Biology subjects

Louis, J. M.

Publications and source records attributed to Louis, J. M..

4 recordsLinked to original sources

bioRxiv2025

TIF1γ drives oral cancer recurrence by the transcriptional regulation of self-renewal genes, such as HES1

TIF1{gamma} is an E3 ubiquitin ligase and key mediator of the noncanonical TGF-{beta} signaling pathway. Initially characterized for its developmental functions, TIF1{gamma} is essential for maintaining the pluripotency of adult stem cells, including long-term hematopoietic stem cells. Although TGF-{beta} signaling contributes to cancer progression and recurrence, TIF1{gamma} has traditionally been regarded as a tumor suppressor due to its inhibition of oncogenes involved in epithelial-mesenchymal transition. However, clinical reports have associated high TIF1{gamma} expression at advanced cancer stages with poor prognosis. To elucidate the mechanism underlying this paradox, we identify a previously unrecognized role of TIF1{gamma} in promoting the self-renewal capacity of oral cancer cells, thereby contributing to disease recurrence. Phosphoproteomic profiling of self-renewal-enriched cells revealed activation of a noncanonical TGF-{beta} pathway. Using extreme limiting dilution assays, an ALDH1A1-DsRed2 cancer stem cell reporter, multiple oral cancer cell lines, primary 3D cultures on alginate matrix, and orthotopic mouse models, we demonstrate that TIF1{gamma} depletion significantly reduces self-renewal and prolongs disease-free survival. Immunoprecipitation (IP)-LC/MS/MS analysis identified transcriptional regulators within the TIF1{gamma} interactome, including TRRAP and H2A.Z, which were validated by IP and FRET assays. ChIP and IP studies further revealed that during self-renewal enrichment, TRRAP acetylates H2A.Z, decreasing the chromatin occupancy of its unacetylated form. Acetylated H2A.Z is subsequently recognized by TIF1{gamma}, which monoubiquitinates H2B at promoters of self-renewal genes such as HES1, initiating transcription. In alignment with findings from mouse neocortical development, where a Notch-independent Hes1-expressing (NIHes1) population defines primitive quiescent stem cells, we show that TIF1{gamma} acts as an acetylation reader specifically at the NIHES1 promoter region of HES1. TIF1{gamma} depletion drives NIHES1 cells toward a Notch-dependent HES1 (NDHES1) identity. RNA-seq confirmed reversal of 100 NIHES1-specific genes following TIF1{gamma} loss, along with downregulation of pluripotency-associated genes found in embryonic stem cells, supporting a critical role for TIF1{gamma} in maintaining primitive cancer stem cell states. Consistent with our in vivo findings, primary oral cancer samples showed that increased frequencies of TIF1{gamma}+/TRRAP+/H2A.Z- cells strongly predict recurrence. Given that the histone acetylation-reader function of TIF1{gamma} drives poor prognosis, our findings suggest the TIF1{gamma} bromodomain as a potential therapeutic target requiring further investigation.

cancer biology↗

bioRxiv2025

EPHA2-dependent Ephrin-B1 signaling supports self-renewal ability and recurrence of oral cancer

Eph-Ephrin pathway that drives a bidirectional signaling regulates a plethora of biological activities with its varied level of complexity. While the canonical trans-interaction of the ligand and receptor on neighboring cells initiates forward signaling that brings about biological activities, their cis-interaction on the same cell attenuates the forward signaling, modulating the biological effects. Yet, non-canonical cis-interactions with heterotypic surface proteins and kinases regulate certain biological effects. In cancer, the canonical signaling is believed to be tumor suppressive, while the ligand-independent non-canonical signaling drives tumor progression and poor prognosis. Self-renewal ability of cancer cells is a major underlying cause of recurrence and poor prognosis of cancer. Using SILAC-based proteomics, we identified Ephrin-B1 signaling as a crucial regulator of oral cancer self-renewal. Though Ephrin-B1 is known to regulate normal stem cells, its role in cancer remains underexplored. Our biochemical analyses show that Ephrin-B1 binds to a nonconventional receptor EPHA2, which is known to regulate cancer stem cells (CSCs), though the mechanism is less explored. Contrary to the belief that the cis-interaction of receptors and ligands is a means to block functional signaling, our immunoprecipitation, FRET facilitated photoswitching analysis, proximity ligation assay, and in vitro kinase assay provide evidence for the Ephrin-B1-EPHA2 cis-interaction leading to the phosphorylation of EphrinB1 at Y324/329 and Y317. Extreme limiting dilution assay in vitro and in vivo confirmed that this cis-interaction promotes CSC enrichment. Substantiating our in vitro results, mouse orthotopic models showed that Ephrin-B1/EPHA2 interaction regulates prognosis. The clinical relevance of the finding was validated using a TCGA data set and immunohistochemical analysis of tissue microarray using samples from oral cancer patients with recurrence in comparison to patients, who showed disease-free survival. Given that Ephrin-B interacts with EphB for normal stem cell homeostasis, this unconventional EPHA2/Ephrin-B1 cis-interaction, specifically manifested in CSC niches, might serve as an attractive target for therapy, warranting further validation.

cancer biology↗

bioRxiv2023

Unusual Structural Properties of Switchback DNA

Synthetic DNA motifs form the basis of nucleic acid nanotechnology, and their biochemical and biophysical properties determine their applications. Here, we present a detailed characterization of switchback DNA, a globally left-handed structure composed of two parallel DNA strands. Compared to a conventional duplex, switchback DNA shows lower thermodynamic stability and requires higher magnesium concentration for assembly but exhibits enhanced biostability against some nucleases. Strand competition and strand displacement experiments show that component sequences have an absolute preference for duplex complements instead of their switchback partners. Further, we hypothesize a potential role for switchback DNA as an alternate structure in sequences containing short tandem repeats. Together with small molecule binding experiments and cell studies, our results open new avenues for switchback DNA in biology and nanotechnology.

bioengineering↗

bioRxiv2019

Dysregulation of HSP27 oligomerization and interactions by a neuropathy-causing mutation in the C-terminal IPV motif

HSP27 (HSPB1) is a systemically expressed human small heat-shock protein that forms large, dynamic oligomers and functions in various aspects of cellular homeostasis. Mutations in HSP27 cause Charcot-Marie-Tooth disease, the most common inherited disorder of the peripheral nervous system. A particularly severe form of the disease is triggered by the P182L mutation within the highly conserved IxI/V motif of HSP27. Here, we observed that the P182L variant of HSP27 lacks the ability to prevent the aggregation of client proteins and formed significantly larger oligomers both in vitro and in vivo. NMR spectroscopy revealed that the P182L IxI/V motif binds its -crystallin domain with significantly lower association rate, and thus affinity, rendering the binding site more available for other interactors. We identified 22 IxI/V-containing proteins that are known to interact with HSP27 and could therefore bind with enhanced affinity to the P182L variant. We validated this hypothesis through co-immunoprecipitation experiments, revealing that the IxI/V motif-bearing co-chaperone BAG3 indeed binds with higher affinity to the P182L variant. Our results provide a mechanistic basis for the impact of the P182L mutation on HSP27, and highlight the general importance of the IxI/V motif and its role in protein-protein interaction networks.

biochemistry↗