Biology subjects

Puig Lombardi, E.

Publications and source records attributed to Puig Lombardi, E..

3 recordsLinked to original sources

bioRxiv2020

Topologically associating domain boundaries are enriched in early firing origins and restrict replication fork progression

Topologically associating domains (TADs) are units of the genome architecture defined by binding sites for the CTCF transcription factor and cohesin-mediated loop extrusion. Genomic regions containing DNA replication initiation sites have been mapped in the proximity of TAD boundaries. However, the factors that determine this positioning have not been identified. Moreover, the impact of TADs on the directionality of replication fork progression remains unknown. Here we use EdU-seq technology to map origin firing sites at 10 kb resolution and to monitor replication fork progression after restart from hydroxyurea arrest. We show that origins firing in early/mid S-phase within TAD boundaries map to two distinct peaks flanking the centre of the boundary, which is occupied by CTCF and cohesin. When transcription is inhibited chemically or deregulated by oncogene overexpression, replication origins become repositioned to the centre of the TAD. Furthermore, we demonstrate the strikingly asymmetric fork progression initiating from origins located within TAD boundaries. Divergent CTCF binding sites and neighbouring TADs with different replication timing (RT) cause fork stalling in regions external to the TAD. Thus, our work assigns for the first time a role to transcription within TAD boundaries in promoting replication origin firing and demonstrates how genomic regions adjacent to the TAD boundaries could restrict replication progression.

genomics↗

bioRxiv2019

G-quadruplexes sequester free heme in living cells

Heme is an essential cofactor for many enzymes, but free heme is toxic and its levels are tightly regulated. G-quadruplexes bind heme avidly in vitro, raising the possibility that they may sequester heme in vivo. If so, then treatment that displaces heme from quadruplexes is predicted to induce expression of genes involved in iron and heme homeostasis. Here we show that PhenDC3, a G-quadruplex ligand structurally unrelated to heme, displaces quadruplex-bound heme in vitro and alters transcription in cultured human cells, up-regulating genes that support heme degradation and iron homeostasis, and most strikingly causing a 30-fold induction of heme oxidase 1, the key enzyme in heme degradation. We propose that G-quadruplexes sequester heme to protect cells from the pathophysiological consequences of free heme. This identifies a new function for G-quadruplexes and a new mechanism for protection of cells from heme.

genomics↗

bioRxiv2018

BRCA2 abrogation triggers innate immune responses potentiated by treatment with PARP inhibitors

Heterozygous germline mutations in BRCA2 predispose to breast and ovarian cancer. Contrary to non-cancerous cells, where BRCA2 deletion causes cell cycle arrest or cell death, BRCA2 inactivation in tumors is associated with uncontrolled cell proliferation. We set out to investigate this conundrum by exploring modalities of cell adaptation to loss of BRCA2 and focused on genome-wide transcriptome alterations. Human cells in which BRCA2 expression was inhibited using a doxycycline (DOX)-inducible shRNA for 4 or 28 days were subjected to RNA-seq analyses. Gene sets differentially expressed in BRCA2-deficient versus -proficient cells revealed a biphasic response to BRCA2 abrogation. The early, acute response consisted of downregulation of genes involved in cell cycle progression, DNA replication and repair and was associated with cell cycle arrest in G1. Surprisingly, the late, chronic response consisted predominantly of upregulation of innate immune response genes controlled by interferon. Activation of the cGAS-STING pathway detected in these cells further substantiated the concept that long-term BRCA2 abrogation triggers cell-intrinsic immune signaling. Importantly, we found that treatment with PARP inhibitors stimulated the interferon response in cells and tumors lacking BRCA2. We propose that PARP inhibitors may suppress growth of BRCA2-deficient cells and tumors, in part, by activating interferon signaling.

cancer biology↗