Biology subjects

Scappini, E.

Publications and source records attributed to Scappini, E..

4 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↗

bioRxiv2023

Unlocking cellular plasticity: Enhancing human iPSC reprogramming through bromodomain inhibition and extracellular matrix gene expression regulation

The transformation of fibroblasts into epithelial cells is critical for iPSC reprogramming. In this report, we describe studies with PFI-3, a small molecule inhibitor that specifically targets the bromodomains of SMARCA2/4 and PBRM1 subunit of SWI/SNF complex, as an enhancer of iPSC reprogramming efficiency. Our findings revealed that PFI-3 induces cellular plasticity in multiple human dermal fibroblasts, leading to a mesenchymal-epithelial transition (MET) during iPSC formation. This transition was characterized by the upregulation of E-cadherin expression, a key protein involved in epithelial cell adhesion. Additionally, we identified COL11A1 as a reprogramming barrier and demonstrated COL11A1 knockdown increased reprogramming efficiency. Notably, we found that PFI-3 significantly reduced the expression of numerous extracellular matrix (ECM) genes, particularly those involved in collagen assembly. Our research provides key insights into the early stages of iPSC reprogramming, highlighting the crucial role of ECM changes and cellular plasticity in this process.

cell biology↗

bioRxiv2019

Non-canonical autophagy in dendritic cells restricts cross-presentation and anti-tumor immunity

Major Histocompatibility Complex I (MHC-I) molecules classically present peptides derived from endogenous antigens, but exogenous antigens can also gain access to the MHC-I machinery in dendritic cells (DCs), which can activate antigen-specific CD8+ T cells. This process, termed cross-presentation, can be triggered by the uptake of dying autologous cells, including tumor cells, by DCs. The molecular mechanisms that underlie efficient cross-presentation remain largely uncharacterized, and an improved understanding of these mechanisms might reveal novel strategies for anti-tumor therapies. Rubicon (RUBCN) is a molecule required for LC3-associated phagocytosis (LAP), but dispensable for canonical autophagy, and mice lacking this protein develop an autoimmune inflammatory pathology with age. Here, we demonstrate that Rubcn-deficient DCs have increased retention of engulfed cellular cargo in immature phagosomes resulting in increased phagosome-to-cytosol escape and antigen access to proteasome-mediated degradation. As a result, mice selectively lacking Rubcn in DCs mount stronger tumor antigen-specific CD8+ T cell responses and exhibit decreased tumor burden compared to wild type littermates. These findings identify LAP as a key regulator of cross-presentation and suggest that targeting RUBCN might represent a novel strategy for anti-tumor therapy.

immunology↗

bioRxiv2019

Two-Step Small Scale Purification of Recombinant Adeno-Associated Viruses

Recombinant adeno-associated viruses (AAVs) are robust and versatile tools for in vivo gene delivery. Natural and designer capsid variations in AAVs allow for targeted gene delivery to specific cell types. Low immunogenicity and lack of pathogenesis also add to the popularity of this virus as an innocuous gene delivery vector for gene therapy. AAVs are routinely used to express recombinases, sensors, detectors, CRISPR-Cas9 components, or to simply overexpress a gene of interest for functional studies. High production demand has given rise to multiple platforms for production and purification of AAVs. However, most platforms rely heavily on large amounts of starting material and multiple purification steps to produce highly purified viral particles. Often, researchers require several small-scale purified AAVs. Here, we describe a simple and efficient technique for purification of recombinant AAVs from small amounts of starting material in a two-step purification method. In this method, AAVs are released into the packaging cell medium using high salt concentration and pelleted by ultracentrifugation to remove soluble impurities. Then, the resuspended pellet is purified using a protein spin-concentrator. The two-step purification consisting of ultracentrifugation and spin-concentration eliminates the need for fraction collection and the time-consuming evaluation of individual fractionated aliquots for titer and purity. In this method, the resulting AAV preparations are comparable in titer and purity to commercially available samples. This simplified process can be used to rapidly generate highly purified AAV particles in small scale, thereby saving resources.

neuroscience↗