Biology subjects

Mariani, L.

Publications and source records attributed to Mariani, L..

6 recordsLinked to original sources

bioRxiv2026

Histone modifications directly modulate transcription factor binding to nucleosomes

Transcription factors (TFs) that can recognize specific DNA sequences within nucleosomes play key roles in regulating cell fate transitions, but what regulates their condition-dependent binding to nucleosomes remains poorly understood. Here, we determined the direct effects of H3K27ac, H3K27me3 and H3K9me3 on binding by 7 human TFs to a highly diverse library of nucleosomes. We found that these marks influence the preference of FOXA1 and PAX7 to bind as monomers versus dimers to nucleosomes. Furthermore, H3K27ac impairs TF binding at internal regions of nucleosomes, enhancing end-binding preferences, for most of the assayed TFs. Our results suggest that histone marks represent an additional layer of cis-regulatory encoding that directly regulates TF interactions with nucleosomal binding sites.

molecular biology↗

bioRxiv2026

Gene-centered representation of coding and regulatory variation enables outcome prediction

Integrating coding and regulatory variation into unified, interpretable representations remains a challenge in functional genomics. Current approaches either focus on common variants or analyze individual variants in isolation, missing the cumulative, cell-type-specific impact of both coding and noncoding variants on each gene. We present Volaria, a computational framework that integrates coding and regulatory genetic variation into unified, gene-centered representations for disease outcome prediction from whole-genome sequencing. Volaria leverages deep learning models to capture variant effects on cell-type-specific gene expression and integrates them with AI-predicted exonic variant pathogenicity to produce representations that capture the cumulative effect of genome-wide rare and common variation. Applied to whole genomes of individuals with rare glomerular diseases, Volaria predicts individual outcomes directly from germline sequence, demonstrating that structured, cell-type-aware representations capture predictive signals beyond population-based polygenic risk scores and unstructured representations. Importantly, the framework identifies context-specific biological mechanisms, providing interpretability that can be aligned with clinical measurements. By encoding genome-wide variation into compact and biologically grounded representations, Volaria provides a scalable foundation for genome interpretation and individualized outcome modeling from germline sequence, complementing phenotypic and clinical information in the future integrative frameworks.

genomics↗

bioRxiv2024

DNA flexibility regulates transcription factor binding to nucleosomes

Cell fate decisions are controlled by sequence-specific transcription factors (TFs), referred to as pioneer factors, that bind their target sites within nucleosomes (pioneer binding) and thus initiate chromatin opening. However, pioneers bind just a minority of their recognition sequences present in the genome, suggesting that local sequence context features may regulate pioneer binding. Here, we developed PIONEAR-seq, a highly parallel sequencing-based biochemical assay for high-throughput analysis of TF binding to nucleosomes on nucleosome positioning sequences. Using PIONEAR-seq, we characterized the pioneer binding of 7 human pioneer TFs. Comparison of TF binding to nucleosomes based on the synthetic Widom 601 (W601) model sequence versus three different genomic sequences revealed that the positional preferences of these TFs binding to nucleosomes (i.e., dyad, periodic and end binding) is determined by the broader sequence context of the nucleosome, rather than being a property intrinsic to the TF. We propose a model where the flexibility and rigidity within nucleosomal DNA regulate where pioneers bind within nucleosomes. Our results suggest that the broader physical properties of nucleosomal DNA represent another layer of cis-regulatory information read out by TFs in eukaryotic genomes.

molecular biology↗

bioRxiv2023

Enhancing anti-EGFRvIII CAR T cell therapy against glioblastoma with a paracrine SIRPγ-derived CD47 blocker

A major challenge for chimeric antigen receptor (CAR) T cell therapy against glioblastoma (GBM) is its immunosuppressive tumor microenvironment (TME), which is densely populated and supported by protumoral glioma-associated microglia and macrophages (GAMs). Targeting of CD47, a "dont-eat-me" signal overexpressed by tumor cells, disrupts the CD47-SIRP axis and induces GAM phagocytic function. However, antibody-mediated CD47 blockade monotherapy is associated with toxicity and low bioavailability in solid tumors. To overcome these limitations, we combined local CAR T cell therapy with paracrine GAM modulation for more effective elimination of GBM. To this end, we engineered a new CAR T cell against epidermal growth factor receptor variant III (EGFRvIII) that constitutively secretes a SIRP{gamma}-related protein (SGRP) with high affinity to CD47. Anti-EGFRvIII-SGRP CAR T cells eliminated EGFRvIII+ GBM in a dose-dependent manner in vitro and eradicated orthotopically xenografted EGFRvIII-mosaic GBM by locoregional application in vivo. This resulted in significant tumor-free long-term survival, followed by partial tumor control upon tumor re-challenge. The combination of anti-CD47 antibodies with anti-EGFRvIII CAR T cells failed to achieve a similar therapeutic effect, underscoring the importance of sustained paracrine GAM modulation. Multidimensional brain immunofluorescence microscopy and in-depth spectral flow cytometry on GBM-xenografted brains showed that anti-EGFRvIII-SGRP CAR T cells accelerated GBM clearance, increased CD68+ cell trafficking to tumor scar sites, and induced myeloid-mediated tumor cell uptake. Additionally, in a peripheral lymphoma mouse xenograft model, anti-CD19-SGRP CAR T cells had superior efficacy compared to conventional anti-CD19 CAR T cells. Validation on human GBM explants revealed that anti-EGFRvIII-SGRP CAR T cells had similar tumor-killing capacity to anti-EGFRvIII CAR monotherapy, but showed a slight improvement in maintenance of tumor-infiltrated CD14+ myeloid cells. Thus, local anti-EGFRvIII-SGRP CAR T cell therapy combines the potent antitumor effect of engineered T cells with the modulation of the surrounding innate immune TME, resulting in the additive elimination of bystander EGFRvIII- tumor cells in a manner that overcomes major mechanisms of CAR T cell therapy resistance, including tumor innate immune suppression and antigen escape.

cancer biology↗

bioRxiv2021

Comparative chromatin accessibility upon BDNF-induced neuronal activity delineates neuronal regulatory elements

Neuronal activity induced by brain-derived neurotrophic factor (BDNF) triggers gene expression, which is crucial for neuronal survival, differentiation, synaptic plasticity, memory formation, and neurocognitive health. However, its role in chromatin regulation is unclear. Here, using temporal profiling of chromatin accessibility and transcription in mouse primary cortical neurons upon either BDNF stimulation or depolarization (KCl), we identify features that define BDNF-specific chromatin-to-gene expression programs. Enhancer activation is an early event in the regulatory control of BDNF-treated neurons, where the bZIP motif-binding Fos protein pioneered chromatin opening and cooperated with co-regulatory transcription factors (Homeobox, EGRs, and CTCF) to induce transcription. Deleting cis-regulatory sequences decreased BDNF-mediated Arc expression, a regulator of synaptic plasticity. BDNF-induced accessible regions are linked to preferential exon usage by neurodevelopmental disorder-related genes and heritability of neuronal complex traits, which were validated in human iPSC-derived neurons. Thus, we provide a comprehensive view of BDNF-mediated genome regulatory features using comparative genomic approaches to dissect mammalian neuronal activity.

genomics↗

bioRxiv2020

Screening of PCRP transcription factor antibodies in biochemical assays

Antibodies offer a powerful means to interrogate specific proteins in a complex milieu. However, antibody availability and reliability are problematic and epitope tagging can be impractical in many cases. In an effort to improve this situation, the Protein Capture Reagents Program (PCRP) generated over a thousand renewable monoclonal antibodies (mAbs) against human-presumptive chromatin proteins. However, these reagents have not been widely field-tested. We therefore performed a screen to test their ability to enrich genomic regions via chromatin immunoprecipitation (ChIP) and a variety of orthogonal assays. 887 unique antibodies against 681 unique human transcription factors (TFs), were assayed by ultra-high resolution ChIP-exo/seq, primarily in a single pass in one cell type (K562). Deep systematic analyses of the resulting [~]1,200 ChIP-exo datasets can be found at www.PCRPvalidation.org. Subsets of PCRP mAbs were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and protein binding microarray (PBM) experiments. About 5% of the tested antibodies displayed target (i.e., cognate antigen) enrichment across at least one assay and are strong candidates for additional validation. An additional 34% produced ChIP-exo data that was distinct from background and thus warrant further testing. The remaining 61% were not substantially different from background, and likely require consideration of a much broader survey of cell types and/or assay optimizations. We demonstrate and discuss the metrics and challenges to antibody validation in chromatin-based assays.

biochemistry↗