Christian Hagemeier

The Discovery of 5‐Formylcytosine in Embryonic Stem Cell DNA

Cellular development requires the silencing and activation of specific gene sequences in a well-orchestrated fashion. Transcriptional gene silencing is associated with the clustered methylation of cytosine bases (C) in CpG units of promoters. The methylation occurs at position C5 of cytosine to give 5methylcytosine (mC) with the help of special DNA methyltransferases (DNMT). [1] The DNA methylome is significantly reprogrammed at various stages during early development, [2] during the development of primordial germ cells, [2c, 3] or later in a locus-specific way at postdevelopmental stages. [4] Decreasing levels of mC can be established passively by successive rounds of DNA replication in the absence of methyltransferases. Active demethylation, in contrast, is proposed to be a process in which the mC bases are directly converted back into unmodified cytosines in the genome. [5] The recent discovery that mC can be further oxidized to hydroxymethylcytosine (hmC) with the help of TET enzymes [6] has led to the idea that hmC is connected to epigenetic reprogramming, [7] maybe as an intermediate in an, as yet controversial, active demethylation process. [4, 5, 8] Indeed recent data suggest that active demethylation in postdevelopmental phases may proceed through deamination of hmC to give 5-hydroxymethyluridine (hmU), which is then removed from the genome with the help of the base excision repair (BER) system. [9] Chemically, an attractive alternative mechanism for a more global active demethylation could be envisioned through further oxidation of hmC to give either 5formylcytosine (fC) or 5-carboxylcytosine (caC) followed by elimination of a formyl or carboxyl group, respectively

Binary specification of nonsense codons by splicing and cytoplasmic translation

Premature translation termination codons resulting from nonsense or frameshift mutations are common causes of genetic disorders. Complications arising from the synthesis of C‐terminally truncated polypeptides can be avoided by ‘nonsense‐mediated decay’ of the mutant mRNAs. Premature termination codons in the β‐globin mRNA cause the common recessive form of β‐thalassemia when the affected mRNA is degraded, but the more severe dominant form when the mRNA escapes nonsense‐mediated decay. We demonstrate that cells distinguish a premature termination codon within the β‐globin mRNA from the physiological translation termination codon by a two‐step specification mechanism. According to the binary specification model proposed here, the positions of splice junctions are first tagged during splicing in the nucleus, defining a stop codon operationally as a premature termination codon by the presence of a 3′ splicing tag. In the second step, cytoplasmic translation is required to validate the 3′ splicing tag for decay of the mRNA. This model explains nonsense‐mediated decay on the basis of conventional molecular mechanisms and allows us to propose a common principle for nonsense‐mediated decay from yeast to man.

Functional interaction between the HCMV IE2 transactivator and the retinoblastoma protein.

The 86 kDa immediate early IE2 protein of human cytomegalovirus (HCMV) can activate transcription of both viral and cellular genes and can repress transcription from its own promoter. Using two in vivo assays, we provide evidence of a functional interaction between IE2 and the retinoblastoma (RB) protein: IE2 alleviates RB‐induced repression of a promoter bearing E2F binding sites and RB alleviates IE2‐mediated repression of its own promoter. These functional effects are likely to be a result of a direct contact between IE2 and RB, which we can demonstrate both in vitro and in HCMV‐infected cells. The interaction between IE2 and RB shows similar characteristics to the interaction between RB and E1A. First, binding to IE2 requires an intact RB pocket domain. Secondly, the binding is sensitive to the phosphorylation state of RB, because cyclin A‐CDK‐induced phosphorylation of RB diminishes IE2 binding. Thirdly, the IE2 domain required for RB binding is separate to the domains necessary for TBP and TFIIB binding. Our results demonstrate that large and small DNA viruses have a common interface with the host cell, namely the association with the RB tumour suppressor protein.

The human cytomegalovirus 86K immediate early (IE) 2 protein requires the basic region of the TATA-box binding protein (TBP) for binding, and interacts with TBP and transcription factor TFIIB via regions of IE2 required for transcriptional regulation

The 86K immediate early (IE) 2 protein of human cytomegalovirus trans-activates a number of homologous and heterologous promoters, including the cellular promoter for the 70K heat-shock protein (hsp70), and the human immunodeficiency virus long terminal repeat. We have previously shown that IE2 trans-activates these two promoters in a TATA-dependent manner, and that IE2 is able to form a direct contact with TATA-box binding protein (TBP) in vitro. We now show that IE2 binds to the basic repeat region of TBP. In addition IE2 can contact a second general transcription factor, TFIIB. We have mapped the TBP- and TFIIB-binding regions within IE2 and show that these regions overlap, and also lie within parts of the protein previously identified as being required for the trans-activation and autoregulation functions of IE2.

An E2F-like repressor of transcription

The activity of a variety of genes whose products are involved in DNA replication and cell-cycle progression are regulated by E2F transcription factors, which bind to E2F sites on DNA in cooperation with members of the DP family of transcription factors. E2F sites can act as both positive and negative control elements. Transcriptional activation from E2F sites is mediated by ‘free’ E2F, whereas repression conventionally requires the formation of a complex with a pocket protein (retinoblastoma protein (RB), p107 or p130). Here we describe an E2F-like protein, termed EMA (for E2F-binding site modulating activity), which can act as a repressor of transcription without a pocket protein.

Mechanism and Stem‐Cell Activity of 5‐Carboxycytosine Decarboxylation Determined by Isotope Tracing

Eraserhead: Stem cells seem to erase epigenetic information by decarboxylation of the newly discovered epigenetic base 5-carboxycytosine (caC; see picture). This reaction is likely to involve a nucleophilic attack of the C5-C6 double bond.

The 72K IE1 and 80K IE2 proteins of human cytomegalovirus independently trans-activate the c-fos, c-myc and hsp70 promoters via basal promoter elements

Growth-regulating cellular genes or genes encoding proteins involved in cell cycle control are likely to be major targets of viral gene products in the establishment of a cellular state favourable for a permissive infection. We have examined whether infection of permissive fibroblasts with human cytomegalovirus (HCMV) results in trans-regulation of such cellular genes. Here we have shown that the proto-oncogenes c-fos and c-myc are specifically induced during immediate early (IE) and early times of HCMV infection, as has recently been shown for the heat shock protein 70 gene (hsp70). Deletion analyses and transfection assays of all three promoters showed that previously defined control sequences upstream of the constitutive promoters and downstream of the mRNA cap site are not required for this up-regulation by HCMV, such that the minimal inducible promoters of c-fos, c-myc and the hsp70 gene contained only 50 to 60 bp upstream of the transcription start site. Cotransfection assays with vectors expressing HCMV major IE cDNAs showed that the 72K IE1 and 80K IE2 proteins are involved in the up-regulation of these promoters. IE1 and IE2 products independently were able to up-regulate the minimal constitutive promoters of the constructs tested here, but trans-activation by IE1 and IE2 together was synergistic. In the case of the hsp70 promoter, promoter constructs containing a variety of different TATA elements could be activated by the 72K IE1 and 80K IE2 proteins.

Mutations and Deletions of the TP53 Gene Predict Nonresponse to Treatment and Poor Outcome in First Relapse of Childhood Acute Lymphoblastic Leukemia

PURPOSE In the clinical management of children with relapsed acute lymphoblastic leukemia (ALL), treatment resistance remains a major challenge. Alterations of the TP53 gene are frequently associated with resistance to chemotherapy, but their significance in relapsed childhood ALL has remained controversial because of small studies. PATIENTS AND METHODS Therefore, we systematically studied 265 first-relapse patients enrolled in the German Acute Lymphoblastic Leukemia Relapse Berlin-Frankfurt-Mü nster 2002 (ALL-REZ BFM 2002) trial for sequence and copy number alterations of the TP53 gene by using direct sequencing and multiplex ligation-dependent probe amplification. RESULTS We observed copy number and sequence alterations of TP53 in 12.4% (27 of 218) of patients with B-cell precursor ALL and 6.4% (three of 47) of patients with T-cell ALL relapse. Backtracking to initial ALL in 23 matched samples revealed that 54% of all TP53 alterations were gained at relapse. Within B-cell precursor ALL, TP53 alterations were consistently associated with nonresponse to chemotherapy (P < .001) and poor event-free survival (P < .001) and overall survival rates (P = .002). TP53 alterations also had a significant impact on survival within intermediate-risk (S2) and high-risk (S3/S4) relapse patients (P = .007 and P = .019, respectively). This prognostic significance of TP53 alterations was confirmed in multivariate analysis. Besides their clinical impact, TP53 alterations were associated with a higher fraction of leukemic cells in S/G(2)-M phase of the cell cycle at relapse diagnosis. CONCLUSION Alterations of the TP53 gene are of particular importance in the relapse stage of childhood ALL, in which they independently predict high risk of treatment failure in a significant number of patients. Therefore, they will aid in future risk assessment of children with ALL relapse.

Novel exon skipping mutation in the fibrillin-1 gene: two 'hot spots' for the neonatal Marfan syndrome.

The Marfan syndrome is an autosomal dominant heritable disorder of connective tissue that involves principally the skeletal, ocular, and cardiovascular systems. The most severe end of the phenotypic spectrum, the neonatal Marfan syndrome (nMFS), is characterized by pronounced atrioventricular valve dysfunction, and death often occurs within the first year of life due to congestive heart failure. Mutations in the gene coding for fibrillin‐1, FBN1, are known to cause Marfan syndrome, and have been identified in almost all exons of FBN1. Here, we describe a novel mutation affecting the invariant +1 position of the splice donor site in intron 31, associated with skipping of exon 31, in a patient with nMFS. Published reports of nMFS are reviewed and a strict definition for nMFS is suggested. If this definition is used, all nMFS mutations reported to date lie in one of two hot spots, comprising mainly missense mutations in FBN1 exons 24–27 and mutations causing skipping of exon 31 or 32.

The human cytomegalovirus immediate early 2 protein dissociates cellular DNA synthesis from cyclin-dependent kinase activation.

Passage through the restriction point late in G1 normally commits cells to replicate their DNA. Here we show that the previously reported cell cycle block mediated by the human cytomegalovirus (HCMV) immediate early 2 (IE2) protein uncouples this association. First, IE2 expression leads to elevated levels of cyclin E‐associated kinase activity via transcriptional activation of the cyclin E gene. This contributes to post‐restriction point characteristics of IE2‐expressing cells. Then these cells fail to undergo substantial DNA replication although they have entered S phase, and the induction of DNA replication observed after overexpression of cyclin E or D can be antagonized by IE2 without impinging on cyclin‐associated kinase activities. These data suggest that IE2 secures restriction‐point transition of cells before it stops them from replicating their genome. Our results fit well with HCMV physiology and support the view that IE2 is part of a viral activity which, on the one hand, promotes cell cycle‐dependent expression of cellular replication factors but, on the other hand, disallows competitive cellular DNA synthesis.