Effect of nutrient deprivation on the expression and the epigenetic signature of sirtuin genes.

BACKGROUND AND AIM: Over the last decades advances in understanding the molecular bases of the close relationship between nutrition, metabolism, and diseases have been impressive. However, there are always novel frontiers coming up and epigenetics is one of these. Sirtuins, are pivotal factors in the control of metabolic pathways according to nutrient availability. In the present study we evaluated the effect of nutrient deprivation on expression, DNA methylation and chromatin status of the sirtuin genes. METHODS AND RESULTS: We performed these studies in mouse hepatoma cells, that were grown in standard medium, or in media containing low glucose concentration, or no glucose, or no amino acids. We applied quantitative real-time PCR to cDNA, methylation-enriched DNA and nuclease-treated DNA in order to evaluate gene expression, DNA methylation, and chromatin condensation, respectively. This study shows that the expression of sirtuin genes varies following nutrient deprivation. Moreover, we observed that changes of DNA methylation and chromatin condensation occur at the transcription start site of sirtuin genes following nutrient deprivation. CONCLUSIONS: Epigenetic mechanisms may have a role in the sirtuin response to nutrient deprivations in cultured hepatoma cells. Replicating these results in vivo to achieve a comprehensive understanding of the epigenetic control of sirtuin expression following nutrient deprivations might open up novel therapeutic possibilities to cure metabolic diseases and promote human health.

Genome-Wide DNA Methylation Analysis Identifies Novel Hypomethylated Non-Pericentromeric Genes with Potential Clinical Implications in ICF Syndrome.

INTRODUCTION AND RESULTS: Immunodeficiency, centromeric instability and facial anomalies syndrome (ICF) is a rare autosomal recessive disease, characterized by severe hypomethylation in pericentromeric regions of chromosomes (1, 16 and 9), marked immunodeficiency and facial anomalies. The majority of ICF patients present mutations in the DNMT3B gene, affecting the DNA methyltransferase activity of the protein. In the present study, we have used the Infinium 450K DNA methylation array to evaluate the methylation level of 450,000 CpGs in lymphoblastoid cell lines and untrasformed fibroblasts derived from ICF patients and healthy donors. Our results demonstrate that ICF-specific DNMT3B variants A603T/STP807ins and V699G/R54X cause global DNA hypomethylation compared to wild-type protein. We identified 181 novel differentially methylated positions (DMPs) including subtelomeric and intrachromosomic regions, outside the classical ICF-related pericentromeric hypomethylated positions. Interestingly, these sites were mainly located in intergenic regions and inside the CpG islands. Among the identified hypomethylated CpG-island associated genes, we confirmed the overexpression of three selected genes, BOLL, SYCP2 and NCRNA00221, in ICF compared to healthy controls, which are supposed to be expressed in germ line and silenced in somatic tissues. CONCLUSIONS: In conclusion, this study contributes in clarifying the direct relationship between DNA methylation defect and gene expression impairment in ICF syndrome, identifying novel direct target genes of DNMT3B. A high percentage of the DMPs are located in the subtelomeric regions, indicating a specific role of DNMT3B in methylating these chromosomal sites. Therefore, we provide further evidence that hypomethylation in specific non-pericentromeric regions of chromosomes might be involved in the molecular pathogenesis of ICF syndrome. The detection of DNA hypomethylation at BOLL, SYCP2 and NCRNA00221 may pave the way for the development of specific clinical biomarkers with the aim to facilitate the identification of ICF patients.

An autoregulatory loop mediated by miR-21 and PDCD4 controls the AP-1 activity in RAS transformation.

The transcription factor AP-1 plays key roles in tumorigenesis, by regulating a variety of protein-coding genes, implicated in multiple hallmarks of cancer. Among non-coding genes, no AP-1 target has been described yet in tumorigenesis. MicroRNAs (miRNAs) are negative post-transcriptional regulators of protein-coding genes. miRNA expression signatures are highly relevant in cancer and several tumor-associated miRNAs (oncomirs) play critical roles in oncogenesis. Here, we show that the miRNA miR-21, which represents the most frequently upregulated oncomir in solid tumors, is induced by AP-1 in response to RAS. By analyzing validated miR-21 targets, we have found that the tumor suppressors PTEN and PDCD4 are downregulated by RAS in an AP-1- and miR-21-dependent fashion. We further show that, given the role of PDCD4 as negative regulator of AP-1, the miR-21-mediated downregulation of PDCD4 is essential for the maximal induction of AP-1 activity in response to RAS. Our data reveal a novel mechanism of positive autoregulation of the AP-1 complex in RAS transformation and disclose the function of oncomirs as critical targets and regulators of AP-1 in tumorigenesis.

Lessons from two human chromatin diseases, ICF syndrome and Rett syndrome.

Spatial organisation of DNA into chromatin profoundly affects gene expression and function. The recent association of genes controlling chromatin structure to human pathologies resulted in a better comprehension of the interplay between regulation and function. Among many chromatin disorders we will discuss Rett and immunodeficiency, centromeric instability and facial anomalies (ICF) syndromes. Both diseases are caused by defects related to DNA methylation machinery, with Rett syndrome affecting the transduction of the repressive signal from the methyl CpG binding protein prototype, MeCP2, and ICF syndrome affecting the genetic control of DNA methylation, by the DNA methyltransferase DNMT3B. Rather than listing survey data, our aim is to highlight how a deeper comprehension of gene regulatory web may arise from studies of such pathologies. We also maintain that fundamental studies may offer chances for a therapeutic approach focused on these syndromes, which, in turn, may become paradigmatic for this increasing class of diseases.

Multiple binding of methyl-CpG and polycomb proteins in long-term gene silencing events.

Epigenetic regulation is involved in the maintenance of long-term silencing phenomena, such as X-inactivation and genomic imprinting in mammals. Gene repression is mediated by several mechanisms, such as histone modifications, DNA methylation, and recruitment of Polycomb proteins. To understand the mechanistic relationships between these mechanisms for stable gene silencing, we analyzed the mechanisms of X- and Y-inactivation of the PAR2 gene SYBL1, previously showed to be regulated by concerted epigenetic mechanisms. Maintenance of stable repression occurs via the recruitment of both MBDPs and PRC2 complexes to SYBL1 promoter. Their binding is equally sensitive to defective DNA methylation seen in cells derived from ICF syndrome patients. Multiple occupancy is a feature shared within long-term repressed genes, such as the X-inactivated PGK1 and the imprinted IGF2. MBD2, MBD3, and MeCP2 occupy SYBL1 promoter simultaneously, as revealed by sequential ChIP. We did not find this co-occurring binding when looked for members of PRC2 complex together with any of the methyl-binding proteins. Furthermore, in co-transfection assays, MECP2 can silence methylated SYBL1 promoter, whereas the mutated protein fails. However, RNA interference of endogenous MECP2 does not induce the expression of the inactive SYBL1 alleles, suggesting that its silencing activity can be replaced by the other methyl-binding proteins. Our data suggest that maintenance of long-term silencing involves multiple layers of epigenetic control functionally redundant. PRC2 and MBD proteins could collaborate to different phases of this process, the former possibly recruiting DNMTs to the silenced promoters, the latter dictating the lock of the transcription.

Maintenance of X- and Y-inactivation of the pseudoautosomal (PAR2) gene SPRY3 is independent from DNA methylation and associated to multiple layers of epigenetic modifications.

Maintenance of X-inactivation is achieved through a combination of different repressive mechanisms, thus perpetuating the silencing message through many cell generations. The second human X-Y pseudoautosomal region 2 (PAR2) is a useful model to explore the features and internal relationships of the epigenetic circuits involved in this phenomenon. Recently, we demonstrated that DNA methylation plays an essential role for the maintenance of X- and Y-inactivation of the PAR2 gene SYBL1; here we report that the silencing of the second repressed PAR2 gene, SPRY3, appears to be independent of DNA methylation. In contrast to SYBL1, the inactive X and Y alleles of SPRY3 are not reactivated in cells treated with a DNA methylation inhibitor and in cells from ICF (immunodeficiency, centromeric instability, facial anomalies) syndrome patients, which have mutations in the DNA methyltransferase gene DNMT3B. SPRY3 X- and Y-inactivation is associated with a differential enrichment of repressive histone modifications and the recruitment of Polycomb 2 group proteins compared to the active X allele. Another major factor in SPRY3 repression is late replication; the inactive X and Y alleles of SPRY3 have delayed replication relative to the active X allele, even in ICF syndrome cells where the closely linked SYBL1 gene is reactivated and advanced in replication. The relatively stable maintenance of SPRY3 silencing compared with SYBL1 suggests that genes without CpG islands may be less prone to reactivation than previously thought and that genes with CpG islands require promoter methylation as an additional layer of repression.

Escape from gene silencing in ICF syndrome: evidence for advanced replication time as a major determinant.

Chromosomal abnormalities associated with hypomethylation of classical satellite regions are characteristic for the ICF immunodeficiency syndrome. We, as well as others, have found that these effects derive from mutations in the DNMT3B DNA methyltransferase gene. Here we examine further the molecular phenotype of ICF cells and report several examples of extensive hypomethylation that are associated with advanced replication time, nuclease hypersensitivity and a variable escape from silencing for genes on the inactive X and Y chromosomes. Our analysis suggests that all genes on the inactive X chromosome may be extremely hypomethylated at their 5' CpG islands. Our studies of G6PD in one ICF female and SYBL1 in another ICF female provide the first examples of abnormal escape from X chromosome inactivation in untransformed human fibroblasts. XIST RNA localization is normal in these cells, arguing against an independent silencing role for this RNA in somatic cells. SYBL1 silencing is also disrupted on the Y chromosome in ICF male cells. Increased chromatin sensitivity to nuclease was found at all hypomethylated promoters examined, including those of silenced genes. The persistence of inactivation in these latter cases appears to depend critically on delayed replication of DNA because escape from silencing was only seen when replication was advanced to an active X-like pattern.

Differentially regulated and evolved genes in the fully sequenced Xq/Yq pseudoautosomal region.

Human sex chromosomes, which are morphologically and genetically different, share few regions of homology. Among them, only pseudoautosomal regions (PARs) pair and recombine during meiosis. To better address the complex biology of these regions, we sequenced the telomeric 400 kb of the long arm of the human X chromosome, including 330 kb of the human Xq/YqPAR and the telomere. Sequencing reveals subregions with distinctive regulatory and evolutionary features. The proximal 295 kb contains two genes inactivated on both the inactive X and Y chromosomes [ SYBL1 and a novel homologue ( HSPRY3 ) of Drosophila sprouty ]. The GC-rich distal 35 kb, added in stages and much later in evolution, contains the X/Y expressed gene IL9R and a novel gene, CXYorf1, only 5 kb from the Xq telomere. These properties make Xq/YqPAR a model for studies of region-specific gene inactivation, telomere evolution, and involvement in sex-limited conditions.

Evolution of the X-specific block embedded in the human Xq21.3/Yp11.1 homology region.

The region Xq21.3/Yp11.1 represents the largest segment of homology between the sex chromosomes in humans, though no recombination occurs in male meiosis. It presumably arose as a transposition from the X to the Y chromosome; the present-day organization in the latter chromosome indicates a paracentric inversion that disrupted its continuity. Moreover, an X-specific block (defined by the marker DXS214) is embedded in the region. Previously, no hypotheses about the length, origin, or evolution of this X-specific segment have been proposed. Here we report on the refinement of the size and the sequence of the distal boundary of the X-specific block. Furthermore, we have tracked by FISH experiments the evolution of this region in primates. This further clarifies the multistep mechanism of origin for the XY homology region, by demonstrating that the X-specific block was deleted from the Y chromosome after the initial transfer from the X chromosome.

Human and mouse SYBL1 gene structure and expression.

SYBL1 is a gene in the 320kb human pseudo-autosomal region at the terminus of Xq and Yq. In contrast to other pseudoautosomal genes, SYBL1 is inactivated on one X in every female cell, and is also inactive on the Y of male cells. Hypermethylation of the CpG island associated with the human gene is involved in this phenomenon. In an attempt to further examine its regulation, the genomic organization of the X-linked mouse Sybl1 homolog was analyzed and compared with the human gene. Human and mouse show the same exon number, exon-intron junctions and a highly conserved basal promoter. The structural and functional conservation of basal regulatory regions suggests that inactivation is imposed by similar auxiliary epistatic regulatory mechanism.

Coding region intron/exon organization, alternative splicing, and X-chromosome inactivation of the KRAB/FPB-domain-containing human zinc finger gene ZNF41.

ZNF41 belongs to a cluster of human zinc finger genes residing within a gene-rich region at Xp11.23. ZNF41 encodes a KRAB/FPB (Krüppel-associated/finger preceding box) domain, a potent transcription repression motif present in hundreds of vertebrate zinc finger protein genes, composed of two protein modules, A and B. Three introns, placed at identical positions in paralogous genes, interrupt four exons encoding the ZNF41 N-terminal amino acids, the KRAB/FPB-A and KRAB/FPB-B modules, and the remaining coding region adjoined to the C-terminal zinc finger domain. Since the KRAB/FPB-A and KRAB/FPB-B modules are encoded by dedicated exons in ZNF41 and paralogous genes, exon skipping may lead to differential usage of these modules in alternative gene products. RT-PCR analysis of ZNF41 mRNAs showed that, while skipping of the KRAB/FPB-A and/or KRAB/FPB-B exons was not detected, the use of alternative donor/acceptor sites upstream of the KRAB/FPB-A exon generates multiple ZNF41 transcripts potentially encoding polypeptides differing in the N-terminal region and expressed in different tissues. The expression pattern in cell hybrids containing either active or inactive X chromosomes indicates that ZNF41, which resides within a region of the X chromosome that includes genes that are both subject to and escape X-inactivation, is susceptible to X-chromosome inactivation.

Differential expression pattern of XqPAR-linked genes SYBL1 and IL9R correlates with the structure and evolution of the region.

The recently discovered second pseudoautosomal region (XqPAR) contains at least two genes, IL9R and SYBL1. Recent findings show that, like XpPAR genes, IL9R escapes X inactivation and its Y allele is also expressed, but SYBL1 seems to act like an X-linked gene, expressed from the active X chromosome but not from the inactive X or Y. Here we show that differences are also seen in the evolution of the sex chromosome locations of IL9R and SYBL1. IL9R is known to be autosomal in mice, and is X-linked only in primates. SYBL1, however, has been found to be on the X chromosome in all mammals tested, from marsupials to humans. Both genes were duplicated on the Y homologue of the terminal portion of the X chromosome during the evolution of Homo sapiens from other higher primates. The inactivation pattern of SYBL1 may be correlated with its longer history of X linkage, and at a more centromeric chromosomal position during evolution; the more recent X linkage and more telomeric position of the IL9R gene may explain its autosomal, 'uninactivated' transcriptional status.

Expressed STSs and transcription of human Xq28.

STSs, which have been used to build and format clone contigs, have been used here to assemble a transcriptional map across a cytogenetic band. Of fifty one STSs in Xq28, 20 were positive by RT-PCR. Thus, an additional 20 possible ESTs were detected among the STSs, and seven of these also identified cDNAs in at least one library. The transcripts confirm the high expression level of this region, correlated with its GC compositional map and CpG island content.