the eukaryotic genome is functionally and spatially organized . during interphase , chromosomes fold into topologically associated domains ( tads ) and divide into heterochromatin and euchromatin . also , chromosomes associate with nuclear structures and occupy distinct territories within the nucleus . within these territories ,
individual genes are positioned with respect to each other and with respect to stable nuclear structures .
for example , in metazoa large transcriptionally repressed lamina associate domains ( lads ) position along the nuclear periphery and interact with the nuclear lamina .
importantly , the spatial organization of the genome is dynamic and the position of individual genes often changes upon activation or repression .
for example , during development , lads can be remodeled to accommodate the repositioning of genes ; the -globin and myod genes move away from the nuclear lamina upon transcriptional activation
. many active genes also interact with nuclear pore proteins ( nups ) in diverse organisms including yeast , flies , worms , and mammalian cells . in both yeast and metazoa
these interactions occur at the nuclear periphery , presumably in contact with the nuclear pore complex ( npc ) , whereas in higher eukaryotes these interactions often occur in the nucleoplasm with soluble nups
active genes colocalize with rnap ii foci in subnuclear compartments called transcription factories . in flies ,
likewise , in yeast , trna genes , silenced telomeres and npc - associated loci each exhibit specific interchromosomal clustering .
these observations support the idea that the spatial organization of the eukaryotic genome compartmentalizes the nucleus into functionally distinct subnuclear environments and that the spatial positioning of a gene both impacts and reflects its transcriptional state .
as a model for these phenomena , we have studied the spatial repositioning of inducible yeast genes from the nucleoplasm to the nuclear periphery .
inducible genes such as ino1 , prm1 and his4 are recruited from the nucleoplasm to the nuclear periphery upon activation .
these genes are inducible under very different conditions ( ino1 is activated by inositol starvation , prm1 is induced by mating pheromone and his4 is induced by amino acid starvation ) and they are targeted to the periphery only under the conditions that lead to their expression .
this recruitment leads to a physical interaction with the nuclear pore complex ( npc ) and promotes stronger expression .
targeting to the periphery is controlled by cis - acting transcription factor ( tf ) binding sites located within the promoters of these genes .
these elements and the tfs that bind to them are not only necessary for recruitment , but are also sufficient to target an ectopic locus to the nuclear periphery .
thus , these cis - acting binding sites function as dna zip codes to control the dynamic , conditional positioning and interchromosomal clustering of genes .
this suggests that one of the functions of tfs is to control the spatial organization of the genome .
targeting of ino1 to the nuclear periphery is mediated by 2 dna elements called grs i and grs ii .
the tfs put3 ( zn - binuclear cluster family ) and cbf1 ( basic helix - loop - helix family ) bind to grsi and grsii , respectively and these tfs are necessary for grs i- and grs ii - mediated targeting to the nuclear periphery .
the grs i element is necessary and sufficient to mediate interchromosomal clustering with other grs i - containing loci .
ino1 transcription is regulated by the ino2/ino4 tfs , which bind to the uasino elements in the promoter .
such a separation between the elements controlling transcription and gene positioning is also seen in other promoters ( our unpublished results ) .
thus , although gene positioning and transcription are coupled , they can be mediated by distinct elements and factors .
in contrast , the positioning of his4 and prm1 is controlled by the same tfs that regulate their expression , gcn4 and ste12 , respectively .
inserting dna elements corresponding to the binding sites of gcn4 ( gcn4bs ) and ste12 ( pheromone - response elements , 3xpre ) at the ectopic site ura3 , which is normally localized to the nucleoplasm , is sufficient to reposition the locus to the nuclear periphery .
furthermore , mutant strains lacking gcn4 or ste12 fail to target his4 or prm1 to the nuclear periphery , respectively . finally , artificially tethering gcn4 or ste12 to the ura3 locus via a lexa dna binding domain is sufficient to cause ura3 to reposition to the nuclear periphery .
therefore , these tfs are both necessary and sufficient to control peripheral localization and interchromosomal clustering .
put3 , cbf1 , gcn4 and set12 represent 4 different families of transcription factors that mediate spatial repositioning and clustering of these genes .
indeed , in erythroid cells , the transcription factor klf1 is necessary for clustering of its target genes into specialized transcription factories and in flies , interactions of genes with nup98 is mediated by the mbd - r2 dna binding factor .
however , not all transcription factors possess this function . as mentioned above , ino2/ino4 binding to the uasino element within the ino1 promoter is neither necessary nor sufficient to recruit chromatin to the nuclear periphery .
the activation domain of put3 and gcn4 is dispensable for targeting to the nuclear periphery ( our unpublished results ) .
furthermore , inactivating rna polymerase ii or promoter mutations that block ino1 transcription does not block zip code - dependent recruitment to the nuclear periphery .
one important , unaddressed question is how these changes in gene positioning are dynamically regulated .
repositioning of ino1 , his4 and prm1 to the nuclear periphery and interchromosomal clustering is conditional and occurs under specific environmental stimuli .
this reflects how each zip code is regulated , which is revealed when the zip code is inserted at an ectopic site .
grsi and grsii are regulated through a context - dependent mechanism : when inserted at an ectopic site , these elements lead to constitutive targeting to the nuclear periphery . in other words , they are negatively regulated in the context of the ino1 promoter and only permitted to function when ino1 is induced . using systematic mutagenesis of cis and trans acting regulators ,
we find that targeting of ino1 to the nuclear periphery by put3 and cbf1 is regulated through local recruitment of rpd3(l ) histone deacetylase complex ( fig .  1 ) .
rpd3(l ) is recruited to the ino1 promoter under repressing conditions by the transcriptional repressors opi1 and ume6 .
rpd3(l ) regulates zip code activity by blocking transcription factor binding through its histone deacetylase activity .
this regulation was abolished by either perturbing the recruitment of rpd3 or inactivating its catalytic activity ( rpd3 h188a ) , resulting in constitutive targeting of ino1 to the nuclear periphery .
furthermore , rpd3(l ) regulation could be recapitulated at the ectopic ura3 locus by artificially tethering either opi1 or ume6 near grs i or grs ii , blocking targeting to the nuclear periphery and interchromosomal clustering ( fig .  1 , left panel ) . to test
if this was general function of repressors in yeast , 21 different repressors were tethered beside the grs i. a majority of repressors could block grs i function ; 16 of the 21 repressors tested blocked put3-mediated recruitment to the nuclear periphery and 11 of these required rpd3 .
thus , 5 repressors block put3 binding by either recruiting a different histone deacetylase or through alternative mechanisms .
these results suggest that put3s ability to reposition genes can be blocked through several different mechanisms .
because put3 controls the positioning of genes that are not perfectly co - regulated , cells could use these different strategies to independently regulate put3-mediated targeting in different contexts , depending on the environmental stimulus . 

figure 1.multiple strategies in regulating transcription factor ( tf)-mediated recruitment to the nuclear periphery over different time scales .
top : ( left ) put3 and cbf1 bind to grsi and grsii respectively in the promoter of ino1 and mediate its recruitment to the nuclear periphery upon inositol starvation .
these tfs are regulated by the local recruitment of rpd3(l ) histone deacetylase by transcriptional repressors opi1 and ume6 .
repression is relieved between 60120  min leading to peripheral localization and interchromosomal clustering of ino1 .
( middle ) ste12 mediates the recruitment of prm1 to the nuclear periphery upon mating pheromone stimulation .
( right ) gcn4-mediated recruitment of his4 to the nuclear periphery is controlled by gcn4 abundance .
gcn4 is translationally regulated . through increased protein levels of gcn4 , maximal peripheral targeting occurs between 3060  min .
multiple strategies in regulating transcription factor ( tf)-mediated recruitment to the nuclear periphery over different time scales .
top : ( left ) put3 and cbf1 bind to grsi and grsii respectively in the promoter of ino1 and mediate its recruitment to the nuclear periphery upon inositol starvation .
these tfs are regulated by the local recruitment of rpd3(l ) histone deacetylase by transcriptional repressors opi1 and ume6 .
repression is relieved between 60120  min leading to peripheral localization and interchromosomal clustering of ino1 .
( middle ) ste12 mediates the recruitment of prm1 to the nuclear periphery upon mating pheromone stimulation .
( right ) gcn4-mediated recruitment of his4 to the nuclear periphery is controlled by gcn4 abundance .
gcn4 is translationally regulated . through increased protein levels of gcn4 , maximal peripheral targeting occurs between 3060  min .
unlike put3 , regulation of ste12- and gcn4-mediated repositioning is context - independent ; inserting the gcn4bs or the 3xpre at ura3 led to repositioning to the nuclear periphery upon histidine starvation or pheromone treatment , respectively .
furthermore , loss of rpd3 had no effect on recruitment of prm1 or his4 to the nuclear periphery and the 3xpre was completely resistant to tethering of opi1 or ume6 . instead
ste12 is constitutively bound and ste12-dependent transcription is inhibited by 2 repressors , dig1 and dig2 . upon mating pheromone stimulation
, dig1 and dig2 are phosphorylated by the mapk fus3 , causing them to dissociate from ste12 .
dissociation of both dig1 and dig2 is required for prm1 transcriptional activation , but loss of dig2 alone led to constitutive ste12-mediated peripheral localization and interchromosomal clustering .
furthermore , mutation of serine 34 to alanine in dig2 - blocking phosphorylation - also blocked targeting to the nuclear periphery . likewise ,
mutation of serine 34 to aspartate - mimicking phosphorylation - led to constitutive targeting to the nuclear periphery .
thus , ste12-mediated gene positioning is regulated through post - translational modification of an inhibitor ( fig .  1 , middle panel ) .
gcn4-mediated targeting to the nuclear periphery is regulated by a third mechanism ; the occupancy of gcn4 binding to its target genes , controlled by its abundance . unlike the other mechanisms discussed above , which behave in a switch - like manner , gcn4-mediated targeting to the nuclear periphery and interchomosomal clustering occurs at a lower level under uninducing conditions and at a maximal level under inducing conditions .
it is well - established that gcn4-mediated transcription is regulated through the abundance of gcn4 . in the presence of amino acids , gcn4 is poorly translated due to several short , upstream open reading frames ( uorfs ) in the 5 end of the gcn4 mrna . when cells are starved for amino acids , translational initiation
is slowed , permitting the ribosome to skip the uorfs and increase the translation of gcn4 .
increasing gcn4 protein production by mutating the uorfs resulted in constitutive recruitment and clustering of his4 .
thus , under uninducing conditions , low levels of gcn4 lead to modest targeting to the nuclear periphery and measurable interchromosomal clustering .
however , under inducing conditions , higher gcn4 protein levels increase the occupancy of the gcn4bs and this leads to maximal peripheral targeting and interchromosomal clustering ( fig .  1
a number of factors have been implicated in the targeting of genes to the npc , including mrna export factors , the saga histone acetyltransferase and components of the npc itself .
we find that the saga complex , for example , is required for targeting of ino1 and his4 to the nuclear periphery , but is dispensable for targeting of prm1 to the nuclear periphery .
if saga functions to bridge the interaction between chromatin and the npc , as has been suggested , this result indicates that transcription factors use more than one mechanism to promote repositioning to the nuclear periphery .
the saga complex is a multi - subunit histone modifying enzyme that has broad impacts on gene expression . because the regulation of put3 , cbf1 and gcn4 occurs at the level of tf occupancy whereas ste12 is regulated downstream of binding , it is possible that saga may either facilitate tf dna binding or alter the abundance of the tfs themselves .
these results highlight the critical role of tfs in controlling gene positioning and interchromosomal interactions .
tf - mediated gene positioning can be regulated through at least 4 different mechanisms : regulation of tf binding by the rpd3(l ) hdac , regulation of tf binding ( or function ) by repressors independent of rpd3(l ) , regulation of tf occupancy through changes in tf abundance and regulation of tf function through post - translational modification of an inhibitor .
these different strategies operate over different time scales to alter the positioning of individual genes and the arrangement of chromosomes with respect to each other : mapk signaling leads to rapid repositioning and clustering of ste12 targets within 1530  min , while changes in gcn4 protein levels leads to a moderate rate of repositioning and clustering within 3060  min and rpd3(l ) regulated gene positioning is derepressed over 60120  min ( fig .  1 ) .
thus , reflecting the regulation of tf function , cells employ different strategies to regulate dynamic tf - mediated gene positioning over very different temporal regimes .
the authors acknowledge members of the brickner lab , past and present , for contributing to the work described and for helpful discussions .
 abstractin yeast , inducible genes such as ino1 , prm1 and his4 reposition from the nucleoplasm to nuclear periphery upon activation . 
 this leads to a physical interaction with nuclear pore complex ( npc ) , interchromosomal clustering , and stronger transcription . 
 repositioning to the nuclear periphery is controlled by cis - acting transcription factor ( tf ) binding sites located within the promoters of these genes and the tfs that bind to them . 
 such elements are both necessary and sufficient to control positioning of genes to the nuclear periphery . 
 we have identified 4 tfs capable of controlling the regulated positioning of genes to the nuclear periphery in budding yeast under different conditions : put3 , cbf1 , gcn4 and ste12 . in each case 
 , we have defined the molecular basis of regulated relocalization to the nuclear periphery . 
 put3- and cbf1-mediated targeting to nuclear periphery is regulated through local recruitment of rpd3(l ) histone deacetylase complex by transcriptional repressors . 
 rpd3(l ) , through its histone deacetylase activity , prevents tf - mediated gene positioning by blocking tf binding . 
 many yeast transcriptional repressors were capable of blocking put3-mediated recruitment ; 11 of these required rpd3 . 
 thus , it is a general function of transcription repressors to regulate tf - mediated recruitment . 
 however , ste12 and gcn4-mediated recruitment is regulated independently of rpd3(l ) and transcriptional repressors . 
 ste12-mediated recruitment is regulated by phosphorylation of an inhibitor called dig2 , and gcn4-mediated gene targeting is up - regulated by increasing gcn4 protein levels . 
 the ability to control spatial position of genes in yeast represents a novel function for tfs and different regulatory strategies provide dynamic control of the yeast genome through different time scales . 
Introduction
Transcription factors reposition genes to the nuclear periphery
Regulation of transcription factor-mediated gene positioning
Disclosure of potential conflicts of interest
Acknowledgments
Funding