Biology subjects

Black, E.

Publications and source records attributed to Black, E..

7 recordsLinked to original sources

bioRxiv2026

Histone H4 acetyl-methyllysine marks accessible chromatin that resists compaction

Certain regulatory DNA regions remain accessible even under conditions of widespread chromatin compaction. These regions are often marked by specific protein factors and histone modifications that help maintain their accessibility. Here, we examine the genomic landscape of acetyl-methyllysine (Kacme), a recently discovered histone post-translational modification. Across multiple systems, Kacme is highly enriched at sites of accessible chromatin, including active promoters, enhancers, silencers, and CTCF-binding sites. We find that Kacme is selectively retained at loci that resist condensation during mitosis, marks XIST and escapee regions on the inactive X chromosome in female cells and demarcates the boundaries of broad heterochromatin domains. Kacme-marked insulator elements block heterochromatin spreading and protect adjacent genes from transcriptional repression, even when H3K27me3 levels are pharmacologically elevated through KDM6A/6B inhibition. Taken together, our findings establish the chromatin features associated with Kacme and support a model in which Kacme helps safeguard chromatin accessibility at loci that resist compaction.

molecular biology↗

bioRxiv2026

5' UTR length regulates alternative N-terminal protein isoform production in health and disease

The 5' untranslated region (5' UTR) of an mRNA is classically viewed as a regulatory region that controls the amount of protein production, but not the resulting protein sequence. Here, we demonstrate that 5' UTR length plays a direct role in alternative N-terminal protein isoform production by controlling start codon selection. We find that very short 5' UTRs enhance leaky ribosome scanning, thereby promoting the production of truncated alternative N-terminal protein isoforms. We also show that endogenous changes in 5' UTR length due to alternative transcription initiation can tune the relative abundance of alternative N-terminal isoforms from the same gene. In addition, we identify mutations in rare genetic diseases that alter 5' UTR length, including a deletion in the VHL 5' UTR in von Hippel-Lindau disease that shifts translation toward the shorter VHLp19 isoform. Together, our results implicate 5' UTR length as a determinant of alternative N-terminal isoform production and reveal an underappreciated mechanism by which noncoding changes can reshape the proteome. HighlightsO_LI5' UTR length affects the landscape of endogenous alternative N-terminal protein isoforms C_LIO_LIGeneration of an alternative truncated AKR7A2 isoform is mediated by short 5' UTR length C_LIO_LIAlternative transcription initiation modulates 5' UTR length to tune N-terminal isoform ratios C_LIO_LIPathogenic VHL 5' UTR variants perturb N-terminal isoform ratios by altering 5' UTR length C_LI

molecular biology↗

bioRxiv2026

Polyclonal and single clonal patient-derived organoid models of Barrett oesophagus and oesophageal adenocarcinoma establish a platform for the analysis of heterogeneity in disease progression and therapy response

SUMMARY/ABSTRACTOesophageal adenocarcinoma (OAC) is a major cause of morbidity and mortality. OAC and its precursor, Barrett oesophagus (BO), are defined by substantial early heterogeneity, complicating prevention and treatment of OAC and remaining poorly recapitulated by current in vitro and animal model systems. We have generated 116 patient- and healthy donor-derived organoids (PDOs) spanning normal oesophagogastric tissue, BO and OAC. These PDOs capture population diversity and recapitulate phenotypic, genomic and transcriptomic features of their respective disease stages. We develop a single cell-derived clonal organoid approach and show that this enables us to capture the heterogeneity and isolate high-risk, subclonal populations that are difficult to discern and maintain in bulk PDO cultures. Using this platform, we demonstrate functional importance of this biobank across the pre-malignant to invasive disease spectrum, including a role for BO in shaping fibroblast phenotype within assembloids, and diverse responses of OAC to chemotherapy, radiotherapy and targeted CDK4/6 inhibition. HIGHLIGHTSO_LIPatient- and healthy donor-derived organoids (PDOs) provide a functional platform of disease progression and heterogeneity across normal gastric, non-dysplastic and dysplastic Barrett oesophagus (BO) and oesophageal adenocarcinoma (OAC). C_LIO_LIWe provide a quantitative phenotypic and molecular framework to assess the provenance and fidelity of each PDO model given the heterogeneity of this disease. C_LIO_LIPDOs recapitulate key features of non-dysplastic and dysplastic BO, as well as invasive OAC. C_LIO_LISingle cell-derived clonal organoids (sc-organoids) isolate and maintain high-risk subclonal populations. C_LIO_LIOAC PDOs mirror known population level variation in response to systemic anti-cancer therapies. C_LI

cancer biology↗

bioRxiv2025

Orthogonal and Robust Analytics Enable Reproducible and Scalable Manufacturing of High Purity Extracellular Vesicles Derived from Mesenchymal Stromal Cells

The development of extracellular vesicle (EV) based therapeutics requires robust analytical assays and scalable downstream processing (DSP) strategies to ensure product quality and reproducibility. In this study, we established an analytical toolbox comprising scattering and fluorescence mode nanoparticle tracking analysis (NTA), fluorescence-based flow cytometry (Fl-FC), and multi-detector analytical size exclusion chromatography. Liposomes, selected for their physicochemical similarity to EVs, were used as reference materials to optimize assay parameters, fluorescence labeling conditions, and dynamic range, minimizing artifacts such as photobleaching and masking effects. Using these optimized analytical tools, we designed a scalable DSP workflow for human bone marrow mesenchymal stromal cell (hBM-MSC) derived EVs, incorporating clarification, tangential flow filtration (TFF), ion exchange chromatography (IEX), buffer exchange, and sterile filtration. IEX chromatography resulted in the elution of two cell-secreted populations, with similar scattering signals while eluate 1 showed approximately 30 times higher absorbance signal compared to eluate 2. Transmission electron microscopy revealed that eluate 1 contained non-vesicular extracellular particles (NVEPs), and eluate 2 was enriched in EVs and showed higher expression of positive markers such as CD81 and CD73 using Simple Western. Additionally, the two IEX eluates showed different proteomic and lipidomic profiles. Then the analytical toolbox was utilized to monitor the DSP process in terms of particle recovery and impurity removal throughout the process determining the high purity level of the final EV preparation. Together, these results demonstrate that orthogonal analytics coupled to a scalable DSP yield reproducible MSC-EV preparations while depleting commonly co-isolated NVEPs. This practical framework advances process analytics of MSC-EV manufacturing and supports reporting of identity, purity, and function aligned with existing guidelines in the field. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/672476v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@142ff8aorg.highwire.dtl.DTLVardef@196c0bforg.highwire.dtl.DTLVardef@485c36org.highwire.dtl.DTLVardef@9dff39_HPS_FORMAT_FIGEXP M_FIG C_FIG Liposomes were used as reference materials to optimize single particle analytics as well as multi detector analytical chromatography to assess particle recovery and impurity removal. This optimized analytical toolbox enabled accurate monitoring of downstream processing steps, establishing a robust workflow for reproducible and scalable mesenchymal stromal derived extracellular vesicles bioprocessing.

bioengineering↗

bioRxiv2022

ATR protects centromere identity by promoting DAXX association with PML nuclear bodies

Centromere protein A (CENP-A) defines centromere identity and nucleates kinetochore formation for mitotic chromosome segregation. Here, we show that Ataxia telangiectasia and Rad3-related (ATR) kinase, a master regulator of the DNA damage response, protects CENP-A occupancy at interphase centromeres in a DNA damage-independent manner. As ATR localizes to promyelocytic leukemia nuclear bodies (PML NBs) in unperturbed cells, we hypothesized that ATR protects CENP-A occupancy by regulating the localization of the histone H3.3 chaperone and PML NB component, DAXX. Indeed, we found that ATR inhibition reduces DAXX association with PML NBs, resulting in the DAXX-dependent loss of CENP-A from interphase centromeres. Lastly, we demonstrate that CENP-A occupancy is not restored until G1 of the following cell cycle, leading to increased mitotic chromosome segregation defects. These findings demonstrate a novel mechanism by which ATR protects centromere identity and genome stability.

cell biology↗

bioRxiv2022

Corruption of DNA End-Joining in Mammalian Chromosomes by Progerin Expression

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic condition characterized by features of accelerated aging and a life expectancy of about 14 years. HGPS is commonly caused by a point mutation in the LMNA gene which codes for lamin A, an essential component of the nuclear lamina. The HGPS mutation alters splicing of the LMNA transcript, leading to a truncated, farnesylated form of lamin A termed "progerin." HGPS is associated with accumulation of genomic DNA double-strand breaks (DSBs), suggesting altered DNA repair. DSB repair normally occurs by either homologous recombination (HR), an accurate, templated form of repair, or by non-homologous end joining (NHEJ), an error-prone non-templated rejoining of DNA ends. Some NHEJ events occur via high-fidelity joining of DNA ends and we refer to such events as precise ligation (PL). Previously, we reported that expression of progerin correlated with increased NHEJ relative to HR. We now report on progerins impact on the nature of DNA end-joining. We used a model system involving a DNA end-joining reporter substrate integrated into the genome of cultured thymidine kinase-deficient mouse fibroblasts. Some cells were engineered to express progerin. DSBs were induced in the substrate through expression of endonuclease I-SceI, and DSB repair events were recovered through selection for thymidine kinase function. Progerin expression correlated with a significant shift away from PL and toward error-prone NHEJ. Our work suggests that progerin suppresses interactions between complementary sequences at DNA termini, shifting DSB repair toward low-fidelity DNA end-joining and perhaps contributing to aging through compromised genome stability.

molecular biology↗

bioRxiv2021

ThruTracker: Open-Source Software for 2-D and 3-D Animal Video Tracking

O_LITracking animal movement patterns using videography is an important tool in many biological disciplines ranging from biomechanics to conservation. Reduced costs of technology such as thermal videography and unmanned aerial vehicles has made video-based animal tracking more accessible, however existing software for processing acquired video limits the application of these methods. C_LIO_LIHere, we present a novel software program for high-throughput 2-D and 3-D animal tracking. ThruTracker provides tools to allow video tracking under a variety of conditions with minimal technical expertise or coding background and without the need for paid licenses. Notable capabilities include calibrating the intrinsic properties of thermal cameras; tracking and counting hundreds of animals at a time; and the ability to make 3-D calibrations without dedicated calibration objects. Automated 2-D and 3-D workflows are integrated to allow for analysis of largescale datasets. C_LIO_LIWe tested ThruTracker with two case studies. The 2-D workflow is demonstrated by counting bats emerging from bridges and caves using thermal Videography. Tests show that ThruTracker has a similar accuracy compared to humans under a variety of conditions. The 3-D workflow is shown for making accurate calibrations for tracking bats and birds at wind turbines using only the wind turbine itself as a calibration object. C_LIO_LIThruTracker is a robust software program for tracking moving animals in 2-D and 3-D. Major applications include counting animals such as bats, birds, and fish that form large aggregations, and documenting movement trajectories over medium spatial scales ([~]100,000 m3). When combined with emerging technologies, we expect videographic techniques to continue to see widespread adoption for an increasing range of biological applications. C_LI

animal behavior and cognition↗