Aus dem Max-Delbrück-Centrum für molekulare Medizin
der Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin

A complex interplay of regulatory domains controls cell cycle dependent subnuclear localization of DNMT1 and is required for the maintenance of epigenetic information

DISSERTATION

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

eingereicht an der

Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin

von
Hariharan P. Easwaran

geb. am 16.09.1974 in Mumbai (Indien)

Präsident der Humboldt-Universität zu Berlin

Prof. Dr. Jürgen Mlynek

Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I

Dekan: Prof. Dr. Michael Linscheid

Gutachter:
1. Gutachter:
2. Prof. Dr. Harald Saumweber
3. Prof. Dr. Heinrich Leonhardt
4. Prof. Dr. Peter B. Becker

Eingereicht: 23. April 2003

Tag der mündlichen Prüfung: 1. October 2003

Summary

DNA methylation constitutes an essential epigenetic mark controlling chromatin organization and gene regulation in higher eucaryotes, which has to be duplicated together with the genetic information at every cell division cycle. In mammals duplication of DNA methylation is mediated by DNA methyltransferase-1 (DNMT1). It associates with sites of nuclear DNA replication, called replication foci (RF), and thereby couples maintenance of DNA methylation to DNA duplication. In this work, we have analyzed the role of regulatory sequences in the N-terminal domain of DNMT1 in controlling its subnuclear localization throughout the cell cycle, and the evolutionary conservation of these sequences and of the mechanisms that mediate association of proteins with RF.

We provide evidence that DNMT1 shows dynamic subnuclear distribution that is controlled by the regulatory sequences depending on the cell cycle stage. To determine the subnuclear distribution of DNMT1 throughout the cell cycle, an RFP-Ligase fusion protein was developed as a marker that allows identification of the cell cycle stage in live cells. Various DNMT1 mutants fused to GFP were coexpressed with RFP-Ligase and imaged by 4-dimensional live cell microscopy during an entire cell cycle. The PBD (PCNA binding domain) drives the localization of DNMT1 at RF throughout S phase and the TS (targeting sequence) mediates retention of DNMT1 only at the late replicating pericentric heterochromatin from late-S phase until early-G1. In contrast, the PBHD (polybromo homology domain) seems to be required for unloading DNMT1 from the pericentric regions in G1. Overexpression of the TS to interfere with this association lowers cell viability and induces the formation of micronuclei and coalescence of centromeric heterochromatin. These results bring forth a novel function of the TS in mediating association of DNMT1 with pericentric heterochromatin from late-S phase through G2 until mitosis, which is important for maintenance of DNA methylation, and heterochromatin structure and function. Database searches indicate that the TS is a domain unique to the DNMT1 family of proteins. Amongst the DNMT1 family, only the metazoan DNMT1 proteins have the PBD. This suggests that coupling of maintenance of DNA methylation with DNA replication occurs only in metazoans, while plants and fungi have alternative mechanisms that maintain DNA methylation patterns, probably mediated by the TS.

The evolutionary conservation of the mechanisms by which proteins associate with RF in mammalian cells was directly tested by analyzing the ability of mammalian replication proteins PCNA and DNA Ligase I as well as DNMT1 to associate with RF in Drosophila cells. Of all the proteins tested, only PCNA associated with RF while the others showed diffused nuclear distribution although they contain a functional PBD. Surprisingly, Drosophila DNA Ligase I associates with RF in mammalian but not in Drosophila cells. These results suggest differences in the dynamics and organization of the replication machinery in these distantly related organisms, which correlates with the increased size and complexity of mammalian genomes.

Zusammenfassung

DNA-Methylierung spielt eine wichtige Rolle bei der Kontrolle der Chromatinorganisation und Genregulation in höheren Eukaryoten und muss zusammen mit der genetischen Information in jedem Zellzyklus dupliziert werden. Bei Mammalia wird DNA durch die DNA-Methyltransferase 1 (DNMT1) methyliert, die dabei mit nukleären Replikationsstellen (RF) assoziiert und so die Erhaltung des Methylierungsmusters mit der Duplikation der DNA verbindet. In dieser Arbeit wurden die Funktion der regulatorischen Sequenzen in der N-terminalen Domäne von DNMT1 bei der Kontrolle ihrer subnukleären Lokalisierung während des Zellzyklus und die evolutionäre Konservierung dieser Sequenzen, sowie die Mechanismen die eine Assoziation von Proteinen mit RF vermitteln, untersucht.

Es konnte gezeigt werden, dass DNMT1 eine dynamische Verteilung im Kern aufweist, die durch regulatorische Sequenzen zellzyklusabhängig gesteuert wird. Um die subnukleäre Verteilung von DNMT1 während des Zellzyklus zu untersuchen, wurden RFP-Ligase Fusionsproteine hergestellt, die als Marker für die Identifikation von Zellzyklusstadien in lebenden Zellen dienen. Verschiedene, mit GFP fusionierte DNMT1 Mutanten wurden zusammen mit RFP-Ligase exprimiert und über einen ganzen Zellzyklus hinweg mit 4-dimensionaler Lebendzellmikroskopie verfolgt. Die PBD (PCNA-Bindungsdomäne) bewirkt die Lokalisierung von DNMT1 an RF während der S-Phase, und die TS ( t argeting s equence) vermittelt die Retention von DNMT1 an spät replizierendem Heterochromatin von der späten S- bis zur frühen G1-Phase. Im Gegensatz dazu scheint die PBHD (Polybromohomologiedomäne) für die Freisetzung von DNMT1 von perizentrischen Regionen während der G1-Phase notwendig zu sein. Eine Überexpression der TS zu Störung dieser Assoziation, senkt die Überlebensrate der Zellen und fördert die Bildung von Mikronuklei sowie die Verschmelzung von zentromerem Heterochromatin. Diese Ergebnisse zeigen eine neue Funktion für die TS bei der Assoziation von DNMT1 mit perizentrischem Heterochromatin von der später S- über die G2-Phase bis hin zur Mitose, die eine wichtige Voraussetzung für die Erhaltung der DNA-Methylierung und Heterochromatinstruktur und –funktion ist. Datenbankanalysen zeigten, dass es sich bei der TS um eine einzigartige Domäne innerhalb der DNMT1 Proteinfamilie handelt. Innerhalb der DNMT1 Familie besitzen nur die DNMT1 Proteine der Metazoen die PBD. Das lässt vermuten, dass die Verknüpfung von Beibehaltung der DNA Methylierung mit der DNA Replikation nur in Metazoen auftritt, während in Pflanzen und Pilzen alternative Mechanismen zur Aufrechterhaltung des Methylierungsmusters, wahrscheinlich vermittelt durch die TS, zur Anwendung kommen.

Die evolutionäre Konservierung von Mechanismen, zur Assoziation von Proteine mit RF in Säugerzellen, wurde durch die Analyse der Säugerproteine PCNA, DNA Ligase I und DNMT1 in Drosophila-zellen direkt getestet. Von allen untersuchten Proteinen assoziiert nur PCNA mit RF, während die anderen nur eine diffuse Verteilung innerhalb des Kerns zeigten, obwohl sie eine funktionale PBD enthalten. Überraschenderweise assoziierte auch die Drosophila DNA Ligase I in Säugerzellen nicht aber in Drosophila-zellen mit RF. Diese Ergebnisse weisen auf Unterschiede in der Dynamik und dem Aufbau der Replikationsmaschinerie in diesen entfernt verwandten Organismen hin, was mit der Vergrösserung und höheren Komplexität des Säugergenoms korreliert.

Inhaltsverzeichnis

• 1.  Introduction
  • 1.1. Genetic and Epigenetic information
  • 1.2. Replication of genetic information
    • 1.2.1. DNA replication origins
    • 1.2.2. DNA replicates in discrete sites in the nucleus called replication foci
    • 1.2.3. Temporal and spatial order of DNA replication
    • 1.2.4. Specific protein sequences mediate assembly of replication factors at RF
  • 1.3. Epigenetic information
    • 1.3.1. Types of epigenetic information
    • 1.3.2. Role of DNA methylation
    • 1.3.3. Regulation of DNA Methylation
    • 1.3.4. Enzymes involved in methylating DNA
    • 1.3.5. Organisms that lack DNA methylation
  • 1.4. Questions addressed in this work
• 2.  Materials and Methods
  • 2.1. Construction of plasmids encoding various fusion proteins
  • 2.2.  Cell culture and transfection
    • 2.2.1. Drosophila cells
    • 2.2.2. Mammalian cells
  • 2.3. Cell extracts and western blot analysis
  • 2.4. Cell cycle and immunofluorescence analysis
    • 2.4.1. BrdU labeling of replication foci and immunostaining
    • 2.4.2. BrdU pulse-chase for identification of cells in G2 phase
    • 2.4.3. Localization of DNMT1 at mitotic chromatin
    • 2.4.4. Obtaining cells in G1
  • 2.5.  Microscopy
  • 2.6.  Live cell microscopy
  • 2.7. Sequence analysis
    • 2.7.1. Search for DNA Ligase I homologue in Drosophila
    • 2.7.2. Multiple sequence alignments, profiles and profile search
    • 2.7.3. PSI-BLAST searches
• 3.  Results
  • 3.1. Association of proteins with RF in Drosophila
    • 3.1.1. PCNA is highly conserved in S. cerevisiae, D. melanogaster and mammals
    • 3.1.2. Human PCNA and Drosophila PCNA can associate with RF across the two organisms
    • 3.1.3.  Subnuclear localization of DNMT1 in Drosophila cells
    • 3.1.4.  Search for an RFTS in Drosophila
  • 3.2.  Regulation of subcellular localization of DNMT1 in mammalian cells throughout the cell cycle
    • 3.2.1. Targeting preference of the three targeting domains of DNMT1
    • 3.2.2. TS associates with late replicating pericentric heterochromatin
    • 3.2.3. Subnuclear localization of DNMT1 throughout the cell cycle
    • 3.2.4. Comparison of dynamics of TS with DNMT1: what determines release of DNMT1 from pericentric heterochromatin during G1?
  • 3.3.  Biological effects of TS overexpression
    • 3.3.1. Effect on cell viability
    • 3.3.2. Effect on nuclear organization and morphology
  • 3.4.  Evolutionary conservation of the regulatory domain of DNMT1
    • 3.4.1. PBD is present only in metazoan DNMT1 family
    • 3.4.2.  TS is a unique domain present only in DNMT1 family from animals, plant and fungi
• 4.  Discussion
  • 4.1. The mammalian DNMT1 does not associate with RF in Drosophila cells
  • 4.2. Targeting of DNMT1 to RF in mammalian cells is driven solely by the PBD
  • 4.3. The TS mediates cell cycle dependent binding of DNMT1 to pericentric heterochromatin
  • 4.4. Alternative mechanism of inheritance of DNA methylation in plants and fungi
  • 4.5. Mechanism of maintenance of epigenetic information by DNMT1 in mammals
• 5.  Outlook
• 6.  Postface
• 7.  Videos
• Abbreviations
• Bibliography
• A) Acknowledgements
• C) Statement

Tabellen

• Table 2.1. Oligonucleotides used for generating PBD-GFP fusions
• Table 2.2. Excitation and emission maxima of various fluorophores used.
• Table 2.3. Excitation and emission filters used for detecting the signals from the different fluorophores and fluorescent proteins.
• Table 3.1. Efficiency of association of various fusion proteins with replication foci in Drosophila and mammalian cells
• Table 3.2: Summary of association (+) or no association (-) of various proteins with RF during different stages of S phase or with pericentric heterochromatin during different stages of the cell cycle. 'Late-S' and 'Throughout' mean association with pericentric heterochromatin occurs only during late-S phase or throughout S phase, respectively.

Bilder

• Fig. 1.1. Distinct spatio-temporal patterns of RF in S phase mammalian nuclei. Mouse cells in S phase displaying the distinct patterns of RF. Euchromatin replicates in early-S phase and heterochromatin replicates in late-S phase The RF are labeled with a GFP-PCNA fusion protein.
• Fig. 1.2. Schematic representation of RF. (A) The three regions in the N-terminal domain that have been shown to independently associate with RF. (B) Schematic of the RF. Various proteins are shown as boxes with protrusions to depict the protein domains that interact with other members of the RF by protein-protein interactions. The different shapes of the protrusions are meant to depict different domains that may play a role in targeting proteins to the RF. The central green ring is the PCNA trimer that encircles DNA (not shown). Association of proteins with the RF is principally mediated by interaction with PCNA. DNMT1 and DNA Lig I are two typical proteins which are targeted to the RF by interaction of their PBD with PCNA. The TS and PBHD are depicted to interact with other unidentified proteins in the complex.
• Fig. 1.3. Mechanism of transfer of methyl group to C5-cytosine based on the mechanism proposed by Wu and Santi (Wu and Santi, 1987) for thymidylate synthase and tRNA-(uracil-5)methyltransferase. A cysteine thiol of the enzyme attacks the 6-carbon of cytosine and forms a covalent DNA-enzyme intermediate. The resulting carbanion at 5-carbon of cytosine then attacks the methyl group of SAM (AdoMet) forming a covalent bond with the methyl group and SAH (AdoHcy) is released. Elimination of the conjugate occurs through abstraction of the proton from carbon-5 by a base (B:) to yield the product 5-methylcytosine.
• Fig. 1.4. Models for effect of methylation on gene activity. Unmethylated DNA is depicted as half circles and methylated DNA as filled circles. (A ) Methylation directly prevents binding of transcription factor (TF) thereby inhibiting transcription. (B) Methyl DNA binding proteins bound to the methylated promoter prevent binding of TF and inhibit transcription. (C) MeCP2 complex containing HDAC binds to methylated
• Fig. 1.5. Processes that change or maintain DNA methylation pattern. In vertebrates DNA methylation occurs mainly in CpG dinucleotides depicted here as CG. Methyl residues are depicted as ‘m’. New methylation patterns are established by the process of de novo methylation (left). Existing methylation pattern can be erased by demethylation (right). During DNA replication (centre), the newly synthesized DNA strand (thin line) is unmethylated while the parent strand (thick line). retains its methylation pattern. The methylation pattern from the parent strand is copied on to the daughter strand by maintenance methyltransferase.
• Fig. 1.6. Five families of eucaryotic MTases. Phylogenetic relationship between known MTases based on comparison of the conserved motifs in the catalytic domains (adapted from (Colot and Rossignol, 1999)). The eucaryotic MTases group into five families (boxed): DNMT1, DNMT2, DNMT3, Chromomethylase (CMT), Masc1 (only one member). Some of the recently identified proteins (CMT3 and DNMT3L) are not shown. MTases from eubacteria and archaebacteria are divergent and lie scattered.
• Fig. 1.7. Domain structure of DNMT1. The somatic long isoform of DNMT1 is shown here. DMAP corresponds to the region in DNMT1 that binds DMAP (Rountree et al., 2000). PBD (PCNA binding domain) (Chuang et al., 1997), TS (targeting sequence) (Leonhardt et al., 1992) and PBHD (polybromo homology domain) (Liu et al., 1998) are reported to target to replication foci. P marks an identified phosphorylation site (Glickman et al., 1997). NLS is the nuclear localization signal (Cardoso and Leonhardt, 1999b). Zn-1 (Bestor, 1992) and Zn-2 (Chuang et al., 1996) are the two Zn binding domains. DB is a DNA binding domain just preceding the TS (Chuang et al., 1996). HDAC1 corresponds to the region in DNMT1 that binds to HDAC1 (Fuks et al., 2000).
• Fig. 1.8. Coupling of DNA methylation with DNA replication. Association of DNMT1 with the replication machinery mediated by RFTS (marked as T) couples maintenance of DNA methylation with DNA replication. The replication machinery is shown tethered to the nuclear matrix. RFTS mediated association of DNA Ligase I with the replication machinery is also shown.
• Fig. 3.1. Drosophila and human PCNA are highly similar. Sequence alignment of HsPCNA (human), DmPCNA (Drosophila) and ScPCNA (budding yeast). The protein sequences were aligned using the Jotun-Hein method (in Lasergene software). Residues identical to HsPCNA are boxed. HsPCNA is about 35% identical to ScPCNA and 70% identical to DmPCNA. Regions in HsPCNA that interacts with the PBD of p21 (Gulbis et al., 1996) are in red.
• Fig. 3.2. A conserved PBD in proteins involved in DNA metabolism. Alignment of the PBD from various homologous proteins from different organisms. Accession numbers are indicated in the second column where available. Asterisk denotes the beginning or end of the protein. Identical residues are highlighted in red and conserved substitution is highlighted in green. Association with replication foci requires both the conserved residues and the stretch of peptide rich in basic amino acids (in blue) (Montecucco 1998). (Hs: Homo sapiens; Mm: Mus musculus; Xl: Xenopus laevis; Dm: Drosophila melanogaster; Ce: Caenorhabditis elegans; Sp: Schizosaccharomyces pombe; Sc: Saccharomyces cerevisiae; Mj: Methanococcus jannaschii).
• Fig. 3.3. Mammalian and Drosophila PCNA associate with RF interchangeably between these divergent organisms. Drosophila S2 and mammalian C2C12 cells were transfected, pulse labeled with BrdU to label sites of active DNA replication, and fixed with formaldehyde followed by immunostaining. (A) S2 cells transfected with plasmid encoding HsPCNA and coimmunostained with anti-PCNA (FL261; Santa Cruz) and anti-BrdU antibodies. Cells positively staining with anti-BrdU were in S phase at the time of BrdU labeling. Anti-PCNA antibody (FL261) specifically stains one transfected cell (arrow) expressing HsPCNA that is in S phase and does not stain endogenous PCNA in the other non-transfected S phase and non-S phase cells. (B) S2 cells transfected with plasmid encoding HsPCNA (top) or GFP-HsPCNA (bottom) and immunostained with anti-BrdU. HsPCNA was detected by coimmunostaining with anti-PCNA antibody (FL261) as in (A). Overlay shows colocalization of HsPCNA and GFP-HsPCNA with BrdU foci. (C) C2C12 cells transfected with plasmid encoding GFP-DmPCNA and immunostained with anti-BrdU. GFP-DmPCNA colocalizes with BrdU foci. Scale bar = 2 µm.
• Fig. 3.4. Deletions of DNMT1 used for evaluating the ability of PBD, TS and PBHD to associate with RF in S2 cells. The somatic isoform, DNMT1(l), is illustrated at the top. Various deletions in the N-terminus fused to GFP/YFP/ß-gal epitope are shown. The predicted molecular weights of the fusion proteins are indicated. See Fig 3.12 for Western blot analysis of the fusion proteins. The boundaries of the different domains and deletions are indicated by the amino acid numbers corresponding to the DNMT(l) isoform. See Fig. 1.7 for description of the domains. An SV40 NLS was added in PBHD-YFP for efficient nuclear targeting.
• Fig. 3.5. Subnuclear localization of NMT 2 -GFP and DNMT1(s) with RF in S2 cells. Cells were transfected and pulse labeled with BrdU 24 hrs later followed by indirect immunostaining for BrdU. (A) C2C12 cells transfected with plasmid encoding NMT2-GFP. Colocalization of NMT2-GFP with BrdU is seen in the overlay as yellow. (B) S2 cells transfected with plasmid encoding NMT2-GFP. NMT2-GFP does not colocalize with BrdU. In most cells it aggregates to form structures (arrows in bottom panel) that are excluded from BrdU incorporation sites. (C) S2 cells transfected with a plasmid encoding DNMT1(s). Cells were immunostained with anti-pATH52 to detect DNMT1. In 80-90 % of cells, DNMT1 is excluded from BrdU sites (top panel). In about 10% of S2 cells, DNMT1 partially colocalizes with BrdU incorporated sites (bottom panel). Scale bar = 2 µm
• Fig. 3.6. GFP-HsLig does not associate with RF in Drosophila S2 cells. Cells were transfected with plasmid encoding GFP-HsLig and pulse labeled with BrdU 24 hrs later followed by indirect immunostaining for BrdU. (A) In S2 cells, GFP-HsLig shows diffused nuclear localization in all cells observed. (B) GFP-HsLig colocalizes with BrdU labeled RF in C2C12 cells seen as yellow colour in overlay. Scale bar = 2 µm
• Fig. 3.7. PBD, PBHD and TS do not mediate association with RF in S2 cells. S2 cells were transfected and pulse labeled with BrdU 24 hrs later followed by indirect immunostaining for BrdU. (A) S2 cells transfected with plasmid encoding MTPBD-GFP (top panel) or HsLigPBD-GFP (bottom panel). Both PBD-GFP fusions show diffused nuclear distribution. (B) S2 cells cotransfected with plasmids encoding MTPBD-GFP and HsPCNA. Presence of HsPCNA does not aid association of MTPBD-GFP with RF as MTPBD-GFP still shows diffused nuclear distribution. (C) S2 cells transfected with plasmid encoding TS-ß-gal. ß-gal epitope was detected by indirect immunostaining with anti-ß-gal antibody. (D) S2 cells transfected with plasmid encoding PBHD-YFP. Both TS-ß-gal and PBHD-YFP show diffused nuclear distribution. Scale Bar = 2 µM.
• Fig. 3.8. Basis for selection of the EST clone LD41868(CG5602) as the putative DNA ligase I homologue in Drosophila. BLAST search of the D. melanogaster genome and EST database with DNA Ligase I (from different species) identified three similar sequences CG5602, CG17227 and CG12176. (A) Each of the putative DNA ligase protein sequences identified in Drosophila is aligned pairwise with HsDNA Lig I. The percentage identity obtained from the alignment is indicated at the right. CG5602 shows maximum identity to HsDNA Lig I compared to the other two sequences. (B) Table showing the percentage identity obtained from pairwise alignment of the three putative DNA ligase homologues in Drosophila with each of HsDNA Ligase I, III and IV. CG17227 and CG12176 show more identity with HsDNA Lig III and IV respectively. (C) Phylogenetic tree from the comparison of HsDNA ligases and the Drosophila homologues created in Lasergene program (J. Hein method with PAM250 residue weight table. Scale represents the amino acid substitution).
• Fig. 3.9. DmDNA Lig I gene, putative protein and potential PBD. (A) Structure of DmDNA Lig I gene and the predicted protein. The gene structure is illustrated at the top and position of exons are marked by nucleotide numbers. The regions shown to be spliced out are the introns (white boxes). A region in the 5-th exon (marked gray) was found to be absent in the cDNA clone. This region was not detected as an intron using programs to detect introns. Middle shows the LD41868 cDNA clone obtained from ResGen. The translational start is shown by the bent arrow and the protein produced from this cDNA clone is illustrated at the bottom. (B) The DmDNA Lig I protein sequence was searched for presence of a PBD (including the deleted region) using ProfileSearch. The sequence which showed all the features of a PBD essential for association with RF is labeled as DmDNA Lig I-PBD. The other two hits show weak similarity. Asterisk indicates the beginning or end of the protein sequence.
• Fig. 3.10. Deletion constructs used for mapping of the RFTS in DmDNA Lig I. (A) Organization of domains in DmDNA Lig I is shown at the top. Various deletions of DmDNA Ligase I fused to GFP at the N-terminus were made as described in Methods. The predicted molecular weight of the fusion proteins is indicated on the left. (B) Western blot analysis of the chimeric proteins confirmed their protein sizes. COS7 or 293T cells were transfected with the fusion constructs shown in (A) and after 24 hrs of incubation the cells were lysed by boiling in Laemmli's loading buffer and analyzed by immunoblotting with rabbit polyclonal antibody against GFP.
• Fig. 3.11. Subcellular localization of the DmDNA Lig I deletions fused to GFP. Cells were transfected with plasmids encoding the various deletions of DmDNA Lig I fused to GFP and pulse labeled with BrdU after 24 hrs followed by indirect immunostaining for BrdU. (A) None of the fusion proteins associate with RF in S2 cells. (B) Only the full length DmDNA Ligase I fused to GFP (GdL) associates with RF in C2C12 cells. GdL-2 and GdL-4 are not efficiently taken up into the nucleus. Scale Bar = 2 µm.
• Fig. 3.12. DmLigPBD-GFP does not associate with RF in S2 cells. Cells were transfected with a plasmid encoding DmLigPBD-GFP and pulse labeled with BrdU 24 hrs later followed by indirect immunostaining for BrdU. (A) Colocalization of DmLigPBD-GFP with BrdU in C2C12 cells. (B) DmLigPBD-GFP does not colocalize with BrdU in S2 cells. Scale Bar = 2 µm
• Fig. 3.13. Different stages during S phase can be identified in an asynchronously growing population of mammalian cells by the pattern of RF in the nucleus. Mouse C2C12 cells were pulse labeled with 10µM BrdU for 10 min and fixed with ice cold methanol and immunostained with FITC conjugated anti-PCNA antibody (Pharmingen). The cells were fixed again with 3.7% formaldehyde and immunostained with anti-BrdU (Harlan Seralab) antibody followed by detection with Cy5 conjugated secondary antibody. Cells showing different patterns of RF typical of early-, mid- and late-S phases are shown. Non-S phase cells are not labeled with BrdU and show diffused distribution of PCNA. Scale Bar = 2 µm
• Fig. 3.14. Deletions of DNMT1 used for studying the preference of PBD, TS and PBHD to associate with RF in mammalian cells. (A) The two isoforms of DNMT1 are shown at the top. DNMT1(l) is the larger somatic isoform and DNMT1(s) is the short isoform expressed in some cells, e.g. oocytes, that differ only in the first 118 amino acids encoding the DMAP binding region. Various deletions of DNMT1(s) were fused to GFP/YFP as listed. The predicted molecular weights of the fusion proteins are indicated. The boundaries of the different domains and deletions are indicated by the amino acid numbers corresponding to the DNMT(l) isoform. The DNMT1(s) isoform was used in making the deletions because the DMAP had no influence on targeting of the protein to RF. See Fig. 1.7 for description of the domains. An SV40 NLS was added in PBHD-YFP for efficient nuclear uptake. F-TS-GFP contains an N-terminal FLAG epitope tag. (B) Western blot analysis of the chimeric proteins showing they are correctly expressed. COS7 or 293T cells were transfected with the fusion constructs shown in (A) and after 24 hrs of incubation the cells were lysed by boiling in Laemmli's loading buffer and analyzed by immunoblotting with the mentioned antibodies.
• Fig. 3.15. Endogenous DNMT1 associates with RF throughout S phase. C2C12 cells were pulse labeled with 100 µM BrdU for 10 min and fixed with 3.7% formaldehyde and immunostained for DNMT1 with anti-PATH52 antibody. In the late-S phase cell, arrowhead illustrates an exemplary late replicating centromeric heterochromatin which colocalizes with the Hoechst bright spots, and the arrow shows smaller foci which do not colocalize with Hoechst spots. Scale Bar = 2 µm.
• Fig. 3.16. Subnuclear localization of various deletions of DNMT1 in early- and late-S phase nuclei. Mouse C2C12 cells were transfected with the indicated plasmid constructs and BrdU labeled 24 hrs later followed by indirect immunostaining for BrdU. Cells transfected with TS-ß-gal were immunostained with antibody against ß-gal. The TS associates with RF specifically during late-S phase. Overlay of green and red images are only shown in cases where there is colocalization of the fusion protein and BrdU foci. Scale bar = 2 µm.
• Fig. 3.17. TS associates with replication foci specifically during late-S phase. Summary of the preference of PBD, TS and PBHD to associate with replication foci is illustrated. ‘+’ indicates association; ‘-‘ indicates no association. PBD associates with RF throughout S phase while TS associates with RF only during late-S phase. PBHD does not associate with RF at any stage during S phase.
• Fig. 3.18. Development of a cell cycle marker for live cell analysis. Characterization of RFP-Ligase fusion protein expression and subnuclear localization. (A) COS7 cells were transfected with the RFP-Ligase fusion plasmid (shown at top) and after 24 hrs the cells were harvested and extracted by boiling in Laemmli’s sample buffer and analyzed by immunoblotting with an antibody against DNA Ligase I (Cardoso et al., 1997). The fusion protein is correctly expressed. Mock represents transfection of cells in which the plasmid DNA is excluded. (B) RFP-ligase behaves like the endogenous DNA Ligase I throughout the cell cycle in mouse cells. On the left, C2C12 cells were transfected with RFP-Ligase fusion plasmid and pulse labeled with BrdU 24 hrs later followed by indirect immunostaining for BrdU. On the right, C2C12 cells were pulse labeled with BrdU and immunostained with antibodies against DNA Ligase I and BrdU.
• Fig. 3.19. Time lapse analysis of mouse cells expressing F-TS-GFP and RFP-Ligase. C2C12 cells were cotransfected with plasmids encoding F-TS-GFP and RFP-Lig. After 24 hrs the cells were imaged at 1 hr intervals in a Zeiss LSM 510. Three Z sections of 1µm each were taken at each time point. Movement in the Z plane was corrected by comparing the Z sections at different time points and arranging them sequentially so as to include the same structures (RF and F-TS-GFP structures) in each sequential image. Only indicated time points are shown here. Overlay shows the merged image of green and red channels. White arrow indicates two exemplary F-TS-GFP structures that existed at 0 hrs and persist throughout the cell cycle. Box shows zoomed regions. Blue arrows in boxed region indicate colocalization of pre-existing F-TS-GFP structure with newly formed RFP-Lig foci. The full time lapse is in Video-1.
• Fig. 3.20. Elucidation of the subnuclear localization of endogenous DNMT1 throughout the cell cycle. (A) Strategy for identifying cells in G1, S and G2. (B) DNMT1 is retained at late replicating centromeric heterochromatin during G2. C2C12 cells were pulse-labeled with 10µM BrdU and chased for 2-3 hrs followed by fixation. Cells were immunostained with anti-BrdU, anti-DNMT1 and anti-PCNA antibodies. G2 cells were identified by their positive staining for BrdU and diffused PCNA. (C) DNMT1 is associated with the chromatin during mitosis. Mitotic cells were identified by their morphology and condensed chromosomes. (D) In early G1 cells, when the daughter cells are still connected (cells marked with arrow), DNMT1 is present at the centromeric heterochromatin. (E) DNMT1 shows a diffused subnuclear localization in G1 cells. G1 cells were obtained by seeding mitotic cells collected by mechanical shake off onto cover slips and allowing them to adhere for 2 hrs. The association of DNMT1 with RF during S phase is shown in Fig 3.15. Scale Bar = 5 µm.
• Fig. 3.21. Time lapse analysis of mouse cells expressing GMT and RFP-Ligase. C2C12 cells were cotransfected with plasmids encoding GMT and RFP-Lig. After 24 hrs the cells were imaged at 1 hr intervals as mentioned in Fig. 3.19. Only indicated time points are shown here. Overlay shows the merged image of green and red channels. White arrowheads point to three exemplary GMT foci which assemble concomitantly with RFP-Lig during late-S phase and are retained through the rest of S phase and in G2. Box in the overlay at 3 hrs shows zoomed region where GMT also associates with smaller foci. Green levels are increased to show this. The images of the M phase to G1 phase are from another cell, hence it starts again from 0 hrs. During M, GMT is associated with the chromatin. The daughter cells in G1 show diffused nuclear distribution of GMT. Full time lapse in Video-2.
• Fig. 3.22. TS is required for retention of DNMT1 at pericentric regions during G2 and M phases. C2C12 cells were transfected with plasmid encoding GMT∆TS. G1 and G2 cells were identified as described in Fig. 3.20. After 24 hrs of transfection, cells were immunostained with antibody against DNA Ligase I to label RF. GMT∆TS colocalizes with sites of DNA replication throughout S phase. During G2, GMT∆TS shows diffused nuclear distribution, and is excluded from mitotic chromatin. G1 cells show diffused nuclear distribution of GMT∆TS. Scale bar = 5 µm
• Fig. 3.23. PBD delays association of TS in DNMT1 with pericentric heterochromatin until late-S phase. (A) C2C12 cells were transfected with plasmids encoding the various mutants of DNMT1 fused to GFP. 24 hrs after transfection the subnuclear localization of the GFP-fusion proteins at S, G2, M and G1 phases were analyzed as described in Fig. 3.20. MTN.1 (ii), which lacks the PBHD, is retained in pericentric heterochromatin during G1. GMT-PBD-H167V (vii) is associated with pericentric heterochromatin right during early- and mid-S phase like the TS-GFP fusion protein. BrdU staining is shown only in the case of GMT-PBD-H167V. Scale Bar = 5 µm. (B) C2C12 cells transfected with various plasmids were pulse labeled with BrdU 24 hrs later and incorporation of BrdU was detected by immunostaining. The percentage of transfected cells in early-/mid-S phase exhibiting colocalization of the GFP-fusion protein with either BrdU labeled RF or pericentric heterochromatin, or showing diffused nuclear distribution was scored. Black is colocalization with RF, gray is colocalization with pericentric heterochromatin, white is diffused nuclear distribution. Only GMT-PBDmut associates with pericentric heterochromatin during early-/mid-S phase.
• Fig. 3.24. Expression of TS reduces viability of cells. (A) Methodology for colony forming assay. (B) Images of Giemsa stained colonies. (C) Colony forming efficiency of C2C12 cells transfected with plasmids encoding the mentioned proteins. The number of colonies in each experiment was normalized to the GFP control of the corresponding experiment and expressed as percentage. Results from three independent experiments were averaged; error bars show standard deviation.
• Fig. 3.25. Micronuclei formation and coalescence of centromeric heterochromatin. Transfected cells expressing the mentioned proteins were fixed and stained for DNA with Hoechst 33258/TOPRO-3 and cells exhibiting micronuclei or coalescence of centromeric heterochromatin were scored. About 200-300 transfected cells were counted in each case and the percent values were calculated. The values obtained from three different transfections were averaged and plotted. Bars indicate standard deviation. (A) Percent transfected cells exhibiting micronuclei formation. Inset shows an image of a cell transfected with plasmid expressing TS-GFP exhibiting several micronuclei. (B) Percent transfected cells in which the centromeric heterochromatin is coalesced. Inset shows an image of a cell transfected with plasmid expressing TS-GFP exhibiting coalesced centromeric heterochromatin and a non-transfected cell showing normal centromeric heterochromatin. (C) C2C12 cells were cotransfected with plasmids encoding TS-GFP and CENPB-RFP to label the centromeres. Coalesced heterochromatin consists of relocated and clustered centromeric regions.
• Fig. 3.26. Search for a PBD in all C-5-methyltransferases. PBDs characterized in different proteins from metazoa were aligned by PileUp and a profile was generated using ProfileMake. This profile, shown at the top, was used to search all the C-5-methyltransferase families in metazoans, plants and fungi using the program ProfileSearch. The PBDs identified were aligned by Clustal and realigned by visual inspection to give maximal alignment. 100% identity is highlighted in red colour, more than 50% identity is highlighted in yellow, residues in blue form the region rich in basic residues. Sequence names in red are from metazoans, in green are from plants and brown is from fungus. Sequence positions are shown in numbers in parentheses. Square brackets at the right indicate protein accession numbers. The sequence of the most conserved residues in the PBD required for binding to PCNA and association with RF is shown at the top. Most of the PBDs identified in the plant and fungal methyltransferases are either in the C-terminal catalytic domain or in the PBHD or chromodomain and they lack the stretch of basic residues. Mm: Mus musculus; Rr: Rattus rattus; Hs: Homo sapiens; Gg: Gallus gallus; Xl: Xenopus laevis; Xm: Xiphophorus maculatus x helleri; Pl: Paracentrotus lividus; Dm: Drosophila melanogaster; At: Arabidopsis thaliana; Dc: Daucus carota; Ps: Pisum sativum; Nt: Nicotiana tabacum; Le: Lycopersicon esculentum; Zm: Zea mays; Nc: Neurospora crassa.
• Fig. 3.27. TS is an unique domain present only in the DNMT1 family. The nr database was searched using PSI-BLAST with the mouse TS as query. Subsequent iterations were performed by including the sequences (which were all C-5 methyltransferases) which showed an E value better than threshold. Following four iterations convergence was attained and no new sequences were retrieved. All the sequences showing significant E value belonged to the DNMT1 family.
• Fig. 3.28. TS domain is duplicated in plants and shows a conserved motif. A typical example of the result of PSI-BLAST showing duplicated TS in plants, in this case A. thaliana ( accession no. AAF14882). Box shows the region that has a high degree of conservation in both TS.
• Fig. 3.29. A conserved motif in the TS from metazoans, plants and fungus. TS from mouse (Leonhardt et al., 1992) was used as a query to search the nr database for similar sequences using PSI-BLAST. All significant hits obtained belonged to the DNMT1 family from metazoans, plants and fungus (see also Fig 3.27). These were aligned using Clustal. Here a region of the alignment with maximum conservation is shown. Residues that are identical to the consensus are shaded black, highly similar residues are shaded grey. Asterisk indicates the phosphorylation site (S at position 514) that is conserved in metazoans. In the consensus sequence, letters in red are 100% identical, uppercase is greater than 80% identity and lowercase is similar residues that are conserved at least between two of the three groups of TS: metazoan TS, TS-1 and TS-2. Conserved changes are shown below the consensus. In sequence names Ai is Ascobolus immersus. Other sequence names are as in Fig 3.26. (AtMET is AAF14882).
• Fig. 4.1. Domain organization in DNMT1 from metazoan, plants and fungi. The TS is duplicated in plants. A TS-like motif identified in N. crassa Dim2 is also illustrated. BAH corresponds to PBHD. Names of organisms are abbreviated as in Fig. 3.26.
• Fig. 4.2. Hypothetical model explaining the mechanism of inheritance of DNA methylation pattern by DNMT1. The events at a replication fork during early-S and late-S are shown. (1) DNMT1 is recruited at early replication foci interacting with PCNA via the PBD. The catalytic domain (cat) in DNMT1 methylates the newly synthesized DNA strand. (2) At late replication foci, DNMT1 is again recruited by PCNA and methylates the newly synthesized strand. (3) At the cessation of DNA replication, PCNA disassembles from the DNA strand but DNMT1 is retained at these sites via the TS. DNMT1 continues methylating the pericentric heterochromatin. (4) During G2, DNMT1 is retained at the pericentric regions where it completes DNA methylation and acts as a docking site for histone modifying enzymes like HDAC1/2 and thereby aids in maturation of newly synthesized heterochromatin.
• Fig. I. Cell cycle marker for live and fixed cells. (A) Only mitosis (M) and interphase can be identified in live cells based on the cellular and nuclear morphology. (B) Expression of RFP-Ligase allows identification of the different cell cycle stages by live cell microscopy. In live cells expressing RFP-Ligase, G2 phase can be identified by following the transition of a cell from S phase (punctate RFP-Ligase pattern corresponding to replication sites) into G2 when RFP-Ligase is diffused in the nucleus. The progression through S phase can be followed by the different patterns of RF. During M phase, the nuclear membrane is broken down and RFP-Ligase is excluded from the chromatin. Cells in G1 phase can be identified by following a cell through mitosis into G1 when RFP-Ligase is diffused in the nucleus. (C) Expression of both GFP-DNMT1 and RFP-Ligase allows identification of the cell cycle stage in fixed as well as live cells. Colocalization of RFP-Ligase and GFP-DNMT1 at RF during S phase is shown in yellow. During G2, only GFP-DNMT1 is bound to pericentric heterochromatin (shown as green "donut" shaped structures) while RFP-Ligase is diffused. During mitosis GFP-DNMT1 is at the chromatin. In G1, both RFP-Ligase and GFP-DNMT1 are diffused (shown as yellow).