The ASCIZ-DYNLL1 axis promotes 53BP1-dependent non-homologous end joining and PARP inhibitor sensitivity

53BP1 controls a specialized non-homologous end joining (NHEJ) pathway that is essential for adaptive immunity, yet oncogenic in BRCA1 mutant cancers. Intra-chromosomal DNA double-strand break (DSB) joining events during immunoglobulin class switch recombination (CSR) require 53BP1. However, in BRCA1 mutant cells, 53BP1 blocks homologous recombination (HR) and promotes toxic NHEJ, resulting in genomic instability. Here, we identify the protein dimerization hub—DYNLL1—as an organizer of multimeric 53BP1 complexes. DYNLL1 binding stimulates 53BP1 oligomerization, and promotes 53BP1’s recruitment to, and interaction with, DSB-associated chromatin. Consequently, DYNLL1 regulates 53BP1-dependent NHEJ: CSR is compromised upon deletion of Dynll1 or its transcriptional regulator Asciz, or by mutation of DYNLL1 binding motifs in 53BP1; furthermore, Brca1 mutant cells and tumours are rendered resistant to poly-ADP ribose polymerase (PARP) inhibitor treatments upon deletion of Dynll1 or Asciz. Thus, our results reveal a mechanism that regulates 53BP1-dependent NHEJ and the therapeutic response of BRCA1-deficient cancers

CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response

Here, we report that genome editing by CRISPR-Cas9 induces a p53-mediated DNA damage response and cell cycle arrest in immortalized human retinal pigment epithelial cells, leading to a selection against cells with a functional p53 pathway. Inhibition of p53 prevents the damage response and increases the rate of homologous recombination from a donor template. These results suggest that p53 inhibition may improve the efficiency of genome editing of untransformed cells and that p53 function should be monitored when developing cell-based therapies utilizing CRISPR-Cas9.Peer reviewe

Rifampicin resistance conferring mutations among Mycobacterium tuberculosis strains in Rwanda

Background: The World Health Organization-endorsed phenotypic and genotypic drug-susceptibility testing (gDST/pDST) assays for the detection of rifampicin-resistant (RR) tuberculosis (TB), may miss some clinically relevant rpoB mutants, including borderline mutations and mutations outside the gDST-targeted hotspot region. Sequencing of the full rpoB gene is considered the reference standard for rifampicin DST but is rarely available in RR-TB endemic settings and when done indirectly on cultured isolates may not represent the full spectrum of mutations. Hence, in most such settings, the diversity and trends of rpoB mutations remain largely unknown. Methods: This retrospective study included rpoB sequence data from a longitudinal collection of RR-TB isolates in Rwanda across 30 years (1991–2021). Results: Of 540 successfully sequenced isolates initially reported as RR-TB, 419 (77.6%) had a confirmed RR conferring mutation. The Ser450 Leu mutation was predominant throughout the study period. The Val170Phe mutation, not covered by rapid gDST assays, was observed in only four patients, three of whom were diagnosed by pDST. Along with the transition from pDST to rapid gDST, borderline RR-associated mutations, particularly Asp435Tyr, were detected more frequently. Borderline mutants were not associated with HIV status but presented lower odds of having rpoA-C compensatory mutations than other resistance-conferring mutations. Conclusion: Our analysis showed changes in the diversity of RR-TB conferring mutations throughout the study period that coincided with the switch of diagnostic tools to rapid gDST. The study highlights the importance of rapid molecular diagnostics reducing phenotypic bias in the detection of borderline rpoB mutations while vigilance for non-rifampicin resistance determinant region mutations is justified in any setting

BRD4-mediated repression of p53 is a target for combination therapy in AML

Acute myeloid leukemia (AML) is a typically lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually, most AML retain wild-type TP53, encoding the pro-apoptotic tumor suppressor p53. MDM2 inhibitors (MDM2i), which activate wild-type p53, and BET inhibitors (BETi), targeting the BET-family co-activator BRD4, both show encouraging pre-clinical activity, but limited clinical activity as single agents. Here, we report enhanced toxicity of combined MDM2i and BETi towards AML cell lines, primary human blasts and mouse models, resulting from BETi’s ability to evict an unexpected repressive form of BRD4 from p53 target genes, and hence potentiate MDM2i-induced p53 activation. These results indicate that wild-type TP53 and a transcriptional repressor function of BRD4 together represent a potential broad-spectrum synthetic therapeutic vulnerability for AML

Molecular regulation of p53-dependent tumour suppressor responses by the p53 binding protein 1

The tumour suppressor p53 binding protein 1 (53BP1), a fundamental node in DNA double strand break (DSB) repair, was identified as a p53-interacting protein over two decades ago. However, its contribution to p53-dependent responses has remained largely enigmatic. Here, using a combination of detailed structure-function approaches and in vivo analyses I aim to unravel this aspect of 53BP1 functionality. I showed 53BP1 to enhance genome-wide p53-dependent transactivation events in response to multiple stress stimuli. Oligomerised 53BP1 relies on the tandem BRCT domain, dispensable for 53BP1-driven DSB repair, to bridge dual interactions with p53 and the ubiquitin specific protease 28 (USP28). These interactions are both essential for 53BP1-dependent modulation of p53 functionality. Indeed, the cooperation between 53BP1 and USP28 is required for proficient p53-dependent G1/S checkpoint and senescence responses. Mechanistically, the action of the USP28-53BP1 complex involves differential ubiquitination events that ultimately stimulate p53’s ability to bind the responsive elements (RE) in its target genes. Furthermore, I demonstrated 53BP1-driven p53 modulation to function independently of 53BP1’s well-described DSB repair roles, and be separable by specific point mutations within 53BP1 architecture. Translation to in vivo mouse models revealed 53BP1-driven DSB repair to be responsible for 53BP1’s role in the physiology of the immune system, while functional 53BP1-p53 connections result in effective quality control of chromosomal dosage upon defective mitotic division. Collectively, my findings define 53BP1 as an integral node in genome stability control, driving efficient DSB repair and mitotic surveillance mechanisms.</p

Molecular regulation of p53-dependent tumour suppressor responses by the p53 binding protein 1

The tumour suppressor p53 binding protein 1 (53BP1), a fundamental node in DNA double strand break (DSB) repair, was identified as a p53-interacting protein over two decades ago. However, its contribution to p53-dependent responses has remained largely enigmatic. Here, using a combination of detailed structure-function approaches and in vivo analyses I aim to unravel this aspect of 53BP1 functionality. I showed 53BP1 to enhance genome-wide p53-dependent transactivation events in response to multiple stress stimuli. Oligomerised 53BP1 relies on the tandem BRCT domain, dispensable for 53BP1-driven DSB repair, to bridge dual interactions with p53 and the ubiquitin specific protease 28 (USP28). These interactions are both essential for 53BP1-dependent modulation of p53 functionality. Indeed, the cooperation between 53BP1 and USP28 is required for proficient p53-dependent G1/S checkpoint and senescence responses. Mechanistically, the action of the USP28-53BP1 complex involves differential ubiquitination events that ultimately stimulate p53âs ability to bind the responsive elements (RE) in its target genes. Furthermore, I demonstrated 53BP1-driven p53 modulation to function independently of 53BP1âs well-described DSB repair roles, and be separable by specific point mutations within 53BP1 architecture. Translation to in vivo mouse models revealed 53BP1-driven DSB repair to be responsible for 53BP1âs role in the physiology of the immune system, while functional 53BP1-p53 connections result in effective quality control of chromosomal dosage upon defective mitotic division. Collectively, my findings define 53BP1 as an integral node in genome stability control, driving efficient DSB repair and mitotic surveillance mechanisms.</p

53BP1 integrates DNA repair and p53-dependent cell fate decisions via distinct mechanisms

The tumor suppressor protein 53BP1, a pivotal regulator of DNA double-strand break (DSB) repair, was first identified as a p53-interacting protein over two decades ago. However, its direct contributions to p53-dependent cellular activities remain undefined. Here, we reveal that 53BP1 stimulates genome-wide p53-dependent gene transactivation and repression events in response to ionizing radiation (IR) and synthetic p53 activation. 53BP1-dependent p53 modulation requires both auto-oligomerization and tandem-BRCT domain-mediated bivalent interactions with p53 and the ubiquitin-specific protease USP28. Loss of these activities results in inefficient p53-dependent cell-cycle checkpoint and exit responses. Furthermore, we demonstrate 53BP1-USP28 cooperation to be essential for normal p53-promoter element interactions and gene transactivation-associated events, yet dispensable for 53BP1-dependent DSB repair regulation. Collectively, our data provide a mechanistic explanation for 53BP1-p53 cooperation in controlling anti-tumorigenic cell-fate decisions and reveal these activities to be distinct and separable from 53BP1’s regulation of DNA double-strand break repair pathway choice

53BP1 integrates DNA repair and p53-dependent cell fate decisions via distinct mechanisms

The tumor suppressor protein 53BP1, a pivotal regulator of DNA double-strand break (DSB) repair, was first identified as a p53-interacting protein over two decades ago. However, its direct contributions to p53-dependent cellular activities remain undefined. Here, we reveal that 53BP1 stimulates genome-wide p53-dependent gene transactivation and repression events in response to ionizing radiation (IR) and synthetic p53 activation. 53BP1-dependent p53 modulation requires both auto-oligomerization and tandem-BRCT domain-mediated bivalent interactions with p53 and the ubiquitin-specific protease USP28. Loss of these activities results in inefficient p53-dependent cell-cycle checkpoint and exit responses. Furthermore, we demonstrate 53BP1-USP28 cooperation to be essential for normal p53-promoter element interactions and gene transactivation-associated events, yet dispensable for 53BP1-dependent DSB repair regulation. Collectively, our data provide a mechanistic explanation for 53BP1-p53 cooperation in controlling anti-tumorigenic cell-fate decisions and reveal these activities to be distinct and separable from 53BP1’s regulation of DNA double-strand break repair pathway choice

MCM8IP activates the MCM8-9 helicase to promote DNA synthesis and homologous recombination upon DNA damage

Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maintain genomic stability. Here we characterize C17orf53/MCM8IP, an OB-fold containing protein that binds ssDNA, as a DNA repair factor involved in HR. MCM8IP-deficient cells exhibit HR defects, especially in long-tract gene conversion, occurring downstream of RAD51 loading, consistent with a role for MCM8IP in HR-dependent DNA synthesis. Moreover, loss of MCM8IP confers cellular sensitivity to crosslinking agents and PARP inhibition. Importantly, we report that MCM8IP directly associates with MCM8-9, a helicase complex mutated in primary ovarian insufficiency, and RPA1. We additionally show that the interactions of MCM8IP with MCM8-9 and RPA facilitate HR and promote replication fork progression and cellular viability in response to treatment with crosslinking agents. Mechanistically, MCM8IP stimulates the helicase activity of MCM8-9. Collectively, our work identifies MCM8IP as a key regulator of MCM8-9-dependent DNA synthesis during DNA recombination and replication.ISSN:2041-172