2. LA EVALUACIÓN DE LOS ESTUDIANTES: ENTRE LO NORMATIVO Y LA
2.3 Dificultades al definir y aplicar el modelo de evaluación por competencias
B cells were isolated from adenoids as depicted in Fig. 4.1 and infected with B95.8 wildtype virus at an MOI of 1.0 to analyze EBV’s methylation profile in the early phase of infection. MeDIP-on-Chip experiments were conducted with infected cells one, two, three, nine, and twelve weeks pi. The cells had different EBV genome loads, especially in the early phase of infection. Thus, the ratio of a constant point of reference was always set to one to normalize and compare the different datasets.
Fig. 4.3 Kinetics of EBV methylation in primary infected B cells
(A) MeDIP on Chip analysis of primary infected B cells at several weeks post infection (wpi). DNA from isolated B cells infected with B95.8 virus at an MOI of 1.0 was prepared after different time points pi. MeDIP analysis was carried out as described in Fig. 4.2, and samples were hybridized to our custom-made EBV tiling microarray. To analyze the kinetics of methylation pattern formation in samples with different genome loads, the
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adjusted data were processed so that the ratio of a constant point of reference, Cp, was set to one in each experiment, because the C promoter region is free of methylated CpG dinucleotides even in established LCLs (Tierney et al., 2000), in contrast to EBV genomes in Raji cells. CpG dinucleotides in EBV DNA were entirely or mostly unmethylated early after infection but became increasingly methylated in resident EBV DNA of latently infected LCLs over time. The results of one of two biological repeats are shown.
(B) The x-axis of the diagram shows the ratio of MeDIP-enriched EBV DNA versus input DNA of cells twelve weeks pi as in (A). The y-axis shows the number of CpG dinucleotides that occur in each of the 285 tiling PCR fragments of the EBV microarray. The regression curve was flat, its coefficient of determination was extremely low (R2 = 5 × 10−6), and the correlation coefficient was <0.003. The results indicated that there is no correlation
between the degree of CpG-methylation in latently infected B cells and the occurrence of CpGs in EBV DNA. (C) RNA isolated from primary B cells infected with B95.8 virus at an MOI of 1.0 was transcribed into cDNA and analyzed by semiquantitative PCR. The expression of DNA methyltransferases (DNMTs) and other genes involved in DNA methylation was assessed, as indicated on the right of the gel image with the number of PCR cycles in brackets. DNMT1 and DNMT3a show an increase in their expression level one week pi, pointing to a role in EBV’s DNA methylation. DNMT3b likewise revealed a slight increase, at much lower levels than the two other DNMTs. Expression levels of NP95, which is a protein involved in maintenance methylation by DNMT1, followed the same pattern seen for DNMT1 and DNMT3a, while PCNA, which is a proliferation marker, showed only a minor increase in expression one week pi. Expression of the housekeeping gene β-glucoronidase (GUSB) was stable in all samples. The results of one representative experiments out of three biological replicates are shown.
In contrast to Raji cells, the C promoter is unmethylated in LCLs (Tierney et al., 2000) and the ratio of MeDIP/input at the C promoter was the lowest value in all experiments. Therefore, this locus served as the reference point. Fig. 4.3 A shows the representative MeDIP-on-Chip result of one out of two biological replicas with two technical repeats each. The genome-wide methylation profile of B cells one week pi did not display a significant enrichment of any locus. This finding indicated that the DNA was unmethylated or only slightly methylated. Two weeks pi, an enrichment of methylated DNA was visible. The most substantial increase in methylation happened between week two and three pi. A manifest DNA methylation profile was visible three weeks pi comprising highly methylated regions and DNA segments that were spared from methylation like the C promoter and the EBER
locus, respectively. This profile did not further change, as the MeDIP-on-Chip profile seen nine or twelve weeks pi did not exhibit any significant difference to the profile at week three. This experiment showed that CpG-methylation of EBV DNA is a slow, but specific process that culminates in a distinct methylation profile after several weeks pi.
The amount of MeDIP DNA is not only a function of the methylation state of the fragment, but also of CpG density in the respective DNA sequence. The ratios of MeDIP/input of each microarray probe at twelve weeks pi was plotted against the number of CpG dinucleotides in the particular probes to test whether the methylation profile seen in LCLs was only dependent on the CpG density. The regression curve was flat, with a very low coefficient of determination (5×10−6), and the correlation coefficient (ranging from one for high correlation to minus one for anti-correlation) was close to zero (Fig. 4.3 B). The analysis indicated no sequence bias in MeDIP enrichment. Other mechanisms but CpG density seem to be
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important to determine the methylation profile of EBV’s DNA in LCLs.
Semiquantitative real-time (RT)-PCR experiments were performed to test, whether de novo
methylation of EBV’s DNA might stem from the upregulation of the expression of DNA methyltransferases (DNMTs) in freshly infected B cells (Fig. 4.3 C). RNA of freshly infected B cells was prepared at different time points pi and transcribed into cDNA.
The maintenance methyltransferase DNMT1 was expressed already at day one pi, but expression increased further, starting seven days pi. DNMT3a and DNMT3b encode for the two de novo methyltransferases. Both genes were weakly expressed at one day pi. The expression level started to rise seven days pi, similar to DNMT1 expression. Previous reports demonstrated that EBV’s latent membrane protein 1 (LMP1) induces the expression of all methyltransferases in nasopharyngal carcinoma cell lines (Tsai et al., 2002) and that DNMT1
expression is upregulated by LMP1 via the JNK pathway (Tsai et al., 2006). These reports suggest that LMP1 is responsible for the upregulation of DNMT expression in my experiments, as it is expressed early after infection. The nuclear protein of 95 kDa (NP95) is a protein that is associated with maintenance and de novo DNA methylation. It recruits DNMT1 to hemimethylated CpG sites in the DNA, but also interacts with de novo DNMTs, histone methyltransferases, and trimethylated H3K9 and connects the DNA methylation pathway to the establishment of repressive histone marks (Meilinger et al., 2009; Rottach et al., 2009).
NP95 was upregulated in EBV infected, primary B cells seven days pi, indicating that this protein is involved in the establishment of epigenetic modifications in EBV as well. The proliferative cell nuclear antigen PCNA is a marker for proliferating cells, but is also involved in maintenance DNA methylation through interactions with DNMT1 at the replication fork. The slight upregulation in primary infected B cells was most likely a consequence of the growth transformation of these cells caused by the EBV infection. The expression of the housekeeping gene β-glucoronidase (GUSB) was stable in primary infected B cells over time. The results of the expression analysis suggested that cellular methyltransferases introduce CpG-methylation at EBV DNA. Upregulation of important genes involved in DNA methylation paralleled the increase in DNA methylation at EBV’s DNA. The slow increase in DNA methylation of EBV’s DNA could probably be the reason for the lack of virion synthesis until day twelve pi (Kalla et al., 2010).
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