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GSBS Dissertations and Theses Graduate School of Biomedical Sciences
1997-07-02
In Vivo Functional Analysis of the Saccharomyces Cerevisiae SWI/SNF Complex: A Dissertation
Loree Griffin Burns University of Massachusetts Medical School
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Repository Citation Burns LG. (1997). In Vivo Functional Analysis of the Saccharomyces Cerevisiae SWI/SNF Complex: A Dissertation. GSBS Dissertations and Theses. https://doi.org/10.13028/2g6f-k063. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/202
This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. In vivo functional analysis of the Saccharomyces cerevisiae
SWI/SNF complex
A Dissertation Presented
Loree Gri Burns
Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfment of the requirements for the degree of:
Doctor of Philosophy
July 1997
Biochemistry Approved as to style and content by:
Martha J. Fedor, Ph.D., Chair of Committee
Kendall L. Knight, Ph.D., Member of Committee
Seth R. Stern, Ph.D., Member of Committee
Richard E. Baker, Ph.D., Member of Committee
Steven Buratowski, Ph.D., Member of Committee
Craig L. Peterson, Ph.D., Dissertation Mentor III DEDICATION
for Gerry, with love and gratit"U ABSTRACT
Chromatin remodelig is crucial to transcriptional regulation in vivo and a number of protein complexes capable of alterig genomic architecture in the
budding yeast Saccaromyces cerevisia have been identifed. Among these, the
SWI/SNF complex, a 2 MDa, eleven subunit protein assembly, has been the most extensively characteried. The SWI/SNF complex is requied for the proper expression of a number of genes in yeast, although it is completely dispensable for the expression of others. Likewise, some, but not al, transcriptional activator proteins require SWI/SNF activity in order to function in vivo. The goal of this thesis work was to identif those components of the transcription process which
dictate this dependence on SWI/SNF activity.
Using the well characterized UASGAL system, we have determined that one of these components is the nucleosome. state of activator binding sites within a
promoter. We fid that whie SWI/SNF activity is not required for the GAL4 activator to bind to and activate transcription from nucleosome-free binding sites, the complex is required for GAL4 to bind and function at low affnity, nucleosomal binding sites in vivo. The SWI/SNF -dependence of these nucleosomal bindig sites can be overcome by 1) replacing the low afnity sites with higher afity, consensus GAL4 binding sequences, or 2) placing the low afnity sites into a nucleos me- ree region. These results provide the fist in vivo evidence that the
SWI/SNF complex can reguate gene expression by modulating the DNA binding of a transcriptional activator protein. To determine whether specifc components of the GAL4 protein are necessary in order for the SWI/SNF complex to modulate binding to nucleosomal sites in our model system , we tested the SWI/SNF dependent DNA binding of various derivative GAL4 proteins. We find that a functional activation domain not required for SWI/SNF to modulate GAL4 binding in vivo. Interestingly, like the ful length protein GAL4 derivatives in which the activation domai has been mutated are able to partialy occupy nucleosomal sites in the absence of SWI/SNF
(bindig in the absence of SWI/SNF is at least forty percent lower than in the presence of SWIISNF), indicating the activation domain is also not required for
SWIISNF-independent DNA bindig.
These results support a model in which the SWI/SNF dependence of a gene reflects the nucleosomal context of its important reguatory sequences, e.g. binding sites for transcriptional regulatory proteins. Although nucleosomal promoter regions have been correlated with SWIISNF -dependence in the past, there has of yet been no gene at which nucleosome location has correlated with a specifc genetic function. In the fial part of this thesis work, we initiated a search for an endogenous SWIISNF -dependent gene for which the nucleosome state of activator binding sites could be determined......
TABLE OF CONTENTS
APPROVAL PAGE...... ii
DEDI CATION ...... iii
AB STRA CT......
LIST OF TABLES......
LIST OF FIGURES......
ABBREVIATIONS USED......
CHATER 1: INTRO D U CTI ON...... Transcription and its regulation
The identifcation of the SWI/SNF complex SWIISNF function in vivo Biochemical properties of the SWIISNF complex Other chromatin remodelig complexes
The GALl, 10 promoter
The galactose utilation pathway ofS. cerevisiae
CHAPTER 2: MATERIALS AND METHODS ...... 32 Strain growth and maintenance Liquid 13- galactosidase assays Filter 13- galactosidase assays Nuclei preparation End-labeling of oligonucleotides Internal labelig of DNA fragments In vivo footprinting Indirect end-labelig RNA isolation Primer extension assays Denaturing gel electrophoresis Preparation of yeast lysates Western blotting Strain construction Plasmid construction
CHATER 3: THE YEAST SWI/SNF COMPLEX FACILITATES BINDING OF A TRANSCRIPTIONAL ACTIVATOR TO NUCLEOSOMA SITES IN VIVO ...... 50 Background ...... ''''''''''''......
Results Discussion'
CHATER 4: CHARACTERIZATION OF THE ROLE OF THE GAL4 ACTIVATION DOMAN IN SWI/SNF TARGETING ...... 82 Background Results Discussion
CHATER 5: CHACTERIZATION OF THE GAL- PHENOTYPE OF swi/ snf MUTANTS ...... 106 Background Results Discussion
CHAPTER 6: SUMMAY...... 118
BIBLIOGRAHY...... 123
APPENDIX A: DETAILED PROTOCOLS...... 135 Isolation of yeast nuclei In vivo DMS footprinting Indirect end-labelig Nystatin permeabilzation of spheroplasts
APPENDIX B: RELATED EXPERIMENTS ...... 151 LIST OF TABLES
Table 1- 1: The subunits of the SWI/SNF complex...... 18
Table 1- 2: Some SWI2/SNF2 ATPase family members and their preferred nucleic acid cofactors...... 24
Table 2- 1: Yeast strains used in this study...... 46
Table 2-2: Yeast strais constructed in this study...... 48
Table 4- 1: GAL4 expression plasmids used in this study...... 93
Table 5- 1: Growth defects of swi/snf mutants ...... 108
Table 5-2: Complementation studies...... 114 ......
LIST OF FIGURES
Figure 1- 1: Preinitiation complex assembly on a eukaryotic promoter ......
Figure 1- 2: The GALl, 10 promoter of Saccharomyces cerevisiae......
Figure 3- 1: GAL4 requires functional SWIISNF complex to activate transcription from two low afity binding sites ...... 53
Figure 3-2: GAL4 can bind its sites in the GALl, 10 UAS region in the presence or
absence of SWI/SNF """""""""""""""""""'''''''''''''''''''''''''''''''''''''''''''''' ...... 5 7
Figure 3- 3: SWI/SNF is required for complete occupancy of two low afity GAL4 binding sites in vivo...... 59
Figure 3-4: Phosphorimager analysis of GAL4 site 3 occupancy...... 64
Figure 3- 5: Nucleosome mapping of the GALl, 10 UAS region...... 67
Figure 3-6: Nucleosome mapping of the two low afity site reporter locus...... 69
Figure 3-7: Nucleosome mapping of the two high afity site reporter locus...... 74
Figure 3-8: Nucleosome mapping of the two low affnity site reporter containing a nucleosome positioning element...... 76
Figure 3-9: Predicted nucleosome structure ofSWIISNF-dependent and SWI/SNF- indep enden t prom oters...... 79
Figure 4- 1: Models for the stabilation of GAL4 binding in vivo...... 84
Figure 4-2: An activation domai is not required for GAL4 bindig to low afnity, nucleosomal sites in vivo...... """""'"'''''''' ...... 86
Figure 4-3: Phosphoriager analysis of site occupancy by full length GAL4 protein and the truncated GAL4 DNA bindig domai...... 88
Figure 4-4: Western analysis of GAL4 derivative proteins ...... 91
Figure 4- 5: The GAL4 derivative CD 19 is stable in a swi/ snf strain ...... 95
Figure 4-6. The activation domain mutant CD19 is able to partialy bind nucleosomal GAL4 binding sites in the absence of swi/ snf...... 98 Figure 4-7. Phosphoriager analysis of site occupancy by full length GAL4 and the activation domain mutant CD19 .....,...... 100
Figure 5- 1: swi/ snf mutants display growth phenotypes...... 109
Figure 5- 2: Analysis of GAL gene transcription in SWI+ and swi- yeast...... 112 g...... """" ...... """""""""""""'"'''' ...... :...... """""""""""""""""''''''''''''''''''''''''''''''......
ABBREVITIONS USED
4 W J ...... four way junction
5- FOA ...... , 5- fl uoroorotic acid
J. C i ...... microCurie
J.l...... ,... microli ter
J.M ...... ,...... micromolar
A...... absorbance
B SA...... bovie serum albumin bp ...... b asep airs cpm ...... counts p er minute
Ci ...... Curies
CTD ...... carboxy-terminal dom ain
dA TP ...... deoxyadenosine triphosphate
dCTP ...... deoxycytosine triphosphate dGTP ...... deoxyguanosine triphosphate
D MS ...... dim ethy lsulfa te
DNA ...... deoxyribonucleic acid dTTP ...... deoxythymidine trip hosp hate
DTT ...... di thiothreitol
exem pli gratia (for example)
EM ...... electron microscopy
ER...... estrogen receptor g...... """...... ""'"'''''''''''''''''''''''''' ...... :......
et al ...... ,...... et albi (and others) ftz...... fushi tarazu
gram
R...... glucocorticoid recep tor
GTF ...... general transcription factor
HEPES...... 4- (2 -hydroxyethyl) - 1- piperazineethanesulfonic acid
e...... id est (that is)
In initiator element
K...... thousand kb ...... kilo- basep airs
L ...... liter
LacZ ...... 13 galactosidase gen
M ...... molar
Mb ... mega - basep airs
MD a ...... me ga - D al tons ml...... milliliter mmol...... millim oles m M...... millimolar
MN as e ...... micrococcal nuclease ng ...... """" ...... nanogram nM ..... nanomolar
OD...... optical density ...... "'"'''' ...... """"""'" ......
PIC...... preini tiation complex
PIPES...... plperazine - N, N' - bis (2-ethanesulonic acid)
PK...... proteinase K
PMSF...... pheny lmethy lsulonyl fluoride
RNAPII RNA polymerase
RN A ...... rib on ucleic acid
RN ase ...... rib on ucle ase
rpm ...... revolutions per minute
SD S ...... sodium dodecy 1 sulfate
SRB ...... suppressor of RNA polymerase b
T AF ...... TBP associated factor
TBP ...... TAT A box binding protein
TCA...... ' trichloroacetic acid
topoII ...... topoisomerase
U AS...... upstream activation sequence w/v...... weight per volume
gal...... 5-bromo-4-chloro-3- indolyl-13- galactopyranoside CHATER I
INTRODUCTION
Transcription and its regulation
The nucleus of the buddig yeast Saccharomyces cerevisiae houses 1400 kb of DNA and over 6500 open readig frames (Guthrie and Fink, 1991). If expressed at the appropriate time durig the lie of the cell and in amounts appropriate to specifc envionmental and developmental cues, these genes alow the yeast cell to grow and reproduce normaly. Any interference in the expression of this genetic information, however, can have disastrous consequences for the cell. It is therefore not surprising that transcription, the process by which al eukaryotic cells access genetic information, is tightly regulated. The mechanisms of this regulation, and of the transcription process itself, are highly conserved among eukaryotes.
The past decade has seen an incredible amount of research in the field of eukaryotic gene expression and the fruits of these labors have iluminated the basic mechanisms of the transcription process for protein -codig (class II) genes
(review:d in Zawel and Reinberg, 1993 and Zawel and Reinberg, 1995). The most crucial genetic component of the class II gene is the open readig frame, which serves as the template for mRNA production by RNA polymerase II (RNAPII), the enzyme responsible for transcribing these genes. In addition to an open readig frame, class II genes contain a number of reguatory sequences which both promote
(promoter regions) and reguate (enhancer elements and silencer elements) their transcription. Minimaly, a promoter region contains a TAT A box and/or an initiator (Inr) element; these DNA sequences direct RNAPII and the rest of the transcription machinery to the proper site for transcription initiation. Enhancers
(often caled upstream activation sequences or simply UAS in lower eukaryotes) are genetic reguatory elements which function as bindig sites for sequence- specifc DNA bindig proteins. Enhancers are often located far from the transcription start site and core promoter elements, yet the proteins which bind to these regions are able to profoundly afect the process of transcription. Whe core promoter elements are universal, the more distal enhancer or UAS elements are gene-specifc. Together these reguatory sequences help the cell' s transcription machinery to express proper amounts of a given gene at the appropriate time in the lie cycle of a cell.
In addition to RNAPII, the transcription apparatus consists of a dizzying array of both general transcription factors (GTFs) and regulatory proteins
(activators, repressors, mediators and coactivators). The proper interplay of these proteins with each other and with the genetic reguatory components described above is crucial to proper gene expression. Furthermore, this myriad of protein- protein and protein-DNA interactions provides ample opportunity for the cell to tightly reguate transcription. ,,,
In vitro, basal gene expression typicaly requires a DNA template (codig
sequence and a core promoter), RNAPII, the GTFs TATA binding protein (TBP),
TFIIB, TFIIF, TFIIE, TFIIH, ribonucleotides and ATP (Zawel and Reinberg, 1995
and references therein). Careful dissection of this in vitro system has determined
that transcription begins with the precisely choreographed assembly of the GTFs
into a preinitiation complex (PIC) at the core promoter (Buratowski et al, 1989;
see Figure l-1A). PIC assenibly is nucleated by the binding ofTFIID (TBP and its
associated TAFs) to the TATA box, which is typicaly located thirty basepairs
upstream of the transcription start site (Buratowski et al, 1989; Kim et al, 1993).
TFIIB enters the complex next by recognzing and bindig to promoter-bound TBP
(Buratowski et al, 1989; Nikolov et al, 1995). TFIIB has been implicated in start
site selection (pinto et al, 1992; Lagrange et al, 1996) and is thought to guide
RNAPII to the 'proper promoter location via a diect interaction with one of
RNAPII' s eleven subunits (Li et al, 1994). TFIIF enters the complex at the same
time as RNAPII, suggesting that it is also involved in RNAPII recruitment
(Buratowski et al, 1989; Kieen et al, 1992). TFIIE and TFIIH appear to enter the
complex afer RNAPII and TFIIF and since transcription initiation can occur in
their absence, these factors are thought to be involved in transcriptional
elongation (reviewed in Drapki and Reinberg, 1994). In support of this notion
TFIIH as a kiase activity capable of phosphorylating the carboxy-terminal
domai (CTD) ofRNAPII and it is the phosphorylated form ofRNAPII which
seems to be involved in elongation (Laybourn and Dahmus, 1990). Furthermore, TATA Inr
TBP Inr
CiFj
TBP Inr
RNAPII , '
/i!::"
. c
TATA Inr
Figure 1-1. Preinitiation complex assembly on a eukaryotic promoter. Panel A (previous page), Stepwise assembly pathway. Panel B Holoenzyme recruitment pathway. Horizontal line depicts a typical eukaryotic promoter with TATA indicating the TATA element, Inr indicating the initiator element, bent arrow indicating the transcription start site and the closed boxes representing upstream activator binding sites. Shapes represent the indicated general trans- cription factors. in vitro studies indicate TFIIE may modulate TFIIH activity (Hua et al, 1992; reviewed in Svejstrup et al, 1996). Finaly, the GTF TFIIA deserves special comment. Although its role in PIC formation has historicaly been enigmatic recent studies indicate TFIIA plays a primariy reguatory role in the initiation of transcription (reviewed in Roeder, 1996 and Nikolov and Burley, 1997). TFIIA recognzes the TBP-TATA element complex in vitro and is required for stable TBP binding at some (but not al) promoters in vivo. This varabilty in TFIIA requirement may reflect the intrinsic TBP-binding properties of individual promoters.
There are two notable exceptions to the PIC assembly pathway described above. First, the promoters of some class II genes do not contain a TATA element.
For these genes, PIC nucleation appears to occur over the Inr element, which encompasses the transcription start site (Smale and Baltimore, 1989). It is believed that TBP as well as its associated factors (TAFs) are recruited to the PIC ofTATA-less promoters by interactions with proteins which bind to the Inr
(reviewed in Weis and Reinberg, 1992 and Roeder, 1996). Secondly, the isolation of stable RNAPII-containg complexes that contai many of the general transcription factors suggests PICs may exist preassembled in vivo and that for some genes this entire assembly may arrive at the promoter at once (Km et al
1994; aldonado et al, 1996; see Figure I- IB).
However the PIC comes to be assembled at the promoter, the end result of its presence in vitro is transcription initiation and basal gene expression. Since the genome is compacted into a chromatin structure which is inhibitory to
transcription (see below), and because the majority of transcription factors are likely to be limiting in the nuclear mileu, it is unlikely that "basal" expression
exists in vivo. Instead, gene expression within the eukaryotic nucleus is thought to be absolutely dependent upon the activity of gene-specifc activator proteins. Activator proteins are typicaly modular: their two essential activities, promoter-binding and transcriptional activation, are conferred by distinct and separable domais (Keegan et al, 1986; Giniger and Ptashne, 1987). In addition to these functional domains, an activator protein requires promoter binding sites in order to properly activate the expression of a gene under its control. These binding sites can be found close to (proximal) or far from (distal) the core promoter. By associating with proximal promoter binding sites, the DNA binding domain wi bring the activation domain into the general vicinity of the core promoter region, where the PIC is being assembled. Once there, the activation domain is thought to inuence the rate and/or effciency of PIC assembly, and therefore of gene expression, by interacting with one or more of the GTFs. These activator-GTF interactions are believed to stimulate transcription even when activator binding sites are located far from the core promoter; in these cases the
DNA located between the distal activator binding sequences and the core promoter region provides a flexible tether which, when looped, can accommodate activator-PIC interactions (ptashne, 1988; Ptashne and Gann, 1997).
Incidentaly, activator-PIC interactions need not be diect; a number of biochemical activities (reviewed in Koleske and Young, 1995) have been found to mediate transcriptional activation and may in fact serve to physicaly lik enhancer-bound activators with promoter-bound GTFs.
Activators have been shown to interact with TFIID (the biochemical fraction containing TBP and its associated TAFs; Stringer et alI990), various TBP associated factors (TAFs; Goodrich et al, 1993; Weinzierl et al, 1993), TFIIB
(Roberts et al, 1993; Choy et al, 1993) and TFIIH (Xao et al, 1994). In some instances, these interactions have b en shown to increase the rate and/or extent of
PIC assembly, resulting in increased levels of transcription (Lin and Green, 1991;
Lieberman and Berk, 1994). However, these experiments have utiled in vitro transcription reactions carried out with partialy puried or recombinant transcription factors and naked DNA templates. In this synthetic situation, the
DNA template is completely accessible to the proteins with which it is incubated.
In contrast, DNA in vivo is tightly associated with cellular proteins to form a compacted, proteinaceous chromatin fiber in which the DNA heli is rather inaccessible. Careful experimentation has verifed that the compaction of DNA into chromatin can impede the transcription process; in vitro, transcription from chromatin templates is markedly decreased when compared to transcription from naked DNA templates (Tsuda et al, 1986; Workman and Roeder, 1987; Lorch et
, 1987). Furthermore, this chromatin-mediated transcriptional repression can be overcome by activator proteins, suggesting that in addition to the so-caled
true" activation described above, activators may also elevate levels of gene expression by servng as "anti-repressors" of chromatin structure (reviewed in
Paranjape et al, 1994).
Chromatin structure
The evolutionary advantage aforded the eukaryotic cell by the compaction of its genome into chromatin is evidenced by the rigid conservation of the structural components of chromatin. The histone proteins, which comprise the nucleosome core (see below) are among the most highly conserved proteins known; the gene encodig histone H4 is over 98% conserved between cows and peas
(Wolfe, 1992). One obvious advantage to a compact chromatin structure is that a large amount of DNA can fit into a very smal area. For example, the 3. bilon basepais of the human genome, equivalent to over two meters of DNA strand, is somehow packaged into a nucleus only 10 J.m, or 10. meters, in size (Wolfe
1995). On the other hand, the formation of a compact chromatin structure is liely to occlude many of the protein-DNA interactions which, as described above, are required for proper transcriptional initiation.
The priary level of chromatin structure in vivo is the nucleosome. A nucleosome consists of 145 bp of DNA wrapped approxiately twice around a core of eight histone proteins (two copies each of his tones H2A, H2B, H3 and H4) to form a globular structure This structure, aptly described as "beads-on-a-string is readiy visible under the electron microscope (EM) when the chromatin fiber is extended by low salt treatment. Interactions between adjacent nucleosomes results in stil further compaction of the genome as the 10 nm "bead-on-a-string structure folds into a thicker 30 nm fiber. The formation of higher order chromatin structures is likely to further occlude reguatory proteins from the DNA. That chromatin structure plays a role in transcriptional regulation has been well established (reviewed in Paranjape et al, 1994). Early experiments in yeast found that misexpression of the genes encoding the core histone proteins could markedly alter gene expression (Clark-Adams et al, 1988; Han and
Grunstein, 1988). Furthermore, in some cases these alterations in gene expression were shown to be accompanied by changes in the chromatin structure of the genes in question (Norris et al, 1988). Careful controls utiling transcriptionaly defective reporters have veried that these changes in chromatin architecture at activated genes are not due to the process of transcription itself
(Hirschhorn et al, 1992), leaving unchalenged the intriguing possibilty that the chromatin alterations are required for (although not necessarily the cause of) gene activation.
The problems posed to transcription by a compacted chromatin conformation are obvious. For example, the formation of a nucleosome directly over a TATA element could stericaly impede TBP bindig if the histone-DNA contact withi the nucleosome core precluded the necessary TBp.TATA box interactions. Indeed, nucleosomes have been shown to inhibit transcription initiation either by blocking access of the general transcription machinery to promoter sequences or by hinderig the binding of upstream activator proteins in vitro (reviewed in Workman and Buchman, 1993). Evidence for nucleosome repression of transcription has been found in vivo as well. In Saccharomyces
nucleosomes located over the UAS of the PH05 cerevisiae, gene under repressing conditions were found to be altered upon gene induction and these alterations are
not dependent on transcription of PH05 (Aler et al, 1986). In contrast, the UAS region of the divergently transcribed GALl and GALlO genes (see below) was found to be constitutively nucleosome-free whie nucleosomes positioned over promoter elements seem to be involved in transcriptional reguation (Fedor and
Kornberg, 1989; Axelrod et al, 1993; see below). Clearly the regulation of gene expression by chromatin, like the other regulatory mechanisms discussed already, are gene-specifc. Advances in the characterization of chromatin structures in
geneticaly amenable systems lie Saccharomyces cerevisiae has alowed researchers to begi to elucidate the mechanisms involved in chromatin-mediated transcriptional reguation. These studies have uncovered a number of proteins and biochemical activities, distinct from those aleady discussed, which appear to regulate gene expression at the level of chromatin structure.
In addition to interfering with transcription, the tight association of DNA with histones and non-histone proteins in vivo is liely to inhibit other DNA- mediat d cellular processes. Possible examples include recombination replication and DNA repair. A number of distinct multisubunit complexes that posses chromatin disruption activity have recently been identifed and mounting evidence suggests these complexes may be involved in mediating these other
DNA-mediated cellular processes in the context of a repressive chromatin structure (reviewed in Peterson, 1996). The SWIISNF complex of the buddig
yeast Saccharomyces cerevisiae appears to represent the yeast homologue of a widely conserved complex which reguates transcriptional activation. This complex is the focus of the research presented in this dissertation.
The identification of the SW/SNF complex
The identifcation of genes that encode subunits of the SWIISNF complex resulted from the convergence of two independent genetic screens in S. cerevisiae:
one for products involved in the expression of the HO gene (SWItch genes), and the
other for gene products involved in SUC2 expression (Sucrose Non-Fermenter
genes). In addition to a number of genes specifcaly involved in HO expression
the former screen identifed mutations in three genes (SVV 1 , SWI2 and SWI3)
which were requied not only for the expression of HO, but also for the expression of a number of other yeast genes (peterson and Herskowitz, 1992; Stern et al
1984). Likewise, a subset of the genes identifed in the SUC2 screen (SNF2
SNF5 and SNF6) seemed to playa global role in transcriptional activation rather
than a specifc role in SUC2 expression (Neigeborn and Carlson, 1984; reviewed in
Winston and Carlson , 1992). The functionaly related SWI and SNF gene sets were found to overlap at the molecular level when cloning and sequencing revealed
SWI2 that SNF2 and were the same gene (Laurent et al, 1991).
Strikingly, most of the SWI/SNF-dependent genes are inducible, i. , the expression is tightly reguated by the growth conditions and developmental state of the cell. Furthermore, the reguation of many of these genes involves a known gene-specifc transcriptional activator protein. These data suggested that the
SWIISNF gene products might function as global activators of transcription by modulating the activity of activator proteins. In fact, a number of transcriptional
activators were found to require SWI SNF genes in order to function in yeast, such as GAL4 and LexA fusion proteins that contain the activation domai of GAL4 or
the activation domai of the Drosophila activater bicoid (peterson and
Herskowitz, 1992; Laurent and Carlson, 1992). Of particular interest was the
SWI- dependence of the Drosophila fushi tarazu (ftz) protein and the mammalan glucocorticoid (GR) and estrogen (ER) receptors (Y oshiaga et al, 1992); the SWI- dependence of these heterologous activators strongly suggested that SWIISNF function is conserved across evolution.
Since deletion of the SWIl, SWI2, SWI3, SNF5 and SNF6 genes independently or in various combinations resulted in identical phenotypes, and since these five genes were required for the expression of an identical set of yeast genes, i seemed liely that these products functioned together, perhaps in a complex. The Peterson and Kornberg laboratories have independently puried this complex and in so doing have verifed that the five SWIISNF gene products isolated geneticaly' do exist in a large multi-component complex in vivo (Cains et al, 1994; Peterson et al, 1994). In addition to the subunits encoded by these five known genes, this 2 MDa assembly contais six additional polypeptides (see below).
SWISNF function in vivo
A clue as to how the SWIISNF gene products function as global activators of
gene expression came from suppressor analyses of swi/snfmutants. In these studies, mutations in genes that encode chromatin components were found to suppress the transcriptional defects caused by the loss of SWI SNF genes
(reviewed in Winston and Carlson, 1992; Wolfe, 1994; Peterson and Tamkun
1995). These suppressors restored the abilty of swi mutants to properly express
SWIISNF-dependent genes , includig HO and SUC2 and thereby conferred a SWI-
INdependent (SIN) phenotype. Mutations in HHTI or HHT2 (encodig histone
H3) and HHFI or HHF2 (encodig histone H4) as well as in the SINI gene
(encodig an HMG I-lie protein which is thought to be a non-histone chromatin
component) were found to aleviate the growth and transcriptional defects of swil
swi2 and swi3 mutants (Kuger and Herskowitz, 1991; Krger et al, 1995). In
additioI?' deletion of one of the two HTAl- HTBl gene clusters (encoding histones
H2A and H2B; Hirschhorn et al, 1992) or mutations in HHTI (prelich and
Winston , 1993) were able to suppress the defects in SUC2 expression of swi2/ snf2, snl5 or snl6 mutants. These genetic interactions suggest that the
SWI/SNF gene products function to activate transcription by relieving the transcriptional repression caused by chromatin structure in vivo.
If the SWIISNF complex functions by aleviating chromatin-mediated transcriptional repression, why does expression of only a subset of genes depend on its activity? For example, although many inducible genes require SWIISNF function in order to be expressed, other genes , like GALl are only slightly afected by the loss of SWIISNF (peterson and Herskowitz, 1992). One possibilty is that
SWIISNF activity is only required when the chromatin structure of a particular promoter is inhibitory to transcription. For example, a strongly positioned nucleosome located over a TATA box might impede the bindingofTBP and thereby inhibit transcription of the adjacent gene. This model predicts that critical promoter elements of SWIISNF-dependent genes exist within positioned
nucleosomes in vivo. The chromatin structure of two SWI- dependent genes, SUC2
and ADH2 have been analyzed. In both cases, positioned nucleosomes appear to playa crucial role in gene expression. The expression of both genes is regulated by carbon source; there is no ADH2 or SUC2 expression when cells are grown in glucose. In this repressed state, nucleosomes are positioned over the TATA box
and UAS region of the SUC2 promoter (Hrschhorn et al, 1992). Upon induction these p sitioned nucleosomes are disrupted and the gene is actively transcribed.
Nucleosome disruption does not require transcription, but does require SWIISNF activity (Hirschhorn et al , 1992). In the case of ADH2 the promoter is also covered by an array of positioned nucleosomes when cells are grown in glucose (Verdone et al, 1996). These precisely positioned nucleosomes cover the RNA initiation sites
and the TATA box of the ADH2 gene, and are destabilzed upon gene induction. It is not known whether the destabilation of these nucleosomes requires SWI/SNF activity. Ifso, the notion that SWIISNF function involves chromatin disruption the vicinity ofnucleosomal protein binding sites becomes even stronger.
It is unlely that the presence of positioned nucleosomes is the sole determinant ofSWIISNF-dependence, because a number of known SWI/SNF- independent genes contai positioned nucleosomes over or near crucial promoter
elements. For example, the SWIISNF -independent PH05 promoter has a precise nucleosome structure, including a nucleosome positioned over the TATA box and
UAS2 (one of two binding sites for the transcriptional activator PH04; Almer et
, 1986). Similarly, nucleosomes are precisely positioned over the divergent
GALl lOpromoter such that the GALlO TATA box and the GALl transcription start site are nucleosomal (Fedor et al, 1988; Fedor and Kornberg, 1989). In these two cases, gene expression is not strongly dependent on SWIISNF activity, despite the presence of these positioned nucleosomes (peterson and Herskowitz, 1992).
Thus, SWIISNF -dependent genes may have a more specifc chromatin architecture or other intrinsic properties which render gene expression dependent on SWI/SNF activity:: In the studies described in this dissertation, we have utilzed the well
characterized GAL regulatory system (see below) to directly test this hypothesis. Biochemical properties of the SWISNF complex
Two groups have puried the 2 MDa SWIISNF complex from S. cerevisiae
(Cairns et al, 1994; Cote et al, 1994). In addition to the subunits encoded by the
five genes isolated geneticaly, SWIl, SWI2/ SNF2, SWI3, SNF5 and SNF6 the complex contains six additional polypeptides (see Table 1- 1). One of these,
SNFll, was isolated in a two-hybrid screen for proteins that interact with the
SWI2/SNF2 protein (Treich, et al, 1995). Another subunit, ANCl, was originaly isolated in a genetic screen for proteins involved in actin cytoskeletal function
(Welch et al, 1993). In addition to being a component of the SWI/SNF complex, the
ANC 1 protein is a subunit of the yeast TFIIF (Henry et al, 1994) and TFIID
(Cairns et al, 1996c) complexes. The 73 kDa subunit , SWP73, has recently been cloned and sequenced; deletions of and some mutations in the gene encoding this
protein result in transcription defects similar to those seen in other swi/ snf mutants (Cairns et al, 1996a). The genes encoding the other three subunits
SWP82, SWP6l and SWP59 have not yet been reported.
The sequence of the SWI2/SNF2 gene, encoding the largest subunit of the complex, contains seven sequence motifs that are characteristic of members of the
DEADIH superfamily of nucleic acid-stimulated ATPases and helicases (Carlson and La rent, 1994). Therefore, it is not surprising that one of the biochemical properties displayed by the SWIISNF complex in vitro is a DNA-stimulated
ATPase activity (Cote et al, 1994). Neither the SWI2/SNF2 protein alone, nor the Subunit Size kDa Other names Human homolo References
SWI2 194 SNF2, GAMl, TYE3, RIC1 hbrm, BRG 1 Laurent et aI, 1991 a SWIl 165 ADR6, GAM3 Peterson and Herskowitz, 1992 SWI3 TYE2 BAFI55, BAF170 Peterson and Herskowitz, 1992 SNF5 103 TYE4 INIl, BAF47 Muchardt et aI, 1995 SWP82 Cairns et al, 1994 SWP73 BAF60 Wang et aI, 1996 SWP61 Cairns et aI, 1994 SWP59 Cairns et al, 1994 SNF6 Estruch and Carlson, 1990 SWP29 ANCl, TFG3, yTAF 30 Welch et aI, 1993 SNFll Treich et ai, 1995
Table 1-1. The subunits of the SWI/SNF complex. The eleven polypeptides which comprise the yeast SWIISNF complex are listed with their size, alases and known human homologs. a, see also Khuvari et al 1993 and Muchardt and Yaniv, 1993; b, see also Taguchi and Young, 1987; c, see also Wang et al, 1996; d see also Laurent et al, 1990 and Kalpana et al, 1994; e, see also Cote et al, 1994; f, see also Cairns et al 1996a; g, see also Cote et al, 1994; h, see also Henry et al, 1994. pured SWI/SNF complex has detectable DNA helicase or DNA trackig activity in vitro (Cote et al, 1994; Laurent et al, 1993; Quinn et al, 1996). As with other members of the DEAD/H superfamily of ATPases, the ATPase activity of the
SWIISNF complex requires a nucleic acid cofactor (Cote et al, 1994; Cairns et al
1994). In particular, the ATPase activity of the complex is stimulated over 30-fold by double-stranded, single-stranded or nucleosomal DNA, but not by RNA (Cote et al, 1994). Interestingly, the presence of synthetic four way junction (4WJ) DNA stimulates the ATPase activity of the complex to levels similar to those seen in the presence of plasmid DNA (Quinn et al, 1996).
The ATPase activity of the SWIISNF complex is crucial to its function; mutation in the putative nucleotide bindig loop of SWI2 obliterates the ATPase activity of the complex in vitro and yeast strains carryig such mutant aleles of
SWI2 display the growth and transcriptional defects of a swi2 deletion mutant
(Laurent et al, 1993; Khuvari et al, 1993). Together with the genetic suppression data (see above), these results suggest that the SWIISNF complex might use the energy of ATP hydrolysis to disrupt chromatin structure.
Consistent with this view, Cote et al (1994) have shown that purifed
SWIISNF is capable of disrupting the rotational positioning of DNA within a mononucleosome in an ATP-dependent manner. Furthermore, this disruption of rotational positioning coincides with an increased afnity of the GAL4- activator for a nucleosomal bindig site. In fact , the afity of GAL4-AH for a nucleosomal site in the presence of SWIISNF and ATP is only about three-fold lower than for naked DNA. Like the disruption of rotational positioning, the abilty of the SWIISNF complex to modulate activator bindig in this in vitro assay is completely dependent on ATP hydrolysis (Cote et al, 1994).
These experiments suggest that the SWIISNF complex is able to use the energy of ATP hydrolysis to modulate transcription factor binding to nucleosomal templates, although the biochemical mechanism by which this occurs is as yet unknown.
Other chromatin remodeling complexes
Since the cloning of the SWI2/SNF2 gene, a number of genes have been identifed that contai ATPase domains which are more homol()gous to that of the
SWI2/SNF2 protein than to other members of the nucleic acid-stimulated ATPase and helicase family (Carlson and Laurent, 1994). These genes have recently been analyzed phylogeneticaly and arranged into eight subfamiles which appear to represent functionaly and evolutionarily distinct classes of proteins (Eisen et al,
1995).
The SWI2/SNF2 subfamily contains S. cerevisiae SWI2/SNF2 as well as
another yeast gene which was identifed based on its homology to SWI2/ SNF2 caled ,!Hl (Laurent et al, 1992). Recently, Cairns et al (1996b) have puried a novel 15-subunit complex from S. cerevisiae which contains the STH1 protein as one of its subunits. In addition, this abundant complex, caled RSC (for its abilty to Remodel the Structure of Chromatin), also contais two other SWIISNF -related subunits: RSC6 is homologous to SWP73 and RSC8 is homologous to SWI3. Like the SWIISNF complex, RSC complex purifed from yeast displays a DNA. dependent ATPase activity and is able to alter nucleosome structure in vitro.
Unlie the SWI/SNF complex, however, the genes encoding the subunits of the
RSC complex are essential for yeast viabilty.
Other members of the SWI2/SNF2 subfamily include the human genes
BRG 1 (Kuvari et al, 1993; Chiba et al , 1994) and hBRM (Chiba et al, 1994;
Muchardt and Yaniv, 1993) and the Drosophila gene brahma (Tamkun et al
1992). The brg1 protein was first isolated from HeLa cells as part of a multimeric protein complex which lie RSC, has many of the same biochemical properties as the yeast SWI/SNF complex (Kuvari et al, 1993; Kwon et al, 1994; Cains et al
1996b). hBRM is also present in a high molecular weight complex in human cells and these two complexes are thought to represent human variants of the yeast
SWIISNF and RSC complexes (Wang et al, 1996). It has further been speculated
that the brahma protein is the catalytic subunit of the Drosophila version of the
SWIISNF complex as brahma can be isolated in a high molecular weight complex with the product of the SNR 1 gene , a homologue of the yeast SNF5 (Dingwal et al,
1995; Muchardt et al, 1995). Domain swapping experiments suggest that the membe:s of the SWI2/SNF2 subfamily function in ways similar to that of the
SWI2/SNF2 protein. In these experiments, the ATPase domain of SWI2/SNF2 was replaced with the corresponding ATPase domai of either brg1 , hBRM or 8TH!. In al cases the chimeric proteins were able to restore proper growth and
transcriptional function in a swi2 mutant (Laurent et al, 1993; Khuvari et al,
1993; Elfing et al, 1994). In contrast, replacement of the ATPase domai of
SWI2/SNF2 with that of ISWI (a member of a distinct subfamily; see below) was
not able to complement a swi2 mutant (Elfring et al, 1994).
The subfamily which is most closely related to the SWI2/SNF2 subfamily
includes the human hSNF2L gene and the Drosophila ISWI gene (this subfamily is referred to as the hSNF2L subfamily). These genes cannot complement the transcriptional defects of a swi2- strain in domain swap experiments, suggesting their roles are functionaly distinct from that of SWI2/ SNF2 (Elfring et al, 1994;
Okabe et al, 1992). However, recent evidence has suggested that the members of this subfamily may also playa role in chromatin remodeling. Tsukiyama and Wu
(1995) have recently reported the purifcation of an ATP-dependent nucleosome
remodelig factor (NURF) from Drosophila embryo extracts. This complex faciltates, in an ATP-dependent manner, the disruption and redistribution of nucleosomes by the GAGA transcription factor in vitro. The NURF complex is
composed of four subunits; the 140 kDa subunit is encoded by the ISWI gene
(Tsukiyama et al, 1995). Although clearly related to the yeast SWIISNF complex the NURF complex has unique cofactor requirements and biochemical characteristics which suggest that it may disrupt chromatin through a distinct biochemical mechanism. For example unle the yeast SWIISNF complex, the
ATPase activity of NURF complex is not stimulated by the presence of double- stranded plasmid DNA, but is stimulated five-fold in the presence of nucleosomal
DNA (Tsukiyama and Wu, 1995).
The Becker laboratory has also identifed an ATP-dependent chromatin
remodeling activity in Drosophila extracts (Varga-Weisz et al, 1995; Wal et al
1995). Molecular analysis of Chromatin Accessibilty Complex (CHRAC) has identifed two of its five subunits as ISWI and topoisomerase II (topo II; Varga-
Weisz et al, In Press). The presence of ISWI in both the CHRAC and NURF complexes lends further support to the notion that members of the SWI2/SNF2
ATPase superfamily are modular components of the cell's chromatin remodeling machiery.
The list of proteins that belong to the SWI2/SNF2 superfamily of ATPases is growing steadiy (Carlson and Laurent, 1994; Eisen et al, 1995). A number of these, including the recombination genes RAD16 and RAD54, are involved in cellular processes other than transcription. These results have led to the speculation that numerous chromatin reconfguration machines, each drven by a distinct SWI2/SNF2-like ATPase motor, may exist in vivo to alow distinct classes of DNA binding proteins access to the DNA at the appropriate time (peterson
1996). Auble et al (1994) proposed that the dierent SWI2/SNF2-lie proteins have unique functions because they are targeted to dierent protein-DNA comple es (see Table 1-2). For example, the yeast SWIISNF complex is targeted to DNA bound histones, RAD 16/RAD54 to repair enzymes bound to sites of DNA damage, and MOT1, a TBP associated factor (TAF), to TBP-bound DNA. Famil member Preferred cofactor SWI2/SNF2 (SW/SNF) Synthetic four-way junctions (strand crossover) DS or SS plasmid DNA Nucleosomal DNA (arrays)
ISW (NURF) Nucleosomal DNA MOTt TBP-DNA complex
Table 1-2. Some SW2/SNF2 ATPase family members and their preferred nucleic acid cofactors. The cofactor requirement for each of the SWI2/SNF2- like proteins listed has been determined and may dictate the specificity of complex activity. DS double-stranded; SS, single-stranded However, the dierences in cofactor requirements between some of these ATPases
(see above) may point to another explanation for their specifcity. Each enzyme may require a unique structured DNA to stimulate its ATPase activity, which may dictate its role in chromatin regulation; the yeast SWI2/SNF2 complex uses
DNA strand crossovers as a cofactor for its ATPase activity, NURF ATPase activity is stimulated by nucleosomal DNA, RAD 16/RAD54 ATPase activities may require damaged DNA as a cofactor and MOT 1 might recognze the bent
DNA structure formed upon TBP bindig to DNA. Cofactor specifcity would liit the catalytic activity of each complex to the appropriate site in the genome.
Testing these hypotheses awaits the purication of these distinct SWI2/SNF2- like ATPase complexes.
The identifcation of multiple chromatin remodelig complexes and the apparent conservation of these complexes from yeasts to mammals underscores the importance of this level of transcriptional regulation. In order to begi a comprehensive analysis of the role of one chromatin remodeling complex (the yeast
SWIISNF complex) in transcriptional reguation, we utilzed the well
characterized GAL gene system of S. cerevisiae.
The GALl, 10 promoter :2' The UAS region which controls expression of the divergently expressed
GALl and GALlO genes of S. cerevisia has been well characteried at a molecular - -
level (West et al, 1984). This region contains four binding sites for the
transcriptional activator GAL4 as well as a site for the abundant cellular protein
GRF2 (Fedor et al, 1988; Chasman et al, 1990). Nucleosome mapping
experiments have clearly demonstrated that this region adopts a precise
chromatin architecture in vivo: using indiect end-labelig, Fedor and Kornberg
(1989) have found that the 150 bp region including and immediately surroundig
the four GAL4 binding sites is nucleosome-free under al growth conditions (See
Figure 1-2). This nucleosome-free region is flanked by precisely positioned
nucleosomes, one between the TATA element and transcription start site of the
GALl gene and the other in the region encompassing the GALlO TATA element.
These strongly positioned nucleosomes serve as a boundary between the
constitutively nucleosome-free GAL4 binding sequences and two surrounding
nucleosome arrays (see Figure 1-2). This architecture is believed to be dependent
upon the protein GRF2, which recognzes a DNA sequence overlapping sites 1 and
2 of the GALl, 10 UAS (Fedor et al, 1988) and may be involved in setting up and/or
maitaing the nucleosome structure of this region.
Peterson et al have found that transcriptional activation by GAL4 on a
LacZ reporter gene whose expression is drven by the GALl, 10 UAS region is
SWIISNF-independent, i. e. in a swi1!1 strain, GAL4 can activate the same high
levels of transcription as it does in a SWI+ strain. Derivatives of this reporter
however, display varyig SWIISNF -dependencies (Craig L. Peterson and Ira
Herskowitz, unpublished observations), suggesting that the determinants of GALlO GALl
Figure 1-2. The GALl, lO promoter of Saccharomyces cerevisiae. Schematic representation of the divergent GALl, 10 promoter demonstrating the nucleosome-free GAL4 binding sites (shaded boxes) and the precisely positioned nucleosomes (ovals). Figure is adapted from Fedor and Kornberg, 1989 and is not drawn to scale. SWI/SNF dependent gene expression lie within the promoter region itself.
Utilzing the GAL reguatory system described below, we set out to identif these determinants.
The galactose utilization pathway of S. cerevisiae
There are nine genes that are necessary for yeast cells to properly utilze galactose as a carbon source (reviewed in Johnston, 1987 and Lohr et al, 1995).
The structural genes GALl (encoding a kiase), GAL2 (encoding a permease),
GAL 7 (encoding a transferase), GALlO (encoding an epimerase), MELI (encoding a
galactosidase) and GAL5 (encoding a mutase) alow the ce to take up galactose from its surroundings and process it into glucose- phosphate, a substrate for glycolysis. The expression of these genes is entirely restricted to those instances when galactose is present in the media and glucose, the preferred carbon source, is
absent. This tight regulation involves the products of the remainig three GAL
genes GAL4 (encoding a transcriptional activator), GALBO (encodig a
transcriptional inhibitor) and GAL3 (encodig a molecule required for rapid induction).
In a widtype yeast cell , the GAL genes can exist in three regulatory states dependi?g on the carbon source. When cells are grown with glucose as the sole
carbon source, expression of al of the GAL genes except GALBO is repressed.
Under these repressing conditions the genes encoding the transcriptional activator GAL4 and the inducer molecule GAL3 are repressed by global transcription repression mechanisms involvig the MIG 1 protein (Johnston et al, 1994). In addition, any residual GAL4 protein present in the cell is prevented from
activating transcription of the other GAL genes by repressive mechanisms involvig inhibitory DNA elements located near the UASGAL region of these genes
(URS element; Johnston et al, 1994).
In the absence of glucose, the global (MIG I-mediated) and specifc (URS- mediated) repression mechanisms just described are circumvented and the
transcription state of the GAL genes now depends on the presence of the inducer galactose. For example, when cells are grown with glycerol as the sole source of carbon (therefore no glucose and no galactose), GAL4 protein is made and can bind its sites in UASGAL regions. Howev , this promoter-bound GAL4 protein is unable to activate transcription because of the presence of the inhibitor GAL80.
GAL80 binds to and masks the C-terminal transactivation domain of GAL4 (Ma and Ptashne, 1987b; Lue et al, 1987), thereby inhibiting its abilty to activate the transcription of downstream genes.
On the other hand, when cells are grown in the absence of glucose (therefore no glucose repression mechanisms are activated) and in the presence of galactose
the GALl, GAL2, GAL7, GALlO andMELl genes are induced 100 to 1000-fold within ~inutes. The abilty of galactose to induce this robust expression requires the product of the GAL3 gene, which is thought to interact with GAL80 in such a way as to inhibit its abilty to interact with the GAL4 activation domain (Suzuki- Fujimoto et al, 1996). The unmasked, UAS-bound GAL4 is therefore able to induce high levels of expression of al the UASGAL-containg genes. Unle the signcant induction of expression seen for the other GAL genes (see above), GAL5
GAL3 and GALBO expression are induced only 3- 10-fold (reviewed in Lohr et al
1995). This varabilty in GAL4 induction may reflect the number and intrinsic effectiveness of the GAL4 binding sites within individual UASGAL regions; endogenous and consensus GAL4 bindig sequences have been found to dier in
GAL4 binding afnity in vitro (Kang et al, 1993, Vashee et al, 1993) and in vivo
(Giniger and Ptashne, 1988).
GAL4 transcriptional activation has been extensively characterized. The
varous UASGAL regions contain from one (in the case of the GALBO, GAL3 and
MELl) to four (in the case of the divergently transcribed GALl and GALlO genes; see above) copies of the GAL4 bindig site. These bindig elements together
defie a consensus GAL4 bindig sequence , 5' - CGG A(C/G)G AC(AI) GTC
(G/C)TC CG - 3' , and a synthetic DNA that diers from this consensus at only two positions (5' - CGGAAGACTCTCCTCCG - 3') has been shown to bind GAL4 protein in vivo (Giniger et al, 1985; this study) and in vitro (Carey et al, 1989) with high afty. Consensus or endogenous GAL4 bindig sites, either singly or in combination, are able to confer galactose-dependent expression onto heterologous genes in vivo (West et al, 1984; Giniger et al, 1985; and for example Martin et al I.e
1990). In general, increased copy number correlates with increased levels of transcriptional activation and cooperativity is thought to playa signcant role in
GAL4 activity.
Since GAL4-responsive promoters which require SWI/SNF activity have been identifed (see previous section) and because the galactose utilzation
pathway is so well characterized , we chose the GAL system of Saccharomyces
cerevisiae to investigate the relationship between transcriptional activation, the
SWIISNF complex and chromatin structure in vivo. In particular, we attempted to determine: 1) which elements , if any, of a GAL gene promoter dictate SWIISNF- dependence, 2) which domais, if any, of the transcriptional activator protein
GAL4 are involved in SWIISNF regulation, and 3) if any endogenous SWI/SNF-
dependent , GAL4-responsive genes exist in Saccharomyces cerevisiae. The results of these lines of inquiry are detaied in the following chapters. CHATER II
MATERIALS AND METHODS
Strain growth and maintenance
Yeast strais were grown in YEP medium (10 g/L yeast extract and 20 g/L peptone) supplemented with 20 mg/L adenine and containing as a carbon source either 2% glucose or 2% galactose with 0.5 % sucrose. To select growth of transformed yeast strais , cells were grown in minimal medium (6.7 g/L yeast nitrogen base without amino acids) supplemented with the appropriate amino acids and as a carbon source either 2% glucose or 2% galactose with
5% sucrose. YEPD and YEPGal plates were made as described above and contained 20 g/L agar. Where appropriate, antimycin was added to a final concentration of 1 J.g/J..
Liquid B-galactosidase assays
13-galactosidase activity of strais harboring LacZ reporter constructs was monitored by liquid 13-galactosidase assay as described (Stern et al,
1984), Strais were grown in the appropriate medium to an ODeoo of 0.
One ml of this log phase culture was harvested by centrigation and the pellet was washed in 1 ml ofZ Buffer (8.53 g/L NazHP04, 5.5 g/L NaHzP04, 75 g/L KCl, 0.246 g/L MgS04). Washed pellet was resuspended in 150 J. of
Z Bufer and cells were treated with 50 J. chloroform and 20 J. of 0. 1 % SDS.
Afer 30 seconds of vigorous vortexig, 700 J. of 0. 1 mg/ml ONPG, 2. 7 J.mlB- mercaptoethanol was added and the sample was mixed by inversion.
Samples were incubated at 30 C until a yellow color was visible. Reactions were stopped with 500 J.IM Na2C03. The absorbance (420 nm) of each sample was determined via spectrophotometry and Mier Units of 13- galactosidase activity was determined with the followig formula:
Units = (1000 X At20) I (ODsoo X time X volume).
Filter f3-galactosidase assays
Yeast cells were grown overnight on fiter paper (Watman 50) placed over the appropriate agar medium. In a sterile petri dish, a second fiter paper (Watman 3) was soaked in 0.03% X-gal (prepared from a stock of
Gal in diethyl formamide) in Z Buffer (8.53 glL N a2HP0 , 5.5 g/L
NaH2P0 , 0.75 g/L KCl, 0.246 g/L MgS0 , 2.7 J./ml13-mercaptoethanol).
Yeast cells were permeabiled on the Whatman 50 fiter by imersion in liquid nitrogen for thirty seconds and the frozen fiter was placed on top of the
gal soaked fiter. Care was taken to remove any air from between the two fiters and plates were incubated at 37 C until a blue color became visible. 34 .
Nuclei preparation
Nuclei were prepared essentialy as described (Shimuzu et al, 1991).
Briefly, a one liter culture was grown in the appropriate medium to an ODsoo ofO. L4. Cells were harvested by centrifgation and washed in S Bufer (1.4
M sorbitol, 40 mM HEPES pH 7. , 0. 5 mM MgCb, 10 mM mercaptoethanol, 1 mM PMSF). Mter washing, cells were resuspended in S
Buffer and incubated for ten minutes at 30 C. Cells were collected by centriugation and resuspended in a volume of S Buffer equal to four times the weight of the pellet. Cells were digested with 1 ml of 10 mg/mllOOT
Zymolyase (Seikagaku Corporation) for 45 minutes at 30 C. Digestion was monitored under the microscope and was considered complete when ::90% of the cells burst upon suspension in 0. 1% SDS. Digestion was stopped by
diution with S Buffer lackig -mercaptoethanol.
Spheroplasts were washed twice in S buffer lacking -mercaptoethanol and disrupted by four passes through a Yamato LS-21 homogenizer (100 rpm) in F Bufer (18% w/v Ficoll 400, 20 mM PIPES pH 6. , 0.5 mM MgCh, ImM
PMSF). Disrupted spheroplasts were carefuly layered on top of GF Buffer
(20% w/v glycerol , 7% w/v Ficoll400, 20 mM PIPES pH 6. , 0.5 mM MgCh,
1mM PMSF) and subjected to high speed centrifugation. The resulting pellet was completely resuspended in F Buffer by vortexing for five minutes at 4
Cellular debris was collected by low speed centrifgation and discarded. Nuclei were isolated from the supernatant by high speed centriugation.
Nuclear pellet was resuspended in D buffer (10mM HEPES, pH 7. , 0.5 mM
MgClz, 0.05 mM CaClz) to make approximately 5 X cell equivalents/ml.
These nuclei were frozen in liquid nitrogen in and stored at -
End-labelling of oligonucleotides
End-labelled oligonucleotides were prepared for use in the in vivo footprinting assay and the high resolution nucleosome mapping experiments.
100 ng of oligonucleotide was labelled in a 25 J. reaction containing 70 mM
Tris-HCl pH 7. , 10 mM MgClz, 5 mM dithiothreitol and 200 J.Ci ATP
(Amersham; 6000 Ci/mmol). Ten units of T4 polynucleotide kinase (New
England BioLabs) was added and the entire reaction was incubated at 37
for 10-30 minutes. Labelled oligonucleotides were purifed over a G-
Sephadex (pharmacia) spin column and incorporation oflabel measured on a
scintilation counter. The specifc activity of probes prepared using this
protocol ranged between 5- 10 X 10 cpm/J.g.
Internal labelling of DNA fragments
Body labelled DNA probes were prepared for use in indirect end-
labeJ.g analyses. Approximately 0. 1 J.g of agarose gel puried DNA
fragment was incubated with random primers and heated to 100 C for two minutes, followed by a brief coolig on ice. Random Priming Bufer (2.
stock: 500 mM HEPES pH 6.6, 125 mM Tris pH 8. , 12. 5 mM MgC12, 28 mM
13-mercaptoethanol, 0.5 mM dTTP, 0.5 mM dGTP, 0.5 mM dATP), 100 J.Ci
dCTP (Amersham, 6000 Ci/mmol) and 5- 10 units DNA polymerase I
Klenow fragment (New England BioLabs) were then added and the entire reaction was incubated at room temperature for one hour. Labelled DNA was puried over a G-25 Sephadex (pharmacia) spin column and incorporation of label measured in a scintilation counter.
In vivo footprinting
A 200 ml or 100 ml yeast culture was grown in the appropriate medium to an OD6oo of 0.75 (minial media) or 1.5-2 (rich media), respectively. Intact cells were treated with dimethyl sulate and the methylated DNA was isolated essentialy as described (Giniger et al, 1985), except fial pellets were dissolved in water and quantitated on a spectrophotometer. DNA
samples were then digested with HaeIII and analyzed via a cyclic primer
extension reaction (Axelrod and Majors, 1989) using a labelled
oligonucleotide. Primer extension products were extracted with
chloroform:isoamyl alcohol (24: 1), precipitated and electrophoresed on a
denaf&rig gel. The gel was dried and exposed to fim for 12-48 hours. As a
control for spurious primer extension products, DNA was also prepared from a , 10 strain that was not treated with DMS. Footprinting at the widtype GALl
UAS was analyzed using an oligonucleotide with the followig sequence: 5' -
GAG CCC CAT TAT CTT AGC - 3' . This oligonucleotide anneals to a
sequence located 60 bp upstream of GAL4 binding site 1. Footprinting at the
integrated GALl, 10 UAS derivatives was analyzed using an oligonucleotide
with the followig sequence: 5' - CCG GCT CGT ATG TTG TGT GG - 3'. This
oligonucleotide anneals to the unique pUC sequences located upstream of the
GAL4 binding sites in these reporters. Footprinting at integrated GALl, 10
UAS derivatives contaiing the nucleosome positioning element (NPE) was
analyzed using an oligonucleotides with the following sequence: 5' - CGG TTA
GTA CTT AAT TCC - 3' (which anneals to basepais 159 through 176 of the
NPE element; numbering of Meersseman et al, 1991) or 5' - CCG GTT CTC
GTC CGA TCA CCG - 3' (which anneals to basepais 118 through 137 of the
NPE element; numbering of Meersseman et al, 1991).
Indirect end-labelling
Nuclei were prepared as described above. To prepare Free DNA, 80
10% SDS and 30 20 mg/ml Proteinase K were added to a 1 ml alquot of
nucl i (approxiately 5 X 10 cell equivalents) and the sample incubated at
C for two hours. Lysed nuclei were treated with 180 5 M potassium
acetate on ice for one hour, subjected to centriugation and the supernatant \.:
precipitated with isopropanol. This DNA sample was resuspended in water and reprecipitated with ethanol. The resultant Free DNA was resuspended in
1 ml Buffer D2 (10 mM HEPES pH 7. , 5 mM MgCb, 2 mM CaCb). Free
DNA and nuclei samples were digested with 0 to 50 Units of MN ase for five minutes at 37 C. MNase reactions were stopped by the addition of SDS and
Proteinase K. Samples were next treated with 5 M potassium acetate on ice for one hour, subjected to centrifugation and the supernatant precipitated
with isopropanol. DNA was then digested with either ClaI (to map
nucleosomes at the integrated GALl , 10 UAS derivative reporters) or EcoRI
(to map nucleosomes at the endogenous GALl, 10 locus) in the presence of
RN ase. Digestions were extracted with bufered phenol and the aqueous layer precipitated with ethanol. Final pellets were electrophoresed on a 1.5% agarose gel overnight. DNA was transferred to a nylon membrane and blots
were probed with an internaly labelled 870 bp LacZ fragment (to map
nucleosomes at the integrated GALl, 10 UAS derivative reporters) or an
internaly labelled 550 bp fragment from the GALl gene (to map nucleosomes
at the endogenous GALl, 10 locus). ,,'
RNA isolation
A saturated yeast culture was diuted to an ODsoo of 0. 15 into 100 ml of the appropriate medium and alowed to grow to an ODsoo of 0.6 - 0. 8. Cells were harvested by centriugation, washed in 5 ml of sterie water and resuspended in 100 J. ofRNA Prep Buffer (0.5 M NaCl, 0.2 M Tris pH 7.
01 M EDTA, 1% SDS). Resuspended cells were added to a tube containing
100 J. of phenol/chloroform Ii so amyl alcohol (25:24:1) and approxiately 0.2 g of glass beads. Samples were vortexed five times in thirty second intervals with incubations on ice between pulses. An additional 150 J. RNA Prep
Bufer and 150 J.l phenol/chloroform/isoamyl alcohol was added to each sample, followed by 15 seconds of mixig. Samples were then spun for minutes at room temperature in a microcentrifuge. 150 J. of the aqueous phase was extracted with phenol/chloroform/isoamly alcohol and precipitated with three volumes of ethanol at - C for at least one hour. RNA was collected by centrifugation and washed with 70% ethanol before resuspension in sterile, RNase-free water. RNA samples were quantitated on a spectrophotometer and integrity was monitored on a 1% TBE agarose gel.
Samples were then stored at - C until needed. Primer extension assays
Transcription levels were monitored using a modied version of a previously described primer extension protocol (McKnght and Kigsbury,
1982). Briefly, 10-20 J.g of total yeast RNA and 1 ng of end-labelled oligonucleotide were boiled in 1.25 M KCl, 10 mM Tris pH 8. , 1 mM EDTA for one minute. Reactions were then incubated for 30-60 minutes at 50
The annealed oligonucleotide primer was extended with 10 Units of
Reverse Transcriptase (promega) in the presence of 2.5 mM deoxynucleotides
(dATP, dCTP, dGTP, dTTP), 50 mM Tris-HCl pH 8. , 5 mM MgClz, 100 J.g/ml
Actinomycin D, 10 mM DTT and 0.25 mM EDT A for 30 minutes at 42
Reactions were halted with 30 J.5 M ammonium acetate pH 7. 5 and precipitated with 180 J.l ethanol at - C. Reaction products were collected by centrifugation , resuspended in 8 l of loading dye (95% formamide, 20 mM
EDTA, 0.05% Bromophenol Blue, 0.05% Xylene Cyanol) and electrophoresed on an 6% denaturing gel. Mter electrophoresis, the gel was dried and exposed to fi. Sequences of the oligonucleotides used are listed below:
GALl 5' - GCG CTA GAA TTG AAC TCA GG - 3'
GAL2 5' - GAT TGT GCG CTT AA TGG G - 3'
GAL 3 5' - CTC TGC CCA GGA GAC CTA GCG - 3'
GAL 5; 5' - GGC ACC TTT AGA ACC CTC TGG - 3'
GAL 7 5' - GGT CTT TTA GCT CTG TGT GG - 3' GALlO 5' - CCA ATG TAT CCA GCA CCA CC - 3'
GAL80 5' - GGG AGC TGC ATT AGG CAC GG - 3'
MEL1 5' - CCC AAC CCA TCT GTG GAG TG - 3'
CLN3 5' - CTA GCG GTA GCA TAC CTT GC - 3' yT AFn90 5' - GGT TGT GGC TGA TGC GTG CC - 3'
Denaturing gel electrophoresis
Denaturing gels of the appropriate percentage were prepared using
National Diagnostics SequagelCI sequencing system accordig to the manufacturer s instructions. Gel solutions were fitered immediately before polymerization.
Preparation of yeast lysates
Yeast cultures were grown to saturation overnight, diuted to an ODsoo of approximately 0. 15 in the appropriate medium and then alowed to grow to an OD6ooofO.5 - 0.9. Five OD units of culture were treated with 1/10 volume of
100% trichloroacetic acid (TCA). Cells were collected by centrifugation resuspended in cold TCA bufer (20 mM Tris pH 8.0, 50 mM NH40Ac, 2 mM
EDT A) and disrupted by vigorous mixing in the presence of glass beads.
Supernatant was transferred to a fresh tube and the beads washed with a fresh 1:1 mixture of20% TCA: TCA Bufer. Wash buffer was added to supernatant and samples spun for five minutes in a microfuge on the highest setting. Pellets were washed with cold acetone, dred and resuspended in
TCA Resuspension Solution (3% SDS, 100 mM Tris, pH 11.0, 3 mM DTT).
Finaly, samples were heated to C for five minutes, boiled for five minutes more and then cooled briefly on ice. Insoluble debris was collected by centriugation and supernatants were frozen at - C until use.
Western blotting
Lysates prepared from yeast strains harboring GAL4 expression plasmids were electrophoresed on 10% SDS-containing, polyacrylamide gels and transferred to a nitrocellulose membrane (Schleicher & Schuell) by standard methods (Maniatis, 1989). Membranes were incubated with agitation in a solution of 10% powdered milk (prepared in TBST; 100 mM
Tris pH 7. , 0.9% NaCl, 0. 1% Tween) for 30 minutes at room temperature to block non-specifc bindig sites on the membrane. Following two ten second rinses in TBST, the membrane was incubated with an antibody directed against the DNA binding domain of GAL4 (a-Gal4 DBD; Santa Cruz) that had been diuted 1:1000 in 2% milklTBST. Best results were obtaied when this incubation was alowed to continue for twelve hours with agitation. Afer washing, the membrane was incubated for 45 minutes with goat anti-mouse secondary antibody that had been diuted 1:10 000 in 2% milkITBST. Finaly, ), ),
the membrane was washed and antibody recognztion was detected with the
Lumi-GloQD chemiluminescence kit accordig to manufacturer s instructions.
Strain construction
Four sets ofisogenic yeast strains were created for the studies described in Chapter 3 of this thesis (see Table 2-2). The four strais composing each of these sets were derived from the yeast strais CY388
(GAL4+, SWIl+ CY398 (gal4Ll, SWIl+ CY399 (GAL4+, swilL1) and CY400
(gal4L1, swilL1; see Table 2- 1). These founder strains contained a reporter
construct integrated at the ura3- 52 locus. In each case, the integration
resulted in a tandem duplication of the URA3 gene (one widtype and one
ura3- 52 alele) with intervening plasmid sequences. To select for cells in which this integrated reporter had been lost (via a genetic recombination event, i. e. "pop-out"), each strain was passaged twice on plates containing 5-
FOA. The 5-FOA resistant strains created in this way were caled CY524
CY525, CY526 and CY527, respectively.
These four new strains were then used to create the four strain sets
mentioned above. In each case , a unique LacZ reporter construct was
targetted for integration at the ura3- 52 locus by digestion with ApaI. CY528
CY529, CY530 and CY531 were created by integrating the plasmid pLB7
(which contains a LacZ reporter driven by a GALl, 10 UAS derivative bearng only two high afnity GAL4 binding sites) into CY524, CY525, CY526 and
CY527, respectively. CY532 , CY533, CY534 and CY535 were created by integrating the plasmid pLB8 (which contains a LacZ reporter drven by a
GALl, 10 UAS derivative bearing only two low afity GAL4 bindig sites) into CY524, CY525, CY526 and CY527, respectively. Similarly, strais
CY586, CY587, CY588 and CY589 were prepared by integrating the plasmid pLB16 (which is identical to pLB8 except for the addition of a nucleosome positioning element upstream of the GAL4 bindig sites) and strains CY594
CY595, CY596 and CY597 were prepar' ed by integrating the plasmid pLB 15
(which is identical to pLB7 except for the addition of a nucleosome positioning element upstream of the GAL4 binding sites). The genotypes of these strains are summarized in Table 2-2. For detais on the plasmids, see below.
Mter the strains described above were prepared, it was discovered that
in the GAL4 deletion strain of each strain set (gal4L1; CY525, CY529, CY533
CY587 and CY595), the reporter gene had been integrated at a site other than ura3- most liely the GALl, 10 locus. This is presumably due to the fact that the founding GAL4 deletion strain, CY398, did not contai an intact
ura3- 52 locus for integration. Plasmid constructions
Plasmids pLB7 and pLB8 were constructed by inserting the 194 bp
SmaI-PvulIfragment ofpUC18 between the URAB gene and the GAL4 binding sites of the plasmids pEG44 and pEG28, respectively. Plasmid pEG44 contains two synthetic, consensus GAL4 binding sites positioned 150
bp upstream of the GALl TATA element. These two sites are separated by
32 bp (center to center distance). Plasmid pEG28 contains GAL4 bindig sites 3 and 4 positioned 190 bp upstream of the GALl TATA element, as they are in the endogenous GALl, 10 locus. Sites 3 and 4 are separated by 62 bp
(center to center distance), Plasmids pLB15 andpLB16 were constructed by inserting a 182 bp PCR fragment encoding the strong nucleosome positioning
element from the 5S RNA genes of the sea urchin Lytechinus variegatus immediately upstream of the GAL4 binding sites in pLB7 and pLB8 respectively. This fragment was amplied from pICN5S182 (git from Jerry
Workman) using the oligonucleotides 5' - CCACGAATAACTTCCAGGG - 3' and 5' - CCCCGAGGAATTAAGTAC - 3' Table 2-1. Yeast strains used in this study.
CY257 MATa swiL1::LEU2 lys2- 80l ade2- l0l leu2-L1l his3-L1200 ura3-L199
CY296 MATa gal4L1::LEU2 lys2-80l ade2- l0l leu2-L1l his3-L1200 ura3-L199
CY297 MATa gal4Li:LEU2 swilL1::LEU2 lys2- 80l ade2- l0l leu2-L1l his3-L1200 ura3-L199
CY340 MATa lys2-L199 ade2- l0l leu2-L1l his3-L1200 ura3-L199 trpl- L199
CY341 MATa lys2-L199 ade2- l0l leu2-L1l his3-L1200 ura3-L199
CY348 swilL1::LEU2 gal80L1 ura3- 52 URA3::pRY171 (GAL1 10)
CY349 gal80L1 ura3- 52 URA3::pRY171 (GALl 10)
CY353 MATa lys2- 80l ade2- l0l leu2-L1l his3-L1200 ura3-
URA3::pRY171 (GAL1 10)
CY366 MATa swilL1::LEU2 lys2-L1l ade2- l0l leu2-L1l his3-
L1200 ura3- 52 URA3::pRY171 (GAL1 10)
CY388 MATa lys2- 80l ade2- l0l leu2-L1l his3-L1200 ura3-
URA3::pEG44 (2 hi)
CY398 MATa gal4L1:: LEU2 lys2- 80l ade2- l0l leu2-L1l his3-
L1200 ura3- 52 URA3::pEG44 (2 hi)
CY399 MATa swilL1::LEU2 lys2- 80l ade2- l0l leu2-L1l his3-
L1200 ura3- 52 URA3::pEG44 (2 hi) Table 2-1, continued
80l ade2- CY400 MATa gal4L1.:: LEU2 swilL1::LEU2 lys2- l0l leu2-L1l his3-L1200 ura3- 52 URA3::pEG44 (2 hi)
L1200 ura3- CY 401 MATa lys2-80l ade2- l0l leu2-L1l his3-
URA3::pEG28 (2lo)
L1l his3- CY422 MATa swi1 ::LEU2 lys2- 80l ade2- l0l leu2-
L1200 ura3- 52 URA3::pEG28 (2lo) '-,
Table 2-2. Yeast strains constructed in this study.
CY524 MATa lys2- 80l ade2- l0lleu2-L1l his3-L1200 ura3-
CY525 MATa gal4L1::LEU2 lys2- 80l ade2- l0lleu2-L1l his3-L1200 ura3-
CY526 MATa swilL1::LEU2 lys2- 80l ade2- l0lleu2-L1l his3-L1200 ura3-
CY527 MATa gal4L1:: LEU2 swilL1::LEU2 lys2- 80l ade2- l0lleu2-L1l his3-L1200 ura3-
CY528 Same as CY524 , but contains URA3::pLB7 (2hi-p UC)
CY529 Same as CY525, but contains URA3::pLB7 (2hi-pUC)
CY530 Same as CY526 , but contains URA3::pLB7 (2hi-pUC)
CY531 Same as CY527, but contains URA3::pLB7 (2hi-p UC)
CY532 Same as CY524 , but contains URA3::pLB8 (2lo-p UC)
CY533 Same as CY525 , but contains URA3::pLB8 (210-p UC)
CY534 Same as CY526 , but contains URA3::pLB8 (2lo-p UC)
CY535 Same as CY527 , but contains URA3::pLB8 (2lo-pUC)
CY586 Same as CY524 , but contains URA3::pLB 16 (2 lo-NPE)
CY587 Same as CY525 , but contains URA3::pLB 16 (2 lo-NPE)
CY588 Same as CY526 , but contains URA3::pLB 16 (2 lo-NPE)
CY58 Same as CY527 , but contains URA3::pLB16 (2 lo-NPE)
CY594 Same as CY524 , but contains URA3::pLB15 (2 hi-NPE) Table 2- , continued
CY595 Same as CY525, but contains URA3::pLB 15 (2 hi-NPE)
CY596 Same as CY526 , but contains URA3::pLB15 (2 hi-NPE)
CY597 Same as CY527 , but contains URA3::pLB15 (2 hi-NPE) CHATER III
THE YEAST SWI/SNF COMPLEX FACILITATES BINDING OF A
TRANSCRIPTIONAL ACTIVATOR TO NUCLEOSOMA SITES IN VIVO
Background
Giniger et al (1985) were the fist to demonstrate that the GAL4 protein is
capable of binding to the four recognition sites found in the GALl, 10 UAS region in vivo. These authors also showed that GAL4 could recognze a synthetic 17 consensus sequence in vivo and that this sequence, when linked to a heterologous reporter gene, could confer high levels of galactose inducibilty (Giniger et al
1985). In subsequent studies, Giniger and Ptashne (1988) demonstrated that
GAL4 bindig can be cooperative in vivo; these authors also suggested that this cooperativity might explai the phenomenon of transcriptional synergy at the
GALl, 10 locus. In addition to providing the early characterization of a genetic system which has since become an indispensable tool for the study of eukaryotic transcriptional regulation, these early studies served as a foundation for the experiments described in the following pages. First, the in vivo footpriting assay developed in this study is based on that used in these earlier studies. In addition
the GALl, 10 UAS derivatives they constructed and characterized provided a crucial starting point for the derivative promoters studied here.
Selleck and Majors (1987) have also monitored GAL4 bindig in vivo.
These authors used a photofootprinting assay (some detais of which were also incorporated into the assay described in this thesis) to veriy GAL4 binding to the
GALl, 10 UAS. In subsequent analyses, Axelrod et al (1993) found that GAL4 binding could induce the disruption of a repressing nucleosome located in the
GALl promoter. This data complements earlier studies on the role ofnucleosome
positioning in transcriptional reguation at the GALl, 10 locus (Fedor and
Kornberg, 1989) and suggests that in addition to interacting with targets in or near the PIC, transcriptional activator proteins may also interact with chromatin components.
Since the SWIISNF complex is believed to be involved in transcriptional regulation at the level of chromatin structure (see Chapter 1), we wondered if this
complex played a role in the l'eguation of the GALl, 10 promoter. Peterson and
Herskowitz (1992) have shown that transcription of GALl is decreased 2- to 6-fold in swi/snlmutants. Although this effect is substantialy smaler than the
SWI/SNF effects seen at other genes (ADH2 expression is decreased 10- to 20-fold and INOI expression is decreased 30-fold; Peterson and Herskowitz, 1992), it
does suggest that SWI/SNF may be involved in reguating the GAL 1 10 promoter. As described in Chapter 1, biochemical experiments have shown that yeast
or human SWIISNF complex can stimulate the binding of an activator protein to a
nucleosomal binding site (Cote et al, 1994; Kwon et al, 1994). Therefore, we began
our studies of SWIISNF function at the GALl, 10 promoter by exploring its effect r on transcriptional activation and DNA bindig by the GAL4 protein. These
experiments utilzed LacZ reporter genes whose expression was controlled by
derivatives of the GALl, 10 UAS region; these UAS derivatives dier in the afnity of the GAL4 binding sites as well as the nucleosomal context of these
sites.
Resul ts
Isogenic SWI+ and swi- reporter strains were constructed as described
Chapter 2 and are shown schematicaly in Figure 3- 1. Strais CY353 (SWI+ and
CY366 (swil- contain a GALl, 10 UAS reporter consisting offour GAL4 bindig
sites upstream of the LacZ gene (reporter a; Guarente et al, 1982). Strains CY 401
(SWI+ and CY 422 (swil- contain a derivative of this reporter that contains only
two of these low afnity GAL4 binding sites (sites 3 and 4; reporter b); strains
CY532 (SWI+ and CY534 (swil- contain a version of reporter b that contais 192
bp of additional plasmid sequence 20 bp upstream of GAL4 site 3 (reporter c);
strains CY528 (SWI+ and CY530 (swil- contain a reporter which contains two pLEXA
SWI+ swil- SWI+ swil-
7218 4691 (65%) n.
390 16(4.1%) 1118 51 (4.5%)
658 30 (4.5%) 2409 74 (3%)
2311 1726 (75%) 2719 1616 (59%)
613 490 (80%)
NPE Figure 3-1. GAL4 requires functional SWI/SNF complex to activate transcription from two low affinity binding sites. f3-galactosidase assays
were performed on isogenic SWI+ or swi- strais harboring the integrated GALl- LacZ UAS reporter plasmid pRY171 (a), pEG28 (b), pLB8 (c), pLB7 (d) or pLB16 (e). GAL4 binding sites are denoted by smal solid boxes; larger solid boxes represent the DNA sequences denoted. GAL4 sites numbered 1-4 represent sites from the GALl, 10 UAS region; GAL4 sites numbered 17 indicate synthetic, consensus GAL4 binding sites. Strains contained plasmids that overexpressed either the bacterial LexA protein (pEG202) or the fu length GAL4 protein (PMA210). Numbers in parentheses show activities as a percentage of the widtype levels. 13- galactosidase activities of al reporters were 0: 1 unit in the absence of GAL4. Drawings are not to scale; n. , not determined. high afity, consensus GAL4 binding sites in place of the two low afnity sites in
reporter c (reporter d; Giniger and Ptashne, 1988). p-galactosidase assays were
performed on these various strains to monitor GAL4 transcriptional activity in
the presence of either endogenous concentrations of GAL4 (column labeled pLEXA)
or under conditions in which GAL4 is overexpressed (column labeled pGAL4).
In the absence of functional SWIISNF complex, transcriptional activation by
GAL4 from the widtype GALl, 10 UAS region is reduced only 1.5-fold compared to
the activity in the respective widtype strai (Figure 3- , reporter a). This result
is consistent with previous studies in which it was found that GALl expression
was only weakly afected by swi/ snl mutations (peterson and Herskowitz, 1992)
In contrast, GAL4 activity on the two low afity GAL4 binding site reporters is
SWI/SNF -dependent; transcriptional activity is I' educed 22- to 32-fold in the swil- strain as compared to the widtype strain (Figure 3- , I'eporters b and c). When
these two low afnity binding sites were replaced with high afity, consensus
GAL4 binding sites, transcriptional activation is only reduced 1.3- fold in the swil-
strain as compared to the wid-type strai (Figure 3- , reporter d).
Strains harboring the two low afnity site constructs were also distinct
from those carryig the two high site construct in that afnity LacZ expression was increased 2. 5 to 3. fold when GAL4 protein was overexpressed (Figure 3-
and data not shown). The simplest interpretation of this result is that physiological levels of GAL4 are not sufcient to fully occupy the two low afty sites (see Giniger and Ptashne, 1988 and see below). Importantly, overexpression
of GAL4 does not overcome the SWIISNF dependence of this reporter (Figure 3-
reporters b and c) It should be noted that the swil mutation does not afect
expression of GAL4 from the overexpression plasmid (peterson and Herskowitz
1992; also see Chapter 5).
To diectly assess the abilty of GAL4 to bind its sites in the dierent
reporter strais in the presence or absence of SWIISNF, we used an in vivo DMS
footprinting assay. GAL4 protects guanine residues at each end of its 17 base- pair recogntion site both in vivo (Giniger et al, 1985) and in vitro (Carey et al
1989). As shown in Figure 3- , these guanine residues within the four GAL4
bindig sites of the GAL 1 10 UAS are protected from DMS methylation in the presence of GAL4 (lane 3), but are accessible to methylation in an isogenic gal4-
strain (lane 2). Introduction of GAL4 expression plasmids into the ga14- strai restores protection of these guanines (lanes 6 and 8), while introduction of a plasmid expressing the bacterial LexA protein does not (lane 7). In the absence of SWIISNF activity, pl' otection of all four sites is retained (lane 4). Furthermore, as shown in Figure 3- , GAL4 also protects the guanine residues at each of the two high afnity binding sites of reporter d in the presence (lane 4) and absence (lane
5) of SWIISNF. These results are consistent with our functional studies (Figure 3-
1) in which SWI/SNF is not required for the activity of GAL4 from either the widtype GALl, 10 UAS or the two high afnity site reporter (Figure 3- , reporters a and d). ..' . .' ' ...
i-)f'
-AI 'o- :.'(;' .,
Figure 3-2. GAL4 can bind its sites in the GALl lO UAS region in the presence or absence of SWI/SNF. In vivo DMS footprinting analysis of the
GALl, 10 UAS region was performed on strains CY341 (SWI+ GAL4+ lane 3),
CY257 (swil- GAL4+ lane 4), CY296 (SWI+ gal4- lane 2), CY296 containig GAL4 expression plasmids (pMA210 or pSD15; lanes 6 and 8), and CY296 contaiing a LexA expression plasmid (pEG202; lane 7). Lane 1 contains a genomic guanne sequencing ladder. Boxes to the left of the figure denote the
GAL4 binding sites of the GALl, 10 UAS and asterisks denote guanine residues I': "1. shown previously to be protected by GAL4 binding. ; , ...... ,' ~~~
.!ft .. ;;" ,. "
. J )\-.. ',j"; : ' ~~~~~~ ,,'#:'; ...,",,.- " ;",,
J:"
_1'
i,:..
v,.
/'c r.,
,N,:
,r Figure 3-3. SWI/SNF is required for complete occupancy of two low affinity GAL4 binding sites in vivo. In vivo DMS footpriting was performed on strains that harbor the two high afnity site reporter (panel A): CY529 (gal4-
SWI+ lane 3), CY528 (GAL4+ SWI+ lane 4) and CY530 (GAL4+ swil- lane 5), or strains that harbor 'the two low afity site reporter (panel B): CY533 (gal4-
SWI+ lane 2), CY532 (GAL4+ SWI+ lane 3), CY534 (GAL4+ swil- lane 4), CY532 harboring a GAL4 expression plasmid (lane 5), and CY534 harboring a GAL4 expression plasmid (lane 6). Arrow denotes the control bands, G449 and G450 by the numbering of Yocum et al, 1984, to which phosphorimager data was normalzed (see Figure 3- 4). As a control, GAL4 bindig in the samples shown in Panel B, lanes 2 and 6 were analyzed for GAL4 binding at the GALl lO UAS locus (panel C). Symbols as described in Figure 3- ).
Figure 3- 3B shows an in vivo footprinting analysis of the two low afnity
site reporter in strains CY532 (SWI+ and CY534 (swil- We detect little
protection of the two low afity sites in our footprinting assay, even in a SWI+
strain (lane 3). As discussed above, this probably reflects the low occupancy of
these sites at physiological GAL41evels, because when GAL4 is overexpressed in
this S1iVZ+ strai protection is restored (lane 5). Overexpression of GAL4 in the
absence of a functional SWIISNF complex, however, does not restore complete
protection of either low afnity bindig site (lane 6). This is most apparent at
GAL4 site 3, where strong protection is observed in the SWI+ strai (lane 5), but
only weak protection in the swil- strain (lane 6). Quantitation of these results
phosphorimager analysis indicates that when GAL4 is overexpressed in the
absence of SWIISNF activity, protection of the relevant guanine residue in site 3 is
decreased 40% as compared to that seen in a wildtype strain (Figure 3-4). :"9 Because al the strains analyzed in this study contai an unaltered copy of
the endogenous GALl, 10 UAS region at its normal location in the genome (in
addition to the reporter locus integrated at the URA310cus), we were able to carry
out an important internal control. The abilty of GAL4 to bind to its sites within
the GALl , 10 locus was analyzed in the same swi- sample which showed poor
protection of the low afnity sites at the reporter locus (Figure 3- , lane 6). As
shown :i Figure 3-3C, we observed complete occupancy of the four GAL4 sites
the endogenous GAL1 10 locus in this sample. Thus, the lack of complete :: : ! j .
55 i
ga14-
25
20
SWI+ pGAL4 i I!!'
00
;Jj.
35
28
21 ! I swi- pGAL4 11: Ii ! 1 j ' " I 00 ,j. . -.., ;:. ;!-
j,,\:F.r;r-,
gal4- SWI+ swi-
(1) G449/G450 control 335 122 146
(2) G438/G439 site 3 399 127
(3) Normalization factor
(4) Adjusted values 399 216 292
(5) % occupancy (0%) (100%) 58%
Figure 3-4. Phosphorimager analysis of GAL4 site 3 occupancy. (panel A; previous page). The gel shown in Figure 3- 3B was scanned on a phosphorimager and the traces for lanes 2 (gal4-) (SWI+ pMA210) and 6
(swi- pMA210) are shown. Top to bottom on the gel is depicted as left to right in the traces. Small arrows denote the peak representing the control doublet G449/G450 (see legend to Figure 3- 3B) and large arrows denote the peaks representing the doublet in site 3 (G438/G439).(panel B). Lines (1) and (2) show the numerical values for the area under the peaks designated in Panel A. For each sample, the values for the control doublet were represented as a fraction of the gal4- control doublet value of 335; this normalization factor is listed in lie (3). This factor was then used to adjust the values for the site 3 doublet; the new values are shown line (4). Line (5) shows the occupancy of site 3 in a swi- stI'ain as a percentage of the occupancy of this site in the gal4- strain
(assigned a value of 0% occupancy) and in the SWI+ strain (assigned a value of 100%) after background had been subtracted. ..;. . ).
occupancy at the two low afnity sites in this strain indicates that the SWI/SNF
complex modulates GAL4 binding in vivo.
To assess whether the chromatin structure of the GAL4 binding sites was
related to their SWI/SNF -dependence, a strong nucleosome positioning element
(NPE) was inserted diectly upstream of the two low afnity bindig sites in
strais CY532 (SWI+) and CY534 (swil- This 182 bp NPE has been shown
previously to translation aly' and rotation aly position a nucleosome in vivo in
yeast (pederson and Fidrych, 1994; see also Figure 3-8). Insertion of the NPE had
a dramatic effect on the SWIISNF dependence of GAL4 function. Whe GAL4
transcriptional activity at the original two low affnity site reporter was decreased
over 20- fold in the absence of SWI/SNF (Figure 3- , reporter b), there was less
than a 1.5-fold decrease when the nucleosome positioning element was inserted
(Figure 3- , reporter e). This effect is not simply due to insertion of foreign DNA
sequences upstream of the GAL4 binding sites because insertion of 192 bp ofpUC
plasmid sequences at an identical position did not alter the SWI/SNF dependence
of GAL4 activity (Figure 1, reporter c).
These results suggest that the SWIISNF -dependence of GAL4 can be
modulated by nucleosome positioning. To confrm this possibilty, we analyzed
'1, l:1 the chromatin structure of some of the reporters tested in Figure 3- 1. As shown
previously by Fedor and colleagues , the four GAL4 binding sites at the GALl, 10
locus are located in a constitutively nucleosome-free region that is flanked by
arrays of positioned nucleosomes (Fedor and Kornberg, 1989; see also Figure 3-5). FreeDNA Chromatin
1921 bp
1110 bp
550 bp Figure 3-5. Nucleosome mapping of the GALl, 10 UAS region. Free DNA and nuclei from the two low afnity site SWI+ reporter strain (CY532) were treated with increasing amounts ofMNase and nucleosome positioning at the GALl locus was analyzed by indiect end-labellng. MNase treatments were as follows: 1 Unit/ml, lane 1; 5 Units/ml, lanes 2 and 3; 25 Units/ml, lane 4. Schematic to the right of each panel depicts mapped locus, black rectangles represent GAL4 binding sites, shaded boxes represent codig sequence of the indicated gene, and shaded ovals represent the predicted position of nucleosomes. Primers used to ampliy the probe fragment by PCR are indicated by black bars. Migration of DNA size standards are noted to the left of each panel. :, ' -/",. ,- ." , "". ' , , " , '
, GALI-LacZ
;"i ''-'':i $'1& , I . '
\C Figure 3-6. Nucleosome mapping of the two low affinity site reporter locus. Free DNA and nuclei from the two low afity site SWI+ reporter strain (CY532) were treated with increasing amounts ofMNase and nucleosome positioning at the reporter locus was analyzed by indiect end-labellng. MNase were as follows: 0 Units/ml, lanes 1 and 7; 0.005 Units/ml, lanes 2 and 8; 0. Units/ml, lanes 3 and 9; 0.5 Units/ml, lanes 4 and 10; 5 Units/ml, lanes 5 and 11; 50 Units/ml, lanes 6 and 12. Restriction sites used to generate the probe fragment are indicated on the map. Symbols as described in Figure 3- The precise nucleosome positioning of this region is believed to require the GRF2
protein, which binds to a sequence that overlaps GAL4 binding sites 1 and 2
(Fedor et al , 1988). The GALl, 10 UAS derivatives that we have analyzed in this
study (Figure 3- , reporters b-e) lack this GRF2 binding site and therefore may
not maitain nucleosome positioning in the region surrounding the GAL4 binding
sites. To test this idea directly, we analyzed the chromatin structure of the two
low afnity site reporters in the SWI+ strains CY532 (without NPE; reporter c in
Figure 3- 1) and CY586 (with NPE; reporter e in Figure 3- 1) and compared these
structures to that of the GAL 1 10 locus in the same strais.
Nuclei and free DNA were prepared from the SWI+ strain CY532, which
contains the parental two low afnity site reporter (reporter c in Figure 3- 1) and
samples were analyzed by MN ase digestion and indirect end-labeling. First, we
analyzed the chromatin structure at the GALl locus of this strain (Figure 3-5).
Comparison of the MN ase cleavage pattern in free and chromatin DNA samples
reveals a repeating pattern ofMNase protections, each approximately 140 bp in
size and flanked by MN ase hypersensitive sites. These results are essentialy
identical to a previous study (Fedor and Kornberg, 1988) and are consistent with an array of positioned nucleosomes downstream of the nucleosome- free GAL4 binding sites.
imilar analysis of the two low afnity site reporter locus in this same strain yielded very dierent results (Figure 3- 6). In the region upstream of the two , \GAL4 binding sites, the MN ase digestion pattern in the chromatin sample is similar to the pattern of digestion in the free DNA, although we do reproducibly
observe several preferred cleavage sites in free DNA that appear to be enhanced in
the chromatin sample. Importantly, the MNase cleavage sites directly upstream
and adjacent to the GAL4 bindig sites are cleaved with equal effciency in the
free and chromatin DNA samples. A region of approxiately 80 bp, which
- contains the GAL4 binding sites, is not cleaved effciently in either the free or
chromatin samples; this is due to the inherent sequence specifcity ofMNase I and
is not due to a positioned nucleosome (Fedor and Kornberg, 1988). In addition
this protected region is not flanked by hypersensitive sites. Directly downstream
: of the GAL4 binding sites, however, a 140 bp region ofMNase resistance which is
flanked by MN ase hypersensitive sites is observed, suggesting the presence of a
positioned nucleosome. The location of this putative nucleosome corresponds to
the positioned nucleosome located between the GAL4 sites and the GALl TATA
box of the endogenous GALl, 10 locus that has been identifed by others (Fedor and
Kornberg, 1988; Axelrod et al, 1993; see also Figure 3-5). Thus, although one
nucleosome appears to be positioned at the GALl , 10 UAS derivative in this strain
(reporter c), the majority ofnucleosome positioning has been lost.
Nucleosome mapping of the SWI+ strai CY528, which harbors the two high
afnity site reporter derivative (reporter d in Figure 3- 1), revealed a pattern very
similar to that seen with the two low afnity site reporter strai just described.
Specifcaly, 1) in the region upstream of the GAL4 binding sites, the MNase
digestion pattern in the chromatin sample is similar to the pattern of digestion . '
observed in the free DNA , 2) as expected, the GAL4 bindig sites themselves are resistant to MNase cleavage, and 3) a protected region the size expected of a
nucleosome was observed between the GAL4 sites and the downstream GAL 1
TATA box (Figure 3-7). Together with the results presented in Figure 3- , these
, results suggest that the removal of the presumptive nucleosome positioning
elements of the GALl, 10 UAS region (i.e. the GRF2 binding sites) can alter
nucleosome positioning over this region in vivo. In the absence of a positioned array ofnucleosomes, these GAL4 binding sites are unlkely to reside in a
constitutively nucleosome-free region.
MNase digestions and indirect end-labeling were also used to confrm that the NPE in strain CY586 (see Figure 3- , reporter e) positioned a nucleosome
upstream of the two low afnity GAL4 binding sites (Figure 3-8). Comparison of
. the free and chromatin DNA samples reveals an MN ase resistant region of about
50 bp (including the NPE sequences as well as the MNase-resistant GAL4
inding site sequences), consistent with positioning of a nucleosome over the NPE equence. In addition, the putative positioned nucleosome located between the
AL4 binding sites and the GALl TATA box that was detected at the GALl ocus (Figure 3-5), at the parental two low afity site reporter (reporter c; see
'Figure 3-6), and at the two high affnity site reporter (reporter d; see Figure 3- 7) is also detected at the NPE-containing reporter locus of CY586 (reporter e; see .. . ',~~~ ' )
Free DNA Chroma tin
MNase
- 2030
, ::rr iL'; 1500 bp
- 1000
.0.
- 500
10 Figure 3-7. Nucleosome mapping of the two high affinity site reporter locus. Free DNA and nuclei from the two high afnity site SWI+ reporter strain (CY528) were treated with increasing amounts ofMNase and nucleosome positioning at the reporter loci was analyzed by indirect end-labellng. MNase treatment was as follows: 0 tJ/ml, lanes 1 and 6; 0.005 U/ml, lanes 2 and 7; 0. U/ml, lane 8; 0. 5 U/ml, lanes 3 and 9; 5 U/ml, lanes 4 and 10; 50 U/ml, lanes 5 and 11. In this map, the two high afity GAL4 binding sites are depicted as open boxes. Migration of DNA size standards are noted to the right. ..
FreeDNA Chromatin MNase
2620 bp
2170bp
870bp Bam
CIa! 3 4 Figure 3-8. Nucleosome mapping of the two low affinity site reporter containing a nucleosome positioning element. Free DNA and nuclei from the
NPE-contaiing, two low afnity site SWI+ reporter strain (CY586) were treated with increasing amounts ofMNase and nucleosome positioning at the reporter locus was analyzed by indirect end-labellng. MN ase treatment was as follows: 5 Units/ml, lanes 1 and 3; 5 Units/ml, lanes 2 and 4. Restriction sites used to generate the probe fragment are indicated on the map accompanying each figure. Symbols as described in Figure 3- In results l'eported elsewhere, Burns and Peterson (1997) have used a restriction enzyme accessibilty assay to monitor the nucleosome state of the
GAL4 bindig sites in some of the reporters described above (reporters a, c and e).
This data is in good agreement with the indirect end-labeling experiments described above and veriy that in reporter c, the two low afity GAL4 binding sites are nucleosomal (as evidenced by their inaccessibilty to restriction enzyme digestion in the accessibilty assay). In this same assay, the four GAL4 binding sites of the endogenous GALl , 10 UAS region as well as the two low affnity sites in the NPE-containing reporter e are completely accessible to enzyme cleavage indicating that these regions are free of nucleosomes (See Appendi B).
Discussion
The results described above suggest that the SWIISNF -dependence of a transcriptional activator protein is not an innate feature of the activator protein per se, but rather reflects the chromosomal context of the activator binding sites.
When the activator binding sites are encompassed in a nucleosome, as in the two low afnity site reporter used in this study, SWIISNF function is necessary for the activator protein to bind to and activate transcription from those sites (see Figure
, top): On the other hand, when binding sites are nucleosome-free, as is the case at the GALl, 10 UAS region or at the NPE-containing two low afnity site reporter, then SWIISNF function is dispensable for GAL4 activity. In this case ,- - - ', ,'.... - - ' , ' ..:,*.:- - "".-,, :'..,.
I"-
SWI/SNF dependent?
" I YES
Figure 3-9. Predicted nucleosome structure ofSWI/SNF-dependent and SWI/SNF-independent promoters. Schematics depicting the predicted nucleosome structure of the two low afnity site l"eporter construct and the same construct containing a nucleosome positioning element directly upstream of the GAL4 binding sites. Closed ovals represent positioned nucleosomes; dashed ovals represent randomly located nucleosomes. Nucleosomes located randomly over the GAL4 binding sites of the former cause the expression of this reporter gene to require SWIISNF activity. In the NPE-containing construct, on the other hand, the GAL4 bindig sites are nucleosome-free and therefore expression of this reporter is SWI/SNF-independent. GAL4 can bind to its sites and activate transcription in the absence of SWIISNF
activity (see Figure 3- , bottom). Furthermore, SWI/SNF dependence does not
appear to correlate with the level of transcriptional activation since we have found
both weak and strong promoters whose activity is dependent upon SWIISNF (see
Figure 3- 1). Thus, based on the results presented here, we predict that the subset
of genes whose expression requires SWIISNF wi have activator bindig sites that
are encompassed in nucleosomes; genes that do not require SWIISNF wi have
such sites positioned between nucleosomes. This positioning may be determined by DNA sequence, as in the NPE-contaiing reporter, or by abundant DNA bindig proteins like GRF2 , as in the case of the intact GALl, 10 reporter.
Our results also require that binding site affnity be incorporated into the
defiition of SWIISNF -dependence. Whle GAL4 requires SWIISNF in order to access low afity, nucleosomal binding sites, high afnity, nucleosomal sites are
occupied even in the absence of SWI/SNF. The abilty of higher afnity binding sites to aleviate SWIISNF -dependence provides strong evidence that the
SWIISNF complex modulates transactivation at the level of DNA binding.
Furthermore, these results suggest that GAL4 binding to high affity nucleosomal sites can occur in vivo via a SWIISNF independent mechanism.
These results are consistent with in vitro studies of Workman and Kingston
(1992) 'Who found that GAL4 can bind to a nucleosomal site to form a tripartite transcription factor-histone-DNA complex. As noted above, we detect a consistent level of GAL4 binding to the low
afnity sites in the swil- strain (Figure 3- , compare lanes 5 and 6);
phosphoriager analysis indicates site 3 may be occupied at 60% of the levels
seen in a SWI+ strain (Figure 3-4). The fact that this level of occupancy results in
negligible levels of transcription (3% of wid type levels, see Figure 3- , line b) may
indicate a role for the SWIISNF complex in transcriptional activation beyond the
modulation of activator bindig. In light of recent reports suggesting that the
SWIISNF complex associates with the RNA polymerase II holoenzyme (Wilson et
al, 1996; but see also Cairris et al, 1996b), it is interesting to consider the
possibilty that an activator-SWIISNF interaction is involved in preinitiation
complex assembly. On the other hand, our in vivo footprinting procedure does not
alow us to determine the extent to which site 3 and site 4 are simultaneously
occupied, nor can we distinguish whether the 60% occupancy in a swi/ snf strai
reflects the stable binding of GAL4 in 60% of the cells, or reflects weak, unstable
binding in 100% of the cells. Therefore, our results may support an additional role
for the SWIISNF complex in faciltating the cooperative bindig of GAL4 or in
enhancing the stabilty of GAL4 binding to low afity, nucleosomal sites iIi vivo.
Finaly, it is important to note that although the protected region identifed
,t" (by indirect end-labelig analysis; see Figures 3- , 3- , 3-7 and 3-8) between site
four an the GALl TATAbox of al the reporters analyzed in this chapter is
consistent with a positioned nucleosome, these results may also indicate the
bindig of a sequence-specifc DNA binding protein to this region in vivo. CHATER IV
CHAACTERIZATION OF THE ROLE OF THE GAL4 ACTIVATION DOMAN
IN SWI/SNF TARGETING
Background
The 888 residue GAL4 protein has been extensively characteried and its modularity well documented (Brent and Ptashne, 1985; Keegan et al, 1986). The
terminall47 amino acids encode the DNA binding and dimerization activities
, " i of the protein (Keegan et al, 1986; Carey et al, 1989). This domain functions independently of the rest of the protein and has been used to target a diverse array of transcription factors and activation domains to promoter regions bearing
GAL4 sequence bindig elements in vivo and in vitro (for example see Barberis et
, 1995; Chatterjee and Struhl, 1995). Two separable activation domains have been identifed within the GAL4 protein, although it is the region of the C- terminus between residues 768 and 881 which, is thought to be the physiologicaly relevant transcriptional activating,region (la and Ptashne, 1987a). This domain is the target of the inhibitor GAL80 (see above) and a number of studies have identif d residues which appear to be crucial for transactivation (Lue et al, 1987;
Leuther et al, 1993). The volume of studies characterizing the structure and function of the GAL4 protein has resulted in the avaiabilty of a library of GAL4 :'
expression plasmids which encode GAL4 derivatives varying in stabilty, DNA
binding abilty and transcriptional activity.
As discussed in detai in Chapters 2 and 3, we have constructed a set of
isogenic yeast strains harboring a LacZ reporter gene whose expression is
dependent upon the presence of 1) the transcriptional activator GAL4 and 2) a
functional SWIISNF complex. In the absence of SWIISNF LacZ expression from
this two low afity site reporter is decreased over 20-fold and GAL4 binding to
one of the two low afnity sites is decreased by 40% (see Chapter 3). In the
experients described below, we have used this reporter gene and the library of
avaiable GAL4 expression plasmids to dissect the functional components of
GAL4 which playa role in the SWIISNF -dependent transcriptional activation.
Resul ts
Although GAL4 binding to the two low affnity site reporter is decreased
substantialy in the swil- strai, we do detect a consistent level of GAL4 binding
to these sites (see Chapter 3, Figure 3- 3B); in fact, phosphorimager analysis
indicates site 3 may be occupied at 60% of the levels seen in a SWI+ strain
(Chapter 3, Figure 3-4). One possible explanation for this finding is that GAL4
binding to nucleosomal sites is stabilzed by two independent pathways (Figure 4-
1). In t e fist pathway, the SWIISNF complex faciltates the interaction between
the activator and the DNA in the context of underlyig nucleosomes. In the
second, interactions between the activation domain of GAL4 and its target(s) in '-"...... - "' "' "". -....".... --.....
SWI/SNF
e__e
1 :
'I..
Figure 4-1. Alternative models for the stabilzation of GAIA binding in vivo. Schematic depiction of a eukaryotic promoter. Shaded boxes represent binding sites for transcriptional activator proteins, bent arrow depicts transcription start site, dashed circles represent nucleosomes. In pathway (1), chromatin remodel- ing machines like the SWIISNF complex stabilze activator binding to nucleosomal sites. In pathway (2), interactions between the activation domain ofthe activator protein and components of the general transcription apparatus stabilze activator binding. These models are not mutually exclusive. :;- . '
the preinitiation complex is able to stabilze its binding to the sites. In order to
address this possibilty, we tested the abilty of a GAL4 derivative lackig an
activation domain (PMA424) to bind to the two low afnity site reporter in the
S"W+ yeast strai CY533.
As shown in Figure 4- , the guanine residues within the two low afity
GAL4 binding sites in this strain are protected when ful length GAL4 protein is
. t'; overexpressed (lane 2), but not when an irrelevant bacterial protein is
overexpressed (lane 4). When the GAL4 DNA binding domain alone is
overexpressed, the relevant guanines are stil protected (lane 3). Phosphorimager
analysis verifes that the protection observed for the DNA bindig domain alone is
similar to that of the full length protein (Figure 4-3). As expected, both fu length
GAL4 and the DNA binding domain alone are able to bind to al four GAL4 sites
found at the endogenous GALl, 10 locus (Figure 4- 2B). These results indicate that
the GAL4 activation domain is not required in order for SWIISNF to modulate
;f; GAL4 bindig in vivo. In the context of the model presented in Figure 4- , these
results suggest that the two pathways for stabilzing GAL4 binding to
nucleosomal sites are redundant; in the absence of either pathway, DNA binding
continues to be stabilzed by the other.
A prediction of this model is that the inactivation of both pathways would F"...
lead to loss of GAL4 bindig to the nucleosomal sites and therefore a loss of
'i" I protection in our in vivo footprinting assay. To test this prediction, we set out to
measure the abilty of the GAL4 DNA binding domain to bind the two low afnity - - ,,,. ;). ,.; .,. -::(;- ,.:, '" ,",., . .:,; -. ,. ,, .::'.:- - -"' '. ' ,"--- ...., " ,", '. '-:: . .-'. :, ".. ,' " .-,., .'". ,,' . ,. - "..."; ,-. -" ;-. ---:' : ,-;,-;:)- --.;-','. .-: ", . _.'.'j,: ''." '- "','\. , , -'. ", "'" ,'"
- I Itlll :. n i ' '; r pMA21 0
il ft. . :'c !' 1 pMA424
t, . ill : j l r " J" )iJ! P EG2 4 -
'i' ' I:, : .1":' . P EG2 j 1 1 '. pMA4 2 4 N I , I J.
I" ,-1:. P MA21 0 '.'8. 1 ..., 17 Figure 4-2. An activation domain is not required for GAL4 binding to low affinity, nucleosomal sites in vivo. In vivo DMS footpriting was performed on strains harborig the two low afnity site reporter and the GAL4 expression plasmids pMA210 (panel A, lane 2; Panel B, lane 3), pMA424 (panel A, lane 3; Panel B, lane 2) or the negative control plasmid pEG202 (panel A, lane 4; Panel B lane 1). In Panel A protection was analyzed at the two low afnity site reporter locus. In Panel B protection was analyzed at the endogenous GALl, 10 locus. Lanes marked G represent genomic guanine sequencing ladders for the respective loci. Schematics to the side of each panel depict the respective loci; open boxes denote GAL4 binding sites and asterisks denote guanine residues shown previously to be protected by GAL4 bindig. \ ! ! ; ~~~! \ ~~~
155
,;i.-. il pMA210 62 I l did ili \ li ir \ /1
pMA424
1\ i I) ' I;: I !: Ii: / \ i i \;\ i
110
pEG202
! : It
:V \ i ?:: ' .
J."/- ;J:('WM
pMA210 pMA424 pEG202
(1) G449/G450 control 740 678 832
(2) G438/G439 site 3 564 380 981
(3) Normalization factor 1.12 1.23
(4) Adjusted values 632 467 981
(5) % occupancy (100%) ::100% (0%)
Figure 4-3. Phosphorimager analysis of site occupancy by full length GAL4 protein and the truncated GAL4 DNA binding domain. (panel A; previous page). The gel shown in Figul'e 4-2A was scanned on a phosphor- imager and the traces for lanes 2 (PMA210), 3 (PMA424) and 4 (pEG202) are shown. Top to bottom on the gel is depicted as left to right in the traces. Small arrows denote the peak representing the control doublet G449/G450 (numbering of Yocum et al, 1984) and large arrows denote the peaks representing the doublet in site 3 (G438/G439). (panel B). Lines (1) and (2) show the numerical values for the area under the peaks designated in Panel A. For each sample, the values for the control doublet were represented as a fraction of the EG202 control value of 832; this normalzation factor is listed in line (3). This factor was then used to adjust thethe values for the site 3 doublet; the new values are shown lie (4). Line(5) shows the occupancy of site 3 in each strain as a percentage of the occupancy of this site in the gal4- strain (assigned a value of 0% occupancy) and in the SVI + strain (assigned a value of 100%) after subtraction of background. );
GAL4 binding sites in our reporter in the absence of functional SWI/SNF complex.
Unfortunately, many fusion proteins and protein derivatives, includig the GAL4
DNA binding domai derivative used in these studies, are notoriously unstable in
swi/ snf mutants (C. Peterson, unpublished results and Figure 4-4). In order to overcome this setback, we set out to identify transactivation-defective GAL4
derivatives which were stable in a swi/snfmutant.
Table 4- 1 lists the GAL4 expression plasmids tested in these studies. Each
expression plasmid was transformed into the isogenic strains CY296 (SWJ+) and
CY297 (swil- in both of these strains the chromosomal GAL4 gene has been
deleted (gal4i'). GAL4levels in lysates prepared from these transform ants was determined by Western blotting with an antibody that recognzes the DNA bindig domain of GAL4. As shown in Figure, , ful length GAL4 protein
(PMA210) was detected in lysates from both SWI+ and swi- cells (lanes 1 and 2);
the apparent increase in GAL4levels in the swi- sample shown in Figure 4-4 is probably not genuine as it is not seen in other experiments (data not shown and
see Figure 4- 5). Lysates prepared from SWI+ andswi- strains transformed with the GAL4 expression plasmids pGG23 (lanes 5 and 6), pGG23 D199Y (lanes 13 and 14), pCD 14XX (lanes 11 and 12) and the control plasmid pEG202 (lanes 3 and
4) contained no detectable GAL4 protein. The absence of detectable GAL4 however. does not suggest a priori that no GAL4 protein is produced in these cells as in some instances the GAL4 derivative is able to activate transcription of reporter genes (for example pGG23: see Table 4- 1 and see text below). ------.---, -- -- - .------..:,,------.' . -',-'.... ' :j."'-, -.-- +
N ' pMA210
pGG23
;1" pGG23 D199Y
pCD19XX
"'Y'.J' t.
It pMA424
pGG23
pEG202
il- pCP175
- u , pCD14XX
A A Figure 4-4. Western analysis ofGAL4 derivative proteins. Lysates prepared from the yeast strains CY296 (SWI+ and CY297 (swil- transformed with the indicated GAL4 expression plasmids were analyzed by Western blot (see Chapter 2). Numbers to the right denote the migration of protein size standards. NOTE: The samples in lanes 9 11 and 13 were prepared from a swi- strain, contrary to the labels in the figure. Likewise, the samples in lane 10, 12 and 14 were prepared from a SWI+ strain. Lanes 1 through 8 and 15 through 18 are labeled properly. ,,!,:!' ., , ("'~~~
O" C" I' ' iR"ii Ij,'
al ex ression Plasmid Residues 210 UAS Reference
pMA210 888 3562 3320 Gill and Ptashne, 1987
pGG23 238 710 Gill and Ptashne, 1987
pGG23 D199Y 238 Gill and Ptashne, 1987
pCD19XX 644, 848-881 Ma and Ptashne, 1987b
pMA424 147 0.43 Giniger and Ptashne, 1987
pEG202 Gyuris et aI, 1993
pCP175 (pEG50) 147 + AH 420 Giniger and Ptashne, 1987
pCDl4X 792, 848-881 467 Ma and Ptashne, 1987b
Table 4-1. GAL4 expression plasmids used in this study. (a) pEG202 is a negative control plasmid encoding the bacterial protein LEXA.
(b) 13- galactosidase expression was measured in yeast strains harboring the indicated expression plasmid and either
the two low afnity site LacZ reporter (2lo) or a GALl, 10 UAS LacZ reporter (UASGAV' The former were performed in this study, the latter were reported in the reference indicated. n. , not determined Only three of the GAL4 expression plasmids tested in this study produced detectable GAL4 derivatives: lysates prepared from cells transformed with pCD 19XX (lane 7), pMA424 (lane 9) and pCP 175 (lane 15) contained GAL4 proteins with approximate molecular weights of 43 kDa (677 amino acids), 17 kDa
(147 amino acids) and 17.5 kDa (162 amino acids), respectively. Of these, the
GAL4 derivatives produced in the pMA424 and pCP175 transform ants were
unstable in the swi- strain (lanes 10 and 16, respectively). On the other hand, the
GAL4 derivative produced in the pCD 19XX transform ant was present in the swil-
strai (lane 8), indicating it was stable in the swil- strain. This derivative
(hereafer referred to as CD19) was chosen for further analysis.
, To verify that the CD 19 protein was defective in transactivation, liquid 13- galactosidase assays were performed. The strain CY533, which harbors the two low afnity site reporter, was transformed with the plasmid pCD 19 and liquid 13- galactosidase assays were performed on three individual transform ants. shown in Table 4- 1 (column labeled 2lo), the overexpression of full-length GAL4 in this strain led to high levels of trans activation, however, very little activity was seen when CD 19 was overexpressed. Interestingly, the GAL4 derivative encoded by the plasmid pGG23, which was completely undetectable by a Western blot of a cell lysate, was able to activate reporter gene expression to low levels (see Table 4-
1 and F gure 4- , lane 4). Western blots were used to veriy the stabilty of CD 19 in this new strain background and the results are shown in Figure 4- 5. Similar to the fuUlength GAL4 protein (lanes 1 and 2), CD 19 protein levels in the swi ;''' " :!..
ZOZD3d
XX61G;)d
OIZVWd
c::
i; ;' Figure 4-5. The GAL4 derivative CD19 is stable in a swi/snfstrain. Western analysis was performed on yeast strains CY533 (SWI+ and CY535 (swil- that had been transformed with the GAL4 expression plasmids pMA210 (lanes 1 and 2), pCD19XX (lanes 3 and 4), or the control plasmid pEG202 (lanes 5 and 6). These strains carry the two low afity site reporter and a deletion of the endogenous GAL4 gene. Numbers to the right indicate migration of protein size standards. mutant are essentialy unchanged in the isogenic SWI+ strain (lanes 3 and 4). As expected, lysates from strains hal'boring the control plasmid pEG202 , which encodes a bacterial protein unrelated to GAL4, contained no detectable GAL4 protein. These results identify a CD 19 as a transcriptionaly defective yet inherently stable GAL4 derivative. We next used this derivative to test the model presented in Figure 4- 1 and described above.
The abilty of CD19 to bind to the GAL4 sites in the two low afnity site reporter in the presence and absence of SWIISNF was monitored by in vivo DMS footpriting. As shown in Figure 4-6, both CD19 and full length GAL4 protein
(M10) were able to occupy low affnity GAL4 binding sites in the presence of
SWI/SNF (panel A, lanes 3 and 4). In the swi- strain, however, neither protein was able to fully occupy these sitescPanel A, lanes 6 and 7). In agreement with the results presented in Chapter 3, we observe a decrease in protection by the ful
length GAL4 protein in the swi- strain as compared to the SWI+ control. In the experiment shown, GAL4 occupancy was decreased by over 60% (Figure 4-7). A similar decrease (53%) was seen in CD19 binding to the site 3 (Figure 4-7).
Discussion
Workman and Kigston (1992) have shown that the transcriptional activator GAL4 can bind to its sites in a nucleosomal DNA template to form a
tripartite DNAlstone/GAL4 complex. These results indicate that GAL4 may
Figure 4-6. The activation domain mutant CD19 is able to partially bind nucleosomal GAL4 binding sites in the absence of swi/snf. In vivo DMS footprinting analysis was performed on strain CY533 (gal4f1 SWI+ that had been transformed with the GAL4 expression plasmids pMA10 (lane 2), pCD19 (lane 3) or the control plasmid pEG202 (lane 4); the same analysis was carried out on the strain CY535 (gal4f1 swi- that had been transformed with the same plasmids (lanes 5, 6 and 7, respectively). GAL4 binding to the two low afity site reporter locus in these strais is shown. Lane 1 contais a genomic guanine sequencing ladder. Symbols as described in the legend to Figure 4- ' "
SWI + pEG202
SWI+ pMA210
\ i SWI+ pCD19 , Y \i! '! 1 . I
I A \ !,1oJ' t I I, :." '" ' .
" I ! I i '
swi- pMA210
swi- pCD19
II: \ I! ! I J!'1'1 ' ' il 1 iII I 11\I I" !\ i ! I JtV 1\' jl!1 11 \1'1; WI ,... -,
;l.",
SWI+ swr
pEG202 pMA210 pCD19 pMA210 pCD19
(1) G449/G450 control 672 546 323 434 334
(2) G438/G439 site 3 717 232 163 337 233
(3) Normalization factor
(4) Adjusted values 717 278 342 506 466
(5) % occupancy (0%) (100%) 85% 49% 58%
Figure 4-7. Phosphorimager analysis of site occupancy by full length GAL4 and the activation domain mutant CD19. (panels A and B, previous pages) The gel shown in Figure 4-6 was scanned on a phosphorimager and the traces for lanes 2 (pEG202, Panel A), 3 (pMA424, Panel A), 4 (pCDI9, Panel A), 5 (pEG202, Panel B), 6 (pMA424, Panel B) and 7 (pCD19, Panel B) are shown. See legend to Figure 4- for detais. 103
recognize its sites in vivo even when they are encompassed in a nucleosomal
structure. The fact that overexpression of GAL4 protein in vivo leads to increased
occupancy ofnucleosomal sites may indicate that in the presence ofnucleosomes,
GAL4 binds its sites weakly and has a high dissociation rate. Increased local
concentrations of GAL4 protein can partialy compensate for this high off rate and
is demonstrated in our footprinting assay by increased protection (see Chapter 3).
Importantly, we fid that the abilty of increased GAL41evels to support
occupancy ofnucleosomal sites requires the SWIISNF complex. This result
indicates that the SWIISNF complex can further stabilze GAL4 binding, although
the mechanisms by which SWIISNF accomplishes this stabilzation remains
unclear. Based on the biochemical and genetic results summarized in Chapter 1
this stabilzation may involve alteration of histone-DNA contacts within the
underlyig nucleosome, resulting in an increased afity of GAL4 for its site (e.
decrease in the dissociation rate).
The data presented in this chapter indicate that the SWIISNF complex can
:"i' also modulate the bindig of a truncated GAL4 derivative consisting solely of the
DNA binding domai. These results indicate that the mechanism by which the
SWIISNF complex stabiles binding to nucleosomal sites does not require the
activation domai. Furthermore, these results leave unanswered the question of
how theJnere one hundred copies of the SWIISNF complex which are thought to
exist within the typical yeast cell are targeted to the sites in the genome at which
its function is required (Dingwal et al, 1994; Cote et al, 1994; Cains et al1994). 104
Even in the absence of SWI/SNF complex, we detect some binding to the nucleosomal GAL4 sites in our two low afnity site reporter. As shown in Figure
, one possible model for this result is that GAL4 bindig to nucleosomal sites is stabilzed by at least two dierent pathways. In the first, the SWIISNF complex modulates binding via an unknown mechanism. In the second, interactions between the activation domain of GAL4 and its downstream targets
(the general transcription machinery) leads to more stable GAL4 binding. If this model is correct, our previous results suggest that these two pathways are not mutualy exclusive and that in the absence of one, the other continues to stabilze
bindig (we see substantial residual GAL4 bindig in swi/ snf mutant; see
Chapter 3). In the studies presented here, we used a GAL4 activation domai mutant, CD19, to test this model further.
We find that in the presence of SWIISNF, CD19 is able to bind to nucleosomal sites as well as the ful length GAL4 protein does. If the model presented above is correct, CD 19 binding in this case is solely the result of
SWI/SNF modulation of binding, since interactions between the bound activator and its downstream targets are disrupted in this activation domain mutant. the other hand, the activation domain of the fu length protein is able to interact with downstream targets and as a result, its bindig to nucleosomal sites is stabilz by both SWIISNF and activator-PIC interactions. If this is true, then one would predict that in the absence of SWIISNF, ful length protein would retai the binding conferred by interactions between its activation domain and its 105 downstream targets; CD19, however, would be unable to bind to the nucleosomal sites at al as both means of stabilzation have been disrupted. This is not the observed result. Instead, we find that CD19 is able to bind the nucleosomal sites in our two low afnity site reporter almost as well as the ful length GAL4 protein
(Figures 4-6 and 4- 7). These results suggest that the residual GAL4 binding seen
in a swi/ snf strain is not due to the stabiling effect of interactions between the activation domain and downstream transcription machinery and that this residual SWIISNF -independent GAL4 binding to low afity, nucleosomal binding sites reflects intrinsic binding properties of the DNA binding domain. 106
CHAPTER V
CHARACTERIZATION OF THE GAL- PHENOTYE OF swi/snfMUTANTS
.a:i '- Background
Yeast strains bearing mutations in genes encoding components of the
SWIISNF complex display a slow growth phenotype on all types of growth media.
This slow growth is exacerbated on media containing galactose as the sole source
of carbon as well as on media lacking inositol; on these medias swi snf mutants
cannot grow at all (Neigeborn and Carlson, 1984, Peterson, et alI992). These
phenotypes have been attributed to reduced transcription of the genes involved in
galactose and inositol utilization in swi/ snf mutants, in part because the defects
can be suppressed by the same chromatin mutations (e.g. deletion of the HTAl-
HTBI gene set) that suppress the characterized transcriptional defects in swi/snf
mutants (Hirschhorn et alI992).
In the experiments described below , an attempt was made to identify genes
in the galactose utilization pathway (GAL genes) which are dependent upon
SWI/SNF activity for expression. If the inabilty of swi/snfmutants to grow on
galactose: media is indeed due to misexpression of one of the GAL genes, we
expected to uncover an endogenous, SWIISNF-dependent, GAL4-responsive
promoter analogous to the synthetic SWIISNF -dependent two low affinity site 107 reporter we have previously characterized (see Chapters 3 and 4). The GAL4 binding sites of such a gene might reside within a region of positioned nucleosomes such that GAL4 cannot occupy the sites in the absence of SWIISNF.
The correlation of SWIISNF -dependence with the nucleosomal state of promoter
binding sites in the promoter region of an endogenous, SWIISNF- dependent GAL gene, would provide strong support for our model.
Results
As shown in Table 5- swil- cells display a growth defect on both glucose- and galactose-containing media. This growth defect is well documented
(Neigeborn and Carlson, 1984; Peterson et al, 1991) and is identical to that
observed in other swilsnfmutants (including swi2, swi3, snf5, snf6 and swp73).
Hirschhorn et al (1992) have shown that this growth defect is exacerbated on galactose plates containing antimycin. In the presence of this electron transport chain inhibitor, yeast cells are forced to rely on glycolysis to provide the energy for cell growth, as the energy normally afforded by oxidative metabolism is disrupted.
In the experiment shown in Figure 5- , a swiL1 strain (CY257) and its isogenic
SWI+ counterpart (CY340) were streaked onto agar plates containing the drug
antimycin. While the SWI+ cells grow at similar rates on glucose-containing
(panel A)' and galactose-containing (panel B) media in the presence of antimycin
the swil- cells are unable to grow at all on galactose plates containing antimycin
(panel B). The swi- cells do grow on glucose-containing media in the presence of - - +++++++
108
Growth
Media SWI+ swi-
YPD
YPD + antimycin YPGal
YPGal + antimycin
Table 5-1. Growth defects of swVsnf mutants. Growth on the indicated media is designated by "+" symbols llo growth is designated by " symbols. ":' , "
Glucose + antimycin
GalaCtose + antimycin
'0; '-'.y.
110
Figure 5- 1. swVsnfmutants display growth phenotypes on antimycin- containing media. SWI+ and swi- yeast strains were streaked onto plates containing the drug antimycin and either glucose (Panel A) or galactose (Panel B) as the sole carbon source. Schematics to the right indicate the genotypes of the strains shown in the plates on the left.
''1
,ji
'J' 111 antimycin (panel A). This sensitivity to antimycin under anaerobic conditions has been used by others to define the Gal- phenotype of swil snf mutants
(Hirschhorn et al, 1992; Cairns et al, 1996a).
In order to identify the gene(s) responsible for the Gal- phenotype of the
GAL gene family members swil- strain CY257 , expression of each of the known Primer was monitored in SWI+ and swi- cells grown under inducing conditions.
extension analysis (Figure 5- 2) was performed on an equal amount of total RNA
and CY257 using primers prepared from the yeast strains CY340 (SWI+ (swil-
GAL2 (lane 5 and 6), specific for the coding sequence of GALl (lanes 3 and 4),
GALBO (lanes 11 and 12), MELI GAL 7 (lanes 7 and 8), GALlO (lanes 9 and 10),
GAL3 (lanes 15 and 16), and GAL5 (lanes 17 and 18). We find (lanes 13 and 14),
, GALBO and MELI transcripts are that the levels of GALl, GAL2, GAL GALlO
not substantially altered in the swil- strain when compared to the levels detected
from the CLN3 gene, which in the respective SWI+ strains. Likewise, transcripts
encodes a mitotic cyclin that is not involved in galactose utilization, are unaltered
in the swil mutant. However, we do detect a consistent decrease in the level of
transcription of the GAL3 and GAL5 genes (compare lane 15 to lane 16 and lane
17 to lane 18). is related To verify that the GAL- phenotype displayed by swilsnfmutants
or GAL5, to the mJsexpression of either GAL3 complementation analyses were
(CY340) and swil- (CY257) carried out (Table 5- 2). In these experiments the SWI+
strains described above were transformed with plasmids directing the expression :;; . .-- ,.. .. , . .:;j :;. - . .. --'- - - ,-- . -
S'IVD 11,
",0 f'IVD
08'IVfl 1:, onvD
L'IVD
nVD
nVD
fN'I :.',
113
Figure 5-2. Analysis of GAL gene transcription in S"W+ and swi- yeast.
Primer extension reactions were performed on total RNA isolated from the SWI+
yeast strain CY340 and the swil- strain CY257 using primers designed to measure the level of transcripts for each of the indicated genes.
:i\ +++
Glucose + antim cin Galactose + antim cin Plasmid or Genot SWI swi- SWI swi-
(a) pMA210 (GAL4)
(b) pTI-3B (GAL5)
(c) pRS426
(d) pGAL3HA (GAL3)
(e) pVTI02UHA
(f) ga180
Table 5-2. Complementation studies. (a-e) SWI+ and swi- yeast strains transformed with the indicated
SWI+ and swi- yeast strains plasmid were streaked onto glucose or galactose medium containing antimycin. (f) were constructed in which the chromosomal GAL80 gene was deleted. These strais were streaked as above. Growth is indicated by "+" symbols, no growth is indicated by " symbols. , -
115
of either GAL5 (line 2; pTI- , gift of D.M. Bedwell, University of Alabama,
Birmingham), GAL3 (line 4; pGAL3HA, gift of Toshio Fukasawa, Kazusa DNA
Research Institute, Japan) or the corresponding control plasmids pRS426 (line 3)
and p VTI02UHA (line 5), respectively. As expected , the SWI+ transform ants
were able to grow on both glucose- and galactose-containing media in the presence
of antimycin (Table 5-2). On the other hand , none of the swil- transformants was able to grow on galactose containing media in the presence of antimycin (Table 5-
2), although they could grow on glucose-containing media in the presence of
antimycin. Overexpression of GAL3 seemed to partially suppress the growth
defect of the swi- strain (Table 5- , line 2), although it did not suppress antimycin
sensitivity.
GAL3 encodes an inducer molecule which is believed to interact with the
GAL80 protein in the presence of galactose (i.e. inducing conditions). This
interaction is thought to alter the interaction between GAL80 and the
transcriptional activator GAL4 such that the latter can now activate the
expression of downstream genes (Suzuki-Fujimoto et aI, 1996). If the Gal defect of swilsnfmutants is indeed due to the inability of these cells to express GAL3 properly, then it is possible that this defect would be overcome by a corresponding
decrease in the expression of GAL80. To test this idea , strains CY349 (gal80L1)
and CY348 (gal80L1, swilL1) were streaked onto glucose and galactose plates containing antimycin and growth after two days at 30 C was monitored. As - .
116
shown in Table 5- , the deletion of the GAL80 gene did not suppress the Gal- I;::
phenotype of the swil- strain. If,
Discussion
The results described above suggest that the Gal defect observed in swil snf
mutants is not simply the result of a transcriptional defect of a single member of
the GAL gene family. While the expression of two GAL genes GAL3 and GAL5
was significantly altered in a swil- strain, the presence of the respective
overexpression plasmid was not able to suppress the Gal defect. It is possible
however, that the Gal- phenotype is due to the combined misexpression of both
GAL3 and GAL5. This model remains to be tested.
The expression plasmid used in the GAL3 complementation experiment
pGAL3HA , consists of the entire GAL3 coding sequence cloned into an ADH2 expression cassette and has been shown to result in the constitutive
overexpression of HA-tagged GAL3 protein (Suzuki-Fujimoto et al, 1996).
However, in its endogenous context , the expression of the ADH2 gene is known to
require SWIISNF, suggesting this promoter is SWI/SNF-dependent. Therefore
the pGAL3HA plasmid may not express the levels of GAL3 that are necessary to
overcome the Gal defect. In addition , the construct used in the GAL5
complementation experiment is a genomic GAL5 clone; the expression of GAL5
this plasmid is controlled by the endogenous GAL5 promoter. Therefore
expression of GAL5 from this plasmid wil be subject to the same constraints as 117
the endogenous GAL5 gene, including SWIISNF- IfF dependence. It should be noted,
however, that both pGAL3HA and pTI-3B are high copy number constructs and
therefore the transformants tested in Table 5-2 should have a copy number 10-
times higher than th nontransformed strain.
We consistently observed small (less than two-fold) decreases in the
expression of all the other GAL genes in swil mutants (see Figure 5-2). It is
possible that the Gal defect results from the combination of these small
transcriptional defects, or from the cumulative defects in the expression of a
unique combination of GAL genes. In either event, our results do indicate that a
single gene is not responsible for the Gal defect observed in swilsnfmutants. ,,/,-j"j' :..
118 :1,
CHAPTER VI
SUMMARY
The yeast SWIISNF complex is emerging as a paradigm for a growing class
of chromatin remodeling assemblies that appear to allow a spectrum of DNA
binding proteins access to the eukaryotic genome. Biochemical experiments
utilizing SWIISNF purified from yeast indicate the complex can modulate the
ability of DNA binding proteins to access nucleosomal binding sites (Cote et aI
1994; see also Kwon et al, 1994; Imbalzano et aI, 1994). Furthermore, the
complex can induce a persistent alteration specifically in that nucleosome within
an array which occludes a protein-DNA interaction (Owen-Hughes et aI, 1996).
More recent results have suggested that the complex may recognize the region of
the nucleosome where the DNA strand crosses over itself to form a four-way
junction-like structure (Quinn et al, 1996). This idea is supported by crosslinking
experiments which demonstrate that certain subunits of the purified complex can
interact closely with specific regions of a nucleosome in vitro and that the
specificity of these interactions is sensitive to the presence of A TP (B.
Bartholemew and C.L. Peterson, unpublished observations). All of this
biochemistry is evidence that the mechanism of SWI/SNF function involves direct -.. g.
119
physical contact with nucleosomes, presumably followed by alterations in histone-
DNA contacts within the nucleosome such that the DNA is more accessible.
Despite rapid progress in understanding the function of purified SWIISNF
complex in vitro, many intriguing questions regarding the function of the complex
in vivo remain unanswered. For example, what dictates the SWIISNF-dependence
of gene expression? How is this relatively low abundance complex targeted to
specific sites in the genome? The work presented in this dissertation addresses
these questions by establishing the molecular basis for SWIISNF function at the
yeast GALl lO promoter. We were able to change this well characterized
SWIISNF-independent promoter into one completely dependent upon the
SWI/SNF complex for proper expression. We find that the SWIISNF-dependence
of GALl lO derivative promoters correlates with the nucleosome state of activator
binding sites in the UAS region, indicating that the role of the SWI/SNF complex
at these promoters is to modulate activator binding to nucleosomal binding sites.
These results complement the biochemical experiments described above and
provide the first evidence that the SWI/SNF complex can modulate activator
binding in vivo.
It is not clear whether the rules for SWIISNF-dependence established here
are applicable to all yeast promoters. Existing data argues that they are not, as
yeast promoters-that contain nucleosomal activator binding sites but are
expressed independently of SWI/SNF have been characterized (e. PH05; see
previous discussion). Instead, SWIISNF-dependence may reflect a combination of 120
nucleosome structure and the intrinsic binding properties of a given activator protein. For example, activators like GAL4, which are unable to stably occupy nucleosomal sites in vivo, wil require SWIISNF activity. In contrast, other
activators may bind nucleosomal sites in vivo without SWI/SNF modulation.
Furthermore, the SWIISNF-dependence of a particular activator may vary depending on the individual promoter binding site. For example, we find
SWIISNF is required for complete occupancy of the two low affnity nucleosomal
GAL4 sites used in our studies but not for binding to other nucleosomal sites which differ in binding sequence.
Our studies also address the targeting question. We find that the ability of
SWIISNF to modulate activator binding does not require a functional activation domain. These results suggest that either the SWIISNF complex is targeted to its site of action solely through the DNA binding domain of the activator, or that it is
not targeted at all. In the former scenario SWI/SNF could recognize some specific feature of the DNA binding domain or, alternatively, the complex might recognize proteins which are loosely associated with their binding sites within the context of nucleosomal DNA. On the other hand, it is possible that SWIISNF interacts with the genome continuously, in a random fashion, such that targeting is not necessary. In this situation, coincidental occupation of a nucleosomal promoter region by SWIISNF and an activator protein would lead to productive activator binding (and eventually to transactivation). This latter model seems unfavorable
in light of the low abundance of SWI/SNF in yeast (estimated 100 copies per 121
nucleus). Interestingly, the relative abundance of SWIISNF complex in human
cells is much higher than in yeast.
Finally, there has been much controversy recently over the observation of
Wilson et al (1996) that the yeast SWI/SNF complex is a component SRB-
containing mediator complex and therefore of the RNAPII holoenzyme itself.
These authors theorize that holoenzyme recruitment, which has been shown to result in transcriptional activation (reveiwed in Ptashne and Gann, 1997) would bring SWIISNF to the promoter where it could then alter chromatin structure.
However, Cairns et al (1996b) were unable to detect an association between
SWIISNF and holoenzyme complexes.
The results presented in Chapter IV of this thesis address this issue. Our results indicate that a GAL4 derivative lacking an activation domain (GAL4 DNA binding domain construct MA424) remains sensitive to SWI/SNF activity, suggesting that this complex is able to reach the promoter independently of holoenzyme. Furthermore, while we do see some co-purification of SRB subunits with SWIISNF complex in our preparations, the amounts represent less than 20% of the SRB subunits in a yeast lysate (L. Boyer and C.L. Peterson, unpublished observations). Taken together, these data suggest that the SWIISNF complex is physically and functionally independent of holoenzyme. It remains possible that the complexes are able to interact in vivo, either directly or indirectly, and that this association results in small amounts of co-purification. 122
Finally, recent studies from a number of groups have verified the importance of protein acetylation/deacetylation in transcription (reviewed in
Pazin and Kadonaga, 1997). Interestingly, some of the gene products found to be involved in histone ac;etylation also interact genetically with the SWIISNF complex (K. Pollard and C. L. Peterson), suggesting that SWIISNF may playa direct or regulatory role in this aspect of transcription regulation. The strains and techniques developed in this thesis will allow a careful analysis of the role of these other factors in transcription factor binding, transactivation and SWIISNF regulation in vivo. 123
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APPENDIX A
DETAILED PROTOCOLS
Isolation of yeast 'nuclei The following protocol was obtained from Randall H. Morse (Molecular Genetic Program, Wadsworth Center, New York State Department of Health and SUNY School of Public Health, Albany, NY 12201-2002) and is based on a procedure published in Shimizu et al (1991) EMBO J. 10, 3033-3041.
1. Grow 1 L of cells to OD600 of 0. 1.4 (The procedure can be scaled up, e. g. grow 4 L of cells and multiply all reagents used by 4.
2. Harvest cells by centrifugation for fifteen minutes at 4K rpm in a J6 rotor.
3. Wash pelleted cells in 50 ml of S Buffer. Spin as above for five minutes.
4. Incubate cells in 25 mL S Buffer at 30 C for ten minutes. This can be done in a shaking incubator (20 rpm) or in a standing incubator. Spin cells as above for five minutes.
5. Drain pellet and weigh. Add volume of S Buffer equal to four times the weight of the pellet (for example, 10 ml to a 2. 5 g pellet).
6. Digest cells with 1 ml10 mg/mll00T Zymolyase.
7. Incubate 30-90 minutes at 30 C with gentle shaking (60 rpm) until cells are well spheroplasted. This can be monitored by lysing the cells in 0. 1% SDS under a microscope; if cells are well digested ::90% of them should lyse under these conditions. I have found that 45 minutes is usually sufficient.
Remaining steps should be carried out in the cold using chilled buffers.
8. Dilute sample to 30 ml with S Buffer. Spin five minutes at 4K.
9. Wash spheroplasts twice more in cold S Buffer, about 30 mL each time. The supernatant from these washes will be progressively cloudy. 136
10. After the second wash, resuspend the pellet in 19 ml F Buffer. Mechanically disrupt the spheroplasts by passing through a homogenizer. I found four passes through a Yamata LH-21 homogenizer set at 100 rpm provided sufficient disruption. Disruption can be monitored under a microscope. Dilute the sample enough to see individual cells -- there should be a lot of smallish debris visible and very few intact, live cells.
11. Layer homogonized sample onto 19 ml ofGF Buffer in an Oak Ridge tube. Spin 13.5K rpm (21 625 X g) in a JA20 rotor for 30 minutes at 4 C. Note: You will need glycerol in a balance tube to achieve sufficient density to balance!
12. Discard supernatant. Resuspend pellet in 20 ml of F Buffer. Mechanically resuspend pellet with a wooden stick to dislodge it from the bottom of the tube. Vortex for five minutes at 4 C to thoroughly resuspend the pellet.
13. Spin sample for fifteen minutes at 4K rpm in J6 rotor at 4
14. Transfer supernatant to a fresh tube. Spin at 13. 5K rpm (21 625 X g) in a JA20 rotor for 25 minutes at 4 C to isolate nuclei. Nuclei should be a white pellet, fairly large and homogeneous looking.
15. Discard supernatant and drain nuclei pellet.
16. Resuspend nuclei pellet in 4 ml of D buffer (to make a final concentration of approximately 6 X cell equivalents/ml). Quick freeze 1 ml aliquots in eppendorf tubes in liquid nitrogen and store at - 137
Solutions
S Buffer 1.4 M Sorbitol 40 mM HEPES pH 7. 5 mM MgCb 10 mM B-mercaptoethanol (added fresh) ImM PMSF (added fresh)
F Buffer 18% w/v Ficoll 400 20 mM PIPES pH 6. 5 mM MgCb 1 mM PMSF (added fresh)
GF Buffer 20% w/v glycerol
7% w/v Fico1l400 20 mM PIPES pH 6. 5 mM MgCb 1 mM PMSF (added fresh)
D Buffer 10 mM HEPES pH 7. 5 mMMgCb 05 mM CaCb 138 i , In vivo DMS footprinting
The following protocol is based on that of Giniger et al (1985 Cell 767-774. ) and a detailed protocol received from Jeff Axelrod a supplement as NAR to the protocols in Axelrod and Majors (1989 : 171- 183.) and Axelrod et al (1993, G&D 1: 857-869).
I. PREPARTION' OF METHYTED GENOMIC DNA
1. Grow 100 ml yeast culture in appropriate rich medium to an OD600 of 1.5- , corresponding to a density of 5 x 10 7 cells/ml. Cells grown in minimal medium should be grown to an OD600 between 0.5 and 0.75. Minimal cultures grown over 0.75 are saturated and consistantly demonstrate poor GAL4 footprinting.
2. Pellet at 4K for 7 minutes.
3. Resuspend in 1.5 ml of the same growth medium. Incubate at room temperature for five minutes.
4. Add 2J.l DMS and vortex well. Incubate two minutes at room temperature.
Note: Use only fresh DMS (less than 4 months old) Also, DMS is nasty stuff. Have a beaker with 100 m15N NaOH (20g NaOH pellets/l00 ml dH20) ready to inactivate DMS tips and waste.
5. Quench reaction with 40 ml of ice cold TEN.
6. Pellet at 4K for 7 minutes.
7. Wash pellet in 1 ml SCED. Spin as above.
8. Resuspend pellet in 1 ml SCED. Transfer to a 1.5 ml Eppendorftube and add 100 J.II0 mg/mll00 T Zymolyase (Seikagaku Corporation; DO NOT USE LESS PURE THAN lOOT!). Invert tubes to mix well.
9. Incubate at 37 C for 25 minutes. Monitor spheroplasting under microscope. When spheroplasting is complete, you should see ::90% lysis when water or 0. 1% SDS is added to spheroplasts.
10. Pellet spheroplasts for 1 minute. Decant supernatant. 139
11. Resuspend spheroplasts in 1 ml of 50mM Tris pH 8. 20 mM EDTA. Resuspension into a homogeneous solution will require careful repeated pipetting. Transfer half of the suspension to a second Eppendorf tube.
12 Lyse spheroplasts by adding 50 J.II0% SDS to each tube. Incubate at C for 30 minutes.
13. Add 3J.l 20mg/ml Proteinase K, mix by inversion and incubate 30 minutes more at 37
14. Add 200 J.l 5M potassium acetate pH 8.0 to each sample and incubate on ice for at least one hour.
15. Spin samples 5 minutes at room temperature. Transfer supernatant to fresh tubes, spin again for 5 minutes at room temperature, transfer supernatant to another fresh tube and preciptate with 700 J.l isopropanol.
16. Pellet nucleic acid in microfuge (3-5 min), rinse pellets with 70% ethanol and air dry approximately 10 minutes.
17. Resuspend pellets in 300 J.l TE. Add 30 J.g RNAse (DNAse-free) to each sample and incubate 37 C for one hour. Pellets may not resuspend readily, but will go into solution after incubation at 37 C with a little intermittent vortexing.
18. Combine samples into one tube. Spin for two minutes on highest setting to remove insoluble debris. Transfer supernatant to a fresh tube.
19. Precipitate DNA by adding 5. 5 ullM spermidine, pH 4. 0. Flick the tube after spermidine addition. A white, threadlike precipitate should form. This may require another 5.5 J.l aliquot of spermidine (final spermidine concentration should be 3-9 mM). Incubate on ice for at least fifteen minutes.
Note: Preciptation of DNA by spermidine is critical to the primer extension assay later. Use a 1M stock of spermidine, pH 4. THE PH OF THE SPERMIDINE IS CRITICAL!
19. Pellet DNA by centrifugation for two minutes. It is a good idea to check the efficiency of the spermidine precipitation on an agarose gel. I typically see 80-90% of the DNA in the pellet. ,!;.;-
d"' 140
20. Resuspend the spermidine pellet in 300 J.l O. IM Tris pHS.O. Incubate at C for two hours with some vortexing. It wil take almost this entire incubation for the pellet to dissolve.
21. Add 165 III 7. 5M ammonium acetate and 1 ml ethanol. Chil on dry ice for 15 minutes. Spin, wash and dry pellet.
22. Resuspend pellet in 100 J.l dH20. Addition of salt will help the dissolution process, so add the salt required of the digestion reaction and incubate at 37 C for a few minutes until the pellet dissolves readily upon vortexing. I typically set up a 200 J.l digestion reaction using the entire sample.
Note: The digestion of the genomic DNA into smaller fragments will facilitate the primer extension reaction. Choose an enzyme which cleaves outside the region you are footprinting, including the primer binding site.
23. Extract the digestion with phenol/chloroform/isoamyl alcohol and precipitate with 1/10 volume 3M sodium acetate pH 5.2 and 2. 5 volumes of ethanol.
24. Spin, wash and dry pellet. Resuspend in 15 J.l dH20; this will give approximately 1J.g/J.l.
NOTE: I have been able to get very clean, reproducible footprints without using a piperidine cleavage step. (In fact, I have had trouble with high background when I use piperidine.) Therefore, proceed directly to the primer extension step.
II. PRIMER EXTENSION
1. Set up primer extension reactions in 0. 5 ml eppendorftubes:
5 J.l methylated, restricted DNA (from step I) 5 J.l 500 J.M dNTP Mix 5 J.l5X Taq Buffer 3 J.l end-labelled oligonucleotide (3ng) 5 J.l (2.5 Units) Sequencing grade Taq Polymerase* (Promega) dH20 to make a 25 J.l reaction
Note: This is the only polymerase that has worked for me. 141
2. To make a guanine ladder, use the same amount ofUNMETHYLATED DNA (isolated exactly as described above, simply omit DMS treatment) and instead of using the 500 J.M dNTP mix, use 5J.l of 5X G Mix in the primer extension reaction.
3. Overlay samples with 50J.l mineral oil and cycle for two minutes each at C/(T m + 2 C)170 C 19 times.
Note: To determine appropriate T m, use the formula below:
Tm = 81.5 + 1.6. (10g M) + 0.41(%GC) - 500/n
M = molarity of salt in buffer, here 0.059 M n = length of oligonucleotide
5. When cycling is finished, add 75 J.l dH20 to each sample and extract with 100 J.l chloroform/isoamyl alcohol. Precipitate with 5 J.l3M sodium acetate pH 5.2 and 165 J.l ethanol. 6. 10Collect sampleminutes. by centrifugation, wash pellet with 70% ethanol and air dry
Note: Do not overdry pellet or it wil be harder to resuspend!
7. Resuspend pellet in 5 J.l Loading Buffer. Vortex well and check resuspension (-c20% of the counts remain in the tube when the sample liquid is removed).
8. Boil samples 5 minutes, remove to ice, and load entire sample on a denaturing gel. Use a 0.4 mm thick gel to increase resolution. With this thickness, low percentage gel can be used, I usually pour a 5% gel.
III. PREPARING A SEQUENCING LADER
While the primer extension reactions are cycling, I usually prepare sequencing markers. This ladder is not necessary if you make a genomic G ladder when you do the primer extension reaction (see above). This is a modification of the Sequenase protocol and uses the same reagents.
1. Add 1 J.l (lJ.g) plasmid DNA and 19 J.l dH20 to an eppendorftube.
2. Add 2 J.12N NaOH and incubate 5 minutes at roo temperature. 142
"I,.3:
iF"' 3. Precipitate with 3 J.l 3M sodium acetate pH 5.2 and 75 J.l ethanol.
4. Chill on dry ice 10 minutes, spin, wash and dry pellet.
5. Resuspend pellet in 10 J.12X Sequenase Buffer. Add 3 J.l end-labelled oligo and 7 J.l dH20:
6. Boil 1 minute and incubate at 37 C for 10 minutes.
7. Spin briefly. Aliquot 4. 5 J.l to each of four tubes containing 3.2 J.l ddNTP mix. Add 1 J.l of diluted SequenaseCI (1:8 in Enzyme Dilution Buffer) to each tube and incubate at 37 C for 5 minutes.
8. Ethanol precipitate with 1 J.13M sodium acetate, pH 5.2 and 30 J.l ethanol. Chill on dry ice and spin. Wash and dry pellet and resuspend as above for primer extension samples. 143
Solutions I
TEN 10 mM Tris pH 8. 1 mM EDTA pH 8. 40mM NaCl
SCED 1 M Sorbitol 100 mM Sodium Citrate pH 5. 10 mM EDTA 2 mM DTT
Solutions II
5X Taq Buffer 250 mM Kcl 50 mM Tris pH 8. 12.5 mM MgCh 850 ug/ml BSA
5X G Mix 1 mM ddCTP 25 J.M dCTP 5 mM dATP 5 mM dTTP 5 mM dGTP
Loading Buffer 95% formamide 20 mM EDTA 05% Bromophenol Blue 05% Xylene Cyanol FF
Solutions In
5X Sequenase Buffer 200 mM Tris-HCl, pH 7. 100 mM MgC12 250 mM NaCl
Enzyme Dilution Buffer 10 mM Tris-HCl, pH 7. 5 mM DTT 5 mg/ml BSA --.
I:. 144
Indirect end-labelling
I. PREPARATION OF FREE DNA
1. Thaw two frozen 1 ml aliquots of nuclei at room temperature.
2. Add 80 J.l10% SDS and 30J.l20 mg/ml Proteinase K to each.
3. Incubate at 37 C for two hours.
4. Add 180 J.I 5 M potassium acetate and incubate on ice for one hour.
5. Spin samples at room temperature in microfuge for 10 minutes. Transfer supernatant to a fresh tube, repeat if necessary (sometimes it is impossible to avoid the pellet after the first spin).
6. Precipitate supernatant with an equal volume of isopropanol.
7. Resuspend the washed pellet in 300 J.l water and reprecipitate.
Note: Do NOT overdry the pellet or it wil be impossible to resuspend.
8. Resuspend the washed pellet in 1 ml of Buffer D2
Note: Free DNA must be resuspended in a buffer containing high cation concentrations to compensate for the loss of these ions due to coating of the phosphate backbone.
II. MNASE DIGESTION
1. Thaw 2 frozen 1 ml aliquots of nuclei at room temperature.
2. Aliquot 325J.l into six eppendorftubes. Do the same for the Free DNA samples from above.
3. Dilute MNase stock to 5 Units/J.l with Buffer D. Serial dilute this 5 Units/J.l stock in Buffer D to make 0. , 0.05 and 0.005 Units/J.l.
4. Prewarm sample tubes at 37 C. Digest with MNase (0 to 50 Units) for five minutes at 37 ..-
145
5. Stop reactions with 26 J.II0% SDS and 10 J.120 mg/ml Proteinase K. 1'' Incubate samples at 37 C for two hours.
6. Add 60 J.l5 M potassium acetate, mix well and incubate samples for at least one hour on ice.
7. Spin samples in' a microfuge for ten minutes at room temperature. Transfer supernatant to a fresh tube and precipitate with 350 J.l isopropanol.
8. Spin, wash and dry pellets. Resuspend in 150 J.I TE pH 8.0. Add 5 J.g RNase and incubate at 37 C for 30-60 minutes.
9. Precipitate DNA with 2.25 J.ll M spermidine pH 4.0. Incubate on ice for at least 15 minutes.
10. Pellet the DNA and resuspend in 150 J.I O. IM Tris pH 8. 0. Incubate at C until pellets are easily resuspended upon vortexing (10-30 minutes).
11. Precipitate DNA with 82. 5 J.l 7.5 M ammonium acetate and 500 J.l ethanoL
12. Spin, wash and dry pellets. Resuspend in 15 J.l of sterile water.
III. SOUTHERN ANALYSIS
1. Electrophorese samples on a 1.5% agarose gel overnight (longest gel size).
2. Soak gel in 0.25% HCL
3. Rinse gel in sterile water.
4. Soak gel in Southern Solution D for 30 minutes at room temperature with gentle agitation.
5. Rinse gel once in Southern Solution N and then soak in this solution for 15 minutes at room temperature with gentle agitation. Discard solution and soak gel in fresh Southern Solution N for 15 more minutes as above.
6. Transfer DNA to nylon membrane (QuiabraneQY) by capilary action (see Maniatis). Transfer should be carried out in 20X SSC for at least 12 hours.
7. When transfer is complete, mark origin in pencil on membrane. Autocrosslink DNA to the membrane (StratalinkerQY) and air dry 15 minutes 146 at room temperature. If necessary, membrane can be stored between two pieces of 3MM filter paper at 4
8. Pre-hybridize membrane for 1-4 hours at 42 C in freshly prepared Prehybridization Solution.
9. Hybridize membrane for at least 12 hours at 42 C in 5 ml Hybridization Solution containing lX10 cpm/mllabelled DNA probe. Be sure to denature double stranded DNA probes by boiling for 5- 10 minutes before use.
10. Wash membrane twice for five minutes at room temperature in 2 X SSC, 1 % SDS.
11. Wash membrane in O. IX SSC, 0. 1% SDS for 15 minutes at hybridization temperature (or higher if necessary). Repeat if necessary. 147
Solutions I
Buffer D2 10 mM HEPES pH 7. 5 mM MgCh 2 mM CaCh
Solutions II
MN ase stock 15 U nits/ml in Digestion Buffer
Digestion Buffer
Buffer D 10 mM HEPES pH 7. 5 mM MgCh 05 mM CaCh
TE pH 8. 10 mM Tris-HCl pH 8. 1 mM EDTA pH 8.
Solutions III
1.5% agarose gel 1.5% agarose in IX TBE (0.089 M Tris base, 0.089 M boric acid, 20 mM EDTA pH 8.
Southern Solution D 0.4 N NaOH 6 M NaCl
Southern Solution N 1.5 M NaCl 5 M Tris-HCl pH 7.
20X SSC 3 M NaCl 3 M sodium citrate Adjust pH to 7.0 with 1 M HCl
Pre-hybridization Solution 6X SSC 50% formamide 5X Denhardt's Solution 50 mM sodium phosphate pH 6. 100 J.g/ml salmon sperm DNA 5% SDS
Hybridization Solution 5X SSC 50% formamide 148 II; ir. IX Denhardt's Solution 50 mM sodium phosphate pH 6. 100 J.g/ml salmon sperm DNA 1% SDS
100X Denhardt's Solution 2% Ficoll 400 2% polyvinylpyrrolidone 2% BSA ---- -_. - -
149
Nystatin permeabilization of spheroplasts This protocol is based on that published in Venditti and Camilloni
(1994 , Mol. Gen. Genet. 242: 100- 104.) and that worked out by Monique Mukrit at UMMC.
1. Grow 200 ml of yeast culture in YEPD.
2. Harvest cells by centrifugation for ten minutes at 4K rpm in a J6 rotor.
3. Resuspend cell pellet in 20 ml Pre-spheroplasting Buffer and add 86 J.l B- mercaptoethanol to make a final concentration of 60 mM.
4. Incubate cells at room temperature for ten minutes.
5. Spin cells as above.
6. Resuspend pellet in 10 ml Spheroplasting Buffer and add 7. 2 J.l B- mercaptoethanol to make a final concentration of 10 mM.
7. Remove 20 J.l of cell suspension, mix with 980 J.l dH20, and record OD6oo.
8. To the remainder, add 200 J.l recombinant lyticase.
9. Incubate at 37 C until OD6oo is decreased by 90% (usually 10- 15 minutes).
10. Spin spheroplasts at 4K rpm for ten minutes in the cold.
11. Wash spheroplasts in 750 J.l Nystatin Buffer.
12. Resuspend washed spheroplasts in 1.5 ml Nystatin Buffer.
13. Add 30 J.l 10 mg/ml nystatin.
14. Aliquot 250 J.l of cell suspension to six eppendorf tubes.
15. Permeabilized spheroplasts can now be digested as desired, i.e. with MNase (for titration or indirect end-labeling experiments) or with restriction enzymes (for accessibility assays).
16. To stop digestion, add 1/10 volume of SDS/EDTA Stop Solution to each sample. 150
Solutions
Pre-spheroplasting Buffer 100 mM Tris-HCl, pH 7.
Spheroplasting Buffer 7 M sorbitol 75% yeast extract 1.5% peptone 10 mM Tris-HCl, pH 7.
Nystatin Buffer 50 mM NaCl 1.5 mM CaC12 20 mM Tris-HCl, pH 8. 1 M sorbitol
SDS/EDTA Stop Solution 10 % SDS 50 mM EDTA i'1 151
APPENDIX B
RELATED EXPERIMENTS
The preprint included in this appendix contains the restriction enzyme accessibility assay experiments described in Chapter 3 (see p. 78). This manuscript is scheduled for publication in the August 1997 issue of Molecuar
and Cellular Biology. While the experiments in question were not performed by the author of this thesis, the results are directly relevant to the work presented herein.
:i;' Vol. 17, No. iOLECULA AND CELLULAR BIOLOGY, Aug. 1997, p. 4811-4819 0270-7306/97/$04.00+0 'Copyright (Q 1997 , American Society for Microbiology
The Yeast SWI -SNF Complex Facilitates Binding of a Transcriptional Activator to Nuc1eosomal Sites In Vivo LOREE GRIFFIN BURNS AND CRAIG L. PETERSON* Program in Molecular Medicine and Department of Biochemistr and Molecular Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01605
Received 2 January 1997/Returned for modification 18 February 1997/Accepted 24 April 1997
The Saccharomyces cerevisiae SWI-SNF complex is a 2-MDa protein assembly that is required for the function of many transcriptional activators. Here we describe experiments on the role of the SWI-SNF complex in activation of transcription by the yeast activator GAL. We find that while SWI-SNF activity is not required for the GAL4 activator to bind to and activate transcription from nucleosome-free binding sites, the complex is required for GAL to bind to and function at low-affnity, nucleosomal binding sites in vivo. This SWI-SNF dependence can be overcome by (i) replacing the low-affnity sites with higher-affnity, consensus GAL binding sequences or (ii) placing the low-affnity sites into a nucleosome-free region. These results define the criteria for the SWI- SNF dependence of gene expression and provide the first in vivo evidence that the SWI-SNF complex can regulate gene expression by modulating the DNA binding of an upstream activator protein.
The SWI-SNF complex is required for the expression of a to facilitate the binding of a transcriptional activator to up- number of diversely regulated genes in the yeast Saccharomy- stream activation sites (UASs). To do this, we monitored the ces cerevisiae. The results of genetic experiments indicate that transcriptional activity and DNA binding of the yeast activator the SWI -SNF requirement reflects the ability of the complex to GAl to derivatives of the UAS of the divergently transcribed antagonize chromatin-mediated transcriptional repression GALl and GALlO genes. These UAS derivatives differ in the (26). In these studies, mutations in genes that encode chroma- affnity of the GAl binding sites as well as the nucleosomal ti components , including HHT1 (encoding histone H3), context of these sites. We find that the SWI-SNF complex is HHFl (encoding histone H4), and SIN1 (encoding an HMG- required for GAl to activate transcription from a promoter like protein which is thought to be a chromatin component), containing two low-affnity, nucleosomal binding sites in vivo. were found to alleviate the transcriptional defects of swi mu- In addition, we show that the SWI-SNF dependence of this tants (14, 15). In addition, alteration of relative histone levels promoter reflects, in part, the ability of the complex to facili- by deletion of one of the two endogenous HTA1-HTB1 gene tate GAl binding to these nucleosomal sites. clusters (encoding histones H2A and H2B) is also able to partially restore transcriptional activity to swi-snf cells (12). MATERIS AND METHODS
These results suggest that the SWI-SNF complex acts to pro- (3-Galactosidase assays. Strains were grown in minimal medium containing mote the expression of genes within the context of a compact 2% galactose, 0,5% sucrose, and all the amino acids except histidine to an optical eukaryotic genome. density at 600 nm (00600) of 0,5 to 0.9. Assays were performed (23) on three transformants, and Miler units (19) were averaged, Standard deviations were The primary level of chromatin architecture in viv() is the 25%, j3-Galactosidase activity from all strains in the absence of GAI expres- cleosome. The results of in vivo and in vitro studies indicate sion was 1 Miler unit. that nucleosomes inhibit transcription by competing with tran- In vivo footprinting. Intact cells were treated with dimethyl sulfate (OMS), scription factors for occupancy of DNA binding sites (for a and the methylated DNA was isolated essentially as previously described (10), except final pellets were dissolved in water and quantitated with a spectropho- review , nucleosomes can inhibit , see reference 27). In vitro tometer. DNA samples were then digested with HaeIII and analyzed via a cyclic transcription initiation either by blocking access of the general primer extension reaction (1) using a labeled oligonucleotide, Primer extension transcription machinery to promoter sequences or by hinder- products were extracted with chloroform-isoamyl alcohol (24:1), precipitated ing the binding of upstream activator proteins. It is not known and electrophoresed on a 5% National Diagnostics sequencing gel. The gel was dried and exposed to film for 12 to 48 h. For a control for spurious primer at present which of these steps might be overcome by the extension products, DNA from a strain that was not treated with OMS was also SWI- SNF complex in vivo. The results of biochemical studies prepared. Footprinting at the wild- type GALl lO VAS (see Fig. 2) was analyzed have shown that yeast or human SWI-SNF complex can stim- by using an oligonucleotide with the following sequence: 5' GAG CCC CAT ulate the binding of an activator protein to a nucleosomal TAT er AGC- , which anneals to a sequence located 60 bp upstream of GAI binding site 1. Footprinting at the integrated reporter loci (see Fig. 3) was binding site (5, 16). Furthermore, the purified human SWI- analyzed by using an oligonucleotide with the following sequence: 5' -CCG GeT SNF complex can facilitate the binding of the general tran- CGT ATG TIG TGT GG- . This oligonucleotide anneals to the unique pVC scription factor TATA box binding protein (TBP) to a posi- sequences located upstream of the GAI binding sites in the reporters (see tioned nucleosome (13). Therefore, the SWI-SNF complex below), may function at one or Indirect end labeling. Nucl i were prepared as described previously (22). To more discrete steps to facilitate gene prepare free DNA, 80 fl1 of 10% sodium dodecyl sulfate (SDS) and 30 fl1 of expression in vivo. 20-mg/ml proteinase K were added to a 1-ml aliquot of nuclei (approximately 5 X In this study, we address the ability of the SWI-SNF complex cell equivalents), and the sample was incubated at 37 C for 2 h. Lysed nuclei were treated with 180 fl1 of 5 M potassium acetate on ice for 1 h and subjected to centrifgation, and the supernatant was precipitated with isopropanol. This . Corresponding author. Mailing address: Program in Molecular DNA sample was resuspended in water and reprecipitated with ethanol. The Medicine and Department of Biochemistry and Molecular Biology, resultant free DNA was resuspended in 1 ml of buffer 02 (10 mM HEPES (pH 7.3), 5 mM MgCl , 2 mM CaCl ). Free DNA and nuclei samples (approximately University of Massachusetts Medical Center, 373 Plantation St. 1.7 X 10 cell equivalents in 300- fl1 aliquots) were digested with 0 to 50 Worcester, MA 01605. Phone: (508) 856-5858. Fax: (508) 856-4289. micrococcal nuclease (MNase) for 5 min at 37 C. Reactions were halted by mail: craig.peterson(fummed.edu. adding 26 fl1 of 10% SDS and 10 fll of 20-mg/ml proteinase K and incubating the
4811 "-'. , ......
i " MoL. CELL. BIOL. I ' 4812 BURNS AND PETERSON
TABLE 1. Yeast strains used in this study I ' I:: mixures at 37 C for 2 h, Samples were treated with 60 fLl of 5 M potassium , and the supernatant was acetate on ice for 1 h and subjected to centrifugation Strain Genotype Clal (see Fig. precipitated with isopropanol. DNA was then digested with either lOlleu2- !: his3, 4A and B) or EcoRl (see Fig, 4C) in the presence of RNase, Digested fragments CY257 MATa swill:::LEU2Iys2-801 ade2, were extracted with buffered phenol, and the aqueous layer was precipitated with 1:200 ura3- !:9 overnight. ethanol. Final pellets were electrophoresed on a 1.5% agarose gel MATa gaI41:::LEU2 lys2-801 ade2- 101Ieu2-1:1 his3- , and blots were probed with an CY296 DNA was transferred to a nylon membrane 1:200 ura3- !:9 internally labeled 870-bp lacZ fragment (see Fig, 4A and B) or an internally 801 ade2-101 GALl gene (see Fig. 4C). CY297 MATa gaI41:::LEU2 swill:::LEU2 lys2, labeled 550-bp fragment from the leu2- 1:1 his3- 1:200 ura3- !:9 HgaI restriction enzyme accessibilty assay. Permeabilized spheroplasts were , in glucose medium) by 801 ade2- 101Ieu2- !: his3, 1:200 ura3- prepared from exponentially growing cells (OD6oo of 0. CY341 MATa lys2- , resuspended in CY353 ...... Same as CY524 but contains URA3::pRY171 treatment with yeast lyticase (24), Briefly, cells were harvested 0), 60 mM 1/10 culture volume of prespheroplasting buffer (100 mM Tris (pH 8. (GALl, 10) were 52 ade2- 101 his3- 1:200 leu2, i3-mercaptoetha l), and shaken for 10 min at room temperature. Cells CY366 MATa swi11:::LEU2 ura3- harvested and resuspended in 1/20 culture volume of spheroplasting buffer (0, 1:1lys2-801 URA3::pRY171 (GALl,lO) , 10 mM Tris (pH 7,5), 10 mM M sorbitol, 0.75% yeast extract, 1.5% peptone URA3::pEG28 (2 low)" , and cells were incubated at 37 C until CY401...... Same as CY524 but contains i3-mercaptocthanol), lyticase was added CY422 ...... ,...Same as CY526 but contains URA3::pEG28 (2 low) the ODooo of cells diluted with water was decreased by 90% (30 to 40 min), 1:1 his3- 1:200 ura3- , washed once with nystatin buffer CY524 MATa lys2-801 ade2- 101Ieu2- Spheroplasts were harvested by centrifugation 801 ade2- 101Ieu2- !: his3- (50 mM NaC!, 1.5 mM CaC!z, 20 mM Tris (pH 8), 1 M sorbitol, 10 mM MgCl CY525 MATa gaI41:::LEU2 lys2- and resuspended in 1/150 culture volume of nystatin buffer. Nystatin was added 1:200 ura3- C for 801 ade2- 101Ieu2-1:1 his3- to a final concentration of 15 fLg/ml, and spheroplasts were incubated at 37 CY526 MATa swi11:::LEU2 lys2- 5 min. Hgal (New England BioLabs) was added to the permeabilized sphero- 1:200 ura3- , and samples were incubated at 37 C for 0 (2 high)b plasts at a concentration of 120 U/ml CY528 ...... Same as CY524 but contains URA3::pLB7 (2 high) to 30 min. Reactions were stopped by the addition of 1/5 volume of 5% SDS- CY529 ...... ,...... Same as CY525 but contains URA3::pLB7 , and DNA was purified as described above (a negative control lacked mM EDTA Same as CY526 but contains URA3::pLB7 (2 high) HindUl (New England BioLabs), elec- CY530 ...... Hgal). Purified DNA was digested with (2 low; , and Same as CY524 but contains URA3::pLB8 trophoresed on a 1.5% agarose gel, Southern blotted to a nylon membrane CY532 ...... fragment from plasmid pLB8 that was internally pUC spacer) probed with a 500-bp EcoRl (2 low; labeled by random priming (see Fig, S). Results were quantitated by phosphor- CY533 ...... Same as CY525 but contains URA3::pLB8 imager analysis, pUC spacer) have been described (2 low; Plasmid and strain construction. The following plasmids CY534 ....,...... Same as CY526 but contains URA3::pLB8 previously: pRY171 (GALl-lacZ fusion (30)), pEG202 (pLEXA (ll)), and pUC spacer) pMA10 (pGAL) and pSD15 (17). Plasmids pLB7 and pLB8 were constructed URA3::pLB16 (2 low; URA3 CY586 ...... Same as CY524 but contains by inserting the 194-bp Smal-Pvull fragment of pUC18 (29) between the NPE) gene and the GAL binding sites of the plasmids pEG44 and pEG28, respec- (2 low; Same as CY525 but contains URA3::pLB16 tively (9). Plasmid pEG28 contains GAL binding sites 3 and 4 positioned 190 bp CY587 ...... upstream of the GALl TATA element. These sites are separated by 62 bp NPE) , consensus URA3::pLB16 (2 low; (center-to-center distance), Plasmid pEG44 contains two synthetic CY588 ...... Same as CY526 but contains TAT A element (9). GAL binding sites positioned 150 bp upstream of the GALl NPE) These two sites are separated by 32 bp ( center-to-center distance), These plas, mids were targeted for integration at the ura3-52 locus of yeast strains CY524 2 low, two low,affnity GAL4 binding sites. CY525 , and CY526 (Table 1) by digestion with ApaI. A DNA fragment contain- " 2 high, two high-affnity GAL4 binding sites, ing the 5S nucleosome positioning element (NPE) of sea urchin was amplified from plasmid plCN5S182 (gift from Jerry Workman) by PCR using the following CCCCGAGGAATTAA primers: 5' CCACGAATAACICCAGGG-3' and 5' , 20 bp GTAC-3' (18). The 182- bp PCR product was inserted into plasmid pLB8 region is reduced only 1.5-fold from the activity in the respec- upstream of GAL binding site 3 (see Fig. 1), This new plasmid, called pLBI6 tive wild-type strain (Fig. lA, reporter a). This result is con- ura3-52 locus of CY524, CY525 , and CY526 GALl was targeted for integration at the sistent with a previous study in which it was found that by digestion with ApaI. Yeast transformations were performed via the lithium swi-snf expression was only weakly affected by mutations (21), acetate method described previously (8). In contrast, GAl activity on the two reporters with two low- affnity GAL4 binding sites is SWI-SNF dependent; transcrip- RESULTS tional activity is reduced 22- to 32-fold in the swir strains from that of the wild-type strain (Fig. lA, reporters b and c), SWI-SNF activity is required for transcriptional activation sites are replaced by two high- some promoters. Isogenic SWl+ and swi- strains When these two low-affnity by GAI at affnity, consensus GAL4 binding sites, transcriptional activa- that contain similar single-copy integrated reporters were con- 1A. Strains tion is reduced only 1.3-fold in the swil- strain from that of the structed and are shown schematically in Fig. , reporter d). contain a GALl lO VAS wild- CY353 (swr+ and CY366 (swir) type strain (Fig. lA Strains harboring the two low-affnity site reporters were reporter consisting of four GAl binding sites upstream of a CY401 (swr+ also distinct from those carrying the two high-affnity site re- GALl-lacZ fusion gene (reporter a). Strains fold porters in that lacZ expression was increased 2.5- to 3. and CY422 (swir) contain a derivative of this reporter that , reporters b contains only two of these low-affnity GAl binding sites when GAL4 protein was overexpressed (Fig. lA (swr+ and CY534 and c; also data not shown). The simplest interpretation of this (sites 3 and 4; reporter b); strains CY532 result is that physiological levels of GAL4 are not suffcient to (swir) contain a version of reporter b that contains 192 bp of site 3 fully occupy the two low-affnity sites (9; also see below). Im- additional plasmid sequences 20 bp upstream of GAl overcome the and CY530 (swir) con- portantly, overexpression of GAL4 does not (reporteJ;)c); strains CY528 (swr+ , reporters b high-affnity, consensus SWI-SNF dependence of this reporter (Fig. lA tain a reporter which contains two swil mutation does not binding sites in place of the two low-affnity sites (re- and c). It should be noted that the GAl affect expression of GAL4 from this overexpression plasmid in porter d) (9). I3-Galactosidase assays were performed on these transcriptional activity either in the this strain background (Fig. lB) (20). strains to monitor GAl SNF is required for GAI occupancy of low-affnity presence of endogenous concentrations of GAl (pLEXA SWI- binding sites in vivo. To directly assess the ability of GAL4 to columns) or under conditions in which GAl is overexpressed columns) (Fig. 1A). bind its sites in the different reporter strains in the presence or (pGAl SNF, we used an in vivo DMS footprinting In the absence of functional SWI-SNF complex, transcrip- absence of SWI- lO VAS assay. GAl protects guanine residues at each end of its 17- tional activation by GAl from the wild-type GALl, ---
BINDING IN VIVO 4813 VOL. 17, 1997 SWI-SNF FACILITATES GAL pLEXA pGAL4
SWJ+ swil- SWJ+ swil-
7218 4691 (65%) (a) LaCZ
C?L4 390 16 (4.1 %) 1118 51 (4.5%) (b) LaCZ
pUC 658 30 (4.5%) 2409 74 (3%) (c) LacZ
pUC 2311 1726 (75%) 2719 1616 (59%) (d) LaCZ
pUC LacZ (e) 613 490 (80%)
NPE Galactosidase assays were performed on FIG, 1. GAL requires functional SWI-SNF complex to activate transcription from two low, affnity binding sites. (A) i3- GAL binding GALl,/acZ UAS reporter plasm ids pRYl71 (a), pEG28 (b), pLB8 (c), pLB7 (d), or pLB16 (e), isogenic SWl+ or swir strains harboring the integrated indicate synthetic GAL sites numbered 1 to 4 indicate sites from the GALl lO UAS region; GAL sites numbered 17 sites are denoted by the small black boxes. DNA binding domain (pEG202) or the full-length GAL4 protein consensus GAL binding sites. Strains contained plasmids that overexpressed either the bacterial LexA Galactosidase activities of all reporters were oC1 Miler unit in the absence (pMA21O), Numbers in parentheses show activities as percentages of the wild-type levels. i3- d" not determined, (B) Western blot analysis of GAL levels, Whole-cell extracts (21) were prepared from equal numbers of of GAL. Drawings are not to scale, n. GAL protein (indicated by the arrow) was identified SWl+ (CY296) and swir (CY297) cells containing the GAL overexpression plasmid pMA21O. Expression of by Western blotting using an antibody directed against the C terminus of GAL (21),
protection in the SYV+ strain (lane 5) and are only weak recognition site both in vivo (10) and in vitro (3). As shown in protection in the swir strain (lane 6) are observed. Quanti- Fig. 2, these guanine residues within the four GAI binding phosphorimager analysis indicates sites of the GAL1 lO VAS are protected from DMS methyl- tation of these results by ation in the presence of GAI (lane 3) but are accessible to that protection of the relevant guanine residue in site 3 in the swir type methylation in an isogenic gal4- strain (lane 2). Introduction strain is decreased (40%) from that seen in a wild- strain restores , we also analyzed these samples of GAI expression plasmids into the gal4- strain. For an internal control GAL1 lO UAS locus. In protection of these guanines (lanes 6 and 8), while introduction for GAI binding at the endogenous GAI of a plasmid expressing the bacterial LexA protein does not all cases , we observe complete occupancy of the four sites in the presence and absence of SWI-SNF (data not (lane 7). In the absence of SWI-SNF activity, protection of all four sites is retained (lane 4). Furthermore, as shown in Fig. shown). The lack of complete occupancy at the two low-affnity sites in the swir strain indicates that the SWI-SNF complex GAI also protects the guanine residues at each of the two modulates GAI binding in vivo. high-affnity binding sites in the presence (lane 4) and absence SNF-dependent pro- SNF. Similar results were obtained in at least Introduction of an NPE into a SWI- (lane 5) of SWI- SNF. The six different experiments with five independent DNA prepara- moter region can suppress the requirement for SWI- tions. These results are consistent with the results of our func- results of genetic and biochemical experiments have indicated SNF is not required that SWI-SNF complex facilitates activator function by disrupt- tional experiments (Fig. 1) in which SWI- assess whether the chromatin for the activity of GAI from either the wild-type GALl, ing chromatin structure. To UAS or the two-high-affnity-site reporter. structure of the GAI binding sites was related to their SWI- SNF dependence, a strong NPE was inserted directly upstream Figure 3B shows the results of an in vivo footprinting anal- (SYV+ ysis of the two-Iow-affnity-site reporter in strains CY532 of the two low-affnity binding sites in strains CY532 and CY534 (swir). This 182-bp NPE sequence has been (SYV+ and CY534 (swir). We detect little protection of the two low-affnity sites in our footprinting assay, even in a SYV+ shown previously to translation ally and rotationally position a , this probably reflects the nuc1eosome in vivo in yeast (20) (Fig. 4B). Insertion of the strain (lane 3). As discussed above SNF dependence of low occupancy of these sites at physiological GAI levels NPE had a dramatic effect on the SWI- GAI function. While GAI transcriptional activity at the because when GAI is overexpressed in this SYV+ strain parental two-Iow-affnity-site reporter was decreased over 20- protection is restored (lane 5). Overexpression of GAI in the SNF complex, however, does not fold in the absence of SWI-SNF (Fig. lA, reporters b and c), absence of a functional SWI- fold decrease when the NPE was restore complete protection of either low-affnity binding site there was less than a 1.5- site 3 , reporter e). This effect of the NPE is not due (lane 6). This is most apparent at GAI , where strong inserted (Fig. lA 4814 BURNS AND PETERSON MoL. CELL. BIOL.
7) (Fig. 4). This precIse nucleosome positioning at the GALl lO locus is believed to require the GRF2 protein which binds to a sequence that overlaps GAL4 binding sites 1 and 2 7). The GALl lO UAS derivatives that we have analyzed in this study (Fig. lA, reporters b to e) lack this GRF2 binding site, and therefore, they may not maintain nucleosome posi- tioning surrounding the GAL binding sites. To confirm this possibility, we analyzed the chromatin structure of the two- low-affnity-site reporters (with and without an NPE) and com- pared these structures to that of the GALl locus in the same strains. Nuclei and free DNA were prepared from the SWI+ strain CY532, which contains the parental two-low-affnity-site re- porter (without an NPE), and samples were analyzed by MNase digestion and indirect end labeling. First, we analyzed the chromatin structure at the GALl locus in this reporter strain (Fig. 4C). Comparison of the MNase cleavage patterns for free and chromatin DNA samples reveals a repeating pat- tern of MN ase protections (each about 140 bp in size) flanked on each side by MNase-hypersensitive sites. These results are essentially identical to a previous study (6) and are consistent with an array of positioned nucleosomes downstream of the nucleosome-free GAL4 binding sites. A similar analysis of the two-low-affnity-site reporter locus (without an NPE) yielded very different results (Fig. 4A). In the region upstream of the two GAL4 binding sites, the MN ase digestion pattern of the chromatin sample is similar to the pattern of digestion of free DNA, although we do reproducibly observe several preferred cleavage sites in free DNA that ap- pear to be enhanced in the chromatin sample. Importantly, the MNase cleavage sites directly upstream and adjacent to the GAL4 binding sites are cleaved with equal effciency in the free and chromatin DNA samples. A region of about 80 bp that contains the GAL binding sites is not cleaved effciently in either the free or chromatin DNA samples; this is due to the inherent sequence specificity of MNase I and is not due to a positioned nucleosome (6). In addition, this protected region is not flanked on both sides by hypersensitive sites. Directly downstream of the GAL4 binding sites, however, a l40- region of MNase resistance is detected, which is flanked on each side by MNase-hypersensitive sites, suggesting the pres- ence of a positioned nucleosome. The location of this nucleo- some corresponds to a positioned nucleosome mapped be- tween the GAL sites and the GALl TATA box of the FIG. 2, GAL can bind its sites in theGALl lO VAS region in the presence endogenous GALl lO locus (2, 6) (Fig. 4C and data not or absence of SWI-SNF. In vivo DMS footprinting analysis of the GALl, 10 VAS shown). Thus, although one nucleosome still appears to be region was performed on strains CY341 (SWI+ GAL+ ) (lane 3), CY257 (swir positioned at this GALl lO UAS derivative, the majority of GAL+ ) (lane 4), CY296 (SWl+ gaI4-) (lane 2), CY296 containing a GAL expression plasmid (pMA10 or pSD15) (lanes 6 and 8), and CY296 containing nucleosome positioning has been lost. Consequently, nucleo- a LexA expression plasmid (pEG202) (lane 7). Lane 1 contains a genomic somes appear to be located randomly upstream of the two guanine sequencing ladder. Boxes to the left of the gel denote the GAL binding remaining GAL4 binding sites. In the absence of a positioned sites of theGALl lO VAS and asterisks denote guanine residues shown previ- , the two low-affnity binding sites ously to be protected by GAL binding, array of nucleosomes GAL are unlikely to reside in a constitutive nucleosome-free region. MNase digestions and indirect end labeling were also used to confirm that the NPE positioned a nucleosome upstream of simply to insertion of for ign DNA sequences directly up- the two low-affnity GAL4 binding sites (Fig. 4B). Comparison stream of the two low-aff ity GAL binding sites, as insertion of the free and chromatin DNA samples reveals an MNase- of 192 bp of pUC plasmid sequences at an identical position protected region of about 250 bp which contains the NPE does not alter the SWI-SNF dependence of GAL activity (Fig. sequences as well as the MNase-resistant GAL binding site , reportersb and c). sequences. This result is consistent with the positioning of a , Nucleosomal context of GAI binding sites dictates SWI- nucleosome over the NPE sequence. In addition, the putative SNF dependence. The results of functional experiments de- positioned nucleosome located directly downstream of the two scribed above suggest that the SWI-SNF dependence of GAL low-affnity GAL binding sites is also detected at the NPE- can be modulated by nucleosome positioning. In the case of containing reporter locus in CY586 (Fig. 4B). the GALl lO locus, the four GAL binding sites are main- These results are consistent with the model shown in Fig. 6. tained in a constitutive nucleosome-free region which In the absence of the NPE, the two low-affnity GAL binding flanked on both sides by an array of positioned nucleosomes (6 sites are encompassed by randomly positioned nucleosomes; VOL. 17, 1997 SWI-SNF FACILITATES GAL BINDING IN VIVO 4815
FIG, 3, SWI-SNF is required for complete occupancy of two low!affnity GAl binding sites in vivo. In vivo DMS footprinting was performed on strains that harbor the two-high-affnity-site reporter (A) or the two-low-affnity-site reporter (B). The strains used were as follows: for panel A, CY529 (galr SWI+ (lane 3), CY528 (lane 3), CY534 (GAL4+ swir) (GAL4+ SWI+ (lane 4), and CY530 (GAL4+ swir) (lane 5); for panel B, CY533 (gaI4- SWI+ (lane 2), CY532 (GAL4+ SWI+ (lane 4), CY532 harboring a GAl expression plasmid (lane 5), and CY534 harboring a GAl expression plasmid (lane 6). For quantitation of lanes 5 and 6 in panel , phosphorimager data were normalized to the doublet band indicated by the arrowhead (G449 and G450 (numbering from reference 30)). Symbols and lane 1 are as described in the legend to Fig. 2,
insertion of the NPE repositions the GAL binding sites into a DNA probe wil detect the HgaI-HindII cleavage products de- nucleosome-free region between two positioned nucleosomes rived from the GALl lO locus (1.6 kb) or the reporter locus (0. (see Fig. 6). To further test the possibility that insertion of the kb) in the same DNA sample (see the schematic in Fig. SA). In NPE changes the accessibility of the GAL binding sites, we the case of the GALl lO locus, we expected that the HgaI site investigated the ability of a restriction enzme to cleave a site within GAl site 3 would be highly accessible in penneabiled within GAL site 3 in permeabilized spheroplasts (Fig. 5). spheroplasts, since this GAl site is known to be nucleosome- Spheroplasts were prepared from the SWI+ strains CY532 free (6). In contrast, the model presented in Fig. 6 predicts that (which contains the parental two-low-affnity-site reporter) and the HgaI site at the parental two-low-affity-site reporter wil be CY586 (which harbors the NPE-containing two-low-affnity- less accessible than the same site present at GALl lO. Further- site reporter). Spheroplasts were made permeable by treat- more, the model predicts that insertion of the NPE wil enhance ment with nystatin (24) and incubated with HgaI restriction the accessibilty of the HgaI site at the reporter locus. endonuclease for 15 to 30 min. Genomic DNA was then Figure 5B shows the phosphorimager quantitation of a typ- purified and digested to completion with HindIII and the ical HgaI accessibility assay. At 15 and 30 min of digestion HgaI-HindII cleavage products were analyzed by Southern blot- HgaI cleavage at the GALl lO locus was nearly complete (val- ting using a DNA probe from the GALl upstream region. Ths ue set at 100%) (data not shown). However, in the case of the .. ..
4816 BURNS AND PETERSON MoL. CELL. BIOL.
FreeDNA Chromatin FreeDNA Chromatin MNase MNase
2620 bp
2170 bp 2620 bp
2170 bp
870 bp Bam
CIa! 7 8 9 10 11 12
FreeDNA Chrmatin
870 bp Bam
1921 bp
1110 bp CIa! 3 4
FIG, 4, Nucleosome mapping of the two-low-affnity-site reporters with and without an NPE, Free DNA and nuclei from the two low-affnity-site swr+ reporter strain (CY532) (A) or the two-low-affnity-site, NPE-containing re- 550 bp porter strain (CY586) (B and C) were treated with increasing amounts of MNase and nucleosome positioning at the reporter loci (A and B), or the GAL1 lO locus (C) was analyzed by indirect end labeling. The following amounts of MNase (in units per mililiter) were added: for panel A, a (lanes 1 and 7), 0.005 (lanes 2 and 8), 0,05 (lanes 3 and 9), 0,5 (lanes 4 and 10), 5 (lanes 5 and 11), 50 (lanes 6 and 12); for panel B, 0.5 (lanes 1 and 3) and 5 (lanes 2 and 4); for panel C, 1 (lane 1), 5 (lanes 2 and 3), and 25 (lane 4). The schematic to the right of each gel depicts the mapped locus, black rectangles represent GAL binding sites, shaded boxes represent coding sequence of the indicated gene, and shaded ovals rep- resent the predicted positions of nucleosomes. Restriction sites used to generate the probe fragment are indicated on the map accompanying each figure, In panel C, primers used to amplify the probe fragment by PCR are indicated by black bars. Migration of DNA size standards are noted to the left of each gel.
parental two-low-affnity-site reporter, cleavage of GAL site DISCUSSION by HgaI was less effcient, indicating that these sequences are SNF less accessible than they are at GAL1 lO. Furthermore, inser- In vitro experiments have demonstrated that the SWI- tion of the NPE upstream of the GAL binding sites had a complex can stimulate the binding of an activator protein to a nucleosomal binding site (5 16). Here we have tested whether dramatic effect on HgaI cleavage. Cleavage at the NPE-con- taining reporter' locus was at least threefold greater than the the complex exhibits this activity in vivo. We have shown that two parental reporter locus, and the cleavage appeared to be even the SWI-SNF complex facilitates the binding of GAL to , we find that the more effcient than cleavage at the GAL1 lO locus. These dif- low-affnity binding sites in vivo. Furthermore ferences in HgaI accessibility are not due to the binding of inability of GAL to occupy these low-affnity sites in the ab- GAL, since similar results were also obtained when perme- sence of SWI-SNF activity can be overcome by (i) replacing the binding sites abilized spheroplasts were prepared from gal4 deletion strains low-affnity binding sites with high-affnity GAL (data not shown). Thus, the accessibility of GAL site 3 to or (ii) placing the low-affnity binding sites into a nucleosome- SNF is able to HgaI cleavage correlates well with the nucleosome mapping free region. These results indicate that SWI- data and the SWI-SNF dependence of GAL activity (Fig. 6). facilitate GAL binding in vivo, perhaps by helping GAL to +/ -
VOL. 17, 1997 SWI-SNF FACILITATES GAL4 BINDING IN VIVO 4817
Hgal
L 10 nnnn GAL1, LOCUS
Hindlll Hindlll Hgal
RA3 L 1 - lacZ REPORTER LOCUS
Hindlll Hindlll
200
150
o GALI, 100 li - NPE Reporter Lous mm + NPE Reporter Locus
TIME (Mil1ulc
FIG, 5, An NPE leads to enhanced accessibility of a GAlA binding site,(A) Schematic of the GALl, 10 and reporter loci. The black bar denotes the DNA fragment used as a probe in Southern blotting, The HgaI site is located at the 3' end of GAI site 3, +/- NPE, with or without an NPE, (B) HgaI accessibility assay,HgaI was added to nystatin-permeabilized spheroplasts prepared from swr' strains harboring either the parental two-low-affnity-site reporter (- NPE; CY532) or the NPE-containing two-low-affnity-site reporter (+ NPE; CY586), DNA digested in situ was purified , digested to completion with HindIII and analyzed by Southern blotting using the DNA probe denoted in panel A. Phosphorimager quantitation of the HgaI-HindIII fragments is shown for the 15- and 30-min HgaI digestion time points, The yields of the O, kb HgaI-HindIII fragments from the two different reporter loci are presented as a perccntage of the 1.6-kb HgaI-HindIII fragment derived from the GAL1 10 locus which was set at 100% accessibility,
compete more effectively with histones for occupancy of its in the absence of SWI-SNF activity. Furthermore, SWI-SNF binding sites. dependence does not appear to correlate with the level of A definition of SWI-SNF dependence. Our results suggest transcriptional activation (i. , promoter strength (Fig. lAD. that the SWI-SNF dependence of a transcriptional activator Thus, based on the results presented here, we predict that the protein is not an innate feature of the activator protein per se subset of genes whose expression requires SWI-SNF wil have but rather reflects the chromosomal context of the activator activator binding sites that are encompassed in nucleosomes; binding sites. We propose that when the activator binding sites genes that do not require SWI-SNF wil have such sites posi- are encompassed in a nucleosome, as in the two-low-affnity- tioned in the linker regions between nucleosomes or in other site reporter used in this study, SWI-SNF function is necessary nucleosome-free regions. This positioning may be determined for the activator protein to bind to and activate transcription by DNA sequence, as in the NPE-containing reporter, or by from those sites (Fig. 6). On the other hand, when binding sites abundant DNA binding proteins like GRF2, as in the case of are nucleosome-free , as is the case at the GALI IO VAS region the intact GALl, IO reporter. or at the NPE-containing two-low-affnity-site reporter, then Our results also require that binding site affnity be incor- SWI-SNF function is dispensable for GAL activity (Fig. 6). In porated intothe definition of SWI-SNF dependence. While this case, GAL4 can bind to its sites and activate transcription GAL4 requires SWI-SNF in order to access two low-affnity ) ( , '
4818 BURNS AND PETERSON MoL. CELL. BIOL.
SWISNF ager analysis. We also acknowledge the initial observation that high- deDendent? affnity GAl binding sites can alleviate the SWI-SNF requirement for GAl enhancement was made by c.L.P. while a postdoctoral fellow with I. Herskowitz. YES This work was supported by NIH grant GM49650 and a Basil Conner Research Starter Scholar Award FY94-0922 to C.L.P. c.L.P. is a scholar of the Leukemia Society of America.
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Soli-line ovals represent ture of UAS and functions as a powerful auxliary gene activator. Genes positioned nucleosomes; broken-line ovals represent randomly located nucleo- Dev, 4:503-514, somes, Nucleosomes located randomly over the GAL binding sites of the 5, Cote, J., J. Quinn, J. L. Workman, and C. L. Peterson. 1994, Stimulation of former cause the expression of this reporter gene to require SWI-SNF activity, In GAL derivative binding to nucleosomal DNA by the yeast SWI/SNF com- the NPE-containing construct, on the other hand, the GAL binding sites are plex. Science 265:53-60. nucleosome-free and therefore reporter gene expression is SWI-SNF indepen, 6, Fedor, M. J., and R. D. Kornberg. 1989. Upstream activation sequence- dent. dependent alteration of chromatin structure and transcription activation of the yeast GALl- GALlO genes. Mol. Cell, BioI. 9:1721-1732. 7. Fedor, M. J., N. F. Lue, and R. D. Kornberg. 1988, Statistical positioning of nucleosomes by specific protein-binding to an upstream activating sequence binding sites, high-affnity sites are occupied even in the ab- in yeast. J. Mol. BioI. 204:109-127. sence of SWI-SNF. The ability of higher-affnity binding sites 8, Gietz, R. D., and R. H. Schiestl. 1991. Applications of high effciency lithium to alleviate SWI- acetate transformation of intact yeast cells using single-stranded nucleic SNF dependence provides strong evidence acids as carrier. Yeast 7:253-263. that the SWI-SNF complex modulates transactivation at the 9, Giniger, E., and M. Ptashne. 1988, Cooperative DNA binding of the yeast level of DNA binding. Furthermore, if these high-affnity bind- transcriptional activator GAL4, Proc. Natl. Acad. Sci. USA 85:382-386, ing sites are encompassed by nuc1eosomes, then our results 10, Giniger, E., S. M. Varnum, and M. ptashne. 1985. Specific DNA binding of GAL, a positive regulator protein of yeast. Cell 40:767- suggest that binding to high-affnity, 774, GAl nuc1eosomal sites 11. Gyuris, J., E. Golemis, H. Chertkov, and R. Brent. 1993, Cdil, a human Gl can occur in vivo via a SWI-SNF-independent mechanism. and S phase protein phosphatase that associates with Cdk2. Cell 75:791-803, These results are consistent with those of Workman and King- 12, Hirshhorn, J. N., S. A. Brown, C. D. Clark, and F. Winston. 1992, Evidence ston (28), who found that GAl can bind to a nuc1eosomal site that SNF/SWI2 and SNF5 activate transcription in yeast by altering chro- to form a tripartite transcription factor-histone- matin structure, Genes Dev. 6:2288-2298. DNA complex. 13, Imbalzano, A. N., H. Kwon, M. R. Green, and R. E. Kingston. 1994. Facil- Does the SWI-SNF complex play an additional role in tran- itated binding of TAT A-binding protein to nucleosomal DNA. Nature 370: scriptional activation? As noted above, we detect a consistent 481-485. 14. Kruger, W., and I. Herskowitz. 1991. A negative regulator of HO transcrip- level of GAl binding to the low-affnity sites in the swil- tion, SIN 1 (SPT2), is a nonspecific DNA-binding protein related to HMG 1. strain (Fig. 3B, compare lanes 5 and 6); phosphorimager anal- Mol. Cell, BioI. 11:4135-4146, ysis indicates site 3 may be occupied at 60% of the level seen 15, Krger, W., C. L. Peterson, A. SiI, C. Coburn, G. Arents, E. N. Moudri- in a swr strain (Fig. 3C). The fact that this level of occupancy anakis, and I. Herskowitz. 1995. Amino acid substitutions in the structured results in negligible levels of transcription (3% of wild-tye domains of histones H3 and H4 partially relieve the requirement of the yeast level (Fig. lA, reporter cD may indicate a role for the SWI- SWI/SNF complex for transcription, Genes Dev, 9:2770-2779. 16, Kwon, H., A. N. Imbalzano, P. A. Khavari, R. E. Kingston, and M. R. Green. SNF complex in transcriptional activation beyond the modu- 1994, Nucleosome disruption and enhancement of activator binding by a lation of activator binding. In light of recent reports suggesting human SWI/SNF complex, Nature 370:477-481. that the SWI-SNF complex may associate with the RNA poly- 17. Ma, J., and M. Ptashne. 1987. Deletion analysis of GAL defines two merase II holoenzye (25), it is interesting to consider the transcriptional activating segments. Cell 48:847-853, 18, Meersseman, G., S. Pennings, and E. M. Bradbury. 1991. Chromatosome possibility that an activator-SWI-SNF interaction is involved in positioning on assembled long chromatin, Linker histones affect nucleosome preinitiation complex assembly. On the other hand, our in vivo placement on 5S rDNA. J, Mol. BioI. 220:89-100, footprinting procedure does not allow us to determine the 19. Miler, J. H. 1972. Experiments in molecular genetics, Cold Spring Harbor extent to which site 3 and site 4 are simultaneously Laboratory, Cold Spring Harbor, N, occupied 20. Pederson, D. S., and T. Fidrych. 1994. Heat shock factor can activate tran- and we cannot distinguish whether the 60% occupancy in a scription while bound to nucleosomal DNA in Saccharomyces cerevisiae, swi-snf strain reflects the stable binding of GAl in 60% of the Mol. Cell. BioI. 14:189-199, cells or reflects weak, unstable binding in 100% of the cells. 21. Peterson, C. L., and I. Herskowitz. 1992, Characterization of the yeast SWIl Therefore SWI2, and SWI3 genes, which encode a global activator of transcription. Cell , our results may support an additional role for the 68:573-583. SWI-SNF complex in facilitating the cooperative binding of 22. Shimizu, M., S. Roth, C. Szent-Gyorgyi, and R. T. Simpson. 1991. Nucleo- GAl or in enhancing the stability of GAl binding to low- somes are positioned with base pair precision adjacent to the a2 operator in affnity, nuc1eosomal sites in vivo. Saccharomyces cerevisiae. EMBO J. 10:3033-3041. 23, Stern, M., R. Jensen, and I. Herskowitz. 1984, Five SWl genes are required for expression of the HO gene in yeast. J. Mol. BioI. 178:853-868. 24. Venditti, S., and G. Camiloni. 1994, In vivo analysis of chromatin following ACKNOWLEDGMENTS nystatin-mediated import of active enzymes into Saccharomyces cerevisiae, We thank 1. Herskowitz, M. Fedor Mol. Gen. Genet. 242:100-104. , L. Apone, and members of the 25, Wilson Peterson laboratory for comments on the manuScript; E. Giniger and , C. J. D. M. Chao, A. N. Imbalzano, G. R. Schnitzler, R. E. Kingston and R. A. Young. 1996, RNA polymerase II holoenzyme contains SWI/SNF J. Workman for plasmids; J. Axelrod and L. Wu for footprinting regulators involved in chromatin remodeling, Cell 84:235-244. protocols; and L. Cerilo and C. Logie for guidance on phosphorim- 26. Winston, F., and M. Carlson. 1992. Yeast SNF/SWI transcriptional activa- VOL. 17, 1997 SWI-SNF FACILITATES GAL4 BINDING IN VIVO 4819
tors and the SPT/SIN chromatin connection, Trends Genet. 8:387-391. 29, Yanisch-Perron, C., J. Vieira, and J. Messing. 1985, Improved M13 J1a 27, Workman, J. L., and A. R. Buchman. 1993, Multiple functions of nucleo, cloning vectors and host strains: nucleotide sequences of the M13mp18 and, somes and regulator factors in transcription, Trends Biochem, 18:90-95, pUC19 vectors, Gene 33:103-119, 28. Workman , J. L., and R. E. Kingston. 1992, Nucleosome core displacement in 30, Yocum, R. R., S. Hanley, R. J. West, and M. Ptashne. 1984, Use of lacZ vitro via a metastable transcription factor-nucleosome complex, Science 258: fusions to delimit regulatory elements of the inducible divergent GALI- 1780-1784, GALlO promoter in Saccharomyces cerevisiae, Mol. Cell. BioI. 4:1985-1998,