Biology subjects

Labrador, M.

Publications and source records attributed to Labrador, M..

8 recordsLinked to original sources

bioRxiv2026

Distinct principles of genome compartmentalization in Drosophila and humans revealed by osmotic stress

The three-dimensional organization of eukaryotic genomes into compartments, topologically associating domains, and loops is mediated by architectural proteins whose organizational principles vary across species. In Drosophila, insulator proteins including Su(Hw) and the histone variant {gamma}H2Av form liquid-liquid phase separation (LLPS) condensates, yet how this phase separation capacity relates to genome compartmentalization has remained unclear. Here we use hyperosmotic stress to simultaneously displace architectural proteins from chromatin in both Drosophila and human cells, enabling a comparative dissection of genome organization principles across species. We find that although human CTCF shares predicted LLPS properties with Drosophila insulator proteins, it does not form condensates upon osmotic stress, while Drosophila insulator proteins do. Hi-C analysis reveals that osmotic stress causes loss of compartments, TAD boundary strength, and loops in both organisms, but genome recovery after stress is rapid and near-complete in human cells while remaining substantially incomplete in Drosophila after one hour. Analysis of this recovery asymmetry reveals a fundamental difference in compartment organization: whereas human A and B compartments engage in robust homotypic long-range interactions, Drosophila B compartments rarely participate in long-range B-to-B contacts, indicating that the Drosophila genome does not replicate canonical A/B compartment organization. Instead, Drosophila genome architecture is dominated by A-to-A interactions, and A compartments are specifically enriched in {gamma}H2Av and Su(Hw), with moderate enrichment of cohesin subunits. Furthermore, loops in Drosophila are mechanistically independent from compartments and TADs, recovering before compartment structure is restored, and are anchored by Su(Hw) and cohesin rather than by dCTCF. Together, these findings suggest that the LLPS properties of {gamma}H2Av and Su(Hw) underlie A compartment formation in Drosophila through a mechanism distinct from the heterochromatin-driven B compartment interactions that predominate in vertebrates, revealing fundamentally different organizational principles between fly and human genomes.

genomics↗

bioRxiv2025

Bedaquiline Amplifies Proteasome Inhibitor Efficacy and Overcomes Resistance in Multiple Myeloma

Proteasome inhibitors (PIs) are cornerstone therapies for multiple myeloma (MM), yet resistance remains a major barrier to durable responses. To identify druggable vulnerabilities that enhance PIs efficacy, we performed a small-molecule chemical screen in the presence of carfilzomib (CFZ). We identified bedaquiline (BDQ), an FDA-approved antimycobacterial agent, as a potent synergistic partner. BDQ and its fumarate salt (BDQ-F) significantly amplified CFZ-induced cytotoxicity in PI-sensitive and PI-resistant MM cells, in AL amyloidosis and other B-cell malignancies, with minimal toxicity toward normal cells. Mechanistic studies confirmed that BDQ specifically targets the ATP5F1C subunit of mitochondrial ATP synthase. BDQ-CFZ combination triggered extensive apoptosis, exacerbating proteotoxic stress and proteasome-associated pathways. BDQ specifically enhanced CFZs inhibition of the proteasomes chymotrypsin-like activity. Importantly, BDQ synergized with multiple proteasome and ubiquitin-activating enzyme inhibitors, but not with other standard MM agents, underscoring its selective interaction with the UPS pathway. BDQ-CFZ co-treatment markedly reduced MM cell viability and tumor burden in patient-derived cells and zebrafish xenograft models. These findings support the therapeutic repurposing of BDQ to potentiate PIs efficacy and overcome resistance in MM and related B-cell malignancies. KEY POINTSO_LIBedaquiline synergizes with proteasome inhibitors in MM and other hematologic malignancies, sparing normal cells C_LIO_LIBedaquiline targets ATP synthase {gamma} and boosts carfilzomib by enhancing chymotrypsin-like proteasome inhibition C_LI NOVELTYWe identify the antimicrobial bedaquiline and its fumarate salt as potent enhancers of proteasome inhibitor efficacy in multiple myeloma and other B-cell malignancies. By inhibiting ATP synthase {gamma}, bedaquiline amplifies chymotrypsin-like proteasome inhibition and overcomes drug resistance. This study reveals a mitochondria- proteasome vulnerability and proposes a clinically actionable strategy to restore sensitivity and reduce toxicity of proteasome-based therapies. VISUAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/661768v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@339391org.highwire.dtl.DTLVardef@1d3271org.highwire.dtl.DTLVardef@daa0faorg.highwire.dtl.DTLVardef@17eb4eb_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Systematic study of hybrid triplex topology and stability suggests a general triplex-mediated regulatory mechanism

By combining in-silico, biophysical and in-cellulo experiments, we decipher the topology, physical and potential biological properties of hybrid-parallel nucleic acids triplexes; an elusive structure at the basis of life. We found that hybrid triplex topology follows a stability order: r(Py)-d(Pu){middle dot}r(Py)> r(Py)-d(Pu){middle dot}d(Py)> d(Py)-d(Pu){middle dot}d(Py)> d(Py)-d(Pu){middle dot}r(Py). The r(Py)-d(Pu){middle dot}d(Py) triplex is expected to be the preferred in the cell as it avoids the need to open the duplex reducing the torsional stress required for triplex formation in the r(Py)-d(Pu){middle dot}r(Py) topology. Upon a massive collection of melting data, we have created the first predictor for hybrid triplex stability. Leveraging this predictor, we conducted a comprehensive scan to assess the likelihood of the human genome and transcriptome to engage in triplex formation. Our findings unveil a remarkable inclination - of both the human genome and transcriptome - to generate hybrid triplex formation, particularly within untranslated (UTRs) and regulatory regions, thereby corroborating the existence of a triplex-mediated regulatory mechanism. Furthermore, we found a correlation between nucleosome linkers and TFS which agree with a putative role of triplexes in arranging chromatin structure and local/global level.

molecular biology↗

bioRxiv2023

Interactions Between the Histone Variant H2Av and the Suppressor of Hairy wing Insulator Influence the DNA Damage Response and Lymph Gland Development in Drosophila

Genome architecture is regulated by chromatin insulator proteins, and misregulation of insulator function is associated with genome instability and transcriptional regulatory defects in both vertebrates and Drosophila. Indeed, mutations of the sole insulator protein in humans, CTCF, are carcinogenic and mutations in the Drosophila insulator protein Suppressor of Hairy wing [Su(Hw)] lead to chromosomal rearrangements. However, the mechanism that links the DNA damage response and the regulation of transcription with insulator function is not yet understood. Here we show that enrichment of Su(Hw) insulator proteins at insulator sites increases after DNA damage. Additionally, Su(Hw) is necessary for phosphorylation of the histone variant H2Av in response to both UV treatment and X-ray irradiation. The requirement of Su(Hw) for H2Av phosphorylation appears to be tissue-specific, since H2Av is phosphorylated in response to DNA damage also in neurons, where Su(Hw) is not normally expressed. Similarly, we provide evidence that Su(Hw) and H2Av work together to ensure proper development of the lymph gland in Drosophila larvae. We show that H2Av regulates formation of the larval lymph gland, and mutation of H2Av causes formation of large melanotic tumors that are rescued by mutation of su(Hw) in the His2Av810 mutant background. Double mutants of su(Hw)- and His2Av810 also form supernumerary lymph gland lobes that are larger than in wild type. Our findings support a role for the Su(Hw) in the DNA damage response through the regulation of H2Av phosphorylation and suggest that Su(Hw) and H2Av also work together to ensure proper development of the lymph gland.

molecular biology↗

bioRxiv2022

Drosophila insulator proteins exhibit in-vivo liquid-liquid phase separation properties

Mounting evidence implicates liquid-liquid phase separation (LLPS), the condensation of biomolecules into liquid-like droplets in the formation and dissolution of membraneless intracellular organelles (MLOs). Eukaryotic cells utilize MLOs or condensates for various biological processes, including emergency signaling, spatiotemporal control over steady-state biochemical reactions and heterochromatin formation. Insulator proteins function as architectural elements involved in establishing independent domains of transcriptional activity within eukaryotic genomes. In Drosophila, insulator proteins coalesce to form nuclear foci known as insulator bodies in response to osmotic stress and during apoptosis. However, the mechanism through which insulator proteins assemble into bodies and whether these bodies confer any genome function are yet to be fully investigated. Here, we identify signatures of liquid-liquid phase separation by insulator bodies, including high disorder tendency in insulator proteins, scaffold-client dependent assembly, extensive fusion behavior, sphericity, and sensitivity to 1,6-hexanediol. We also show that the cohesin subunit Rad21 is a component of insulator bodies adding to the known insulator proteins and the histone variant {gamma}H2Av constituents. Our data suggest a concerted role of cohesin and insulator proteins in insulator body formation and under physiological conditions. We propose a mechanism whereby these architectural proteins modulate 3D genome organization through LLPS.

molecular biology↗

bioRxiv2021

A Phosphorylated Histone H2A Variant Displays Properties of Chromatin Insulator Proteins in Drosophila

Chromatin insulators are responsible for mediating long-range interactions between enhancers and promoters throughout the genome and align with the boundaries of topologically associating domains (TADs). Here, we demonstrate an interaction between proteins that associate with the gypsy insulator and the phosphorylated histone variant H2Av ({gamma}H2Av), a marker of DNA double strand breaks. Gypsy insulator components colocalize with {gamma}H2Av throughout the genome. Mutation of insulator components prevents stable H2Av phosphorylation in polytene chromatin. Phosphatase inhibition strengthens the association between insulator components and {gamma}H2Av and rescues {gamma}H2Av localization in insulator mutants. We also show that {gamma}H2Av is a component of insulator bodies, and that phosphatase activity is required for insulator body dissolution after recovery from osmotic stress. We further demonstrate a tight association between {gamma}H2Av and TAD boundaries. Together, our results indicate a novel mechanism linking insulator function with a histone H2A variant and with genome stability.

molecular biology↗

bioRxiv2020

THE IMPACT OF THE HYDROXYMETHYLCYTOSINE EPIGENETIC SIGNATURE ON DNA STRUCTURE AND FUNCTION

We present a comprehensive, experimental and theoretical study of the impact of 5-hydroxymethylation of DNA cytosine. Using molecular dynamics, biophysical experiments and NMR spectroscopy, we found that Ten-Eleven translocation (TET) dioxygenases generate an epigenetic variant with structural and physical properties not too different to those of 5-methylcytosine. Experiments and simulations demonstrate that 5-methyl-cytosine (mC) and 5-hydroxymethyl-cytosine (hmC) generally lead to more rigid duplexes with poorer circularization efficiencies and lower ability to form nucleosomes. In particular, we can rule out the hypothesis that hydroxymethylation reverts to unmodified cytosine physical properties, as hmC is even more rigid than mC. Thus, we do not expect dramatic changes in the chromatin structure induced by differences in physical properties between d(mCpG) and d(hmCpG). On the contrary, our simulations suggest that methylated-DNA binding domains (MBD), associated with repression activities, are very sensitive to the substitution d(mCpG)[->] d(hmCpG), while MBD3 which has a dual activation/repression activity is not sensitive to the d(mCpG) [->] d(hmCpG) change. Overall, while changes in gene activity due to cytosine methylation are the result of the combination of stiffness-related chromatin reorganization and MBD binding, those associated to 5-hydroxylation of methylcytosine could be explained by a change in the balance of repression/activation pathways related to differential MBD binding.

biochemistry↗

bioRxiv2019

A Drosophila Insulator Interacting Protein Suppresses Enhancer-Blocking Function and Modulates Replication Timing

Insulators play important roles in genome structure and function in Drosophila and mammals. More than six different insulator proteins are required in Drosophila for normal genome function, whereas CTCF is the only identified protein contributing to insulator function in mammals. Interactions between a DNA binding insulator protein and its interacting partner proteins define the properties of each insulator site. The different roles of insulator protein partners in the Drosophila genome and how they confer functional specificity remain poorly understood. Functional analysis of insulator partner proteins in Drosophila is necessary to understand how genomes are compartmentalized and the roles that different insulators play in genome function. In Drosophila, the Suppressor of Hairy wing [Su(Hw)] insulator is targeted to the nuclear lamina, preferentially localizes at euchromatin/heterochromatin boundaries, and is associated with the Gypsy retrotransposon. The properties that the insulator confers to these sites rely on the ability of the Su(Hw) protein to bind the DNA at specific sites and interact with Mod(mdg4)-67.2 and CP190 partner proteins. HP1 and insulator partner protein 1 (HIPP1) is a recently identified partner of Su(Hw), but how HIPP1 contributes to the function of Su(Hw) insulators has not yet been elucidated. Here, we find that mutations in the HIPP1 crotonase-like domain have no impact on the function of Su(Hw) enhancer-blocking activity but do exhibit an impaired ability to repair double-strand breaks. Additionally, we find that the overexpression of each HIPP1 and Su(Hw) causes defects in cell proliferation by limiting the progression of DNA replication. We also find that HIPP1 overexpression suppresses the Su(Hw) insulator enhancer-blocking function.

molecular biology↗