Gene-specific RNA homeostasis revealed by perturbation of coactivator complexes.

Transcript buffering entails the reciprocal modulation of mRNA synthesis and degradation rates to maintain stable RNA levels under varying cellular conditions. Current research supports a global, non-sequence-specific connection between mRNA synthesis and degradation, but the underlying mechanisms are still unclear. In this study, we investigated changes in RNA metabolism following acute depletion of TIP60/KAT5, the acetyltransferase subunit of the NuA4 transcriptional coactivator complex, in mouse embryonic stem cells.

Optimal AID expression and efficient immunoglobulin class switch recombination are dependent on the hypoxia-inducible factor.

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.

Heterochromatic repeat clustering imposes a physical barrier on homologous recombination to prevent chromosomal translocations.

Mouse pericentromeric DNA is composed of tandem major satellite repeats, which are heterochromatinized and cluster together to form chromocenters. These clusters are refractory to DNA repair through homologous recombination (HR). The mechanisms by which pericentromeric heterochromatin imposes a barrier on HR and the implications of repeat clustering are unknown.

MOSPD2 is an endoplasmic reticulum-lipid droplet tether functioning in LD homeostasis.

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.

Evolutionary conservation of centriole rotational asymmetry in the human centrosome.

Centrioles are formed by microtubule triplets in a ninefold symmetric arrangement. In flagellated protists and 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.

Activation of homologous recombination in G1 preserves centromeric integrity.

Centromeric integrity is key for proper chromosome segregation during cell division. Centromeres have unique chromatin features that are essential for centromere maintenance. Although they are intrinsically fragile and represent hotspots for chromosomal rearrangements, little is known about how centromere integrity in response to DNA damage is preserved.

Fam72a enforces error-prone DNA repair during antibody diversification.

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) and by the subsequent excision of the resulting uracils by uracil DNA glycosylase (UNG) and by mismatch repair proteins. Although uracils arising in DNA are accurately repaired, 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.

The related coactivator complexes SAGA and ATAC control embryonic stem cell self-renewal through acetyltransferase-independent mechanisms.

SAGA (Spt-Ada-Gcn5 acetyltransferase) and ATAC (Ada-two-A-containing) are two related coactivator complexes, sharing the same histone acetyltransferase (HAT) subunit. The HAT activities of SAGA and ATAC are required for metazoan development, but the role of these complexes in RNA polymerase II transcription is less understood. To determine whether SAGA and ATAC have redundant or specific functions, we compare the effects of HAT inactivation in each complex with that of inactivation of either SAGA or ATAC core subunits in mouse embryonic stem cells (ESCs).

Integrity of the short arm of the nuclear pore Y-complex is required for mouse embryonic stem cell growth and differentiation.

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.

AHNAK controls 53BP1-mediated p53 response by restraining 53BP1 oligomerization and phase separation.

p53-binding protein 1 (53BP1) regulates both the DNA damage response and p53 signaling. Although 53BP1's function is well established in DNA double-strand break repair, how its role in p53 signaling is modulated remains poorly understood. Here, we identify the scaffolding protein AHNAK as a G1 phase-enriched interactor of 53BP1.

Loss of ZBTB24 impairs nonhomologous end-joining and class-switch recombination in patients with ICF syndrome.

The autosomal recessive immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is a genetically heterogeneous disorder. Despite the identification of the underlying gene defects, it is unclear how mutations in any of the four known ICF genes cause a primary immunodeficiency. Here we demonstrate that loss of ZBTB24 in B cells from mice and ICF2 patients affects nonhomologous end-joining (NHEJ) during immunoglobulin class-switch recombination and consequently impairs immunoglobulin production and isotype balance.

hVFL3/CCDC61 is a component of mother centriole subdistal appendages required for centrosome cohesion and positioning.

Background: The centrosome regulates cell spatial organisation by controlling the architecture of the microtubule (MT) cytoskeleton. Conversely, the position of the centrosome within the cell depends on cytoskeletal networks it helps organizing. In mammalian cells, centrosome positioning involves a population of MT stably anchored at centrioles, the core components of the centrosome.

COSPLAY: An expandable toolbox for combinatorial and swift generation of expression plasmids in yeast.

A large number of genetic studies in yeast rely on the use of expression vectors. To facilitate the experimental approach of these studies, several collections of expression vectors have been generated (YXplac, pRS series, etc.).

Allele-Specific CRISPR/Cas9 Correction of a Heterozygous DNM2 Mutation Rescues Centronuclear Myopathy Cell Phenotypes.

Genome editing with the CRISPR/Cas9 technology has emerged recently as a potential strategy for therapy in genetic diseases. For dominant mutations linked to gain-of-function effects, allele-specific correction may be the most suitable approach. In this study, we tested allele-specific inactivation or correction of a heterozygous mutation in the Dynamin 2 (DNM2) gene that causes the autosomal dominant form of centronuclear myopathies (CNMs), a rare muscle disorder belonging to the large group of congenital myopathies.

PARP3, a new therapeutic target to alter Rictor/mTORC2 signaling and tumor progression in BRCA1-associated cancers.

PARP3 has been shown to be a key driver of TGFβ-induced epithelial-to-mesenchymal transition (EMT) and stemness in breast cancer cells, emerging as an attractive therapeutic target. Nevertheless, the therapeutic value of PARP3 inhibition has not yet been assessed. Here we investigated the impact of the absence of PARP3 or its inhibition on the tumorigenicity of BRCA1-proficient versus BRCA1-deficient breast cancer cell lines, focusing on the triple-negative breast cancer subtype (TNBC).

Nup133 Is Required for Proper Nuclear Pore Basket Assembly and Dynamics in Embryonic Stem Cells.

Nup133 belongs to the Y-complex, a key component of the nuclear pore complex (NPC) scaffold. Studies on a null mutation in mice previously revealed that Nup133 is essential for embryonic development but not for mouse embryonic stem cell (mESC) proliferation. Using single-pore detection and average NE-fluorescence intensity, we find that Nup133 is dispensable for interphase and postmitotic NPC scaffold assembly in pluripotent mESCs.

Trib1 Is Overexpressed in Systemic Lupus Erythematosus, While It Regulates Immunoglobulin Production in Murine B Cells.

Systemic lupus erythematosus (SLE) is a severe and heterogeneous autoimmune disease with a complex genetic etiology, characterized by the production of various pathogenic autoantibodies, which participate in end-organ damages. The majority of human SLE occurs in adults as a polygenic disease, and clinical flares interspersed with silent phases of various lengths characterize the usual evolution of the disease in time. Trying to understand the mechanism of the different phenotypic traits of the disease, and considering the central role of B cells in SLE, we previously performed a detailed wide analysis of gene expression variation in B cells from quiescent SLE patients.

Imaging of native transcription factors and histone phosphorylation at high resolution in live cells.

Fluorescent labeling of endogenous proteins for live-cell imaging without exogenous expression of tagged proteins or genetic manipulations has not been routinely possible. We describe a simple versatile antibody-based imaging approach (VANIMA) for the precise localization and tracking of endogenous nuclear factors. Our protocol can be implemented in every laboratory allowing the efficient and nonharmful delivery of organic dye-conjugated antibodies, or antibody fragments, into different metazoan cell types.

Med1 deficiency alters the somatic hypermutation spectrum in murine B cells.

The Mediator complex is known to orchestrate transcription. Here we show that B cell conditional deficient mice for the Med1 subunit display robust somatic hypermutation. Nevertheless, the mutation frequency at A residues is decreased and the expected A/T ratio is abolished, implicating Mediator in the second phase of somatic hypermutation.

DNA end resection requires constitutive sumoylation of CtIP by CBX4.

DNA breaks are complex DNA lesions that can be repaired by two alternative mechanisms: non-homologous end-joining and homologous recombination. The decision between them depends on the activation of the DNA resection machinery, which blocks non-homologous end-joining and stimulates recombination. On the other hand, post-translational modifications play a critical role in DNA repair.

Robust immunoglobulin class switch recombination and end joining in Parp9-deficient mice.

To mount highly specific and adapted immune responses, B lymphocytes assemble and diversify their antibody repertoire through mechanisms involving the formation of programmed DNA damage. Immunoglobulin class switch recombination (CSR) is triggered by DNA lesions induced by activation-induced cytidine deaminase, which are processed to double-stranded DNA break (DSB) intermediates. These DSBs activate the cellular DNA damage response and enroll numerous DNA repair factors, involving poly(ADP-ribose) polymerases Parp1, Parp2, and Parp3 to promote appropriate DNA repair and efficient long-range recombination.