Targets and dynamics of promoter DNA methylation during early mouse development

DNA methylation is extensively reprogrammed during the early phases of mammalian development, yet genomic targets of this process are largely unknown. We optimized methylated DNA immunoprecipitation for low numbers of cells and profiled DNA methylation during early development of the mouse embryonic lineage in vivo. We observed a major epigenetic switch during implantation at the transition from the blastocyst to the postimplantation epiblast. During this period, DNA methylation is primarily targeted to repress the germline expression program. DNA methylation in the epiblast is also targeted to promoters of lineage-specific genes such as hematopoietic genes, which are subsequently demethylated during terminal differentiation. De novo methylation during early embryogenesis is catalyzed by Dnmt3b, and absence of DNA methylation leads to ectopic gene activation in the embryo. Finally, we identify nonimprinted genes that inherit promoter DNA methylation from parental gametes, suggesting that escape of post-fertilization DNA methylation reprogramming is prevalent in the mouse genome.     

Regulation of DNA methylation of Rasgrf1.

In mammals, DNA is methylated at cytosines within CpG dinucleotides. Properly regulated methylation is crucial for normal development. Inappropriate methylation may contribute to tumorigenesis by silencing tumor-suppressor genes or by activating growth-stimulating genes. Although many genes have been identified that acquire methylation and whose expression is methylation-sensitive, little is known about how DNA methylation is controlled. We have identified a DNA sequence that regulates establishment of DNA methylation in the male germ line at Rasgrf1. In mice, the imprinted Rasgrf1 locus is methylated on the paternal allele within a differentially methylated domain (DMD) 30 kbp 5' of the promoter. Expression is exclusively from the paternal allele in neonatal brain. Methylation is regulated by a repeated sequence, consisting of a 41-mer repeated 40 times, found immediately 3' of the DMD. This sequence is present in organisms in which Rasgrf1 is imprinted. In addition, DMD methylation is required for imprinted Rasgrf1 expression. Together the DMD and repeat element constitute a binary switch that regulates imprinting at the locus.     

The role of DNA methylation in setting up chromatin structure during development

DNA methylation inhibits gene expression in animal cells, probably by affecting chromatin structure. Biochemical studies suggest that this process may be mediated by methyl-specific binding proteins that recruit enzymatic machinery capable of locally altering histone modification. To test whether DNA methylation actually has a role in the assembly of chromatin during normal development, we used cell transfection and a transgene construct genetically programmed to be either methylated or unmethylated in all cell types of the mouse. Chromatin immunoprecipitation (ChIP) analysis shows that the presence of DNA methylation brings about the deacetylation of histone H4 and methylation of Lys9 of histone H3 (H3 Lys9) and prevents methylation of Lys4 of histone H3 (H3 Lys4), thus generating a structure identical to that of methylated sequences in the genome. These results indicate that the methylation pattern established in early embryogenesis is profoundly important in setting up the structural profile of the genome.     

Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome

To gain insight into the function of DNA methylation at cis-regulatory regions and its impact on gene expression, we measured methylation, RNA polymerase occupancy and histone modifications at 16,000 promoters in primary human somatic and germline cells. We find CpG-poor promoters hypermethylated in somatic cells, which does not preclude their activity. This methylation is present in male gametes and results in evolutionary loss of CpG dinucleotides, as measured by divergence between humans and primates. In contrast, strong CpG island promoters are mostly unmethylated, even when inactive. Weak CpG island promoters are distinct, as they are preferential targets for de novo methylation in somatic cells. Notably, most germline-specific genes are methylated in somatic cells, suggesting additional functional selection. These results show that promoter sequence and gene function are major predictors of promoter methylation states. Moreover, we observe that inactive unmethylated CpG island promoters show elevated levels of dimethylation of Lys4 of histone H3, suggesting that this chromatin mark may protect DNA from methylation.     

Virus epstein-barr et système immunitaire

Epstein-Barr virus persists in CD27+ resting memory B cells of infected people. In lymph nodes, expression of latency II EBV program allows long term survival of EBV infected B cells. Sometimes a lymphoproliferation occurs due to the expression of latency III program which is associated with viral lytic cycle. This escape is usually controlled by the immune system and leads only to asymptomatic viral excretion in immunocompetent people. The control of EBV infection is mainly achieved with specific cellular immune response. Cytotoxic T CD8 lymphocytes (CTLs) were the first studied and their clinical significance has been demonstrated in transplanted patients in whom they prevent or allow the regression of EBV associated lymphoproliferations.CTLs detect lytic antigens with a high frequency during acute mononucleosis and persist thereafter during the latent infection. CTLs also detect latent antigens, however the frequency and intensity of response are lower in that case. Many epitopes have been described for the EBNA3 group proteins while LMP1, LMP2 a et b and EBNA LP proteins present just a few epitopes and induce moderate responses. EBNA 1 protein escapes to CTLs detection because it is not presented by the major histocompatibility complex class I. EBNA1 seems, however, to induce Th1 TCD4 response. T CD4 lymphocytes (TCD4) detect also EBNA3 proteins. Furthermore TCD4 play another role in EBV cycle, and this through cooperation with B lymphocyte (LB); indeed the CD40ligand of TCD4 interacts with the CD40 of LB.This interaction is necessary to obtain specific TCD4 and TCD8 responses and seems to play a key role in the reactivation of the EBV lytic cycle. Further studies should shed light on the interactions between EBV and TCD4 and determine the respective roles of responses toward latent and lytic antigens. Current data on the EBV specific immune response have already allowed to consider new therapeutic strategies for EBV associated lymphoproliferations by the restoration of cytotoxic responses against one or many epitopes.     

Global assessment of promoter methylation in a mouse model of cancer identifies ID4 as a putative tumor-suppressor gene in human leukemia

DNA methylation is associated with malignant transformation, but limitations imposed by genetic variability, tumor heterogeneity, availability of paired normal tissues and methodologies for global assessment of DNA methylation have limited progress in understanding the extent of epigenetic events in the initiation and progression of human cancer and in identifying genes that undergo methylation during cancer. We developed a mouse model of T/natural killer acute lymphoblastic leukemia that is always preceded by polyclonal lymphocyte expansion to determine how aberrant promoter DNA methylation and consequent gene silencing might be contributing to leukemic transformation. We used restriction landmark genomic scanning with this mouse model of preleukemia reproducibly progressing to leukemia to show that specific genomic methylation is associated with only the leukemic phase and is not random. We also identified Idb4 as a putative tumor-suppressor gene that is methylated in most mouse and human leukemias but in only a minority of other human cancers.     

Differentially methylated forms of histone H3 show unique association patterns with inactive human X chromosomes.

Studies of histone methylation have shown that H3 can be methylated at lysine 4 (Lys4) or lysine 9 (Lys9). Whereas H3-Lys4 methylation has been correlated with active gene expression, H3-Lys9 methylation has been linked to gene silencing and assembly of heterochromatin in mouse and Schizosaccharomyces pombe. The chromodomain of mouse HP1 (and Swi6 in S. pombe) binds H3 methylated at Lys9, and methylation at this site is thought to mark and promote heterochromatin assembly. We have used a well-studied model of mammalian epigenetic silencing, the human inactive X chromosome, to show that enrichment for H3 methylated at Lys9 is also a distinguishing mark of facultative heterochromatin. In contrast, H3 methylated at Lys4 is depleted in the inactive X chromosome, except in three 'hot spots' of enrichment along its length. Chromatin immunoprecipitation analyses further show that Lys9 methylation is associated with promoters of inactive genes, whereas Lys4 methylation is associated with active genes on the X chromosome. These data demonstrate that differential methylation at two distinct sites of the H3 amino terminus correlates with contrasting gene activities and may be part of a 'histone code' involved in establishing and maintaining facultative heterochromatin.     

Polycomb-mediated methylation on Lys27 of histone H3 pre-marks genes for de novo methylation in cancer

Many genes associated with CpG islands undergo de novo methylation in cancer. Studies have suggested that the pattern of this modification may be partially determined by an instructive mechanism that recognizes specifically marked regions of the genome. Using chromatin immunoprecipitation analysis, here we show that genes methylated in cancer cells are specifically packaged with nucleosomes containing histone H3 trimethylated on Lys27. This chromatin mark is established on these unmethylated CpG island genes early in development and then maintained in differentiated cell types by the presence of an EZH2-containing Polycomb complex. In cancer cells, as opposed to normal cells, the presence of this complex brings about the recruitment of DNA methyl transferases, leading to de novo methylation. These results suggest that tumor-specific targeting of de novo methylation is pre-programmed by an established epigenetic system that normally has a role in marking embryonic genes for repression.     

Le virus epstein-barr : structure et cycle biologique

The Epstein-Barr virus (EBV) is an oncogene virus which is widely spread in the human populations. Following infection, EBV establishes a latent infection which allows its persistence during the entire life of the infected host. Most often viral persistence is asymptomatic. Nevertheless Epstein-Barr virus is also associated with various malignancies of epithelial (nasopharygeal carcinoma) and B-cell origin (Burkitt's lymphoma, B-cell lymphomas and lymphoproliferative diseases of immunocompromised patients). An important part of our current knowledge provides from studies which have been conducted on EBV-infected cell lines. This review aimed at presenting these data and will most especially stress on various aspects of the biological cycle of the virus and its ability to transform cells.     

Diagnostic des pathologies liées au virus Epstein-Barr

Epstein-Barr virus (EBV), a member of the Herpesviridae familly, infects most human (95 % of the world population). In immunocompetent hosts primary infection results in acute infectious mononucleosis. In immunocompromised hosts, EBV is associated with lymphoproliferative disorders which occur with an incidence 30 to 50 higher than in the immunocompetent host. The laboratory diagnosis of an EBV infection is based on two techniques : demonstration of the virus by viral antigen or viral DNA detection and serologic responses. Direct culture of the virus on B lymphocytes is time consuming and is not of general use.     

Epigenome analyses using BAC microarrays identify evolutionary conservation of tissue-specific methylation of SHANK3

CpG islands are present in one-half of all human and mouse genes and typically overlap with promoters or exons. We developed a method for high-resolution analysis of the methylation status of CpG islands genome-wide, using arrays of BAC clones and the methylation-sensitive restriction enzyme NotI. Here we demonstrate the accuracy and specificity of the method. By computationally mapping all NotI sites, methylation events can be defined with single-nucleotide precision throughout the genome. We also demonstrate the unique expandability of the array method using a different methylation-sensitive restriction enzyme, BssHII. We identified and validated new CpG island loci that are methylated in a tissue-specific manner in normal human tissues. The methylation status of the CpG islands is associated with gene expression for several genes, including SHANK3, which encodes a structural protein in neuronal postsynaptic densities. Defects in SHANK3 seem to underlie human 22q13 deletion syndrome. Furthermore, these patterns for SHANK3 are conserved in mice and rats.     

Apoptose et virus (hormis les rétrovirus)

Apoptosis is a genetically preprogrammed cellular event which can be repressed by survival genes or activated by death genes. Numerous viral gene products bind to these genes or are homologous in sequence and function with them and block or mimic their activities, for example proteins of adenovirus and of Epstein-Barr virus (EBV). First, the initial signal for apoptosis activation, which is mediated by Fas/Apo1/TNFR complex, can be inhibited by proteins from adenovirus (E1B 19 kDa and E3), from myxomavirus (MT2), from baculovirus (iap), from herpes simplex virus and cytomegalovirus, and induced by myxoviruses, hepatitis C virus and cytomegalovirus. Secondly, the transduction of the signal to the genes of the cell death machinery (p53, pRB, bcl-2) and the transactivation of the cellular protooncogenes (c-myc, c-fos, c-jun) can be inhibited by proteins from adenovirus (E1B 19 kDa), from EBV (BHRF1, LMP1, BZLF1, EBNA-5LP), from herpesvirus (orf16), from baculovirus (p35), from cytomegalovirus (IE1/IE2/IE86), from SV40 (T), from hepatitis B virus (pX) and from papillomavirus (E6) or induced by proteins from adenovirus (E1A), from herpes simplex type 1 (VP16), from papillomavirus (E7), from polyomavirus (T), from EBV (EBNA-5), from chicken anemia virus (VP3) and from B19 parvovirus (NS1). Third, the effector phase of apoptosis which includes the proteases (caspases) can be inhibited by proteins from cowpoxvirus (crmA) and from baculovirus (p53) or induced by Sindbis virus, coxsackievirus B3, arteriviridae and dengue virus. Early cell death limits virus production, reduces spread of progeny viruses and results in virus clearance under CTL activity. Delayed apoptosis at late stages of infection, allows production and spread of high yields of progeny viruses, evading host immune inflammatory responses and protecting progeny viruses from host enzymes and antibodies. Inhibition of apoptosis contributes to the maintenance of viral latency or to cell transformation. Such virally induced apoptosis may also contribute to pathogenesis and treatment of viral diseases.     

Virus epstein-barr et pathologies épithéliales et conjonctives

Besides the lymphoid proliferations, the Epstein-Barr virus (EBV) has been detected within a wide variety of pathological tissues over the last years, due largely to the application of modern viral detection techniques. We present here the epithelial andmesenchymal EBV-associated neoplasms.