Journal of Bacteriology, August 2001, p. 4636-4642, Vol. 183, No. 15
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.15.4636-4642.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Green Fluorescent Protein-Dal80p Illuminates up to 16 Distinct
Foci That Colocalize with and Exhibit the Same Behavior as Chromosomal
DNA Proceeding through the Cell Cycle of Saccharomyces
cerevisiae
MacKenzie
Distler,
Ajit
Kulkarni,
Rajendra
Rai, and
Terrance G.
Cooper*
Department of Microbiology and Immunology,
University of Tennessee, Memphis, Tennessee 38163
Received 20 February 2001/Accepted 28 April 2001
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ABSTRACT |
Four GATA family DNA binding proteins mediate nitrogen catabolite
repression-sensitive transcription in Saccharomyces
cerevisiae. Gln3p and Gat1p are transcriptional activators,
while Dal80p and Deh1p repress Gln3p- and Gat1p-mediated transcription
by competing with these activators for binding to DNA. Strong Dal80p
binding to DNA is thought to result from C-terminal leucine
zipper-mediated dimerization. Many Dal80p binding site-homologous
sequences are relatively evenly distributed across the S.
cerevisiae genome, raising the possibility that Dal80p might be
able to "stain" DNA. We demonstrate that cells containing enhanced
green fluorescent protein-Dal80p (EGFP-Dal80p) exhibit up to 16 fluorescent foci that colocalize with DAPI
(4',6'-diamidino-2-phenylindole)-positive material and follow DNA
movement through the cell cycle, suggesting that EGFP-Dal80p may indeed
be useful for monitoring yeast chromosomes in live cells and in real time.
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TEXT |
The Saccharomyces
cerevisiae GATA family transcription factors are responsible
for nitrogen catabolite repression (NCR)-sensitive gene
expression (for reviews, see references 6, 18, and
24). Gln3p and Gat1p are transcriptional activators,
while Dal80p and Deh1p (Gzf3p) are repressors of Gln3p- and
Gat1p-mediated transcription. The functionality of Gln3p and Gat1p in
response to nitrogen availability is controlled by their access to the
nucleus. In cells provided with a poor nitrogen source (e.g.,
proline), Gln3p and Gat1p are predominantly nuclear, and NCR-sensitive
gene expression is high (2-4, 8, 9, 17). In contrast,
when excess nitrogen is present (e.g., with glutamine), these
transcription factors are cytoplasmic, and NCR-sensitive gene
expression is repressed (2-4, 8, 9, 17).
In addition to NCR, Gln3p- and Gat1p-mediated transcription is
regulated by the repressors, Dal80p and Deh1p (7, 18, 24).
Several lines of evidence suggest that Dal80p down-regulates transcription by competing with Gln3p and Gat1p for binding to their
target GATA sequences upstream of NCR-sensitive genes (1, 10-12,
14). Although Dal80p and Gln3p both bind to GATAs
(13), their binding sites differ significantly. Gln3p
binds to single GATAs, while two GATAs oriented head to tail or tail to
tail, but not head to head, 15 to 40 bp apart are required for Dal80p binding (10). The observed strength of Dal80p binding to
DNA has been suggested to derive from the fact that it does so as a
dimer, which forms through C-terminal leucine zipper motifs (23). In addition, Dal80p appears to possess unlimited
access to the nucleus independent of nutritional conditions (M. Distler and T. G. Cooper, unpublished data).
A homology search, conducted as part of a genomic analysis of
Dal80p-regulated genes (7), resulted in identification of an unusually large number of Dal80p binding site-homologous sequences relatively evenly distributed throughout the genome. This raised the
possibility that enhanced green fluorescent protein-Dal80p (EGFP-Dal80p) might be used as a probe to monitor chromosome movement in yeast. Although DAPI (4',6'-diamidino-2-phenylindole) staining and
fluorescent in situ hybridization analysis have been used to visualize
chromosomes, both methods are ill-suited for use with live
preparations. Therefore, we determined whether Dal80p possessed
any potential in this regard. Our data suggest that EGFP-Dal80p may
indeed be a useful probe with which to monitor yeast chromosomes in
live cells and in real time.
Multiple fluorescent foci appear in cells containing
GFP-Dal80p.
When wild-type S. cerevisiae is transformed
with plasmid-borne GAL1,10-GFP-GLN3 or
GAL1,10-GFP-GAT1, fluorescence is uniformly distributed
in the nucleus (8, 9). In contrast, nuclear fluorescence
is punctate when a GFP-DAL80 plasmid is used (Fig. 1). As the cell cycle progresses, these foci move within
the cell and between mother and daughter cells as does DAPI-positive
material. At the early bud stage, the foci cluster adjacent to the
forming bud (Fig. 1A, B, K, and L). As the bud grows to the same size as the mother cell, fluorescent foci span the neck, often lined up like
beads on a string (Fig. 1C to F). As cytokinesis occurs, fluorescent
foci are situated in both mother and daughter cells (Fig. 1G to J, M,
and N). Occasionally, the fluorescent foci form a circle or part of one
(Fig. 1C and I).
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FIG. 1.
GAL1,10-EGFP-DAL80 in wild-type GYC86 transformed with
pNVS80. All strains, methods, and culture conditions were described
earlier (8, 9, 19). Neither the nitrogen source nor
overproduction of Ure2p affects the pattern or distribution of
fluorescence (data not shown). The nitrogen sources were 0.1% proline
(A to E and G to J), glutamine (K to N), or ammonia (F and O to R).
pNVS80 was constructed by cloning the
NdeI-HindIII fragment of pTSC416 into the
NdeI and HindIII sites of pNVS2.
GAL1,10-EGFP-DAL80 in pNVS80 was able to complement the
GAT1 overproduction defect in a
dal80::hisG mutant (R. Andhare and T. G. Cooper, data not shown). Higher-quality images of all color photographs
are available upon request.
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GFP-Dal80p-generated fluorescence colocalizes with DAPI-positive
material.
Since GFP-Dal80p-generated fluorescent foci move through
the cell cycle in the same way as DAPI-stained material, we determined whether GFP-fluorescence and DAPI-positive material colocalize and
found that they do (Fig. 2, rows 1 and 2). Further,
occasionally DAPI-stained images exhibit defined foci as seen with
GFP-Dal80p (Fig. 2, frames 2B and 2C). This is more clearly seen at
higher magnification (Fig. 2, frames 3A and 3B). Note that this was
observed only when unfixed cells were stained with DAPI.
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FIG. 2.
(Rows 1 and 2) Colocalization of DAPI-positive material
and GFP-Dal80p fluorescence in cells prepared as described in the
legend to Fig. 1. Frames A to C were made using white light, DAPI, and
GFP filter sets, respectively. Frames of DAPI-stained cells (B frames)
were superimposed on frames of GFP-Dal80p-generated fluorescence (C
frames) after the DAPI-positive material was pseudocolored red (D
frames). In all cases colocalization was observed (indicated as yellow
in the D frames). (Row 3) Enlargements of the B and C frames of row 2. (Row 4) GYC86 transformed with pNVS82, containing
GAL1,10-EGFP-DAL82, prepared as described earlier
(22). The slide in frame 4A was illuminated with a small
amount of white as well as fluorescent light; in frame 4B only
fluorescent light was used.
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Confocal analysis of GFP-Dal80-generated fluorescent foci.
If
the GFP-Dal80p fluorescent foci are associated with yeast chromosomes,
one would expect to observe up to 16 of them. Therefore, we used
confocal microscopy to unambiguously monitor the individual foci,
thereby permitting us to determine their number (Fig.
3); foci are lettered to facilitate tracking them
through the optical sections. We observed 16 distinct foci that differ
in size and fluorescence intensity, and one of them, P, appears to
possess a distinctive shape, which has been noticed in a number of
confocal image sets. Computer reconstruction of the 11 confocal images from Fig. 3 is shown in Fig. 4. Although this result has
been replicated multiple times, we cannot resolve 16 foci in every set
of confocal images. We more often can unequivocally distinguish only 13 to 15 foci. We also performed deconvolution reconstructions of cell
images. Here, 15 discrete foci can be distinguished, although one is
quite faint (Fig. 5F).
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FIG. 3.
Z sections generated by confocal microscopy (Zeiss
Axiovert) of GYC86 transformed with GAL1,10-GFP-DAL80
pNVS80. Unique foci are lettered A to P.
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FIG. 4.
Computer reconstruction of information derived from the
Z sections depicted in Fig. 3 that are superimposed on a light
micrograph of the cell analyzed. This reconstruction was performed
using the Zeiss LSM510 reconstruction program.
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FIG. 5.
Expression of GAL1,10-EGFP-DAL80 pNVS80
in wild-type GYC86 (A to C) and a bim1 strain
(Research Genetics BY20147 [MATa
his3D1 leu2D0 met15D0
ura3D0 bim1::kan1/MAT
his3D1 leu2D0 lys2DO ura3D
bim1::kan1]) (D and E). Patterns of EGFP-Dal80p
localization are the same in GYC86 and BY4743, the isogenic wild-type
strain of BY20147. (F) Deconvolution reconstruction of a GYC86 cell
transformed with GAL1,10-EGFP-DAL80 pNVS80. The image was
prepared using Deltavision software.
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GFP-Dal80p in a bim1 mutant.
The above data
prompted us to compare the distributions of fluorescent foci in
wild-type and bim1 cells. Bim1p is required to correctly
position the mitotic spindle (19, 20). In about 20 to 25%
of bim1 cells, the mitotic spindle misaligns such that its axis is perpendicular to the normal axis running from mother to
daughter cell (19). As a result, DAPI-positive material
moves to opposite sides of the mother cell rather than entering the daughter cell (see Fig. 3A in reference 19). In wild-type
cells, GFP-Dal80p fluorescent foci collect near the neck between mother and daughter cells (Fig. 5A to C). In the bim1 mutant, at
an equal or slightly later stage of the cell cycle (Fig. 5D and E), the
fluorescent foci are situated quite removed from the neck, in a pattern
identical to that observed with DAPI-stained bim1 cells
(19).
GFP-Dal82p yields punctate fluorescent foci.
If
GFP-Dal80p is visualizing yeast chromosomes, one might see a
similar pattern of fluorescence with other molecules that bind tightly
to DNA. Dal82p is the UISALL-binding
protein required for inducer (OXLU)-mediated DAL gene
expression (6, 15). Footprinting experiments demonstrate
that Dal82p binds avidly to its target site, and the genome contains
many UISALL-homologous sequences
(25). Therefore, we reviewed micrographs made during a
study of Dal82p localization (22). Although less clearly
defined, a punctate pattern of fluorescence is evident (Fig. 2, row 4). The GFP-Dal82p punctate foci appear superimposed on a background of
nuclear fluorescence similar to that observed with the DAPI-stained image mentioned above (compare rows 3 and 4 in Fig. 2).
Since Dal80p is a GATA binding transcription factor, it is not
difficult to envision that the fluorescent foci represent Dal80p-DNA complexes, thereby arguing that GFP-Dal80p may be visualizing yeast
chromosomes. However, two questions lead to skepticism about such an
interpretation: (i) whether the genome contains sufficient GFP-Dal80p
sites to illuminate individual chromosomes and (ii) why similar images
are not observed with GFP-Gln3p, especially since Gln3p binds to a
simpler structure (10, 13), i.e., a single GATA. Using
previously documented characteristics of the Dal80p binding site, the
genome contains 3,388 homologous sequences, randomly distributed across
all chromosomes. The number of sites per chromosome ranged from 63 (chromosome I) to 439 (chromosome IV); 84% of them are within open
reading frames. Since two Dal80p molecules bind per site, the overall
number of Dal80p molecules that potentially bind may be as high as
6,776, assuming of course that all of the identified sites are able to
bind Dal80p.
The second question is more difficult to answer, but two reasons may
account for the difference. (i) Dal80p, probably as a result of
dimerization (21), binds to DNA more avidly than Gln3p or
Gat1p. The phenotypes of dal80 and gln3 deletions
argue that Dal80p binds to promoter GATA elements in preference to
Gln3p or Gat1p. If, on average, the GATA sequences are occupied
relatively more often with Gln3p and Gat1p than with Dal80p, then
dal80 mutants would not exhibit the strong phenotype
reported (5, 6, 14, 18, 22, 24). Also correlating with
this explanation is that the Dal82p footprint (25) is
stronger than that obtained with Gln3p (21). (ii) It is
conceivable that more than two Dal80p molecules bind to some sites.
When DNA fragments containing "strong" Dal80p binding sites are
used as probes in electrophoretic mobility shift assays, a second,
much-higher-molecular-weight species is observed in addition to the
normally expected band (10). If this slowly migrating
species represents some level of further Dal80p polymerization, it
would increase the fluorescent yield of each "good" Dal80p binding site.
Mammalian erythroid cell-specific GATA-1, GATA-2, and GATA-3 generate
punctate staining in erythroblasts and megakaryocytes (16). The pattern of fluorescence, however, is different
from what we describe here. Anti-GATA-1 antibody illuminates one or two
foci per C88 cell and up to five foci per Buf707 cell. However, there
is no evidence that they move during the cell cycle, and their number
does not correlate with chromosome number (16).
In the past, it has not been possible to take full advantage of the
large number of S. cerevisiae mutants with defects in normal
chromosome movement and cell biology due to an inability to visualize
them individually except in fixed preparations using fluorescent in
situ hybridization or scanning or transmission electron
microscopic procedures. DAPI staining visualizes DNA, but as
little more than an amorphous spot. EGFP-Dal80p may offer an
alternative methodology, providing a more detailed view of DNA movement
in wild-type and mutant cells using real-time microscopy in living cells.
| |
ACKNOWLEDGMENTS |
We thank Lorraine Albritton for bringing the Bim1p papers to our
attention, Tim Higgins for preparing the artwork, and the University of
Tennessee Yeast Group for suggestions to improve the manuscript.
This work was supported by NIH grant GM-35642.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, University of Tennessee, Memphis, TN
38163. Phone: (901) 448-6179. Fax: (901) 448-8462. E-mail:
tcooper{at}utmem.edu.
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Journal of Bacteriology, August 2001, p. 4636-4642, Vol. 183, No. 15
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.15.4636-4642.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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