Biology subjects

Anderson, S. M.

Publications and source records attributed to Anderson, S. M..

11 recordsLinked to original sources

bioRxiv2025

Divergence between transcriptomes and chromatin accessibility during differentiation from a bipotential progenitor cell population to erythroblasts and megakaryocytes

Changes in gene expression drive differentiation along distinct cell lineages, and these shifts in gene expression are associated with alterations in chromatin accessibility and modifications reflecting activation or repression. We used deep sequencing of polyA+ RNA to map the transcriptomes of the megakaryocyte-erythroid progenitor (MEP) and cells of its two daughter lineages, erythroblasts (ERY) and megakaryocytes (MEG) in mice to reveal insights into differentiation. Transcriptome comparisons revealed that MEPs already expressed much of the MEG program while continuing to express genes associated with parallel myeloid lineages. By contrast, ERY underwent an extensive program of gene induction along with repression of pan-hematopoietic and MEG genes. Maps of transcription factor (TF) occupancy also indicated distinct modes of regulation for the MEG and ERY programs, with MEG genes preferentially occupied by hematopoietic TFs in multipotent progenitors and continued occupancy post-commitment, in contrast to erythroid genes that were primarily occupied in committed ERY. Previous work had indicated a surprising discordance in the clustering of MEP with other hematopoietic cell types by RNA-seq versus chromatin states. We combined the differential expression data with chromatin accessibility across blood cell types to identify trends that contribute to this discordance. Specifically, candidate cis-regulatory elements (cCREs) in some ERY-specific genes were precociously actuated in the bipotential cell populations, and some other genes were expressed in both the MEP population and MEG but their cCREs have less chromatin accessibility in MEP. This discordance in cell type clustering by different modalities of functional genomics may reflect the different contributions of subpopulations in the MEP to the different modalities measured.

genomics↗

bioRxiv2025

High-throughput discovery of transmembrane helix dimers from human single-pass membrane proteins with TOXGREEN sort-seq

The oligomerization of the transmembrane helices of single-pass membrane proteins is crucial to biological function and its misregulation can lead to many diseases. The study of transmembrane helix oligomerization is facilitated by the availability of genetic reporter assays, which are essential tools for understanding the organization and biology of single-pass systems. In particular, reporter assays are crucial for mapping the oligomerization interfaces of transmembrane helices through scanning mutagenesis but their application is limited by the need to clone and measure each construct individually. Here, we present "TOXGREEN sort-seq", a high-throughput version of the TOXGREEN assay that enables the direct measurement of transmembrane helix oligomerization in large libraries using fluorescence-activated cell sorting and next-generation sequencing. We show that TOXGREEN sort-seq is robust and reproduce the direct measurements of individual constructs with good accuracy and sensitivity. The method produced high-quality mutational profiles from a library of 17,400 constructs designed to probe the interface of 100 potential GASright dimers predicted from sequences of human single-pass membrane proteins. We report the validated structural model of twelve dimers involved in a variety of biological functions, including immune response (interleukin-22 receptor subunit alpha-1, butyrophilin-like protein 3, hepatitis A virus cellular receptor 2), transport (transferrin receptor protein 1), and cell-surface signaling and proliferation (syndecan-3; semaphorins 5A, 6B and 6D). Remarkably, all three semaphorins in the dataset formed strong dimers and produced mutational profiles consistent with the computational structure. These findings open the possibility that dimerization may be relevant to these proteins activity and provide a validated interface for assessing their biological role.

biophysics↗

bioRxiv2023

Glioblastoma cells use an integrin- and CD44-mediated motor-clutch mode of migration in brain tissue

Glioblastoma (GBM) is an aggressive malignant brain tumor with 2-year survival rates of 6.7% [1], [2]. One key characteristic of the disease is the ability of glioblastoma cells to migrate rapidly and spread throughout healthy brain tissue[3], [4]. To develop treatments that effectively target cell migration, it is important to understand the fundamental mechanism driving cell migration in brain tissue. Here we utilized confocal imaging to measure traction dynamics and migration speeds of glioblastoma cells in mouse organotypic brain slices to identify the mode of cell migration. Through imaging cell-vasculature interactions and utilizing drugs, antibodies, and genetic modifications to target motors and clutches, we find that glioblastoma cell migration is most consistent with a motor-clutch mechanism to migrate through brain tissue ex vivo, and that both integrins and CD44, as well as myosin motors, play an important role in constituting the adhesive clutch.

cancer biology↗

bioRxiv2023

Cross-species regulatory landscapes and elements revealed by novel joint systematic integration of human and mouse blood cell epigenomes

Knowledge of locations and activities of cis-regulatory elements (CREs) is needed to decipher basic mechanisms of gene regulation and to understand the impact of genetic variants on complex traits. Previous studies identified candidate CREs (cCREs) using epigenetic features in one species, making comparisons difficult between species. In contrast, we conducted an interspecies study defining epigenetic states and identifying cCREs in blood cell types to generate regulatory maps that are comparable between species, using integrative modeling of eight epigenetic features jointly in human and mouse in our Validated Systematic Integration (VISION) Project. The resulting catalogs of cCREs are useful resources for further studies of gene regulation in blood cells, indicated by high overlap with known functional elements and strong enrichment for human genetic variants associated with blood cell phenotypes. The contribution of each epigenetic state in cCREs to gene regulation, inferred from a multivariate regression, was used to estimate epigenetic state Regulatory Potential (esRP) scores for each cCRE in each cell type, which were used to categorize dynamic changes in cCREs. Groups of cCREs displaying similar patterns of regulatory activity in human and mouse cell types, obtained by joint clustering on esRP scores, harbored distinctive transcription factor binding motifs that were similar between species. An interspecies comparison of cCREs revealed both conserved and species-specific patterns of epigenetic evolution. Finally, we showed that comparisons of the epigenetic landscape between species can reveal elements with similar roles in regulation, even in the absence of genomic sequence alignment.

genomics↗

bioRxiv2023

Morphological profiling by cell painting in human neural progenitor cells classifies hit compounds in a pilot drug screen for Alzheimer's disease

1Alzheimers disease (AD) accounts for 60-70% of dementia cases. Current treatments are inadequate and there is a need to develop new approaches to AD drug discovery. We chose to develop a cell phenotype-based drug screen centred on the AD-risk gene, SORL1, which encodes the protein SORLA. Increased AD risk has been repeatedly linked to variants in SORL1, particularly those that confer loss of, or decreased, SORLA. This is consistent with the lower SORL1 levels observed in post-mortem brain samples from individuals with AD. Consistent with its role in the endolysosomal pathway, deletion of SORL1 is associated with enlarged endosomes in neural progenitor cells (NPCs) and neurons. We, therefore, hypothesised that multiparametric, image-based phenotyping would identify features characteristic of SORL1 deletion. An automated morphological profiling assay (known as Cell Painting) was adapted to wild-type and SORL1-/- NPCs. This methodology was used to determine the phenotypic response of SORL1-/- NPCs to treatment with compounds from a small FDA/internationally-approved drug library (TargetMol, 330 compounds). We detected distinct phenotypic signatures for SORL1-/- NPCs compared to isogenic wild-type controls. Furthermore, we identified 16 approved drugs that reversed the mutant morphological signatures in NPCs derived from 3 SORL1-/- subclonal iPSC lines. Network pharmacology analysis revealed the 16 compounds belonged to five mechanistic groups: 20S proteasome, aldehyde dehydrogenase, topoisomerase I and II, and DNA synthesis inhibitors. Enrichment analysis confirmed targeting to gene sets associated with these annotated targets, and to pathways/biological processes associated with DNA synthesis/damage/repair, Proteases/proteasome and metabolism._Prediction of novel targets for some compounds revealed enrichment in pathways associated with neural cell function and AD. The findings suggest that image-based phenotyping by morphological profiling distinguishes SORL1-/- NPCs from isogenic wild-type lines, and predicts treatment responses that rescue SORL1-/--associated cellular signatures that are relevant to both SORLA function and AD. Overall, this work suggests that i) a quantitative phenotypic metric can distinguish iPSC-derived SORL1-/- NPCs from isogenic wild-type control and ii) phenotypic screening combined with multiparametric high-content image analysis is a viable option for drug repurposing and discovery in this human neural cell model of Alzheimers disease.

neuroscience↗

bioRxiv2022

RAD-TGTs: Measurement of cellular tensions via flow cytometry and DNA sequencing enabled by force-dependent rupture and delivery of DNA tension probes

Mechanical force is a key driver of cellular processes and is dysregulated in many diseases. Measuring cellular tensions to elucidate mechanotransduction pathways typically involves high-resolution but low throughput imaging of surfaces and arduous experimental preparation of materials. We present here Rupture and Deliver DNA-duplex based molecular tension sensors-RAD-TGTs. RAD-TGTs consist of immobilized DNA duplexes conjugated to a ligand and indicator (fluorophore, barcode etc) which rupture in a force-dependent manner when cells are bound. Readout of rupture is performed in cells of interest using high throughput methods such as flow cytometry and leveraging covalent DNA-protein linking HUH-tags simplifies the preparation of the tension sensor to allow use of "off-the-shelf" oligos. We demonstrate that rupture and delivery is decreased by inhibitors of cytoskeletal dynamics and knockout of mechanosensing proteins. We also show that rupture and delivery correlates with ligand affinity. Excitingly, we demonstrate that rupture and delivery of barcoded DNA-duplexes can be quantified using DNA sequencing, propelling cellular force measurements into the -omics era.

biophysics↗

bioRxiv2021

Preventing ovariectomy-induced weight gain decreases tumor burden in rodent models of obesity and postmenopausal breast cancer.

Obesity and adult weight gain are linked to increased breast cancer risk and poorer clinical outcomes in postmenopausal women, particularly for hormone-dependent tumors. Menopause is a time when significant weight gain occurs in many women, and clinical and preclinical studies have identified menopause (or ovariectomy) as a period of vulnerability for breast cancer development and promotion. We hypothesized that preventing weight gain after ovariectomy (OVX) may be sufficient to prevent the formation of new tumors and decrease growth of existing mammary tumors. Here, we tested this hypothesis in a rat model of obesity and carcinogen-induced postmenopausal mammary cancer and validated our findings in a murine xenograft model with implanted human tumors. In both models, preventing weight gain after OVX significantly decreased obesity-associated tumor development and growth. Importantly, we did not induce weight loss in these animals, but simply prevented weight gain. In both lean and obese rats, preventing weight gain reduced visceral fat accumulation and associated insulin resistance. Similarly, the intervention decreased circulating tumor-promoting growth factors and inflammatory cytokines (ie, BNDF, TNF, FGF2), with greater effects in obese compared to lean rats. In obese rats, preventing weight gain decreased adipocyte size, adipose tissue macrophage infiltration, reduced expression of the tumor-promoting growth factor FGF-1, and reduced phosphorylated FGFR in tumors. Together, these findings suggest that the underlying mechanisms associated with the anti-tumor effects of weight maintenance are multifactorial, and that weight maintenance during the peri-/post-menopausal period may be a viable strategy for reducing obesity-associated breast cancer risk and progression in women.

cancer biology↗

bioRxiv2021

Generation and diversification of recombinant monoclonal antibodies for studying mitosis

Antibodies are indispensable tools used for a large number of applications in both foundational and translational bioscience research; however, there are drawbacks to using traditional antibodies generated in animals. These include a lack of standardization leading to problems with reproducibility, high costs of antibodies purchased from commercial sources, and ethical concerns regarding the large number of animals used to generate antibodies. To address these issues, we have developed practical methodologies and tools for generating low-cost, high-yield preparations of recombinant monoclonal antibodies and antibody fragments directed to protein epitopes from primary sequences. We describe these methods here, as well as approaches to diversify monoclonal antibodies, including customization of antibody species specificity, generation of genetically encoded small antibody fragments, and conversion of single chain antibody fragments (e.g. scFv) into full-length, bivalent antibodies. This study focuses on antibodies directed to epitopes important for mitotic cell division; however, the methods and reagents described here are applicable to antibodies and antibody fragments for use in any field.

cell biology↗

bioRxiv2021

Loss of SORCS2 is associated with neuronal DNA double-strand breaks

SORCS2 is one of five proteins that constitute the Vps10p-domain receptor family. Members of this family play important roles in cellular processes linked to neuronal survival, differentiation and function. Genetic and functional studies implicate SORCS2 in cognitive function, as well as in neurodegenerative and psychiatric disorders. DNA damage and DNA repair deficits are linked to ageing and neurodegeneration, and transient neuronal DNA double-strand breaks (DSBs) also occur as a result of neuronal activity. Here, we report a novel role for SORCS2 in DSB formation. We show that SorCS2 loss is associated with elevated DSB levels in the mouse dentate gyrus and that knocking out SORCS2 in a human neuronal cell line increased Topoisomerase II{beta}-dependent DSB formation and reduced neuronal viability. Neuronal stimulation had no impact on levels of DNA breaks in vitro, suggesting that the observed differences may not be the result of aberrant neuronal activity in these cells. Our findings are consistent with studies linking the VPS10 receptors and DNA damage to neurodegenerative conditions.

neuroscience↗

bioRxiv2020

Effects of sheared chromatin length on ChIP-seq quality and sensitivity

Chromatin immunoprecipitation followed by massively parallel, high throughput sequencing (ChIP-seq) is the method of choice for identifying, on a genome-wide scale, the segments of DNA bound by specific transcription factors (TFs) or in chromatin with particular histone modifications. However, the quality of ChIP-seq datasets vary over a wide range, with a substantial fraction being of intermediate to poor quality. Such experimental variability can lead to many false positives or false negatives, impairing the ability to interpret the data. Thus, it is important to discern and control the factors that contribute to variation in ChIP-seq. In this study, we focus on the sonication step to produce sheared chromatin, a variable controllable by the user and applicable to all ChIP-seq protocols. We systematically varied the amount of shearing of fixed chromatin from a mouse erythroid cell line, carefully measured the distribution of resultant fragment lengths using the Agilent Bioanalzyer 2100, and then immunoprecipitated these batches of chromatin using highly specific antibodies against either TAL1 or CTCF. We found that the level of sonication, which was affected by both the number of sonication cycles, as well as the starting cell number, had a pronounced impact on the quality of resulting ChIP-seq signals. Specifically, over-sonication led to degradation of quality (e.g. increased background and reduction in signal), while the impact of under-sonication of chromatin differed between the two transcription factors, leading to the loss of sites occupied by TAL1 but not those bound by CTCF. We leveraged these findings to produce a set of CTCF ChIP-seq datasets in primary hematopoietic progenitor cells, including several rare cell types. Together, these results suggest that the amount of sonication is a key variable in success of ChIP-seq experiments, and that carefully monitoring the level of chromatin sonication is one way to improve ChIP-seq quality and reproducibility, which in turn facilitates low input ChIP-seq in rare cell types.

genomics↗

bioRxiv2019

Regulation and dynamics of force transmission at individual cell-matrix adhesion bonds

Integrin-based adhesion complexes link the cytoskeleton to the extracellular matrix (ECM) and are central to the construction of multicellular animal tissues. How biological function emerges from the 10s-1000s of proteins present within a single adhesion complex has remained unclear. We used fluorescent molecular tension sensors to visualize force transmission by individual integrins in living cells. These measurements revealed an underlying functional modularity in which integrin class controlled adhesion size and ECM ligand specificity, while the number and type of connections between integrins and F-actin determined the force per individual integrin. In addition, we found that most integrins existed in a state of near-mechanical equilibrium, a result not predicted by existing models of cytoskeletal force transduction. A revised model that includes reversible crosslinks within the F-actin network accounts for this result, and suggests how cellular mechanical homeostasis can arise at the molecular level.

cell biology↗