Search

p57 Kip2 is a member of the cyclin-dependent kinase (CDK) Interacting Protein/Kinase Inhibitory Protein (CIP/Kip) family that also includes p21 Cip1/WAF1 and p27 Kip1 . Different from its siblings, few data are available about the p57 Kip2 protein, especially in humans. Structurally, p57 Kip2 is an intrinsically unstructured protein, a characteristic that confers functional flexibility with multiple transient interactions influencing the metabolism and roles of the protein. Being an IUP, its localization, stability, and binding to functional partners might be strongly modulated by post-translational modifications, especially phosphorylation. In this work, we investigated by two-dimensional analysis the phosphorylation pattern of p57 Kip2 in different cellular models, revealing how the human protein appears to be extensively phosphorylated, compared to p21 Cip1/WAF1 and p27 Kip1 . We further observed clear differences in the phosphoisoforms distributed in the cytosolic and nuclear compartments in asynchronous and synchronized cells. Particularly, the unmodified form is detectable only in the nucleus, while the more acidic forms are present in the cytoplasm. Most importantly, we found that the phosphorylation state of p57 Kip2 influences the binding with some p57 Kip2 partners, such as CDKs, LIMK1 and CRM1. Thus, it is necessary to completely identify the phosphorylated residues of the protein to fully unravel the roles of this CIP/Kip protein, which are still partially identified.

Rett syndrome (RTT, online MIM 312750) is a devastating neurodevelopmental disorder characterized by motor and cognitive disabilities. It is mainly caused by pathogenetic variants in the X-linked MECP2 gene, encoding an epigenetic factor crucial for brain functioning. Despite intensive studies, the RTT pathogenetic mechanism remains to be fully elucidated. Impaired vascular function has been previously reported in RTT mouse models; however, whether an altered brain vascular homeostasis and the subsequent blood-brain barrier (BBB) breakdown occur in RTT and contribute to the disease-related cognitive impairment is still unknown. Interestingly, in symptomatic Mecp2 -null ( Mecp2 -/y , Mecp2 tm1.1Bird ) mice, we found enhanced BBB permeability associated with an aberrant expression of the tight junction proteins Ocln and Cldn -5 in different brain areas, in terms of both transcript and protein levels. Additionally, Mecp2 -null mice showed an altered expression of different genes encoding factors with a role in the BBB structure and function, such as Cldn3 , Cldn12 , Mpdz , Jam2 , and Aqp4 . With this study, we provide the first evidence of impaired BBB integrity in RTT and highlight a potential new molecular hallmark of the disease that might open new perspectives for the setting-up of novel therapeutic strategies.

It is now definitively established that a large part of the human genome is transcribed. However, only a scarce percentage of the transcriptome (about 1.2%) consists of RNAs that are translated into proteins, while the large majority of transcripts include a variety of RNA families with different dimensions and functions. Within this heterogeneous RNA world, a significant fraction consists of sequences with a length of more than 200 bases that form the so-called long non-coding RNA family. The functions of long non-coding RNAs range from the regulation of gene transcription to the changes in DNA topology and nucleosome modification and structural organization, to paraspeckle formation and cellular organelles maturation. This review is focused on the role of long non-coding RNAs as regulators of cyclin-dependent kinase inhibitors' (CDKIs) levels and activities. Cyclin-dependent kinases are enzymes necessary for the tuned progression of the cell division cycle. The control of their activity takes place at various levels. Among these, interaction with CDKIs is a vital mechanism. Through CDKI modulation, long non-coding RNAs implement control over cellular physiology and are associated with numerous pathologies. However, although there are robust data in the literature, the role of long non-coding RNAs in the modulation of CDKIs appears to still be underestimated, as well as their importance in cell proliferation control.

The Cyclin-dependent kinase (CDK) regulator p27 Kip1 is a gatekeeper of G1/S transition. It also regulates G2/M progression and cytokinesis completion, via CDK-dependent or -independent mechanisms. Recently, other important p27 Kip1 functions have been described, including the regulation of cell motility and migration, the control of cell differentiation program and the activation of apoptosis/autophagy. Several factors modulate p27 Kip1 activities, including its level, cellular localization and post-translational modifications. As a matter of fact, the protein is phosphorylated, ubiquitinated, SUMOylated, O -linked N-acetylglicosylated and acetylated on different residues. p27 Kip1 belongs to the family of the intrinsically unstructured proteins and thus it is endowed with a large flexibility and numerous interactors, only partially identified. In this review, we look at p27 Kip1 properties and ascribe part of its heterogeneous functions to the ability to act as an anchor or scaffold capable to participate in the construction of different platforms for modulating cell response to extracellular signals and allowing adaptation to environmental changes.

p57 Kip2 protein is a member of the CIP/Kip family, mainly localized in the nucleus where it exerts its Cyclin/CDKs inhibitory function. In addition, the protein plays key roles in embryogenesis, differentiation, and carcinogenesis depending on its cellular localization and interactors. Mutations of CDKN1C , the gene encoding human p57 Kip2 , result in the development of different genetic diseases, including Beckwith-Wiedemann, IMAGe and Silver-Russell syndromes. We investigated a specific Beckwith-Wiedemann associated CDKN1C change (c.946 C>T) that results in the substitution of the C-terminal amino acid (arginine 316) with a tryptophan (R316W-p57 Kip2 ). We found a clear redistribution of R316W-p57 Kip2 , in that while the wild-type p57 Kip2 mostly occurs in the nucleus, the mutant form is also distributed in the cytoplasm. Transfection of two expression constructs encoding the p57 Kip2 N- and C-terminal domain, respectively, allows the mapping of the nuclear localization signal(s) (NLSs) between residues 220-316. Moreover, by removing the basic RKRLR sequence at the protein C-terminus (from 312 to 316 residue), p57 Kip2 was confined in the cytosol, implying that this sequence is absolutely required for nuclear entry. In conclusion, we identified an unreported p57 Kip2 NLS and suggest that its absence or mutation might be of relevance in CDKN1C -associated human diseases determining significant changes of p57 Kip2 localization/regulatory roles.

Rett syndrome (RTT) is an extremely invalidating, cureless, developmental disorder, and it is considered one of the leading causes of intellectual disability in female individuals. The vast majority of RTT cases are caused by de novo mutations in the X-linked Methyl-CpG binding protein 2 ( MECP2 ) gene, which encodes a multifunctional reader of methylated DNA. MeCP2 is a master epigenetic modulator of gene expression, with a role in the organization of global chromatin architecture. Based on its interaction with multiple molecular partners and the diverse epigenetic scenario, MeCP2 triggers several downstream mechanisms, also influencing the epigenetic context, and thus leading to transcriptional activation or repression. In this frame, it is conceivable that defects in such a multifaceted factor as MeCP2 lead to large-scale alterations of the epigenome, ranging from an unbalanced deposition of epigenetic modifications to a transcriptional alteration of both protein-coding and non-coding genes, with critical consequences on multiple downstream biological processes. In this review, we provide an overview of the current knowledge concerning the transcriptomic and epigenomic alterations found in RTT patients and animal models.

The ongoing pandemic due to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has so far infected about 2.42 × 10 7 (as at 27 August 2020) subjects with more than 820,000 deaths. It is the third zoonotic coronavirus-dependent outbreak in the last twenty years and represents a major infective threat for public health worldwide. A main aspect of the infection, in analogy to other viral infections, is the so-called "cytokine storm", an inappropriate molecular response to virus spread which plays major roles in tissue and organ damage. Immunological therapies, including vaccines and humanized monoclonal antibodies, have been proposed as major strategies for prevention and treatment of the disease. Accordingly, a detailed mechanistic knowledge of the molecular events with which the virus infects cells and induces an immunological response appears necessary. In this review, we will report details of the initial process of SARS-CoV-2 cellular entry with major emphasis on the maturation of the spike protein. Then, a particular focus will be devoted to describe the possible mechanisms by which dendritic cells, a major cellular component of innate and adaptive immune responses, may play a role in the spread of the virus in the human body and in the clinical evolution of the disease.

Pericentric heterochromatin (PCH) is a particular form of constitutive heterochromatin that is localized to both sides of centromeres and that forms silent compartments enriched in repressive marks. These genomic regions contain species-specific repetitive satellite DNA that differs in terms of nucleotide sequences and repeat lengths. In spite of this sequence diversity, PCH is involved in many biological phenomena that are conserved among species, including centromere function, the preservation of genome integrity, the suppression of spurious recombination during meiosis, and the organization of genomic silent compartments in the nucleus. PCH organization and maintenance of its repressive state is tightly regulated by a plethora of factors, including enzymes (e.g., DNA methyltransferases, histone deacetylases, and histone methyltransferases), DNA and histone methylation binding factors (e.g., MECP2 and HP1), chromatin remodeling proteins (e.g., ATRX and DAXX), and non-coding RNAs. This evidence helps us to understand how PCH organization is crucial for genome integrity. It then follows that alterations to the molecular signature of PCH might contribute to the onset of many genetic pathologies and to cancer progression. Here, we describe the most recent updates on the molecular mechanisms known to underlie PCH organization and function.

Facioscapulohumeral muscular dystrophy (FSHD) is characterized by incomplete penetrance and intra-familial clinical variability. The disease has been associated with the genetic and epigenetic features of the D4Z4 repetitive elements at 4q35. Recently, D4Z4 hypomethylation has been proposed as a reliable marker in the FSHD diagnosis. We exploited the Italian Registry for FSHD, in which FSHD families are classified using the Clinical Comprehensive Evaluation Form (CCEF). A total of 122 index cases showing a classical FSHD phenotype (CCEF, category A) and 110 relatives were selected to test with the receiver operating characteristic (ROC) curve, the diagnostic and predictive value of D4Z4 methylation. Moreover, we performed DNA methylation analysis in selected large families with reduced penetrance characterized by the co-presence of subjects carriers of one D4Z4 reduced allele with no signs of disease or presenting the classic FSHD clinical phenotype. We observed a wide variability in the D4Z4 methylation levels among index cases revealing no association with clinical manifestation or disease severity. By extending the analysis to family members, we revealed the low predictive value of D4Z4 methylation in detecting the affected condition. In view of the variability in D4Z4 methylation profiles observed in our large cohort, we conclude that D4Z4 methylation does not mirror the clinical expression of FSHD. We recommend that measurement of this epigenetic mark must be interpreted with caution in clinical practice.

Lithium salt is the first-line therapeutic option for bipolar disorder and has been proposed as a potential antitumoral drug. The effects of LiCl treatment were investigated in SH-SY5Y, a human neuroblastoma cell line and an in vitro model of dopaminergic neuronal differentiation. LiCl, at the dosage used in psychiatric treatment, does not affect cell proliferation, while at higher doses it delays the SH-SY5Y cell division cycle and for prolonged usage reduces cell viability. Moreover, the ion treatment affects DNA integrity as demonstrated by accumulation of p53 and γH2AX (the phosphorylated form of H2AX histone), two important markers of genome damage. p57 Kip2 , a CIP/Kip protein, is required for proper neuronal maturation and represents a main factor of response to stress including genotoxicity. We evaluated the effect of lithium on p57 Kip2 levels. Unexpectedly, we found that lithium downregulates the level of p57 Kip2 in a dose-dependent manner, mainly acting at the transcriptional level. A number of different approaches, mostly based on p57 Kip2 content handling, confirmed that the CKI/Kip reduction plays a key role in the DNA damage activated by lithium and suggests the unanticipated view that p57 Kip2 might be involved in DNA double-strand break responses. In conclusion, our study identified novel roles for p57 Kip2 in the molecular mechanism of lithium at high concentration and, more in general, in the process of DNA repair.

Methyl-CpG binding protein 2 (MeCP2) is a multi-function factor involved in locus-specific transcriptional modulation and the regulation of genome architecture, e.g., pericentric heterochromatin (PCH) organization. MECP2 mutations are responsible for Rett syndrome (RTT), a devastating postnatal neurodevelopmental disorder, the pathogenetic mechanisms of which are still unknown. MeCP2, together with Alpha-thalassemia/mental retardation syndrome X-linked protein (ATRX), accumulates at chromocenters, which are repressive PCH domains. As with MECP2 , mutations in ATRX cause ATR-X syndrome which is associated with severe intellectual disability. We exploited two murine embryonic stem cell lines, in which the expression of MeCP2 or ATRX is abolished. Through immunostaining, chromatin immunoprecipitation and western blot, we show that MeCP2 and ATRX are reciprocally dependent both for their expression and targeting to chromocenters. Moreover, ATRX plays a role in the accumulation of members of the heterochromatin protein 1 (HP1) family at PCH and, as MeCP2, modulates their expression. Furthermore, ATRX and HP1 targeting to chromocenters depends on an RNA component. 3D-DNA fluorescence in situ hybridization (FISH) highlighted, for the first time, a contribution of ATRX in MeCP2-mediated chromocenter clustering during neural differentiation. Overall, we provide a detailed dissection of the functional interplay between MeCP2 and ATRX in higher-order PCH organization in neurons. Our findings suggest molecular defects common to RTT and ATR-X syndrome, including an alteration in PCH.

The CDKN1C gene encodes the p57 Kip2 protein which has been identified as the third member of the CIP/Kip family, also including p27 Kip1 and p21 Cip1 . In analogy with these proteins, p57 Kip2 is able to bind tightly and inhibit cyclin/cyclin-dependent kinase complexes and, in turn, modulate cell division cycle progression. For a long time, the main function of p57 Kip2 has been associated only to correct embryogenesis, since CDKN1C -ablated mice are not vital. Accordingly, it has been demonstrated that CDKN1C alterations cause three human hereditary syndromes, characterized by altered growth rate. Subsequently, the p57 Kip2 role in several cell phenotypes has been clearly assessed as well as its down-regulation in human cancers. CDKN1C lies in a genetic locus, 11p15.5, characterized by a remarkable regional imprinting that results in the transcription of only the maternal allele. The control of CDKN1C transcription is also linked to additional mechanisms, including DNA methylation and specific histone methylation/acetylation. Finally, long non-coding RNAs and miRNAs appear to play important roles in controlling p57 Kip2 levels. This review mostly represents an appraisal of the available data regarding the control of CDKN1C gene expression. In addition, the structure and function of p57 Kip2 protein are briefly described and correlated to human physiology and diseases., Competing Interests: The authors declare no conflict of interest.