Establishment and mitotic stability of an extra-chromosomal mammalian replicon

<p>Abstract</p> <p>Background</p> <p>Basic functions of the eukaryotic nucleus, like transcription and replication, are regulated in a hierarchic fashion. It is assumed that epigenetic factors influence the efficiency and precision of these processes. In order to uncouple local and long-range epigenetic features we used an extra-chromosomal replicon to study the requirements for replication and segregation and compared its behavior to that of its integrated counterpart.</p> <p>Results</p> <p>The autonomous replicon replicates in all eukaryotic cells and is stably maintained in the absence of selection but, as other extra-chromosomal replicons, its establishment is very inefficient. We now show that following establishment the vector is stably associated with nuclear compartments involved in gene expression and chromosomal domains that replicate at the onset of S-phase. While the vector stays autonomous, its association with these compartments ensures the efficiency of replication and mitotic segregation in proliferating cells.</p> <p>Conclusion</p> <p>Using this novel minimal model system we demonstrate that relevant functions of the eukaryotic nucleus are strongly influenced by higher nuclear architecture. Furthermore our findings have relevance for the rational design of episomal vectors to be used for genetic modification of cells: in order to improve such constructs with respect to efficiency elements have to be identified which ensure that such constructs reach regions of the nucleus favorable for replication and transcription.</p

snRNA and Heterochromatin Formation Are Involved in DNA Excision during Macronuclear Development in Stichotrichous Ciliates

Several models for specific excision of micronucleus-specific DNA sequences during macronuclear development in ciliates exist. While the template-guided recombination model suggests recombination events resulting in specific DNA excision and reordering of macronucleus-destined sequences (MDS) guided by a template, there is evidence that an RNA interference-related mechanism is involved in DNA elimination in holotrichous ciliates. We describe that in the stichotrichous ciliate Stylonychia, snRNAs homologous to micronucleus-specific sequences are synthesized during macronuclear differentiation. Western and in situ analyses demonstrate that histone H3 becomes methylated at K9 de novo during macronuclear differentiation, and chromatin immunoprecipitation revealed that micronucleus-specific sequences are associated with methylated H3. To link both observations, expression of a PIWI homolog, member of the RNA-induced silencing complex, was silenced. In these cells, the methylated micronucleus-specific histone H3 variant “X” is still present in macronuclear anlagen and no K9 methylation of histone H3 is observed. We suggest that snRNA recruits chromatin-modifying enzymes to sequences to be excised. Based on our and earlier observations, we believe that this mechanism is not sufficient for specific excision of sequences and reordering of MDS in the developing macronucleus and propose a model for internal eliminated sequence excision and MDS reordering in stichotrichous ciliates

snRNA and Heterochromatin Formation Are Involved in DNA Excision during Macronuclear Development in Stichotrichous Ciliates

Several models for specific excision of micronucleus-specific DNA sequences during macronuclear development in ciliates exist. While the template-guided recombination model suggests recombination events resulting in specific DNA excision and reordering of macronucleus-destined sequences (MDS) guided by a template, there is evidence that an RNA interference-related mechanism is involved in DNA elimination in holotrichous ciliates. We describe that in the stichotrichous ciliate Stylonychia, snRNAs homologous to micronucleus-specific sequences are synthesized during macronuclear differentiation. Western and in situ analyses demonstrate that histone H3 becomes methylated at K9 de novo during macronuclear differentiation, and chromatin immunoprecipitation revealed that micronucleus-specific sequences are associated with methylated H3. To link both observations, expression of a PIWI homolog, member of the RNA-induced silencing complex, was silenced. In these cells, the methylated micronucleus-specific histone H3 variant “X” is still present in macronuclear anlagen and no K9 methylation of histone H3 is observed. We suggest that snRNA recruits chromatin-modifying enzymes to sequences to be excised. Based on our and earlier observations, we believe that this mechanism is not sufficient for specific excision of sequences and reordering of MDS in the developing macronucleus and propose a model for internal eliminated sequence excision and MDS reordering in stichotrichous ciliates

Establishment and mitotic stability of an extra-chromosomal mammalian replicon-2

<p><b>Copyright information:</b></p><p>Taken from "Establishment and mitotic stability of an extra-chromosomal mammalian replicon"</p><p>http://www.biomedcentral.com/1471-2121/8/33</p><p>BMC Cell Biology 2007;8():33-33.</p><p>Published online 6 Aug 2007</p><p>PMCID:PMC1959191.</p><p></p>omosomes (A), and the equal distribution of vector molecules was monitored in postmitotic nuclei of dividing cells (B). The episome (green) was visualized by pEPI FISH. To-Pro-3 was used for DNA counterstaining (red). A maximum intensity projection was rendered from a set of 5 mid serial sections in (B). Arrows in (A) indicate a pEPI signal pair, each signal localized on a sister chromatid. For further details see Additional file

Establishment and mitotic stability of an extra-chromosomal mammalian replicon-3

<p><b>Copyright information:</b></p><p>Taken from "Establishment and mitotic stability of an extra-chromosomal mammalian replicon"</p><p>http://www.biomedcentral.com/1471-2121/8/33</p><p>BMC Cell Biology 2007;8():33-33.</p><p>Published online 6 Aug 2007</p><p>PMCID:PMC1959191.</p><p></p>d using pEPI FISH and BrdU pulse labeling. Maximum intensity projections were rendered from a set of serial sections. The nuclear contour was outlined with a white line. (A) Replication foci were labeled during early S-phase by incorporation of BrdU (1 h) into newly synthesized DNA. Subsequently nuclei were fixed and BrdU (red) was visualized by antibody staining. The episome (green) was visualized by pEPI FISH. Co-localizing voxels were highlighted in white color. (B) Alternatively nascent DNA was pulse-labeled with BrdU for 1 h during early S-phase, fixing and subsequent immunolocalization was done following cell division. The episome (green) was visualized by pEPI FISH. Co-localizing voxels were highlighted in white color

Establishment and mitotic stability of an extra-chromosomal mammalian replicon-5

<p><b>Copyright information:</b></p><p>Taken from "Establishment and mitotic stability of an extra-chromosomal mammalian replicon"</p><p>http://www.biomedcentral.com/1471-2121/8/33</p><p>BMC Cell Biology 2007;8():33-33.</p><p>Published online 6 Aug 2007</p><p>PMCID:PMC1959191.</p><p></p>ondensed/inactive chromatin: mid columns), H3 trimethyl-K4 (open/active chromatin: right handed columns) or using a control IgG (left handed columns). The percentage of either pEPI-1 (gray columns) or the linearized control vector (striped columns) precipitated from input was quantified using real time PCR. Since no significant amount of the vector molecules was pulled down with the control IgG, it could be clearly demonstrated that about 30 times more vector molecules were precipitated using the anti-H3 trimethyl-K9 antibody in a precipitate from cells containing the integrated vector compared to cells containing the vector in its episomal form. When the same chromatin preparations were precipitated with an antibody directed against H3 trimethyl-K4 over 20 times more vector molecules were obtained from cells containing the episome compared to cells containing the integrated vector

Establishment and mitotic stability of an extra-chromosomal mammalian replicon-0

<p><b>Copyright information:</b></p><p>Taken from "Establishment and mitotic stability of an extra-chromosomal mammalian replicon"</p><p>http://www.biomedcentral.com/1471-2121/8/33</p><p>BMC Cell Biology 2007;8():33-33.</p><p>Published online 6 Aug 2007</p><p>PMCID:PMC1959191.</p><p></p>terphase nuclei was analyzed using pEPI FISH (A-J). In some experiments pEPI FISH was used in combination with immunofluorescence techniques to analyse the co-localization of pEPI with subnuclear marker proteins (E-J). Co-localizing voxels of two channels being analyzed were highlighted in white color in (A, B, C, D, H). The episome (green) was visualized by pEPI FISH. To-Pro-3 was used for DNA counterstaining (red) in (A, B, C, D, E, G, I). Maximum intensity projections were rendered from a set of 5 mid serial sections (A, B, C, D, E, G, H, I). For further details see Additional file . (A, B) Co-localization analyses showed that cells transfected with the linearized vectors displayed FISH signals consistent with integration of the vector at a single chromosomal locus as the vectors completely co-localized with domains containing condensed chromatin. Equal results were obtained when hyper-condensation of chromatin was induced prior to fixation (B). (C, D) Analyses of single pEPI signals revealed that the vector did not (green) or at most incompletely (white/green) colocalize to condensed chromatin regions (red). Insets are 400% magnifications of white or blue framed sectors in C and D. Such representative pEPI signals consist of green voxels (negative co-localization with condensed chromatin) or are composed of white and green voxels (incomplete co-localization with condensed chromatin). Negative or incomplete co-localization suggests that the episome is localized within the IC or at perichromatin regions bordering the IC at condensed chromatin surfaces. Equal results were obtained when hyper-condensation of chromatin was induced prior to fixation (B). (For selected light optical sections corresponding to the nuclei displayed in C and D as maximum intensity projections see the Additional file .) (E, F) Co-localization analyses of the episome (green) and SC35 (blue) showed that the episome occured co-localized or in close proximity to nuclear speckles, a structure which is found within the IC. (F) A 3D reconstruction was rendered from the same nucleus as displayed in (E). (G, H) Nuclear localization of the episome (green) and histone3 acetylated at lysines 9/14 (H3 acetyl-K9/K14) (blue). The nuclear counterstain (red) channel was hidden in (H) to facilitate co-localization analysis of the episome and H3 acetyl-K9/K14 showing that the episome occurred co-localized or in close proximity to sites of active transcription. (I, J) Co-localization analyses of the episome (green) and histone3 trimethylated at lysine9 (H3 trimethyl-K9) (blue) showed that the episome did not co-localize with such sites. (J) A 3D reconstruction was rendered from the same nucleus as displayed in (I). Bar in (A) is representative for (A-J)

Establishment and mitotic stability of an extra-chromosomal mammalian replicon-1

<p><b>Copyright information:</b></p><p>Taken from "Establishment and mitotic stability of an extra-chromosomal mammalian replicon"</p><p>http://www.biomedcentral.com/1471-2121/8/33</p><p>BMC Cell Biology 2007;8():33-33.</p><p>Published online 6 Aug 2007</p><p>PMCID:PMC1959191.</p><p></p>ondensed/inactive chromatin: mid columns), H3 trimethyl-K4 (open/active chromatin: right handed columns) or using a control IgG (left handed columns). The percentage of either pEPI-1 (gray columns) or the linearized control vector (striped columns) precipitated from input was quantified using real time PCR. Since no significant amount of the vector molecules was pulled down with the control IgG, it could be clearly demonstrated that about 30 times more vector molecules were precipitated using the anti-H3 trimethyl-K9 antibody in a precipitate from cells containing the integrated vector compared to cells containing the vector in its episomal form. When the same chromatin preparations were precipitated with an antibody directed against H3 trimethyl-K4 over 20 times more vector molecules were obtained from cells containing the episome compared to cells containing the integrated vector