Untitled Document

Induction of Interphase Chromosome Movement by Transcriptional Activators

Reference:

Tumbar, T. and Belmont, A.S. (2001) "Interphase movements of a DNA chromosome region modulated by VP16 transcriptional activator," Nature Cell Biology 3:134-139.

Background:

Increasing evidence suggests nonrandom localization of DNA sequences in the cell nucleus. In particular, transcriptionally active versus inactive regions, in a number of specific examples, have been shown to have differential intranuclear locations. Early replicating, gene rich R bands localize preferentially to the nuclear interior while gene poor, late replicating G/Q bands predominate in the nuclear periphery. Several specific active genes showed preferential interior nuclear localization in mammalian cells. Polyadenylated RNA colocalized with high concentrations of splicing factors and was not present in an area ~1mm wide adjacent to the nuclear envelope in human diploid fibroblasts. In yeast, increased gene silencing was observed for a weak silencer when it was targeted to the nuclear envelope experimentally. These findings have suggested a possible functional role for targeting to the nuclear periphery versus interior in gene regulation.

A number of silent genes associate with centromeric heterochromatin, suggesting the recruitment of these genes to nuclear compartments non-permissive to transcription. Mutations in the HS2 enhancer from the beta-globin LCR resulted in increased transgene silencing and association with centromeres. Insertion of a heterochromatic block in the Drosophila brown locus caused both variegated expression of the modified gene and an association with centromeric heterochromatin. Finally it has been shown that chromosomes occupy defined territories and some active and inactive genes localize preferentially at the periphery of these territories.

Results:

Analysis of chromosome movements associated with transcriptional activation and/or repression has been complicated by the complexity of the available model systems. We created a simplified, flexible system to study the cell cycle dependent nuclear localization of a chromosome site with or without a strong transcriptional activator bound to it. Targeting of the acidic activation domain of the strong transcriptional activator, VP16, resulted in a repositioning of the chromosome site from the nuclear periphery to the nuclear interior. Differential intranuclear positioning of the chromosome site to the nuclear interior by tethering of VP16 was established soon after mitosis. In contrast in control cells, the chromosome site examined was localized preferentially to the nuclear periphery within the first few hours of G1, moving to the nuclear interior 1-2 hours into S phase.

Histogram (Left): . A histogram of distances of the labeled spot from the nuclear edge in cross-section shows a high percentage of cells within a half a micron from the edge in control C614-LacI cells expressing the dimer lac repressor-EGFP. Cells expressing the dimer lac repressor-EGFP-VP16 AAD showed a more interior distribution as shown by stable clones C7-VP16 and C4-VP16 and a mixture of stable clones Mix-VP16. Top of histogram shows numbers of cells for each data point. Numbers on left are percentages of cells.

Image Montage (Right): Times shown are minutes after cell division. Top panel- In cells expressing GFP-lac repressor-VP16, chromosome site moves to nuclear interior in early G1 and remains there. Bottom panel- In cells expressing the control GFP-lac repressor construct, chromosome site locates to periphery within first several hours into G1. A significant fraction of cells show a transient extension or elongation of chromosome region during this early G1 period.

Details:

The C6 cell line was isolated containing a localized insertion of 10-20 copies of a transgene containing lac operator direct repeats. In control cells not expressing lac repressor, or expressing a GFP-lac repressor fusion protein, this site localized preferentially to the 3-dimensional nuclear periphery in roughly 75% of log phase cells. In 2-D projected images the chromosome site localized to the nuclear edge in ~50% of cells. This peripheral localization visualized in 2-D images greatly facilitated acquisition of statistics from large numbers of cells.

Southern analyses revealed a maximum of 10-20 copies of vector DNA integrated in the CHO genome (data not shown). We determined the chromosome location in C6-14 cells, expressing GFP-lac repressor, using a modified chromosome spreading procedure, which preserved GFP fluorescence. The vector insertion site is located on the largest metacentric chromosome in the CHO DG44 genome, far from the centromere and just proximal to a bright band of DAPI staining.

To test whether transcriptional activation of this locus would cause a change in nuclear localization, we examined C6 cells 72 hours after transfection of either the control GFP-lac repressor or the GFP-lac repressor-VP16 AAD vector constructs. To simplify the statistical analysis, we determined the nuclear positioning of the chromosome site using a 2D analysis, with the cross-sectional area of the nucleus outlined by the background staining of lac repressor-GFP fluorescence signal and/or the DAPI staining. We defined the 0.5 mm wide area immediately adjacent to the 2-D projected nuclear edge as the nuclear "periphery" and the rest of the cross-sectional area of the nucleus as the nuclear "interior". This analysis method allowed rapid scoring of hundreds of nuclei and was designed to exploit the nonrandom distribution of the C6 vector insertion site. Chromosome sites were peripheral in 55% of GFP-lac repressor-VP16 AAD expressing cells versus only 27% of control cells expressing GFP-lac repressor. This analysis was repeated, with similar results (see histogram above), using stably transformed cells expressing GFP-lac repressor-VP16 AAD.

To determine whether intranuclear positioning of the chromosome site in C6 cells was cell cycle dependent, we analyzed chromosome site location as a function of S phase progression. Log phase C6 cells stably expressing GFP-lac repressor were pulsed labeled for 15 minutes with BrdU. Nuclei with no BrdU incorporation identify cells in G1 or G2. Labeled cells were classified using five patterns of nuclear nucleotide incorporation corresponding to different S phase periods. Using this approach we found that in control cells the chromosome site moved to the interior by pattern 2 of S phase, implying movement during the first 1-2 hours of S phase. This movement was confirmed by in vivo visualization of cells blocked in early S phase and then released (See What We've Done- Chromosome Movements During S phase).

In contast, in stable clones expressing GFP-lac repressor- VP16, cells blocked in late G1/early S phase showed predominately interior localization of the chromosome site. Direct observation of a large number of cells during the first several hours after mitosis showed that in cells expressing GFP-lac repressor-VP16 the chromosome site localized to the interior within the first 1-2 hours after cell division. In contrast, as shown above, the chromosome site localized to the periphery in control cells during the first several hours of G1.

Conclusions:

We used an engineered, artificial system to analyze the role of a transcriptional activation in determining the nuclear localization of a specific chromosome site. By targeting a specific transcriptional activator to a specific chromosome site, we reprogrammed the normal S phase repositioning to the nuclear interior for this site to a more permanent positioning within the nuclear interior during the first few hours of G1. These changes in positioning occured within a large proportion of cells.

Previous work in our laboratory has demonstrated that targeting the lac repressor -VP16 AAD fusion protein leads to histone hyperacetylation, recruitment of histone acetyltransferases (GCN5, P300/CBP, and PCAF), and increased transcription (See What We've Done- Direct visualization of chromatin modifications and recruitment of coactivators). Recruitment of other components of the transcriptional machinery by the VP16 AAD is now being characterized. Through the combined use of VP16 AAD mutants, dominant negative mutants of HAT and chromatin remodeling complex components, and additional lac repressor fusions, our system will provide a simplified experimental approach to dissect mechanisms underlying the reprogramming of cell cycle intranuclear chromosome movement.

				BELMONT LAB
				BELMONT LAB

BELMONT LAB HOME WHO WE ARE WHAT WE ARE ASKING WHAT WE'VE DONE WHAT WE ARE DOING WHAT WE WOULD LIKE TO DO POSITION OPENINGS PICTURE GALLERY MOVIES				Induction of Interphase Chromosome Movement by Transcriptional Activators Reference: Tumbar, T. and Belmont, A.S. (2001) "Interphase movements of a DNA chromosome region modulated by VP16 transcriptional activator," Nature Cell Biology 3:134-139. Background: Increasing evidence suggests nonrandom localization of DNA sequences in the cell nucleus. In particular, transcriptionally active versus inactive regions, in a number of specific examples, have been shown to have differential intranuclear locations. Early replicating, gene rich R bands localize preferentially to the nuclear interior while gene poor, late replicating G/Q bands predominate in the nuclear periphery. Several specific active genes showed preferential interior nuclear localization in mammalian cells. Polyadenylated RNA colocalized with high concentrations of splicing factors and was not present in an area ~1mm wide adjacent to the nuclear envelope in human diploid fibroblasts. In yeast, increased gene silencing was observed for a weak silencer when it was targeted to the nuclear envelope experimentally. These findings have suggested a possible functional role for targeting to the nuclear periphery versus interior in gene regulation. A number of silent genes associate with centromeric heterochromatin, suggesting the recruitment of these genes to nuclear compartments non-permissive to transcription. Mutations in the HS2 enhancer from the beta-globin LCR resulted in increased transgene silencing and association with centromeres. Insertion of a heterochromatic block in the Drosophila brown locus caused both variegated expression of the modified gene and an association with centromeric heterochromatin. Finally it has been shown that chromosomes occupy defined territories and some active and inactive genes localize preferentially at the periphery of these territories. Results: Analysis of chromosome movements associated with transcriptional activation and/or repression has been complicated by the complexity of the available model systems. We created a simplified, flexible system to study the cell cycle dependent nuclear localization of a chromosome site with or without a strong transcriptional activator bound to it. Targeting of the acidic activation domain of the strong transcriptional activator, VP16, resulted in a repositioning of the chromosome site from the nuclear periphery to the nuclear interior. Differential intranuclear positioning of the chromosome site to the nuclear interior by tethering of VP16 was established soon after mitosis. In contrast in control cells, the chromosome site examined was localized preferentially to the nuclear periphery within the first few hours of G1, moving to the nuclear interior 1-2 hours into S phase. Histogram (Left): . A histogram of distances of the labeled spot from the nuclear edge in cross-section shows a high percentage of cells within a half a micron from the edge in control C614-LacI cells expressing the dimer lac repressor-EGFP. Cells expressing the dimer lac repressor-EGFP-VP16 AAD showed a more interior distribution as shown by stable clones C7-VP16 and C4-VP16 and a mixture of stable clones Mix-VP16. Top of histogram shows numbers of cells for each data point. Numbers on left are percentages of cells. Image Montage (Right): Times shown are minutes after cell division. Top panel- In cells expressing GFP-lac repressor-VP16, chromosome site moves to nuclear interior in early G1 and remains there. Bottom panel- In cells expressing the control GFP-lac repressor construct, chromosome site locates to periphery within first several hours into G1. A significant fraction of cells show a transient extension or elongation of chromosome region during this early G1 period. Details: The C6 cell line was isolated containing a localized insertion of 10-20 copies of a transgene containing lac operator direct repeats. In control cells not expressing lac repressor, or expressing a GFP-lac repressor fusion protein, this site localized preferentially to the 3-dimensional nuclear periphery in roughly 75% of log phase cells. In 2-D projected images the chromosome site localized to the nuclear edge in ~50% of cells. This peripheral localization visualized in 2-D images greatly facilitated acquisition of statistics from large numbers of cells. Southern analyses revealed a maximum of 10-20 copies of vector DNA integrated in the CHO genome (data not shown). We determined the chromosome location in C6-14 cells, expressing GFP-lac repressor, using a modified chromosome spreading procedure, which preserved GFP fluorescence. The vector insertion site is located on the largest metacentric chromosome in the CHO DG44 genome, far from the centromere and just proximal to a bright band of DAPI staining. To test whether transcriptional activation of this locus would cause a change in nuclear localization, we examined C6 cells 72 hours after transfection of either the control GFP-lac repressor or the GFP-lac repressor-VP16 AAD vector constructs. To simplify the statistical analysis, we determined the nuclear positioning of the chromosome site using a 2D analysis, with the cross-sectional area of the nucleus outlined by the background staining of lac repressor-GFP fluorescence signal and/or the DAPI staining. We defined the 0.5 mm wide area immediately adjacent to the 2-D projected nuclear edge as the nuclear "periphery" and the rest of the cross-sectional area of the nucleus as the nuclear "interior". This analysis method allowed rapid scoring of hundreds of nuclei and was designed to exploit the nonrandom distribution of the C6 vector insertion site. Chromosome sites were peripheral in 55% of GFP-lac repressor-VP16 AAD expressing cells versus only 27% of control cells expressing GFP-lac repressor. This analysis was repeated, with similar results (see histogram above), using stably transformed cells expressing GFP-lac repressor-VP16 AAD. To determine whether intranuclear positioning of the chromosome site in C6 cells was cell cycle dependent, we analyzed chromosome site location as a function of S phase progression. Log phase C6 cells stably expressing GFP-lac repressor were pulsed labeled for 15 minutes with BrdU. Nuclei with no BrdU incorporation identify cells in G1 or G2. Labeled cells were classified using five patterns of nuclear nucleotide incorporation corresponding to different S phase periods. Using this approach we found that in control cells the chromosome site moved to the interior by pattern 2 of S phase, implying movement during the first 1-2 hours of S phase. This movement was confirmed by in vivo visualization of cells blocked in early S phase and then released (See What We've Done- Chromosome Movements During S phase). In contast, in stable clones expressing GFP-lac repressor- VP16, cells blocked in late G1/early S phase showed predominately interior localization of the chromosome site. Direct observation of a large number of cells during the first several hours after mitosis showed that in cells expressing GFP-lac repressor-VP16 the chromosome site localized to the interior within the first 1-2 hours after cell division. In contrast, as shown above, the chromosome site localized to the periphery in control cells during the first several hours of G1. Conclusions: We used an engineered, artificial system to analyze the role of a transcriptional activation in determining the nuclear localization of a specific chromosome site. By targeting a specific transcriptional activator to a specific chromosome site, we reprogrammed the normal S phase repositioning to the nuclear interior for this site to a more permanent positioning within the nuclear interior during the first few hours of G1. These changes in positioning occured within a large proportion of cells. Previous work in our laboratory has demonstrated that targeting the lac repressor -VP16 AAD fusion protein leads to histone hyperacetylation, recruitment of histone acetyltransferases (GCN5, P300/CBP, and PCAF), and increased transcription (See What We've Done- Direct visualization of chromatin modifications and recruitment of coactivators). Recruitment of other components of the transcriptional machinery by the VP16 AAD is now being characterized. Through the combined use of VP16 AAD mutants, dominant negative mutants of HAT and chromatin remodeling complex components, and additional lac repressor fusions, our system will provide a simplified experimental approach to dissect mechanisms underlying the reprogramming of cell cycle intranuclear chromosome movement.