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Regulating the HO endonuclease in yeast
Regulating
#he HO endonuclease
in yeast
Kim Nasmyth Research
Institute
of Molecular
Pathology,
Vienna,
Austria
The pedigree of mating-type switching in yeast is determined by the transcription pattern of the HO endonuclease gene, which is expressed during late Cl in mother cells but not at all in daughter cells. The late-Cl specificity of HO transcription depends on a heteromeric factor, SBF, which is composed of the Swi4 and Swi6 proteins. Mother-cell specificity involves a second site-specific DNA-binding factor, Swi5, which is synthesized in the C2 and M phases and only enters the nucleus at the end of mitosis. *Swi5 enters mother and daughter nuclei in equal amounts and most is then rapidly degraded. It has been suggested that in mothers but not in daughters some Swi5 protein escapes degradation and persists until SBF is activated in late Cl. This subset of Swi5 molecules may constitute a mother cell’s memory.
Current
Opinion
in Genetics
and Development
Introduction
How haploid yeast spores become diploid vegetative cells was first investigated by Winge nearly sixty years ago [ 1 I. Spores germinate and rapidly undergo two cell divisions by budding, but then all four cells suddenly stop budding, turn into gametes and conjugate in pairs to form two zygotes. This remarkable sequence of events illustrates the choice that most eukaryotic cells make between proliferation and differentiation. Why do the progeny of spores renounce ceU division at the four-cell stage and turn instead into gametes? We now know that conjugation is controlled by the mating-type (MA73 locus, whose a and a alleles [2] contain different DNA sequences that encode regulators (al, al and a2) of the genes involved in cell signalling [3,4]. MATa cells secrete the a-factor pheromone and express a receptor for a factor, whereas MA7& cells secrete a factor and express a receptor for a factor. Pheromones cause cells with the appropriate receptors to arrest in the Gl phase of the cell cycle and to induce genes involved in conjugation. Thus, conjugation only occurs between cells of opposite mating type. The diploidization observed by Wiige is known as homothallism. It arises because cells switch their mating types (Hawthrone DC, abstract 3.49, 11th International Congress of Genetics, Seattle, 1963) in a specific pattern upon germination. Using an assay for mating type that measures the response of individual cells to a factor (see Fig. l), it has been established that spores divide once without switching their mating type to produce a daughter ceU (derived from the bud), which also divides without switching, and a mother ceU that switches
MAT-mating-type; UAS-upstream
1993, 3:286-294
its mating type and thereby produces two progeny with a mating type opposite to those of the daughter cell [ 51. By this means, two pairs of a and a cells are produced that cause each other to arrest cell division and to differentiate into gametes that conjugate. The heteromeric al-a2 repressor formed from products encoded by M4Ta and I%&I~&,respectively, prevents further mating-type switching and conjugation in the zygote and its progeny. The efficient formation of a pair of zygotes at the four-cell stage is only possible because switching is confined to mother cells and because the budding process is spatially regulated so that both progeny of the mother cells find themselves opposite the progeny of daughter cells with whom they can conjugate (see Fig. 2) [ 61. Matingtype switching restricted to mother cells can be sustained through every division if cells are prevented from conjugating by micro-manipulation [5]; daughter cells have never been observed to switch. Mating-type switching occurs through the replacement of a- or a-specific DNA sequences at MAT by ‘silent’ copies of the opposite mating type that reside elsewhere in the yeast genome (at the HMLcr and fiY4Ra loci) [7]. The mechanism appears similar to that of gene conversion, although it is much more frequent (occurring during virtually every cell cycle in the mother cells). The switch takes place when a silent copy containing opposite mating-type DNA is used to repair a double-strand break at WIT made by an endonuclease encoded by the HO gene [8]. Mutation of HO prevents switching and allows the propagation of ‘heterothallic’ cells with stable mating
types[91.
Abbreviations SCB-Swi4/6 cell-cycle box; SPB-spindle activating
@ Current
sequence;
Biology
URS-upstream
regulatory
Ltd ISSN 0959437X
pole body; sequence.
Regulating
the
HO
endonuclease
in yeast
Nasmyth
curs transiently in late Gl during the mother cell cycle (i.e., when cells become committed to cell division at Start) but never in daughter [lo] or in diploid cells [ 111. Both the HO endonuclease and its transcript are unstable so that enzyme made in mothers is not inherited by daughters. A variant of the HO promoter that is transcribed in late Gl in daughter cells at a level normally seen in mother cells causes daughter cells to switch mating type 1121.
(a)
HO activation in late Gl requires the Cdc28 protein kinase [IO], which when inactivated by pheromones causes the Gl arrest required for conjugation. The endonuclease cannot therefore be made in cells that have embarked on conjugation; indeed, it would be disastrous if such cells were to switch mating types. Activation of HO in late Gl suggests that the switching process starts before replication of the MAT locus, which may help ensure that both progeny of the mother cell inherit a new mating type [ 131. Another aspect of I+0 regulation is HOs failure to be expressed as cells undergo Start following release from a Gl arrest induced by pheromone. This situation may be analogous to re-entry of zygotes into the cell cycle following karyogamy; another circumstance in which mating-type switching would be deleterious.
fb)
The slightest disruption of HOs regulation could disrupt the yeast life cycle. This may explain why as much as two kilobases of promoter DNA [ 141, and at least at least eleven genes (SWI-IO and SfVFO are necessary for HO activation [15,16] and why several other genes (occurring in the gene families SIN and/or SLYI are required for the promoter to remain fully dependent on these activators [ 17,181. The juxtaposition of positive and negative controls may contribute to HO’s sensitivity to multiple developmental states; for example, the cellcycle stage, whether a cell is a mother or daughter, and whether the cell is a haploid or diploid.
The HO promoter Fig. 1. Mating-type
switches are manifested by a cell’s response to pheromone. A single spore was germinated in medium containing a factor. (a) The spore budded and must therefore be of the a mating type. (b) Both progeny resulting from the first division also budded and have therefore retained the spore’s a mating type, (c) The mother but not the daughter cell from the first division gives rise to a pair of cells that no longer bud and instead turn into pear-shaped gametes; that is, the mother but not the daughter cell switched mating type and produced a pair of cells expressing the a mating type.
Regulation
of the HO gene
The pedigree of mating-type switching is largely determined by the pattern of HO transcription, which oc-
The analysis of HO promoter deletions suggests that mother/daughter dependence and cell-cycle control are conferred by separate DNA sequences and depend on different transacting factors. A key discovery was the finding that removal of DNA between -900 and -150 upstream of the transcription initiation point causes the promoter to become constitutive throughout Gl and independent of Cdc28, without destroying mother-cell specificity [ 12,141. In contrast, the promoter is inactivated by removal of sequences between -1000 and -1400, and their replacement by the upstream activation sequences (UASs) from the CXLl-IO promoter creates a GAL-HO hybrid promoter whose mother cell dependence is replaced by galactose dependence [ 121.
287
288
Gene expression
and differentiation
Switching
Medial
pedigree
(a)
a
0
Polar
D
I\
budding
a/a
0
a
o/\J\ a
a or a
(b)
0 M
budding
a
co or
0 a
a
0 a
a/a
The promoter can therefore be crudely divided into a distant upstream region- known as upstream regulatory sequence (URS)-l- concerned with mother-cell specificity, and a more proximal region (known as URS2), which is concerned with Cdc28 dependence. A fragment containing two kilobases of promoter DNA is sufficient for mother/daughter control [ 191. No single part of URS2 is required for Cdc28 dependence because the oligonucleotide sequence responsible, CACGAAAA, is present in multiple (up to ten) copies [ 201. A tandem array of this oligonucleotide activates transcription from an unrelated reporter gene in a Cdc2Sdependent manner [16], but because the elkt of mutating individual elements in situ has never been analyzed, it is still not clear whether it merely acts positively. Repression in diploids is the result of multiple binding sites for the al-u2 repressor within the HO promoter [21].
The transcription and Swi6
factor
SBF: composed
of Swi4
The biological activity of CACGAAAA elements depends on the binding of a heteromeric transcription factor called SBF, which is composed of proteins encoded by the SW4 and SWIG genes [16,22,23,24*-l. Sequences bound by this factor are now known as Swi4/6 cell-cycle boxes @CBS). Mutation of either SW’4 or SWIG abolishes transcription from the intact HO promoter, but not from deleted versions lacking URS2 [16]. SBF can be detected in crude extracts using a gel retardation as-
Zygotes
Fig. 2. The regulation of mating-type &itching and bud positioning fackates zygote formation in homothallic yeast. (a) Mother(M) but not daughter (D) cells switch mating type in pairs. (b) New buds are produced close to old ones in haploid (a or a) but not in diploid (a/a) cells. (c) These two properties facilitate zygote formation at the four-cell stage.
say and oligonucleotide probes containing two or more SCBs [22,23,24.*]. SBF-SCB complexes cannot form in extracts made from swi4 or s&G mutants [22], and are recognized by antibodies specihc for Swi4 [23] or ~wi6 [24.*]. Swi4 and Swi6 may be the sole constituents of SBF because a binding activity with very similar electrophoretic properties can be reconstituted by co-translation of SW4 and SWIG transcripts in reticulocyte lysates [ 25**]. SBF can bind to at least three different regions of URS2 [ 24**].
~wi4 and .%vi6 are large proteins with molecular weights of 124 and 91 kD respectively, and are partly homologous [ 23,261. The central regions of both contain two copies of a 33 amino-acid motif now known as the ankyrin repeat [27], which in other proteins has been implicated in protein-protein and protein-DNA interactions, but whose function in Swi4 and ~wi6 is not yet understood. SBF binds to SCBs through a novel (110 ammo acid) site-specific DNA-binding domain located at the amino terminus of Swi4 [ 250.1. As a consequence, Swi4, but not Swib, forms binary complexes with SCB DNA The binding of ~wi6 to Swi4, both on [25**] and off DNA [ 28~1, is mediated by their carboxyl-terminal 120 amino acids and does not require their ankyrin repeats. swi6, but not Swi4, possesses a leucine zipper, which is not needed for its interaction with Swi4, but is nevertheless required for SBF to bind SCBs (L Breeden, personal communication). SBF has never been shown to bind oligonucleotides containing a single SCB, and it is therefore conceivable that SBF binds DNA as a tetramer whose two Swi4/Swi6 subunits interact through Swib’s leucine zipper. Although ZfO normally needs both proteins to be transcribed, overproduction of Swi4 can
Regulating
overcome the need for Swi6 but not vice versa; that is, in sufficient concentration, Swi4 can bind HO SCBs in vivo and activate transcription without &vi6 [29].
Cell-cycle
control
of SBF
SBF is required for transcribing not only HO but also the CLhV, CLN2 and HC52G Gl cyclin genes [30ww,31**]. Transcription of all four genes (and that due to isolated SCB elements) begins in late Gl and depends on the Cdc28 kinase [32**,33**]. A potential mechanism for the activation of SBF’s by Cdc28 is an increase in SW4 transcription, which is also maximal in late Gl [34**]. However, there are no large fluctuations in the level of SBF-binding activity during the cell cycle, suggesting that Swi4 protein, at least when complexed with Swib, may be quite stable [24**]. The variation in SW74 transcrlption could cause modest (up to twofold) changes in the concentration of SBF. It seems unlikely that this would be sufficient to activate HO, unless newly symhesized SBF behaved differently from SBF inherited from the previous cell cycle. SW74 expression from the GALI-20 promoter throughout the cell cycle appears to disturb the repression of HO in G2 [34**], but this also causes HO to become independent of SWTGwhich could be the primary cause of deregulation [35**]. There is evidence that post-transcriptional events could regulate SBF activity. A change in the electrophoretic properties of SBF at the time of HO activation has been detected using a gel retardation assay, but this change persists throughout the rest of the cell cycle [24**]. If the change is related to the formation of an active form of SBF, then HO repression during G2 must occur by a process that is not simply a reversal of its activation. This could well be the case, because gene activation by SBF in late Gl depends on forms of the Cdc28 kinase associated with the Gl-specific cyclins Clnl, -2 and -3, whereas repression in G2 requires forms of the kinase associated with the GZ-speciiic cyclins Clbl, -2, -3 and -4 (A Amon, personal communication). Whether SBF activation in Gl or its inhibition in G2 is the result of SBF’s phosphorylation by Cdc28 is not known. Swi6 changes its localization during the cell cycle; it is concentrated in the nucleus for most of the cell cycle but then accumulates in the cytoplasm of mitotic cells [ 24**]. This could play some role in the inhibition of SBF during G2, but is unlikely to be important for its activation at Start because &vi6 is found concentrated in the nucleus as soon as cells enter Gl; that is, long before gene activation.
Differential control daughter cells
of the mother
and
The mechanism responsible for differential expression in mother and daughter cells is one of the great mysteries of HO regulation. Physiological differences between mother and daughter cells that may be relevant to asym-
the HO endonuclease
in yeast Nasmyth
289
metric expression of HO have barely been addressed. Instead, work has concentrated on which NO transcription factor might differ between mother and daughter cells. The hybrid CAL-HO promoter remains dependent on SWI, -2, -3, -4, -6 -7, -8, -9 and -20, even though it is expressed equally in mother and daughter cells, suggesting that the activities of the above genes are not exclusive to mother cells [ 121. It is now known that SW& -2, -3, -4, 4 and -10 activate genes other than HO in both mother and daughter cells [30**,31°*,36*]. SW5 is the only gene that becomes redundant when UPS1 is replaced by the GiiLI- lOUAS [ 121, and this has provoked a close scrutiny of its potential mother-cell specificity. SW25 encodes an 85 kD protein [37] that binds two sites (called A and B) within URSl [38**] through a three zinc-linger DNA-binding domain near its carboxyl terminus [391. Mutation of either site alone reduces HO transcription (site B more so than site A) and mutation of both abolishes transcription [ 381. HO activation therefore involves the binding of at least two sequence-specific factors: Swi5 to two sites within URSl, and SBF to three sites within URS2. More evidence that asymmetric HO expression involves Swi5 stems from the analysis of suppressor mutations that inactivate the SLW.3/SLW gene and allow HO to be at least partially expressed in the absence of Swi5 [ 17,181. SIN3 possibly affects the activity of repressors that inhibit HO activator proteins in such a way that HO then requires SW5 expression. The precise role of Sin3 is unclear, but it is pertinent that HO is expressed equally in mothers and daughters of sin.3 swi5 double mutants, that is, there is no HO asymmetry in the absence of SW5 dependence. There is no evidence that SW is required directly for mother/daughter control. The daughter cell switching that results from loss of SfWl function can be explained by HO’s loss of SW15 dependence [ 181. Furthermore, Sin3 protein is found in both mothers and daughters [40]. If Swi5 is a factor whose inheritance only by mother cells determines the lack of HO expression in daughters, then one would not expect it to be synthesized by daughter cells until after HO transcription is triggered in late Gl [lo]; indeed, not until SBF is inactivated by a rise in Cdc28 kinase activity associated with the G2 cyclins Clbl and Clb2. It is therefore remarkable that SW75 transcripts are regulated in this fashion; they are absent throughout Gl and are not synthesized until CLBZ and CLB2 transcripts appear in G2 [41]. This cell-cycle regulation is determined by an oligonucleotide sequence that forms ternary complexes between Mcml (which is also involved in the regulation by the MATlocus of genes encoding pheromones and their receptors) and the factor SFFwhose gene has not yet been characterized [ 42.1. Thus, if Swi5 is present at the time of HO activation (which seems likely), it would have to be inherited from the previous cell cycle. Despite indications that Swi5 has a special role in HOs mother-cell specticity, the bulk of Swig is evenly segregated to mother and daughter cells at cell division. Swi5 protein accumulates in the cytoplasm during the G2 and M phases but translocates to both mother and daughter
.
. 290
Gene exmssion
and differentiation
nuclei in roughly equal amounts following inactivation of the Cdc28 protein kinase at the end of mitosis [43]. This cell cycle regulated nuclear uptake is dependent upon serine residues within or close to the Swi5 nuclear localization signal, whose phosphorylation by the Cdc28 protein kinase prevents nuclear uptake [44**]. It should be emphasized that nuclear uptake of Swi5 does not immediately ttigger HO transcxiption, as this cannot occur until later in Gl when SBF becomes active as a result of the re-activation of Cdc28. Most Swi5 molecules are rapidly degraded in both mother and daughter cells soon after (and possibly as a consequence of) entry into the nucleus [ 38**]. Despite the apparently equal segregation of Swi5 to mothers and daughters, there is evidence that a lack of Swi5 is at least partly responsible for daughters’ failure to express HO. Expression of SW’5 throughout Gl at a level comparable to normal G2specillc expression causes daughter cells to switch mating type with a frequency that is 40% of the rate observed in mother cells [42*]. This suggests that Swi5 can in principle bind to and activate the HO promoter in daughter cells, but that it normally does not because most if not all the Swi5 that enters daughter-cell nuclei is degraded before SBF is activated. The deletion of specific sequences located centrally in Swi5 (called the inhibitory region) delays its degradation and causes efficient daughter cell switching (it remains, nevertheless, at least twofold less than the observed rate in mothers) [38=*]. Whether this daughter cell switching is due entirely to an increased concentration of Swi5 in late Gl cells, or to a change in its ability to bind DNA or activate transcription is not certain. In either case, a lack of Swi5 function in daughter cells is implicated in HO inactivity. The lack of parity between the rates of mother and daughter cell switching caused by de-regulating Swi5 suggests that Swi5 might not be the only factor that differs in mothers and daughters. SwiS’s instability and lack of synthesis following mitosis raise the possibility that differences in the interval between a cell’s entry into Gl and SBF activation could contribute to the di@erential expression of HO in mothers and daughters. Daughters are usually born smaller than mothers and must therefore spend longer growing to the minimum size needed for Start. Thus, expression of HO could be decided by a race between Swi5 degradation and SBF activation. Might only mother cells with their shorter Gl periods have enough Swi5 at Start to activate HO? It is important to note that such a mechanism would only work if Swi5 must be bound to HO at the time of SBF activation (i.e., that Swi5 does not execute its function appreciably before SBF does). In fact, neither shortening Gl in daughters [41] nor lengthening it in mothers [38**] has much effect on the pattern of HO transcription, implying that Gl length is not an important parameter. It is interesting that mother cells that are arrested in Gl for several hours by depriving them of Gl cyclins retain the abiity to express HO when Gl cyclin activity is restored, despite SWI5 not being synthesized during the arrest period [38*=]; that is, mother cells remember
their identity, they have a ‘memory’. This contrasts with the lack of HOexpression during release from a Gl arrest resulting from pheromone treatment [41]. The implication is that pheromone treatment and not Gl arrest per se destroys a mother cell’s memory. The loss of memory as induced by pheromone could result from the destruction of Swi5 (which cannot be resynthesized), because constitutive SW5 expression allows HO to be activated following a pheromone-induced Gl arrest [24**]. How can the evidence that Swi5 is limiting in daughter cells be reconciled with its even segregation at cell division? It is clear that the differential expression of HO in mother and daughter cells must stem from the asymmetric segregation of a factor other than Swi5. Nevertheless, it seems that the role of such factor might be to facilitate Swi5 function in mothers or impede it in daughters. If we assume that Swi5 must be present at the time of HO activation, then the factor (or its absence) must enable Swi5 function to be retained during the Gl period in mothers. Most of the Swi5 inherited even by mother cells is destroyed during Gl, suggesting that only a limited quantity of Swi5 molecules need be retained for full activity [ 380.1. The asymmetrically segregated factor might therefore determine the formation at HO of stable transcription complexes containing Swi5 (see Fig. 3). Such complexes could constitute a mother cell’s memory. Although daughter cells cannot form these stable complexes (i.e. they cannot acquire memory), Swi5 can nevertheless be delivered to the HO promoter in daughter cells, either by de novo synthesis [41,42*] at the time of SBF activation or by mutation of its inhibitory domain [38**]. Thus far, there is little indication whether the asymmetric factor acts positively or negatively. Despite extensive searches for HO activators [15,16], no obvious gene candidate for a mother cell specific factor has been identified. Furthermore, mutants in which HO loses its differential expression have never been sought for, mainly for lack of an easy assay. What determines the different fate of Swi5 in mothers and daughters? Preferential segregation of a parental DNA strand associated with a ‘competent’ HO gene to the mother pole at mitosis is ruled out. Neither the orientation of HO within the chromosome nor its chromosomal location are important for mother-cell specificity [ 191. The asymmetric segregation of a nuclear factor is another possibility. Autonomously replicating plasmids that lack centromeres are preferentially inherited by mother cells [45]. Furthermore, the yeast spindle pole body (SPB) appears to be replicated by a conservative mechanism with the old and new SPBs segregating to mother and daughter nuclei respectively [46**]. This organelle could conceivably have a role in the asymmetric segregation of factors that facilitate or antagonize Swi5 function. HO continues to be expressed exclusively in mother cells even when SW5 has been mutated so that the Swi5 protein enters the nucleus constitutively [44**]. What then might be the function of SwiS’s regulated nuclear uptake? In addition to not transcribing HO, swi5 mutants have a mild defect in cell separation. The cytological regulation of Swi5 may therefore be more important for the
Regulating
c2
M
Early
T
in yeast Nasmyth
the HO endonuclease
Cl
late
291
Cl
SBF-I
SBF-A
Cdc284b inactivation m Swi5 synthesis and accumulation in the cytoplasm
+
Cdc28-Cln activation
D Swi5 both
enters nuclei
SwiS
SBF-A
URSI
URS2
,d>X> URSl
URS2
HO
Fig. 3, Steps leading to HO activation in mother (M) but not daughter (D) cells. It is suggested that stable complexes containing Swi5 form on the HO promoter only in mother cells. Swi5 is synthesized and accumulates in the cytoplasm during the G2 and M phases. Destruction of Cdc28 kinase associated with the Clb cyclins at telophase 0 signals the entry of Swi5 into both the mother and daughter nuclei (indicated by black shading). Most Swi5 molecules are then degraded during early Cl, but some form stable complexes on the HO promoter in mother but not in daughter cells (indicated by the light-shaded nucleus of the mother cell). It is therefore suggested that HO chromatin differs between the mother and daughter cells (represented by the black-shaded square). The formation of stable Swi5 complexes at HO are not sufficient for its transcriptional activation farrowed), which must await the activation of SBF (SBF-I converted to SBF-A) by Cdc28 kinase associated with Cln cyclins (indicated by the dashed arrows). URS indicates upstream regulatory regions.
cell cycle dependent activation of cell separation genes than it is for HO. c7sZ encodes a chitinase needed for cell separation and is activated at the time Swi5 enters the nucleus, which is appreciably earlier than HO activation (R Siegmund, personal communication). C7SZ transctiptlon is not in fact dependent on Swi5 but instead on the factor Ace2, which exhibits extensive homology to Swi5 and whose entry into the nucleus is similarly regulated [47]. Deletion of URS2 places the SwiS-binding sites within URSl close to the HO TATA box, and thereby allows HO to be expressed in the absence of SBF. This deleted promoter is not expressed during G2 and is only activated upon entry of Swi5 into the nucleus at the end of mitosis [43,44**]; that is, its regulation resembles that of ml.
How does the HO promoter
work?
This article has concentrated on HOs cell cycle dependence and its mother-cell specificity, which concern the functions of SBF and Swl5, respectively. What is the function of all the other genes required for HO transcription; genes such as SWl, -2, -3 - 7, -8, -9 and -IQ The discovery that SWZ2 is probably allelic to the SNF2 gene required for transcription of WC2 (K Nasmyth, unpublished data) led to the finding that other genes such as SNFS and SNFG,which are required for SLJC2expression, are also required for HO expression (SNFS is probably allellc with SW10 [K Nasmyth, unpublished data]). SWZ2/SNF2 [ 481 encodes a protein with homology to helicases [49,50] and is highly homologous to the Brahma protein from Drosophila [51]. SW3 also seems have a homologue in Drasophih and has been shown to interact with steroid receptors [ 52.1. Thus far, none of these proteins have been shown to bind specllic DNA sequences, but chimeras of Swi2/Snf2, Snf6, and SnWSwilO with the bacterial 1exA protein activate transcription from a leti operator/promoter [48]. Swil, -2 and -3, Snf6, D
and SnfS/SwilO may form a large complex of highly conserved proteins that exerts gene-specific activation through interaction with site-specific transcription factors like Swi5 or SBF. It seems likely that Sw17, -8 and -9 will also prove to be general transcription factors. The transcriptional defects of swil, -2 and -3 mutants are partially suppressed by mutations in genes that encode histones or HMG-like proteins [ 531, and it has therefore been suggested that helicase activity associated with Swi2/Snt2 could be involved in regulating chromatln structure [ 541. An alternative is that such a helicase is involved in melting promoters prior to the initiation of transcription. Why HO needs at least two DNA sequence-specific factors, and how they interact with more general transcription factors in the context of chromatin may need to be &r&d before we can comprehend how the arrival of Swi5 in the nucleus at the end of mitosis is a pre-condition for HO activation, and why a change associated with SBF later in Gl eventually triggers the activation. Swi5, al-c&? and SBF all have multiple binding sites within the HO promoter; a redundancy that may contribute to the explosive activation of HO in late Gl, and to preventing expression at times when it would be disastrous for the yeast life cycle.
Conclusion The HO endonuclease gene is transcribed transiently during the late Gl period in only one of the two progeny of a yeast cell division; that is, in mother but not in daughter cells. Iate Gl specificity is conferred by the SBF transcription factor, which also activates genes for Gl cyclins. It seems likely that SBF is activated by the Cdc28 protein kinase. The mechanism of activation and subsequent repression are not yet understood. The mother ceil specificity of HO involves a second transctiption factor called Swl5, which is synthesized during G2 but does not enter nuclei until the end of mitosis. Swi5 enters both mother and daughter nuclei and most is then rapidly degraded.
.
Howevqr, some appears to survive in mother cells until SBF is activated by Cdc28 in late Gl. The challenge now is to determine in what form SwiS survive3 in mother cells and why this process cannot occur in daughters. A further objective will be to elucidate why the arrival of SwiS, the activation of SBF, and the involvement of other more general transcription factors are all needed for the initiation of HO transcription.
15.
STERN MR, JENSEN R, HERSKOWITZ I: Five quired for Expression of the HO Gene 1984, 178:853-868.
16.
BREEDEN 1 HO Gene: 48:389-397.
17.
STERNBERG PW. STERN MJ, Cwuc I, HE~KO~ITX I: Activation of the Yeast HO Gene by Release from Multiple Negative Controls. Cell 1987, 48:567-577.
18.
NAShnTH K, STILL&IAN D. KIPIJNG D: Both Positive and Neg ative Regulators of HO Transcription are Required for Mother Cell-specific Mating Type Switching in Yeast. Cell 1987, 48:579-587.
19.
K~AR AJS: The Mother-Daughter Asymmetry of Budding Yeast gation of Parental HO Gene 1:1059-1064.
20.
NASMYTH, K: A Repetitive DNA Sequence that cycle START (CDCZS)-dependent Transcription Gene in Yeast. Cell 1985, 42:225-235.
21.
MILIIR AM, MCKAY VL, NX+WH KA: Identification and Comparison of Two Sequence Elements that Confer Cell-type Specific Transcription in Yeast. Nalrrre 1985, 314:598-603.
22.
ANDRE BJ, HERSKO~ITZ I: Identification of a DNA Binding Factor Involved in Cell-cycle Control of the Yeast HO Gene. Cell 1989, 57:21-29.
23.
A~WKRYIS BJ, HEHSKOWIIX 1: The Yeast SWI4 Protein Contains a Motif Present in Developmental Regulators and is Part of a Complex Involved in Cell-cycle-dependent Transcription. Nature 1989. 342830-833.
Acknowledgements I would like to thank Gustav Ammerer and Celia Dower for comments while preparing this manuscript, and Hannes Tkadletz help preparing the figures.
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Base Pairs Expression
AW)JI(
in Yeasts. 1949. 24:
Lineage.
Nature
Cell-specific Regulator
Matof HO
Monitoring cerevisiae.
of 5’-Flanking of the HO
DNA Gene
Control Regulators.
of
the Yeast Ce// 1987,
Mating Type Switching is not Conferred by the SegreDNA Strands. Genies DW 1987.
Confers Cellof the HO
24. ..
HERSKOWITZ I: Asymmetry and Directionality of New Cell Types During Clonal Growth: Pattern of Homothallic Yeast. Cell 1979.
of Yeast
NA%NIH K: Cell Cycle Cisand Transacting
SWI Genes are Rein Yeast. J MO/ Rio1
of AIo/ is in
TMA MRM. M~IKOFF I, LSDAU. D, TEBB G, NASCnTH K: Changes in a sw14,6 DNA-binding Complex Occur at the Time of HO Gene Activation in Yeast. Genes Del* 1991. 5:200&2013. This paper addresses the cell-cycle regulation of SBF activity. Two fomis of SBF’, which are distinguished hy their electrophoretic mobility, are detected. One form is absent in early Gl cells, appears at the time of HO actiMtion, hut persists until the end of mitosis. The appearance of this form could therefore play a role in the activation of HO. The other form is found at all stages of the cell cycle. Thus, SBF of one form or another can be detected throughout the cell-cycle. suggesting that the cell cycle regulation of SW4 transcription described in 134**] does not ause major fluctuations in SBF concentration during the cell cycle. The Swi6 and Swi4 proteins are shown to be part of SBF-SCB complexes, which form on three different fragments derived from URSZ. The intro. cellular location of ~wi6 varies during the cell cycle: ir accumu&s in the cytoplasm of mitotic cells hut is concentrated in the nucleus at all other cell-cycle stages. 25. ..
PRI~IIG M, SOC~ATHAN S. ALZH H. N,\SbnTH K: Anatomy of a Transcriprion Factor Important for the Start of the CeU Cy cle in Saccharomyces cereoisiae. Nature 1992, 358~593-597. This paper shows that SBF can he reconstituted by translating SW74 and SW6 mRNAs in a reticu&yte lysate, that SBF binds to SC8 DNA through a 110 amino.acid domain located near %4‘s amino terminus. and that Swi4 and ~wi6 interact through sequences near their carboxyl termini. 26.
BREEDEN L. NAS~~TH K: Similarity Between Cell-cycle of Budding Yeast and Fission Yeast and the notch Drosophila. Nature 1987h. 329:651-654.
27.
MICHAEL~ P, BENNET V: The tif Involved in Macromolecular 1992, 2:127-129.
28. .
ANK Repeat: Recognition.
Genes Gene of
A Ubiquitous MoTrefr& Ce// Biol
ANDREWS BJ, MOORE LA: Interaction of the Yeast Swi4 and Swi6 Regulatory Proteins in Vitro. Proc Nafl Acad Sci USA 1992, 89:11852-l 1856. The interaction of Swi4 and Swi6 proteins translated in reticulocyte lysates in vitro is dependent upon their carboxyl~terminal sequences but not their ankyrin motifs.
Regulating 29.
SOCKANATHAN
Specific University
S: Characterization Transcriptional Activator of Cambridge; 1990.
of Swi4: A Yeast CeU Cycle [PhD Thesis]. Cambridge:
NAS~H K, DIRICK L The Role of SW14 and SlV76 in the 30. .. Activity of Gl CycUns in Yeast. Cell 1991, 66995-1013. This paper shows that SW14 and S1P76are needed for the transcription and function of the Gl cyclin genes CLNl and CLN2, that SBF binds to SCBs within the UN2 promoter, and that the lethality of scui4 su+G double mutants can be rescued by expression of CLNI? from a foreign promoter. Thus, SBF is a Start-dependent activator not only of HO but also of CLh’I and CLh’2. OCAS J. ANDEWS BJ, HIX!~KOWITZ I: Transcriptional Activation of CLNl, CLNZ, and a Putative New Gl Cyclin (HCS26) by SWl4, a Positive Regulator of Gl-specific Transcription. Cell 1991, 66:1015-1026. SW14 is an essential gene in diploids (in some but not all genetic backgrounds). Multicopy plasmids that suppress the lethality are found to contain the CLNI, CLN2 and HCS26 genes. HCS26 encodes a novel cy clin whose promoter contains SCBs and its expression, like CLNl and CLN2, is dependent on SW14 and is triggered in late Gl. 31. ..
DIRICK L NASMYIH K: Positive Feedback in the Activation of Gl Cyclins in Yeast. Nulrtre 1991, 351:754-757. &ivation of HO by SBF is Cdc28 dependent. To reconcile this with SBF also being an activator of Gl cyclins, which are themselves activators of Cdc28, it wzzs suggested that activation of CLNl and CLN2 by SBF occurs through a positive feedback loop. As predicted by this hy pothesis. the transcription of the CLNI and CLN2 genes is shown to depend on CLX28 and on Gl cyclin activity. CROSS F, TINKHJZ~ERG AH: A Potential Positive Feedback Loop Controlling CLNl and CLN2 Gene Expression at the Start of the Yeast Cell Cycle.&// 1991, 65:875-&%3. The Start’ of the yeast cell cycle is thought to result from activation of the Cdc28 protein kinase by the appearance of Gl cyclins. This paper addresses the mechanism by which the transcription of the G1 cyclin genes CLNI and CLN, is activated in late Gl. It is shown that their activation is stimulated by Gl cyclin activity and depends on the Cdc28 kinase, implying that a positive feedback loop is involved.
39.
DNA
Finger Direct
NAS~V~~~H K. SEDDON A, AMMERER G: CeU Cycle Regulation SWI5 is Required for Mother-cell-specific HO Transcription in Yeast. CelI 1987, 49549558
42. .
Lyow Yeast:
43.
NA~MVH K, ADOLF G, LYDW D, SEDD~N A: The Identification of a Second CeU Cycle Control on the HO Promoter in Yeast: CeU Cycle Regulation of SWl5 Nuclear Entry. CelI 1990, 62:631-647.
of
D, AMMEKER G, NA%IIIH K: A New Role for MCMl ln CeU Cycle Regulation of SW-5 Transcription. Genes Der 1991, 5:2405-2419. Expression of SW15 in G2 is mediated by the binding of Mcml, in complex with the novel factor SFF, to the SW15 promoter. This paper also presents the clearest evidence to date that the absence of SW15 transcription in Gl is necessary to prevent HO expression in daughter cells.
45.
STILLMAN DJ, BANKIER AT, SEDDON A, GROENHOLIT G. NA%M-H KA: Characterization of a Transcription Factor Involved in Mother CeU Specific Transcription of the Yeast HO Gene. EMBO J 1988, 7:485-494.
to
K, RHODES D: Zinc and Folded in Vihv Nafure 1988, 332284-286.
E. coli
41.
DIRICK I., MOLL T. AUER H, NASWIH K: A Central Role for SWIG in Modulating CeU Cycle START Specific Transcrip tion in Yeast. Nurure 1992, 357:50%513. Swi6 protein is a component of two different late Gl-specific transcrlption factors. It complexes with Swi4 to form SBF. which regulates HO and Gl cyclins. and with a 12OkD protein that is distinct from Swi4 to form MBF, which regulates most genes involved in DNA replication. Neither set of genes are transcribed efficiently in the absence of swi6. Moreover, cellcycle regulation of SBF.actlvared genes is impaired and that of MBF-activated genes is abolished in suli6 mutants.
37.
Y, N~s~vm in
WANG H, CLARK I, NICHOLSON PR, HERSKO~I’I~! I, SnllMAN DJ: The Saccharomyces cerevisiae SIN3 Gene, a Negative Regulator of HO, Contains Four Paired Amphipathic Helix Motifs. &lo/ Cell Biol 1990, 10:5927-5936.
44. ..
PE’IIXSON CL, HERSKOWII-,! 1: Characterization of the Yeast SWll. SWIZ, and SWI3 Genes, which Encode a Global Activator of Transcription. Cell 1992, 68:773-583. SWII. SWi’2 and SWI.? are required for the expression of many genes other than HO, but the pleiotrophies of double mutants are no more severe than those of single mutants, suggesting that all three genes are involved in the same function.
NAGAI K, NMEKO Motifs Expressed Specific Binding
40.
BREEDEN L, MIKES~U. GE: Cell Cycle-specific Expression of the SWI4 Transcription Factor is Required for the Cell Cycle Regulation of HO Transcription. Genes Del* 1991, 5:1183-l 190. SW74 transcripts fluctuate during the cell cycle; being most abundant in late Gl at the time of HO activation, and least abundant in early Gl and in G2, when HO is not transcribed. How important this regulation is for confining SBF activity to late Gl and S phase is not clear. SBF-binding activity does not vary much during the cell cycle (see ]24**]). Highlevel expression of SW14 from the C&U! promoter seems to cause HO to be expressed in G2, but this effect could at leasr partly be because SII’IG becomes redundant for f-/O transcription under these conditions.
36. .
293
TEBB G, Mou T, D~WLER C, NASMYIH K: SWI5 Instabiity may be Necessary but is not Sufficient for Asymmetric HO Expression in Yeast. Genes Deu 1993, in press. HO activation by Swl5 is mediated by Swi5 binding to two sites within URSl. Most of the Swi5 protein that enters mother and daughter nuclei at the end of mitosis is rapidly degraded by a process that requires spe cific sequences located centrally in the Swl5 protein. Deletion of these sequences causes HO to be expressed in daughter cells, which raises the possibility that differences between mothers and daughters in the time between Sti5 entty into the nucleus at the end of mitosis, and activation of SBF at Start could contribute to HCh asymmetric expression. However, greatly extending this intetval in mother cells has little effect on HO tntnsctiption, refuting the hypothesis. It is proposed that mother and daughter cells differ in their ability to trap Swl5 in a form that persists until SBF is activated in late Gl.
34. ..
35. ..
in yeast Nasmyth
38. ..
32.
33. . .
the HO endonuclease
MOU T, TEBB G, SURANA U, ROBI~SCH H, NA%IYIH K: The Role of Phosphorylation and the CDC28 Protein Kinase in CeU Cycle-regulated Nuclear Import of the S. cereuisiue Transcription Factor SWI5. Gel/ 1991, 66:743-758. The cell cycle regulated entry of Sti5 into the nucleus is shown to depend on phosphotylation sites within or near the Swi5 nuclear localixation signal. It is suggested that destruction of the Cdc28 protein klnase at the end of mitosis causes Swl5 de-phosphotylation and thereby activation of its nuclear localization signal. Regulated Swi5 nuclear entry is not necessary for mother cell specific HO expression but probably regulates other genes that, unlike HO, are expressed as soon as cells enter Gl. MLIRRAY AW. SZOSTAK Jw: Pedigree Analysis regation in Yeast. Cell 1983, 35:167-174.
of
Plasmid
Seg-
46. ..
VALIIN EA, S~HERXIN n, ROBER‘I~ T, VAN ZEE K, Rosa MD: Asymmetric Mitotic Segregation of the Yeast Spindle Pole Body. Cell 1992, 69505-515. It appears that the asymmetric segregation of some factor other than Swi5 must be responsible for HCYs mother-cell specificity. SPBs in yeast are probably duplicated by a conservative mechanism. This paper indicates that old and new spindle pole bodies may be segregated to mother and daughter cells, respectively. Something associated with only one of the two SPBs might be responsible for the asymmetry of HO. D~HR~IANN PR. BUTLER G, TAMAI K, D~RIAND S, GREENE JR, THIELE DJ, Snmm DJ: Parallel Pathways of Gene Regulation: Homologous Regulators SWI5 and ACE2 Dilferentially Control Transcription of HO and Chitinase. Genes Deu 1992, 6:93-104. AC..~ encodes a protein that is closely related to SWl5, and which is required for the activation of the CTXI chitlnase gene but not for HO. Ace2 and Swl5 have very similar DNA-binding domains and nuclear localization signals. Furthermore, their synthesis and entry into the nucleus are similarly cell cycle regulated. This paper suggests that c7sl 47. ..
294
Gene expression
and differentiation
expressiop is also dependent on the SWI4 and SU’%encoded func tions, and that its regulation is therefore similar to that of HO. These last two assertions may be incorrect because CIsh is transcribed nor malty in swi4 and stui6 mutants and unlike HO is activated as soon as cells enter Gl; presumably resulting from the regulated entry of Ace2 into the nucleus (R Siegmund, T Schuster, K Nasmyth, unpublished data). 48.
LAURENT BC, TRE~L MA, Cluuso~ M: pendence of the Yeast SNF2, SNFS, in- Transcription Activation. Proc Natl
Functional lnterdeand .SNF~ Proteins Acad SciUSA 1991,
88:2&7-2691. 49.
DAVIS JI,
KUNISAWA
(MOT1 Gene’Product) quired for Viabiity 1992, 12:1879-1892.
R, Tttottmttt
JA Presumptive Helicase Affects Gene Expression and is Rein the Yeast S. cerevisiae. IBOI Cell Biol
IAURE~ BC, YANG X, COON M: An Essential S. cerevisiae Gene Homologous to SNF2 Encodes a Helicase-related Protein in a New Famjly. Mel Cell Biol 1992, 12:1893-1902. This paper, together with 1511, suggests that the SwiZ/SnR protein con. tains motifs characteristic of helicases. Whether the protein is a helicase and, if so, what its function might be are as yet unknown.
50. .
51.
TAMKUN JW, DEURING R, Sco7-r AM, KAUFMAN TC, KENNIXIN of
Drosophila
Homeotic
Genes
M. I&INGER JA: Brahms: Structurally
the Yeast 68:561-572.
52. .
Transcriptional
Activator
YOSHINAGA SK, PETERSON K: Roles of SWIl, SWl2,
SNF2/SWl2
CL,
HER~KOVQITZ
SWI3 Steroid
Proteins Receptors.
Gz//
1992,
1, Y~IOTO for Transcrip Scierrce 1992,
tional Enhancement by 258:1598-1604. Activity of the rat glucocorticoid receptor in yeast requires the SWII, -2 and .3 genes. It is hypothesized that the Swil and Swi2 proteins may fomi a complex with Swi3, which interacts v&h the glucocorticoid re ceptor. None of these Swi proteins have been shown to bind specific DNA sequences and the authors suggest that their gene-specific action might result from interaction with sequencespecific tnnscription fat tots. Previously, a similar conclusion was reached for Snf2, which is probably identical to Swi2 (K Nasmyth, unpublished data), in the case of WC2 acthation (see 1481). The sequencespecitic factor with which the putative complex comprising Swil, .2 and .3 interacts on the HO promoter is not yet known; Swi5 and SBF are possibilities.
53.
KRUGER W. HERFKOWITZ I: A Negative Regulator of HO Transcription, SIN1 (SPITZ), is a Nonspecific DNA-binding Protein Related to HMGl. ,Mol CeN Biol 1991, 11:41354146.
54.
WINSTON tivators Genet
F, ORISON M: Yeast SNF/SWl and the SPT/SlN Chromatin 1332, 8:387-391.
Transcriptional Connection.
AcTWI&
M, PAI-FAT~CCI A
Regulator Related to
K Nasmyth, Research Gasse 7. A-1030 Vienna,
Institute Austria.
of
Molecular
Patholog),
Dr.
Bohr-
#he HO endonuclease
in yeast
Kim Nasmyth Research
Institute
of Molecular
Pathology,
Vienna,
Austria
The pedigree of mating-type switching in yeast is determined by the transcription pattern of the HO endonuclease gene, which is expressed during late Cl in mother cells but not at all in daughter cells. The late-Cl specificity of HO transcription depends on a heteromeric factor, SBF, which is composed of the Swi4 and Swi6 proteins. Mother-cell specificity involves a second site-specific DNA-binding factor, Swi5, which is synthesized in the C2 and M phases and only enters the nucleus at the end of mitosis. *Swi5 enters mother and daughter nuclei in equal amounts and most is then rapidly degraded. It has been suggested that in mothers but not in daughters some Swi5 protein escapes degradation and persists until SBF is activated in late Cl. This subset of Swi5 molecules may constitute a mother cell’s memory.
Current
Opinion
in Genetics
and Development
Introduction
How haploid yeast spores become diploid vegetative cells was first investigated by Winge nearly sixty years ago [ 1 I. Spores germinate and rapidly undergo two cell divisions by budding, but then all four cells suddenly stop budding, turn into gametes and conjugate in pairs to form two zygotes. This remarkable sequence of events illustrates the choice that most eukaryotic cells make between proliferation and differentiation. Why do the progeny of spores renounce ceU division at the four-cell stage and turn instead into gametes? We now know that conjugation is controlled by the mating-type (MA73 locus, whose a and a alleles [2] contain different DNA sequences that encode regulators (al, al and a2) of the genes involved in cell signalling [3,4]. MATa cells secrete the a-factor pheromone and express a receptor for a factor, whereas MA7& cells secrete a factor and express a receptor for a factor. Pheromones cause cells with the appropriate receptors to arrest in the Gl phase of the cell cycle and to induce genes involved in conjugation. Thus, conjugation only occurs between cells of opposite mating type. The diploidization observed by Wiige is known as homothallism. It arises because cells switch their mating types (Hawthrone DC, abstract 3.49, 11th International Congress of Genetics, Seattle, 1963) in a specific pattern upon germination. Using an assay for mating type that measures the response of individual cells to a factor (see Fig. l), it has been established that spores divide once without switching their mating type to produce a daughter ceU (derived from the bud), which also divides without switching, and a mother ceU that switches
MAT-mating-type; UAS-upstream
1993, 3:286-294
its mating type and thereby produces two progeny with a mating type opposite to those of the daughter cell [ 51. By this means, two pairs of a and a cells are produced that cause each other to arrest cell division and to differentiate into gametes that conjugate. The heteromeric al-a2 repressor formed from products encoded by M4Ta and I%&I~&,respectively, prevents further mating-type switching and conjugation in the zygote and its progeny. The efficient formation of a pair of zygotes at the four-cell stage is only possible because switching is confined to mother cells and because the budding process is spatially regulated so that both progeny of the mother cells find themselves opposite the progeny of daughter cells with whom they can conjugate (see Fig. 2) [ 61. Matingtype switching restricted to mother cells can be sustained through every division if cells are prevented from conjugating by micro-manipulation [5]; daughter cells have never been observed to switch. Mating-type switching occurs through the replacement of a- or a-specific DNA sequences at MAT by ‘silent’ copies of the opposite mating type that reside elsewhere in the yeast genome (at the HMLcr and fiY4Ra loci) [7]. The mechanism appears similar to that of gene conversion, although it is much more frequent (occurring during virtually every cell cycle in the mother cells). The switch takes place when a silent copy containing opposite mating-type DNA is used to repair a double-strand break at WIT made by an endonuclease encoded by the HO gene [8]. Mutation of HO prevents switching and allows the propagation of ‘heterothallic’ cells with stable mating
types[91.
Abbreviations SCB-Swi4/6 cell-cycle box; SPB-spindle activating
@ Current
sequence;
Biology
URS-upstream
regulatory
Ltd ISSN 0959437X
pole body; sequence.
Regulating
the
HO
endonuclease
in yeast
Nasmyth
curs transiently in late Gl during the mother cell cycle (i.e., when cells become committed to cell division at Start) but never in daughter [lo] or in diploid cells [ 111. Both the HO endonuclease and its transcript are unstable so that enzyme made in mothers is not inherited by daughters. A variant of the HO promoter that is transcribed in late Gl in daughter cells at a level normally seen in mother cells causes daughter cells to switch mating type 1121.
(a)
HO activation in late Gl requires the Cdc28 protein kinase [IO], which when inactivated by pheromones causes the Gl arrest required for conjugation. The endonuclease cannot therefore be made in cells that have embarked on conjugation; indeed, it would be disastrous if such cells were to switch mating types. Activation of HO in late Gl suggests that the switching process starts before replication of the MAT locus, which may help ensure that both progeny of the mother cell inherit a new mating type [ 131. Another aspect of I+0 regulation is HOs failure to be expressed as cells undergo Start following release from a Gl arrest induced by pheromone. This situation may be analogous to re-entry of zygotes into the cell cycle following karyogamy; another circumstance in which mating-type switching would be deleterious.
fb)
The slightest disruption of HOs regulation could disrupt the yeast life cycle. This may explain why as much as two kilobases of promoter DNA [ 141, and at least at least eleven genes (SWI-IO and SfVFO are necessary for HO activation [15,16] and why several other genes (occurring in the gene families SIN and/or SLYI are required for the promoter to remain fully dependent on these activators [ 17,181. The juxtaposition of positive and negative controls may contribute to HO’s sensitivity to multiple developmental states; for example, the cellcycle stage, whether a cell is a mother or daughter, and whether the cell is a haploid or diploid.
The HO promoter Fig. 1. Mating-type
switches are manifested by a cell’s response to pheromone. A single spore was germinated in medium containing a factor. (a) The spore budded and must therefore be of the a mating type. (b) Both progeny resulting from the first division also budded and have therefore retained the spore’s a mating type, (c) The mother but not the daughter cell from the first division gives rise to a pair of cells that no longer bud and instead turn into pear-shaped gametes; that is, the mother but not the daughter cell switched mating type and produced a pair of cells expressing the a mating type.
Regulation
of the HO gene
The pedigree of mating-type switching is largely determined by the pattern of HO transcription, which oc-
The analysis of HO promoter deletions suggests that mother/daughter dependence and cell-cycle control are conferred by separate DNA sequences and depend on different transacting factors. A key discovery was the finding that removal of DNA between -900 and -150 upstream of the transcription initiation point causes the promoter to become constitutive throughout Gl and independent of Cdc28, without destroying mother-cell specificity [ 12,141. In contrast, the promoter is inactivated by removal of sequences between -1000 and -1400, and their replacement by the upstream activation sequences (UASs) from the CXLl-IO promoter creates a GAL-HO hybrid promoter whose mother cell dependence is replaced by galactose dependence [ 121.
287
288
Gene expression
and differentiation
Switching
Medial
pedigree
(a)
a
0
Polar
D
I\
budding
a/a
0
a
o/\J\ a
a or a
(b)
0 M
budding
a
co or
0 a
a
0 a
a/a
The promoter can therefore be crudely divided into a distant upstream region- known as upstream regulatory sequence (URS)-l- concerned with mother-cell specificity, and a more proximal region (known as URS2), which is concerned with Cdc28 dependence. A fragment containing two kilobases of promoter DNA is sufficient for mother/daughter control [ 191. No single part of URS2 is required for Cdc28 dependence because the oligonucleotide sequence responsible, CACGAAAA, is present in multiple (up to ten) copies [ 201. A tandem array of this oligonucleotide activates transcription from an unrelated reporter gene in a Cdc2Sdependent manner [16], but because the elkt of mutating individual elements in situ has never been analyzed, it is still not clear whether it merely acts positively. Repression in diploids is the result of multiple binding sites for the al-u2 repressor within the HO promoter [21].
The transcription and Swi6
factor
SBF: composed
of Swi4
The biological activity of CACGAAAA elements depends on the binding of a heteromeric transcription factor called SBF, which is composed of proteins encoded by the SW4 and SWIG genes [16,22,23,24*-l. Sequences bound by this factor are now known as Swi4/6 cell-cycle boxes @CBS). Mutation of either SW’4 or SWIG abolishes transcription from the intact HO promoter, but not from deleted versions lacking URS2 [16]. SBF can be detected in crude extracts using a gel retardation as-
Zygotes
Fig. 2. The regulation of mating-type &itching and bud positioning fackates zygote formation in homothallic yeast. (a) Mother(M) but not daughter (D) cells switch mating type in pairs. (b) New buds are produced close to old ones in haploid (a or a) but not in diploid (a/a) cells. (c) These two properties facilitate zygote formation at the four-cell stage.
say and oligonucleotide probes containing two or more SCBs [22,23,24.*]. SBF-SCB complexes cannot form in extracts made from swi4 or s&G mutants [22], and are recognized by antibodies specihc for Swi4 [23] or ~wi6 [24.*]. Swi4 and Swi6 may be the sole constituents of SBF because a binding activity with very similar electrophoretic properties can be reconstituted by co-translation of SW4 and SWIG transcripts in reticulocyte lysates [ 25**]. SBF can bind to at least three different regions of URS2 [ 24**].
~wi4 and .%vi6 are large proteins with molecular weights of 124 and 91 kD respectively, and are partly homologous [ 23,261. The central regions of both contain two copies of a 33 amino-acid motif now known as the ankyrin repeat [27], which in other proteins has been implicated in protein-protein and protein-DNA interactions, but whose function in Swi4 and ~wi6 is not yet understood. SBF binds to SCBs through a novel (110 ammo acid) site-specific DNA-binding domain located at the amino terminus of Swi4 [ 250.1. As a consequence, Swi4, but not Swib, forms binary complexes with SCB DNA The binding of ~wi6 to Swi4, both on [25**] and off DNA [ 28~1, is mediated by their carboxyl-terminal 120 amino acids and does not require their ankyrin repeats. swi6, but not Swi4, possesses a leucine zipper, which is not needed for its interaction with Swi4, but is nevertheless required for SBF to bind SCBs (L Breeden, personal communication). SBF has never been shown to bind oligonucleotides containing a single SCB, and it is therefore conceivable that SBF binds DNA as a tetramer whose two Swi4/Swi6 subunits interact through Swib’s leucine zipper. Although ZfO normally needs both proteins to be transcribed, overproduction of Swi4 can
Regulating
overcome the need for Swi6 but not vice versa; that is, in sufficient concentration, Swi4 can bind HO SCBs in vivo and activate transcription without &vi6 [29].
Cell-cycle
control
of SBF
SBF is required for transcribing not only HO but also the CLhV, CLN2 and HC52G Gl cyclin genes [30ww,31**]. Transcription of all four genes (and that due to isolated SCB elements) begins in late Gl and depends on the Cdc28 kinase [32**,33**]. A potential mechanism for the activation of SBF’s by Cdc28 is an increase in SW4 transcription, which is also maximal in late Gl [34**]. However, there are no large fluctuations in the level of SBF-binding activity during the cell cycle, suggesting that Swi4 protein, at least when complexed with Swib, may be quite stable [24**]. The variation in SW74 transcrlption could cause modest (up to twofold) changes in the concentration of SBF. It seems unlikely that this would be sufficient to activate HO, unless newly symhesized SBF behaved differently from SBF inherited from the previous cell cycle. SW74 expression from the GALI-20 promoter throughout the cell cycle appears to disturb the repression of HO in G2 [34**], but this also causes HO to become independent of SWTGwhich could be the primary cause of deregulation [35**]. There is evidence that post-transcriptional events could regulate SBF activity. A change in the electrophoretic properties of SBF at the time of HO activation has been detected using a gel retardation assay, but this change persists throughout the rest of the cell cycle [24**]. If the change is related to the formation of an active form of SBF, then HO repression during G2 must occur by a process that is not simply a reversal of its activation. This could well be the case, because gene activation by SBF in late Gl depends on forms of the Cdc28 kinase associated with the Gl-specific cyclins Clnl, -2 and -3, whereas repression in G2 requires forms of the kinase associated with the GZ-speciiic cyclins Clbl, -2, -3 and -4 (A Amon, personal communication). Whether SBF activation in Gl or its inhibition in G2 is the result of SBF’s phosphorylation by Cdc28 is not known. Swi6 changes its localization during the cell cycle; it is concentrated in the nucleus for most of the cell cycle but then accumulates in the cytoplasm of mitotic cells [ 24**]. This could play some role in the inhibition of SBF during G2, but is unlikely to be important for its activation at Start because &vi6 is found concentrated in the nucleus as soon as cells enter Gl; that is, long before gene activation.
Differential control daughter cells
of the mother
and
The mechanism responsible for differential expression in mother and daughter cells is one of the great mysteries of HO regulation. Physiological differences between mother and daughter cells that may be relevant to asym-
the HO endonuclease
in yeast Nasmyth
289
metric expression of HO have barely been addressed. Instead, work has concentrated on which NO transcription factor might differ between mother and daughter cells. The hybrid CAL-HO promoter remains dependent on SWI, -2, -3, -4, -6 -7, -8, -9 and -20, even though it is expressed equally in mother and daughter cells, suggesting that the activities of the above genes are not exclusive to mother cells [ 121. It is now known that SW& -2, -3, -4, 4 and -10 activate genes other than HO in both mother and daughter cells [30**,31°*,36*]. SW5 is the only gene that becomes redundant when UPS1 is replaced by the GiiLI- lOUAS [ 121, and this has provoked a close scrutiny of its potential mother-cell specificity. SW25 encodes an 85 kD protein [37] that binds two sites (called A and B) within URSl [38**] through a three zinc-linger DNA-binding domain near its carboxyl terminus [391. Mutation of either site alone reduces HO transcription (site B more so than site A) and mutation of both abolishes transcription [ 381. HO activation therefore involves the binding of at least two sequence-specific factors: Swi5 to two sites within URSl, and SBF to three sites within URS2. More evidence that asymmetric HO expression involves Swi5 stems from the analysis of suppressor mutations that inactivate the SLW.3/SLW gene and allow HO to be at least partially expressed in the absence of Swi5 [ 17,181. SIN3 possibly affects the activity of repressors that inhibit HO activator proteins in such a way that HO then requires SW5 expression. The precise role of Sin3 is unclear, but it is pertinent that HO is expressed equally in mothers and daughters of sin.3 swi5 double mutants, that is, there is no HO asymmetry in the absence of SW5 dependence. There is no evidence that SW is required directly for mother/daughter control. The daughter cell switching that results from loss of SfWl function can be explained by HO’s loss of SW15 dependence [ 181. Furthermore, Sin3 protein is found in both mothers and daughters [40]. If Swi5 is a factor whose inheritance only by mother cells determines the lack of HO expression in daughters, then one would not expect it to be synthesized by daughter cells until after HO transcription is triggered in late Gl [lo]; indeed, not until SBF is inactivated by a rise in Cdc28 kinase activity associated with the G2 cyclins Clbl and Clb2. It is therefore remarkable that SW75 transcripts are regulated in this fashion; they are absent throughout Gl and are not synthesized until CLBZ and CLB2 transcripts appear in G2 [41]. This cell-cycle regulation is determined by an oligonucleotide sequence that forms ternary complexes between Mcml (which is also involved in the regulation by the MATlocus of genes encoding pheromones and their receptors) and the factor SFFwhose gene has not yet been characterized [ 42.1. Thus, if Swi5 is present at the time of HO activation (which seems likely), it would have to be inherited from the previous cell cycle. Despite indications that Swi5 has a special role in HOs mother-cell specticity, the bulk of Swig is evenly segregated to mother and daughter cells at cell division. Swi5 protein accumulates in the cytoplasm during the G2 and M phases but translocates to both mother and daughter
.
. 290
Gene exmssion
and differentiation
nuclei in roughly equal amounts following inactivation of the Cdc28 protein kinase at the end of mitosis [43]. This cell cycle regulated nuclear uptake is dependent upon serine residues within or close to the Swi5 nuclear localization signal, whose phosphorylation by the Cdc28 protein kinase prevents nuclear uptake [44**]. It should be emphasized that nuclear uptake of Swi5 does not immediately ttigger HO transcxiption, as this cannot occur until later in Gl when SBF becomes active as a result of the re-activation of Cdc28. Most Swi5 molecules are rapidly degraded in both mother and daughter cells soon after (and possibly as a consequence of) entry into the nucleus [ 38**]. Despite the apparently equal segregation of Swi5 to mothers and daughters, there is evidence that a lack of Swi5 is at least partly responsible for daughters’ failure to express HO. Expression of SW’5 throughout Gl at a level comparable to normal G2specillc expression causes daughter cells to switch mating type with a frequency that is 40% of the rate observed in mother cells [42*]. This suggests that Swi5 can in principle bind to and activate the HO promoter in daughter cells, but that it normally does not because most if not all the Swi5 that enters daughter-cell nuclei is degraded before SBF is activated. The deletion of specific sequences located centrally in Swi5 (called the inhibitory region) delays its degradation and causes efficient daughter cell switching (it remains, nevertheless, at least twofold less than the observed rate in mothers) [38=*]. Whether this daughter cell switching is due entirely to an increased concentration of Swi5 in late Gl cells, or to a change in its ability to bind DNA or activate transcription is not certain. In either case, a lack of Swi5 function in daughter cells is implicated in HO inactivity. The lack of parity between the rates of mother and daughter cell switching caused by de-regulating Swi5 suggests that Swi5 might not be the only factor that differs in mothers and daughters. SwiS’s instability and lack of synthesis following mitosis raise the possibility that differences in the interval between a cell’s entry into Gl and SBF activation could contribute to the di@erential expression of HO in mothers and daughters. Daughters are usually born smaller than mothers and must therefore spend longer growing to the minimum size needed for Start. Thus, expression of HO could be decided by a race between Swi5 degradation and SBF activation. Might only mother cells with their shorter Gl periods have enough Swi5 at Start to activate HO? It is important to note that such a mechanism would only work if Swi5 must be bound to HO at the time of SBF activation (i.e., that Swi5 does not execute its function appreciably before SBF does). In fact, neither shortening Gl in daughters [41] nor lengthening it in mothers [38**] has much effect on the pattern of HO transcription, implying that Gl length is not an important parameter. It is interesting that mother cells that are arrested in Gl for several hours by depriving them of Gl cyclins retain the abiity to express HO when Gl cyclin activity is restored, despite SWI5 not being synthesized during the arrest period [38*=]; that is, mother cells remember
their identity, they have a ‘memory’. This contrasts with the lack of HOexpression during release from a Gl arrest resulting from pheromone treatment [41]. The implication is that pheromone treatment and not Gl arrest per se destroys a mother cell’s memory. The loss of memory as induced by pheromone could result from the destruction of Swi5 (which cannot be resynthesized), because constitutive SW5 expression allows HO to be activated following a pheromone-induced Gl arrest [24**]. How can the evidence that Swi5 is limiting in daughter cells be reconciled with its even segregation at cell division? It is clear that the differential expression of HO in mother and daughter cells must stem from the asymmetric segregation of a factor other than Swi5. Nevertheless, it seems that the role of such factor might be to facilitate Swi5 function in mothers or impede it in daughters. If we assume that Swi5 must be present at the time of HO activation, then the factor (or its absence) must enable Swi5 function to be retained during the Gl period in mothers. Most of the Swi5 inherited even by mother cells is destroyed during Gl, suggesting that only a limited quantity of Swi5 molecules need be retained for full activity [ 380.1. The asymmetrically segregated factor might therefore determine the formation at HO of stable transcription complexes containing Swi5 (see Fig. 3). Such complexes could constitute a mother cell’s memory. Although daughter cells cannot form these stable complexes (i.e. they cannot acquire memory), Swi5 can nevertheless be delivered to the HO promoter in daughter cells, either by de novo synthesis [41,42*] at the time of SBF activation or by mutation of its inhibitory domain [38**]. Thus far, there is little indication whether the asymmetric factor acts positively or negatively. Despite extensive searches for HO activators [15,16], no obvious gene candidate for a mother cell specific factor has been identified. Furthermore, mutants in which HO loses its differential expression have never been sought for, mainly for lack of an easy assay. What determines the different fate of Swi5 in mothers and daughters? Preferential segregation of a parental DNA strand associated with a ‘competent’ HO gene to the mother pole at mitosis is ruled out. Neither the orientation of HO within the chromosome nor its chromosomal location are important for mother-cell specificity [ 191. The asymmetric segregation of a nuclear factor is another possibility. Autonomously replicating plasmids that lack centromeres are preferentially inherited by mother cells [45]. Furthermore, the yeast spindle pole body (SPB) appears to be replicated by a conservative mechanism with the old and new SPBs segregating to mother and daughter nuclei respectively [46**]. This organelle could conceivably have a role in the asymmetric segregation of factors that facilitate or antagonize Swi5 function. HO continues to be expressed exclusively in mother cells even when SW5 has been mutated so that the Swi5 protein enters the nucleus constitutively [44**]. What then might be the function of SwiS’s regulated nuclear uptake? In addition to not transcribing HO, swi5 mutants have a mild defect in cell separation. The cytological regulation of Swi5 may therefore be more important for the
Regulating
c2
M
Early
T
in yeast Nasmyth
the HO endonuclease
Cl
late
291
Cl
SBF-I
SBF-A
Cdc284b inactivation m Swi5 synthesis and accumulation in the cytoplasm
+
Cdc28-Cln activation
D Swi5 both
enters nuclei
SwiS
SBF-A
URSI
URS2
,d>X> URSl
URS2
HO
Fig. 3, Steps leading to HO activation in mother (M) but not daughter (D) cells. It is suggested that stable complexes containing Swi5 form on the HO promoter only in mother cells. Swi5 is synthesized and accumulates in the cytoplasm during the G2 and M phases. Destruction of Cdc28 kinase associated with the Clb cyclins at telophase 0 signals the entry of Swi5 into both the mother and daughter nuclei (indicated by black shading). Most Swi5 molecules are then degraded during early Cl, but some form stable complexes on the HO promoter in mother but not in daughter cells (indicated by the light-shaded nucleus of the mother cell). It is therefore suggested that HO chromatin differs between the mother and daughter cells (represented by the black-shaded square). The formation of stable Swi5 complexes at HO are not sufficient for its transcriptional activation farrowed), which must await the activation of SBF (SBF-I converted to SBF-A) by Cdc28 kinase associated with Cln cyclins (indicated by the dashed arrows). URS indicates upstream regulatory regions.
cell cycle dependent activation of cell separation genes than it is for HO. c7sZ encodes a chitinase needed for cell separation and is activated at the time Swi5 enters the nucleus, which is appreciably earlier than HO activation (R Siegmund, personal communication). C7SZ transctiptlon is not in fact dependent on Swi5 but instead on the factor Ace2, which exhibits extensive homology to Swi5 and whose entry into the nucleus is similarly regulated [47]. Deletion of URS2 places the SwiS-binding sites within URSl close to the HO TATA box, and thereby allows HO to be expressed in the absence of SBF. This deleted promoter is not expressed during G2 and is only activated upon entry of Swi5 into the nucleus at the end of mitosis [43,44**]; that is, its regulation resembles that of ml.
How does the HO promoter
work?
This article has concentrated on HOs cell cycle dependence and its mother-cell specificity, which concern the functions of SBF and Swl5, respectively. What is the function of all the other genes required for HO transcription; genes such as SWl, -2, -3 - 7, -8, -9 and -IQ The discovery that SWZ2 is probably allelic to the SNF2 gene required for transcription of WC2 (K Nasmyth, unpublished data) led to the finding that other genes such as SNFS and SNFG,which are required for SLJC2expression, are also required for HO expression (SNFS is probably allellc with SW10 [K Nasmyth, unpublished data]). SWZ2/SNF2 [ 481 encodes a protein with homology to helicases [49,50] and is highly homologous to the Brahma protein from Drosophila [51]. SW3 also seems have a homologue in Drasophih and has been shown to interact with steroid receptors [ 52.1. Thus far, none of these proteins have been shown to bind specllic DNA sequences, but chimeras of Swi2/Snf2, Snf6, and SnWSwilO with the bacterial 1exA protein activate transcription from a leti operator/promoter [48]. Swil, -2 and -3, Snf6, D
and SnfS/SwilO may form a large complex of highly conserved proteins that exerts gene-specific activation through interaction with site-specific transcription factors like Swi5 or SBF. It seems likely that Sw17, -8 and -9 will also prove to be general transcription factors. The transcriptional defects of swil, -2 and -3 mutants are partially suppressed by mutations in genes that encode histones or HMG-like proteins [ 531, and it has therefore been suggested that helicase activity associated with Swi2/Snt2 could be involved in regulating chromatln structure [ 541. An alternative is that such a helicase is involved in melting promoters prior to the initiation of transcription. Why HO needs at least two DNA sequence-specific factors, and how they interact with more general transcription factors in the context of chromatin may need to be &r&d before we can comprehend how the arrival of Swi5 in the nucleus at the end of mitosis is a pre-condition for HO activation, and why a change associated with SBF later in Gl eventually triggers the activation. Swi5, al-c&? and SBF all have multiple binding sites within the HO promoter; a redundancy that may contribute to the explosive activation of HO in late Gl, and to preventing expression at times when it would be disastrous for the yeast life cycle.
Conclusion The HO endonuclease gene is transcribed transiently during the late Gl period in only one of the two progeny of a yeast cell division; that is, in mother but not in daughter cells. Iate Gl specificity is conferred by the SBF transcription factor, which also activates genes for Gl cyclins. It seems likely that SBF is activated by the Cdc28 protein kinase. The mechanism of activation and subsequent repression are not yet understood. The mother ceil specificity of HO involves a second transctiption factor called Swl5, which is synthesized during G2 but does not enter nuclei until the end of mitosis. Swi5 enters both mother and daughter nuclei and most is then rapidly degraded.
.
Howevqr, some appears to survive in mother cells until SBF is activated by Cdc28 in late Gl. The challenge now is to determine in what form SwiS survive3 in mother cells and why this process cannot occur in daughters. A further objective will be to elucidate why the arrival of SwiS, the activation of SBF, and the involvement of other more general transcription factors are all needed for the initiation of HO transcription.
15.
STERN MR, JENSEN R, HERSKOWITZ I: Five quired for Expression of the HO Gene 1984, 178:853-868.
16.
BREEDEN 1 HO Gene: 48:389-397.
17.
STERNBERG PW. STERN MJ, Cwuc I, HE~KO~ITX I: Activation of the Yeast HO Gene by Release from Multiple Negative Controls. Cell 1987, 48:567-577.
18.
NAShnTH K, STILL&IAN D. KIPIJNG D: Both Positive and Neg ative Regulators of HO Transcription are Required for Mother Cell-specific Mating Type Switching in Yeast. Cell 1987, 48:579-587.
19.
K~AR AJS: The Mother-Daughter Asymmetry of Budding Yeast gation of Parental HO Gene 1:1059-1064.
20.
NASMYTH, K: A Repetitive DNA Sequence that cycle START (CDCZS)-dependent Transcription Gene in Yeast. Cell 1985, 42:225-235.
21.
MILIIR AM, MCKAY VL, NX+WH KA: Identification and Comparison of Two Sequence Elements that Confer Cell-type Specific Transcription in Yeast. Nalrrre 1985, 314:598-603.
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ANDRE BJ, HERSKO~ITZ I: Identification of a DNA Binding Factor Involved in Cell-cycle Control of the Yeast HO Gene. Cell 1989, 57:21-29.
23.
A~WKRYIS BJ, HEHSKOWIIX 1: The Yeast SWI4 Protein Contains a Motif Present in Developmental Regulators and is Part of a Complex Involved in Cell-cycle-dependent Transcription. Nature 1989. 342830-833.
Acknowledgements I would like to thank Gustav Ammerer and Celia Dower for comments while preparing this manuscript, and Hannes Tkadletz help preparing the figures.
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PRI~IIG M, SOC~ATHAN S. ALZH H. N,\SbnTH K: Anatomy of a Transcriprion Factor Important for the Start of the CeU Cy cle in Saccharomyces cereoisiae. Nature 1992, 358~593-597. This paper shows that SBF can he reconstituted by translating SW74 and SW6 mRNAs in a reticu&yte lysate, that SBF binds to SC8 DNA through a 110 amino.acid domain located near %4‘s amino terminus. and that Swi4 and ~wi6 interact through sequences near their carboxyl termini. 26.
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DIRICK L NASMYIH K: Positive Feedback in the Activation of Gl Cyclins in Yeast. Nulrtre 1991, 351:754-757. &ivation of HO by SBF is Cdc28 dependent. To reconcile this with SBF also being an activator of Gl cyclins, which are themselves activators of Cdc28, it wzzs suggested that activation of CLNl and CLN2 by SBF occurs through a positive feedback loop. As predicted by this hy pothesis. the transcription of the CLNI and CLN2 genes is shown to depend on CLX28 and on Gl cyclin activity. CROSS F, TINKHJZ~ERG AH: A Potential Positive Feedback Loop Controlling CLNl and CLN2 Gene Expression at the Start of the Yeast Cell Cycle.&// 1991, 65:875-&%3. The Start’ of the yeast cell cycle is thought to result from activation of the Cdc28 protein kinase by the appearance of Gl cyclins. This paper addresses the mechanism by which the transcription of the G1 cyclin genes CLNI and CLN, is activated in late Gl. It is shown that their activation is stimulated by Gl cyclin activity and depends on the Cdc28 kinase, implying that a positive feedback loop is involved.
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STILLMAN DJ, BANKIER AT, SEDDON A, GROENHOLIT G. NA%M-H KA: Characterization of a Transcription Factor Involved in Mother CeU Specific Transcription of the Yeast HO Gene. EMBO J 1988, 7:485-494.
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DIRICK I., MOLL T. AUER H, NASWIH K: A Central Role for SWIG in Modulating CeU Cycle START Specific Transcrip tion in Yeast. Nurure 1992, 357:50%513. Swi6 protein is a component of two different late Gl-specific transcrlption factors. It complexes with Swi4 to form SBF. which regulates HO and Gl cyclins. and with a 12OkD protein that is distinct from Swi4 to form MBF, which regulates most genes involved in DNA replication. Neither set of genes are transcribed efficiently in the absence of swi6. Moreover, cellcycle regulation of SBF.actlvared genes is impaired and that of MBF-activated genes is abolished in suli6 mutants.
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Y, N~s~vm in
WANG H, CLARK I, NICHOLSON PR, HERSKO~I’I~! I, SnllMAN DJ: The Saccharomyces cerevisiae SIN3 Gene, a Negative Regulator of HO, Contains Four Paired Amphipathic Helix Motifs. &lo/ Cell Biol 1990, 10:5927-5936.
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PE’IIXSON CL, HERSKOWII-,! 1: Characterization of the Yeast SWll. SWIZ, and SWI3 Genes, which Encode a Global Activator of Transcription. Cell 1992, 68:773-583. SWII. SWi’2 and SWI.? are required for the expression of many genes other than HO, but the pleiotrophies of double mutants are no more severe than those of single mutants, suggesting that all three genes are involved in the same function.
NAGAI K, NMEKO Motifs Expressed Specific Binding
40.
BREEDEN L, MIKES~U. GE: Cell Cycle-specific Expression of the SWI4 Transcription Factor is Required for the Cell Cycle Regulation of HO Transcription. Genes Del* 1991, 5:1183-l 190. SW74 transcripts fluctuate during the cell cycle; being most abundant in late Gl at the time of HO activation, and least abundant in early Gl and in G2, when HO is not transcribed. How important this regulation is for confining SBF activity to late Gl and S phase is not clear. SBF-binding activity does not vary much during the cell cycle (see ]24**]). Highlevel expression of SW14 from the C&U! promoter seems to cause HO to be expressed in G2, but this effect could at leasr partly be because SII’IG becomes redundant for f-/O transcription under these conditions.
36. .
293
TEBB G, Mou T, D~WLER C, NASMYIH K: SWI5 Instabiity may be Necessary but is not Sufficient for Asymmetric HO Expression in Yeast. Genes Deu 1993, in press. HO activation by Swl5 is mediated by Swi5 binding to two sites within URSl. Most of the Swi5 protein that enters mother and daughter nuclei at the end of mitosis is rapidly degraded by a process that requires spe cific sequences located centrally in the Swl5 protein. Deletion of these sequences causes HO to be expressed in daughter cells, which raises the possibility that differences between mothers and daughters in the time between Sti5 entty into the nucleus at the end of mitosis, and activation of SBF at Start could contribute to HCh asymmetric expression. However, greatly extending this intetval in mother cells has little effect on HO tntnsctiption, refuting the hypothesis. It is proposed that mother and daughter cells differ in their ability to trap Swl5 in a form that persists until SBF is activated in late Gl.
34. ..
35. ..
in yeast Nasmyth
38. ..
32.
33. . .
the HO endonuclease
MOU T, TEBB G, SURANA U, ROBI~SCH H, NA%IYIH K: The Role of Phosphorylation and the CDC28 Protein Kinase in CeU Cycle-regulated Nuclear Import of the S. cereuisiue Transcription Factor SWI5. Gel/ 1991, 66:743-758. The cell cycle regulated entry of Sti5 into the nucleus is shown to depend on phosphotylation sites within or near the Swi5 nuclear localixation signal. It is suggested that destruction of the Cdc28 protein klnase at the end of mitosis causes Swl5 de-phosphotylation and thereby activation of its nuclear localization signal. Regulated Swi5 nuclear entry is not necessary for mother cell specific HO expression but probably regulates other genes that, unlike HO, are expressed as soon as cells enter Gl. MLIRRAY AW. SZOSTAK Jw: Pedigree Analysis regation in Yeast. Cell 1983, 35:167-174.
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Plasmid
Seg-
46. ..
VALIIN EA, S~HERXIN n, ROBER‘I~ T, VAN ZEE K, Rosa MD: Asymmetric Mitotic Segregation of the Yeast Spindle Pole Body. Cell 1992, 69505-515. It appears that the asymmetric segregation of some factor other than Swi5 must be responsible for HCYs mother-cell specificity. SPBs in yeast are probably duplicated by a conservative mechanism. This paper indicates that old and new spindle pole bodies may be segregated to mother and daughter cells, respectively. Something associated with only one of the two SPBs might be responsible for the asymmetry of HO. D~HR~IANN PR. BUTLER G, TAMAI K, D~RIAND S, GREENE JR, THIELE DJ, Snmm DJ: Parallel Pathways of Gene Regulation: Homologous Regulators SWI5 and ACE2 Dilferentially Control Transcription of HO and Chitinase. Genes Deu 1992, 6:93-104. AC..~ encodes a protein that is closely related to SWl5, and which is required for the activation of the CTXI chitlnase gene but not for HO. Ace2 and Swl5 have very similar DNA-binding domains and nuclear localization signals. Furthermore, their synthesis and entry into the nucleus are similarly cell cycle regulated. This paper suggests that c7sl 47. ..
294
Gene expression
and differentiation
expressiop is also dependent on the SWI4 and SU’%encoded func tions, and that its regulation is therefore similar to that of HO. These last two assertions may be incorrect because CIsh is transcribed nor malty in swi4 and stui6 mutants and unlike HO is activated as soon as cells enter Gl; presumably resulting from the regulated entry of Ace2 into the nucleus (R Siegmund, T Schuster, K Nasmyth, unpublished data). 48.
LAURENT BC, TRE~L MA, Cluuso~ M: pendence of the Yeast SNF2, SNFS, in- Transcription Activation. Proc Natl
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DAVIS JI,
KUNISAWA
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JA Presumptive Helicase Affects Gene Expression and is Rein the Yeast S. cerevisiae. IBOI Cell Biol
IAURE~ BC, YANG X, COON M: An Essential S. cerevisiae Gene Homologous to SNF2 Encodes a Helicase-related Protein in a New Famjly. Mel Cell Biol 1992, 12:1893-1902. This paper, together with 1511, suggests that the SwiZ/SnR protein con. tains motifs characteristic of helicases. Whether the protein is a helicase and, if so, what its function might be are as yet unknown.
50. .
51.
TAMKUN JW, DEURING R, Sco7-r AM, KAUFMAN TC, KENNIXIN of
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52. .
Transcriptional
Activator
YOSHINAGA SK, PETERSON K: Roles of SWIl, SWl2,
SNF2/SWl2
CL,
HER~KOVQITZ
SWI3 Steroid
Proteins Receptors.
Gz//
1992,
1, Y~IOTO for Transcrip Scierrce 1992,
tional Enhancement by 258:1598-1604. Activity of the rat glucocorticoid receptor in yeast requires the SWII, -2 and .3 genes. It is hypothesized that the Swil and Swi2 proteins may fomi a complex with Swi3, which interacts v&h the glucocorticoid re ceptor. None of these Swi proteins have been shown to bind specific DNA sequences and the authors suggest that their gene-specific action might result from interaction with sequencespecific tnnscription fat tots. Previously, a similar conclusion was reached for Snf2, which is probably identical to Swi2 (K Nasmyth, unpublished data), in the case of WC2 acthation (see 1481). The sequencespecitic factor with which the putative complex comprising Swil, .2 and .3 interacts on the HO promoter is not yet known; Swi5 and SBF are possibilities.
53.
KRUGER W. HERFKOWITZ I: A Negative Regulator of HO Transcription, SIN1 (SPITZ), is a Nonspecific DNA-binding Protein Related to HMGl. ,Mol CeN Biol 1991, 11:41354146.
54.
WINSTON tivators Genet
F, ORISON M: Yeast SNF/SWl and the SPT/SlN Chromatin 1332, 8:387-391.
Transcriptional Connection.
AcTWI&
M, PAI-FAT~CCI A
Regulator Related to
K Nasmyth, Research Gasse 7. A-1030 Vienna,
Institute Austria.
of
Molecular
Patholog),
Dr.
Bohr-