BACKGROUND
the gaga factor  of drosophila is a ubiquitous transcription factor that plays important roles in multiple processes ranging from regulation of gene expression to the structural organization of heterochromatin and chromatin remodeling  <cit> .
genetically, gaf is classified as a member of the trithorax group proteins  counteracting the silencing of polycomb group proteins  by maintaining an epigenetically heritable active state of gene expression  <cit> .
however, the identified biochemical interactions with a variety of chromatin remodeling complexes and mutant analyses indicate a much broader role for gaf  <cit> .
how such divergent functions of gaf might be acquired and controlled still remains elusive.
in drosophila, multiple gaf isoforms are encoded from a single gene termed trithorax-like  <cit> .
all trl splice forms characterized so far, contain two open reading frames for a protein of  <dig>  or  <dig> amino acid  residues, respectively  <cit> .
both gaf isoforms contain  <dig> recognizable and evolutionarily highly conserved domains, a poz/btb domain, a zinc finger dna binding domain and a glutamine-rich region referred to here as the gaf-q or polyglutamine  domain  <cit> .
the isoforms are highly identical in their n-terminal part but they differ in the length of the c-terminal glutamine-rich regions   <cit> .
the poz/btb domain has been shown to function as a protein-protein interaction domain and the dbd  domain is important for sequence recognition and dna binding activity  <cit> .
although q domains found in different transcription factors were suggested to be associated with transcriptional control  <cit> , in one study gaf-q was shown to be dispensable for chromatin binding and transcriptional activation  <cit> .
however, other studies assign the transcriptional activity to the c-terminal polyq domain  <cit> .
interestingly, in vitro studies have suggested that the gaf-q domain of gaf <dig> facilitates multimerization, which may explain the multimeric distribution of gaf observed in vivo <cit> .
additionally, the gaf <dig> polyq domain was shown to be essential for the formation of long unbranched amyloid fibers in vitro <cit> .
the amyloid fibers formed by the gaf <dig> resemble fibers formed by sup <dig>  the prion determinant of the yeast prion   <cit> .
like most yeast prions, sup <dig> has a well characterized glutamine or asparagine  rich region, which is known as the prion domain   <cit> .
it enables sup <dig> to exist in distinct physical and functional states that are interconvertible and heritable, i.e.
a  soluble, functional state and a  aggregated, non-functional state  <cit> .
in  cells, the sup <dig> protein acts as a translation termination factor that, together with sup <dig>  recognizes stop codons and terminates translation.
in contrast, in  cells the sup <dig> is unable to efficiently participate in translation termination as most of sup <dig> protein is sequestered in self-replicating prion multimers  <cit> .
interestingly, the existence of chromatin associated proteins swi <dig> and cyc <dig> in distinct physical and functional states, reminiscent of prion-like behavior, was discovered in yeast  <cit> .
the glutamine-rich regions of swi <dig> and cyc <dig> were shown to be essential for prion-like behavior of these proteins and modulation of global gene expression patterns which are epigenetically inherited  <cit> .
although a large number of proteins in eukaryotic proteins have long q-rich tracts, similar to those found in the prion domains of yeast prions  <cit> , only a few are characterized to have properties similar to prions  <cit> .
expansion of polyq domains is known to contribute to heritable alterations of protein conformation which is associated with prion proteins  <cit> .
hence, we tested whether the q domains of the gaf isoforms, gaf <dig> and gaf <dig>  could act as prion-like domains using established validation tools in the yeast saccharomyces cerevisiae.
we replaced the n-term prion-forming domain of sup <dig> with the gaf <dig> and gaf <dig> q domains  and fused it to the c-terminal portion of the sup <dig> in order to test whether the chimeric proteins were able to induce a prion-like state as demonstrated for a variety of other potential prion forming domains  <cit> .
the resulting gafq-supc fusion proteins were able to rescue the lethality of a sup <dig> deletion strain.
expression of gafq-supc led to the appearance of a stably maintained nonsense suppression phenotype in a small proportion of cells, in a manner similar to the appearance of  in cells over-expressing sup <dig>  this prion-like phenotype of the gafq-supc expressing cells could be reversibly cured in the same manner as the  prion by growing the cells in the presence of guanidine hcl  or by over-expression of the hsp <dig> chaperone protein.
furthermore, when fused to gfp, the gaf <dig> and gaf <dig> polyq domains formed aggregates in yeast cells.
in corroboration, sedimentation analysis using differential centrifugation revealed that gafq-supc fusions are aggregated in the same manner as the  prion.
these aggregates were cured by growing the cells in the presence of guhcl.
finally, meiotic segregation analysis through tetrad dissection also revealed that the prion-like phenotype of gaf582supc segregated in a non-mendelian fashion.
importantly, prion-like behavior exhibited by meiotic progeny was also cured when grown in the presence of guhcl.
such prion-like behavior of gaf polyq domains in yeast suggest that polyq domains in gaf may render a conformational switch which may help gaf perform its versatile functions in maintaining different epigenetic states of gene expression.
RESULTS
the gaf isoforms gaf <dig> and gaf <dig> share the same n terminal domain but have distinct glutamine -rich c termini of different lengths.
the c terminal  <dig> amino acids of the gaf <dig> contains 39% q residues whereas the c terminal  <dig> amino acids of the gaf <dig> comprises of 41% of q residues in multiple repeats .
the well characterized genetic assays based on the  reporter system  <cit>  for evaluation of prions in yeast were employed to examine if gaf <dig> and gaf <dig> polyq domains  can act as prion-like domains.
a colony color assay based on  and  states of sup <dig> can be employed to reproducibly monitor the prion-like behavior of q-rich regions  <cit> .
the non-prion, functional part of sup35p i.e.
supc needed for translation termination has been used with fusions of q-rich yeast protein regions to detect prion-like behavior  <cit> .
in the case of the  state, cells that carry a premature stop codon in their ade <dig> gene  do not make functional ade <dig> and accumulate a metabolite making the colonies appear red on complete medium.
in contrast, the  cells are characterized by a read-through of the premature stop codon  in ade1- <dig>  functional ade <dig> is produced as most of sup <dig> protein is sequestered in self-replicating prion aggregates and is unable to participate in translation termination.
cells therefore produce white colonies and can grow on adenine-deficient medium  <cit> .
we generated dna constructs replacing the prion domain of sup <dig> with either gaf <dig> or gaf <dig> q domains  to monitor if gaf-q can substitute for the sup <dig> prion domain .
the non-prion, functional part of sup35p  was fused with gaf <dig> and gaf <dig> q-rich regions  and the resultant fusion constructs were named as 519q-supc and 582q-supc  .
the gafq-supc fusions and supc alone  under a constitutive promoter were transfected in a diploid  yeast strain gt <dig>  <cit>  in which we introduced a deletion of one copy of endogenous sup <dig> .
in strain gt <dig>  the  suppressible marker is ade1- <dig> with a uga premature stop codon.
cells produce white colonies on complete media  and grow without adenine supplementation.
in contrast  cells do not form colonies on adenine-deficient medium  and are red on ypd  <cit> .
the gafq-supc containing diploid cells heterozygous for sup <dig> were induced for sporulation  and resulting sup35∆ haploids were specifically monitored for viability by selecting on media supplemented with g <dig> and lacking histidine , indicators for the sup <dig> deletion and plasmids carrying gafq-supc fusions, respectively .
further, the sup35∆ haploids were confirmed by detecting expression of endogenous sup <dig> using antibody raised against the n-terminal  of sup <dig> .
the haploids confirmed for deletion of sup <dig> were also probed for expression of fusion proteins with an antibody raised against the c terminus of sup <dig> , which detected signals at 57 kda and 67 kda, the expected sizes of the 519q-supc and 582q-supc fusions, respectively .
the growth of sup35∆ haploids on ypd indicated that lethality based on the absence of sup <dig> was rescued by both 519q-supc and 582q-supc fusions .
importantly, the haploids expressing 519q-supc and 582q-supc fusions produced white colonies on ypd and were viable on -ade medium .
this is indicative of a nonsense suppression prion-like phenotype caused by the read-through of a nonsense codon in ade1- <dig> marker , similar to  cells.
in contrast, sup35∆ haploids expressing supc∆nm  produced red colored colonies and did not grow on medium lacking adenine .
this clearly illustrates that polyq domains of gaf <dig> and gaf <dig> may substitute the prion domain of sup <dig> and exhibit behavior similar to the sup <dig> prion domain.
because all yeast prions characterized so far exhibit the ability to exist in two functionally distinct states that are heritable and interconvertible at low frequency  <cit>  we next monitored the metastable behavior of gafq-supc containing sup35∆ haploids.
the existence of gafq-supc in two different functional states could be visualized by the colony color assay by repeatedly streaking individual haploids on ypd plates and monitoring the appearance of red and white colored colonies.
gafq-supc expressing fusions in sup35∆ haploids led to the appearance of colonies which were metastable, resulting in the appearance of some red colonies after several generations, similar to the metastability of some  prion phenotypes  <cit> .
reversible curing of prion phenotype is an important genetic criterion for analysis of prion proteins in yeast.
the known yeast prions , , ,  and  are cured by growth in the presence of guanidine hcl , which is suggested to inhibit heat shock protein  <dig>   <cit> .
the reversible curing of the prion-like phenotype of gafq-supc expressing cells was demonstrated by the conversion of colonies from a white color on ypd to a red color on ypd plates supplemented with 5 mm guanidine hcl  .
similar to the reversible curing of the  phenotype  <cit> , the gafq-supc nonsense suppression phenotype  was cured by growth in the presence of guhcl, resulting in the appearance of red colored colonies mimicking the  phenotype , which remained stably cured when grown in the absence of guhcl .
the colony color assay as well as reversible curing by guhcl clearly illustrates that the gaf-q domains have the ability to confer upon sup35c a capacity to exist in distinct physical and functional states that are interconvertible and heritable.
a small percentage of gafq-sup35c expressing cells that exhibited nonsense suppressor phenotype were reverted back to the non-supressor state with each subsequent passage on rich medium.
this phenomena was more pronounced at later passages  where each haploid stably exhibiting nonsense suppression phenotype showed high frequency of metastable behavior i.e.
appearance of many red colonies in the progeny after streaking .
however, this red and white colored colony phenotype was again subsequently maintained stable, generation after generation, upon re-streaking individual colonies picked up from the progeny.
importantly, the white colored colonies from the progeny exhibited nonsense suppression  like phenotype  as they could grow on -ade medium , which was reversible by growth in the presence of guhcl .
the curing of  nonsense suppression phenotype may be achieved through inactivation of hsp <dig> by guhcl treatment  <cit>  or by hsp <dig> mutation or by over-expression of hsp <dig>  <cit> .
indeed the nonsense suppression prion-like phenotype caused by the gafq-sup35c fusions was also cured by over-expression of hsp <dig>  when compared to control  cells, hsp <dig> over-expressing cells appeared red on rich medium  and were unable to grow on -ade medium, indicative of a curing of 519q-supc and 582q-supc nonsense suppression phenotype .
we determined if we could visually detect different aggregative states of gaf-q fusion proteins using previously employed fluorescent microscopy techniques for yeast prion characterization .
we fused the gaf <dig> and gaf <dig> q domains to green fluorescent protein  under an inducible promoter .
the prion-forming domain of sup <dig>  fused to gfp , shown to form punctate aggregates in  cells  <cit> , was used as a positive control.
the gafq-gfp and nm-gfp constructs were transformed in four different yeast strains.
these vary in strength of the prion phenotype due to presence or absence of either one of  and  or both prions.
gfp is normally soluble in yeast, but as expected the fusion to the nm domain conferred for gfp a capacity to exist in distinct states: a few large aggregates or a soluble protein .
as described for previously characterized prion determining regions of yeast proteins  <cit> , both the 519q and 582q also conferred upon gfp a capacity to exist in distinct states and showed aggregation pattern similar to nm-gfp .
like nm-gfp, the aggregation of the gaf-gfp fusions was dependent on the presence of , the prion form of the rnq <dig> protein of yeast required for sup <dig> aggregation and prion formation  <cit> .
inducible expression of both 519q-gfp and 582q-gfp showed aggregation  in 12-15% cells.
we also used q-rich region of drosophila zeste protein   <cit>  fused to gfp to monitor if any q-rich region may lead to aggregation of gfp independent of  or .
the inducible expression of zeste-gfp  in any of the yeast strains mentioned above showed no aggregation , which is similar to the pattern exhibited by gfp alone .
this reveals that aggregation patterns observed for gafq-gfp, similar to nm-gfp , is specific, further substantiating that gaf-q domains exhibit prion-like behavior in yeast.
we also employed differential centrifugation assay to explore the possibility of aggregation of 519q-supc and 582q-supc fusions that exhibited non-sense suppression phenotype in sup35∆ haploids.
total cell extracts from 519q-supc and 582q-supc expressing cells , prepared under non-denatured conditions, were fractionated by high speed centrifugation.
supernatant  and pellet  fractions were probed with anti-sup <dig> antibody .
unlike supc alone , both 519q-supc and 582q-supc were present in insoluble  fraction primarily , which is similar to the behavior of sup <dig> protein in a  strain .
moreover, 519q-supc was always equally present in both supernatant and pellet fractions.
however, guhcl treated populations of same cells revealed 519q-supc and 582q-supc in soluble  fraction , which is also similar to the sup <dig> protein behavior from  cells which were treated with guhcl .
these results concur with our observations with gafq-gfp fusions  and corroborate with the notion that 519q and 582q domains exhibit prion-like behavior and exist in distinct physical states.
finally, dominant behavior of prion-like gafq-supc  was demonstrated by mating 582q + mata  haploid with a 582q- matα haploid, i.e.
582q- represents a red colony indicating non-prion form of 582q .
the resultant diploid mata/α  not only displayed 582q + phenotype  but also when sporulated produced haploid  progeny.
these exhibited prion like behavior displaying meiotic inheritance of 582q + phenotype in a dominant, non-mendelian manner.
all  <dig> haploid  products of meiosis showed stable non-sense suppression phenotype by their growth on -ade plates, which was curable when grown in the presence of guhcl .
by making use of well-characterized genetic assays determining prion-like characteristics of glutamine-rich domains in different proteins, we have identified the q domains of both the gaf isoforms as prion-like domains.
the fusion proteins in which gaf-q domains were introduced in place of the sup35p prion domain could support distinct physical and functional prion states that recapitulated the translation termination defect associated with .
importantly, the nonsense suppression prion-like state exhibited by the gafq-supc fusion was cured by growth in the presence of guhcl.
similar to the  prion state of sup35p, the nonsense suppression phenotype by gafq-supc could also be cured by the over-expression of hsp <dig>  the gaf-q domains fused to gfp also formed visible aggregates resembling those of gfp labeled sup35p in , which also depended on   <cit> .
many sequences with high q content  including human polyglutamine expansion disease proteins, form visible aggregates when over-expressed in yeast as gfp fusions  <cit> .
however, only a limited number of q/n rich sequences are bone fide prion domains capable of propagating these aggregates over multiple cell generations even when expressed at low levels  <cit> .
construction of a synthetic prion revealed that pathogenically expanded stretch of  <dig> qs  fused to sup35c or gfp could mimic prion-like behavior, however, 22q did not show such characteristics  <cit> .
the prion-like behavior of 519q similar to sup <dig> is of significance because it contains only a short q stretch as compared to q <dig>  importantly, computational assessment of gaf <dig> and gaf <dig> using prion aggregation prediction algorithm reveals that both proteins have propensity to make prions  <cit> .
as compared to other eukaryotes analyzed, a surprisingly large number of proteins in drosophila have extended q-rich tracts, remarkably similar to those found in the prion-forming domains of yeast proteins  <cit> .
in in vitro studies and in transient assays in cell culture fusions of the q domains with the gal <dig> dna binding domain activate by stabilizing the transcriptional complex  <cit> .
however, in transgenic flies chromatin binding and transcriptional activation activity by gaf was found to be independent of q domains, leaving open the designation of the exact molecular function  <cit> .
so far, the combined results suggest that the q domains are mostly involved in the formation of larger gaf complexes.
the associated prion-like activity might thus provide an ability to gaf to attain distinct conformational states that may be heritable.
the high conservation of the c terminal q-rich domain of gaf in insects, suggests that there is a strong evolutionary preference to maintain such associated structure and function.
CONCLUSIONS
the analysis of gafq-supc fusions in yeast provides an interesting analogy between gaf-q and the sup <dig> prion domain, consistent with the previous findings, revealing that the gaf-q domain is essential for the formation amyloid fibers in vitro <cit> .
our results also support the previous findings that oligomerization of gaf found in drosophila cells may be facilitated by the long q stretches in gaf  <cit> .
we emphasize that gaf may not be a bone fide prion but q domains in gaf may induce conformational switch reminiscent of prion-like behavior.
in yeast, prions are not pathogenic but rather act as an epigenetic regulator of cell physiology and several epigenetically heritable traits are found to depend on a prion mechanism  <cit> .
evidence for regulation of gene expression patterns by propagation of swi <dig> and cy <dig> proteins as prions has provided a novel link between chromatin remodeling proteins and prion formation  <cit>  and it has revealed an additional mechanism for controlling global gene transcription that is based on an inherited self-perpetuating change in the conformation.
our results indicate that the possibility of such an intricate link between chromatin associated proteins, prion formation and epigenetic inheritance of gene expression might also apply in higher eukaryotes.
intriguingly, a large majority of the identified drosophila proteins with q-rich domains are essential developmental proteins including chromatin regulating proteins from pcg and trxg involved in epigenetic inheritance  <cit> .
it could be envisaged that gaf-q domains provide an inherent plasticity which may lead to a conformational switch in gaf in a changing environment.
such a q domain dependent conformation switch in gaf may be regulated by some specific post-translational modifications of gaf and facilitated by molecular chaperones.
this could result in modulated gene expression patterns that may contribute to phenotypic variation.
we suggest that gaf-q domain may act as prion-like domain in drosophila and support the notion that oligomeriztaion of gaf and other pcg/trxg proteins, which is known to be crucial for the function of these proteins, may be facilitated by such prion-like domains  <cit> .

 the gaga factor of drosophila is a ubiquitous transcription factor that plays important roles in multiple processes ranging from regulation of gene expression to the structural organization of heterochromatin and chromatin remodeling. genetically, gaf is classified as a member of the trithorax group proteins counteracting the silencing of polycomb group proteins by maintaining an epigenetically heritable active state of gene expression. however, the identified biochemical interactions with a variety of chromatin remodeling complexes and mutant analyses indicate a much broader role for gaf. how such divergent functions of gaf might be acquired and controlled still remains elusive. in drosophila, multiple gaf isoforms are encoded from a single gene termed trithorax-like. all trl splice forms characterized so far, contain two open reading frames for a protein of or amino acid residues, respectively. both gaf isoforms contain recognizable and evolutionarily highly conserved domains, a poz/btb domain, a zinc finger dna binding domain and a glutamine-rich region referred to here as the gaf-q or polyglutamine domain. the isoforms are highly identical in their n-terminal part but they differ in the length of the c-terminal glutamine-rich regions. the poz/btb domain has been shown to function as a protein-protein interaction domain and the dbd domain is important for sequence recognition and dna binding activity. although q domains found in different transcription factors were suggested to be associated with transcriptional control, in one study gaf-q was shown to be dispensable for chromatin binding and transcriptional activation. however, other studies assign the transcriptional activity to the c-terminal polyq domain. interestingly, in vitro studies have suggested that the gaf-q domain of gaf facilitates multimerization, which may explain the multimeric distribution of gaf observed in vivo. additionally, the gaf polyq domain was shown to be essential for the formation of long unbranched amyloid fibers in vitro. the amyloid fibers formed by the gaf resemble fibers formed by sup the prion determinant of the yeast prion. like most yeast prions, sup has a well characterized glutamine or asparagine rich region, which is known as the prion domain. it enables sup to exist in distinct physical and functional states that are interconvertible and heritable, i.e. a soluble, functional state and a aggregated, non-functional state. in cells, the sup protein acts as a translation termination factor that, together with sup recognizes stop codons and terminates translation. in contrast, in cells the sup is unable to efficiently participate in translation termination as most of sup protein is sequestered in self-replicating prion multimers. interestingly, the existence of chromatin associated proteins swi and cyc in distinct physical and functional states, reminiscent of prion-like behavior, was discovered in yeast. the glutamine-rich regions of swi and cyc were shown to be essential for prion-like behavior of these proteins and modulation of global gene expression patterns which are epigenetically inherited. although a large number of proteins in eukaryotic proteins have long q-rich tracts, similar to those found in the prion domains of yeast prions, only a few are characterized to have properties similar to prions. expansion of polyq domains is known to contribute to heritable alterations of protein conformation which is associated with prion proteins. hence, we tested whether the q domains of the gaf isoforms, gaf and gaf could act as prion-like domains using established validation tools in the yeast saccharomyces cerevisiae. we replaced the n-term prion-forming domain of sup with the gaf and gaf q domains and fused it to the c-terminal portion of the sup in order to test whether the chimeric proteins were able to induce a prion-like state as demonstrated for a variety of other potential prion forming domains. the resulting gafq-supc fusion proteins were able to rescue the lethality of a sup deletion strain. expression of gafq-supc led to the appearance of a stably maintained nonsense suppression phenotype in a small proportion of cells, in a manner similar to the appearance of in cells over-expressing sup this prion-like phenotype of the gafq-supc expressing cells could be reversibly cured in the same manner as the prion by growing the cells in the presence of guanidine hcl or by over-expression of the hsp chaperone protein. furthermore, when fused to gfp, the gaf and gaf polyq domains formed aggregates in yeast cells. in corroboration, sedimentation analysis using differential centrifugation revealed that gafq-supc fusions are aggregated in the same manner as the prion. these aggregates were cured by growing the cells in the presence of guhcl. finally, meiotic segregation analysis through tetrad dissection also revealed that the prion-like phenotype of gaf582supc segregated in a non-mendelian fashion. importantly, prion-like behavior exhibited by meiotic progeny was also cured when grown in the presence of guhcl. such prion-like behavior of gaf polyq domains in yeast suggest that polyq domains in gaf may render a conformational switch which may help gaf perform its versatile functions in maintaining different epigenetic states of gene expression.  the gaf isoforms gaf and gaf share the same n terminal domain but have distinct glutamine -rich c termini of different lengths. the c terminal amino acids of the gaf contains 39% q residues whereas the c terminal amino acids of the gaf comprises of 41% of q residues in multiple repeats. the well characterized genetic assays based on the reporter system for evaluation of prions in yeast were employed to examine if gaf and gaf polyq domains can act as prion-like domains. a colony color assay based on and states of sup can be employed to reproducibly monitor the prion-like behavior of q-rich regions. the non-prion, functional part of sup35p i.e. supc needed for translation termination has been used with fusions of q-rich yeast protein regions to detect prion-like behavior. in the case of the state, cells that carry a premature stop codon in their ade gene do not make functional ade and accumulate a metabolite making the colonies appear red on complete medium. in contrast, the cells are characterized by a read-through of the premature stop codon in ade1- functional ade is produced as most of sup protein is sequestered in self-replicating prion aggregates and is unable to participate in translation termination. cells therefore produce white colonies and can grow on adenine-deficient medium. we generated dna constructs replacing the prion domain of sup with either gaf or gaf q domains to monitor if gaf-q can substitute for the sup prion domain. the non-prion, functional part of sup35p was fused with gaf and gaf q-rich regions and the resultant fusion constructs were named as 519q-supc and 582q-supc. the gafq-supc fusions and supc alone under a constitutive promoter were transfected in a diploid yeast strain gt in which we introduced a deletion of one copy of endogenous sup. in strain gt the suppressible marker is ade1- with a uga premature stop codon. cells produce white colonies on complete media and grow without adenine supplementation. in contrast cells do not form colonies on adenine-deficient medium and are red on ypd. the gafq-supc containing diploid cells heterozygous for sup were induced for sporulation and resulting sup35∆ haploids were specifically monitored for viability by selecting on media supplemented with g and lacking histidine, indicators for the sup deletion and plasmids carrying gafq-supc fusions, respectively. further, the sup35∆ haploids were confirmed by detecting expression of endogenous sup using antibody raised against the n-terminal of sup. the haploids confirmed for deletion of sup were also probed for expression of fusion proteins with an antibody raised against the c terminus of sup, which detected signals at 57 kda and 67 kda, the expected sizes of the 519q-supc and 582q-supc fusions, respectively. the growth of sup35∆ haploids on ypd indicated that lethality based on the absence of sup was rescued by both 519q-supc and 582q-supc fusions. importantly, the haploids expressing 519q-supc and 582q-supc fusions produced white colonies on ypd and were viable on -ade medium. this is indicative of a nonsense suppression prion-like phenotype caused by the read-through of a nonsense codon in ade1- marker, similar to cells. in contrast, sup35∆ haploids expressing supc∆nm produced red colored colonies and did not grow on medium lacking adenine. this clearly illustrates that polyq domains of gaf and gaf may substitute the prion domain of sup and exhibit behavior similar to the sup prion domain. because all yeast prions characterized so far exhibit the ability to exist in two functionally distinct states that are heritable and interconvertible at low frequency we next monitored the metastable behavior of gafq-supc containing sup35∆ haploids. the existence of gafq-supc in two different functional states could be visualized by the colony color assay by repeatedly streaking individual haploids on ypd plates and monitoring the appearance of red and white colored colonies. gafq-supc expressing fusions in sup35∆ haploids led to the appearance of colonies which were metastable, resulting in the appearance of some red colonies after several generations, similar to the metastability of some prion phenotypes. reversible curing of prion phenotype is an important genetic criterion for analysis of prion proteins in yeast. the known yeast prions,,, and are cured by growth in the presence of guanidine hcl, which is suggested to inhibit heat shock protein. the reversible curing of the prion-like phenotype of gafq-supc expressing cells was demonstrated by the conversion of colonies from a white color on ypd to a red color on ypd plates supplemented with 5 mm guanidine hcl. similar to the reversible curing of the phenotype, the gafq-supc nonsense suppression phenotype was cured by growth in the presence of guhcl, resulting in the appearance of red colored colonies mimicking the phenotype, which remained stably cured when grown in the absence of guhcl. the colony color assay as well as reversible curing by guhcl clearly illustrates that the gaf-q domains have the ability to confer upon sup35c a capacity to exist in distinct physical and functional states that are interconvertible and heritable. a small percentage of gafq-sup35c expressing cells that exhibited nonsense suppressor phenotype were reverted back to the non-supressor state with each subsequent passage on rich medium. this phenomena was more pronounced at later passages where each haploid stably exhibiting nonsense suppression phenotype showed high frequency of metastable behavior i.e. appearance of many red colonies in the progeny after streaking. however, this red and white colored colony phenotype was again subsequently maintained stable, generation after generation, upon re-streaking individual colonies picked up from the progeny. importantly, the white colored colonies from the progeny exhibited nonsense suppression like phenotype as they could grow on -ade medium, which was reversible by growth in the presence of guhcl. the curing of nonsense suppression phenotype may be achieved through inactivation of hsp by guhcl treatment or by hsp mutation or by over-expression of hsp. indeed the nonsense suppression prion-like phenotype caused by the gafq-sup35c fusions was also cured by over-expression of hsp when compared to control cells, hsp over-expressing cells appeared red on rich medium and were unable to grow on -ade medium, indicative of a curing of 519q-supc and 582q-supc nonsense suppression phenotype. we determined if we could visually detect different aggregative states of gaf-q fusion proteins using previously employed fluorescent microscopy techniques for yeast prion characterization. we fused the gaf and gaf q domains to green fluorescent protein under an inducible promoter. the prion-forming domain of sup fused to gfp, shown to form punctate aggregates in cells, was used as a positive control. the gafq-gfp and nm-gfp constructs were transformed in four different yeast strains. these vary in strength of the prion phenotype due to presence or absence of either one of and or both prions. gfp is normally soluble in yeast, but as expected the fusion to the nm domain conferred for gfp a capacity to exist in distinct states: a few large aggregates or a soluble protein. as described for previously characterized prion determining regions of yeast proteins, both the 519q and 582q also conferred upon gfp a capacity to exist in distinct states and showed aggregation pattern similar to nm-gfp. like nm-gfp, the aggregation of the gaf-gfp fusions was dependent on the presence of, the prion form of the rnq protein of yeast required for sup aggregation and prion formation. inducible expression of both 519q-gfp and 582q-gfp showed aggregation in 12-15% cells. we also used q-rich region of drosophila zeste protein fused to gfp to monitor if any q-rich region may lead to aggregation of gfp independent of or. the inducible expression of zeste-gfp in any of the yeast strains mentioned above showed no aggregation, which is similar to the pattern exhibited by gfp alone. this reveals that aggregation patterns observed for gafq-gfp, similar to nm-gfp, is specific, further substantiating that gaf-q domains exhibit prion-like behavior in yeast. we also employed differential centrifugation assay to explore the possibility of aggregation of 519q-supc and 582q-supc fusions that exhibited non-sense suppression phenotype in sup35∆ haploids. total cell extracts from 519q-supc and 582q-supc expressing cells, prepared under non-denatured conditions, were fractionated by high speed centrifugation. supernatant and pellet fractions were probed with anti-sup antibody. unlike supc alone, both 519q-supc and 582q-supc were present in insoluble fraction primarily, which is similar to the behavior of sup protein in a strain. moreover, 519q-supc was always equally present in both supernatant and pellet fractions. however, guhcl treated populations of same cells revealed 519q-supc and 582q-supc in soluble fraction, which is also similar to the sup protein behavior from cells which were treated with guhcl. these results concur with our observations with gafq-gfp fusions and corroborate with the notion that 519q and 582q domains exhibit prion-like behavior and exist in distinct physical states. finally, dominant behavior of prion-like gafq-supc was demonstrated by mating 582q + mata haploid with a 582q- matα haploid, i.e. 582q- represents a red colony indicating non-prion form of 582q. the resultant diploid mata/α not only displayed 582q + phenotype but also when sporulated produced haploid progeny. these exhibited prion like behavior displaying meiotic inheritance of 582q + phenotype in a dominant, non-mendelian manner. all haploid products of meiosis showed stable non-sense suppression phenotype by their growth on -ade plates, which was curable when grown in the presence of guhcl. by making use of well-characterized genetic assays determining prion-like characteristics of glutamine-rich domains in different proteins, we have identified the q domains of both the gaf isoforms as prion-like domains. the fusion proteins in which gaf-q domains were introduced in place of the sup35p prion domain could support distinct physical and functional prion states that recapitulated the translation termination defect associated with. importantly, the nonsense suppression prion-like state exhibited by the gafq-supc fusion was cured by growth in the presence of guhcl. similar to the prion state of sup35p, the nonsense suppression phenotype by gafq-supc could also be cured by the over-expression of hsp the gaf-q domains fused to gfp also formed visible aggregates resembling those of gfp labeled sup35p in, which also depended on. many sequences with high q content including human polyglutamine expansion disease proteins, form visible aggregates when over-expressed in yeast as gfp fusions. however, only a limited number of q/n rich sequences are bone fide prion domains capable of propagating these aggregates over multiple cell generations even when expressed at low levels. construction of a synthetic prion revealed that pathogenically expanded stretch of qs fused to sup35c or gfp could mimic prion-like behavior, however, 22q did not show such characteristics. the prion-like behavior of 519q similar to sup is of significance because it contains only a short q stretch as compared to q importantly, computational assessment of gaf and gaf using prion aggregation prediction algorithm reveals that both proteins have propensity to make prions. as compared to other eukaryotes analyzed, a surprisingly large number of proteins in drosophila have extended q-rich tracts, remarkably similar to those found in the prion-forming domains of yeast proteins. in in vitro studies and in transient assays in cell culture fusions of the q domains with the gal dna binding domain activate by stabilizing the transcriptional complex. however, in transgenic flies chromatin binding and transcriptional activation activity by gaf was found to be independent of q domains, leaving open the designation of the exact molecular function. so far, the combined results suggest that the q domains are mostly involved in the formation of larger gaf complexes. the associated prion-like activity might thus provide an ability to gaf to attain distinct conformational states that may be heritable. the high conservation of the c terminal q-rich domain of gaf in insects, suggests that there is a strong evolutionary preference to maintain such associated structure and function.  the analysis of gafq-supc fusions in yeast provides an interesting analogy between gaf-q and the sup prion domain, consistent with the previous findings, revealing that the gaf-q domain is essential for the formation amyloid fibers in vitro. our results also support the previous findings that oligomerization of gaf found in drosophila cells may be facilitated by the long q stretches in gaf. we emphasize that gaf may not be a bone fide prion but q domains in gaf may induce conformational switch reminiscent of prion-like behavior. in yeast, prions are not pathogenic but rather act as an epigenetic regulator of cell physiology and several epigenetically heritable traits are found to depend on a prion mechanism. evidence for regulation of gene expression patterns by propagation of swi and cy proteins as prions has provided a novel link between chromatin remodeling proteins and prion formation and it has revealed an additional mechanism for controlling global gene transcription that is based on an inherited self-perpetuating change in the conformation. our results indicate that the possibility of such an intricate link between chromatin associated proteins, prion formation and epigenetic inheritance of gene expression might also apply in higher eukaryotes. intriguingly, a large majority of the identified drosophila proteins with q-rich domains are essential developmental proteins including chromatin regulating proteins from pcg and trxg involved in epigenetic inheritance. it could be envisaged that gaf-q domains provide an inherent plasticity which may lead to a conformational switch in gaf in a changing environment. such a q domain dependent conformation switch in gaf may be regulated by some specific post-translational modifications of gaf and facilitated by molecular chaperones. this could result in modulated gene expression patterns that may contribute to phenotypic variation. we suggest that gaf-q domain may act as prion-like domain in drosophila and support the notion that oligomeriztaion of gaf and other pcg/trxg proteins, which is known to be crucial for the function of these proteins, may be facilitated by such prion-like domains. 
the prion-like phenotype can be reversibly cured in the presence of guanidine hcl or by over-expression of the hsp chaperone protein. in an established yeast test system the two gaf q domains convey prion activities comparable to well known yeast prions. additionally, when fused to gfp, the q domains form aggregates in yeast cells. here we show that the q domains exhibit prion-like properties. the q domains stably maintain two distinct conformational states imposing functional constraints on the fused yeast reporter protein. we conclude that prion-like behavior of the gaf q domain suggests that this c-terminal structure may perform stable conformational switches. gaf functions are intimately associated to chromatin-based epigenetic control, linking basic transcriptional regulation to heritable long-term maintenance of gene expression.
 the drosophila gaga factor participates in nucleosome remodeling to activate genes, acts as an antirepressor and is associated with heterochromatin, contributing to gene repression. gaf functions are intimately associated to chromatin-based epigenetic control, linking basic transcriptional regulation to heritable long-term maintenance of gene expression. these diverse functions require gaf to interact with different partners in different multiprotein complexes. the two isoforms of gaf depict highly conserved glutamine-rich c-terminal domains, which have been implicated in complex formation.  here we show that the q domains exhibit prion-like properties. in an established yeast test system the two gaf q domains convey prion activities comparable to well known yeast prions. the q domains stably maintain two distinct conformational states imposing functional constraints on the fused yeast reporter protein. the prion-like phenotype can be reversibly cured in the presence of guanidine hcl or by over-expression of the hsp chaperone protein. additionally, when fused to gfp, the q domains form aggregates in yeast cells.  we conclude that prion-like behavior of the gaf q domain suggests that this c-terminal structure may perform stable conformational switches. such a self-perpetuating change in the conformation could assist gaf executing its diverse epigenetic functions of gene control in drosophila. gaga factorepigeneticsprion-likechromatin 