Biology subjects

Tillo, D.

Publications and source records attributed to Tillo, D..

9 recordsLinked to original sources

bioRxiv2026

BPabZIP, a new bZIP protein motif that promotes binding near, and displacement of, nucleosomes

Many transcription factors (TFs) bind only a subset of their canonical binding sites in mammalian cells. To identify differences between bound and unbound sites we examined Zta(N182S), a mutant of the Epstein Barr Virus (EBV)-encoded Zta bZIP protein that binds distinct DNA sequences that are not strongly bound by any known human or viral TF, reducing the effects of selective pressure on endogenous genomic binding sites. We stably expressed Zta(N182S) in human HEK293 cells and monitored protein binding (ChIP-seq) and effects on chromatin accessibility (ATAC-seq). Zta(N182S) binds ~10% of the 14,979 genomic occurrences of the canonical 9-mer ATCACTCAT, creating stronger overall ATAC-seq signal compared to control cells, suggesting nucleosome displacement. Nucleosome occupancy, either predicted or experimentally determined (MNase), indicates that canonical Zta and Zta(N182S) sites are more strongly bound when they are ~60bp from a positioned nucleosome dyad. These data suggest that Zta and Zta(N182S) binding results in nucleosome remodeling, consistent with pioneer-like activity. Examination of amino acids across Zta and human bZIPs identifies four conserved basic amino acids, a proline, and acidic amino acids immediately N-terminal of the basic amino acids of the bZIP domain (PARRTRKPQQPESLEECDSELEIKRYKN). We term this new protein motif "BPabZIP" (Basic-Proline-acidic bZIP). Molecular structure predictions for both Zta and human Fos/Jun reveal the basic amino acids interacting with the acidic patch on the nucleosome. The acidic amino acids act as an a-helical extension of the basic region that mimics DNA by interacting with histones H2A and H2B. Taken together, our analyses of this synthetic TF reveal a pioneer-like mechanism that is present in both human and viral bZIP proteins.

genomics↗

bioRxiv2026

STING-STAT3-SOX18 Axis Drives EndMT and Epigenetic Reprogramming in SAVI Lung Fibrosis

A high prevalence of early-onset interstitial lung disease, including pulmonary fibrosis, in pediatric patients with Stimulator of interferon genes (STING)-Associated Vasculopathy with onset in infancy (SAVI) suggests a critical role for the cGAS-STING pathway in the pathogenesis of pulmonary fibrosis. We identified an endothelial-to-mesenchymal transition (EndMT) signature in lesional lung biopsies from SAVI patients, marked by a loss of endothelial and acquisition of mesenchymal markers. Consistently, induced pluripotent stem cell-derived endothelial cells (iECs) from SAVI patients harboring gain-of-function STING1 mutations spontaneously undergo EndMT, a process rescued in isogenic-correction. In endothelial cells, STING activation induces IRF3-independent STAT3 phosphorylation, initiating a SLUG-dependent mesenchymal transcriptional program while repressing SOX18 and an epigenetically-regulated endothelial maintenance network. Our studies define a non-canonical cGAS-STING-STAT3 signaling axis that couples a mesenchymal transcriptional program with epigenetic silencing of an endothelial maintenance program, promoting TGF{beta}-independent STING-mediated EndMT and endothelial dysfunction, and suggesting STING as a therapeutic target for inflammatory pulmonary fibrosis.

cell biology↗

bioRxiv2025

Plasma membrane accessible cholesterol is regulated by ACC1 and lipid droplets

Accessible cholesterol, the pool of membrane cholesterol with high chemical activity, regulates vital processes including vertebrate development and pathogen evasion. The mechanisms that govern plasma membrane (PM) cholesterol accessibility are incompletely understood. Using a genome-wide screen we find that acetyl-CoA carboxylase alpha (ACC1) loss causes a ~10-fold increase in PM accessible cholesterol in cells and a male mouse model. We demonstrate that reduced fatty acyl-CoA levels, achieved by targeting metabolic enzymes including ACC1, fatty acid synthase (FASN) or acyl-CoA synthetases long chain (ACSL1, 3 and 4), results in the activation of adipose triacylglycerol lipase (ATGL). ATGL activation elevates polyunsaturated diacylglycerols even in triacylglycerol-deficient backgrounds, suggesting that ATGL acts independently of triacylglycerol hydrolysis. These diacylglycerol species are utilized to generate polyunsaturated phosphatidylcholine and phosphatidylethanolamine, which raises PM fluidity and cholesterol accessibility. Conversely, ATGL inhibition rigidifies the PM, reducing PM accessible cholesterol. Increased PM fluidity impairs cholesterol transport, triggering SREBP2 activation. This study reveals a surprising link between fatty acid metabolism and cholesterol homeostasis, demonstrating how ATGL-dependent lipid remodeling tunes membrane fluidity to regulate intracellular cholesterol signaling.

cell biology↗

bioRxiv2024

Microenvironment Shapes Cell State, Plasticity, and Heterogeneity of Small Cell Lung Cancer

Small-cell lung cancer (SCLC) is the most fatal form of lung cancer. Intra-tumoral heterogeneity, marked by neuroendocrine (NE) and non-neuroendocrine (non-NE) cell states, defines SCLC, but the drivers of SCLC plasticity are poorly understood. To map the landscape of SCLC tumor microenvironment (TME), we apply spatially resolved transcriptomics and quantitative mass spectrometry-based proteomics to metastatic SCLC tumors obtained via rapid autopsy. The phenotype and overall composition of non-malignant cells in the tumor microenvironment (TME) exhibits substantial variability, closely mirroring the tumor phenotype, suggesting TME-driven reprogramming of NE cell states. We identify cancer-associated fibroblasts (CAF) as a crucial element of SCLC TME heterogeneity, contributing to immune exclusion, and predicting exceptionally poor prognosis. Together, our work provides a comprehensive map of SCLC tumor and TME ecosystems, emphasizing their pivotal role in SCLCs adaptable nature, opening possibilities for re-programming the intercellular communications that shape SCLC tumor states.

cancer biology↗

bioRxiv2023

Direct ionic stress sensing and mitigation by the transcription factor NFAT5

Homeostatic control of intracellular ionic strength is essential for protein, organelle and genome function, yet mechanisms that sense and enable adaptation to ionic stress remain poorly understood in animals. We find that the transcription factor NFAT5 directly senses solution ionic strength using a C-terminal intrinsically disordered region. Both in intact cells and in a purified system, NFAT5 forms dynamic, reversible biomolecular condensates in response to increasing ionic strength. This self-associative property, conserved from insects to mammals, allows NFAT5 to accumulate in the nucleus and activate genes that restore cellular ion content. Mutations that reduce condensation or those that promote aggregation both reduce NFAT5 activity, highlighting the importance of optimally tuned associative interactions. Remarkably, human NFAT5 alone is sufficient to reconstitute a mammalian transcriptional response to ionic or hypertonic stress in yeast. Thus NFAT5 is both the sensor and effector of a cell-autonomous ionic stress response pathway in animal cells.

cell biology↗

bioRxiv2023

DECODING COMPLEXITY IN BIOMOLECULAR RECOGNITION OF DNA I-MOTIFS

DNA i-motifs (iMs) are non-canonical C-rich secondary structures implicated in numerous cellular processes. Though iMs exist throughout the genome, our understanding of iM recognition by proteins or small molecules is limited to a few examples. We designed a DNA microarray containing 10,976 genomic iM sequences to examine the binding profiles of four iM-binding proteins, mitoxantrone, and the iMab antibody. iMab microarray screens demonstrated that pH 6.5, 5% BSA buffer was optimal, and fluorescence was correlated with iM C-tract length. hnRNP K broadly recognizes diverse iM sequences, favoring 3-5 cytosine repeats flanked by thymine-rich loops of 1-3 nucleotides. Array binding mirrored public ChIP-Seq datasets, in which 35% of well-bound array iMs are enriched in hnRNP K peaks. In contrast, other reported iM-binding proteins had weaker binding or preferred G-quadruplex (G4) sequences instead. Mitoxantrone broadly binds both shorter iMs and G4s, consistent with an intercalation mechanism. These results suggest that hnRNP K may play a role in iM-mediated regulation of gene expression in vivo, whereas hnRNP A1 and ASF/SF2 are possibly more selective in their binding preferences. This powerful approach represents the most comprehensive investigation of how biomolecules selectively recognize genomic iMs to date.

biochemistry↗

bioRxiv2022

The homeobox transcription factor DUXBL controls exit from totipotency

Upon exit from the totipotent 2-cell (2C) embryo stage, the 2C-associated transcriptional program needs to be efficiently silenced. However, the molecular mechanisms involved in this process remain mostly unknown. Here, we demonstrate that the 2C-specific transcription factor DUX directly induces the expression of DUXBL to promote this silencing. Indeed, DUX expression in Duxbl-knockout ESC causes increased induction of the 2C-transcriptional program, whereas DUXBL overexpression impairs 2C-associated transcription. CUT&RUN analyses show that DUXBL gains accessibility to DUX-bound regions in DUX-induced ESC while it is unable to bind those regions in uninduced cells. Mechanistically, we determined that DUXBL interacts with TRIM24 and TRIM33, two members of the tripartite motif superfamily involved in gene silencing and co-localizes with them in nuclear foci upon DUX expression. Furthermore, DUXBL downregulation in mouse zygotes leads to a penetrant 2C-stage arrest. Our data reveals an unexpected role for DUXBL in controlling the exit from totipotency.

developmental biology↗

bioRxiv2021

The ETS Transcription Factor ERF controls the exit from the naive pluripotent state

The naive epiblast undergoes a transition to a pluripotent primed state during embryo implantation. Despite the relevance of the FGF pathway during this period, little is known about the downstream effectors regulating this signaling. Here, we examined the molecular mechanisms coordinating the naive to primed transition by using inducible ESC to genetically eliminate all RAS proteins. We show that differentiated RASKO ESC remain trapped in an intermediate state of pluripotency with naive-associated features. Elimination of the transcription factor ERF overcomes the developmental blockage of RAS-deficient cells by naive enhancer decommissioning. Mechanistically, ERF regulates NANOG expression and ensures naive pluripotency by strengthening naive transcription factor binding at ESC enhancers. Moreover, ERF negatively regulates the expression of the de novo methyltransferase DNMT3B, which participates in the extinction of the naive transcriptional program. Collectively, we demonstrated an essential role for ERF controlling the exit from naive pluripotency during the progression to primed pluripotency. TeaserERF is the MAPK-dependent switch controlling the transition between naive and primed pluripotency during embryonic development.

developmental biology↗

bioRxiv2020

CTCF is a Barrier for Totipotent-like Reprogramming

Totipotent cells have the ability of generating embryonic and extra-embryonic tissues1,2. Interestingly, a rare population of cells with totipotent-like potential was identified within ESC cultures3. These cells, known as 2 cell (2C)-like cells, arise from ESC and display similar features to those found in the totipotent 2 cell embryo2-4. However, the molecular determinants of 2C-like conversion have not been completely elucidated. Here, we show that CTCF is a barrier for 2C-like reprogramming. Indeed, forced conversion to a 2C-like state by DUX expression was associated with DNA damage at a subset of CTCF binding sites. Endogenous or DUX-induced 2C-like ESC showed decreased CTCF enrichment at known binding sites, suggesting that acquisition of a totipotent-like state is associated with a highly dynamic chromatin architecture. Accordingly, depletion of CTCF in ESC efficiently promoted spontaneous and asynchronous conversion to a totipotent-like state. This phenotypic reprogramming was reversible upon restoration of CTCF levels. Furthermore, we showed that transcriptional activation of the ZSCAN4 cluster was necessary for successful 2C-like reprogramming. In summary, we revealed the intimate relation between CTCF and totipotent-like reprogramming.

developmental biology↗