Open Access

The nuclear localization of SWI/SNF proteins is subjected to oxygen regulation

  • Ranita Ghosh Dastidar1,
  • Jagmohan Hooda1,
  • Ajit Shah1,
  • Thai M Cao1,
  • Robert Michael Henke1 and
  • Li Zhang1Email author
Contributed equally
Cell & Bioscience20122:30

DOI: 10.1186/2045-3701-2-30

Received: 13 July 2012

Accepted: 17 August 2012

Published: 29 August 2012

Abstract

Background

Hypoxia is associated with many disease conditions in humans, such as cancer, stroke and traumatic injuries. Hypoxia elicits broad molecular and cellular changes in diverse eukaryotes. Our recent studies suggest that one likely mechanism mediating such broad changes is through changes in the cellular localization of important regulatory proteins. Particularly, we have found that over 120 nuclear proteins with important functions ranging from transcriptional regulation to RNA processing exhibit altered cellular locations under hypoxia. In this report, we describe further experiments to identify and evaluate the role of nuclear protein relocalization in mediating hypoxia responses in yeast.

Results

To identify regulatory proteins that play a causal role in mediating hypoxia responses, we characterized the time courses of relocalization of hypoxia-altered nuclear proteins in response to hypoxia and reoxygenation. We found that 17 nuclear proteins relocalized in a significantly shorter time period in response to both hypoxia and reoxygenation. Particularly, several components of the SWI/SNF complex were fast responders, and analysis of gene expression data show that many targets of the SWI/SNF proteins are oxygen regulated. Furthermore, confocal fluorescent live cell imaging showed that over 95% of hypoxia-altered SWI/SNF proteins accumulated in the cytosol in hypoxic cells, while over 95% of the proteins were nuclear in normoxic cells, as expected.

Conclusions

SWI/SNF proteins relocalize in response to hypoxia and reoxygenation in a quick manner, and their relocalization likely accounts for, in part or in whole, oxygen regulation of many SWI/SNF target genes.

Keywords

Hypoxia response Oxygen regulation SWI/SNF Live cell imaging Protein localization

Background

Living organisms ranging from yeast to mammals use oxygen to generate their cellular energy supply and to synthesize important biomolecules. Hence, they need to respond effectively to changes in oxygen levels in the environment, particularly to hypoxia[1, 2]. In humans, hypoxia is responsible for death or damage by the ischemia accompanying heart attack, stroke, and traumatic injuries[35]. The molecular and cellular events induced by changes in oxygen levels are very broad in eukaryotes. For example, over 20% of yeast genes change their transcript levels in response to hypoxia[6]. In the human arterial endothelial cells, more than 8% of all genes alter their transcript levels by at least 1.5-fold in response to hypoxia[7]. In the human primary astrocytes, more than 5% of the genes alter their transcript levels by at least 2-fold in response to hypoxia[8]. Such broad changes in gene expression likely involve coordinated actions of multiple pathways and regulators.

Previous studies have identified several transcriptional regulators, including Mga1 and Rox1, that can mediate oxygen regulation of gene expression in the yeast Saccharomyces cerevisiae[9, 10]. However, these regulators can account for the regulation of only a fraction of hypoxia-regulated genes[6]. Many other regulators are likely involved in mediating oxygen regulation. Recently, in an effort to systematically identify proteins that can mediate oxygen regulation and signaling, we performed a genome-wide screen for proteins that exhibit altered cellular distribution patterns in response to hypoxia and reoxygenation[11]. We found that over 200 proteins alter their cellular locations in response to hypoxia. Particularly, under hypoxia, a good number (at least 121) of nuclear proteins do not localize to the nucleus, but accumulate in the cytosol. In response to reoxygenation, they readily localize to the nucleus. Notably, many of these hypoxia-redistributed nuclear proteins are subunits of key regulatory complexes involved in chromatin remodeling (such as the SWI/SNF complex)[1214], in transcriptional regulation (such as the SAGA complex)[15], and in splicing (such as the MRP complex)[16]. Hence, it is conceivable that some of these complexes can play a dominant role in mediating oxygen regulation of gene expression.

To further assess the roles of these regulators in mediating oxygen signaling and regulation, we examined the time course characteristics of the relocalization of these proteins in response to hypoxia and reoxygenation. We found a small group of nuclear proteins relocalized in a significantly shorter time period in response to both hypoxia and reoxygenation, when compared to other proteins. These proteins include three components of the SWI/SNF complex. Furthermore, using confocal fluorescent imaging of live cells, we quantitatively characterized the effect of hypoxia on the distribution of SWI/SNF proteins. We found that in live hypoxic cells, over 95% of Swi3, Snf5, Snf6, Snf11, Snf12 and Swp82 were in the cytosol, while over 95% of hypoxia-unaffected proteins, such as Swi2 and Taf14, were in the nucleus. These results suggest that hypoxia can significantly alter the composition and property of the SWI/SNF complex and mediate oxygen regulation of gene expression.

Results

Among the hypoxia-redistributed nuclear proteins we previously identified, some are likely involved in mediating oxygen signaling and regulation of gene expression. Particularly, proteins that change their locations in relatively shorter time periods are likely the regulators that initiate further downstream events in responses to hypoxia and reoxygenation. In other words, they are likely to be positioned in the upstream of the hierarchy of the molecular events elicited by hypoxia or reoxygenation, and are responsible for initiating downstream changes such as those in gene expression. We therefore decided to characterize the time course response of the hypoxia-redistributed nuclear proteins in response to hypoxia and reoxygenation.

First, we examined the time course characteristics of nuclear proteins in response to hypoxia. We found that all proteins became predominantly cytosolic after exposure to hypoxia for 12 hours; see Snf11 in Figure1A for an example. One group of these proteins became predominantly cytosolic after only 6 hours or shorter times; see Swp82 in Figure1A for an example. This group has 48 proteins (see Table1). They include several transcriptional regulators and regulators of chromatin, DNA replication and repair, and RNA processing (Figure2). Notably, five components of the SWI/SNF complex relocalized in 6 hours (see Table1 and Figure2), suggesting that they may have a signaling role in initiating downstream events.
Figure 1

Time course characteristics of protein relocalization elicited by hypoxia or reoxygenation. (A) The time courses of relocalization of Snf11 and Swp82 in response to hypoxia. Cells expressing Snf11-GFP or Swp82-GFP were grown in air and then shifted to hypoxic growth conditions. At various time points, cells were imaged, and the number of cells showing GFP-tagged proteins in the nucleus (N) or cytosol (C) was counted. The percentage of cells showing nuclear locations is calculated and plotted. (B) The time courses of relocalization of Snf5 and Swi3 in response to reoxygenation. Cells expressing Snf5-GFP or Swi3-GFP were grown under hypoxia and then shifted to normoxic growth conditions. At various time points, cells were imaged, and the number of cells showing GFP-tagged proteins in the nucleus (N) or cytosol (C) was counted. The percentage of cells showing nuclear localization is plotted here.

Table 1

Nuclear proteins that relocalized to the cytosol in response to hypoxia in a shorter time period

ORF name

Gene name

Description

YOR113W

AZF1

Involved in glucose induction of CLN3 transcription

YML102W

CAC2

Component of the chromatin assembly complex

YKL022C

CDC16

Subunit of the anaphase-promoting complex/cyclosome

YFR036W

CDC26

Subunit of the Anaphase-Promoting Complex/Cyclosome

YIL036W

CST6

Member of the ATF/CREB family

YML113W

DAT1

DNA binding protein that recognizes oligo(dA).oligo(dT) tracts

YIL131C

FKH1

Forkhead family transcription factor

YNL068C

FKH2

Forkhead family transcription factor

YDR096W

GIS1

JmjC domain-containing histone demethylase

YDR295C

HDA2

Subunit of a class II histone deacetylase complex

YPR179C

HDA3

Subunit of a class II histone deacetylase complex

YOR038C

HIR2

Subunit of the HIR nucleosome assembly complex

YDL108W

KIN28

Subunit of the transcription factor TFIIH

YNR024W

MPP6

RNA binding protein that associates with the exosome

YGL013C

PDR1

Master regulator of multidrug resistance genes

YDL106C

PHO2

Homeobox transcription factor

YJR006W

POL31

DNA polymerase III (delta) subunit

YNL282W

POP3

Subunit of both RNase MRP

YBL018C

POP8

Subunit of both RNase MRP

YKL113C

RAD27

5' to 3' exonuclease, 5' flap endonuclease

YPL153C

RAD53

Required for cell-cycle arrest in response to DNA damage

YMR182C

RGM1

Putative transcriptional repressor

YBR095C

RXT2

Subunit of the histone deacetylase Rpd3L complex

YDR180W

SCC2

Subunit of cohesin loading factor (Scc2p-Scc4p)

YGL066W

SGF73

Subunit of SAGA histone acetyltransferase complex

YIL104C

SHQ1

Required for the assembly of box H/ACA snoRNPs

YHR206W

SKN7

Regulator for optimal induction of heat-shock genes

YDR073W

SNF11

Subunit of the SWI/SNF chromatin remodeling complex

YNR023W

SNF12

73 kDa subunit of the SWI/SNF chromatin remodeling complex

YBR289W

SNF5

Subunit of the SWI/SNF chromatin remodeling complex

YHL025W

SNF6

Subunit of the SWI/SNF chromatin remodeling complex

YCR033W

SNT1

Subunit of the Set3C deacetylase complex

YPL138C

SPP1

Subunit of the COMPASS complex

YBR152W

SPP381

Component of U4/U6.U5 tri-snRNP

YDR464W

SPP41

Negative regulator of expression of PRP4 and PRP3

YDR392W

SPT3

Subunit of the SAGA and SAGA complexes

YJL176C

SWI3

Subunit of the SWI/SNF chromatin remodeling complex

YFL049W

SWP82

Subunit of the SWI/SNF chromatin remodeling complex

YDR334W

SWR1

Component of the SWR1 complex

YDR416W

SYF1

Component of the spliceosome complex

YDR079C-A

TFB5

Component of TFIIH

YNL273W

TOF1

Subunit of a replication-pausing checkpoint complex

YPL203W

TPK2

cAMP-dependent protein kinase catalytic subunit

YBR030W

YBR030W

Putative ribosomal lysine methyltransferase

YGR093W

YGR093W

Putative debranching enzyme associated ribonuclease

YLR455W

YLR455W

Putative protein of unknown function

YNL035C

YNL035C

Putative protein of unknown function

YPR107C

YTH1

Component of cleavage and polyadenylation factor

Figure 2

Graphical representation of protein-protein interaction networks for the nuclear proteins that localized to the cytosol in response to hypoxia in a shorter time period. The information on the biochemical interactions and complex formation of the 48 faster responding nuclear proteins (listed in Table1) was downloaded from the SGD database, and then imported to Cytoscape for network construction. The proteins are shown as round nodes in different colors based on their cellular functions. The GO terms for protein complexes or functional categorizations are indicated and are shown in square nodes. Nodes of the same sub-networks are colored similarly, and a key for the coloring of the nodes is shown. Lines represent an association of the protein to a particular complex or functional GO term.

Second, we characterized the changes in protein distribution when cells grown under hypoxia were exposed to oxygen. We found that 76 hypoxia-redistributed nuclear proteins (see Table2) recovered their nuclear locations in the majority of cells in one hour; see Swi3 in Figure1B for an example. The rest of the proteins recovered their nuclear location in the majority of the cells in 2 or more hours; see Snf5 in Figure1B for an example. Among these nuclear proteins, 17 of them responded to both hypoxia and reoxygenation in shorter times than the rest of the proteins (see Figure3). Notably, 3 of these faster responding proteins are components of the SWI/SNF complex (Figure3). These and previous results strongly suggest that the SWI/SNF proteins play regulatory roles in mediating oxygen regulation and hypoxia response. Given their roles in chromatin remodeling and transcriptional regulation[17, 18], they are likely responsible for initiating certain changes in gene expression in response to changes in oxygen levels. Although 14 other proteins also responded to hypoxia and reoxygenation in shorter times, they are generally not components of one regulatory complex (Figure3).
Table 2

Proteins that recovered their nuclear locations in response to oxygen in a shorter time period

ORF name

Gene name

Description

YBR236C

ABD1

Methyltransferase

YPR180W

AOS1

Smt3p (SUMO) activator

YJL115W

ASF1

Nucleosome assembly factor

YNR010W

CSE2

Subunit of the RNA polymerase II mediator complex

YIL036W

CST6

Member of the ATF/CREB family

YJL006C

CTK2

Beta subunit of C-terminal domain kinase I

YEL018W

EAF5

Subunit of the NuA4 acetyltransferase complex

YMR277W

FCP1

Carboxy-terminal domain (CTD) phosphatase

YCL011C

GBP2

Poly(A+) RNA-binding protein

YGR252W

GCN5

Subunit of the ADA and SAGA complexes

YDR096W

GIS1

JmjC domain-containing histone demethylase

YDR174W

HMO1

Chromatin associated high mobility group family member

YFL013C

IES1

Subunit of the INO80 chromatin remodeling complex

YHR085W

IPI1

Essential component of the Rix1 complex

YIL026C

IRR1

Subunit of the cohesin complex

YDL108W

KIN28

Subunit of the transcription factor TFIIH

YDL087C

LUC7

Associated with the U1 snRNP complex

YMR043W

MCM1

Involved in cell-type-specific transcription

YDL005C

MED2

Subunit of the RNA polymerase II mediator complex

YMR070W

MOT3

Nuclear transcription factor mediating hypoxia response

YKL059C

MPE1

Essential conserved subunit of CPF

YNR024W

MPP6

Nuclear RNA binding protein

YLR116W

MSL5

Component of the commitment complex

YPR144C

NOC4

Mediating maturation and nuclear export of 40S

YHR133C

NSG1

Regulator of sterol biosynthesis

YKR082W

NUP133

Subunit of the nuclear pore complex

YAR002W

NUP60

Subunit of the nuclear pore complex

YJL061W

NUP82

Nucleoporin, subunit of the nuclear pore complex (NPC)

YOL115W

PAP2

Catalytic subunit of TRAMP

YDR228C

PCF11

mRNA 3' end processing factor

YMR076C

PDS5

Required for sister chromatid condensation and cohesion

YNL282W

POP3

Subunit of both RNase MRP

YGR030C

POP6

Subunit of both RNase MRP

YBL018C

POP8

Subunit of both RNase MRP

YLL036C

PRP19

Splicing factor associated with the spliceosome

YGR156W

PTI1

Pta1p Interacting protein

YKL113C

RAD27

5' to 3' exonuclease, 5' flap endonuclease

YGL246C

RAI1

Required for pre-rRNA processing

YNL216W

RAP1

Involved in either activation or repression of transcription

YDR195W

REF2

RNA-binding protein

YAR007C

RFA1

Subunit of heterotrimeric Replication Protein A

YNL290W

RFC3

Subunit of heteropentameric Replication factor C

YOL094C

RFC4

Subunit of heteropentameric Replication factor C

YHR197W

RIX1

Essential component of the Rix1 complex

YMR061W

RNA14

Cleavage and polyadenylation factor I (CF I) component

YJL011C

RPC17

RNA polymerase III subunit C17

YER117W

RPL23B

Component of the large (60S) ribosomal subunit

YDR427W

RPN9

Non-ATPase regulatory subunit of the 26S proteasome

YHR062C

RPP1

Subunit of both RNase MRP

YBR095C

RXT2

Subunit of the histone deacetylase Rpd3L complex

YIL084C

SDS3

Component of the Rpd3p/Sin3p deacetylase complex

YJL168C

SET2

Histone methyltransferase

YIL104C

SHQ1

Required for the assembly of box H/ACA snoRNPs

YHR206W

SKN7

Regulator of heat-shock genes

YGR074W

SMD1

Core Sm protein Sm D1

YHL025W

SNF6

Subunit of the SWI/SNF chromatin remodeling complex

YMR016C

SOK2

Involved in the cAMP-dependent protein kinase signaling

YBR152W

SPP381

mRNA splicing factor

YER161C

SPT2

Involved in negative regulation of transcription

YDR392W

SPT3

Subunit of the SAGA and SAGA-like complexes

YIL143C

SSL2

Component of RNA polymerase transcription factor TFIIH

YBR231C

SWC5

Component of the SWR1 complex

YJL176C

SWI3

Subunit of the SWI/SNF chromatin remodeling complex

YFL049W

SWP82

Subunit of the SWI/SNF chromatin remodeling complex

YGR129W

SYF2

Component of the spliceosome complex

YGR274C

TAF1

TFIID subunit (145 kDa)

YGL112C

TAF6

Subunit (60 kDa) of TFIID and SAGA complexes

YDR311W

TFB1

Subunit of TFIIH and nucleotide excision repair factor complexes

YPL203W

TPK2

cAMP-dependent protein kinase catalytic subunit

YDR165W

TRM82

Subunit of a tRNA methyltransferase complex

YNL246W

VPS75

NAP family histone chaperone

YOR229W

WTM2

Regulator of meiosis, silencing, and expression of RNR genes

YHR090C

YNG2

Subunit of the NuA4 histone acetyltransferase complex

YIL063C

YRB2

Involved in nuclear processes of the Ran-GTPase cycle

YGR270W

YTA7

Regulator of histone gene expression

YPR107C

YTH1

Component of cleavage and polyadenylation factor

Figure 3

Graphical representation of protein-protein interaction networks for the nuclear proteins that changed their locations in response to hypoxia and reoxygenation in shorter time periods. The information on the biochemical interactions and complex formation of the 17 faster responding nuclear proteins was downloaded from the SGD database, and then imported to Cytoscape for network construction. The proteins are shown as round nodes in different colors based on their cellular functions. The GO terms for protein complexes or functional categorizations are indicated and shown in square nodes. Nodes of the same sub-networks are colored similarly, and a key for the coloring of the nodes is shown. Lines represent an association of the protein to a particular complex or functional GO term.

Therefore, we decided to further characterize the effect of hypoxia on the SWI/SNF proteins. First, we examined if changes in oxygen levels affect the protein levels of SWI/SNF proteins. To this end, we detected and compared the levels of SWI/SNF proteins in hypoxic and normoxic cells. We used yeast strains expressing the SWI/SNF proteins with the TAP tag at the C-terminus from the natural chromosomal locations[19]. We found that the levels of all detected SWI/SNF proteins were not significantly affected by hypoxia (Figure4). The variations in the ratios of protein levels in hypoxic vs. normoxic cells were generally less than 30%, suggesting that hypoxia did not cause significant degradation of the Swi/Snf proteins during the time period when the proteins would be relocalized to the cytosol. These proteins include those whose cellular location was affected by hypoxia, such as Snf6, Swi3, Swp82 and Snf11 (see Figure4). They also include all those SWI/SNF proteins whose localization was not affected by hypoxia. These results show that the levels of SWI/SNF proteins are not regulated by oxygen levels.
Figure 4

Western blot showing TAP-tagged proteins in extracts prepared from normoxic and hypoxic cells. Shown here are proteins in extracts from the parent BY4741 cells without any TAP-tagged proteins expressed (N), and from cells grown in air (A) or under hypoxia (H) which expresses Snf6-TAP (molecular mass: 58 kDa), Swi3-TAP (113 kDa), Swp82-TAP (90 kDa), Snf11-TAP (40 kDa), Swi1-TAP (168 kDa), Swi2-TAP (214 kDa), Arp7-TAP (74 kDa), Arp9-TAP (73 kDa), Taf14-TAP (47 kDa), respectively. For the hypoxic condition, cells were placed in a hypoxia chamber for up to 12 hours (the time period necessary for the proteins to relocate to the cytosol). The intensity of bands representing the Swi/Snf proteins was quantified, and the intensity ratios of the bands representing the Swi/Snf proteins in hypoxic vs. normoxic cells were plotted and shown below the Western blot images. The data plotted are averages of three replicates.

Therefore, the regulation of nuclear localization is likely the dominant mechanism mediating oxygen regulation of SWI/SNF proteins and the regulation of their targets. To further confirm the regulation of nuclear localization of the SWI/SNF proteins by oxygen, we quantitatively examined and compared their distribution in live hypoxic and normoxic cells, by using confocal fluorescent live cell imaging. As expected, for the SWI/SNF proteins whose localization was not affected by oxygen levels, over 95% of the proteins was present in the nucleus in both normoxic and hypoxic cells (see Figure5). Figure5A shows the distribution of Taf14 in air and under hypoxia, while Figure5B shows the distribution of Swi2. For those proteins whose localization was affected by oxygen, over 95% of the proteins was present in the nucleus in air, whereas over 95% of the proteins was present in the cytosol under hypoxia (Figures6 and7). Figure6A shows the distribution of Swi3 in normoxic cells, while Figure6B shows the distribution of Swi3 in hypoxic cells. We also quantified the distribution of other hypoxia-relocalized SWI/SNF proteins (Figure7). Figure7A-E show the distribution of Snf5, Snf6, Snf11, Snf12 and Swp82 in hypoxic cells (The images for normoxic cells invariably showed nuclear localization, as expected and as shown in Figures5 and6, and are therefore omitted). Clearly, Swi2, Snf5, Snf6, Snf11, Snf12 and Swp82 proteins were transported to the nucleus in normoxic cells, but they accumulated in the cytosol in hypoxic cells.
Figure 5

Examples of GFP, DAPI and merged confocal fluorescent images of cells expressing proteins whose cellular localization is not affected by hypoxia. Cells expressing Taf14-GFP (A) or Swi2-GFP (B) were grown in air or under hypoxia (Hyp), and the images were captured. The percentages of GFP fluorescence in the nucleus (N) or cytosol (C) were quantified and plotted here. The scale bar represents 1 μm.

Figure 6

DAPI and merged confocal fluorescent images of cells expressing Swi3-GFP. Cells were grown in air or under hypoxia (Hyp), and the images were captured. The percentages of GFP fluorescence in the nucleus (N) or cytosol (C) was quantified and plotted here. The scale bar represents 1 μm.

Figure 7

DAPI and merged confocal fluorescent images of cells expressing SWI/SNF proteins whose cellular location is affected by hypoxia. Cells expressing Snf5-GFP, Snf6-GFP, Snf11-GFP, Snf12-GFP and Swp82-GFP were grown in air or under hypoxia (Hyp), and the images were captured. Only the images of hypoxic cells are shown, because the normoxic cells all exhibit the same nuclear pattern as shown in Figures5 and6. The percentages of GFP fluorescence in the nucleus (N) or cytosol (C) were quantified and plotted here. The scale bar represents 1 μm.

To further ascertain the role of SWI/SNF proteins in oxygen regulation of gene expression, we determined if and how many oxygen-regulated genes are SWI/SNF protein targets as well. To this end, we used our previous microarray and computational work analyzing genes regulated by oxygen/ and Δ hap1 cells[6]. We also used the previously identified targets of 263 transcription factors[20]. Using these two sets of data and the R program, we identified those hypoxia-regulated genes that are targets of SWI/SNF proteins and calculated the p-values. Table3 shows that in the wild type HAP1 cells, 95, 112, 67, 109, 9 and 19 target genes of Swi2, Swi3, Snf5, Snf6, Snf11 and Taf14, respectively, are oxygen regulated. In Δ hap1 cells, similar numbers of these SWI/SNF targets are hypoxia altered. These results strongly suggest that SWI/SNF proteins play a major role in mediating oxygen regulation and hypoxia responses. Furthermore, the changes in the relocalization of SWI/SNF proteins in response to hypoxia are completed between 6–12 hours or 1–2 generations; and the changes in the relocalization of SWI/SNF proteins in response to reoxygenation are completed in less than one generation. In contrast, the transcriptome response to hypoxia are completed after 5–6 generations; and the transcriptome response to reoxygenation are completed in 2 generations[21]. These results show that changes in SWI/SNF protein localization precede transcriptome responses. They therefore strongly suggest that oxygen regulation of SWI/SNF protein localization contribute to, at least in part, oxygen regulation of gene expression.
Table 3

The number of Swi/Snf targets whose transcript level is regulated by oxygen

 

HAP1 cells

Δ hap1 cells

 

Targets

p-value

Targets

p-value

Swi2

95

7.55E-69

119

2.87E-69

Swi3

112

6.14E-94

118

7.31E-102

Snf5

67

5.24E-49

71

1.05E-53

Snf6

109

8.06E-73

139

1.10E-107

Snf11

9

6.79E-10

6

7.45E-06

Taf14

19

8.09E-19

20

3.0E-20

Discussion

The SWI/SNF complex is an ATP-dependent chromatin remodeling complex[22]. Its composition and function are conserved from yeast to humans[13]. In yeast, more than 10% of the genes are the targets of SWI/SNF proteins, although the targets of different SWI/SNF proteins are different[20]. Hypoxia and reoxygenation induce changes in gene expression in over 20% of yeast genes[6]. Such broad changes in gene expression involve the action of an array of regulators. Previous studies have shown that Mga2, Rox1, Hap1 and Mot3 are all involved in mediating oxygen regulation of several subsets of genes[6, 21, 23, 24]. In this report, we show that several SWI/SNF proteins alter their subcellular localization readily in response to hypoxia or reoxygenation and that this change in subcellular localization likely contributes to oxygen regulation of SWI/SNF target genes.

Because the targets of SWI/SNF proteins overlap but are not identical[20], it is likely that different SWI/SNF proteins act on different groups of genes and control their expression. Here, we show that six SWI/SNF proteins accumulate in the cytosol in hypoxic cells, and relocalize to the nucleus in response to reoxygenation. Furthermore, several of the SWI/SNF proteins respond to hypoxia or reoxygenation and relocalize in a relatively quick manner. The redistribution of the six SWI/SNF proteins in the cytosol should presumably change the composition, and thereby the function or selectivity of the SWI/SNF complexes in the nucleus. Hence, the relocalization can affect the target expression of not only these SWI/SNF proteins whose localization is altered by hypoxia, such as Swi3, but also those whose localization is not affected by hypoxia, such as Swi2 and Taf14 (Table3). Very likely, under hypoxia, because Swi3 and other proteins are predominantly present in the cytosol, the nuclear SWI/SNF proteins, such as Swi2, are likely complexed with other proteins, and act as chromatin remodelers on different sets of target genes. This explains why many SWI/SNF target genes are altered by hypoxia/reoxygenation (Table3). Previous studies showed that Swi2, Arp7 and Arp9 form a core subcomplex possessing the ATP-dependent remodeling activity[25], while Swi3 controls SWI/SNF assembly, ATP-dependent H2A-H2B displacement, as well as recruitment to target genes[25, 26]. Notably, the nuclear localization of Swi2, Arp7 and Arp9 is not affected by hypoxia, while Swi3 is affected. This supports the idea that the core complex can associate with other as yet unidentified proteins and form a different kind of SWI/SNF complexes in the nucleus in hypoxic cells.

These results suggest a model for how oxygen may modulate SWI/SNF complex composition and function (Figure8). In normoxic cells, SWI/SNF components form complexes in the nucleus and remodel chromatin structure at their target genes. In hypoxic cells Swi3 and other five SWI/SNF proteins accumulate in the cytosol, leaving the Swi2-Arp7-Arp9 core subcomplex available to interact with other proteins. Consequently, a different kind of SWI/SNF complex containing the core complex and other proteins (A, B and C in Figure8) can be formed and act to remodel chromatin and control gene expression in different sets of genes. In response to reoxygenation, Swi3 and other proteins can readily relocalize to the nucleus, forming the normoxic SWI/SNF complexes, and re-establish gene expression patterns under normoxic conditions.
Figure 8

A cartoon illustrating how oxygen may affect SWI/SNF composition and function. In normoxic cells, the components form the SWI/SNF complex in the nucleus, enabling it to remodel chromatin at the target genes. In hypoxic cells, Swi3 and five other components are retained in the cytosol, perhaps due to modifications of these components and/or interactions with unidentified factor(s) X. In the nucleus, Swi2 and other remaining components may interact with some other proteins (marked as A, B, and C), forming complexes with different composition and targeting different sets of genes.

This model is also consistent with a recent study showing that Swi3 is a key regulator in controlling respiration genes[27]. The authors used a computational approach to analyze modules of genes with a common regulation that are affected by specific DNA polymorphisms. They integrated genotypic and expression data for individuals in a segregating population with complementary expression data of strains mutated in a variety of regulatory proteins, in order to identify regulatory-linkage modules. In so doing, they found that Swi3 is a dominant regulator in the control of respiratory gene expression[27]. The effect of swi3 deletion is stronger than that of known respiratory regulators, including Hap2/3/4/5, Mot3 and Rox1. This is in complete agreement with our results showing that hundreds of SWI/SNF targets are altered by hypoxia (Table3), and supports our model (Figure8). The regulation of SWI/SNF protein localization may also occur in other eukaryotes. For example, in mammalian cells, recent studies showed that SWI/SNF proteins are important for oxygen regulation in mammalian cells[28, 29]. It is likely that SWI/SNF proteins can respond to changes in oxygen levels and regulate gene expression in diverse eukaryotes.

Conclusions

Several SWI/SNF proteins, including Swi3, Snf6 and Swp82, respond to hypoxia or reoxygenation and alter their subcellular distribution in a relatively quick manner. This change in localization likely contributes to oxygen regulation of SWI/SNF target genes.

Methods

Yeast strains and antibodies

The yeast GFP clone collection of 4159 strains expressing GFP-tagged proteins[30] was purchased from Invitrogen Corp. The anti-TAP monoclonal antibody was purchased from Open Biosystems.

The creation of hypoxic growth conditions

Hypoxic (~10 ppb O2) growth condition was created by using a hypoxia chamber (Coy Laboratory, Inc.) and by filling the chamber with a mixture of 5% H2 and 95% N2 in the presence of a palladium catalyst[31]. The oxygen level in the chamber was monitored by using the Model 10 gas analyzer (Coy Laboratory, Inc.). The precise level of oxygen was also estimated by using a CHEMetrics rhodazine oxygen detection kit (K-7511) with the minimum detection limit at 1 ppb, and a range of 0–20 ppb. The hypoxic state was further confirmed by measuring oxygen-controlled promoter activities, including UAS1/CYC1, ANB1 and OLE1[9, 10, 31].

Time course characterization of cellular localization of SWI/SNF proteins

For a time course characterization of SWI/SNF protein relocalization in response to hypoxia or reoxygenation, we used a previously defined nuclear protein import assay in yeast[3234]. Briefly, cells expressing GFP-tagged proteins at various time points of hypoxia or reoxygenation treatment were collected, and images were acquired. At least 25 cells were counted at each time point, and three sets of cells were counted. A particular cell was counted as having the GFP-tagged protein in the nucleus if the nucleus was much brighter than the surrounding cytoplasm and a clear nuclear-cytoplasmic boundary was visible. Cells with excessive bright or weak fluorescence or with aberrant morphology were not scored.

Confocal fluorescent live cell imaging and quantitation

GFP-tagged strains were grown in synthetic complete media in air or in a hypoxia chamber. Cells were collected and subjected to confocal fluorescent imaging and quantitation. Image acquisition of live cells was performed by using a Perkin Elmer UltraView ERS Spinning Disc Confocal Microscope (Perkin Elmer, Waltham, MA) with a Zeiss 100x/1.4 Oil Immersion objective (Carl Zeiss, Thornwood, NY). High speed images were captured by using a Hamamatsu EMCCD C9100 digital camera (Hamamatsu Corporation, Bridgewater, NJ). Z-stacks were recorded for the DAPI channel (EX 405nm) and the GFP channel (EX 488nm) by moving the objective turret with a UltraView z-focus drive (Perkin Elmer, Waltham, MA). Volocity 5.4.2 (Improvision, Perkin Elmer, MA, USA) was used for image acquisition. The 3D confocal images were analyzed, and statistical data were collected by using Imaris 7.4.0 (Bitplane, South Windsor, CT).

Preparation of yeast cell extracts and Western blotting

Yeast cells expressing various TAP-tagged proteins were grown to an optical density (OD600) of approximately 0.8. Cells were harvested and resuspended in 3 packed cell volumes of buffer (20 mM Tris, 10 mM MgCl2, 1 mM EDTA, 10% glycerol, 1 mM dithiothreitol, 0.3 M NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 mg of pepstatin per ml, 1 mg of leupeptin per ml). Cells were then permeabilized by agitation with 4 packed cell volumes of glass beads, and extracts were collected as described previously[35]. Protein concentrations were determined by the BCA (bicinchoninic acid) protein assay kit (Pierce).

For Western blotting, approximately 100 μg of whole-cell extracts were first separated on 8% sodium dodecyl sulfate (SDS)–polyacrylamide gels and then transferred to polyvinylidene difluoride or nitrocellulose membranes (Bio-Rad Laboratories). TAP-tagged proteins were detected by using a monoclonal antibody against TAP and a chemiluminescence Western blotting kit (Roche Diagnostics). The signals were detected and quantified by using a Kodak image station 4000MM Pro with the molecular imaging software, version 4.5.

Protein GO analysis and construction of the network map

The analysis of functional categories of relocalized proteins was performed on Funspec (http://funspec.med.utoronto.ca/). For constructing the protein network map, the Cytoscape application program (http://www.cytoscape.org/) was used. The faster responding proteins identified were mapped according to their GO terms. They were obtained by using the SGD Gene Ontology Slim Mapper Web Tool set to "Macromolecular Complex terms: Components" on the SGD website. The mapped output file was reformatted into a Cytoscape compatible network file, and the network map was created. The network map was further graphically refined by using the Canvas application program. The subcellular compartments of these proteins in normoxic cells were designated based on data from the O'Shea lab[30].

Notes

Declarations

Acknowledgments

This work was supported by NIH grant GM62246 (LZ). We would like to acknowledge the assistance of the UT Southwestern Live Cell Imaging Facility, a Shared Resource of the Harold C. Simmons Cancer Center, supported in part by an NCI Cancer Center Support Grant, 1P30 CA142543-01.

Authors’ Affiliations

(1)
Department of Molecular and Cell Biology, Center for Systems Biology, University of Texas at Dallas

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© Ghosh Dastidar et al.; licensee BioMed Central Ltd. 2012

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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