EZH2, JMJD3 and UTX epigenetically regulate hepatic plasticity inducing retro-differentiation and proliferation of liver cells

Modification of histones by lysine methylation plays a role in many biological processes, and it is dynamically regulated by several histone methyltransferases and demethylases. The polycomb repressive complex contains the H3K27 methyltransferase EZH2 and controls dimethylation and trimethylation of H3K27 (H3K27me2/3), which trigger gene suppression. JMJD3 and UTX have been identified as H3K27 demethylases that catalyze the demethylation of H3K27me2/3, which in turns lead to gene transcriptional activation. EZH2, JMJD3 and UTX have been extensively studied for their involvement in development, immune system, neurodegenerative disease, and cancer. However, their role in molecular mechanisms underlying the differentiation process of hepatic cells is yet to be elucidated. Here, we show that EZH2 methyltransferase and JMJD3/UTX demethylases were deregulated during hepatic differentiation of human HepaRG cells resulting in a strong reduction of H3K27 methylation levels. Inhibition of JMJD3 and UTX H3K27 demethylase activity by GSK-J4 epi-drug reverted phenotype of HepaRG DMSO-differentiated cells and human primary hepatocytes, drastically decreasing expression of hepatic markers and inducing cell proliferation. In parallel, inhibition of EZH2 H3K27me3 activity by GSK-126 epi-drug induced upregulation of hepatic markers and downregulated the expression of cell cycle inhibitor genes. To conclude, we demonstrated that modulation of H3K27 methylation by inhibiting methyl-transferase and dimethyl-transferase activity influences the differentiation status of hepatic cells, identifying a possible new role of EZH2, JMJD3 and UTX epi-drugs to modulate hepatic cell plasticity

Insights into the tumor suppressor KCASH2: new functions and mechanisms of regulation

Medulloblastoma (MB) is the most common malignant childhood brain tumor. About 30% of all MBs belong to the I molecular subgroup, characterized by constitutive activation of the Sonic Hedgehog (Hh) pathway. The Hh pathway is involved in several fundamental processes during embryogenesis and in adult life and its deregulation may lead to cerebellar tumorigenesis. Indeed, Hh activity must be maintained via a complex network of activating and repressor signals. One of these repressor signals is KCASH2, belonging to the KCASH family of protein, which acts as negative regulators of the Hedgehog signaling pathway during cerebellar development and differentiation. KCASH proteins possess a well conserved N-terminal BTB domain but an heterogenous C-terminus, suggesting the presence of peculiar functions or different mechanisms of individual KCASHs functional regulation. In order to better characterize the physiologic role and modulation mechanisms of KCASH2, we have searched through a proteomic approach for new KCASH2 interactors, identifying Potassium Channel Tetramerization Domain Containing 15 (KCTD15) and Mitotic Arrest Deficient2-like 1 (MAD2). KCTD15 is able to directly interact with KCASH2, through its BTB/POZ domain. This interaction leads to increase KCASH2 stability which implies a reduction of the Hh pathway activity and a reduction of Hh-dependent MB cells proliferation. Here, we report the identification of KCTD15 as a novel player in the complex network of regulatory proteins which modulate Hh pathway, this could be a promising new target for therapeutic approach against MB. MAD2 is the main player in the spindle assembly complex (SAC), essential for chromosomal stability during cell mitosis, preventing defected cellular divisions that may lead to aneuploidy. Nowadays, no mechanisms have been provided for the MAD2 regulation, although it has been suggested that MAD2 may be degraded following ubiquitination by an unknown E3 ligase. Our work fills this gap, identifying in Cul3-KCASH2 the E3 ligase involved in MAD2 degradation process. Our data suggested that KCASH2 overexpression, affecting MAD2 protein levels, alters SAC formation during cell cycle, promoting mitotic defects that may give rise to chromosomal aberration and aneuploidy. The discovery of a mechanism able to modulate MAD2 protein levels and, indirectly, SAC checkpoint functionality and cell cycle progression may have important implications both in therapeutic approaches directed to the reconstitution of a normal SAC function and in approaches aiming to increase chromosomal instability to a level not sustainable by tumor cells, leading to their death. Finally, we have investigated the KCASH2 transcriptional regulation aimed to discover new potential therapeutics mechanisms for all that tumor types in which KCASH2 is low expressed. We have analyzed its proximal promoter region and performed bioinformatics analyses in order to identify putative transcription factors involved in KCASH2 regulation. Here, we have identified SP1, considered unanimously a hallmark of cancer, as a key transcriptional regulator that is involved in KCASH2 expression modulation in different cancers. The work presented here draw a more complex, although not yet complete, picture of the biological role of KCASH2, unveiling its additional functions as a new putative “guardian” of genomic stability and identifying two novel mechanism of regulation of its expression in KCTD15 and SP1

Aorta Structural Alterations in Term Neonates: The Role of Birth and Maternal Characteristics

Aim. To evaluate the influence of selected maternal and neonatal characteristics on aorta walls in term, appropriately grown-for-gestational age newborns. Methods. Age, parity, previous abortions, weight, height, body mass index before and after delivery, smoking, and history of hypertension, of diabetes, of cardiovascular diseases, and of dyslipidemia were all assessed in seventy mothers. They delivered 34 males and 36 females healthy term newborns who underwent ultrasound evaluation of the anteroposterior infrarenal abdominal aorta diameter (APAO), biochemical profile (glucose, insulin, total cholesterol, HDL and LDL cholesterol, triglycerides, fibrinogen, and D-dimers homeostasis model assessment [HOMAIR]index), and biometric parameters. Results. APAO was related to newborn length (r=+0.36; P=0.001), head circumference (r=+0.37; P=0.001), gestational age (r=+0.40, P=0.0005), HOMA index (r=+0.24; P=0.04), and D-dimers (r=+0.33, P=0.004). Smoke influenced APAO values (odds ratio: 1.80; confidence interval 95%: 1.05–3.30), as well as diabetes during pregnancy (r=+0.42, P=0.0002). Maternal height influenced neonatal APAO (r=+0.47, P=0.00003). Multiple regression analysis outlined neonatal D-dimers as still significantly related to neonatal APAO values. Conclusions. Many maternal and neonatal characteristics could influence aorta structures. Neonatal D-dimers are independently related to APAO

Specialized replication mechanisms maintain genome stability at human centromeres

The high incidence of whole-arm chromosome aneuploidy and translocations in tumors suggests instability of centromeres, unique loci built on repetitive sequences and essential for chromosome separation. The causes behind this fragility and the mechanisms preserving centromere integrity remain elusive. We show that replication stress, hallmark of pre-cancerous lesions, promotes centromeric breakage in mitosis, due to spindle forces and endonuclease activities. Mechanistically, we unveil unique dynamics of the centromeric replisome distinct from the rest of the genome. Locus-specific proteomics identifies specialized DNA replication and repair proteins at centromeres, highlighting them as difficult-to-replicate regions. The translesion synthesis pathway, along with other factors, acts to sustain centromere replication and integrity. Prolonged stress causes centromeric alterations like ruptures and translocations, as observed in ovarian cancer models experiencing replication stress. This study provides unprecedented insights into centromere replication and integrity, proposing mechanistic insights into the origins of centromere alterations leading to abnormal cancerous karyotypes

KCTD15 inhibits the Hedgehog pathway in Medulloblastoma cells by increasing protein levels of the oncosuppressor KCASH2

Medulloblastoma (MB) is the most common malignant childhood brain tumor. About 30% of all MBs belong to the I molecular subgroup, characterized by constitutive activation of the Sonic Hedgehog (Hh) pathway. The Hh pathway is involved in several fundamental processes during embryogenesis and in adult life and its deregulation may lead to cerebellar tumorigenesis. Indeed, Hh activity must be maintained via a complex network of activating and repressor signals. One of these repressor signals is KCASH2, belonging to the KCASH family of protein, which acts as negative regulators of the Hedgehog signaling pathway during cerebellar development and differentiation. KCASH2 leads HDAC1 to degradation, allowing hyperacetylation and inhibition of transcriptional activity of Gli1, the main effector of the Hh pathway. In turn, the KCASH2 loss leads to persistent Hh activity and eventually tumorigenesis. In order to better characterize the physiologic role and modulation mechanisms of KCASH2, we have searched through a proteomic approach for new KCASH2 interactors, identifying Potassium Channel Tetramerization Domain Containing 15 (KCTD15). KCTD15 is able to directly interact with KCASH2, through its BTB/POZ domain. This interaction leads to increase KCASH2 stability which implies a reduction of the Hh pathway activity and a reduction of Hh-dependent MB cells proliferation. Here we report the identification of KCTD15 as a novel player in the complex network of regulatory proteins, which modulate Hh pathway, this could be a promising new target for therapeutic approach against MB