Biology subjects

Bennett, B.

Publications and source records attributed to Bennett, B..

16 recordsLinked to original sources

bioRxiv2025

H3K27me3 chromatin heterogeneity reveals variable cell responses to estrogen and endocrine treatment

Gene expression heterogeneity generates subpopulations of tumor cells that can evade therapeutic pressure. This heterogeneity has been observed in both primary Estrogen Receptor alpha positive (ER+) breast tumors and cell lines. Therefore, understanding the mechanisms regulating expression heterogeneity is critical towards developing effective therapies. A key contributor to gene expression variability is the stochastic nature of transcription. Transcription occurs in a probabilistic, burst-like manner, in which gene activation occurs intermittently, producing RNA in pulses and interspersed with transcriptional off-periods. The estrogen-responsive gene TFF1 is expressed in the majority of ER breast tumors and exemplifies such heterogeneity, with transcriptional inactivity ranging from minutes to several days. Here, we identify the molecular mechanism underlying the wide range in TFF1 expression by analyzing cells sorted based on their TFF1 activity levels. We observed that TFF1 inactive (TFF1low) cells exhibit a repressive chromatin state marked by H3K27me3 at the TFF1 promoter and enhancer. Despite global similarity in ER binding, occupancy at the TFF1 regulatory elements was selectively reduced in TFF1low cells, resulting in fewer active alleles and diminished transcriptional bursting frequency. Conversely, TFF1high cells exhibited more active TFF1 alleles and hyperbursting. These cells also retained sensitivity to endocrine therapy, while TFF1low cells displayed reduced drug responsiveness. Genome-wide, differentially enriched H3K27me3 regions correlated with variable expression of estrogen-responsive genes, highlighting a broader regulatory mechanism that links chromatin state to expression variability. Together, our findings establish how repressive chromatin dynamics contribute to gene expression heterogeneity and endocrine resistance in ER breast cancer. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/677184v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1194eedorg.highwire.dtl.DTLVardef@8cb67aorg.highwire.dtl.DTLVardef@202ad9org.highwire.dtl.DTLVardef@1dd222a_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

SPEECHLESS duplication in grasses expands potential for environmentalregulation of stomatal development

Plants regulate stomatal development and function to acquire atmospheric carbon dioxide for photosynthesis while minimizing water loss. The ancestral basic helix-loop-helix transcription factor (TF) gene that drove stomata production in early land plants diversified to become paralogs SPEECHLESS (SPCH), MUTE, and FAMA. Extant grasses exhibit a particularly interesting set of duplications and losses of SPCH. Using phylogenetic methods, we tracked the history of SPCH duplications. Brachypodium distachyon and Oryza sativa plants bearing mutations in either SPCH1 or SPCH2, and B. distachyon plants with SPCH1 or SPCH2 translational reporters were assayed under different environmental conditions for their effects on stomatal development. We identified the Poaceae-specific rho whole genome duplication as the origin of SPCH1 and SPCH2 and demonstrated that both paralogs remain under selection. We found paralog-specific divergence in response to two environmental perturbations in both B. distachyon and O. sativa. Plausible molecular mechanisms underpinning paralog divergence, and cellular mechanisms driving the stomatal phenotypes are supported by analyses of BdSPCH1 and BdSPCH2 RNA and protein expression and by sequence variation among grasses. These studies suggest ways in which a duplication of a key stomatal regulator enables adaptation and could inform genetic strategies to mitigate anticipated stressors in agronomically important plants.

plant biology↗

bioRxiv2025

Trimethylamine-N-oxide affects cell type-specific pathways and networks in mouse aorta to promote atherosclerotic plaque vulnerability

BackgroundTrimethylamine-N-oxide (TMAO) has been significantly linked to atherosclerosis via several mechanisms, but its direct effect on the atherosclerosis-prone vasculature remains unclear. The objective of this study was to characterize the cell type-dependent and independent effects of TMAO on key vascular cell types involved in atherosclerosis progression in vivo. MethodsWe performed single cell RNA-sequencing (scRNAseq) on aortic athero-prone regions of female Ldlr-/- mice fed control Chow, high-cholesterol (HC), or HC+TMAO diets for three months to identify which aortic cell types, differentially expressed genes, and biological pathways are affected by TMAO. We also modeled cell-cell communications and intracellular gene regulatory networks to identify gene networks perturbed by TMAO feeding. Key genes and pathways were validated using primary human smooth muscle cells exposed to TMAO. Changes in the thickness of lesional fibrous caps in response to TMAO in female Ldlr-/- mice fed HC+TMAO versus HC diets were measured using transgelin immunostaining. ResultsOur scRNAseq analysis revealed that TMAO supplementation upregulated apoptotic gene signatures and downregulated extracellular matrix (ECM) organization and collagen formation genes in a subset of atherosclerosis-specific modulated vascular smooth muscle cells (vSMCs). We also identified "degradation of the ECM" as a top pathway for SMC-derived macrophage DEGs in response to TMAO. Network analyses support that macrophage-vSMC communication mediates ECM remodeling. Using human smooth muscle cells exposed to TMAO in vitro, we confirmed the direct effect of TMAO on regulating collagen and apoptotic genes. In agreement with the changes in these pathways that affect plaque stability, we observed a significant decrease in fibrous cap thickness in mice supplemented with TMAO. ConclusionsOur results reveal the effects of TMAO on vSMCs to promote apoptosis and decrease ECM formation, and on macrophage-mediated ECM degradation in atherosclerotic lesions to in concert enhance plaque instability. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/640205v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@100254org.highwire.dtl.DTLVardef@109155aorg.highwire.dtl.DTLVardef@158d280org.highwire.dtl.DTLVardef@63d544_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIscRNAseq of the aortic athero-prone regions in female Ldlr-/- mice supplemented with TMAO in the diet revealed the effect of TMAO across cell types, particularly in SMC-derived macrophages and atheroprotective modulated vSMCs. C_LIO_LITMAO increases apoptotic gene signatures and reduces ECM organization and collagen formation gene signatures in modulated vSMCs in vivo, and in vitro exposure studies support a direct effect of TMAO on these genes. C_LIO_LIModulated vSMC-specific gene regulatory networks enriched for apoptotic genes and ECM organization genes were organized by intracellular regulators such as Ccl19 and Tnn and extracellular regulators such as Mmp9 and Spp1 from macrophages. C_LIO_LIFibrous cap thickness, a marker of atherosclerotic plaque stability, was significantly reduced in female Ldlr-/- mice fed HC+TMAO versus HC diets for five months. C_LI

physiology↗

bioRxiv2024

mRNA stability fine tunes gene expression in the developing cortex to control neurogenesis

RNA expression levels are controlled by the complementary processes of synthesis and degradation. Although mis-regulation of RNA turnover is linked to neurodevelopmental disorders, how it contributes to cortical development is largely unknown. Here, we profile the RNA stability landscape of the cortex across development and demonstrate that control of stability by the CCR4-NOT complex is essential for corticogenesis in vivo. First, we use SLAM-seq to measure RNA half-lives transcriptome-wide across multiple stages of cortical development. We characterize cis-acting features associated with RNA stability and find that RNAs that are upregulated across development tend to be more stable, while downregulated RNAs are less stable. To probe how disruption of RNA turnover impacts cortical development, we assess developmental requirements of CNOT3, a core component of the CCR4-NOT deadenylase complex. Mutations in CNOT3 are associated with human neurodevelopmental disorders, however its role in cortical development is unknown. Conditional knockout of Cnot3 in neural progenitors and their progeny in the developing mouse cortex leads to severe microcephaly due to reduced neuron production and p53-dependent apoptosis. Collectively, our findings demonstrate that fine-tuned control of RNA turnover is crucial for brain development.

neuroscience↗

bioRxiv2024

Cryo-EM captures the coordination of long-range allostery and asymmetric electron transfer by a bi-copper cluster in the nitrogenase-like DPOR complex

Enzymes that catalyze long-range electron transfer reactions are often structurally evolved to possess two symmetrical halves. The functional advantages and mechanistic principles for such architecture remain a mystery. Using Cryo-EM we capture snapshots of the nitrogenase-like Dark-operative Protochlorophyllide Oxidoreductase (DPOR) enzyme during substrate recognition and turnover. The structures reveal that asymmetry is enforced upon substrate binding and leads to an allosteric inhibition of protein-protein interactions and electron transfer in one half. Residues that form a conduit for electron transfer are aligned in one half while misaligned in the other. An ATP-turnover coupled switch is triggered once electron transfer is accomplished in one half and relayed through a bi-copper cluster at the oligomeric interface, leading to activation of enzymatic events in the other. The findings provide a mechanistic blueprint for regulation of asymmetric long-range electron transfer. One-Sentence SummaryA bi-copper cluster coordinates electron transfer for substrate reduction in the nitrogenase-like DPOR enzyme and the structures reveal how allostery and asymmetry are enacted over 100[A] and utilized for sequential electron transfer.

biochemistry↗

bioRxiv2023

ERα mediated gene state switching regulates the extent of the single-cell estrogen response

Gene regulation is complex, involving the coordination of hundreds of proteins that function to control genome accessibility, mediate enhancer-promoter interactions, and initiate transcription. At individual loci, transcriptional initiation is stochastic, resulting in short periods of nascent RNA synthesis known as transcriptional bursts. To understand how altered Estrogen Receptor function and cofactor recruitment regulates transcriptional bursting, we used single molecule imaging of estrogen responsive genes in Bisphenol A (BPA) treated cells. Using live cell imaging of the estrogen responsive TFF1 gene, we observe that cells treated with BPA exhibited burst initiation kinetics and burst sizes which were indistinguishable from cells induced with Estradiol (E2). However, we observed a 50% reduction in the number of active alleles in BPA treated cells. This effect is gene specific, as GREB1 was unperturbed. Although we observed no difference in chromatin accessibility, the TFF1 promoter exhibited an altered structure which coincided with reduced ER and cofactor binding. Lastly, deletion of the enhancer locus removed the BPA effect, indicating that enhancer function was perturbed. Our results demonstrate gene specific effects of altered ER recruitment and function which lead to a reduction of transcriptionally permissive states. Our work supports the model that the early estrogen response occurs from alleles in primed transcriptionally permissive states with additional inactive alleles contributing to the response over time.

cell biology↗

bioRxiv2023

Cnot3 is required for male germ cell development and spermatogonial stem cell maintenance

The foundation of spermatogenesis and lifelong fertility is provided by spermatogonial stem cells (SSCs). SSCs divide asymmetrically to either replenish their numbers (self-renewal) or produce undifferentiated progenitors that proliferate before committing to differentiation. However, regulatory mechanisms governing SSC maintenance are poorly understood. Here, we show that the CCR4-NOT mRNA deadenylase complex subunit CNOT3 plays a critical role in maintaining spermatogonial populations in mice. Cnot3 is highly expressed in undifferentiated spermatogonia, and its deletion in spermatogonia resulted in germ cell loss and infertility. Single cell analyses revealed that Cnot3 deletion led to the de-repression of transcripts encoding factors involved in spermatogonial differentiation, including those in the glutathione redox pathway that are critical for SSC maintenance. Together, our study reveals that CNOT3 - likely via the CCR4-NOT complex - actively degrades transcripts encoding differentiation factors to sustain the spermatogonial pool and ensure the progression of spermatogenesis, highlighting the importance of CCR4-NOT-mediated post-transcriptional gene regulation during male germ cell development. HighlightsO_LICnot3 is predominantly expressed in undifferentiated spermatogonia, and its expression decreases in later stages. C_LIO_LIConditional deletion of Cnot3 leads to total germ cell loss and male infertility. C_LIO_LICNOT3 represses transcripts encoding factors involved in spermatogonial differentiation. C_LIO_LICNOT3 protects spermatogonial stem cells maintenance via GSH/redox pathway. C_LI Graphical AbstractCnot3 promotes SSC self-renewal by balancing ROS production via the regulation of GSH production O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/562256v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@3afef9org.highwire.dtl.DTLVardef@139cb19org.highwire.dtl.DTLVardef@1450c47org.highwire.dtl.DTLVardef@293300_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2023

Genetic Screen Identified Prmt5 as a Neuroprotection Target against Cerebral Ischemia

Epigenetic regulators present novel opportunities for both ischemic stroke research and therapeutic interventions. While previous work has implicated that they may provide neuroprotection by potentially influencing coordinated sets of genes and pathways, most of them remains largely uncharacterized in ischemic conditions. In this study, we used the oxygen-glucose deprivation (OGD) model in the immortalized mouse hippocampal neuronal cell line HT-22 and carried out an RNAi screen on epigenetic regulators. We identified Prmt5 as a novel negative regulator of neuronal cell survival after OGD, which presented a phenotype of translocation from the cytosol to the nucleus upon oxygen and energy depletion both in vitro and in vivo. Prmt5 bound to the chromatin and a large number of promoter regions to repress downstream gene expression. Silencing Prmt5 significantly dampened the OGD-induced changes for a large-scale of genes, and gene ontology analysis showed that Prmt5-target genes were highly enriched for Hedgehog signaling. Encouraged by the above observation, we treated mice with middle cerebral artery occlusion (MCAO) with the Prmt5 inhibitor EPZ015666 and found that Prmt5 inhibition sustain protection against neuronal death in vivo. Together, our findings revealed a novel epigenetic mechanism of Prmt5 in cerebral ischemia and uncovered a potential target for neuroprotection.

cell biology↗

bioRxiv2021

Revisiting the Hayflick Limit: Insights from an Integrated Analysis of Changing Transcripts, Proteins, Metabolites and Chromatin

The process wherein dividing cells exhaust proliferative capacity and enter into replicative senescence has become a prominent model for cellular aging in vitro. Despite decades of study, this cellular state is not fully understood in culture and even much less so during aging. Here, we revisit Leonard Hayflicks original observation of replicative senescence in WI-38 human lung fibroblasts equipped with a battery of modern techniques including RNA-seq, single cell RNA-seq, proteomics, metabolomics, and ATAC-seq. We find evidence that the transition to a senescent state manifests early, increases gradually, and corresponds to a concomitant global increase in DNA accessibility in nucleolar and lamin associated domains. Furthermore, we demonstrate that senescent WI-38 cells acquire a striking resemblance to myofibroblasts in a process similar to the epithelial to mesenchymal transition (EMT) that is regulated by the transcription factors YAP1/TEAD1 and TGF-{beta}2. Lastly, we show that verteporfin inhibition of YAP1/TEAD1 activity in aged WI-38 cells robustly attenuates this gene expression program.

genomics↗

bioRxiv2020

INO80 promotes H2A.Z occupancy to regulate 1 cell fate transition in pluripotent stem cells

The INO80 chromatin remodeler is involved in many chromatin-dependent cellular functions. However, its role in pluripotency and cell fate transition is not fully defined. We examined the impact of Ino80 deletion in the naive and primed pluripotent stem cells. We found that Ino80 deletion had minimal effect on self-renewal and gene expression in the naive state, but led to cellular differentiation and de-repression of developmental genes in the transition toward and maintenance of the primed state. Mechanistically, INO80 pre-marked gene promoters that would adopt the H3K4me3 and H3K27me3 bivalent histone modifications. It promoted H2A.Z occupancy at these future bivalent domains to facilitate H3K27me3 installation and maintenance as well as downstream gene repression. Thus, INO80-dependent H2A.Z occupancy is a critical a licensing step for bivalency and poised gene expression in pluripotent stem cells. Our results uncovered an unexpected function of INO80 in H2A.Z deposition and gene repression, and an epigenetic mechanism by which chromatin remodeling, histone variant and modification coordinately control cell fate.

developmental biology↗

bioRxiv2020

Precise Estimation of In Vivo Protein Turnover Rates

Isotopic labeling with deuterium oxide (D2O) is a common technique for estimating in vivo protein turnover, but its use has been limited by two long-standing problems: (1) identifying non-monoisotopic peptides; and (2) estimating protein turnover rates in the presence of dynamic amino acid enrichment. In this paper, we present a novel experimental and analytical framework for solving these two problems. Peptides with high probabilities of labeling in many amino acids present fragmentation spectra that frequently do not match the theoretical spectra used in standard identification algorithms. We resolve this difficulty using a modified search algorithm we call Conditional Ion Distribution Search (CIDS). Increased identifications from CIDS along with direct measurement of amino acid enrichment and statistical modeling that accounts for heterogeneous information across peptides, dramatically improves the accuracy and precision of half-life estimates. We benchmark the approach in cells, where near-complete labeling is possible, and conduct an in vivo experiment revealing, for the first time, differences in protein turnover between mice and naked mole-rats commensurate with their disparate longevity.

bioinformatics↗

bioRxiv2020

HbtR, a heterofunctional homolog of the virulence regulator TcpP, facilitates the transition between symbiotic and planktonic lifestyles in Vibrio fischeri

The bioluminescent bacterium Vibrio fischeri forms a mutually beneficial symbiosis with the Hawaiian bobtail squid, Euprymna scolopes, in which the bacteria, housed inside a specialized light organ, produce light used by the squid in its nocturnal activities. Upon hatching, E. scolopes juveniles acquire V. fischeri from the seawater through a complex process that requires, among other factors, chemotaxis by the bacteria along a gradient of N-acetylated sugars into the crypts of the light organ, the niche in which the bacteria reside. Once inside the light organ, V. fischeri transitions into a symbiotic, sessile state in which the quorum-signaling regulator LitR induces luminescence. In this work we show that expression of litR and luminescence are repressed by a homolog of the V. cholerae virulence factor TcpP, which we have named HbtR. Further, we demonstrate that LitR represses genes involved in motility and chemotaxis into the light organ and activates genes required for exopolysaccharide production. ImportanceTcpP homologs are widespread throughout the Vibrio genus; however, the only protein in this family described thus far is a V. cholerae virulence regulator. Here we show that HbtR, the TcpP homolog in V. fischeri, has both a biological role and regulatory pathway completely unlike that in V. cholerae. Through its repression of the quorum-signaling regulator LitR, HbtR affects the expression of genes important for colonization of the E. scolopes light organ. While LitR becomes activated within the crypts, and upregulates luminescence and exopolysaccharide genes and downregulates chemotaxis and motility genes, it appears that HbtR, upon expulsion of V. fischeri cells into seawater, reverses this process to aid the switch from a symbiotic to a planktonic state. The possible importance of HbtR to the survival of V. fischeri outside of its animal host may have broader implications for the ways in which bacteria transition between often vastly different environmental niches.

microbiology↗

bioRxiv2019

The flexible N-terminus of BchL protects its cluster in oxygenic environments and autoinhibits activity

The dark-operative protochlorophyllide oxidoreductase (DPOR) enzyme contains two [4Fe-4S]- containing component proteins (BchL and BchNB) that assemble in an ATP-dependent fashion to coordinate electron transfer and reduction of protochlorophyllide to chlorophyllide. Photosynthesis generates an oxygenic environment that is non-optimal for [Fe-S] clusters and we here present an elegant evolutionarily conserved mechanism in BchL to protect its [4Fe-4S] cluster. We present a crystal structure of BchL in the nucleotide-free form with an ordered N-terminus that shields the [4Fe-4S] cluster at the docking interface between BchL and BchNB. Amino acid substitutions that perturb the shielding of the [4Fe-4S] cluster produce an unstable, but hyper-active enzyme complex, suggesting a role for the N-terminus in both auto-inhibition and enzyme stability. Upon ATP binding, a patch of amino acids, Asp-Phe-Asp ( DFD patch), situated at the mouth of the BchL ATP-binding pocket promotes inter-subunit cross stabilization of the two subunits and relieves the auto-inhibition by the N-terminus. A linked BchL dimer with one defective ATP-binding site does not support substrate reduction, illustrating that nucleotide binding to both subunits is a prerequisite for the inter-subunit cross stabilization. We propose that ATP-binding produces a conformational compaction of the BchL homodimer leading to a release of the flexible N-terminus from blocking the [4Fe-4S] cluster and promotes complex formation with BchNB to drive electron transfer. The auto-inhibitive feature and release mechanism appear unique to DPOR and is not found in the structurally similar nitrogenase.

biochemistry↗

bioRxiv2018

NF-Y Controls Fidelity of Transcription Initiation at Gene Promoters Through Maintenance of the Nucleosome-Depleted Region

Faithful transcription initiation is critical for accurate gene expression, yet the mechanisms underlying specific transcription start site (TSS) selection in mammals remain unclear. Here, we show that the histone-fold domain protein NF-Y, a ubiquitously expressed transcription factor, controls the fidelity of transcription initiation at gene promoters. We report that NF-Y maintains the region upstream of TSSs in a nucleosome-depleted state while simultaneously protecting this accessible region against aberrant and/or ectopic transcription initiation. We find that loss of NF-Y binding in mammalian cells disrupts the promoter chromatin landscape, leading to nucleosomal encroachment over the canonical TSS. Importantly, this chromatin rearrangement is accompanied by upstream relocation of the transcription preinitiation complex and ectopic transcription initiation. Further, this phenomenon generates aberrant extended transcripts that undergo translation, disrupting gene expression profiles. These results establish NF-Y as a central player in TSS selection in metazoans and highlight the deleterious consequences of inaccurate transcription initiation.

genomics↗

bioRxiv2018

The anthelmintic niclosamide is a potent TMEM16A antagonist that fully bronchodilates airways

There is an unmet need in severe asthma where approximately 40% of patients exhibit poor {beta}-agonist responsiveness, suffer daily symptoms and show frequent exacerbations. Antagonists of the Ca2+-activated-Cl- channel, TMEM16A, offers a new mechanism to bronchodilate airways and block the multiple contractiles operating in severe disease. To identify TMEM16A antagonists we screened a library of ~580,000 compounds. The anthelmintics niclosamide, nitazoxanide and related compounds were identified as potent TMEM16A antagonists that blocked airway smooth muscle depolarization and contraction. To evaluate whether TMEM16A antagonists resist use- and inflammatory-desensitization pathways limiting {beta}-agonist action, we tested their efficacy under harsh conditions using maximally contracted airways or airways pretreated with a cytokine cocktail. Stunningly, TMEM16A antagonists fully bronchodilated airways, while the {beta}-agonist isoproterenol showed only partial effects. Thus, antagonists of TMEM16A and repositioning of niclosamide and nitazoxanide represent an important additional treatment for patients with severe asthma and COPD that is poorly controlled with existing therapies. It is of note that drug repurposing has also attracted wide interest in niclosamide and nitazoxanide as a new treatment for cancer and infectious disease. For the first time we identify TMEM16A as a molecular target for these drugs and thus provide fresh insights into their mechanism for the treatment of these disorders in addition to respiratory disease.

physiology↗

bioRxiv2017

High-Resolution Maps of Mouse Reference Populations

Genetic reference panels are widely used to map complex, quantitative traits in model organisms. We have generated new high-resolution genetic maps of 259 mouse inbred strains from recombinant inbred strain panels (C57BL/6J x DBA/2J, ILS/IbgTejJ x ISS/IbgTejJ, C57BL/6J x A/J) and chromosome substitution strain panels (C57BL/6J-Chr# , C57BL/6J-Chr# , C57BL/6JChr# ). We genotyped all samples using the Affymetrix Mouse Diversity Array with an average inter-marker spacing of 4.3kb. The new genetic maps provide increased precision in the localization of recombination breakpoints compared to the previous maps. Although the strains were presumed to be fully inbred, we found residual heterozygosity in 40% of individual mice from five of the six panels. We also identified de novo deletions and duplications, in homozygous or heterozygous state, ranging in size from 21kb to 8.4Mb. Almost two-thirds (46 out of 76) of these deletions overlap exons of protein coding genes and may have phenotypic consequences. Twenty-nine putative gene conversions were identified in the chromosome substitution strains. We find that gene conversions are more likely to occur in regions where the homologous chromosomes are more similar. The raw genotyping data and genetic maps of these strain panels are available at http://churchill-lab.jax.org/website/MDA.

genetics↗