Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2011 Dec 16.
Published in final edited form as: Cell Cycle. 2008 Dec 10;7(24):3928–3934. doi: 10.4161/cc.7.24.7380

Positional analyses of BRCA1-dependent expression in Saccharomyces cerevisiae

Robert V Skibbens 1,*, Danielle N Ringhoff 1, Jutta Marzillier 1, Lynne Cassimeris 1, Laura Eastman 1
PMCID: PMC3241008  NIHMSID: NIHMS341691  PMID: 19098428

Abstract

Mutations in BRCA1 account for a significant proportion of hereditary breast and ovarian cancers, but analysis of BRCA1 function is complicated by pleiotropic effects and binding partners (Pol II holoenzyme and transcription factors, chromatin remodelers, recombination complexes and E3 ligases). In vertebrate cells, efforts to elucidate BRCA1 transcriptional effects have focused on specific genes or restricted portions of the genome—limiting analyses of BRCA1 effects on adjoining DNA sequences and along chromosome lengths. Here, we use microarray analyses on the genetically tractable yeast cell system to elucidate BRCA1-dependent genome-wide positional effects on both gene induction and repression. Yeast responses may be of clinical relevance based on findings that BRCA1 severely diminishes yeast growth kinetics but that BRCA1 mutated at sites identified from breast tumors is no longer able to retard yeast cell growth kinetics. Our analysis suggests that BRCA1 acts through both transcription factors to upregulate specific loci and chromatin remodeling complexes to effect global changes in gene expression. BRCA1 also exhibits gene repression activities. Cluster-functional analysis reveals that these repressed factors are required for mitotic stability and provide a novel molecular explanation for the conditional lethality observed between BRCA1 and chromosome segregation genes.

Keywords: BRCA1, gene expression, chromosome segregation, aneuploidy, microarray, chromatin remodeling

Characteristics of BRCA1-Dependent Gene Upregulation

Mutations in BRCA1 account for a significant proportion of hereditary breast and ovarian cancers.1 While recent reports have focused on BRCA1-dependent expression effects within specific subsets of genes,2,3 the role that BRCA1 plays in a both a positional and genome-wide context has yet to be performed. Such an analyses would be greatly simplified in yeast—given that yeast gene nomenclature provides for unambiguous positional and strand utilization cues along the chromosome length.4 Importantly, expression of human BRCA1 in budding yeast appears to provide a clinically relevant readout since BRCA1 severely diminishes yeast growth kinetics but BRCA1 mutated at sites identified from breast tumors is no longer able to retard yeast cell growth.5,6 In support of this, yeast mutated in CHL1 (homolog of human BACH1/BRIP1/FANJ DNA helicase that binds BRCA1 and is required for BRCA1-dependent double strand break repair) suppress BRCA1-dependent growth defects.7-9 Thus, BRCA1-targeted pathways are highly conserved in yeast. To capitalize on this conservation of function and to provide a unique positional context for BRCA1 function along the length of yeast chromosomes, we used human-assisted search methods to assess BRCA1 affects on mRNA levels for both individual genes and extended chromatin domains.

Recent reports document that BRCA1 genetically effects both transcription and chromosome segregation pathways in yeast,9-12 the latter of which directly produces aneuploidy when mutated. We decided to focus on the C-terminal BRCT domain of BRCA1 because it is both necessary and sufficient to elicit the yeast small colony phenotype and because of its relevance to cancer progression. 5,6,10-14 To elucidate BRCA1 effects on gene expression, vector or vector containing the BRCT domain of BRCA1 (herein termed BRCA1) was transformed into wildtype yeast, RNA extracted from log phase yeast grown at either 23° or 30°C and genome-wide changes in expression levels analyzed by microarray hybridization. We limited our analyses to those genes whose expression was altered two-fold or greater. Results show that mRNA levels of 461 genes were altered beyond this threshold in response to BRCA1 at 23°C relative to vector controls: 307 of which were upregulated and 154 which were downregulated (Suppl. Table 1). mRNA levels of 430 genes were altered two-fold or greater by BRCA1 expression at 30°C relative to vector controls: 350 of which were upregulated and 80 of which were downregulated (Suppl. Table 2).

We identified both discrete genes and contiguous multi-gene domains that were significantly upregulated in response to BRCA1 expression. Of 307 upregulated loci (23°C), 35 instances (11%) were identified in which the affected areas encompassed 2 or more adjacent open reading frames. Of 350 upregulated loci (30°C), 38 instances (11%) were identified in which the affected areas encompassed 2 or more adjacent open reading frames. Independent analyses of both data sets revealed instances in which positively affected areas encompassed 4 adjacent open reading frames to span up to 12 kb of contiguous DNA (Suppl. Table 3). Often, one actively transcribed domain was separated from a similarly upregulated domain by only a single-intervening locus. When we allowed for single locus gaps, upregulated regions that encompassed up to 10 loci and spanned over 23 kb were identified (Suppl. Table 4). Under this criterion, a total of 109 genes (roughly 1/3) of all positively affected genes may be attributable to global changes in gene expression. In summary, these results provide novel information that BRCA1 may associate with both yeast transcription factors and chromatin remodeling complexes, similar to those interactions observed in human cells, and that BRCA1-activated complexes elicit global and extensive increases in Saccharomyces cerevisiae mRNA levels (Suppl. Fig. 1).

Characteristics of BRCA1-Dependent Gene Repression

In human cells, BRCA1 blocks the assembly of pre-initiation transcription complexes—providing one mechanism of gene repression.12 As noted above, 154 of the 461 BRCA-affected loci were downregulated 2-fold or greater (23°C), revealing a role for BRCA1 in yeast gene repression. This data set also provided an opportunity to quantify extended regions of BRCA1-dependent repressed domains. Thus, we tabulated by hand all incidences in which repressed genes occurred immediately adjacent to one another. 10 instances were identified that encompassed a total of 20 genes (13% of total repressed genes) in which one repressed gene was immediately juxtaposed to another repressed gene (Suppl. Table 5). Independent analyses (30°C) identified 2 such instances, involving a total of 4 loci (5%), in which repressed genes were immediately juxtaposed (Suppl. Table 6). No instances of 3 adjacent repressed loci were observed in either data set. In combination, these findings reveal that while low incidences of multi-gene repression can occur, the role for BRCA1 in repression predominantly occurs in a locus-specific manner and, once established, infrequently spreads to repress adjoining domains.

We next characterized the boundaries between repressed and upregulated BRCA1-affected genes. Of the 5749 verified and expressed yeast genes,2 genes unaffected by BRCA1 expression (5442) outnumber genes upregulated by BRCA1 (307) roughly 20:1. Thus, the predicted incidence of finding a downregulated locus situated next to an upregulated locus would be at most 5%. We further reasoned that since approximately 1/3 of genes upregulated by BRCA1 appear to occur through a global-acting mechanism, the frequency of finding adjoining but oppositely regulated loci would decrease below 2%. In contrast, however, 18 examples (12%) of the 154 BRCA1-dependent repressed genes (23°C) were positioned immediately adjacent to an upregulated gene (Suppl. Table 7). Similarly, 8 examples (10%) of 80 repressed genes (30°C) were identified in which a downregulated gene was next to an upregulated gene (Suppl. Table 8). In combination, these results reveal that a surprisingly high percentage of repressed genes are situated immediately adjacent to upregulated genes. The boundary elements that establish and then maintain these transcriptional states remain an important but as yet uncharacterized facet of BRCA1-dependent gene regulation.

To better understand these transition states, we tested whether the ability to juxtapose oppositely regulated genes depended on DNA strand context. Out of the 18 adjacent but oppositely affected gene pairs, 5 were comprised of gene pairs situated on the Crick strand (C), 4 were comprised of gene pairs situated on the Watson (W) strand and 9 involved gene pairs in which one was located on the Watson while the other was located on the Crick strand (including both C→W and W→C orientations). Thus, BRCA1-dependent transition states between adjacent but oppositely affected genes appear to occur independent of strand bias (data not shown).

Functional-Cluster Analyses of BRCA1-Affected Genes

BRCA1 is conditionally lethal when expressed in yeast strains mutated in various kinetochore or cohesion factors.9 Thus, the second major goal of this study was to elucidate the molecular pathways through which BRCA1 expression promotes lethality in these mutants. Venn analysis was performed to identify, out of the 461 genes (23°C) and 430 genes (30°C) altered by BRCA1 expression, a high confidence level of genes whose temperature-independent regulation depended on BRCA1. The resulting analysis produced a list of 222 genes whose expression was uniformly altered 2-fold or greater in a temperature-independent manner. Of these, 183 genes were upregulated (Table 1) and 39 genes were downregulated (Table 2) in response to BRCA1 expression. For each category, we clustered together genes involved in similar pathways or function.

Table 1.

BRCA1-dependent upregulation at both 23° and 30°

Fold change 23° Up Fold change 30° Up Systematic Common
3.4433699 Up 2.156699 Up YMR056C AAC1
2.840843 Up 2.936136 Up YJR155W AAD10
3.4504972 Up 3.541029 Up YOL165C AAD15
2.2339227 Up 2.7618916 Up YER045C ACA1
2.1536045 Up 2.8568575 Up YFL055W AGP3
3.3267636 Up 4.341087 Up YDR242W AMD2
2.7140546 Up 2.2674854 Up YGL156W AMS1
2.1114006 Up 2.661742 Up YOL058W ARG1
3.0350552 Up 2.6912796 Up YML116W ATR1
2.1226618 Up 2.1849225 Up YOR011W AUS1
4.675042 Up 4.2784038 Up YNR058W BIO3
3.1359777 Up 2.564124 Up YNR057C BIO4
4.0968795 Up 3.8620148 Up YNR056C BIO5
2.5255096 Up 2.0384958 Up YJR025C BNA1
7.379563 Up 8.30086 Up YLR267W BOP2
2.327485 Up 3.3769863 Up YML042W CAT2
4.509865 Up 3.985083 Up YPR001W CIT3
2.0578852 Up 3.9770317 Up YHL048W COS8
4.398049 Up 2.9582012 Up YOR100C CRC1
5.347875 Up 2.6645525 Up YMR094W CTF13
7.80064 Up 11.025473 Up YML054C CYB2
2.5044303 Up 3.0635834 Up YIR027C DAL1
2.550858 Up 5.6318183 Up YIR028W DAL4
4.4843516 Up 4.3695993 Up YDL024C DIA3
2.1668363 Up 2.9939582 Up YDR403W DIT1
6.571231 Up 7.6273603 Up YDL174C DLD1
2.6791651 Up 2.0592175 Up YJR137C ECM17
2.2003522 Up 2.5504754 Up YMR062C ECM40
2.4166193 Up 3.266436 Up YBR033W EDS1
3.1554222 Up 2.536958 Up YOR393W ERR1
3.7436044 Up 2.9526684 Up YPL281C ERR2
2.269241 Up 2.2603095 Up YLR377C FBP1
2.2849061 Up 2.2530105 Up YJL221C FSP2
7.659798 Up 7.1114554 Up YIL054W FYV2
76.735115 Up 14.4041395 Up YBR020W GAL1
76.735115 Up 166.01247 Up YBR019C GAL10
78.75131 Up 53.76265 Up YLR081W GAL2
101.56116 Up 93.95381 Up YPL248C GAL4
36.34818 Up 46.29145 Up YBR018C GAL7
5.330396 Up 4.0937595 Up YDR019C GCV1
2.5090592 Up 2.682127 Up YPR184W GDB1
4.222917 Up 3.8256583 Up YAL062W GDH3
2.9879272 Up 8.363512 Up YCR098C GIT1
2.694075 Up 2.6447406 Up YDL223C HBT1
2.7697783 Up 2.462242 Up YOR202W HIS3
2.4303727 Up 2.9735975 Up YCL030C HIS4
2.7684693 Up 2.5967464 Up YIL116W HIS5
3.793316 Up 5.640973 Up YFL011W HXT10
2.7209184 Up 2.324241 Up YHR096C HXT5
3.2667696 Up 3.6192696 Up YJL219W HXT9
2.1079352 Up 2.2038836 Up YMR108W ILV2
3.0951784 Up 8.414132 Up YKL217W JEN1
2.4908004 Up 2.6344728 Up YGL009C LEU1
2.373958 Up 2.3708763 Up YNL104C LEU4
2.5583594 Up 2.961486 Up YIR034C LYS1
2.2018661 Up 6.01615 Up YGR292W MAL12
2.286683 Up 6.6888905 Up YBR299W MAL32
3.6775527 Up 2.285604 Up YKR069W MET1
3.3087142 Up 3.3314137 Up YFR030W MET10
3.7851515 Up 3.1707487 Up YPR167C MET16
2.9498475 Up 2.9523308 Up YLR303W MET17
12.952447 Up 6.8508286 Up YNL277W MET2
4.7936215 Up 3.8631165 Up YIR017C MET28
2.9084132 Up 2.3667684 Up YDR253C MET32
3.1603968 Up 2.1044753 Up YNL036W NCE103
5.837712 Up 11.026299 Up YDL085W NDE2
2.7281747 Up 2.2748764 Up YHR124W NDT80
2.418558 Up 2.8711798 Up YIL164C NIT1
2.4033759 Up 2.2632685 Up YKL120W OAC1
3.7699878 Up 4.3708344 Up YPR194C OPT2
2.0619988 Up 2.2916145 Up YAL064W ORF:YAL064W
4.7190886 Up 5.521752 Up YBL048W ORF:YBL048W
4.3965955 Up 5.554456 Up YBL049W ORF:YBL049W
4.4264603 Up 5.0889173 Up YBR047W ORF:YBR047W
4.103467 Up 6.7295485 Up YCL048W ORF:YCL048W
2.192048 Up 4.3749895 Up YCR099C ORF:YCR099C
2.638211 Up 2.9866683 Up YCR101C ORF:YCR101C
18.36149 Up 5.4998984 Up YCR105W ORF:YCR105W
2.1239603 Up 2.6114385 Up YDL241W ORF:YDL241W
2.5549884 Up 2.244851 Up YDR223W ORF:YDR223W
2.150253 Up 2.8765693 Up YEL057C ORF:YEL057C
3.0552046 Up 6.2938676 Up YEL070W ORF:YEL070W
2.087863 Up 2.793788 Up YER185W ORF:YER185W
3.748168 Up 18.387726 Up YFL052W ORF:YFL052W
2.7145524 Up 6.0371685 Up YFL061W ORF:YFL061W
3.2444153 Up 2.1592965 Up YFR012W-A ORF:YFR012W-A
3.7236464 Up 3.2456906 Up YGL024W ORF:YGL024W
2.2985814 Up 2.8179967 Up YGL059W ORF:YGL059W
2.628734 Up 3.4248686 Up YGL117W ORF:YGL117W
3.6972415 Up 4.12001 Up YGL230C ORF:YGL230C
3.283544 Up 2.5214186 Up YGR043C ORF:YGR043C
2.307301 Up 2.472971 Up YGR050C ORF:YGR050C
4.205944 Up 5.055826 Up YGR110W ORF:YGR110W
3.769867 Up 3.490024 Up YGR161C ORF:YGR161C
2.0598817 Up 2.2321963 Up YHL044W ORF:YHL044W
4.1469936 Up 2.850817 Up YHR029C ORF:YHR029C
2.4945712 Up 2.215926 Up YHR048W ORF:YHR048W
2.241283 Up 3.735952 Up YHR209W ORF:YHR209W
2.6221356 Up 3.084134 Up YIL121W ORF:YIL121W
3.1692755 Up 2.6197135 Up YIL165C ORF:YIL165C
2.2139091 Up 2.1267798 Up YIL172C ORF:YIL172C
2.5514972 Up 5.3436313 Up YIR043C ORF:YIR043C
3.59348 Up 2.4726734 Up YJL045W ORF:YJL045W
5.3583336 Up 8.838197 Up YJL160C ORF:YJL160C
3.1550093 Up 3.1441882 Up YJL213W ORF:YJL213W
2.1492753 Up 3.9401343 Up YKL071W ORF:YKL071W
6.108961 Up 6.504658 Up YKL107W ORF:YKL107W
3.2176416 Up 3.032829 Up YKL162C-A ORF:YKL162C-A
2.549582 Up 2.4074943 Up YKR046C ORF:YKR046C
2.3823843 Up 3.5281072 Up YLR054C ORF:YLR054C
2.1908948 Up 3.9726424 Up YLR194C ORF:YLR194C
5.400229 Up 9.768513 Up YLR311C ORF:YLR311C
4.7420163 Up 4.786415 Up YLR312C ORF:YLR312C
2.472059 Up 2.5937066 Up YMR007W ORF:YMR007W
2.1991053 Up 2.0030406 Up YMR057C ORF:YMR057C
22.084738 Up 15.958245 Up YMR107W ORF:YMR107W
3.4861953 Up 11.092461 Up YMR118C ORF:YMR118C
4.8684373 Up 3.8995376 Up YMR322C ORF:YMR322C
2.6965623 Up 5.0872335 Up YMR323W ORF:YMR323W
2.0763237 Up 2.021945 Up YNL114C ORF:YNL114C
4.7190886 Up 5.7443166 Up YNL335W ORF:YNL335W
2.286996 Up 4.8582363 Up YNR064C ORF:YNR064C
2.2116995 Up 5.510426 Up YNR066C ORF:YNR066C
2.7544036 Up 10.333814 Up YNR073C ORF:YNR073C
2.2676046 Up 3.1759288 Up YOL047C ORF:YOL047C
2.622635 Up 2.360953 Up YOL157C ORF:YOL157C
2.3561645 Up 2.3535578 Up YOL159C-A ORF:YOL159C-A
2.809437 Up 4.3108993 Up YOL162W ORF:YOL162W
2.8775344 Up 4.160822 Up YOL163W ORF:YOL163W
2.7927597 Up 5.7056117 Up YOL166C ORF:YOL166C
3.3838398 Up 2.3424292 Up YOR203W ORF:YOR203W
3.9368253 Up 2.5369966 Up YOR289W ORF:YOR289W
3.0312536 Up 2.4283605 Up YOR338W ORF:YOR338W
2.6611648 Up 3.1574056 Up YOR345C ORF:YOR345C
13.983435 Up 8.293946 Up YPL033C ORF:YPL033C
2.1662319 Up 2.6543095 Up YPL110C ORF:YPL110C
4.4853578 Up 3.4309742 Up YPL280W ORF:YPL280W
2.6383016 Up 3.1795871 Up YPR015C ORF:YPR015C
2.6498392 Up 2.718381 Up YPR061C ORF:YPR061C
3.1841009 Up 3.0100422 Up YPR077C ORF:YPR077C
2.7551093 Up 2.236529 Up YOR130C ORT1
2.897362 Up 2.426087 Up YKR097W PCK1
3.158231 Up 3.0981598 Up YHR071W PCL5
2.2268972 Up 4.8464212 Up YPR002W PDH1
3.7822063 Up 7.592838 Up YBR296C PHO89
3.041848 Up 8.434918 Up YKL163W PIR3
3.034698 Up 4.1046576 Up YIL160C POT1
3.4939804 Up 4.4523406 Up YIL117C PRM5
2.0202703 Up 2.4520059 Up YGL062W PYC1
6.2763557 Up 3.4175785 Up YGL158W RCK1
3.5144181 Up 2.9104614 Up YCR106W RDS1
4.28649 Up 5.964159 Up YBR050C REG2
2.9678848 Up 2.7935257 Up YBR256C RIB5
2.1734767 Up 2.2412622 Up YGL224C SDT1
2.8915923 Up 4.9005575 Up YAL067C SEO1
2.2840748 Up 4.980292 Up YJL089W SIP4
8.228353 Up 6.9054456 Up YMR095C SNO1
7.2946773 Up 5.99051 Up YMR096W SNZ1
2.3738842 Up 2.5341263 Up YNL012W SPO1
2.8698087 Up 2.3937123 Up YMR017W SPO20
2.821707 Up 3.10122 Up YOL091W SPO21
2.4767158 Up 2.5756717 Up YIL073C SPO22
2.4217575 Up 3.1003695 Up YPR007C SPO69
3.9720883 Up 3.8201165 Up YKL218C SRY1
3.1808107 Up 3.0908365 Up YPL092W SSU1
2.3064563 Up 2.933995 Up YKL178C STE3
2.6517718 Up 3.5276618 Up YJR130C STR2
4.557136 Up 4.7047777 Up YGL184C STR3
24.49464 Up 10.887441 Up YBR294W SUL1
4.3167863 Up 2.0504751 Up YLR092W SUL2
5.3514595 Up 7.343781 Up YJR156C THI11
4.995868 Up 5.263618 Up YNL332W THI12
7.22231 Up 5.4404936 Up YDL244W THI13
5.0305867 Up 5.6415343 Up YFL058W THI5
3.988496 Up 4.3984036 Up YER175C TMT1
2.9507484 Up 3.4170206 Up YDR059C UBC5
2.2585113 Up 2.0042167 Up YLL039C UBI4
2.1997151 Up 2.295983 Up YDL170W UGA3
2.5031931 Up 2.1808121 Up YGR065C VHT1
6.214656 Up 2.4561589 Up YAR035W YAT1
4.987914 Up 3.2750573 Up YER024W YAT2
2.3179839 Up 5.0609155 Up YNR065C YSN1
2.4574826 Up 2.6144147 Up YBR046C ZTA1
Open in a new tab

Genes upregulated 2-fold or greater in response to BRCA1 expression at both 23°C (column A) and at 30°C (column B). To facilitate cluster-function analyses, genes are presented alphabetically based on standard gene nomenclature (column F). Systematic gene nomenclature is also provided (column E).

Table 2.

BRCA1-dependent repression at 23° and 30°

Fold change 23° Down Fold change 30° Down Systematic Common
2.6663523 Dn 2.5401378 Dn YNL141W AAH1
2.348434 Dn 2.1471424 dn YNR044W AGA1
2.1432571 Dn 2.8746247 Dn YPL061W ALD6
3.9368253 Dn 3.039458 Dn YGR177C ATF2
2.205411 Dn 3.4943202 Dn YGR108W CLB1
2.640706 Dn 2.4160028 Dn YPR119W CLB2
2.5200815 Dn 5.1660275 Dn YGR109C CLB6
2.0592175 Dn 2.0186563 Dn YMR215W GAS3
2.0306427 Dn 2.456279 Dn YCL036W GFD2
2.2512314 Dn 2.1754 Dn YBR244W GPX2
2.209742 Dn 2.8485 Dn YOL151W GRE2
9.850322 Dn 6.3388696 Dn YOR032C HMS1
3.0393798 Dn 3.3753817 Dn YDL227C HO
2.352046 Dn 2.2479405 Dn YMR032W HOF1
18.128693 Dn 12.658796 Dn YJL153C INO1
2.695608 Dn 2.2437422 Dn YDL003W MCD1
3.2499297 Dn 2.2911003 Dn YAR047C ORF:YAR047C
4.06215 Dn 2.2012846 Dn YDL038C ORF:YDL038C
2.295059 Dn 3.2390332 Dn YDR222W ORF:YDR222W
2.3097382 Dn 2.936668 Dn YER053C-A ORF:YER053C-A
6.423311 Dn 2.7086365 Dn YGR052W ORF:YGR052W
2.2692592 Dn 2.2693837 Dn YHL026C ORF:YHL026C
2.037372 Dn 2.1799862 Dn YIL158W ORF:YIL158W
2.5801733 Dn 2.7344072 Dn YMR088C ORF:YMR088C
2.198507 Dn 2.792885 Dn YNL134C ORF:YNL134C
2.718264 Dn 2.8701663 Dn YNL194C ORF:YNL194C
2.1698606 Dn 2.656128 Dn YNR009W ORF:YNR009W
3.4301567 Dn 3.1999304 Dn YOR315W ORF:YOR315W
3.8612068 Dn 2.2171853 Dn YPL095C ORF:YPL095C
2.0609047 Dn 2.051279 Dn YHR215W PHO12
2.4203475 Dn 2.2458477 Dn YBR092C PHO3
5.1002965 Dn 4.6914625 Dn YMR006C PLB2
2.530907 Dn 2.1181576 Dn YDR501W PLM2
2.8532867 Dn 2.4002085 Dn YNL301C RPL18B
2.2034757 Dn 2.053107 Dn YHR172W SPC97
3.815995 Dn 3.740144 Dn YOR313C SPS4
2.6739023 Dn 2.9553387 Dn YHL028W WSC4
3.9242835 Dn 7.133377 Dn YNL160W YGP1
2.2180047 Dn 5.0412126 Dn YGL255W ZRT1
Open in a new tab

Genes downregulated 2-fold or greater in response to BRCA1 expression at both 23°C and 30°C. Column designations are identical to Table 1.

Repressed genes

BRCA1-deficient human cells exhibit dramatic chromosome segregation defects, inter-sister chromatid gaps and translocations.1,15 Thus, we first wanted to uncover how BRCA1 might affect pathways that contribute to conditional lethality in yeast chromosome segregation mutants. Of the 39 downregulated loci (Table 2), 13 are termed dubious open reading frames or un-annotated and thus are not considered further.4 Importantly, functional-cluster analysis revealed that the largest class of genes downregulated by BRCA1 (6 of the remaining 26) play essential roles in mitosis. CLB1, CLB2 and CLB6 (all three encode different B-type cyclins) were identified as well as MCD1/SCC1 (encoding the key structural sister chromatid cohesion factor). We note that BRCA1 ectopic expression in colon cancer cells similarly showed significant reduction of B-type cyclin and cohesion regulators17,18—attesting to the efficacy of the current approach. Two other factors downregulated in this class are SPC97 (encodes a spindle pole body component associated with microtubule nucleation)19 and HOF1 (encodes a cytokinesis regulatory factor).20 While speculative, a plausible model is that BRCA1-expressing yeast cells are deficient in maintaining both a mitotic state and sister chromatid identity—coupling BRCA1 to aneuploidy pathways (Fig. 1). That these yeast pathways are conserved through evolution and of clinical relevance is supported by findings in vertebrate cell studies that BRCA1 regulates numerous aspects of mitosis that include kinetochore, spindle checkpoint, cyclin-dependent kinase, cohesion and cytokinesis pathways.16-18 In summary, BRCA1 represses a suite of mitotic regulators and structural components that are conserved through evolution, suggesting that the chromosome aberrations and aneuploidy observed in breast/ovarian cancer cells may arise in part through defects in chromosome segregation pathways.

Figure 1.

Figure 1

Open in a new tab

The combination of genetic and microarray analyses indicate that BRCA1 may contribute to cell aneuploidy and chromosomal aberrations via a two-hit mechanism. BRCA1 drives inappropriate elevated expression of CTF13, adversely effecting kinetochore assembly (revealed in the context of COMA kinetochore mutants such as ctf19). BRCA1 reduces expression of genes required for mitotic stability (numerous B-type cyclins, cohesin factor MCD1/SCC1 and spindle pole component SPC97)—all of which are required for high fidelity chromosome segregation.

The remaining 20 downregulated genes fall into 6 other functional clusters (Fig. 2), four of which include phospholipid metabolism and phosphate utilization factors (INO1, PHO3, PHO12 and PBL2), stress responders (ALD6, ATF2, GPX2 and GRE2), cell wall components and plasma membrane transporter (AGA1, GAS3, YPG1 and ZRT1) and ribosome subunits and translation factors (RPL18B, WSC4 and GFD2). The fifth cluster is comprised of transcription regulators (HMS1 and PLMS2). This latter functional-cluster contains a surprisingly small number of genes, given prior studies that mutations in transcriptional responders suppress BRCA1-induced lethality.10,11 The sixth cluster is comprised of orphan genes of unrelated functions (SPS4, HO and AAH1).

Figure 2.

Figure 2

Open in a new tab

Schematic highlighting cluster-function analyses of genes downregulated in response to BRCA1. Cluster defined as ‘other’ not shown. See text for details.

Upregulated genes

We next performed a functional-cluster analysis on the 183 genes that were upregulated in response to BRCA1 (Table 1). 70 un-annotated loci (and an additional 7 genes for which only putative or implied functions exist) were removed from the data set. Functional-cluster analysis of the remaining 106 genes revealed that only a single chromosome segregation gene—encoding the kinetochore factor Ctf13p, was upregulated in response to BRCA1. This observation couples together previously disparate reports that (1) elevated levels of CTF13 are conditionally lethal in ctf19 mutant strains and (2) BRCA1 expression is conditionally lethal in strains mutated in COMA kinetochore components including Ctf19p.9,21,22 Since kinetochore assembly is uniquely sensitive to increased CTF13 dosage, CTF13 upregulation by BRCA1 accounts for the conditional lethality of BRCA1 in ctf19 mutants (Fig. 1)—validating the current study and providing a genetically closed loop of BRCA1 function in yeast through microarray analyses. From these and other results, we posit a two-hit mechanism by which BRCA1 contributes to cell aneuploidy: BRCA1 may drive inappropriate expression of highly dosage-sensitive kinetochore factors and does so in the context of reduced mitotic genes that include B-type cyclins (CLBs) and cohesion factors (MCD1/SCC1).

Of further interest are the roughly 8 genes involved in meiosis and sporulation (including SPO1, SPO20-22 and SPO69), suggesting that BRCA1 expression may inappropriately activate recombination or synapsis pathways that contribute to aneuploidy in cancer cells. The bulk of genes upregulated by BRCA1 function either as permeases/transporters (14 loci) or related biosynthetic pathways (59 loci). In many cases, multiple members of a single pathway were identified (BIO3-BIO5; GAL1, GAL2, GAL4, GAL7 and GAL10; HIS3-HIS5; THI5, THI11-13 and MET1, MET2, MET10, MET16, MET17, MET28 and MET32). Our analyses also revealed 13 loci that contribute to mitochondrial function—which may contribute to the small (petite-like) colony phenotype observed in BRCA1-expressing cells.5,6 We note that many mitochondrial genes are also regulated by BRCA1 in vertebrate cells, while the extent that these genes effect apoptotic responses remains unclear.17,23 Surprisingly, very few upregulated genes (5 loci) could be classified as transcriptional regulators or in modifying transcript stability. Thus, the BRCA1 affects observed in yeast most likely occur directly through interactions with transcription factors and chromatin remodelers—as opposed to BRCA1 upregulation of transcription factors that contribute secondary and thus indirect effects on gene expression.

In summary, the current study addresses key and novel aspects of BRCA1 function in a genetically amenable and conserved response system. Positional analyses made accessible by yeast gene nomenclature illustrates that both individual loci and large and contiguous multi-loci DNA tracts are positively upregulated in response to BRCA1. In contrast, few adjacent genes are downregulated in tandem, suggesting that BRCA1-dependent transcriptional repression in yeast occurs predominantly (but not exclusively) in a gene-specific fashion. We also found that many more than predicted repressed genes are situated immediately adjacent to upregulated genes. Yeast thus provides a powerful avenue to pursue further the chromatin basis of these transition zones.

Labor-intensive vertebrate cell studies previously demonstrated that BRCA1 alters the expression of mitotic components including kinetochore, checkpoint, CDK and cohesion factors.1 The current study reveals that BRCA1 affects identical pathways in yeast. Furthermore, our data show that the kinetochore-encoding locus CTF13 was uniquely upregulated in response to BRCA1. Kinetochore assembly in key mutant backgrounds is highly sensitive to elevated levels of Ctf13p and provides a molecular explanation regarding the conditionally lethality of BRCA1 in kinetochore COMA mutant cells.9 This finding raises the possibility that inappropriate BRCA1 expression in human cells may similarly induce elevated levels of dosage-critical factors and contribute to aberrant chromosome structures observed in breast cancer cells.

Experimental Procedures

10 ml of log growth cultures harboring vector alone or vector harboring BRCA1 were harvested by centrifugation and RNA extracted from the resulting pellets using either hot phenol or RNeasy (Qiagen) procedures.24 In all cases, RNA quality was first assessed by A240/A260 ratio (Nanodrop) and further validated by Agilent 2100 Bioanalyzer. Hybridized one-color samples were prepared using Agilent Yeast Oligo Microarrays (V2) 4X44k format (G2519F), which includes >6,200 ORFs with a total of 45,018 features of 60-mer controls and gene probes, according to Agilent instructions and using Agilent reagents. Paired comparisons were made between Control-23°C and BRCA1-23°C and between Control-30°C and BRCA1-30°C RNA extracts. BRCA1 effects on yeast cell growth is temperature independent.9

One-color microarrays were scanned with an Agilent Microarray Scanner System, which generated the TIFF images of low and high intensity scans utilized by Agilent Feature Extraction Software (v9.5). Feature Extraction processing of fluorescent data corrected signals for background noise, foreground intensities, positive and negative spot controls, background subtraction, and signal normalization. Tab delimited text files generated for each of the four experimental arrays were then analyzed using Agilent Technologies software GeneSpring GX (v9.0.5). Data were processed in GeneSpring GX (v9.0.5) by first filtering on expression intensities to retain features within the 20.0 to 100.0 percentile range followed by filtering on flags for features either present or marginal in at least 1 of the 2 arrays juxtaposed. A fold change threshold of 2.0 was imposed for each pairing. During final manuscript preparations, version GeneSpring (v9.5) was released. Venn re-analyses of our data sets using this updated software identified an additional 54 genes (primarily un-annotated ORFs) common to all data sets with only minor modifications to identified genes.

Supplementary Material

Supplemental Data

Acknowledgments

The authors thank the Cassbens lab group and Dr. Kerry Bloom for comments and Dr. Ken Belanger for sharing of reagents. This work was supported by an award to R.V.S. from the Susan G. Komen for the Cure Foundation (BCTR0707708) and to L.C. from the National Institutes of Health (GM058025). Any opinions, conclusions or recommendations are those of the authors and do not necessarily reflect the views of either Komen for the Cure or N.I.H.

Footnotes

Note Supplementary materials can be found at: www.landesbioscience.com/supplement/SkibbensCC7-24-Sup.pdf

References

  • 1.Murray MM, Mullan PB, Harkin DP. Role played by BRCA1 in transcriptional regulation in response to therapy. Biochem Soc Trans. 2007;35:1342–6. doi: 10.1042/BST0351342. [DOI] [PubMed] [Google Scholar]
  • 2.Aiyar SE, Cho H, Lee J, Li R. Concerted transcriptional regulation by BRCA1 and COBRA1 in breast cancer cells. Int J Biol Sci. 2007;3:486–92. doi: 10.7150/ijbs.3.486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Hosey AM, Gorski JJ, Murray MM, Quinn JE, Chung WY, Stewart GE, et al. Molecular basis for estrogen receptor alpha deficiency in BRCA1-linked breast cancer. J Natl Cancer Inst. 2007;99:1683–94. doi: 10.1093/jnci/djm207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Saccharomyces Genome Database. www.yeastgenome.org.
  • 5.Humphrey JS, Salim A, Erdos MR, Collins FS, Brody LC, Klausner RD. Human BRCA1 inhibits growth in yeast: Potential use in diagnostic testing. Proc Nat Acad Sci. 1997;94:5820–5. doi: 10.1073/pnas.94.11.5820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Coyne RS, McDonald HB, Edgemon K, Brody LC. Functional characterization of BRCA1 sequence variants using a yeast small colony phenotype assay. Cancer Biol Ther. 2004;3:453–7. doi: 10.4161/cbt.3.5.809. [DOI] [PubMed] [Google Scholar]
  • 7.Cantor SB, Bell DW, Ganesan S, Kass EM, Drapkin R, Grossman S, et al. BACH1, a novel helicase-like protein, interacts directly with BRCA1 and contributes to its DNA repair function. Cell. 2001;105:149–60. doi: 10.1016/s0092-8674(01)00304-x. [DOI] [PubMed] [Google Scholar]
  • 8.Skibbens RV. Chl1p, a DNA helicase-like protein in budding yeast, functions in sister chromatid cohesion. Genetics. 2004;166:33–42. doi: 10.1534/genetics.166.1.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Skibbens RV, Sie C, Eastman L. Role of chromosome segregation genes in BRCA1-dependent lethality. Cell Cycle. 2008;7:1–2. doi: 10.4161/cc.7.13.6165. [DOI] [PubMed] [Google Scholar]
  • 10.Westmoreland TJ, Olson JA, Saito WY, Huper G, Marks JR, Bennett CB. DHH1 regulates the G1/S-checkpoint following DNA damage or BRCA1 expression in yeast. J Surg Res. 2003;113:62–73. doi: 10.1016/s0022-4804(03)00155-0. [DOI] [PubMed] [Google Scholar]
  • 11.Bennett CB, Westmoreland TJ, Verrier CS, Blanchette CAB, Sabin TL, Phatnani P, et al. Yeast screens identify the RNA polymeriase II CTD and SPT5 as relevant targets of BRCA1 interaction. PLoS ONE. 2008;1:1448. doi: 10.1371/journal.pone.0001448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Horwitz AA, Afar EB, Heine GF, Shi Y, Parvin JD. A mechanism for transcriptional repression dependent on the BRCA1 E3 ubiquitin ligase. Proc Nat Acad Sci. 2007;104:6614–9. doi: 10.1073/pnas.0610481104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Gough CA, Gojobori T, Imanishi T. Cancer-related mutations in BRCA1-BRCT cause long-range structural changes in protein-protein binding sites: a molecular dynamics study. Proteins. 2007;66:69–86. doi: 10.1002/prot.21188. [DOI] [PubMed] [Google Scholar]
  • 14.Tischkowitz M, Hamel N, Carvalho MA, Birrane G, Soni A, van Beers EH, et al. Pathogenicity of the BRCA1 missense variant M1775K is determined by the disruption of the BRCT phosphopeptide-binding pocket: a multi-modal approach. Eur J Hum Genet. 2008;16:820–32. doi: 10.1038/ejhg.2008.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Skibbens RV. Cell biology of cancer: BRCA1 and sister chromatid pairing reactions? Cell Cycle. 2008;7:449–52. doi: 10.4161/cc.7.4.5435. [DOI] [PubMed] [Google Scholar]
  • 16.Wang R-H, Yu H, Deng C-X. A requirement for breast-cancer-associated gene 1 (BRCA1) in the spindle checkpoint. Proc Nat Acad Sci. 2004;101:17108–13. doi: 10.1073/pnas.0407585101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Bae I, Rih JK, Kim HJ, Kang HJ, Haddad B, Kirilyuk A, Fan S, Avantaggiati ML, Rosen EM. BRCA1 regulates gene expression for orderly mitotic progression. Cell Cycle. 2005;4:1641–66. doi: 10.4161/cc.4.11.2152. [DOI] [PubMed] [Google Scholar]
  • 18.MacLachlan TK, Somasundaram K, Sgagias M, Shifman Y, Muschel RJ, Cowan KH, El-Deiry WS. BRCA1 effects on the cell cycle and the DNA damage response are linked to altered gene expression. J Biol Chem. 2000;275:2777–85. doi: 10.1074/jbc.275.4.2777. [DOI] [PubMed] [Google Scholar]
  • 19.Knop M, Pereira G, Geissler S, Grein K, Schiebel E. The spindle pole body component Spc97p interacts with the gamma-tubulin of Saccharomyces cerevisiae and functions in microtubule organization and spindle pole body duplication. EMBO. 1997;16:1550–64. doi: 10.1093/emboj/16.7.1550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Vallen EA, Caviston J, Bi E. Roles of Hof1p, Bni1p, Bnr1p and myo1p in cytokinesis in Saccharomyces cerevisiae. Mol Biol Cell. 2000;11:593–611. doi: 10.1091/mbc.11.2.593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Hyland KM, Kingsbury J, Koshland D, Hieter P. Ctf19p: A novel kinetochore protein in Saccharomyces cerevisiae and a potential link between the kinetochore and mitotic spindle. J Cell Biol. 1999;145:15–28. doi: 10.1083/jcb.145.1.15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.De Wulf P, McAinsh AD, Sorger PK. Hierarchical assembly of the budding yeast kinetochore from multiple subcomplexes. Genes Dev. 2003;17:2902–21. doi: 10.1101/gad.1144403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Borst P, Jonkers J, Rottenberg What makes tumors multidrug resistant? Cell Cycle. 2007;22:2782–7. doi: 10.4161/cc.6.22.4936. [DOI] [PubMed] [Google Scholar]
  • 24.Schmitt ME, Brown TA, Trumpower BL. A rapid and simple method for preparation of RNA from Saccharomyces cerevisae. Nuc Acids Res. 1990;18:3091–2. doi: 10.1093/nar/18.10.3091. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Data
NIHMS341691-supplement-Supplemental_Data.pdf (157.9KB, pdf)

RESOURCES