Paused Pol II Captures Enhancer Activity and Acts As a Potent Insulator

Home , Derepression, Insulator (genetics)

Downloaded from genesdev.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press

PERSPECTIVE

Paused Pol II captures enhancer activity and acts as a potent insulator

Leighton J. Core and John T. Lis1 Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA

Enhancers act over many kilobase pairs to activate target Interplay of enhancers and insulators promoters, but their activity is constrained by insulator Enhancers are powerful regulatory elements that can elements that prevent indiscriminate activation of nearby reside many thousands of base pairs upstream of or genes. In the July 1, 2009, issue of Genes & Development, downstream from a target gene and strongly stimulate Chopra and colleagues (pp. 1505–1509) report that pro- its transcription. Enhancers are comprised of clusters of moters containing a stalled Pol II are activated by short regulatory elements to which a collection of tran- enhancers, but these promoters also serve as insulators scription factors bind. In some cases, the mixture of that block enhancers from reaching more distal genes. proteins includes not only transcription activators, but This new class of insulators provide critical clues to reg- also architectural factors that allow the collection of ulatory mechanisms. proteins to assemble into a stable structure called the enhanceosome (Merika and Thanos 2001). Enhancers are numerous and distributed broadly over the genome. This Precise regulation of transcription by RNA polymerase II broad distribution and ability of enhancers to broadcast (Pol II) is critical for the proper growth, development, and their activation signals over long distances is ostensibly survival of an organism. Transcription can be regulated at a reckless mechanism of gene regulation that potentially several stages, and current data increasingly support that could lead to widespread misregulation of gene transcrip- the transition from initiation to elongation is frequently tion. Fortunately, enhancers are kept in check by in- exploited as a regulatory target that can govern the level sulator elements that are also numerous and broadly of gene transcription in response to developmental pro- distributed and provide boundaries that confine the gramming (Muse et al. 2007; Wang et al. 2007; Zeitlinger action of enhancer to particular gene targets. Insulators et al. 2007). During this transition, Pol II can pause or stall block the ability of enhancers to stimulate a promoter proximal to the promoter, where it awaits the proper when placed between an enhancer and its targeted pro- cellular cues and undergoes physical and chemical changes moter, and can also function as barrier elements that that are necessary for producing a processive elongation block the localized spread of active or repressive chroma- complex that is capable of faithfully transcribing the tin (Geyer 1997; Maeda and Karch 2007). Interestingly, full length of a gene (Saunders et al. 2006; Gilmour the molecular compositions of insulators show consider- 2008). This mechanism has been proposed to provide able variety, and their mechanism of action remains a number of advantages that result in the rapid, synchro- mysterious. The sophisticated and conserved arrangement nous activation and repression of transcription in response of these elements in cis at the ancient homeotic gene to environmental or developmental stimuli (Lis 1998). It is cluster pays heed to the importance of local chromatin also proposed to provide a checkpoint that allows time for architecture in the regulation of gene transcription. In the Pol II to mature into a productive elongation complex, July 1, 2009, issue of Genes & Development,Chopraetal. and coordinate multiple pre-mRNA processing events (2009) propose an intimate link between Pol II stalling and that occur cotranscriptionally. Control of transcription in enhancer function by demonstrating an intrinsic insulator response to developmental signals is also known to be activity associated with stalled promoters. mediated by short and long-range interactions of promoters with complex cis-element regulatory regions that A model for precise and responsive include enhancers, insulators, and repressive elements transcriptional regulation (Maeda and Karch 2007). The regulation of the Drosophila melanogaster Anten- napedia (ANT-C) and bithorax (BX-C) clusters of homeotic genes provide a classic example of the critical need for precise transcription regulation. During early embryo- [Keywords: Hox; Pol II stalling; insulator; NELF; DSIF; long range] genesis, expression of the genes within the ANT-C and 1Corresponding author. E-MAIL [email protected]; FAX (607) 255-2428. BX-C dictate the identity of the anterior/thoracic and Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1827709. thoracic/posterior parasegments (for reviews, see Akbari

1606 GENES & DEVELOPMENT 23:1606–1612 Ó 2009 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/09; www.genesdev.org Downloaded from genesdev.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press

Paused Pol II has insulator activity et al. 2006; Maeda and Karch 2007). These patterning events occur in a narrow time frame and in segments that are only several cells in width; thus, tight control of gene expression in time and space is crucial for normal development. In the case of the BX-C, promoter regulation is carried out by multiple cis-acting enhancer, repressor, and insulator elements within the 300-kb region of the gene complex. For the well-studied Abd-B gene, gene activation is controlled by a number of iab (infra-abdominal) regions that are situated 39 of the gene in a linear fashion that mirrors the ordering of the segments within which they activate gene expression (Akbari et al. 2006; Maeda and Karch 2007). The iab regions contain the enhancer Figure 1. Summary of results from Chopra et al (2009). Chopra and repressor elements, and insulators have been identi- et al. (2009) demonstrate enhancer-blocking activity of stalled fied that separate the iab regions. The insulating ele- promoters. (A) Enhancers (blue trapezoid) can activate Pol II (red) ments can confine both repressor and enhancer activities, at distal as well as nearby genes when the proximal promoter is and are proposed to interact with each other and/or other not stalled. (B) However, distal promoters are insulated from elements to create looped chromatin domains (Maeda and enhancer-directed activation when an intervening promoter contains a stalled Pol II. Components of the stalled complex—NELF Karch 2007). It is believed that these looped domains are (yellow pentagon), DSIF (dark-blue oval), and the short, nascent responsible for restricting certain IAB–promoter interac- RNA (blue line)—are shown. tions within a domain. Considerable research has focused on determining the molecular mechanisms involved in this intricate system of gene regulation. in between the Abd-B or Ubx stalled promoters and the white reporter, it was able to activate both promoters. Although a promoter-tethering element was identified Promoter-paused/stalled Pol II has insulator activity recently immediately upstream of the Abd-B gene (Akbari In collaboration with the Young and Adelman laborato- et al. 2008), the entire tethering element was not inculded ries, Mike Levine’s laboratory (Zeitlinger et al. 2007) in the Abd-B promoter construct (VS Chopra, pers. comm.). demonstrated recently that many homeotic genes likely These results suggest that stalled promoters do not effec- harbor a promoter-proximal stalled Pol II. Interestingly, of tively compete with other promoters via a tethering mech- the eight genes contained within the ANT-C and BX-C, anism. Together, these observations suggest that the stalled only the two promoters that flank each cluster apparently promoters flanking the hox clusters are important for show characteristics of a stalled Pol II. Running with restricting enhancer function to genes within the Hox this observation, Chopra et al. 2009 hypothesized that cluster and for inhibiting enhancer elements outside the the stalled Pol II could define the functional domains of cluster from regulating Hox genes that reside within the each hox cluster. To test this, they turned to classic complex. enhancer-blocking assays that use promoter–reporter Stalling of Pol II at promoters is controlled by the gene fusions placed in various configurations relative to differential activities of multiple elongation factors. The elements of interest, such as enhancers or insulators. DSIF and NELF complexes help maintain Pol II in These assays revealed that when any of the four stalled the stalled conformation, while the activity of P-TEFb promoters controlling a lacZ reporter gene were placed kinase stimulates the escape of Pol II from the paused in between the IAB5 enhancer element and a white state and into productive elongation (Saunders et al. 2006; reporter gene, only the stalled promoter was activated. Gilmour 2008). To test whether the role of these factors In contrast, when a nonstalled promoter was placed in in pausing is also important for enhancer-blocking activ- between the enhancer and the white reporter, both genes ity, Chopra et al. (2009) then tested the properties of were activated. These results indicate that the stalled stalled promoters in genetic backgrounds, where each of Pol II promoters respond to enhancers and contain an these factors and an additional elongation factor, Elongin enhancer-blocking activity that prevents enhancers from A, were reduced. As expected, reduction in P-TEFb or reaching over these stalled promoters to target other Elongin A did not alter the enhancer-blocking properties genes (Fig. 1). of the stalled Abd-B or Ubx promoters that flank the Certain promoter elements can effectively tether an BX-C. However, reduction of the components in the DSIF enhancer’s activity. For instance, a TATA element has or NELF complexes results in a depletion of enhancer been shown to compete for a functional interaction with- blocking. NELF depletion has been shown previously to in an enhancer when an intervening promoter is TATA- reduce the level of pausing on several genes in Drosophila. less, or if the enhancer is placed in between the two pro- Thus, the above results are consistent with a general moters (Ohtsuki et al. 1998). In contrast, whereas stalled requirement for stalling to confer enhancer-blocking prop- promoters are targets of enhancer activity to block down- erties to a promoter. stream activity, they do not inactivate enhancer action on Interestingly, Chopra et al. (2009) also show that neg- neighboring promoters that reside on the opposite side of ative elongation factors are necessary for the insulator the enhancer (Fig. 1). When the IAB5 enhancer was placed activities of the Fab-7 and Fab-8 elements contained

GENES & DEVELOPMENT 1607 Downloaded from genesdev.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press

Core and Lis within BX-C. These last results bring forth an interesting The shared features of insulators and promoters might question: Why are transcription elongation factors that also suggest that insulators are specialized promoters. are necessary for stalling at promoters also necessary for Indeed, several Drosophila insulators have been shown to the activity of known insulators? Chopra et al. (2009) contain promoters, and the iab regions within the BX-C speculate that insulators interact with stalled promoters do support transcription (Geyer 1997; Bae et al. 2002; to form higher-order chromatin loop domains similar to Drewell et al. 2002). Transcription occurs within and is known insulator–insulator interactions (Blanton et al. restricted to the iab domains that will eventually activate 2003; Cleard et al. 2006; Kyrchanova et al. 2007). Al- transcription of their target gene (Bae et al. 2002; Drewell though this idea is yet to be tested directly, physical and et al. 2002). Transcription through these regions is pro- functional interactions between the Fab-7 and Fab-8 in- posed to promote an open chromatin organization that sulators and the stalled Abd-B promoter have been de- allows for the enhancer elements to become functional. scribed (Cleard et al. 2006; Kyrchanova et al. 2007). They Interestingly, removal of an insulator within the BX-C further suggest that proteins that bind insulators interact has been shown to abrogate transcription of the neigh- with components of the Pol II complex at stalled genes, boring iab domain, resulting in a transformation of the which is consistent with the recent observation that the corresponding segment to the anterior one (Drewell et al. BEAF insulator protein tends to colocalize with NELF 2002). Thus, it is possible that the unidentified promoter (Jiang et al. 2009). The resulting loops may produce re- is related to the insulator element. However, whether or stricted structures that prevent cross-talk between do- not intergenic transcription within the BX-C control remains, but could also be important for targeting enhanc- gions originates from the insulator elements or other ers to certain promoters. For instance, the 39-distal IAB5 cryptic promoters within the iab regions remains to be and IAB6 enhancers must traverse both the Fab-7 and determined. Fab-8 insulator elements in order to interact with the The looping domain model proposes that insulators can Abd-B promoter. Perhaps direct interaction of Fab-7 with interact with each other, specialized promoter targeting the Abd-B promoter can specifically target these en- sequences, and promoters themselves to create looped hancers to the Abd-B promoter (Cleard et al. 2006). domains that both target enhancers to specific promoters This new function of stalled promoters raises several and restrict enhancer activity to particular domains interesting questions regarding the mechanisms through (Maeda and Karch 2007). This idea is supported by many which both stalled promoters and insulators contribute to physical and functional interactions between insulator regulation of gene expression. Specifically, it is impor- elements and insulator-binding proteins with promoters tant to ask why stalled promoters are bestowed with (Cleard et al. 2006; Holohan et al. 2007; Kyrchanova et al. enhancer-blocking activities, while nonstalled promoters 2007). For instance, the insulator-binding protein CTCF are not. Also, a wealth of data point to several equiv- has been shown to interact with several known and alencies between insulators and stalled promoters; thus, putative insulator regions within the BX-C, but is also it is practical to consider the similarities and differences found at the Abd-B promoter (Holohan et al. 2007). It is in order to unravel the meaning of promoter–insulator proposed that CTCF acts as a boundary that restricts the interactions. initial activation signal within each iab region, but it is also possible that homo- or hetero-typic interactions between CTCF at insulators and promoters could direct Insights from the similarities between promoters active enhancers to their target. and insulators

Insulators can be promoter mimics or interact directly The generality of promoter–insulator interactions with promoters Recent evidence suggests that insulator–promoter inter- Promoters and insulators display similarities in DNase actions are a general means for regulating transcrip- I-hypersensitive site enrichment, GAGA factor (GAF) bind- tion of a wide variety of genes. Several insulator-binding ing, histone turnover, H3.3 depostition, H2A.Z deposi- proteins—CP190, dCTCF, GAF, and BEAF—frequently tion, H3K4me1,2,3, H3ac, and low H3k27me3 (Mutskov map by chromatin immunoprecipitation (ChIP) proximal et al. 2002; Barski et al. 2007; Mito et al. 2007; Mavrich to transcription start sites (TSSs). CP190 and dCTCF sites et al. 2008). Either very similar mechanisms are occurring correlate with active genes, and also mark a boundary at promoters and insulators independently, or these prop- between repressive marks (h3k27me3) and active pro- erties are ‘‘shared’’ between the two due to a close prox- moters (Mohan et al. 2007). BEAF-binding sites have imity brought on by a looping interaction. The former a higher correlation with TSSs than CP190 or dCTCF suggests that insulators may block enhancer/repressor sites, and these interactions could be a consequence of interaction with a promoter by acting as a promoter de- both direct binding to promoters and looping between coy (Geyer 1997). In the promoter decoy or mimicry insulators and promoters. Interestingly, the NELF elon- model, insulators are proposed to recruit components of gation factor, which is important for promoter stalling the transcription machinery that effectively trap enhanc- and insulator function (Chopra et al. 2009), overlaps with ers into a nonproductive interaction. Thus, by mimicking ;40% of identified BEAF sites in a genome-wide analysis a promoter-bound complex, insulators can protect pro- (Jiang et al. 2009). In addition, 90% of BEAF-associated moters from enhancers when placed in between them. genes are contained within the upper half of genes ranked

1608 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press

Paused Pol II has insulator activity by their level of expression, suggesting that BEAF plays chromatin barriers in the absence of bona fide insulator- a role in high-level transcription of genes. As demon- binding activities. It is also possible that insulator-binding strated by Chopra et al. (2009), stalled promoters have proteins could act in concert with a stalled promoter to insulator function, and it has also been documented that enhance barrier formation. NELF binding (Lee et al. 2008) and stalling/pausing correlate with gene activity levels (Core et al. 2008). GAF ‘makes the connection’ These observations suggest a tight link between Pol II stalling, insulator function, known insulator proteins, Perhaps the most striking similarity between stalled and gene activity. An intriguing extension of these results promoters and insulators is their frequent association is that insulator–promoter interactions could potentially with GAF (Maeda and Karch 2007; Lee et al. 2008). GAF is mediate high levels of transcription by creating localized encoded by Trithorax-like (Trl) gene (Farkas et al. 1994), chromatin domain compartments that concentrate tran- and is named for the (GA)n consensus site that it binds scription factors within regions where high levels of gene (Adkins et al. 2006). It has three major domains: a single expression are needed (Yao et al. 2007). In contrast to zinc finger DNA-binding domain, a BTB/POZ domain, what one might expect if insulators interact with stalled and a glutamine-rich domain (Q domain). The BTB/POZ promoters, specific interaction of BEAF-containing insu- domain is found in a wide variety of proteins and is be- lators with stalled promoters is less evident, since only lieved to mediate homo- or hetero-protein interactions ;25% of BEAF-occupied sites contain a stalled Pol II. (Mahmoudi et al. 2002; Adkins et al. 2006). The Q domain This may be a result of underestimation of the number of is believed to function as a multimerization domain that stalled genes, but could also be due to differences in the can also bind to ssDNA (Wilkins and Lis 1999). specific composition of certain insulators, evidenced by GAF has multiple functions. It plays an intricate role the incomplete overlap of insulator protein-binding sites in both promoter stalling (Lee et al. 1992, 2008) and (Mohan et al. 2007; Jiang et al. 2009). interactions between cis-regulatory regions. GAF binding is associated with transcription derepression/activation (Biggin and Tjian 1988; Tsukiyama et al. 1994), repression Promoters and insulators can act as barriers (Hagstrom et al. 1997; Mishra et al. 2001), insulator Insulators originally were isolated based on their ability activity (Schweinsberg et al. 2004), barrier formation to act as barriers that prevent localized spreading of (O’Donnell and Wensink 1994), enhancer blocking (Ohtsuki chromatin states associated with activation or insertional and Levine 1998), and insulator bypass (Melnikova et al. position effects (Kellum and Schedl 1991, 1992). It is 2004). GAF’s primary role in these processes is thought to believed that barrier function comes from the ability of be its ability to recruit the ATP-dependent chromosome insulators to maintain a localized open chromatin con- remodeler NURF, which remodels nucleosomes to create figuration involving nucleosome remodeling and histone an open chromatin configuration (Adkins et al. 2006). modifications. Promoters occasionally have been as- Based on this, the complex and often opposing array of cribed with the same properties (Geyer 1997). Moreover, activities listed above likely result from the DNA-bind- stalling at promoters has been proposed to help maintain ing specificities of protein complexes that bind to sequen- promoters in a nucleosome-free state (Shopland et al. ces exposed by GAF. Two examples of the diverse 1995; Shopland and Lis 1996), and recent experiments activities of GAF that are relevant to our discussion show that depletion of NELF can result in decreased Pol II are observations that GAF is necessary but not com- occupancy and a more repressive chromatin structure at pletely sufficient for insulator activity (Melnikova et al. promoters (Muse et al. 2007; Gilchrist et al. 2008). A 2004) or promoter-proximal pausing (Lee et al. 1992). recent observation that supports this can be found at the However, given the similarities and physical contacts 87A heat-shock locus of D. melanogaster. This locus between promoters and insulators and enhancers, the contains two divergently transcribed Hsp70 genes that role of GAF in these seemingly separate events deserves are flanked by the first insulator elements to be described: re-examination. scs and scs9 (Kellum and Schedl 1991, 1992). These In addition to its role in creating an open chromatin elements interact to form a looped domain that pre- architecture, GAF can support bridging interactions be- sumably acts as a barrier to the propagation of changes tween insulators that result in insulator bypass. For ex- in chromatin structure. (Blanton et al. 2003). A recent ample, GAF in one insulator has been shown to interact study from our laboratory (Petesch and Lis 2008) has with the su(Hw) insulator-binding protein Mod(mgd4) shown that a rapid, transcription-independent loss of nu- and lead to an enhancer bypass of both insulators and cleosomes across the 87A locus is indeed restricted to the activation of a downstream promoter (Fig. 2A; Melnikova regions within the scs and scs9 elements. However, RNAi et al. 2004). Notably, Mod(mgd4) also has a BTB domain. depletion of either BEAF or Zw5, which bind the scs and It is proposed that the interaction loops out the interven- scs9 elements and mediate the looping interaction, does ing DNA, and somehow allows an interaction between not result in nucleosome loss beyond the insulators for the enhancer and promoter. GAF can also form homo- Hsp70 gene activation. Intriguingly, both insulators over- typic interactions through its BTP/POZ or Q domains lap with promoters that exhibit Pol II enrichment at the that are capable of allowing enhancer-dependent gene promoter (Muse et al. 2007). This leads to the hypothesis activation in both cis and trans when expressed heterol- that the stalled promoters are capable of enforcing local ogously in yeast (Wilkins and Lis 1999; Mahmoudi et al.

GENES & DEVELOPMENT 1609 Downloaded from genesdev.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press

Core and Lis

loops between cis-acting elements that result in insulator bypass, enhancer blocking, or promoter targeting, de- pending on the context. Third, a combination of the above models could exist, whereby looping interactions stimulated by GAF could, in concert with its promoter- opening activities, stabilize an interaction between cis- elements and stalled Pol II complexes. The Levine laboratory (Ohtsuki and Levine 1998) previously uncovered an example of a GAF-mediated enhancer–promoter interaction. GAF-binding sites proximal to the eve promoter block the IAB5 enhancer when inserted between the enhancer and another promoter. Similar to the stalled promoters used in Chopra et al. (2009) the GAF sites were not strictly involved in com- petition with other promoters. Rather, promoter compe- tition was mediated by the presence of a TATA element in the eve promoter. This last result implies that enhancer– promoter pairing is a function of both their arrangement and local sequence elements; thus, it is important to ask how sequence elements and stalling might control en- Figure 2. Possible mechanisms of GAF-mediated looping, and possible interactions between enhancers and a stalled Pol II hancer specificity. complex. (A) A model depicting GAF-mediated insulator bypass by the pairing of two insulators through interactions be- Specificity of stalled promoters for enhancer blocking tween GAF (green) and the Su(Hw) insulator-binding protein Mod(mgd4) (yellow). (B) A speculative model of how interac- Why do stalled promoters act as enhancer blockers, while tions between GAF proteins (green) or other proteins (yellow) nonstalled promoters do not? One possibility is that the present within enhancers or insulators could stimulate forma- increased dwell time of Pol II at stalled promoters allows tion of chromatin domains that could direct enhancers to a greater window of opportunity for enhancer–promoter certain promoters, while bypassing others. These interactions interactions to take place (Core and Lis 2008). This sim- could also be mediated through specific enhancer interactions ple interpretation could be combined with the hypothesis with stalled Pol II (red). These models have not been tested. (C) It is possible for enhancers (blue trapezoid) to interact with that enhancers preferably interact with some component GAF (green) at promoters, components of the stalled complex, of the stalled complex, or preferably interact with a Pol II or Pol II (red) itself. Specific interactions with a stalled Pol II that is not yet modified with the characteristics of a pro- could be mediated through the C-terminal domain, which is ductive elongation complex (Fig. 2C). For instance, an phosphorylated at Ser5 (purple P) of the heptad repeat. Changes enhancer could interact with a stalled Pol II regardless of in the transcription complex associated with elongation, such the modification state of Pol II, or the composition as Ser2 phosphorylation of the C-terminal domain (blue P), of factors associated with it. A stalling event would phosphorylation of Spt5 (blue P), or loss of NELF (yellow then create a nonmoving target that might be bound pentagon) could prevent enhancers from interacting with non- more easily by an enhancer, since Pol II is not undergoing stalled Pol II. dynamic conformational changes associated with transcription elongation. If the enhancer–promoter interactions were dependent on the modification state of 2002; Petrascheck et al. 2005). Given the high coinci- the polymerase (such as Ser5 phosphorylation of the dence of GAF at promoters and insulators, and its ability C-terminal domain), or the composition of factors asso- to forge homo- or hetero-typic interactions, it seems rea- ciated with the Pol II (such as NELF), stalling would again sonable to surmise that long-range interactions between allow more time for the interaction to occur. Conversely, insulators and stalled promoters or stalled promoters and Pol II at nonstalled promoters would be a moving target, enhancers could also be mediated by the presence of GAF and/or spend less time in the state that allows enhancer at one or both sites. interaction. Finally, Chopra et al. (2009) demonstrate this Beyond its role in insulator bypass, we envision several preference for several promoters; however, the nonstalled ways that GAF could promote these interactions (Fig. 2B). promoters tested appear to be less active or nonactive First, GAF-dependent stalling at promoters could medi- prior to enhancer function. It will be interesting to see if ate enhancer blocking by allowing efficient enhancer– the specificity of enhancer blocking for stalled promoters promoter interaction as a result of the open chromatin holds up in this type of analysis when using a gene that is configuration. Second, GAF’s role in stalling and in- constitutively active in all cells and shows no signs of sulator activity are separable, such that GAF bound at stalling in its regulation. a promoter can, as proposed for dCTCF, form homo- or Another possibility that may underlie the mechanisms hetero-typic interactions with components of insulators, described above is the inherent selectivity of enhancers independent of the transcription machinery. In this case, for certain types of promoters. Enhancer–promoter speci- the promoter-bound GAF could mediate the formation of ficity has been documented for different types of core

1610 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press

Paused Pol II has insulator activity promoters. Promoters consist of various core elements that References dictate the location and strength with which transcription Adkins NL, Hagerman TA, Georgel P. 2006. GAGA protein: A will initiate (Sandelin et al. 2007; Juven-Gershon et al. multi-faceted transcription factor. Biochem Cell Biol 84: 2008b). The most abundant core elements identified thus 559–567. far include the TATA box, the TFIIB recognition element Akbari OS, Bousum A, Bae E, Drewell RA. 2006. Unraveling cis- (BRE), the initiator (Inr), the motif 10 element (MTE), and regulatory mechanisms at the abdominal-A and abdominal-B the downstream promoter element (DPE) (Juven-Gershon genes in the Drosophila bithorax complex. Dev Biol 293: et al. 2008b). They adhere to a consensus sequence to 294–304. varying degrees and all are rarely found together at a single Akbari OS, Bae E, Johnsen H, Villaluz A, Wong D, Drewell RA. promoter. Some Drosophila enhancers have a preference 2008. A novel promoter-tethering element regulates enhancer- for activating a promoter containing a DPE (Butler and driven gene expression at the bithorax complex in the Drosophila embryo. Development 123–131. Kadonaga 2001; Juven-Gershon et al. 2008a), whereas 135: Bae E, Calhoun VC, Levine M, Lewis EB, Drewell RA. 2002. others appear to prefer a promoter with a TATA box, or Characterization of the intergenic RNA profile at abdominal- are not discriminatory (Ohtsuki et al. 1998). A recent A and abdominal-B in the Drosophila bithorax complex. Proc analysis identified that stalled promoters are linked with Natl Acad Sci 99: 16847–16852. the presence of a DPE or newly identified element, the Barski A, Cuddapah S, Cui K, Roh TY, Schones DE, Wang Z, Wei pause button (Hendrix et al. 2008). The DPE or the pause G, Chepelev I, Zhao K. 2007. High-resolution profiling of button appears to synergize with an Inr and GAF sites to histone methylations in the human genome. Cell 129: 823– provoke stalling at promoters (Hendrix et al. 2008; Lee 837. et al. 2008). These observations, in light of the results Biggin MD, Tjian R. 1988. Transcription factors that activate the from Chopra et al. (2009), may provide clues as to how the ultrabithorax promoter in developmentally staged extracts. promoter DNA sequence elements result in enhancer Cell 53: 699–711. Blanton J, Gaszner M, Schedl P. 2003. Protein:protein interac- specificity with promoters. GAF sites might be consid- tions and the pairing of boundary elements in vivo. Genes & ered in the future as another element that can dictate Dev 17: 664–675. some level of promoter specificity by the mechanisms Butler JE, Kadonaga JT. 2001. Enhancer–promoter specificity outlined above, or through its known role in insulator mediated by DPE or TATA core promoter motifs. Genes & bypass. It will be interesting to see if the presence of GAF Dev 15: 2515–2519. or a stalled Pol II at promoters are specific targets for Chopra VS, Cande J, Hong J-W, Levine M. 2009. Stalled Hox different types of enhancers, as seen with several pro- promoters as chromosomal boundaries. Genes & Dev 23: moter elements. Furthermore, if stalling is involved in 1505–1509. promoter/enhancer selectivity, it will be interesting to Cleard F, Moshkin Y, Karch F, Maeda RK. 2006. Probing long- examine the activators at enhancers in order to gain distance regulatory interactions in the Drosophila mela- information about what specific steps of transcription nogaster bithorax complex using dam identification. Nat Genet 38: 931–935. the individual activators stimulate. Core LJ, Lis JT. 2008. Transcription regulation through promoter– proximal pausing of RNA polymerase II. Science 319: 1791– 1792. Conclusion Core LJ, Waterfall JJ, Lis JT. 2008. Nascent RNA sequencing In summary, the results from Chopra et al. (2009) unveil reveals widespread pausing and divergent initiation at hu- an exciting new dimension of how developmental gene man promoters. Science 322: 1845–1848. programs are regulated. Regulation of long-range enhancer– Drewell RA, Bae E, Burr J, Lewis EB. 2002. Transcription defines the embryonic domains of cis-regulatory activity at the Dro- promoter communication by a stalled Pol II challenges us sophila bithorax complex. Proc Natl Acad Sci 99: 16853– to rethink how promoter regulation is carried out, and 16858. how chromatin domains are formed to affect cis-regulatory Farkas G, Gausz J, Galloni M, Reuter G, Gyurkovics H, Karch F. trafficking. We attempted here to put the two modes of 1994. The trithorax-like gene encodes the Drosophila GAGA regulation into context by examining the similarities factor. Nature 371: 806–808. between the mechanisms at hand in each process. There Geyer PK. 1997. The role of insulator elements in defining appear to be several links between the promoter func- domains of gene expression. Curr Opin Genet Dev 7: 242–248. tion and regulation of chromatin interactions in cis; Gilchrist DA, Nechaev S, Lee C, Ghosh SK, Collins JB, Li L, namely, paused/stalled Pol II and the presence of GAF Gilmour DS, Adelman K. 2008. NELF-mediated stalling and insulator-binding proteins at promoters and distal of pol II can enhance gene expression by blocking pro- regulatory sites. These connections can possibly lead to moter–proximal nucleosome assembly. Genes & Dev 22: 1921–1933. future experiments aimed at determining how stalled Pol Gilmour DS. 2008. Promoter proximal pausing on genes in II might direct certain chromatin domain formation, or metazoans. Chromosoma 118: 1–10. enhancer-specific interactions. Hagstrom K, Muller M, Schedl P. 1997. A polycomb and GAGA dependent silencer adjoins the fab-7 boundary in the Dro- Acknowledgments sophila bithorax complex. Genetics 146: 1365–1380. Hendrix DA, Hong JW, Zeitlinger J, Rokhsar DS, Levine MS. We thank Michael Levine, Vivek S. Chopra, Steven J. Petesch, 2008. Promoter elements associated with RNA pol II stalling and Abbie Saunders for providing critical feedback on this in the Drosophila embryo. Proc Natl Acad Sci 105: 7762– manuscript. Our research is supported by grants from the NIH. 7767.

GENES & DEVELOPMENT 1611 Downloaded from genesdev.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press

Core and Lis

Holohan EE, Kwong C, Adryan B, Bartkuhn M, Herold M, Mutskov VJ, Farrell CM, Wade PA, Wolffe AP, Felsenfeld G. Renkawitz R, Russell S, White R. 2007. CTCF genomic 2002. The barrier function of an insulator couples high binding sites in Drosophila and the organisation of the histone acetylation levels with specific protection of pro- bithorax complex. PLoS Genet 3: e112. doi: 10.1371/jorunal. moter DNA from methylation. Genes & Dev 16: 1540–1554. pgen.0030112. O’Donnell KH, Wensink PC. 1994. GAGA factor and TBF1 bind Jiang N, Emberly E, Cuvier O, Hart CM. 2009. Genome-wide DNA elements that direct ubiquitous transcription of the mapping of BEAF binding sites in Drosophila links BEAF to Drosophila a 1-tubulin gene. Nucleic Acids Res 22: 4712– transcription. Mol Cell Biol. doi: 10.1128/MCB.01748-08. 4718. Juven-Gershon T, Hsu JY, Kadonaga JT. 2008a. Caudal, a key Ohtsuki S, Levine M. 1998. GAGA mediates the enhancer developmental regulator, is a DPE-specific transcriptional blocking activity of the eve promoter in the Drosophila factor. Genes & Dev 22: 2823–2830. embryo. Genes & Dev 12: 3325–3330. Juven-Gershon T, Hsu JY, Theisen JW, Kadonaga JT. 2008b. The Ohtsuki S, Levine M, Cai HN. 1998. Different core promoters RNA polymerase II core promoter—The gateway to tran- possess distinct regulatory activities in the Drosophila em- scription. Curr Opin Cell Biol 20: 253–259. bryo. Genes & Dev 12: 547–556. Kellum R, Schedl P. 1991. A position-effect assay for boundaries Petesch SJ, Lis JT. 2008. Rapid, transcription-independent loss of of higher order chromosomal domains. Cell 64: 941–950. nucleosomes over a large chromatin domain at Hsp70 loci. Kellum R, Schedl P. 1992. A group of scs elements function as Cell 134: 74–84. domain boundaries in an enhancer-blocking assay. Mol Cell Petrascheck M, Escher D, Mahmoudi T, Verrijzer CP, Schaffner Biol 12: 2424–2431. W, Barberis A. 2005. DNA looping induced by a transcrip- Kyrchanova O, Toshchakov S, Parshikov A, Georgiev P. 2007. tional enhancer in vivo. Nucleic Acids Res 33: 3743–3750. Study of the functional interaction between mcp insulators Saunders A, Core LJ, Lis JT. 2006. Breaking barriers to transcrip- from the Drosophila bithorax complex: Effects of insulator tion elongation. Nat Rev Mol Cell Biol 7: 557–567. pairing on enhancer-promoter communication. Mol Cell Biol Sandelin A, Carninci P, Lenhard B, Ponjavic J, Hayashizaki Y, 27: 3035–3043. Hume DA. 2007. Mammalian RNA polymerase II core pro- Lee C, Li X, Hechmer A, Eisen M, Biggin MD, Venters BJ, Jiang amoters: Insights from genome-wide studies. Nat Rev Genet C, Li J, Pugh BF, Gilmour DS. 2008. NELF and GAGA factor 8: 424–436. Schweinsberg S, Hagstrom K, Gohl D, Schedl P, Kumar RP, are linked to promoter-proximal pausing at many genes in Mishra R, Karch F. 2004. The enhancer-blocking activity of Drosophila. Mol Cell Biol 28: 3290–3300. the fab-7 boundary from the Drosophila bithorax complex Lee H, Kraus KW, Wolfner MF, Lis JT. 1992. DNA sequence requires GAGA-factor-binding sites. Genetics 168: 1371– requirements for generating paused polymerase at the start of 1384. hsp70. Genes & Dev 6: 284–295. Shopland LS, Lis JT. 1996. HSF recruitment and loss at most Lis J. 1998. Promoter-associated pausing in promoter architec- Drosophila heat shock loci is coordinated and depends on ture and postinitiation transcriptional regulation. Cold Spring proximal promoter sequences. Chromosoma 105: 158–171. Harb Symp Quant Biol 63: 347–356. Shopland LS, Hirayoshi K, Fernandes M, Lis JT. 1995. HSF access Maeda RK, Karch F. 2007. Making connections: Boundaries and to heat shock elements in vivo depends critically on pro- insulators in Drosophila. Curr Opin Genet Dev 17: 394–399. moter architecture defined by GAGA factor, TFIID, and RNA Mahmoudi T, Katsani KR, Verrijzer CP. 2002. GAGA can medi- polymerase II binding sites. Genes & Dev 9: 2756–2769. ate enhancer function in trans by linking two separate DNA Tsukiyama T, Becker PB, Wu C. 1994. ATP-dependent nucleo- molecules. EMBO J 1775–1781. 21: some disruption at a heat-shock promoter mediated by bind- Mavrich TN, Jiang C, Ioshikhes IP, Li X, Venters BJ, Zanton SJ, ing of GAGA transcription factor. Nature 367: 525–532. Tomsho LP, Qi J, Glaser RL, Schuster SC, et al. 2008. Wang X, Lee C, Gilmour DS, Gergen JP. 2007. Transcription Nucleosome organization in the Drosophila genome. Nature elongation controls cell fate specification in the Drosophila 453: 358–362. embryo. Genes & Dev 21: 1031–1036. Melnikova L, Juge F, Gruzdeva N, Mazur A, Cavalli G, Georgiev Wilkins RC, Lis JT. 1999. DNA distortion and multimerization: P. 2004. Interaction between the GAGA factor and mod(mdg4) Novel functions of the glutamine-rich domain of GAGA proteins promotes insulator bypass in Drosophila. Proc Natl factor. J Mol Biol 285: 515–525. Acad Sci 101: 14806–14811. Yao J, Ardehali BM, Fecko CJ, Webb WW, Lis JT. 2007. Intra- Merika M, Thanos D. 2001. Enhanceosomes. Curr Opin Genet nuclear distribution and local dynamics of RNA polymerase Dev 11: 205–208. II during transcription activation. Mol Cell 28: 978–990. Mishra RK, Mihaly J, Barges S, Spierer A, Karch F, Hagstrom K, Zeitlinger J, Stark A, Kellis M, Hong JW, Nechaev S, Adelman K, Schweinsberg SE, Schedl P. 2001. The iab-7 polycomb re- Levine M, Young RA. 2007. RNA polymerase stalling at sponse element maps to a nucleosome-free region of chro- developmental control genes in the Drosophila melanogaster matin and requires both GAGA and pleiohomeotic for embryo. Nat Genet 39: 1512–1516. silencing activity. Mol Cell Biol 21: 1311–1318. Mito Y, Henikoff JG, Henikoff S. 2007. Histone replacement marks the boundaries of cis-regulatory domains. Science 315: 1408–1411. Mohan M, Bartkuhn M, Herold M, Philippen A, Heinl N, Bardenhagen I, Leers J, White RA, Renkawitz-Pohl R, Saumweber H, et al. 2007. The Drosophila insulator proteins CTCF and CP190 link enhancer blocking to body patterning. EMBO J 26: 4203–4214. Muse GW, Gilchrist DA, Nechaev S, Shah R, Parker JS, Grissom SF, Zeitlinger J, Adelman K. 2007. RNA polymerase is poised for activation across the genome. Nat Genet 39: 1507–1511.

1612 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press

Paused Pol II captures enhancer activity and acts as a potent insulator

Leighton J. Core and John T. Lis

Genes Dev. 2009, 23: Access the most recent version at doi:10.1101/gad.1827709

Related Content Stalled Hox promoters as chromosomal boundaries Vivek S. Chopra, Jessica Cande, Joung-Woo Hong, et al. Genes Dev. July , 2009 23: 1505-1509

References This article cites 54 articles, 28 of which can be accessed free at: http://genesdev.cshlp.org/content/23/14/1606.full.html#ref-list-1

Articles cited in: http://genesdev.cshlp.org/content/23/14/1606.full.html#related-urls

License

Email Alerting Receive free email alerts when new articles cite this article - sign up in the box at the top Service right corner of the article or click here.