioRxiv2022

Single-cell transcriptome reveals the redifferentiation trajectories of the early stage of de novo shoot regeneration in Arabidopsis thaliana

De novo shoot regeneration from a callus plays a crucial role in both plant biotechnology and the fundamental research of plant cell totipotency. Recent studies have revealed many regulatory factors involved in this developmental process. However. our knowledge of the cell heterogeneity and cell fate transition during de novo shoot regeneration is still limited. Here, we performed time-series single-cell transcriptome experiments to reveal the cell heterogeneity and redifferentiation trajectories during the early stage of de novo shoot regeneration. Based on the single-cell transcriptome data of 35,669 cells at five-time points, we successfully determined seven major cell populations in this developmental process and reconstructed the redifferentiation trajectories. We found that all cell populations resembled root identities and undergone gradual cell-fate transitions. In detail, the totipotent callus cells differentiated into pluripotent QC-like cells and then gradually developed into less differentiated cells that have multiple root-like cell identities, such as pericycle-like cells. According to the reconstructed redifferentiation trajectories, we discovered that the canonical regeneration-related genes were dynamically expressed at certain stages of the redifferentiation process. Moreover, we also explored potential transcription factors and regulatory networks that might be involved in this process. The transcription factors detected at the initial stage, QC-like cells, and the end stage provided a valuable resource for future functional verifications. Overall, this dataset offers a unique glimpse into the early stages of de novo shoot regeneration, providing a foundation for a comprehensive analysis of the mechanism of de novo shoot regeneration.

plant biology↗

bioRxiv2021

Structural and functional characterizations of altered infectivity and immune evasion of SARS-CoV-2 Omicron variant

The SARS-CoV-2 Omicron with increased fitness is spreading rapidly worldwide. Analysis of cryo-EM structures of the Spike (S) from Omicron reveals amino acid substitutions forging new interactions that stably maintain an "active" conformation for receptor recognition. The relatively more compact domain organization confers improved stability and enhances attachment but compromises the efficiency of viral fusion step. Alterations in local conformation, charge and hydrophobic microenvironments underpin the modulation of the epitopes such that they are not recognized by most NTD- and RBD-antibodies, facilitating viral immune escape. Apart from already existing mutations, we have identified three new immune escape sites: 1) Q493R, 2) G446S and 3) S371L/S373P/S375F that confers greater resistance to five of the six classes of RBD-antibodies. Structure of the Omicron S bound with human ACE2, together with analysis of sequence conservation in ACE2 binding region of 25 sarbecovirus members as well as heatmaps of the immunogenic sites and their corresponding mutational frequencies sheds light on conserved and structurally restrained regions that can be used for the development of broad-spectrum vaccines and therapeutics.

biophysics↗

bioRxiv2021

Monospecific and bispecific monoclonal SARS-CoV-2 neutralizing antibodies that maintain potency against B.1.617

COVID-19 pathogen SARS-CoV-2 has infected hundreds of millions and caused over 5 million deaths to date. Although multiple vaccines are available, breakthrough infections occur especially by emerging variants. Effective therapeutic options such as monoclonal antibodies (mAbs) are still critical. Here, we report the development, cryo-EM structures, and functional analyses of mAbs that potently neutralize SARS-CoV-2 variants of concern. By high-throughput single cell sequencing of B cells from spike receptor binding domain (RBD) immunized animals, we identified two highly potent SARS-CoV-2 neutralizing mAb clones that have single-digit nanomolar affinity and low-picomolar avidity, and generated a bispecific antibody. Lead antibodies showed strong inhibitory activity against historical SARS-CoV-2 and several emerging variants of concern. We solved several cryo-EM structures at [~]3 [A] resolution of these neutralizing antibodies in complex with prefusion spike trimer ectodomain, and revealed distinct epitopes, binding patterns, and conformations. The lead clones also showed potent efficacy in vivo against authentic SARS-CoV-2 in both prophylactic and therapeutic settings. We also generated and characterized a humanized antibody to facilitate translation and drug development. The humanized clone also has strong potency against both the original virus and the B.1.617.2 Delta variant. These mAbs expand the repertoire of therapeutics against SARS-CoV-2 and emerging variants.

immunology↗

bioRxiv2021

Aquaglyceroporin AQP7's affinity for its substrate glycerol

AQP7 is one of the four human aquaglyceroporins that facilitate glycerol transport across the cell membrane, a biophysical process that is essential in human physiology. Therefore, it is interesting to compute AQP7s affinity for its substrate (glycerol) with reasonable certainty to compare with the experimental data suggesting high affinity in contrast with most computational studies predicting low affinity. In this study aimed at computing the AQP7-glycerol affinity with high confidence, we implemented a direct computation of the affinity from unbiased equilibrium molecular dynamics (MD) simulations of three all-atom systems constituted with 0.16M, 4.32M, and 10.23M atoms, respectively. These three sets of simulations manifested a fundamental physics law that the intrinsic fluctuations of pressure in a system are inversely proportional to the system size (the number of atoms in it). These simulations showed that the computed values of glycerol-AQP7 affinity are dependent upon the system size (the inverse affinity estimations were, respectively, 47.3 mM, 1.6 mM, and 0.92 mM for the three model systems). In this, we obtained a lower bound for the AQP7-glycerol affinity (an upper bound for the dissociation constant). Namely, the AQP7-glycerol affinity is stronger than 1087/M (the dissociation constant is less than 0.92 mM). Additionally, we conducted hyper steered MD (hSMD) simulations to map out the Gibbs free-energy profile. From the free-energy profile, we produced an independent computation of the AQP7-glycerol dissociation constant being approximately 0.18 mM. Table of contents entry O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/469753v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@420c90org.highwire.dtl.DTLVardef@46d27borg.highwire.dtl.DTLVardef@1a4b269org.highwire.dtl.DTLVardef@1bd3979_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

A live attenuated influenza virus-vectored intranasal COVID-19 vaccine provides rapid, prolonged, and broad protection against SARS-CoV-2 infection

Remarkable progress has been made in developing intramuscular vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); however, they are limited with respect to eliciting local immunity in the respiratory tract, which is the primary infection site for SARS-CoV-2. To overcome the limitations of intramuscular vaccines, we constructed a nasal vaccine candidate based on an influenza vector by inserting a gene encoding the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, named CA4-dNS1-nCoV-RBD (dNS1-RBD). A preclinical study showed that in hamsters challenged 1 day and 7 days after single-dose vaccination or 6 months after booster vaccination, dNS1-RBD largely mitigated lung pathology, with no loss of body weight, caused by either the prototype-like strain or beta variant of SARS-CoV-2. Lasted data showed that the animals could be well protected against beta variant challenge 9 months after vaccination. Notably, the weight loss and lung pathological changes of hamsters could still be significantly reduced when the hamster was vaccinated 24 h after challenge. Moreover, such cellular immunity is relatively unimpaired for the most concerning SARS-CoV-2 variants. The protective immune mechanism of dNS1-RBD could be attributed to the innate immune response in the nasal epithelium, local RBD-specific T cell response in the lung, and RBD-specific IgA and IgG response. Thus, this study demonstrates that the intranasally delivered dNS1-RBD vaccine candidate may offer an important addition to fight against the ongoing COVID-19 pandemic, compensating limitations of current intramuscular vaccines, particularly at the start of an outbreak.

pathology↗

bioRxiv2021

Effector loss drives adaptation of Pseudomonas syringae pv. actinidiae to Actinidia arguta

A pandemic isolate of Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) has devastated kiwifruit orchards growing cultivars of Actinidia chinensis. In contrast, A. arguta (kiwiberry) is resistant to Psa3. This resistance is mediated via effector-triggered immunity, as demonstrated by induction of the hypersensitive response in infected A. arguta leaves, observed by microscopy and quantified by ion-leakage assays. Isolates of Psa3 that cause disease in A. arguta have been isolated and analyzed, revealing a 49 kb deletion in the exchangeable effector locus (EEL). This natural EEL-mutant isolate and strains with synthetic knockouts of the EEL were more virulent in A. arguta plantlets than wild-type Psa3. Screening of a complete library of Psa3 effector knockout strains identified increased growth in planta for knockouts of four effectors - AvrRpm1a, HopF1c, HopZ5a, and the EEL effector HopAW1a - suggesting a resistance response in A. arguta. Hypersensitive response (HR) assays indicate that three of these effectors trigger a host species-specific HR. A Psa3 strain with all four effectors knocked out escaped host recognition, but a cumulative increase in bacterial pathogenicity and virulence was not observed. These avirulence effectors can be used in turn to identify the first cognate resistance genes in Actinidia for breeding durable resistance into future kiwifruit cultivars.

plant biology↗

bioRxiv2021

T1SEstacker: A tri-layer stacking model effectively predicts bacterial type 1 secreted proteins based on C-terminal non-RTX-motif sequence features

The proteins secreted through type 1 secretion systems often play important roles in pathogenicity of various gram-negative bacteria. However, the type 1 secretion mechanism remains unknown. In this research, we observed the sequence features of RTX proteins, a major class of type 1 secreted substrates. We found striking non-RTX-motif amino acid composition patterns at the C-termini, most typically exemplified by the enriched [FLI][VAI] at the most C-terminal two positions. Machine-learning models, including deep-learning models, were trained using these sequence-based non-RTX-motif features, and further combined into a tri-layer stacking model, T1SEstacker, which predicted the RTX proteins accurately, with a 5-fold cross-validated sensitivity of ~0.89 at the specificity of ~0.94. Besides substrates with RTX motifs, T1SEstacker can also well distinguish non-RTX-motif type 1 secreted proteins, further suggesting their potential existence of common secretion signals. In summary, we made comprehensive sequence analysis on the type 1 secreted RTX proteins, identified common sequence-based features at the C-termini, and developed a stacking model that can predict type 1 secreted proteins accurately.

microbiology↗

bioRxiv2021

The TFIIH Complex is Required to Establish and Maintain Mitotic Chromosome Structure

Condensins compact chromosomes to promote their equal segregation during mitosis, but the mechanism of condensin engagement with and action on chromatin is incompletely understood. Here, we show that the general transcription factor TFIIH complex is continuously required to establish and maintain a compacted chromosome structure in transcriptionally silent Xenopus egg extracts. Inhibiting the DNA-dependent ATPase activity of the TFIIH complex subunit XPB rapidly and reversibly induces a complete loss of chromosome structure and prevents the enrichment of condensins I and II, but not topoisomerase II, on chromatin. In addition, inhibiting TFIIH prevents condensation of both mouse and Xenopus nuclei in Xenopus egg extracts, which suggests a fundamental mechanism of TFIIH action. Reducing nucleosome density through partial histone depletion restores chromosome structure and condensin enrichment in the absence of TFIIH activity. We propose that the TFIIH complex promotes mitotic chromosome condensation by dynamically altering chromatin structure to facilitate condensin loading and condensin-dependent loop extrusion.

cell biology↗

bioRxiv2021

The Ets protein Pointed P1 represses Asense expression in type II neuroblasts by activating Tailless

Intermediate neural progenitors (INPs) boost the number and diversity of neurons generated from neural stem cells (NSCs) by undergoing transient proliferation. In the developing Drosophila brains, INPs are generated from type II neuroblasts (NBs). In order to maintain type II NB identity and their capability to produce INPs, the proneural protein Asense (Ase) needs to be silenced by the Ets transcription factor pointed P1 (PntP1), a master regulator of type II NB development. However, the molecular mechanisms underlying the PntP1-mediated suppression of Ase is still unclear. In this study, we utilized genetic and molecular approaches to determine the transcriptional property of PntP1 and identify the direct downstream effector of PntP1 and the cis-DNA elements that mediate the suppression of ase. Our results demonstrate that PntP1 directly activates the expression of the transcriptional repressor, Tailless (Tll), by binding to seven Ets-binding sites, and Tll in turn suppresses the expression of Ase in type II NBs by binding to two hexameric core half-site motifs. We further show that Tll provides positive feedback to maintain the expression of PntP1 and the identity of type II NBs. Thus, our study identifies a novel direct target of PntP1 and reveals mechanistic details of the specification and maintenance of the type II NB identity by PntP1. Author summaryType II neuroblasts (NBs) are the neural stem cells (NSCs) in Drosophila central brains that produce neurons by generating intermediate neural progenitors (INPs) to boost brain complexity, as mammalian NSCs do during the development of neocortex. The key to the generation of INPs from type II NBs is the suppression of proneural protein Asense (Ase) in type II NBs by the Ets family transcription factor Pointed P1 (PntP1), but how PntP1 suppresses Ase expression remains unclear. In this study, we provided evidence to demonstrate that PntP1 directly activates the orphan nuclear receptor Tailless (Tll), which in turn suppresses Ase expression to maintain the capability of type II NBs to produce INPs. Meanwhile, Tll provides positive feedback to maintain the expression of PntP1 and type II NB identity. We further identified seven PntP1 binding sites in the tll enhancer regions and two Tll binding sites in the ase regulatory regions that mediate the activation of tll and the suppression of ase, respectively. Our work reveals detailed mechanisms of the specification and maintenance of the type II NB identity by PntP1.

developmental biology↗