Biology subjects

Reina, J.

Publications and source records attributed to Reina, J..

5 recordsLinked to original sources

bioRxiv2026

The Drosophila FET orthologue Cabeza is an essential cofactor for ETV4-mediated activation of GGAA microsatellite neoenhancers

The conversion of transcriptionally silent GGAA microsatellites (GGAA{micro}Sats) into functional enhancers by FET::ETS oncogenic fusions is a hallmark of Ewing sarcoma. However, emerging evidence implicates non-fused, full-length oncogenic ETS transcription factors in activating these repeats in other malignancies. Evaluating the in vivo transcriptional requirements of various human ETS factors in Drosophila, we found that human ETV4 uniquely binds and robustly activates GGAA{micro}Sats in a tissue-specific manner. This activation is strongly inhibited by the human ETS repressor ETV6. Taking advantage of low genetic redundancy in Drosophila, we identified Cabeza (Caz), the single fly FET orthologue, as a necessary cofactor for ETV4-mediated transcription at GGAA{micro}Sats. Conversely, EWS::FLI1-mediated transcriptional activation of GGAA{micro}Sats is entirely independent of endogenous Caz, highlighting the distinct mechanics of covalent tethering versus non-covalent physical complexes. Collectively, our findings provide definitive in vivo evidence that non-fused ETS factors cooperate with endogenous FET proteins to drive transcription from silent GGAA repeats, mechanistically validating this regulatory transformation known to operate as an oncogenic mechanism beyond Ewing sarcoma.

genetics↗

bioRxiv2025

Human Oncogene EWS::FLI1 Functions as a Pioneer Factor in Saccharomyces cerevisiae.

Ewing sarcoma (EwS) is an aggressive, human-exclusive tumor typically driven by the EWS::FLI1 fusion protein. To assess whether the neomorphic functions of EWS::FLI1 are fundamentally dependent on evolutionarily recent cofactors such as ETS transcription factors (ETS-TFs), Plycomb group (PcG) proteins, CBP/p300, or specific subunits of the BAF complex, we expressed EWS::FLI1 in the model organism Saccharomyces cerevisiae. This minimal system was chosen because several key EWS::FLI1s cofactors possess greatly reduced sequence homology (e.g., BAF) or are lacking altogether (e.g., ETS-TFs, PcG, or CBP/p300). We used co-IP/MS to map the yeast interactome, Chip-Seq to identify gDNA binding sequences, RNA-Seq for global gene expression, and engineered reporters to test conversion of (GGAA) tandem repeats (GGAASat) into neoenhancers. We found that the yeast EWS::FLI1 interactome was more limited and qualitatively distinct from its human counterpart, sharing core machinery (e.g. RNA Polymerase II, FACT) but lacking the BAF/SWI-SNF and spliceosome complexes, and showing strong enrichment for the SAGA chromatin remodeling complex. We also found that EWS::FLI1 binds to hundreds of sites in the yeast genome with a clear preference for putative ETS-TF consensus sequences and (CA) dinucleotide repeats. Yet, EWS::FLI1 expressing cells presented only minimal transcriptional dysregulation, a stark contrast to the extensive changes observed in humans and Drosophila cells. Finally, we found that EWS::FLI1 successfully converted silent GGAASat sequences into active enhancers in yeast. This remarkable result occurs despite the absence of homologs for key human activators, such as CBP/p300, strongly suggesting that EWS::FLI1 can mobilize functionally related, non-homologous pathways to establish neoenhancers at GGAASat sites. Altogether, our results indicate that EWS::FLI1s core ability to drive GGAASat-dependent gene expression is a conserved, ancient property, while GGAASat-independent extensive transcriptome reprogramming is dependent on co-factors and pathways specific to animal cells.

cancer biology↗

bioRxiv2024

LHPP expression in triple-negative breast cancer promotes tumor growth and metastasis by modulating the tumor microenvironment

Triple-negative breast cancer (TNBC) is a highly aggressive and metastatic form of breast cancer that lacks an effective targeted therapy. To identify new therapeutic targets, we investigated the phosphohistidine phosphatase, LHPP, which has been implicated in the development of several types of cancer. However, the full significance of LHPP in cancer progression remains unclear due to our limited understanding of its molecular mechanism. We found that levels of the LHPP phosphohistidine phosphatase were significantly increased in human breast cancer patients compared to normal adjacent tissues, with the highest levels in the TNBC subtype. When LHPP was knocked out in the MDA-MB-231 human TNBC cell line, cell proliferation, wound healing capacity, and invasion were significantly reduced. However, LHPP knockout in TNBC cells did not affect the phosphohistidine protein levels. Interestingly, LHPP knockout in MDA-MB-231 cells delayed tumor growth and reduced metastasis when orthotopically transplanted into mouse mammary glands. To investigate LHPPs role in breast cancer progression, we used next-generation sequencing and proximity-labeling proteomics, and found that LHPP regulates gene expression in chemokine-mediated signaling and actin cytoskeleton organization. Depletion of LHPP reduced the presence of tumor-infiltrating macrophages in mouse xenografts. Our results uncover a new tumor promoter role for LHPP phosphohistidine phosphatase in TNBC and suggest that targeting LHPP phosphatase could be a potential therapeutic strategy for TNBC.

cancer biology↗

bioRxiv2023

The Drosophila cancer-germline, head-to-head gene pair TrxT and dhd is dispensable for normal brain development but plays a major role in l(3)malignant brain tumour growth.

Drosophila thioredoxins Deadhead (Dhd) and Thioredoxin-T (TrxT) are encoded by a head-to-head gene pair in the X chromosome. Akin to human cancer-germline (CG) genes, dhd and TrxT expression is normally sex and germline specific, but becomes upregulated in brain tumours of either sex caused by mutation in l(3)malignant brain tumour (l(3)mbt). Using CRISPR/Cas9-mediated knock-out alleles and RNA-seq, we show that while both TrxT and dhd are dispensable for normal brain development, each of them has a significant but partial effect on l(3)mbt brain tumour development as well as on tumour-linked transcriptomic signatures. Moreover, this effect is enhanced by the concomitant removal of both thioredoxins that strongly inhibits l(3)mbt larval brain tumour traits, reduces gene expression differences between tumour and wild-type tissue, and essentially erases all traces of gene expression differences between tumours from larvae of different sex. These results show that TrxT and dhd play a major synergistic role in the emergence of l(3)mbt tumour-linked transcriptomic signatures and tumour development, which is remarkable taking into account that these two genes are never expressed together under normal conditions. We have also found that TrxT is crucial but dhd has no effect on the growth of male-derived l(3)mbt allografts, hence suggesting that the initial stages of tumour development and sustained, long-term tumour growth may depend upon different molecular pathways. In humans, head-to-head inverted gene pairs are abundant among the CG genes that map to the X chromosome, some of which have been shown to work as a unit cancer. Our results identify the first instance of an X-linked, head-to-head CG gene pair in Drosophila and underpin the potential of such CG genes that are dispensable for normal development and homeostasis of somatic tissue, as targets to curtail malignant growth with minimal impact on overall health.

cancer biology↗

bioRxiv2018

Identification of a putative nuclear localization signal in maspin protein shed light into its nuclear import regulation

Maspin (SERPINB5) is a potential tumor suppressor gene with pleiotropic biological activities, including regulation of cell proliferation, death, adhesion, migration and gene expression. Several studies suggest that subcellular localization plays an essential role on maspin tumor suppression activity. In this study we investigated the molecular mechanisms underlying maspin nucleocytoplasmic shuttling. An in vitro nuclear-import assay using digitonin-permeabilized HeLa cells demonstrated that maspin enters the nucleus by an energy-and carrier-independent mechanism. However, previous studies indicated that maspin subcellular localization is regulated in the cell. Using a nuclear localization signal (NLS) prediction software, we identified a putative NLS in the maspin amino acid sequence. To distinguish between passive and regulated nuclear translocation, maspinNLS or the full-length protein (MaspinFL) were fused to 5GFP, rendering the construct too large to enter the nucleus passively. Unexpectedly, 5GFP-maspinNLS, but not maspinFL-5GFP, entered the nucleus of HeLa cells. Dominant-negative Ran-GTPase mutants RanQ69L or RanT24N, suppressed 5GFP-maspinNLS nuclear localization. In summary, we provide evidence that maspin translocates to the nucleus passively. In addition, we identified a peptide in the maspin protein sequence, which is able to drive a 5GFP construct to the nucleus in an energy-dependent manner.

cell biology↗