Targeted DNA methylation by homology-directed repair in mammalian cells. Transcription reshapes methylation on the repaired gene.

We report that homology-directed repair of a DNA double-strand break within a single copy Green Fluorescent Protein (GFP) gene in HeLa cells alters the methylation pattern at the site of recombination. DNA methyl transferase (DNMT)1, DNMT3a and two proteins that regulate methylation, Np95 and GADD45A, are recruited to the site of repair and are responsible for selective methylation of the promoter-distal segment of the repaired DNA. The initial methylation pattern of the locus is modified in a transcription-dependent fashion during the 15\u201320 days following repair, at which time no further changes in the methylation pattern occur. The variation in DNA modification generates stable clones with wide ranges of GFP expression. Collectively, our data indicate that somatic DNA methylation follows homologous repair and is subjected to remodeling by local transcription in a discrete time window during and after the damage. We propose that DNA methylation of repaired genes represents a DNA damage code and is source of variation of gene expression

Un metodo per la diagnosi , il monitoraggio dell'efficacia di una terapia e per lo sviluppo di un trattamento per la Sclerosi Multipla Method for diagnosis, monitoring the efficacy of a therapy and for development of treatment for Multiple Sclerosis

The present invention relates to a method for diagnosis and/or prognosis of multiple sclerosis or to monitor the efficacy of a therapy and/or to screen for a treatment for multiple sclerosis comprising measuring the amount or assessing the cellular localization of one or more specific molecular species in stimulated oligodendrocyte cells. Changes of expression or localization of specific proteins involved in oligodendrocyte differentiation are measured after incubation of differentiating cells with cerebrospinal fluid, immunoglobulins extracted from blood serum or blood of patients with multiple sclerosis

Scleroderma

Scleroderma (systemic sclerosis) is a complex disease in which extensive fibrosis, vascular alterations, and autoantibodies against various cellular antigens are among the principal features (Fig. 1 and 2).1 There are two major subgroups in the commonly accepted classification of scleroderma: limited cutaneous scleroderma and diffuse cutaneous scleroderma.2 In limited cutaneous scleroderma, fibrosis is mainly restricted to the hands, arms, and face. Raynaud’s phenomenon is present for several years before fibrosis appears, pulmonary hypertension is frequent, and anticentromere antibodies occur in 50 to 90% of patients. Diffuse cutaneous scleroderma is a rapidly progressing disorder that affects a large area of the skin and compromises one or more internal organs. We believe that the acronym CREST (calcinosis, Raynaud’s phenomenon, esophageal motility dysfunction, sclerodactyly, and telangiectasia) is obsolete, since it cannot be assigned to only one subgroup of patients with the disease and does not sufficiently indicate the burden of internal-organ involvement. In rare cases, patients with scleroderma have no obvious skin involvement. Patients with scleroderma plus evidence of systemic lupus erythematosus, rheumatoid arthritis, polymyositis, or Sjögren’s syndrome are considered to have an overlap syndrome. This classification can be useful, but none of the proposed classifications sufficiently reflect the heterogeneity of the clinical manifestations of scleroderma. Scleroderma can lead to severe dysfunction and failure of almost any internal organ. Here, too, there is considerable heterogeneity (Table 1). Involvement of visceral organs is a major factor in determining the prognosis. The kidneys, esophagus, heart, and lungs are the most frequent targets. Renal involvement can be controlled by angiotensin-converting–enzyme inhibitors. Severely debilitating esophageal dysfunction is the most common visceral complication, and lung involvement is the leading cause of death. The mechanisms underlying visceral involvement in scleroderma are unclear, despite progress in the treatment of these complications. Relevant data on mechanisms are limited, since most of the available information is derived from crosssectional studies and from patients in various stages of the disease, often after treatment; moreover, there are no satisfactory animal models of scleroderma. Nevertheless, a critical evaluation of the available experimental and clinical data will help reduce ambiguity and may provide the basis for future studies of scleroderma

Pathogenic autoantibodies in systemic sclerosis.

Systemic sclerosis, scleroderma, is a disease characterized by widespread vascular injury and fibrosis of the skin and visceral organs. Circulating autoantibodies against several intracellular antigens are common in scleroderma patients. The specificities of such autoantibodies correlate with distinct clinical manifestations. However, till date there is no evidence that these autoantibodies, though helpful in diagnosis and prognosis, are linked to the pathogenesis of scleroderma nor that they may cause any feature of the disease. Recently, the discovery of novel agonistic autoantibodies targeting the PDGF receptor has provided important insight into the molecular pathogenesis of scleroderma and the intracellular mechanisms leading to fibrosis. Although their pathogenic role awaits validation in in vivo models, these antibodies represent the molecular link between the immune system and fibrosis

Pathogenic autoantibodies in systemic sclerosis

Systemic sclerosis, scleroderma, is a disease characterized by widespread vascular injury and fibrosis of the skin and visceral organs. Circulating autoantibodies against several intracellular antigens are common in scleroderma patients. The specificities of such autoantibodies correlate with distinct clinical manifestations. However, till date there is no evidence that these autoantibodies, though helpful in diagnosis and prognosis, are linked to the pathogenesis of scleroderma nor that they may cause any feature of the disease. Recently, the discovery of novel agonistic autoantibodies targeting the PDGF receptor has provided important insight into the molecular pathogenesis of scleroderma and the intracellular mechanisms leading to fibrosis. Although their pathogenic role awaits validation in in vivo models, these antibodies represent the molecular link between the immune system and fibrosis

Stimulatory autoantibodies to the PDGF receptor: a link to fibrosis in scleroderma and a pathway for novel therapeutic targets.

Systemic sclerosis (scleroderma) is a complex disease characterized by excessive deposition of collagen and abnormalities of blood vessels. In addition, activation of the immune system is a central feature of scleroderma as shown by mononuclear cell infiltration of the skin, autoantibody production and release of inflammatory cytokines. The pathogenesis of the disease is poorly understood and the molecular events underlying the main clinical features are not known. The detection of agonistic autoantibodies targeting PDGF receptor in serum of patients with scleroderma may indicate a novel link between phenotypic features of the disease and a specific signalling pathway. Agonistic PDGF receptor antibodies induce in vitro the scleroderma phenotype in normal human fibroblasts and, thus, link autoimmunity to fibrosis. These findings pave the way to novel therapeutic strategies

Role for cyclin-dependent kinase 2 in mitosis exit.

Mitosis requires cyclin-dependent kinase (cdk) 1-cyclin B activity [1]. Exit from mitosis depends on the inactivation of the complex by the degradation of cyclin B [2]. Cdk2 is also active during mitosis [3, 4]. In Xenopus egg extracts, cdk2 is primarily in complex with cyclin E, which is stable [5]. At the end of mitosis, downregulation of cdk2-cyclin E activity is accompanied by inhibitory phosphorylation of cdk2 [6]. Here, we show that cdk2-cyclin E activity maintains cdk1-cyclin B during mitosis. At mitosis exit, cdk2 is inactivated prior to cdk1. The loss of cdk2 activity follows and depends upon an increase in protein kinase A (PKA) activity. Prematurely inactivating cdk2 advances the time of cyclin B degradation and cdk1 inactivation. Blocking PKA, instead, stabilizes cdk2 activity and inhibits cyclin B degradation and cdk1 inactivation. The stabilization of cdk1-cyclin B is also induced by a mutant cdk2-cyclin E complex that is resistant to inhibitory phosphorylation. P21-Cip1, which inhibits both wild-type and mutant cdk2-cyclin E, reverses mitotic arrest under either condition. Our findings indicate that the proteolysis-independent downregulation of cdk2 activity at the end of mitosis depends on PKA and is required to activate the proteolysis cascade that leads to mitosis exit