Aguirre-Arteta, Ana Maria: REGULATION OF DNA METHYLATION DURING DEVELOPMENT: ALTERNATIVE ISOFORMS OF DNA METHYLTRANSFERASE

Aus dem Institut für Biologie der Humboldt-Universität Berlin


DISSERTATION

zur Erlangung des akademischen Grades
doctor rerum naturalium
(Dr. rer. nat. )
im Fach BIOLOGIE

REGULATION OF DNA METHYLATION DURING DEVELOPMENT:
ALTERNATIVE ISOFORMS OF DNA METHYLTRANSFERASE

eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin

von Ana Maria Aguirre-Arteta,
geb. am 02.01.1968 in Bilbao (Spanien)

Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Dr. hc. Hans Meyer

Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. Bernhard Ronacher

Gutachter:
1. Prof. Dr. Harald Saumweber
2. PD Dr. Jörn Walter
Prof. Dr. Franz Theuring

Tag der mündlichen Prüfung: 28.06.00

DISSERTATION

To obtain the academic degree
doctor rerum naturalium
(Dr. rer. nat. )
in the field of BIOLOGY

REGULATION OF DNA METHYLATION DURING DEVELOPMENT:
ALTERNATIVE ISOFORMS OF DNA METHYLTRANSFERASE

submitted at the
Faculty of Mathematics and Natural Sciences I
Of the Humboldt-University Berlin

von Ana Maria Aguirre-Arteta,
geb. am 02.01.1968 in Bilbao (Spain)

Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Dr. hc. Hans Meyer

Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. Bernhard Ronacher

Gutachter:
1. Prof. Dr. Harald Saumweber
2. PD Dr. Jörn Walter
3. Prof. Dr. Franz Theuring

Tag der mündlichen Prüfung: 28.06.00

DIE WISSENSCHAFT FAENGT EIGENTLICH ERST DA AN INTERESSANT ZU WERDEN, WO SIE AUFHOERT
JUSTUS VON LIEBIG (1803-1873)

Abstract

DNA methyltransferases (DNA MTases) are enzymes responsible for DNA methylation (transfer of methyl groups to a base in the DNA) and are vital for the development of mammals. Several MTases have been identified in eukaryotes but the most abundant is Dnmt1. Furthermore, many pathological conditions are often attributed to an altered availability or function of this enzyme, however the understanding of the regulation of Dnmt1 and the concomitant relationship to diseases is far from being complete. In mammals the methylation of DNA correlates with gene activity, and methylation patterns change dramatically during early development when the genome of the mammalian embryo undergoes consecutive waves of demethylation (loss of methylation) and de novo methylation (methylation of DNA sites that have not been previously methylated). The hypothesis of this study was that alternative Dnmt1 isoforms are expressed at specific developmental stages and thus contribute to changes in the DNA methylation pattern. To study this regulation the structure of the Dnmt1 gene was determined. In this work, the tissue distribution and abundance of Dnmt1 mRNA was analyzed by Northern blot and a new, longer transcript was identified that is present in testis and skeletal muscle tissue. The novel isoform was cloned by a combination of RT-PCR and RACE techniques and found to be identical in both tissues. This new isoform differs from the ubiquitous cDNA in the 5‘ end, utilizing a new transcriptional start site and an 800 bp long alternative first exon. The cellular localization of this new transcript was determined by in situ hybridization and found to be present in the more specialized haploid spermatogenic cells, spermatids and at lower level in skeletal muscle. During muscle differentiation, the ubiquitous isoform is downregulated while the alternative isoform is upregulated. Although this mRNA codes for several short upstream ORFs which could prevent translation of the Dnmt1-specific ORF, it was found by immunofluorescence and Western blot analyses that this transcript can be translated in vivo producing a shorter Dnmt1 protein. The results shown here indicate that alternative Dnmt1 isoforms are expressed in vivo and might play an active role in the regulation of DNA methylation.

Zusammenfassung

Die DNA Methyltransferasen sind verantwortlich für die spezifische Methylierung von DNA-Basen. Mehrere DNA Methyltransferasen sind bekannt, wobei die Dnmt1 das hauptsächlich vorkommende Enzym ist. Bei Säugetieren korreliert die DNA-Methylierung mit der Genaktivität und ist essentiell für die Embryonalentwicklung. Eine beeinträchtigte Funktion oder Verfügbarkeit des Enzyms kann zu pathologisch veränderten Zuständen führen. Die Regulation der Dnmt1 und die damit verbundene Bedeutung bei der Entstehung von Krankheiten ist bisher nur unvollständig untersucht. In der Frühphase der Embryonalentwicklung von Säugetieren ändert sich das Methylierungsmuster des Genoms dramatisch. In zeitlich aufeinander folgenden Phasen wird die DNA demethyliert (Verlust der Methylgruppen) und neu methyliert (De-Novo Methylierung). Die Hypothese dieser Arbeit ist, dass verschiedene Isoformen der Dnmt1 in spezifischen Entwicklungsstadien exprimiert werden und zu Veränderungen des Methylierungsmusters der DNA beitragen. Um diese Regulation zu untersuchen, wurde die Struktur der Maus Dnmt1-Gens bestimmt. Außerdem wurde in verschiedenen Gewebetypen die Transkriptionsgröße und die Transkriptionsintensität der mRNA mit Hilfe von Northern-Blots quantifiziert. Mit diesen Experimenten konnte im Hoden- und Skelettmuskelgewebe ein längeres Dnmt1-Transkript als in anderen Geweben identifiziert werden. Dieses neue Dnmt1-Transkript wurde mit Hilfe von RT-PCR und RACE-Techniken kloniert und ist in beiden Geweben identisch. Es unterscheidet sich auf DNA-Ebene in der Sequenz des 5‘-Endes von der bisher bekannten Form der Dnmt1 und besitzt einen anderen Startpunkt für die Transkription. Darüber hinaus besitzt das neue Dnmt1-Transkript ein 800 Basenpaar großes erstes Exon, welches sich von dem des bekannten Dnmt1-Transkripts unterscheidet. Die spezifische zelluläre Lokalisation des neuen Transkripts wurde mit Hilfe der In-Situ-Hybridisierung analysiert. Mit dieser Technik wurde das alternative Transkript in stärker spezialisierten, haploiden spermatogenen Zellen (Spermatiden) und zu einem geringen Maß im Skelettmuskel nachgewiesen. Während der Differenzierung von Muskelzellen wurde eine verminderte Expression des bereits bekannten mRNA-Transkripts und eine verstärkte Expression des neu identifizierten mRNA-Transkripts festgestellt. Obwohl die mRNA der alternativen Isoform verschiedene, kurze offene Leserahmen enthält, welche die Translation eines spezifischen Dnmt1 Proteins verhindern könnten, wurde durch Immunofluoreszenz- und Western-Blot Analysen ein Translationsprodukt nachgewiesen. Nach den hier aufgezeigten Ergebnissen werden alternative Dnmt1 Isoformen in vivo exprimiert, welche eine aktive Rolle bei der Regulation der DNA-Methylierung spielen könnten.

Keywords:
DNA methyltransferase, Dnmt1, methylation, isoform

Schlagwörter:
DNA ‚Methyltransferase, Dnmt1, Methylierung, Isoform


Seiten: [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [II] [III] [IV] [i] [ii] [iii] [iv] [v]

Inhaltsverzeichnis

TitelseiteREGULATION OF DNA METHYLATION DURING DEVELOPMENT: ALTERNATIVE ISOFORMS OF DNA METHYLTRANSFERASE
TitelseiteREGULATION OF DNA METHYLATION DURING DEVELOPMENT: ALTERNATIVE ISOFORMS OF DNA METHYLTRANSFERASE
Widmung
1 Introduction
1.1DNA METHYLATION
1.1.1TYPES OF DNA METHYLATION
1.1.2DNA METHYLATION REACTION
1.1.3STRUCTURE OF A C5-CYTOSINE METHYLTRANSFERASE
1.1.4DNA METHYLATION AND GENE EXPRESSION
1.2DNA METHYLATION IN NORMAL DEVELOPMENT AND DISEASE
1.2.1DNA METHYLATION AND DEVELOPMENT
1.2.2DNA METHYLATION AND DISEASE
1.2.3DNA METHYLATION AND IMPRINTING
1.3REGULATION OF DNA METHYLATION
1.4MAMMALIAN DNA MTases
2 Materials and Methods
2.1RNA ISOLATION
2.2REVERSE TRANSCRIPTION
2.3POLYMERASE CHAIN REACTION
2.4PLASMID DNA TRANSFORMATION INTO ESCHERICHIA COLI AND SELECTION OF RECOMBINANTS
2.5SEQUENCING
2.6NORTHERN HYBRIDIZATION
2.6.1Probe labeling
2.6.2Hybridization
2.7IN SITU HYBRIDIZATION
2.7.1PROBE LABELLING
2.7.2SAMPLE PREPARATION
2.7.3HYBRIDIZATION
2.8CELL CULTURE AND TRANSFECTION
2.9IMMUNOFLUORESCENCE ANALYSIS
2.9.1TISSUE SECTIONS
2.9.2CULTURED CELLS
2.9.3IMMUNOSTAINING
2.10MICROSCOPY
2.11CELL EXTRACTS AND WESTERN BLOT ANALYSIS
3 Results
3.1GENOMIC STRUCTURE OF DNMT1 GENE
3.2ALTERNATIVE DNMT1 ISOFORMS DURING GAMETOGENESIS
3.2.1CLONING OF ALTERNATIVE DNMT1 ISOFORMS FROM TESTIS
3.2.2EXPRESSION OF THE DNMT1 ISOFORMS BY NORTHERN BLOT ANALYSIS
3.2.3LOCALIZATION OF THE DNMT1 ISOFORMS BY IN SITU HYBRIDIZATION ANALYSIS
3.2.4LOCALIZATION OF THE DNMT1 PROTEIN IN MALE AND FEMALE GERM CELLS
3.3EXPRESSION OF DNMT1 ISOFORMS DURING MYOGENESIS
3.3.1CLONING OF THE ALTERNATIVE DNMT1 ISOFORM FROM SKELETAL MUSCLE
3.3.2THE ALTERNATIVE MUSCLE ISOFORM IS EXPRESSED SPECIFICALLY IN DIFFERENTIATED MYOTUBES
3.4GENOMIC ORGANIZATION OF THE DNMT1 ISOFORMS
3.5TRANSLATION EFFICIENCY OF THE TESTIS/ SKELETAL MUSCLE ISOFORM
4 Discussion
5 List of references
Danksagung
Lebenslauf
Selbständigkeitserklärung
Abkürzungsverzeichnis Abbreviations

Tabellenverzeichnis

Table 3.1. The start codon corresponds to the third ATG (ATG3) in exon 1 (Gaudet et al., 1998). The stop codon (TAG) is located 39 bp into exon 39 and is followed bz a 311 bp untranslated region. The lines separating exon 30 and 31 show the separation between catalytic and regulatory domains. Intron sizes were determined by sequencing (GenBank accession numbers: AF 175410-175431, AF 234317-234318) and/or gel electrophoresis (Fig. 3.1). Intron phases are defined as the position of the intron within the codon.

Abbildungsverzeichnis

Fig.1.1. Reaction pathway for C5-cytosine methyltransferase based on the mechanism proposed by Wu and Santi for thymidylate synthase and tRNA-(uracil-5)methyltransferase. The attack on carbons 5 and 6 are shown in the figure. The methyl group is transferred to carbon-5 of cytosine, and the hydrogen located on carbon-5 is released as H+. During this reaction SAM is coverted to SAH. A base (B:) that abstracts a proton from carbon-5 is needed for the elimination step .
Fig.1.2. Graphic representation of the complex of the MTase HhaI covalently bound to a 13-mer DNA duplex containing its recognition sequence. The end product of the reaction, SAH, is also present (yellow). The protein is shown in brown, the DNA backbone in magenta, the DNA bases in green, and the active-site loop and the recognition loops are represented in white (right panel). The left panel is a view looking down the DNA helix axis, in which the DNA can be seen to lie between the large domain (on the right) and the small domain (on the left). On the right panel a side view from the minor groove is shown. (Fig. from ).
Fig.1.3. A model to explain the effects of DNA methylation on transcription. The relevant parameters are proximity and methylation of CpG sites. The MeCP molecules are shown as ovals, the CpG sites as filled circles (methylated) or half circles (non-methylated) circles, the arrows represent transcription from the promoter, TF1 and 2 represent transcription factors 1 and 2, sensitive or insensitive to methylation, respectively and HDAC represents the histone deacetylase. Active transcription is indicated with an arrow.
Fig.1.4. Methylation and histone deacetylation. MeCP2, a protein that binds to methylated DNA, exists in a complex with histone deacetylase and mSin3a. Nucleosomes (as several connecting disks) with their histone tails (as thin lines coming out of the nucleosomes) are shown. The two mechanisms in which histone deacetylation changes the structure of the nucleosomes are also depicted. The deacetylation by the MeCP2 complex (MeCP2, mSin3a and histone deacetylase) of the lysine amino groups on the histone tails might allow the interaction of the histone tails and the DNA backbone or might lead to compaction of the chromatin by allowing the interactions between the nucleosomes (Figure from (.
Fig.1.5. Methylation during gametogenesis and early development in mammals. The overall methylation increases during gametogenesis being higher in the male gametes. During cleavage, the genome undergoes global demethylation and after the blastocyst stage the overall methylation level remains low in the germ line and increases in the somatic lineage. There are therefore two waves of de novo methylation (grey boxes), one is during gametogenesis and the other is after implantation when tissue specific patterns are being established (Fig. modified from T. H. Bestor).
Fig.1.6. Establishment and maintenance of DNA methylation patterns. The establishment of DNA methylation patterns involves a number of processes. Left, de novo methylation: when a potential methylatable site (indicated as a circle) that is not methylated on both strands of the DNA undergoes methylation (indicated as M outside the circles). Center, maintenance methylation: the DNA molecule is composed of a parental methylated strand (thick line) and a nascent unmethylated strand (thin line). Maintenance DNA MTases add methyl groups to the nascent unmethylated cytosines that reside opposite to methylated sites on the parental strand and thus the pattern of methylation is replicated. Right, demethylation: when a methyl group (M) is removed from methylated CpG sites.
Fig.1.7. Structure of the mouse Dnmt1 protein. The Dnmt1 protein contains an N-terminal domain of 1051 amino-acids which is joined to the 570 amino-acid C-terminal domain by a run of 13 alternating lysyl and glycyl residues, shown in the figure as a thick connecting line. Binding site for PCNA: proliferating cell nuclear antigen ; NLS: nuclear localization sequence ; Ser: phosphorylation site at serine 514 ; RFTS: replication foci targeting sequence ; Zn binding: in vitro zinc binding (Cys-rich) region ; PBHD: polybromo-1 protein homologous domain ; ProCys: dipeptide part of the catalytic site ; TRD: target recognition domain ; filled boxes: highly conserved sequence motifs (I, II, IV, V, VI, VII, IX, X); grey boxes: less conserved sequence motifs (III, V, VII) between the mammalian and the bacterial MTases.
Fig. 3.1. Genomic organization of the mouse Dnmt1 gene. At the top of the figure are depicted the exons and introns, the restriction map and the localization of the cloned genomic DNA fragments within the Dnmt1 locus. B: BamHI; E: EcoRI; S: SalI; X: XbaI. The clones denominated pSP72S5 and pSP72S11 share the SalI site in exon 17. Plasmid insert sizes are indicated in italics. At the bottom of the figure the intron size determinations by agarose gel electrophoresis are shown. MTase specific primers were selected within 50 bp on both sides of the putative intron boundary and used for PCR amplification. The PCR products were separated on an agarose gel and intron sizes were determined from the standard curve of the molecular weight markers. The results are summarized in the table 3.1.
Fig. 3.2. Alternative dnmt1 isoforms in testis. (A) Short Dnmt1 genomic scheme with the location of the oligos used. (B) Agarose gels showing the products from the RT-PCR (this reaction was carried out in duplicate) and the RACE reactions, the oligos used are indicated on top of each of the gels. The PCR control without cDNA is indicated with a minus sign (-). (C) Two new alternative Dnmt1 isoform are depicted. By RT-PCR, with random oligos and subsequent nested PCR with the oligos indicated, a new alternative splicing form was found starting with exon 1 and continuing with 88 nucleotides of the testis specific exon (dark blue). By RACE analysis an extension (light blue) of the 5’ end of the t-exon was obtained using the oligos depicted in the panel B. A first RACE reaction was done with oligo 347 in combination with the adaptor primer 1 (AP1). An aliquot from this first reaction was then used in a second nested RACE reaction with oligo 348 in combination with adaptor primer 2 (AP2).
Fig. 3.3. Expression of Dnmt1 mRNA in different mouse tissues. The figure shows Northern blots containing polyA RNA from the mouse tissues indicated. The blots were hybridized with three probes as indicated by the color coding. The black vertical bar indicates the separation between the N- and C-terminal domains. In the case of brain and heart tissue where Dnmt1 mRNA levels were high, a shorter exposure is shown underneath the corresponding blot. All blots were reprobed with a beta-actin probe as a loading control.
Fig.3.4. Map of the cycle of the seminiferous epithelium of the mouse . The developmental progression of a cell is followed horizontally until the right hand border of the cycle map is reached. The cell progression continues at the left of the cycle map one row up. The vertical columns, designated by Roman numbers, depict cell associations (stages), meaning the different cell types that can be found in a testis cross section. In, intermediate spermatogonia; B, type B cells; Pl, preleptotene; L, leptotene; Z, zygotene; P, pachytene; D, diplotene.
Fig. 3.5. Localization of Dnmt1 mRNA in different adult mouse tissues and embryo. (A) and (B) show cryo sections of mouse testis, skeletal muscle and 13 day-old embryo. The hybridizations were done using probes as indicated by the color coding. Sagittal sections were used for skeletal muscle and embryo and for testis cross sections. Very weak signals (black dots) are seen in skeletal muscle (A) using the probe (depicted in blue) which also highlights the 6.2 kb band corresponding to the testis mRNA in the Northern blots (Fig. 3.3). When using the same probe in testis sections clear signals are localized on the cells closest to the lumen (spermatids). However, when using a probe that spans most of the cDNA (depicted in green), the signals are seen in most of the cells. The probe, depicted in blue, was used for embryo sections (B). Areas localized in the ear such as the lateral wall of right posterior semicircular canal (1) and right tubo tympanic recess (2) and also areas on the hip joint such as acetabular fossa (3) showed the presence of this transcript in embryo sections. The exposure times were 2 weeks for testis and embryo and 4 weeks for skeletal muscle. Negative controls (sense probe) were hybridized to adjacent sections. (C) Cross section of a seminiferous tubule. The schematic outline was taken from Clermont et al., and the diagram showing the different types of cells on a cross section from testis was taken from Setchell et al., . SG; spermatogonia, SC; primary spermatocyte, SD; spermatid, S; sertoli cell. (D)The ear and the hip anatomy are shown and the schematic outlines were taken from http://www.bcm.tcm.edu/oto/studs/anat/tbone.html and http://www.acay.com. au/~dissi/sbc/hipdys.htm , respectively. Ear anatomy, (1) Semicircular canals and (2) Tympanic membrane are indicated in the figure. Hip anatomy, (1): Centre of the femoral head; (2): Cranial acetabular edge; (3): Acetabular fossa; (4): Cranial effective acetabular edge; (5): Dorsal acetabular edge; (6): Caudal acetabular edge; (7): Femoral head/neck reconturing; (8): Femoral ahead reconturing.
Fig. 3.6. Localization of Dnmt1 during oogenesis. (A) Scheme of the development of an ovarian follicle is depicted (figure modified from R. Rugh). (B) Localization of the Dnmt1 protein by immunofluorescence staining with the rabbit polyclonal pATH5 antibody, detected with the Rhodamine conjugated anti-rabbit IgG antibody. The DNA counterstaining was performed with Hoechst 33258 to locate cell nuclei. The Dnmt1 protein is localized on the cytoplasm of the oocyte as indicated by the intense staining with the pATH5 antibody. - Ab control: represents the control without the first antibody.
Fig. 3.7. Localization of Dnmt1 in seminiferous tubules. The figure shows the localization of the Dnmt1 protein by inmunofluorescence staining with the rabbit polyclonal pATH5 antibody, detected with the Rhodamine conjugated anti rabbit IgG antibody. The DNA counterstaining was performed with Hoechst 33258 to locate the cell nuclei. The Dnmt1 protein is localized in spermatogonial cells which are the cells with a basal localization. The Dnmt1 is also seen in spermatids which are the cells closer to the lumen. -Ab control: indicates the control without the first antibody.
Fig. 3.8. Cloning of the skeletal muscle Dnmt1 isoform. (A) 3‘ RACE was used to screen for differences at the 3‘ end which could account for the novel Dnmt1 transcript in skeletal muscle. A forward oligo in exon 37 (371) in combination with the adaptor primer (AP) were used. The PCR reaction was carried out in duplicate and the agarose shows the PCR products obtained whose size corresponds to approximately 800 bp. The PCR products were then cloned into the pCRII vector (sequence in capital letters) and sequenced. sk.m, corresponds to PCR product from skeletal muscle cDNA; -, represents the minus cDNA control PCR. The sequence obtained shows no new exons (from exon 37 to 39) and utilization of the first polyadenylation signal (indicated by a red box)(section 3.1 and ). (B) Screening for the presence of alternative exons by comparing RT-PCR products from skeletal muscle cDNA with a plasmid containing the ubiquitously expressed Dnmt1 cDNA. Skeletal muscle RNA was reverse transcribed using random primers. Different PCR reactions were done using oligo pairs as indicated, spanning the known Dnmt1 cDNA from exon 5 (ex. 5) to exon 37 (ex. 37). -RT, corresponds to the negative control without reverse transcriptase; -, corresponds to the negative control for the PCR reaction without cDNA template; C, PCR product from the plasmid; sk. m, PCR product from skeletal muscle cDNA. (C) To screen for new 5‘ end and/or alternative exons, a combination of oligos were used in 5‘ RACE and RT-PCR reactions using skeletal muscle mRNA. The corresponding location of the primers used and the 5‘ genomic structure (see also Fig. 3.10) of the ubiquitous Dnmt1 (exon 1 to 9) as well as the testis specific isoform (indicated in blue) are depicted in the diagram. Reverse transcriptase reactions were done both with random and with and oligo (403) located in exon 9, and the RACE reaction with an oligo positioned in exon 4. -RT, as in (B); -, as in (B); +, positive PCR control. All the PCR products were cloned into pCRII and sequenced. From the RACE reactions two isoforms were obtained multiple times, which contain either exon 1 or the isoform isolated from testis (section 3.2.1). RT-PCR analysis using primers at different positions of the testis specific exon further confirmed the presence of this isoform in skeletal muscle mRNA. The sequence of the longest PCR product (GenBank accession number AF 175432) is shown underneath and the different exons are color coded as in the genomic structure.
Fig.3.9. (A) The images and diagram above illustrate the myogenic differentiation system. Upon serum deprivation myoblasts (MB) fuse to form multinucleated myotubes (MT) which express several muscle-specific proteins such as the basic helix-loop-helix transcription factor myogenin. (B) Location of the oligos used for RT-PCR. (C) RT-PCR products (with and without reverse transcriptase, + and -) were analyzed in agarose gels. A 343 bp fragment was obtained in MT but not in MB using oligos located in the testis/skeletal muscle exon (depicted in blue) and in exon 4. For the ubiquitously expressed isoform, oligos located in exon 1 and exon 4 were used in the RT-PCR reaction, and the expected fragment of 264 bp was obtained in MB and at lower level in MT. RT-PCR with GAPDH specific oligos was used as a loading control giving rise to the 463 bp product seen at equal amounts in MB and MT. As a control for myogenic differentiation oligos that amplify a 450 bp fragment of the myogenin transcription factor were used and, as expected, a product is obtained in MT and not in MB.
Fig. 3.10. The mouse Dnmt1 gene contains at least three alternative exons at the 5‘ end. The diagram depicts the genomic structure of the 5‘ end of murine Dnmt1. Three alternative 5‘ exons have been identified: one is specific to the oocyte, one to the testis and skeletal muscle and one to somatic cells. The exons of Dnmt1 are represented to scale with respect to their size and relative position in the gene (GenBank accession number AF 175410-175412). The exons present in each isoform are indicated and are renamed following the chronological order of identification: 1a, exon 1 ; 1b, spermatocytes , spermatids and skeletal muscle exon (this work); 1c, oocyte exon . CpG and GpC incidence diagrams are plotted below to scale. The presence of a CpG island around exon 1a is a typical feature of housekeeping genes and fits well with the ubiquitous expression of this isoform.
Fig. 3.11. The skeletal muscle Dnmt1 transcript is translatable. Different expression constructs were designed to test whether the skeletal muscle isoform could generate a truncated Dnmt1 protein. (A)The strategy followed to generate some of the Dnmt1 expression constructs is depicted. In the first PCR cycle all the reagents necessary for a PCR reaction but the flanking oligos were included. The first cycle allows the annealing and extension of the overlapping area between the two denaturated DNAs (DNA1 and DNA2). In the next cycles the oligos were added and amplification of the desired area occurs. (B) The different epitope tagged Dnmt1 expression constructs used to transfect COS-7 cells; 1a-9, 1b-9, 3-9, 1b‘-9 and 1b‘‘-9 (b‘ and b‘‘-9 correspond to nucleotides 651 to 1418, respectively from GenBank sequence accession number AF 175432). The exons included in each of the constructs are indicated (see Fig. 3.10 for exon nomenclature). The dotted lines in exon 1b, which is found in testis and skeletal muscle cDNA, represent the areas which were deleted in the specific construct. The small flags at the end of each construct represent the 8-amino acid FLAG coding sequence which was added to each of them. ORFs starting at ATG2 and ATG4 in exons 1a and 4 respectively of the ubiquitous Dnmt1 isoform cDNA and finishing in the FLAG tag are shown in thick lines and the short ORFs are shown in thin lines. The short vertical lines correspond to ATGs in the particular reading frame and the stars represent stop codons. The different expression plasmids were used for transient transfection of COS-7 cells. (C) The two types of Dnmt1 protein bands obtained are shown in the Western blot and were detected with the anti-FLAG M2 mouse monoclonal antibody, using the Dnmt1 constructs 1a-9 (lane1) and 1b‘‘-9 (lane2). (D) Expression of the FLAG tagged Dnmt1 protein (thick lines) was analyzed by immunofluorescence staining with the anti-Flag M2 mouse monoclonal antibody. The DNA counterstaining was performed with Hoechst 33258 and the corresponding phase contrast (PC) images are also shown. The mock transfection is depicted at the top using similar conditions.

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