Bernardo Reina-San-Martin

Hypoxia has been shown to shape the humoral immune response within germinal centers, where B cells diversify their receptors through somatic hypermutation (SHM) and class switch recombination (CSR). Both processes are initiated by activation‐induced cytidine deaminase (AID), which deaminates cytosines into uracils in immunoglobulin genes, leading to point mutations during SHM or to double‐stranded DNA breaks during CSR. Although AID's function is well characterized, the mechanisms governing its expression remain ill‐defined. We previously identified the hypoxia‐inducible transcription factor (HIF) transcription complex (comprised of HIF‐1α, HIF‐1β, and the auxiliary subunits HIF‐2α and HIF‐3α) as a regulator of optimal AID expression during CSR. Indeed, loss of HIF‐1α or HIF‐1β delays AID expression and impairs CSR. Here, we examine the contribution of HIF‐2α and HIF‐3α. We show that deficiency in HIF‐2α, but not HIF‐3α, impairs CSR, whereas combined loss of HIF‐1α and HIF‐2α does not exacerbate the defect, indicating that HIF‐1α is the dominant subunit driving this process. Importantly, we demonstrate that hypoxia can induce AID expression, independently of additional stimuli. Collectively, these findings reveal that hypoxia regulates AID expression in a context‐ and time‐dependent manner through HIF activation. This underscores the central role of hypoxic signaling in antibody diversification and suggests broader implications for immune regulation and the onset of B‐cell malignancies.

During immune responses, B cells engaging a cognate antigen are recruited to GCs in secondary lymphoid organs where they will diversify their BCR to generate highly specific and adapted humoral responses. They do so, by inducing the expression of activation‐induced cytidine deaminase (AID), which initiates somatic hypermutation (SHM) and class switch recombination (CSR). AID deaminates cytosines in ss DNA, generating U:G mismatches that are processed to induce ds DNA break intermediates during CSR that result in the expression of a different antibody isotype. Interestingly, hypoxia regions have been reported in GCs and suggesting that hypoxia could modulate the humoral response. Furthermore, hypoxia inducible transcription factor (HIF) can bind to the AID promoter and induce AID expression in a non‐B‐cell setting, suggesting that it might be involved in the transcriptional induction of AID in B cells, hence, regulating SHM and CSR. We, thus, hypothesized that HIF could regulate the efficiency of CSR. Here, we show that the inactivation of both the HIF‐1α and HIF‐1β subunits of the HIF transcription factor in murine CH12 B cells results in defective CSR and that this is due to the suboptimal induction of AID expression.

Membrane contact sites between organelles are organized by protein bridges. Among the components of these contacts, the VAP family comprises ER–anchored proteins, such as MOSPD2, that function as major ER–organelle tethers. MOSPD2 distinguishes itself from the other members of the VAP family by the presence of a CRAL-TRIO domain. In this study, we show that MOSPD2 forms ER–lipid droplet (LD) contacts, thanks to its CRAL-TRIO domain. MOSPD2 ensures the attachment of the ER to LDs through a direct protein–membrane interaction. The attachment mechanism involves an amphipathic helix that has an affinity for lipid packing defects present at the surface of LDs. Remarkably, the absence of MOSPD2 markedly disturbs the assembly of lipid droplets. These data show that MOSPD2, in addition to being a general ER receptor for inter-organelle contacts, possesses an additional tethering activity and is specifically implicated in the biology of LDs via its CRAL-TRIO domain.

Membrane contact sites between organelles are organized by protein bridges. Among the components of these contacts, the VAP family comprises endoplasmic reticulum (ER)-anchored proteins, such as MOSPD2, functioning as major ER-organelle tethers. MOSPD2 distinguishes itself from the other members of the VAP family by the presence of a CRAL-TRIO domain. In this study, we show that MOSPD2 forms ER-LD contacts thanks to its CRAL-TRIO domain. MOSPD2 ensures the attachment of the ER to LDs through a direct protein-membrane interaction involving an amphipathic helix that has an affinity for lipid packing defects present at the surface of LDs. Remarkably, the absence of MOSPD2 markedly disturbs the assembly of lipid droplets. These data show that MOSPD2, in addition to being a general ER receptor for inter-organelle contacts, possesses an additional tethering activity and is specifically implicated in the biology of LDs via its CRAL-TRIO domain.

Centromeric integrity is key for proper chromosome segregation during cell division¹. Centromeres have unique chromatin features that are essential for centromere maintenance². Although they are intrinsically fragile and represent hotspots for chromosomal rearrangements³, little is known about how centromere integrity in response to DNA damage is preserved. DNA repair by homologous recombination requires the presence of the sister chromatid and is suppressed in the G1 phase of the cell cycle⁴. Here we demonstrate that DNA breaks that occur at centromeres in G1 recruit the homologous recombination machinery, despite the absence of a sister chromatid. Mechanistically, we show that the centromere-specific histone H3 variant CENP-A and its chaperone HJURP, together with dimethylation of lysine 4 in histone 3 (H3K4me2), enable a succession of events leading to the licensing of homologous recombination in G1. H3K4me2 promotes DNA-end resection by allowing DNA damage-induced centromeric transcription and increased formation of DNA–RNA hybrids. CENP-A and HJURP interact with the deubiquitinase USP11, enabling formation of the RAD51–BRCA1–BRCA2 complex⁵ and rendering the centromeres accessible to RAD51 recruitment and homologous recombination in G1. Finally, we show that inhibition of homologous recombination in G1 leads to centromeric instability and chromosomal translocations. Our results support a model in which licensing of homologous recombination at centromeric breaks occurs throughout the cell cycle to prevent the activation of mutagenic DNA repair pathways and preserve centromeric integrity.

Efficient humoral responses rely on DNA damage, mutagenesis and error-prone DNA repair. Diversification of B cell receptors through somatic hypermutation and class-switch recombination are initiated by cytidine deamination in DNA mediated by activation-induced cytidine deaminase (AID)¹ and by the subsequent excision of the resulting uracils by uracil DNA glycosylase (UNG) and by mismatch repair proteins^1,2,3. Although uracils arising in DNA are accurately repaired^1,2,3,4, how these pathways are co-opted to generate mutations and double-strand DNA breaks in the context of somatic hypermutation and class-switch recombination is unknown^1,2,3. Here we performed a genome-wide CRISPR–Cas9 knockout screen for genes involved in class-switch recombination and identified FAM72A, a protein that interacts with the nuclear isoform of UNG (UNG2)⁵ and is overexpressed in several cancers⁵. We show that the FAM72A–UNG2 interaction controls the levels of UNG2 and that class-switch recombination is defective in Fam72a^−/− B cells due to the upregulation of UNG2. Moreover, we show that somatic hypermutation is reduced in Fam72a^−/− B cells and that its pattern is skewed upon upregulation of UNG2. Our results are consistent with a model in which FAM72A interacts with UNG2 to control its physiological level by triggering its degradation, regulating the level of uracil excision and thus the balance between error-prone and error-free DNA repair. Our findings have potential implications for tumorigenesis, as reduced levels of UNG2 mediated by overexpression of Fam72a would shift the balance towards mutagenic DNA repair, rendering cells more prone to acquire mutations.

Centrioles are formed by microtubule triplets in a nine-fold symmetric arrangement. In flagellated protists and in animal multiciliated cells, accessory structures tethered to specific triplets render the centrioles rotationally asymmetric, a property that is key to cytoskeletal and cellular organization in these contexts. In contrast, centrioles within the centrosome of animal cells display no conspicuous rotational asymmetry. Here, we uncover rotationally asymmetric molecular features in human centrioles. Using ultrastructure expansion microscopy, we show that LRRCC1, the ortholog of a protein originally characterized in flagellate green algae, associates preferentially to two consecutive triplets in the distal lumen of human centrioles. LRRCC1 partially co-localizes and affects the recruitment of another distal component, C2CD3, which also has an asymmetric localization pattern in the centriole lumen. Together, LRRCC1 and C2CD3 delineate a structure reminiscent of a filamentous density observed by electron microscopy in flagellates, termed the ‘acorn’. Functionally, the depletion of LRRCC1 in human cells induced defects in centriole structure, ciliary assembly and ciliary signaling, supporting that LRRCC1 cooperates with C2CD3 to organizing the distal region of centrioles. Since a mutation in the LRRCC1 gene has been identified in Joubert syndrome patients, this finding is relevant in the context of human ciliopathies. Taken together, our results demonstrate that rotational asymmetry is an ancient property of centrioles that is broadly conserved in human cells. Our work also reveals that asymmetrically localized proteins are key for primary ciliogenesis and ciliary signaling in human cells.

Many cellular processes, ranging from cell division to differentiation, are controlled by nuclear pore complexes (NPCs). However, studying the contributions of individual NPC subunits to these processes in vertebrates has long been impeded by their complexity and the lack of efficient genetic tools. Here, we use genome editing in mouse embryonic stem cells (mESCs) to characterize the role of NPC structural components, focusing on the short arm of the Y-complex that comprises Nup85, Seh1 and Nup43. We show that Seh1 and Nup43, although dispensable in pluripotent mESCs, are required for their normal cell growth rates, their viability upon differentiation and for the maintenance of proper NPC density. mESCs with an N-terminally truncated Nup85 mutation (in which interaction with Seh1 is greatly impaired) feature a similar reduction of NPC density. However, their proliferation and differentiation are unaltered, indicating that it is the integrity of the Y-complex, rather than the number of NPCs, that is critical to ensure these processes.

Efficient humoral responses rely on DNA damage, mutagenesis and error-prone DNA repair. B cell receptor diversification through somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by cytidine deamination in DNA mediated by activation induced cytidine deaminase (AID) ¹ and by the subsequent excision of the resulting uracils by Uracil DNA glycosylase (UNG) and by mismatch repair (MMR) proteins ^2–4 . Although uracils arising in DNA are faithfully repaired ^2–7 , it is not known how these pathways are co-opted to generate mutations and double stranded DNA breaks (DSBs) in the context of SHM and CSR ^2,4,8 . Here we have performed a genome-wide CRISPR/Cas9 knockout screen for genes involved in CSR. The screen identified FAM72A, a protein that interacts with the nuclear isoform of UNG (UNG2) ⁹ and that is overexpressed in several cancers ⁹ . We show that the FAM72A-UNG2 interaction controls the protein levels of UNG2 and that CSR is defective in Fam72a ^−/− B cells due to the specific upregulation of UNG2. Moreover, we show that in Fam72a ^−/− B cells SHM is reduced by 5-fold and that upregulation of UNG2 results in a skewed mutation pattern. Our results are consistent with a model in which FAM72A interacts with UNG2 to control its physiological level by triggering its degradation. Consequently, deficiency in Fam72a leads to supraphysiological levels of UNG2 and enhanced uracil excision, shifting the balance from error-prone to error-free DNA repair. Our findings have potential implications for tumorigenesis, as Fam72a overexpression would lead to reduced UNG2 levels, shifting the balance toward mutagenic DNA repair and rendering cells more prone to acquire mutations.

Many cellular processes, ranging from cell division to differentiation, are controlled by nuclear pore complexes (NPCs). However studying contributions of individual NPC subunits to these processes in vertebrates has long been impeded by their complexity and the lack of efficient genetic tools. Here we use genome editing in mouse embryonic stem cells (mESCs) to characterize the role of NPC structural components, focusing on the short arm of the Y-complex that comprises Nup85, Seh1 and Nup43. We show that Seh1 and Nup43, although dispensable in pluripotent mESCs, are required for their normal cell growth rates, their viability upon differentiation, and for the maintenance of proper NPC density. mESCs with an N-terminally truncated Nup85 mutation (in which interaction with Seh1 is greatly impaired) feature a similar reduction of NPC density. However, their proliferation and differentiation are unaltered, indicating that it is the integrity of the Y-complex, rather than the number of NPCs, that is critical to ensure these processes. Summary statement: Seh1 and Nup43, although dispensable in pluripotent mouse embryonic stem cells, are required for normal cell growth, viability upon differentiation, and maintenance of proper NPC density.