Giacomo Buscemi

Chromatin is a dynamic cellular structure basically constituted by nucleosomes, which consist of a DNA sequence wrapped around an octameric histones core. Histone synthesis and transport, nucleosome formation and proper chromatin assembly is an ordered and stepwise process guided by histone chaperones. Several families of histone chaperones have been identified and one of them is the nucleosome assembly protein (NAP) superfamily. Members of this family have been involved not only in chromatin constitution and regulation but also in several other cellular processes, such as nucleocytoplasmic shuttling, DNA replication, transcription and cell-cycle regulation. Testis specific protein Y-like 2 (TSPYL2) is a peculiar member of the NAP superfamily of histone chaperone. This protein has been initially isolated as a nuclear antigen in patients affected by discoid lupus erythematosus and as a TGF-β target. Its ability to bind histones has been demonstrated. In addition, TSPYL2 has been reported to regulate transcription, cell-cycle progression and the DNA-damage response, independently of its role in chromatin organization. In accordance with its multiple functions, defects in TSPYL2 have been associated with different diseases, mainly cancer and neurodevelopmental abnormalities. In this review we summarize and discuss the multiple cellular functions of TSPYL2, pointing out new and unexpected aspects like a sex-related activity and their relationship with different diseases.

DNA double-strand breaks (DSBs) can be induced by cellular byproducts or genotoxic agents. Improper processing of these lesions leads to increased genome instability, which constitutes a hallmark of pathological conditions and fuels carcinogenesis. DSBs are primarily repaired by homologous recombination (HR) and non-homologous end joining (NHEJ) and the proper balance between these two pathways is finely modulated by specific molecular events. Here, we report that the histone chaperone DAXX plays a fundamental role in the response to DSBs. Indeed, in human cells, DSBs induce ATM/ATR-dependent phosphorylation of DAXX on serine 424 and 712 and promote its binding to chromatin and the deposition of the histone variant H3.3 in proximity to DNA breaks. Enrichment of H3.3 at DSBs promotes 53BP1 recruitment to these lesions and the repair of DNA breaks by HR pathways. Moreover, H3.3-specific post translational modifications, particularly K36 tri-methylation, play a key role in these processes. Altogether, these findings indicate that DAXX and H3.3 mutations may contribute to tumorigenesis-enhancing genome instability.

Epidemiological studies have revealed significant sex differences in the incidence of tumors unrelated to reproductive functions, with females demonstrating a lesser risk and a better response to therapy than males. However, the reasons for these disparities are still unknown and cancer therapies are generally sex-unbiased. The tumor-suppressor protein p53 is a transcription factor that can activate the expression of multiple target genes mainly involved in the maintenance of genome stability and tumor prevention. It is encoded by TP53, which is the most-frequently mutated gene in human cancers and therefore constitutes an attractive target for therapy. Recently, evidence of sex differences has emerged in both p53 regulations and functions, possibly providing novel opportunities for personalized cancer medicine. Here, we will review and discuss current knowledge about sexual disparities in p53 pathways, their role in tumorigenesis and cancer progression, and their importance in the therapy choice process, finally highlighting the importance of considering sex contribution in both basic research and clinical practice.

Senataxin is an evolutionarily conserved DNA/RNA helicase, whose dysfunctions are linked to neurodegeneration and cancer. A main activity of this protein is the removal of R-loops, which are nucleic acid structures capable to promote DNA damage and replication stress. Here we found that Senataxin deficiency causes the release of damaged DNA into extranuclear bodies, called micronuclei, triggering the massive recruitment of cGAS, the apical sensor of the innate immunity pathway, and the downstream stimulation of interferon genes. Such cGAS-positive micronuclei are characterized by defective membrane envelope and are particularly abundant in cycling cells lacking Senataxin, but not after exposure to a DNA breaking agent or in absence of the tumor suppressor BRCA1 protein, a partner of Senataxin in R-loop removal. Micronuclei with a discontinuous membrane are normally cleared by autophagy, a process that we show is impaired in Senataxin-deficient cells. The formation of Senataxin-dependent inflamed micronuclei is promoted by the persistence of nuclear R-loops stimulated by the DSIF transcription elongation complex and the engagement of EXO1 nuclease activity on nuclear DNA. Coherently, high levels of EXO1 result in poor prognosis in a subset of tumors lacking Senataxin expression. Hence, R-loop homeostasis impairment, together with autophagy failure and unscheduled EXO1 activity, elicits innate immune response through micronuclei formation in cells lacking Senataxin.

Protein acetylation and deacetylation events are finely regulated by lysine-acetyl-transferases and lysine-deacetylases and constitute an important tool for the activation or inhibition of specific cellular pathways. One of the most important lysine-acetyl-transferases is p300, which is involved in the regulation of gene expression, cell growth, DNA repair, differentiation, apoptosis, and tumorigenesis. A well-known target of p300 is constituted by the tumor suppressor protein p53, which plays a critical role in the maintenance of genomic stability and whose activity is known to be controlled by post-translational modifications, among which acetylation. p300 activity toward p53 is negatively regulated by the NAD-dependent deacetylase SIRT1, which deacetylates p53 preventing its transcriptional activation and the induction of p53-dependent apoptosis. However, the mechanisms responsible for p53 regulation by p300 and SIRT1 are still poorly understood. Here we identify the nucleosome assembly protein TSPY-Like 2 (TSPYL2, also known as TSPX, DENTT, and CDA1) as a novel regulator of SIRT1 and p300 function. We demonstrate that, upon DNA damage, TSPYL2 inhibits SIRT1, disrupting its association with target proteins, and promotes p300 acetylation and activation, finally stimulating p53 acetylation and p53-dependent cell death. Indeed, in response to DNA damage, cells silenced for TSPYL2 were found to be defective in p53 activation and apoptosis induction and these events were shown to be dependent on SIRT1 and p300 function. Collectively, our results shed new light on the regulation of p53 acetylation and activation and reveal a novel TSPYL2 function with important implications in cancerogenesis.

Human CCAR2 has recently emerged as having a pivotal role in the DNA damage response, promoting apoptosis and repair of heterochromatic DNA breaks. However, less is known about the function of CCAR2 in tumor formation and cancer progression. Here, we demonstrate, for the first time, that CCAR2 loss inhibits the proliferation of cancer cells, but preserves the growth of normal cells. Investigating the mechanisms responsible for this differential effect, we found that CCAR2 depletion specifically impairs the activation of AKT pathway in cancer cells, but not in normal cells, by reducing AKT phosphorylation on Ser473. This effect is achieved through the transcriptional upregulation ofTRB3gene and accumulation of TRB3 protein, which then binds to and inhibits the phosphorylation and activation of AKT. The defective activation of AKT finally results in reduced GSK3βphosphorylation, prevention of G1/S transition and inhibition of cancer cell growth. These results establish an important role for CCAR2 in cancer cells proliferation and could shed new light on novel therapeutic strategies against cancer, devoid of detrimental side effects.