Mouse Ribosomal RNA Genes Contain Multiple Differentially Regulated Variants

Previous cytogenetic studies suggest that various rDNA chromosomal loci are not equally active in different cell types. Consistent with this variability, rDNA polymorphism is well documented in human and mouse. However, attempts to identify molecularly rDNA variant types, which are regulated individually (i.e., independent of other rDNA variants) and tissue-specifically, have not been successful. We report here the molecular cloning and characterization of seven mouse rDNA variants (v-rDNA). The identification of these v-rDNAs was based on restriction fragment length polymorphisms (RFLPs), which are conserved among individuals and mouse strains. The total copy number of the identified variants is less than 100 and the copy number of each individual variant ranges from 4 to 15. Sequence analysis of the cloned v-rDNA identified variant-specific single nucleotide polymorphisms (SNPs) in the transcribed region. These SNPs were used to develop a set of variant-specific PCR assays, which permitted analysis of the v-rDNAs' expression profiles in various tissues. These profiles show that three v-rDNAs are expressed in all tissues (constitutively active), two are expressed in some tissues (selectively active), and two are not expressed (silent). These expression profiles were observed in six individuals from three mouse strains, suggesting the pattern is not randomly determined. Thus, the mouse rDNA array likely consists of genetically distinct variants, and some are regulated tissue-specifically. Our results provide the first molecular evidence for cell-type-specific regulation of a subset of rDNA

Parp1 Localizes within the Dnmt1 Promoter and Protects Its Unmethylated State by Its Enzymatic Activity

Aberrant hypermethylation of CpG islands in housekeeping gene promoters and widespread genome hypomethylation are typical events occurring in cancer cells. The molecular mechanisms behind these cancer-related changes in DNA methylation patterns are not well understood. Two questions are particularly important: (i) how are CpG islands protected from methylation in normal cells, and how is this protection compromised in cancer cells, and (ii) how does the genome-wide demethylation in cancer cells occur. The latter question is especially intriguing since so far no DNA demethylase enzyme has been found.Our data show that the absence of ADP-ribose polymers (PARs), caused by ectopic over-expression of poly(ADP-ribose) glycohydrolase (PARG) in L929 mouse fibroblast cells leads to aberrant methylation of the CpG island in the promoter of the Dnmt1 gene, which in turn shuts down its transcription. The transcriptional silencing of Dnmt1 may be responsible for the widespread passive hypomethylation of genomic DNA which we detect on the example of pericentromeric repeat sequences. Chromatin immunoprecipitation results show that in normal cells the Dnmt1 promoter is occupied by poly(ADP-ribosyl)ated Parp1, suggesting that PARylated Parp1 plays a role in protecting the promoter from methylation.In conclusion, the genome methylation pattern following PARG over-expression mirrors the pattern characteristic of cancer cells, supporting our idea that the right balance between Parp/Parg activities maintains the DNA methylation patterns in normal cells. The finding that in normal cells Parp1 and ADP-ribose polymers localize on the Dnmt1 promoter raises the possibility that PARylated Parp1 marks those sequences in the genome that must remain unmethylated and protects them from methylation, thus playing a role in the epigenetic regulation of gene expression

Fractal Characteristics of May-Grünwald-Giemsa Stained Chromatin Are Independent Prognostic Factors for Survival in Multiple Myeloma

The use of computerized image analysis for the study of nuclear texture features has provided important prognostic information for several neoplasias. Recently fractal characteristics of the chromatin structure in routinely stained smears have shown to be independent prognostic factors in acute leukemia. In the present study we investigated the influence of the fractal dimension (FD) of chromatin on survival of patients with multiple myeloma.We analyzed 67 newly diagnosed patients from our Institution treated in the Brazilian Multiple Myeloma Study Group. Diagnostic work-up consisted of peripheral blood counts, bone marrow cytology, bone radiograms, serum biochemistry and cytogenetics. The International Staging System (ISS) was used. In every patient, at least 40 digital nuclear images from diagnostic May-Grünwald-Giemsa stained bone marrow smears were acquired and transformed into pseudo-3D images. FD was determined by the Minkowski-Bouligand method extended to three dimensions. Goodness-of-fit of FD was estimated by the R(2) values in the log-log plots. The influence of diagnostic features on overall survival was analyzed in Cox regressions. Patients that underwent autologous bone marrow transplantation were censored at the day of transplantation.Median age was 56 years. According to ISS, 14% of the patients were stage I, 39% were stage II and 47% were stage III. Additional features of a bad prognosis were observed in 46% of the cases. When stratifying for ISS, both FD and its goodness-of-fit were significant prognostic factors in univariate analyses. Patients with higher FD values or lower goodness-of-fit showed a worse outcome. In the multivariate Cox-regression, FD, R(2), and ISS stage entered the final model, which showed to be stable in a bootstrap resampling study.Fractal characteristics of the chromatin texture in routine cytological preparations revealed relevant prognostic information in patients with multiple myeloma

Epigenetic control of nuclear architecture

The cell nucleus is a highly structured compartment where nuclear components are thought to localize in non-random positions. Correct positioning of large chromatin domains may have a direct impact on the localization of other nuclear components, and can therefore influence the global functionality of the nuclear compartment. DNA methylation of cytosine residues in CpG dinucleotides is a prominent epigenetic modification of the chromatin fiber. DNA methylation, in conjunction with the biochemical modification pattern of histone tails, is known to lock chromatin in a close and transcriptionally inactive conformation. The relationship between DNA methylation and large-scale organization of nuclear architecture, however, is poorly understood. Here we briefly summarize present concepts of nuclear architecture and current data supporting a link between DNA methylation and the maintenance of large-scale nuclear organization

MeCP2 binds to nucleosome free (linker DNA) regions and to H3K9/H3K27 methylated nucleosomes in the brain

Methyl-CpG-binding protein 2 (MeCP2) is a chromatin-binding protein that mediates transcriptional regulation, and is highly abundant in brain. The nature of its binding to reconstituted templates has been well characterized in vitro. However, its interactions with native chromatin are less understood. Here we show that MeCP2 displays a distinct distribution within fractionated chromatin from various tissues and cell types. Artificially induced global changes in DNA methylation by 3-aminobenzamide or 5-aza-2′-deoxycytidine, do not significantly affect the distribution or amount of MeCP2 in HeLa S3 or 3T3 cells. Most MeCP2 in brain is chromatin-bound and localized within highly nuclease-accessible regions. We also show that, while in most tissues and cell lines, MeCP2 forms stable complexes with nucleosome, in brain, a fraction of it is loosely bound to chromatin, likely to nucleosome-depleted regions. Finally, we provide evidence for novel associations of MeCP2 with mononucleosomes containing histone H2A.X, H3K9me2 and H3K27me3 in different chromatin fractions from brain cortex and in vitro. We postulate that the functional compartmentalization and tissue-specific distribution of MeCP2 within different chromatin types may be directed by its association with nucleosomes containing specific histone variants, and post-translational modifications

Revising Accounting and Branding of Italian Cultural Firms: Intangible Assets

What can be a strong signal in order to increase ticket revenues and attract private contributions in modern cultural firms? Intangible Assets. Though ticket revenues have not declined, the Italian State has not lost his aptitude of social father, taking care of merit goods such as Performing and Visual Arts. Though they are encouraging grant-making initiatives and stakeholders such as Banking Foundations, private citizens and enterpreneurs, Italian Cultural Firms are getting on a sweet privatization. Not confident of the Italian private vocation for arts financing and always estimating cultural goods as fundamentals of the Italian tradition and history, they don\u2019t strongly invest in fundraising and advertising campaigns, in ultimate sense, intangibles and they only trust State and Local Administrations Funding. Revising communication and accounting practices could be a virtuous track in order to get on a tangible privatization. Here, it is given evidence of strategies and performance of Italian Cultural Foundations