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Background: Accumulating evidence suggests that Diabetes mellitus type 2 affects the angiogenic functions of Endothelial Progenitor Cells and a good glycemic control is not enough to restore these functions. Therefore, it is necessary to investigate if there are changes at the epigenetic level that can explain this phenomenon. Results: We isolated EPC from mononuclear cells and cultured them in 5 mM of D-glucose equivalent to normal glycemia, 10 mM equivalent to a glycemia clinically acceptable for a diabetic patient, and 20 mM of D-glucose corresponding to a value of glycemia in a patient with poor glycemic control; and we evaluated the adhesion capacity of EPC to fibronectin matrix. We also determined global methylation levels by slot blot for the 5-methylcytocine and 5-hydroxymethylcytocine and the expression levels of the TET1 and TET2 genes using qPCR. We found that high glucose concentrations do not affect EPC differentiation. Cells grown at 10 mM D-glucose do not decrease their adhesion to fibronectin matrix but at 20 mM culture we observed decrease in their adhesion (p> 0.05), but interesting, in both culture conditions we observed hypomethylation of the genome (p> 0.05) accompanied with an increase in 5-hydroxymethylcytocine in cells treated with 20mM D-glucose (p TET1 and TET2 genes. Treatment with demethylating agent 5-aza-2'-deoxycytidine, shows that adhesion failure is dependent on genome demethylation (p> 0.001).Conclusions: High concentrations of D-glucose (20mM) generate an active hypomethylation of the hEPC genome and a decrease in their adhesion capacity. Although the concentration of D-Glucose considered as "clinically acceptable" glycemia in a diabetic patient (10mM), do not produce significant changes in hEPC adhesion to fibronectin matrix, a global hypomethylation of the genome, was observed, which could be responsible for impair angiogenic functions of the hEPC in patients with good glycemic control.

Heterotopic ossification (HO) is a condition in wich extra-skeletal bone formation occurs in soft tissues of the body. HO is formed in numerous clinical situations such as burns, neurological damage, post-combat trauma, hip surgery, etc. The most severe condition of heterotopic ossification is the pathology called Fibrodysplasia Ossificans Progressiva (FOP), wich is produced by a point mutation in the ACVR1 receptor gene, triggering its constitutive activation and a hyper-activation of the SMAD pathway. Different pharmacological treatments have been used to inhibit the progression of ossification outbreaks, but a low effectiveness has been reported. An alternative therapy is the use of nicotinamide where a delay in the appearance of ossification flare ups has been observed. However, its effect on the SMAD-mediated signaling pathway is not known. In this work we use nicotinamide in culture of C2C12 cells in the presence or absence of the osteoinductive BMP-2 to investigate its effect on osteoblastogenesis. Through western blot, Inmmunocytochemistry and Real time PCR we investigated the effect of nicotinamide on the SMAD pathway and gene expression of genes involved in the pathology. Our results show that in the presence of nicotinamide a decrease in the osteoblast differentiation and in SMAD complex phosphorylation levels while expression of the ACVR1 BMP-2 receptor and Runx2 are not affected by the treatment. New evidence is provided regarding the mechanism by which nicotinamide acts. These findings could be helpful in the clinic for the treatment of diseases related to HO bone formation.

1α,25-Dihydroxyvitamin D3-mediated transcriptional control of the bone-specific osteocalcin (OC) gene requires the integration of regulatory signals at the vitamin D-responsive element (VDRE) and flanking tissue-specific sequences. The 1α,25-dihydroxyvitamin D3 receptor (VDR) is a member of the nuclear receptor superfamily and forms a heterodimeric complex with the receptor for 9-cis retinoic acid (RXR) that binds to the VDRE sequence. We have demonstrated previously that changes in chromatin structure at the VDRE region of the rat OC gene promoter accompany transcriptional enhancement in vivo, suggesting a requirement for chromatin remodelling. Here we show that the VDRE in the distal region of the OC gene promoter is refractory to binding of the VDR—RXR complex when organized in a nucleosomal context. Addition of the ligand 1α,25-dihydroxyvitamin D3 or the presence of other transcription factors, such as YY1 and Runx/Cbfa (core-binding factor α), which also bind to sequences partially overlapping or near the VDRE, is not sufficient to render the VDRE accessible. Thus the VDR—RXR, unlike other steroid receptors, such as glucocorticoid receptor, progesterone receptor and thyroid receptor, is unable to bind its target sequence within a nucleosomal context. Taken together these results demonstrate that nucleosomal remodelling is required for in vivo occupancy of binding sites in the distal region of the OC gene promoter by the regulatory factors responsible for 1α,25-dihydroxyvitamin D3-dependent enhancement of transcription.

The One of the most frequent chromosomal translocation found in patients with acute myeloid leukemia (AML) is the t(8;21). This translocation involves the RUNX1 and ETO genes. The breakpoints regions for t(8;21) are located at intron 5 and intron 1 of the RUNX1 and ETO gene respectively. To date, no homologous sequences have been found in these regions to explain their recombination. The breakpoint regions of RUNX1 gene are characterized by the presence of DNasaI hypersensitive sites and topoisomerase II cleavage sites, but no information exists about complementary regions of ETO gene. Here, we report analysis of chromatin structure of ETO breakpoint regions. Chromatin immunoprecipitation (ChIP) were performed with antibodies specific to acetylated histone H3, H4, and total histone H1. Nucleosomal distribution at the ETO locus was evaluated by determining total levels of histone H3. Our data show that in myeloid cells, the breakpoint regions at the ETO gene are enriched in hyperacetylated histone H3 compared to a control region of similar size where no translocations have been described. Moreover, acetylated H4 associates with both the whole ETO breakpoint regions as well as the control intron. Interestingly, we observed no H1 association either at the breakpoint regions or the control region of the ETO gene. Our data indicate that a common chromatin structure enriched in acetylated histones is present in breakpoint regions involved in formation of (8;21) leukemic translocation.

There is growing recognition that the organization of nucleic acids and regulatory proteins is functionally linked to the assembly, localization and activity of gene regulatory machinery. Cellular, molecular, biochemical and in-vivo genetic evidence support an obligatory relationship between nuclear microenvironments where regulatory complexes reside and fidelity of transcriptional control. Perturbations in mechanisms governing the intranuclear trafficking of transcription factors and the temporal/spatial organization of regulatory proteins within the nucleus occur with compromised gene expression that abrogates skeletal development and mediates leukemogenesis.

Development of the osteoblast phenotype requires transcriptional mechanisms that regulate induction of a program of temporally expressed genes. Key components of gene activation, repression, and responsiveness to physiologic mediators require remodeling of the chromatin structure of a gene that renders promoter elements competent for the assembly of macromolecular transcriptional complexes. Here we review evidence that the Runx transcription factors support tissue-specific gene expression and bone formation by contributing to promoter structure, chromatin remodeling, and the integration of independent signaling pathways. In addition, we discuss the role of Runx2 in both activation and negative regulation of gene promoters (osteocalcin, bone sialoprotein, and Runx2/Cbfa1) in relation to the interaction of Runx with co-regulatory proteins in distinct subnuclear foci. The modifications in chromatin organization and transcription of the osteocalcin gene that are influenced by the activities of Runx2/Cbfa1 mediated by interacting proteins (YAP, TLE, SMAD, C/EBP) are emphasized. These functional properties of Runx2 provide novel insights into the requirements for multiple levels of transcriptional control within the context of nuclear architecture to support the convergence of regulatory signals that control tissue-specific gene expression.

Three sets of histone variants are coexisting in the embryo at larval stages of sea urchin's development: the maternally inherited cleavage stage variants (CS) expressed during the two initial cleavage divisions, the early histone variants, which are recruited into embryonic chromatin from middle cleavage stages until hatching and the late variants, that are fundamentally expressed from blastula stage onward. Since the expression of the CS histones is confined to the initial cleavage stages, these variants represent a very minor proportion of the histones present in the plutei larvae, whereas the late histone variants are predominant. To determine the position of these CS in the embryonic territories, we have immunolocalized the CS histone variants in plutei larvas harvested 72 h post-fertilization. In parallel, we have pulse labeled the DNA replicated during the initial cleavage cycle with bromodeoxyuridine (BrdU) and its position was further determined in the plutei larvas by immunofluorescence. We have found that the CS histone variants were segregated to specific territories in the plutei. The position in which the CS histone variants were found to be segregated was consistent with the position in which the DNA molecules that were replicated during the initial cleavage divisions were localized. These results strongly suggest that a specification of embryonic nuclei occurs at the initial cleavage divisions which is determined by a chromatin organized by CS histone variants.

The architecturally associated subnuclear organization of nucleic acids and cognate regulatory factors suggest functional interrelationships between nuclear structure and gene expression. Mechanisms that contribute to the spatial distribution of transcription factors within the three-dimensional context of nuclear architecture control the sorting of regulatory information as well as the assembly and activities of sites within the nucleus that support gene expression. Vitamin D control of gene expression serves as a paradigm for experimentally addressing mechanisms that govern the intranuclear targeting of regulatory factors to nuclear domains where transcription of developmental and tissue-specific genes occur. We will present an overview of molecular, cellular, genetic, and biochemical approaches that provide insight into the trafficking of regulatory factors that mediate vitamin D control of gene expression to transcriptionally active subnuclear sites. Examples will be presented that suggest modifications in the intranuclear targeting of transcription factors abrogate competency for vitamin D control of skeletal gene expression during development and fidelity of gene expression in tumor cells.