Biology subjects

Aguirrebengoa, M.

Publications and source records attributed to Aguirrebengoa, M..

9 recordsLinked to original sources

bioRxiv2026

Direct effects on gene transcription revealed by global and CRISPR-targeted oxidative DNA damage

Oxidative stress induces rapid expression changes of specific genes. However, whether these changes are due to direct and local DNA oxidation and whether they depend on the repair machinery is still debated. Here, we show by total RNA-Seq that upon oxidative stress, many genes are either rapidly repressed or activated. The early transcriptional response also includes the activation of many read-through RNAs. Importantly, degradation of the endogenous OGG1 DNA glycosylase showed that the early repression of these genes partially depends on OGG1, therefore demonstrating that these genome expression changes are due to the processing of oxidized DNA. We further demonstrate by ChIPseq against total or S5-phosphorylated RNA polymerase II that transcriptional regulation by oxidative stress involves changes in the recruitment of RNA polymerase II to transcription start sites. Finally, through the use of a chemoptogenetic system allowing the targeting of ROS to chosen genomic loci, we were able to reproduce repression of some genes through the local induction of oxidative DNA damage at their promoters, demonstrating that local DNA oxidation directly affects transcription of genes. Altogether, our results show that local oxidative DNA damage leads to alterations in the activity of specific promoters by a mechanism dependent on repair processes.

genomics↗

bioRxiv2026

Loss of p27Kip1 causes metabolic reprogramming and is sufficient to induce a Warburg effect and glutamine addiction in untransformed cells

The metabolic needs of a cell are tightly linked to its proliferative state and increasing evidence indicate an extensive bidirectional crosstalk between metabolic pathways and cell cycle regulators. In cancer cells, metabolism is reprogrammed to couple energetic needs and relentless proliferation. The cyclin/CDK inhibitor p27Kip1 (p27) is frequently inactivated in cancers. p27 is also involved in multiple cellular processes, including transcriptional regulation or autophagy induction. Herein, we investigated the effect of p27 loss on cell metabolism. The knockout of p27 in immortalized mouse fibroblasts increases glucose uptake and glycolysis, while decreasing mitochondrial ATP production, consistent with induction of a Warburg effect, and this was accompanied by an increased glutamine dependency to feed the TCA cycle. Our data suggest that p27 loss causes this phenotype through extensive transcriptional remodeling of metabolic gene expression. Importantly, p27 silencing in human retinal RPE1-hTERT cells was sufficient to induce a Warburg effect. Together, these results reveal a new function of p27 in regulating energy metabolism and that loss of p27 expression is sufficient to induce metabolic reprogramming and a Warburg effect, suggesting that p27 inactivation in cancer cells not only results in the loss of cell cycle inhibition but also enables the metabolic rewiring needed for increased proliferation.

cell biology↗

bioRxiv2025

Sni445 recruits box C/D snoRNPs snR4 and snR45 to guide ribosomal RNA acetylation by Kre33

Eukaryotic ribosome synthesis is a highly complex, multistep process that is best characterized in the yeast Saccharomyces cerevisiae. It is orchestrated by over 200 ribosome assembly factors and 75 small nucleolar ribonucleoproteins (snoRNPs), which guide site-specific chemical modifications of precursor ribosomal RNA (pre-rRNA). While canonical box C/D snoRNPs direct 2-O-methylation, the atypical box C/D snoRNPs snR4 and snR45 mediate acetylation of 18S rRNA residues C1280 and C1773, respectively, catalyzed by the acetyltransferase Kre33. Here, we identify and characterize Ynl050c/Sni445 as a novel ribosome assembly factor and previously unrecognized auxiliary component of the snR4 and snR45 box C/D snoRNPs. Sni445 associates with snR4 and snR45 in their free form and is required for their stable incorporation into 90S pre-ribosomes. Genetic interactions link Sni445 and the snR4 and snR45 snoRNAs to ribosomal proteins Rps20 (uS10) and Rps14 (uS11), which are positioned near the respective acetylation sites in the 40S subunit. Moreover, Sni445 physically interacts with Kre33 within the 90S pre-ribosome, and its absence abolishes acetylation of C1280 and C1773. Our findings suggest that Sni445 facilitates the recruitment of snR4 and snR45 snoRNPs to 90S particles and might promote their interaction with Kre33, thereby enabling the site-specific acetylation of 18S rRNA by Kre33.

molecular biology↗

bioRxiv2023

Ribosomal RNA synthesis by RNA polymerase I is regulated by premature termination of transcription.

The RNA polymerase I (Pol I) enzyme that synthesizes large rRNA precursors, exhibits high rate of pauses during elongation, indicative of a discontinuous process. We show here that Premature Termination of Transcription (PTT) by Pol I is a critical regulatory step limiting rRNA production in vivo. The Pol I mutant, SuperPol (RPA135-F301S), produces 1.5-fold more rRNA than the wild type (WT). Combined CRAC and rRNA analysis link increased rRNA production in SuperPol to reduced PTT, resulting in shifting polymerase distribution toward the 3 end of rDNA genes. In vitro, SuperPol shows reduced nascent transcript cleavage, associated with more efficient transcript elongation after pauses, to the detriment of transcriptional fidelity. Notably, SuperPol is resistant to BMH-21, a drug impairing Pol I elongation and inducing proteasome-mediated degradation of Pol I subunits. Compared to WT, SuperPol maintains subunit stability and sustains high transcription levels upon BMH-21 treatment. These comparative results show that PTT is alleviated in SuperPol while it is stimulated by BMH-21 in WT Pol I.

molecular biology↗

bioRxiv2023

Circadian PERIOD complex regulates TC-DSB repair through anchoring to the nuclear envelope

Repair of DNA Double-Strand Breaks (DSBs) produced in transcriptionally active chromatin occurs through a poorly characterized pathway called Transcription-Coupled DSB repair (TC-DSBR). Here, using a screening approach scoring multiple outputs in human cells, we identified proteins from the PERIOD complex, a key module ensuring circadian oscillations, as novel TC-DSBR players. We show that the core PER complex protein PER2 is recruited at TC-DSBs and that it contributes to the targeting of TC-DSBs at the nuclear envelope (NE). At the NE, SUN1 and the Nuclear Pore Complex (NPC) act as docking sites for TC-DSBs and TC-DSB anchoring fosters RAD51 assembly. Impaired DSB localization at the NE results in elevated DSB clustering and translocation rate. In agreement, the circadian clock regulates TC-DSB anchoring to the NE, RAD51 assembly, and DSB clustering. Our study shows that DSB localization to the NPC is a conserved molecular pathway that also occurs in human cells and provides a direct link between the circadian rhythm and the response to DSBs occurring in active genes. This opens new therapeutic strategies for chemotherapies based on drugs that are inducing DSBs in active loci such as topoisomerase poisons.

molecular biology↗

bioRxiv2022

p57Kip2 acts as a transcriptional corepressor to regulate intestinal stem cell fate and proliferation

p57Kip2 is a cyclin/CDK inhibitor and a negative regulator of cell proliferation. Remarkably, p57 is the only CDK inhibitor required for embryonic development and p57 knockout mice display multiple developmental anomalies, including intestinal shortening. Here, we report that p57 regulates intestinal stem cell (ISC) fate and proliferation in a CDK-independent manner during intestinal development. In absence of p57, proliferation in intestinal crypts is markedly increased and genetic labelling experiments revealed an amplification of transit amplifying cells and of Hopx+ ISCs, which are no longer quiescent. On the other hand, Lgr5+ crypt-base columnar (CBC) cells were unaffected. RNA-Seq analyses of Hopx+ ISCs show major changes in gene expression in absence of p57. We found that p57 binds to and inhibits the activity of Ascl2, a transcription factor critical for ISC specification and maintenance, by participating in the recruitment of a corepressor complex to Ascl2 target gene promoters. Thus, our data suggests that during intestinal development, p57 plays a key role in maintaining Hopx+ stem cell quiescence and repressing the ISC phenotype outside of the crypt bottom by inhibiting the transcription factor Ascl2 in a CDK-independent manner.

cell biology↗

bioRxiv2020

Transcriptome profiling of the Olig2-expressing astrocyte subtype reveals their unique molecular signature

Astrocytes are recognized to be a heterogeneous population of cells that differ morphologically, functionally and molecularly. Whether this heterogeneity results from generation of distinct astrocyte cell lineages, each functionally specialized to perform specific tasks, remains an open question. In this study, we used RNA-seq analysis to determine the global transcriptome profile of the Olig2-expressing astrocyte subtype (Olig2-AS), a specific spinal astrocyte subtype which segregates early during development from Olig2 progenitors and differs from other spinal astrocytes by the expression of Olig2. We identified 245 differentially expressed genes. Among them, 135 exhibit higher levels of expression when compared to other populations of spinal astrocytes, indicating that these genes can serve as a uniquefunctional signature of Olig2-AS. Further analysis highlighted, in particular, enrichment in Olig2-AS of a set of genes specialized in regulating synaptic activity. Our work thus reveals that Olig2 progenitors produce a unique astrocyte subtype specialized to perform certain specific functions.

developmental biology↗

bioRxiv2020

BLM-dependent Break-Induced Replication handles DSBs in transcribed chromatin upon impaired RNA:DNA hybrids dissolution.

Transcriptionally active loci are particularly prone to breakage and mounting evidence suggest that DNA Double-Strand Breaks arising in genes are handled by a dedicated repair pathway, Transcription-Coupled DSB Repair (TC-DSBR), that entails R-loops accumulation and dissolution. Here, we uncovered a critical function of the Bloom RecQ DNA helicase (BLM) in TC-DSBR in human cells. BLM is recruited in a transcription dependent-manner at DSBs where it fosters resection, RAD51 binding and accurate Homologous Recombination repair. However, in a R-loop dissolution-deficient background BLM switches from promoting Homologous Recombination to promoting Break-Induced Replication (BIR), which strongly impairs cell viability. Altogether our work unveils a role for BLM in BIR at DSBs in active chromatin, and highlights the toxic potential of RNA:DNA hybrids that accumulate at these transcription-associated DSBs.

molecular biology↗

bioRxiv2020

Circular ANRIL isoforms switch from repressors to activators of p15/CDKN2B expression during RAF1 oncogene-induced senescence

Long non-coding RNAs (ncRNAs) are major regulators of gene expression and cell fate. The INK4 locus encodes the tumour suppressor proteins p15INK4b, p16INK4a and p14ARF required for cell cycle arrest and whose expression increases during senescence. ANRIL is a ncRNA antisense to the p15 gene. In proliferative cells, ANRIL prevents senescence by repressing INK4 genes through the recruitment of Polycomb-group proteins. In models of replicative and RASval12 oncogene-induced senescence (OIS), the expression of ANRIL and Polycomb proteins decreases, thus allowing INK4 derepression. Here, we found in a model of RAF1 OIS that ANRIL expression rather increases, due in particular to an increased stability. This led us to search for circular ANRIL isoforms, as circular RNAs are rather stable species. We found that the expression of two circular ANRIL increases in several OIS models (RAF1, MEK1 and BRAF). In proliferative cells, they repress p15 expression, while in RAF1 OIS, they promote full induction of p15, p16 and p14ARF expression. Further analysis of one of these circular ANRIL shows that it interacts with Polycomb proteins and decreases EZH2 Polycomb protein localization and H3K27me3 at the p15 and p16 promoters, respectively. We propose that changes in the ratio between Polycomb proteins and circular ANRIL isoforms allow these isoforms to switch from repressors of p15 gene to activators of all INK4 genes in RAF1 OIS. Our data reveal that regulation of ANRIL expression depends on the senescence inducer and underline the importance of circular ANRIL in the regulation of INK4 gene expression and senescence.

molecular biology↗