Thesis (PhD)--University of Stellenbosch, 2002 / ENGLISH ABSTRACT: Upon nutrient limitation, normal cells of the budding yeast, Saccharomyces cerevisiae,
undergo a transition from ovoid cells that bud in an axial (haploid) or bipolar (diploid) fashion
to elongated cells that bud in a unipolar fashion. The daughter cells stay attached to the
mother cells, resulting in chains of cells referred to as pseudohyphae. These filaments can
grow invasively into the growth substrate (haploid), or away from the colony (diploid), and
are hypothesised to be an adaptation of yeast cells that enables them to search for nutrientrich
substrates. This filamentous growth response to nutrient limitation was shown to be
dependent on the expression of, amongst others, the MUC1 gene.
MUC1 (also known as FL011) encodes a large, cell wall-associated, GPI-anchored
threonine/serine-rich protein that bears structural resemblance to mammalian mucins and to
the yeast flocculins. Deletion and overexpression studies demonstrated that it is critical for
pseudohyphal differentiation and invasive growth, and that overexpression of the gene also
results in strongly flocculating yeast strains. The upstream regulatory region of MUC1
comprises the largest yeast promoter identified to date and areas as far as 2.4 kb upstream of
the translational start site have been shown to confer regulation on MUC1 expression. The
large promoter region is not unique to MUC1, however, since it is almost identical to that of
the functionally unrelated STA2 gene. The STA2 gene, as well as the identical STA1 and STA3
genes, encodes extracellular glucoamylase isozymes that enable the yeast cell to utilise
starch as a carbon source. Glucoamylases liberate glucose residues from the non-reducing end
of the starch molecule, thereby making it accessible to yeast cells.
The high identity between the promoters of MUC1 and STA1-3 suggests that the two genes
are co-regulated. In addition, several transcription factors that regulate the transcriptional
levels of both MUC1 and STA2 have been identified and include Msn1p and the previously
uncharacterised Mss11p. Overexpression of either Msn1p or Mss11p results in elevated levels
of MUC1 and STA2 transcription and a dramatic increase in flocculation, invasive growth,
pseudohyphal differentiation and the ability to utilise starch, suggesting that the two genes
are indeed co-regulated. The main objective of this study was to characterise Mss11p and its
role in the co-regulation of MUC1 and STA2 (as a representative member of the STA gene
family).
A detailed expression analysis, using Northern blots and Lacl reporter gene expression
studies in different media, confirmed that these genes are indeed co-regulated to a large
extent. MUC1 and STA2 are also regulated by the same transcriptional regulators, which
include not only Msn1pand Mss11p, but also Ste12p, the transcription factor of the mating
pheromone/filamentous growth signalling cascade, and Flo8p, a transcriptional activator of
the flocculation genes. Overexpression of the genes encoding these factors results in elevated
expression levels of both MUC1 and STA2 in most nutritional conditions and enhances the filamentous growth phenotypes of the strain, as well as the ability to degrade starch. On the
other hand, the deletion thereof results in severe reductions in the transcription levels of
MUC1 and STA2, with equally severe reductions in filamentous growth and the ability to
hydrolyse starch. These expression studies also showed that the repressive effect of STA10, a
previously uncharacterised negative regulator of STA2, is actually a phenotype conferred by a
FLOB mutation in some laboratory strains of S. cerevisiae.
The upstream regulatory regions of MUC1 and STA2 are the largest promoters in the yeast
genome. By sequencing the upstream areas of STA2 and STA3 and comparing them to the
sequence of MUC 1, it was shown that these upstream areas are 99.7%identical over more
than 3 900 base pairs (bp) upstream of the translational start. With the exception of a few
minor substitutions, the only significant difference between the MUC1 and STA2 promoters is
the presence of a 20-bp and a 64-bp sequence found in the MUC1 promoter, but not in the
promoters of any of the STA1-3 genes.
Through a promoter-deletion analysis, it was shown that Mss11p, Msn1pand Flo8p exert
their control over the transcription of MUC1 and STA2 from an 90-bp sequence located at
-1 160 to -1 070 in the STA2 and -1 210 to -1 130 in the MUC1 promoters. This sequence also
mediates the effect of carbon catabolite repression on the transcription of STA2 and MUC1.
Despite the similarities in the expression patterns of MUC1 and STA2, some discrepancies
also exist. The most significant difference is that, in wild-type cells and under all nutritional
conditions tested, MUC1 transcription is reduced significantly if compared to the transcription
levels of STA2. This was attributed to the presence of the 20- and 64-bp sequences, that are
present in the promoter region of MUC1, but absent from that of STA2.
To place the transcriptional regulators of MUC1 and STA2 in the context of known signal
transduction pathways, an epistasis analysis was conducted between MSN1, MSS11 and
components of the mating pheromone/filamentous response MAPkinase cascade and cAMPPKA
pathway that were shown to be required for the filamentous growth response. This
analysis revealed that Msn1p functions in a third, as yet uncharacterised, signal transduction
pathway, also downstream of Ras2p,but independent of the two identified pathways, i.e. the
cAMP-PKA and pheromone response/filamentous growth response MAP kinase pathways.
However, Mss11p seems to function downstream of all three the identified pathways. This
suggestsa critical and central role for Mss11p in determining the transcription levels of MUC1
and STA2.
To further characterise Mss11p and its role in the transcriptional regulation of MUC1 and
STA2, it was also subjected to a detailed deletion and mutation analysis. Mss11p was shown
to harbour two distinct activation domains required for the activation of MUC1 and STA2, but
also able to activate a reporter gene expressed from under the GALl promoter. The more
prominent of the activation domains of Mss11p was shown to be one of the domains with
homology to Flo8p, designated H2. The H2 domain has significant homology to a number of proteins of unknown function from a range of different organisms. A multi-sequence
alignment allowed the identification of conserved amino acids in this domain. Mutations in
two of the four conserved amino acid pairs in the H2 domain completely eliminated the
activation function of Mss11p.
The poly-glutamine and poly-asparagine domains of Mss11p are not required for its
activation function. The deletion of these domains has no impact on the ability of Mss11p to
activate MUC1 or STA2 or of the Gal4p-Mss11p fusion to activate the lacl reporter gene
expressed from under the GAL7 promoter. Gal4p fusions of either of these domains were also
unable to trans-activate the PGAL7-lacl reporter gene. As such, it was concluded that neither
of these domains performs a function in the role of Mss11p as a transcriptional activator. We
also demonstrated that the putative ATP/GTP-binding domain (P-loop) is not required for the
transcriptional activation function of Mss11p.
In an attempt to identify other target genes of Mss11p, the use of micro-arrays was
employed to assessthe impact of the overexpression and deletion of MSS11 on the total yeast
transcriptome. These results showed that MUC1 and STA2 are the only two genes in the ISP15
genetic background that are significantly (more than 15-fold) enhanced by the overexpression
of MSS11. Mss11p therefore seemsto playa very specific or dedicated role in MUC1 and STA2
transcription. This analysis also identified several genes (DBP2, ROM2, YPLOBOC, YGR053C,
YNL179C, YGR066C) that are repressed by overexpression of MSS11 and activated when MSS11
is deleted.
To integrate all the results, three possible models for the activation of MUC1 and STA2
transcription by Mss11p are proposed: (i) Mss11p performs the role of a transcriptional
mediator, possibly in a protein complex, to convey information from upstream regulatory
elements to the transcription machinery assembledat the core promoters of MUC1 and STA2;
(ii) Mss11p plays a more direct role in transcriptional activation, possibly as a transcription
factor itself; and (iii) Mss11p facilitates transcription of the MUC1 and STA2 promoters as part
of a larger complex that removes or releases the chromatin barrier over the MUC1 and STA2
promoters in responseto specific nutritional signals. / AFRIKAANSE OPSOMMING: Wanneer voedingstowwe beperkend raak, ondergaan selle van die botselvormende gis,
Saccharomyces cerevisiae, fn transformasie vanaf ronde selle, wat in fn aksiale (haploïede) of
bipolêre (diploïede) patroon bot, tot verlengde selle, wat slegs op een punt bot. Die
dogterselle blyaan die moederselle geheg, sodat kettings van selle, wat as pseudohifes
bekend staan, gevorm word. Hierdie filamente kan fn groeisubstraat binnedring (haploïede) of
vanaf die kolonie weggroei (diptoïede), en is moontlik fn aanpassing van die gisselle wat hulle
in staat stelom na meer voedingstofryke substrate te groei. Die vermoë om filamente in
respons tot voedingstoftekorte te vorm, is onderhewig aan die uitdrukking van, onder meer,
die MUC1-geen.
MUC1 (ook bekend as FL011) kodeer vir fn selwand-geassosieerde treonien/serien-ryke
proteten met fn GPI-anker wat strukturele verwantskappe met die mukiene van soogdiere en
die flokkuliene van giste toon. Delesie- en ooruitdrukkingstudies het bewys dat dit krities is
vir die ontwikkeling van pseudohifes en penetrerende groei, terwyl die ooruitdrukking
daarvan ook tot sterk flokkulerende gisrasse lei. Die stroom-op regulatoriese area van MUC1
vorm die grootste promotor wat tot dusver in gis geïdentifiseer is, en daar is bewys dat areas
so ver as 2.4 kb stroom-op van die translasie-inisiëringsetel die regulering van MUC1
beïnvloed. Hierdie groot promotor is egter nie uniek tot MUC1 nie, aangesien fn amper
identiese promotor die regulering van die funksioneelonverwante STA2-geen beheer. Die
STA2-geen, asook die identiese STA1- en STA3-gene, kodeer vir ekstrasellulêre glukoamilase
isosieme wat die gis in staat stelom stysel as koolstofbron te benut. Dit bevry
glukosemolekules vanaf die nie-reduserende punt van die styselmolekuul en stel dit sodoende
aan gisselle beskikbaar.
Die hoë vlak van eendersheid tussen dié twee promotors veronderstel dat die twee gene op
soortgelyke wyse gereguleer word. Verskeie transkripsiefaktore wat die transkripsievlakke van
beide MUC1 en STA2 beheer, is ook geïdentifiseer, Dit sluit Msn1p en die tot dusver
ongekarakteriseerde Mss11p in. Ooruitdrukking van Msn1p of Mss11p lei tot verhoogde vlakke
van MUC1 en STA2 se transkripsie en fn dramatiese toename in flokkulasie, asook die vermoë
om penetrerend te groei, pseudohifes te vorm en stysel te benut. Dit bevestig dat die twee
gene wel tot fn groot mate op dieselfde wyse gereguleer word. Die hoofdoel van hierdie
studie was om Mss11p en die rol daarvan in die regulering van MUC1 en STA2 te karakteriseer.
Gedetailleerde uitdrukkingsanalises met behulp van die Northern-kladtegniek en facZverklikkergeeneksperimente
in verskillende media het bevestig dat die gene wel tot fn groot
mate op dieselfde wyse gereguleer word. Transkripsie van MUC1 en STA2 word ook deur
dieselfde transkripsionele reguleerders beheer, wat nie net Msn1pen Mss11p insluit nie, maar
ook Ste12p, die transkripsiefaktor van die paringsferomoon/filamentagtige groei
seintransduksiekaskade, en Fl08p, fn transkripsionele aktiveerder van die flokkulasiegene. Ooruitdrukking van die gene wat vir hierdie faktore kodeer, veroorsaak verhoogde
uitdrukkingsvlakke van beide MUC1 en STA2 onder die meeste groeitoestande en verbeter die
vermoë van die gisras om filamentagtig te groei en om stysel te benut. Andersyds veroorsaak
delesies van die gene 'n dramatiese afname in die transkripsievlakke van MUC1 en STA2, met
vergelykbare afnames in die vermoë van die gisras om filamentagtig te groei en om stysel te
benut. Hierdie uitdrukkingstudies het ook bewys dat die onderdrukkingseffek van STA10, 'n
tot dusver ongekarakteriseerde, negatiewe reguleerder van STA2, aan 'n mutasie in FLOB in
sekere laboratoriumrasse van S. cerevisiae toegeskryf kan word.
Die stroom-op regulatoriese areas van MUC1 en STA2 is die grootste promotors in die gis se
genoom. Deur die nukleotiedvolgordes van die ver stroom-op areas van STA2 en STA3 te
bepaal en hulle met dié van MUC1 te vergelyk, is daar vasgestel dat die stroom-op areas van
die gene 99.7% identies is oor meer as 3 900 basispare (bp) stroom-op van die beginsetel van
translasie. Met die uitsondering van enkele basispaarverskille, is die enigste noemenswaardige
verskil tussen die promotors van MUC1 en STA2 die teenwoordigheid van 'n 20 bp- en 'n
64 hp-fragment wat in die MUC1-promotor aangetref word, maar nie in die promotors van die
STA1-3 gene nie.
Deur 'n promotordelesie-analise kon daar bewys word dat Mss11p, Msn1p en Flo8p beheer
uitoefen oor die transkripsie van MUC1 en STA2 vanaf 'n 90-bp-fragment, wat by posisie
-1 160 tot -1 070 in die STA2-promotor en posisie -1 210 tot -1 130 in die MUC1-promotor
aangetref word. Koolstofkatabolietonderdrukking van MUC1 en STA2 se transkripsie geskied
ook deur middel van hierdie fragment.
Ten spyte van die ooreenkomste in die uitdrukkingspatrone van MUC1 en STA2, kom daar
tog ook verskille voor. Die mees opvallende verskil is dat, in wilde-tipe selle en onder alle
toestande tot dusver getoets, die transkripsievlakke van MUC1 aansienlik laer is as dié van
STA2. Dit word toegeskryf aan die teenwoordigheid van die 20 bp- en 64 bp-fragmente, wat in
die promotor van MUC1 teenwoordig is, maar in die promotor van STA2 afwesig is.
Om die transkripsionele reguleerders van MUC1 en STA2 in die konteks van bekende
seintransduksieweë te plaas, is 'n epistase-analise gedoen tussen MSN1, MSS11 en
komponente van die paringsferomoon/filamentagtige groei MAP-kinasekaskade en die cAMPPKA-
weg wat uitgewys het dat dit 'n rol in die filamentagtige groeirespons speel. Hierdie
analise het onthul dat Msn1p in 'n derde, tot dusver onbeskryfde, seintransduksieweg
funksioneer, wat ook stroom-af van Ras2p is, maar wat onafhanklik funksioneer van die twee
bekende weë, die cAMP-PKA-weg en die paringsferomoon/filamentagtige groei MAPkinasekaskade.
Mss11p blyk egter stroom-af van al drie dié weë te funksioneer. Dit wys dat
Mss11p 'n kritiese en sentrale rol in die bepaling van MUC1 en STA2 se transkripsievlakke
speel.
Om Mss11p en die rol daarvan in die regulering van MUC1 en STA2 se transkripsie verder te
karakteriseer, is dit aan 'n volledige delesie- en mutasie-analise onderwerp. Dit het gewys dat Mss11p twee verskillende aktiveringsdomeine bevat wat vir die transkripsionele aktivering van
STA2 en MUC1 benodig word, maar wat ook 'n verklikkergeen kon aktiveer wat onder die
GAL7-promotor uitgedruk word. Die prominentste van die twee aktiveringsdomeine van
Mss11p is een van die domeine wat homologie toon met 'n soortgelyke domein van Flo8p, die
sogenaamde H2-domein. Die H2-domein toon hornologie met 'n verskeidenheid van
organismesse proteïene, waarvan die funksie onbekend is. 'n Vergelyking van al die relevante
aminosuurvolgordes uit dié proteïene het gehelp om 'n aantal gekonserveerde aminosure te
identifiseer. Mutasies van twee van die vier gekonserveerde aminosuurpare het die vermoë
van Mss11p om transkripsie te aktiveer, heeltemal geëlimineer.
Die poliglutamien- en poliasparagiendomeine van Mss11p word nie vir die aktiveringsfunksie
benodig nie. Die delesie van die domeine het geen impak gehad op die vermoë van Mss11p om
die transkripsie van MUC1 en STA2 te aktiveer nie, of op die vermoë van die Gal4p-Mss11p
fusie om die lacZ-verklikkergeen onder regulering van die GAL7-promotor te aktiveer nie.
Gal4p-fusies met enige van die domeine was ook nie in staat om die PGAL7-lacZverklikkergeen
te aktiveer nie. Daar kan dus afgelei word dat nie een van die twee domeine
'n funksie in die rol van Mss11p as transkripsionele aktiveerder het nie. Soortgelyke
eksperimente het bewys dat die moontlike ATP/GTP-bindingsdomein (P-lus) nie vir die
transkripsionele aktiveringsfunksie van Mss11p benodig word nie.
In 'n poging om ander teikengene van Mss11p te identifiseer, is mikro-ekspressieroosters
gebruik om die impak van die ooruitdrukking en delesie van MSS11 op die totale
transkriptoom van die gis te bepaal. Dié resultate het gewys dat MUC1 en STA2 die enigste
twee gene in die ISP15genetiese agtergrond is waarvan transkripsie noemenswaardig (meer
as 15-voudig) deur die ooruitdrukking van MSS11 verhoog word. Dit wil dus voorkom asof
Mss11p 'n baie spesifieke rol in die transkripsie van MUC1 en STA2 speel. Hierdie analise het
ook verskeie gene (DBP2, ROM2, YPLOBOC,YGR053C, YNL179C, YGR066C) geïdentifiseer wat
deur die ooruitdrukking van MSS11 onderdruk word en deur die delesie van MSS11 geaktiveer
word.
Ten einde al die resultate te integreer, word drie moontlike modelle vir die aktivering van
MUC1- en STA2-transkripsie deur Mss11p voorgestel: (i) Mss11p vervul die rol van 'n
transkripsionele tussenganger, moontlik as deel van 'n proteïenkompleks, om die inligting van
die stroom-op regulatoriese elemente aan die transkripsiemasjinerie wat oor die
kernpromotor van MUC1 en STA2 gebind is, oor te dra; (ii) Mss11p speel 'n meer direkte rol in
transkripsionele aktivering, moontlik as 'n transkripsiefaktor self; en (iii) Mss11p maak die
transkripsie van MUC1 en STA2 moontlik as deel van 'n groter kompleks wat die
chromatienblokkade oor die promotors van STA2 en MUC1 in respons tot spesifieke seine
verslap of verwyder.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53138 |
| Date | 03 1900 |
| Creators | Gagiano, Marco, 1971- |
| Contributors | Pretorius, I. S., Bauer, F. F., Stellenbosch University. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 201 p. : ill. |
| Rights | Stellenbosch University |
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