InteractiveFly: GeneBrief

enhancer of yellow 3: Biological Overview | References

The novel regulator of metazoan development SAYP organizes a nuclear coactivator supercomplex

SAYP is a dual-function transcription coactivator of RNA polymerase II. It is a metazoan-specific factor with regulated expression that is apparently involved in signaling pathways controlling normal development. In Drosophila, SAYP is maternally loaded into the embryo, participates in cell cycle synchronization in early syncytial embryos, and is indispensible for early embryogenesis. SAYP is abundant in many embryonic tissues and imaginal discs in larvae and is crucial for oogenesis in adults. PHF10 is a mammalian homologue of SAYP whose expression is confined to certain tissues in adults. The molecular mechanism of the SAYP function is related to the conserved domain SAY, which assembles a nuclear supercomplex BTFly consisting of Brahma and TFIID coactivators. It is suggested that nuclear supercomplexes may be important means of gene-specific regulation of transcription during development (Vorobyeva, 2009b; full text of article).

SAYP interacts with DHR3 nuclear receptor and participates in ecdysone-dependent transcription regulation

The role of metazoan coactivator SAYP in nuclear receptor-driven gene activation in the ecdysone cascade of Drosophila is considered. SAYP interacts with DHR3 nuclear receptor and activates the corresponding genes by recruiting the BTFly (Brahma and TFIID) coactivator supercomplex. The knockdown of SAYP leads to a decrease in the level of DHR3-activated transcription. DHR3 and SAYP interact during development and have multiple common targets across the genome (Vorobyeva, 2011).

This study analyzed the role of transcription coactivator SAYP and the SAYP-assembled complex in the transcription activation of several genes activated by ecdysone in Drosophila. The results obtained by different methods show that SAYP interacts with the DHR3 nuclear receptor, a component of the ecdysone cascade. In particular, their direct interaction was demonstrated in the yeast two-hybrid system. In gel filtration and co-IP experiments, a significant proportion of DHR3 proved to be associated with the high-MW SAYP-containing protein complex. The association of these factors is confirmed by data on their colocalization on polytene chromosomes as well as their coexpression and cooperation during development. These data indicate that DHR3 interacts with SAYP both in embryos and in pupae (Vorobyeva, 2011).

DHR3 and SAYP are coordinately recruited onto promoters in pupae and S2 cells. SAYP knockdown has a negative effect on the level of DHR3-driven transcription, which indicates that SAYP mediates the action of DHR3 in transcription activation. Thus, SAYP operates as a classic coactivator, which is recruited by an activator and is important for full-level gene activity. This is in agreement with the previously demonstrated mechanism of SAYP action as a component of the BTFly coactivator complex (Vorobyeva, 2009a). As suggested previously, BTFly possesses specific features allowing its employment as a specific and efficient molecular machine for activation of genes in development. DHR3 acts together with other components of the ecdysone pathway to establish a specific pattern of gene activity in a restricted time window. It is suggested that the DHR3-SAYP interaction may be important for such specificity of DHR3 action (Vorobyeva, 2011).

The only known target gene activated by DHR3 is ftz-f1, but the current data indicate that DHR3 together with SAYP may be important for the expression of many other genes, since both these proteins co-occupy multiple sites on the polytene chromosomes. This study has directly shown that DHR3, via the interaction with SAYP, drives the expression of several SAYP-dependent genes in cell culture. These genes have not been recognized as components of the ecdysone cascade, which is evidence that the effect of ecdysone stimulation may be much wider than expected previously (Vorobyeva, 2011).

SAYP and Brahma are important for 'repressive' and 'transient' Pol II pausing

Drosophila SAYP, a homologue of human PHF10/BAF45a, is a metazoan coactivator associated with Brahma and essential for its recruitment on the promoter. The role of SAYP in DHR3 activator-driven transcription of the ftz-f1 gene, a member of the ecdysone cascade was studied. In the repressed state of ftz-f1 in the presence of DHR3, the Pol II complex is pre-recruited on the promoter; Pol II starts transcription but is paused 1.5 kb downstream of the promoter, with SAYP and Brahma forming a 'nucleosomal barrier' (a region of high nucleosome density) ahead of paused Pol II. SAYP depletion leads to the removal of Brahma, thereby eliminating the nucleosomal barrier. During active transcription, Pol II pausing at the same point correlates with Pol II CTD Ser2 phosphorylation. SAYP is essential for Ser2 phosphorylation and transcription elongation. Thus, SAYP as part of the Brahma complex participates in both 'repressive' and 'transient' Pol II pausing (Vorobyeva, 2012).

The mechanism of ftz-f1 transcription activation has been analyzed in S2 cells. Sequential addition and removal of ecdysone allows the DHR3 and ftz-f1 genes in these cells to be activated in accordance with their expression pattern in vivo. This system is of considerable interest, since only a few Drosophila models of activated transcription are available. It also provides the possibility of studying the mechanism of pausing in the active and repressed transcription states of the same gene, whereas previous such studies have been performed with different genes (Vorobyeva, 2012).

Pol II pausing on ftz-f1 occurs at about 1.5 kb downstream of the promoter, i.e. at a much greater distance than that described for other genes (from +30 to +100 nt). Future studies will show how widespread is this mode of pausing. It is of interest in this context that a case of Pol II pausing at 800 bp downstream of the promoter was described for the β-actin gene (Vorobyeva, 2012).

The ftz-f1 activation at the molecular level is a several-stage process. At the first stage, when the ecdysone titer and DHR3 expression are high, DHR3, SAYP, TFIID, Brahma and Pol II accumulate at the promoter. Transcription is initiated, but Pol II is paused 1.5 kb downstream of the promoter; DHR3, SAYP and Brahma are also present at this site, where a nucleosomal barrier is formed. At the next stage, ~1 h after ecdysone removal, promoter-bound factors remain at the same levels, except for SAYP (its level on the promoter decreases). Pol II and associated factors disappear from the site of pausing, and the nucleosomal barrier is eliminated, but the transcription level does not increase. The following stage is characterized by rapid intensification of transcription, which reaches a maximum within several hours; the level of Pol II increases in the body of the gene, and its pausing is observed again, with SAYP and Brahma being present at the corresponding position. In addition, the level of SAYP on the promoter is recovered, indicating that it is highly regulated at different transcription stages. The DHR3 activator is present at the site of pausing, and its level does not change upon SAYP knockdown. This is evidence that DHR3 may participate in SAYP recruitment for subsequent nucleosomal barrier formation and Pol II pausing (Vorobyeva, 2012).

The region of high nucleosome density (nucleosomal barrier) is specific for the repression stage, at which the DHR3 activator induces the assembly of the Pol II preinitiation complex on the promoter and makes paused Pol II competent for transcription initiation. Nucleosomal barrier disruption by SAYP knockdown leads to the full-length transcript synthesis, indicating that the nucleosomal barrier contributes to preventing the entry of Pol II to the transcribed region. The data show that SAYP and Brahma play the crucial role in organization of the nucleosomal barrier: this barrier coincides in location with the peak of these coactivators and disappears after SAYP knockdown, which leads to elimination of Brahma from the gene. Thus, SAYP and Brahma at the stage of repressed transcription have an important role in blocking the synthesis of full-length transcripts. Although the transcription increases upon SAYP depletion and elimination of the nucleosomal barrier, its level remains low, compared with that in the permissive state. This is evidence for the existence of different mechanisms of Pol II pausing regulation, which also correlates with the fact that the depletion of NELF, an important factor of Pol II pausing, causes a 2.5-fold increase in the transcription of hsp70 or hsp26 gene in the repressed state, which, however, does not reaches the level characteristic of a fully activated gene (Vorobyeva, 2012).

The question arises as to the structure of the nucleosomal barrier. As shown previously, the human SWI/SNF complex can not only erase nucleosomes from the template but also produce a stable remodeled dimer of mononucleosome core, with this complex being also needed for converting this product back to the cores. One may suggest that the Drosophila Brahma complex operates in the same way. In the current experiments, the level of histone H3 increased ~2-fold in the region of the nucleosomal barrier, compared with its general level on the gene, which agrees with the assumption concerning the presence of a nucleosome dimer. The fact that the region of nucleosomal barrier is significantly enriched in sequences with a high nucleosome-positioning probability indicates that DNA sequences probably contribute to organization of this barrier (Vorobyeva, 2012).

Previous experiments have revealed a relationship between Pol II pausing and the nucleosomal structure of the template. It has been shown that Pol II stops at the site where the nucleosome density is restored to the average level characteristic of the gene. However, no specific nucleosome-dense regions preventing Pol II transcription have been described as yet (Vorobyeva, 2012).

The transition to the transcription-permissive state correlates with significant rearrangements in the promoter-distal region (disappearance of Brahma, SAYP, Pol II and nucleosomal barrier at the site of Pol II pausing). However, no increase in the ftz-f1 transcription level has been observed within the first 30 min after this transition. As shown in the study on estradiol (ER)-mediated gene expression, productive transcription is preceded by an unproductive cycle (~40 min) that is necessary for promoter preparation to this process. This may be the case for ftz-f1, with a certain period of time being required for rearrangements preceding its active transcription (Vorobyeva, 2012).

At the (+;-) stage, the level of SAYP on the promoter is recovered within 2-3 h after the onset of transcription, with SAYP RNAi influencing the Brahma and TFIID levels on the promoter. Pol II pausing correlating with its Ser2 modification is again observed as the transcription level increases. Although SAYP and Brahma occur again together with paused Pol II, their function appears to be different from that at the repression stage. The nucleosomal barrier is not restored, and SAYP depletion has only a slight effect on chromatin structure (Vorobyeva, 2012).

However, SAYP depletion severely disturbs transient pausing, interfering with Ser2 phosphorylation. This impairs proper transition to productive elongation and leads to a decrease in Pol II level on the body of the gene. Thus, SAYP knockdown not only affects the level of ftz-f1 activation but also shifts the timing of its expression. The slower kinetics of transcription induction together with the slight decrease in the Pol II level on the promoter upon SAYP knockdown are evidence for the retarded Pol II passage in the coding region of the gene and, hence, for disturbances in the elongation mechanisms. Similar consequences are observed for other genes regulating on pausing mechanisms (Vorobyeva, 2012).

The results of this study show that SAYP is important for proper timing of ftz-f1 transcription during Drosophila metamorphosis. The ftz-f1 gene is a major regulator of metamorphosis, that is why its precise activation in time is crucial during development. On the whole, the data provide evidence for the important role of pausing in sequential activation of genes in cascades and indicate that this mechanism may have a general role in development (Vorobyeva, 2012).

In addition, these results also support the idea that Pol II pausing may require not only NELF and DSIF but also other factors, such as nucleosome-remodeling complexes. Interestingly, the depletion of NELF proved to result in an increased nucleosome occupancy at the promoters of some genes (Vorobyeva, 2012).

In summary, this study has found that Pol II pausing is dependent on the interplay of several molecular mechanisms, including the formation of a specific chromatin structure via the action of coactivators. These results indicate that, although Pol II pausing is a genome-wide phenomenon, the specific molecular mechanism controlling paused Pol II activity on individual genes may vary significantly (Vorobyeva, 2012).

Transcription co-activator SAYP mediates the action of STAT activator

Jak/STAT is an important signaling pathway mediating multiple events in development. This study describes participation of metazoan co-activator SAYP/PHF10 in this pathway downstream of STAT. The latter, via its activation domain, interacts with the conserved core of SAYP. STAT is associated with the SAYP-containing co-activator complex BTFly (Vorobyeva, 2009a) and recruits BTFly onto genes. SAYP is necessary for stimulating STAT-driven transcription of numerous genes. Mutation of SAYP leads to developmental defects similar to those observed in STAT mutants. Thus, SAYP is a novel co-activator mediating the action of STAT (Panov, 2012).

This study analyzed the role of co-activator SAYP in the Jak/Stat pathway in Drosophila and found that SAYP interacts with STAT and mediates its activation potential. SAYP operates as a component of large protein complex BTFly, which also contains Brahma and TFIID, and mediates subsequent recruitment of PolII onto the promoter. The results of polytene chromosome staining and measurement of multiple STAT-dependent genes testify to genome-wide cooperation of STAT and SAYP in gene activation (Panov, 2012).

The activation domain of STAT interacts with the SAY-PHD fragment of SAYP within the BTFly, and the STAT-BTFly association is not mediated by Brahma or TFIID. It is noteworthy that the interacting fragment of SAYP belongs to its conserved core, which is also found in vertebrate homologues of SAYP. Therefore, the above interaction may also take place in other species (Panov, 2012).

This finding broadens the known spectrum of transcription factors mediating the effect of STAT on gene expression. Such a diversity of cooperating factors appears to provide a basis for the specificity and strength of Jak/Stat-driven response in different cell types. In particular, BTFly may serve for rapid induction of transcription, as proposed previously. Indeed, the positive effect of SAYP content on short-term induction after STAT activation was observed. SAYP is also important for PolII stalling, which may provide for the precision of responses of target genes in the signaling pathway. One more point of interest is the putative role of SAYP in coordinating the crosstalk between Jak/Stat and other signaling pathways, which follows from the fact that SAYP is also involved in the ecdysone cascade and probably in some other cascades (Panov, 2012).

Mutation of both STAT and SAYP leads to formation of excess numbers of ovarian follicular cells and ectopic wing veins. SAYP is abundant in cells with high proliferative potential, and its mutation results in overproliferation of polar cells in embryos. Therefore, SAYP may participate in regulation of proliferation of certain cell types. Interestingly, PHF10-a vertebrate homologue of SAYP-is required for proliferation of stem/progenitor cells (Lessard, 2007) and fibroblasts (Banga, 2009). On the other hand, it has been shown that STAT also has a role in proliferation and growth control. Therefore, it appears that SAYP and STAT are jointly involved in regulation of proliferation and differentiation of certain cell types during metazoan development (Panov, 2012).

The transcriptional coactivator SAYP is a trithorax group signature subunit of the PBAP chromatin remodeling complex

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SWI/SNF ATP-dependent chromatin remodeling complexes (remodelers) perform critical functions in eukaryotic gene expression control. BAP and PBAP are the fly representatives of the two evolutionarily conserved major subclasses of SWI/SNF remodelers. Both complexes share seven core subunits, including the Brahma ATPase, but differ in a few signature subunits; POLYBROMO and BAP170 specify PBAP, whereas OSA defines BAP. This study shows that the transcriptional coactivator and PHD finger protein SAYP is a novel PBAP subunit. Biochemical analysis established that SAYP is tightly associated with PBAP but absent from BAP. SAYP, POLYBROMO, and BAP170 display an intimately overlapping distribution on larval salivary gland polytene chromosomes. Genome-wide expression analysis revealed that SAYP is critical for PBAP-dependent transcription. SAYP is required for normal development and interacts genetically with core- and PBAP-selective subunits. Genetic analysis suggested that, like BAP, PBAP also counteracts Polycomb silencing. SAYP appears to be a key architectural component required for the integrity and association of the PBAP-specific module. It is concluded that SAYP is a signature subunit that plays a major role in the functional specificity of the PBAP holoenzyme (Chalkley, 2008).

Gene expression control is one of the most fundamental biological processes and, to a large extent, occurs at the transcriptional level. The transcription of a single protein-encoding eukaryotic gene involves a stunning plethora of regulating factors comprising some 100 or so distinct polypeptides. These can be classified as sequence-specific DNA-binding transcription factors that initiate the recruitment of positive or negative coregulatory complexes and the basal transcription machinery. Coactivators include a variety of proteins performing distinct functions during the transcription cycle such as the opening up of chromatin structure, mediating posttranslational histone modifications or bridging between activators and the basal transcription machinery. It has become clear that the diversity among gene-specific activators and repressors is complemented by functional specification among coregulatory complexes and even the core transcription machinery. One important class of coregulators is formed by the ATP-dependent chromatin-remodeling factors (remodelers) (Chalkley, 2008).

Remodelers are large multisubunit complexes defined by the presence of an ATPase 'engine' subunit. These proteins act like DNA translocases and use the energy derived from ATP hydrolysis to change the DNA-histone contacts, thus remodeling chromatin structure. Based on the identity of their central ATPase, four major classes of remodelers have been recognized: SWI/SNF, ISWI, CHD/Mi2, and Ino80/Swr1 (Lall, 2007). Different remodelers are not exchangeable; rather, each executes unique biological functions. An early example of functional diversification was the finding that the Drosophila SWI/SNF class Brahma (BRM) remodelers, but not the ISWI remodelers, act as chromatin-specific coactivators for the transcription factor Zeste (Kal, 2000; Chalkley, 2008 and references therein).

SWI/SNF class remodelers perform broad yet gene-selective transcription regulatory functions during development, cell cycle control, and tumor suppression. There are two major SWI/SNF subclasses, conserved evolutionarily from yeast to humans. The first subclass includes yeast SWI/SNF (ySWI/SNF), fly BAP, and mammalian BAF, whereas the second subfamily includes yeast RSC, fly PBAP, and mammalian PBAF (Mohrmann, 2005). The corresponding multiprotein complexes are composed of highly related paralogs or identical subunits and a limited number of subclass-specific proteins. For example, Drosophila BAP and PBAP share seven core subunits, but each is defined by unique signature subunits: the BAP-specific OSA and the PBAP-specific POLYBROMO and BAP170. In this study the term SWI/SNF is used when making general statements that apply to both subcomplexes (Chalkley, 2008).

Previous structure-function dissection of fly SWI/SNF revealed that the common core subunits play architectural and enzymatic roles, whereas the signature subunits are key to the functional specificity of BAP and PBAP holoenzymes. In particular, BRM and MOR are critical for the structural integrity of both BAP and PBAP (Moshkin, 2007). Regulation of the majority of target genes required the signature subunit OSA, PB, or BAP170, suggesting that SWI/SNF remodelers function mostly as holoenzymes (Moshkin, 2007). BAP and PBAP regulate distinct but overlapping transcriptional circuitries, acting either independently, similarly, or antagonistically. Likewise, BAP and PBAP direct convergent as well as distinct biological processes. BAP, but not PBAP, is required for cell cycle progression through mitosis. BAP mediates G2/M transition through direct regulation of the cell cycle regulator string/cdc25. OSA is required for targeting BAP to the string/cdc25 promoter (Chalkley, 2008).

The genes encoding BRM, MOR, and OSA were originally discovered in screens for dominant suppressors of Polycomb (Pc) mutations and therefore were classified as trithorax group (trxG) proteins. The trxG of activators, together with their antagonists, the Pc group (PcG) of repressors, maintain correct expression of many developmental regulators. So far, no other core- or PBAP-selective subunits have been identified as trxG proteins. Thus, whether PBAP, like BAP, acts as a trxG suppressor of Pc remains unclear (Chalkley, 2008).

SAYP is a chromatin-associated transcriptional coactivator that was originally identified as the enhancer of yellow, e(y)3, gene (Shidlovskii, 2005). SAYP contains two PHD fingers, an AT hook and a highly conserved SAY domain that is essential for transcription coactivation. Analysis of mutants revealed that SAYP is essential for oogenesis and early development (Shidlovskii, 2005). However, the molecular functioning of SAYP remained completely unclear (Chalkley, 2008).

Because distinct SWI/SNF subunits each provide unique functionalities, the complete determination of the BAP and PBAP composition is an important objective. In this study PBAP was purified and the coactivator SAYP was identified as a novel subunit. SAYP was found to be an essential and distinctive subunit of PBAP, which is absent from BAP. A variety of genomic, biochemical, and genetic approaches were used to dissect the role of SAYP. It is concluded that the transcriptional coactivator SAYP is a novel signature subunit that is essential for the functional specificity of the PBAP holoenzyme (Chalkley, 2008).

This study shows that SAYP is a novel PBAP signature subunit. The human homologue of SAYP has been shown to be a subunit of PBAF. Moreover, genes homologous to sayp were identified in the genomes of sequenced metazoans, but no clear homologues were detected in, e.g., Neurospora spp., Saccharomyces cerevisiae, or Schizosaccharomyces pombe, suggesting that SAYP might be a metazoan-specific subunit (Chalkley, 2008).

Immunodepletion of a nuclear extract using antibodies directed against MOR led to the concomitant loss of SAYP in the unbound fraction. This result suggests that the majority of SAYP exists as part of the PBAP complex. Previous results have suggested that SWI/SNF subunits that are not incorporated in a complex are unstable and degraded. Moreover, this study established that particular subunits, e.g., MOR, are essential for the architectural integrity of BAP and PBAP. This analysis found that SAYP is required for the incorporation of BAP170 and POLYBROMO into PBAP. This relationship was not reciprocal: neither BAP170 nor POLYBROMO was required for SAYP assembly (Chalkley, 2008).

Whereas the BAP and PBAP signature modules require the core, removal of the PBAP signature subunits or OSA depletion did not affect the core complex. However, in the absence of the signature subunits, the SWI/SNF core alone turned out to be largely dysfunctional in global gene expression control. This finding highlights the interesting issue that untargeted chromatin remodeling, which can be mediated efficiently by the SWI/SNF core by itself does not suffice for transcription control. Therefore, it is suggested that SWI/SNF remodelers act as holoenzymes, in which the core subunits provide key architectural and enzymatic functions, but the signature subunits determine most of the transcriptional specificity (Chalkley, 2008).

Another interesting outcome from this analysis is that there is no evidence for BAP/PBAP hybrid complexes. Thus, the docking of OSA and the PBAP signature module appear to be mutually exclusive. Although there are likely to be additional sites of contact, SAYP appears to be particularly important for the stable association of POLYBROMO and BAP170 with the core complex. BAP170 stabilizes POLYBROMO binding (Moshkin, 2007). Most likely, there is a hierarchy of incorporation in which SAYP and OSA compete for an overlapping docking site within the core complex. The association of POLYBROMO and BAP170 is stabilized by SAYP but blocked by OSA, preventing the formation of hybrid complexes (Chalkley, 2008).

These results and earlier studies (Moshkin, 2007) demonstrate the value of epistasis analysis combined with whole-genome expression profiling for the structure-function analysis of multisubunit regulatory complexes. Principal component analysis (PCA) of gene expression profiles after BAP- or PBAP-selective depletion revealed that BAP and PBAP regulate distinct but overlapping transcriptional circuitries, acting either together, independently, or antagonistically. Likewise, BAP and PBAP execute related but also distinct biological functions. Previously, it was found that BAP, but not PBAP, controls cell cycle progression (Moshkin, 2007). BAP mediates G2/M transition through direct regulation of the cell cycle regulator string/cdc25. BAP recruitment to the stg/cdc25 promoter was critically dependent on OSA. In contrast, PBAP neither bound nor activated the stg/cdc25 promoter. Deciphering the role of SWI/SNF in cell proliferation and differentiation is of particular interest because of the association between SWI/SNF malfunction and human cancer. For example, the loss of the human homologue of SNR1, hSNF5, causes a defective cell cycle and the loss of ploidy control in malignant rhabdoid tumor cells (Chalkley, 2008).

Previously, the genes encoding the BAP-signature subunit OSA and two core subunits, BRM and MOR, were identified as trxG members because they act as dominant suppressors of Pc. However, it remained unclear whether PBAP also antagonizes Pc silencing. This study found that mutations in the genes coding for the PBAP signature subunits suppress the leg transformations caused by Pc mutations. Thus, both BAP and PBAP can be classified as trxG transcriptional coactivator complexes (Chalkley, 2008).

This study has also implicated PBAP in additional developmental control pathways. Mutations in genes encoding PBAP-specific subunits were shown to cause characteristic leg malformations and microcephaly, which are reminiscent of phenotypes caused by defective ecdysone signaling. These observations suggest that PBAP might be involved in ecdysone-inducible gene regulation in vivo. Previous in vitro results suggested that human PBAF is selectively required for ligand-dependent transactivation by nuclear hormone receptors (Lemon, 2001). Therefore, it will be interesting to investigate the role of BAP and PBAP in nuclear hormone receptors signaling during development (Chalkley, 2008).

In summary, BAP and PBAP are essential chromatin remodeling factors that perform cooperative and unique functions during development. Because distinct subunits appear to be dedicated to specific regulatory pathways, a complete structure-function analysis is required to gain insight into the roles of SWI/SNF remodelers in development and disease. This study has identified the coactivator SAYP as novel PBAP subunit. It is concluded that SAYP is a novel signature subunit that is essential for the functional specificity of the PBAP holoenzyme. Furthermore, this analysis of SWI/SNF remodelers has suggested that they are dedicated to specific transcriptional pathways, rather than acting as true general factors. Future studies will aim at dissecting the gene-selective functions of remodelers during development and disease (Chalkley, 2008).

A novel multidomain transcription coactivator SAYP can also repress transcription in heterochromatin

Enhancers of yellow (e(y)) is a group of genetically and functionally related genes for proteins involved in transcriptional regulation. The e(y)3 gene of Drosophila, considered in this study, encodes a ubiquitous nuclear protein that has homologues in other metazoan species. The protein encoded by e(y)3, named Supporter of Activation of Yellow Protein (SAYP), contains an AT-hook, two PHD fingers, and a novel evolutionarily conserved domain with a transcriptional coactivator function. Mutants expressing a truncated SAYP devoid of the conserved domain die at a midembryonic stage, which suggests a crucial part for SAYP during early development. SAYP binds to numerous sites of transcriptionally active euchromatin on polytene chromosomes and coactivates transcription of euchromatin genes. Unexpectedly, SAYP is also abundant in the heterochromatin regions of the fourth chromosome and in the chromocenter, and represses the transcription of euchromatin genes translocated to heterochromatin; its PHD fingers are essential to heterochromatic silencing. Thus, SAYP plays a dual role in transcription regulation in euchromatic and heterochromatic regions (Shidlovskii, 2005).

Two mutant alleles of e(y)3 genetically mapped to 19C of the X chromosome have been isolated. The viable e(y)3^u1 allele was induced by insertion of a Stalker mobile element. The lethal allele e(y)3^EMSl was found in the progeny of ethylmethanesulfonate-treated (EMS) males (Shidlovskii, 2005).

To isolate the e(y)3 gene, the sequences surrounding the Stalker in e(y)3^u1 flies were cloned. Sequencing demonstrated that Stalker insertion occurred in the genetic locus encoding a protein with two PHD fingers at the C terminus (FlyBase report CG12238). To prove that e(y)3 mutations really influence CG12238, the corresponding genomic region including the predicted promoter sequences was cloned in CaSpeR3 vector and used to rescue the e(y)3^u1 and e(y)3^EMSl mutants. Each of five independently obtained transgenes completely restored the wild-type phenotype, demonstrating that the isolated gene was really e(y)3 (Shidlovskii, 2005).

Several cDNA clones corresponding to e(y)3 were isolated from a cDNA library prepared from strain Oregon R. This gene has 12 exons containing an open reading frame (ORF) for a protein of 2008 amino acids, that is, 165 residues longer than the one presented in FlyBase. The main e(y)3 mRNA detected by Northern blot hybridization was 10 kb long. However, three additional weaker transcripts of lower molecular weight were also found. Analysis of the cDNA clones showed that three mRNAs of e(y)3 were identical in their coding sequences but different in their 3′-untranslated regions. The 6.5-kb transcript was found to have an alternative start of transcription at exon 2 and thus to contain the coding region, which is 163 amino acids shorter (Shidlovskii, 2005).

Next, polyclonal antibodies against two different peptides from the SAYP N-proximal region were raised in rabbits. Antiserum 1 and affinity-purified Ab1 and Ab2 detected the same two closely migrating protein species (about 270 and 250 kDa) in a nuclear extract from Drosophila embryos. Moreover, the bands specifically disappeared if the antiserum was incubated with the peptides used for immunization before Western blotting. The lower band seems to represent a version of SAYP lacking the N-terminal stretch and synthesized from the 6.5-kb transcript, as the difference between two bands (about 20 kDa) is quite close to the expected one (18 kDa). The molecular weight of SAYP proteins is higher than that calculated from the amino-acid sequence, which may be explained by post-translational modifications. Also, it cannot be excluded that the two bands detected on Western blot represent differently modified SAYP (Shidlovskii, 2005).

SAYP contains four serine-rich regions, a proline-rich region, two glutamine stretches, and two positively charged clusters. It also contains seven putative nuclear localization signals in different parts of the molecule (Shidlovskii, 2005).

An AT-hook motif is present in the central part of SAYP. The AT-hook, first described in HMGA proteins, is a small motif recognizing AT-rich sequences and binding to the minor groove of the DNA helix. Multiple or single AT-hook motifs have been detected in a wide range of nuclear proteins from different species, including multidomain chromatin-associated proteins involved in transcription activation or repression Shidlovskii, 2005).

Near the carboxy terminus of SAYP, there are two PHD fingers. PHD is an orphan conserved Cys₄-His-Cys₃ Zn-finger domain found in many chromatin-associated proteins, including several transcription factors, for example, TrxG and PcG; acetyltransferase CBP/p300; Mi-2 protein, a component of the histone deacetylating complex; and the chromatin remodeling protein Acf1 (Shidlovskii, 2005).

A database search revealed sequences homologous to SAYP in human (hypothetical protein XAP135/PHF10) and several other metazoan species including mouse Mus musculus, zebrafish Danio rerio, and nematode Caenorhabditis elegans as well as mosquito Anopheles gambiae. The vertebrate proteins are very close in sequence (75% identity between human and fish) and length, representing the core of evolutionary conservation. Both insect and nematode proteins are considerably longer, extending in both directions (Shidlovskii, 2005).

The comparison indicated that all homologues contained a highly conserved domain, referred to as the Supporter of Activation of Yellow (SAY) domain. The SAY domain (30% identity and 45% similarity between Drosophila and human proteins) is followed, after a short low-homology region, by PHD fingers. It is noteworthy that the two putative PHD fingers of SAYP share the conserved Cys residue, implying that only one PHD may function at a moment. The same is observed for SAYP homologues from other species, except for the mosquito protein, which has only one finger. The Drosophila protein, like the vertebrate homologues, has a serine-rich region in the spacer. The pronounced conservation of this domain arrangement from insects to mammals strongly suggests that both domains are essential to the function of these factors (Shidlovskii, 2005).

The 10-kb e(y)3 mRNA was detected at all stages of insect development as well as were all weaker transcripts. However, the most intense transcription was observed in adult females. The highest content of e(y)3 mRNA was detected in ovaries. It was present in the cytoplasm of nursing cells and growing oocytes at all stages of development and accumulated in mature oocytes (Shidlovskii, 2005).

Immunostaining also revealed SAYP in the nuclei of syncytium blastoderm of early embryos and in the nuclei of different tissues of late embryos, larvae, and adults. According to the in situ hybridization data, SAYP is abundant in the nuclei of various ovary cells. Thus, SAYP is a ubiquitous nuclear protein, expressed at all stages of development and in different tissues of flies. Interestingly, the cDNAs of XAP135/PHF10, the human counterpart of SAYP, were found in EST databases prepared from various tissues, suggesting that XAP135/PHF10 is also a ubiquitous protein (Shidlovskii, 2005).

In line with the essential role of SAYP in oogenesis, the major phenotypic manifestation of the e(y)3^u1 mutation was female sterility. The e(y)3^u1 mutation also decreased fly viability -- by 50% hemizygous males and by 20% in homozygous females -- and caused disturbances in the development of femur, shortened body, and expanded wings. All these features were weak and were observed in 15%-20% of flies (Shidlovskii, 2005).

Homozygous e(y)3^EMSl females and hemizygous e(y)3^EMSl males died at a midembryonic stage. Their survival at earlier stages appears to be due to the maternal effect of the e(y)3 gene. Examination of e(y)3^EMSl embryos revealed multiple and variable disturbances in development including the formation of head, midgut, malpigian tubes, and embryonic gonads. The mutations of genes encoding several different transcription factors have similar manifestations, suggesting that these genes probably interact with e(y)3 in development. Of particular interest is the cut locus, whose function is necessary for specification of a large number of cell types. Previously, the genetic interaction of e(y)3 and the cut locus (Melnikova, 1996) has been reported. However, the interaction of e(y)3 with other genes needs further careful investigation. The manifestations of e(y)3 mutations suggest SAYP to be indispensable for oogenesis and early stages of development (Shidlovskii, 2005).

To assess the distribution of SAYP in chromatin, immunostaining of polytene chromosomes from Drosophila salivary glands was undertaken. Affinity-purified antibodies Ab1 or Ab2 were used for immunostaining. Both antibodies were shown to be specific: they selectively recognize on Western blots the wild-type and mutated versions of SAYP and do not recognize any unspecific bands. About 150 sites of SAYP binding were detected; the two antibodies recognized the same sites in the arms of polytene chromosomes. Most of them coincided with those containing Pol II and were localized in the less compact regions of chromatin poorly stained with DAPI. In contrast, Pol II was revealed at many more sites than SAYP, indicating that SAYP is present at only a certain fraction of the transcribed genes (Shidlovskii, 2005).

Unexpectedly, immunostaining of polytene chromosomes also revealed SAYP in heterochromatic regions: in the chromocenter and at chromosome 4, most of which is represented by heterochromatin. Antibodies against SAYP strongly decorated these regions, while only weak staining of the fourth chromosome was observed with antibodies against Pol II. The heterochromatic nature of the sites of SAYP binding is further proved by comparison of the distribution of SAYP with that of heterochromatin protein 1 (Shidlovskii, 2005).

To verify the observed pattern, transgenic flies bearing the construct expressing FLAG-tagged SAYP were obtained. The transgene was able to rescue the e(y)3^u1 and e(y)3^EMSl mutations, testifying that tagged SAYP is functional. The antibodies against FLAG stained euchromatic and heterochromatic regions on polytene chromosomes of the transgenic flies, demonstrating complete colocalization with SAYP both in the sites on the arms of chromosomes and on the fourth chromosome and chromocenter (Shidlovskii, 2005).

The results obtained implicate SAYP in the organization of heterochromatin structure and hence in regulation of gene expression in heterochromatin (Shidlovskii, 2005).

To study the function of SAYP in heterochromatin, the effect was investigated of the e(y)3^u1 mutation on expression of transgenes located in different heterochromatic regions of the fourth chromosome and in the chromocenter. The transgenic lines of flies bearing the P-element vector P[hsp26-pt, hsp70-w] have been described. This vector contained the white gene driven by the hsp70 promoter, which produced a convenient marker to monitor gene expression both visually and by quantitating the amount of pigment accumulated in eyes (Shidlovskii, 2005).

The transgenic flies with inserts in heterochromatic domains demonstrated a variegating eye phenotype due to silencing of the transgene. The effect of SAYP on transgene expression was investigated in females heterozygous for e(y)3^u1 and P insertion and in males hemizygous for e(y)3^u1 and heterozygous for P insertion. Significantly increased transgene expression was observed in males of most of the tested lines. Even in females, the effect, although less prominent, could be observed despite the presence of a wild-type e(y)3 allele. Increased expression was observed in lines in which the P insertion occurred in the fourth chromosome and in the centromeric region. The extent of the observed effect of e(y)3^u1 was similar to that shown previously for a missense mutation of HP1 (Su(var)2-5) (Shidlovskii, 2005).

The influence of e(y)3^u1 was tested on the natural white-mottled (w^m4h) mutation, which leads to variegated color of eyes because of X-chromosome inversion bringing the white gene in proximity to the centromere. Introduction of the e(y)3^u1 in w^m4h flies tripled the white expression in females heterozygous for e(y)3^u1 and w^m4h mutations (Shidlovskii, 2005).

Thus, like the known mutations of HP1 and other proteins shown to participate in heterochromatin silencing, the e(y)3^u1 mutation is a dominant suppressor of PEV. It represses transcription of euchromatic genes brought into heterochromatin surroundings, affecting transgenes as well as a natural w^m4h mutation. However, the influence of SAYP on the expression of genes originally residing in heterochromatin may be different. It may activate them, just as HP1 activates transcription of light and rolled genes of heterochromatin. Importantly, recent findings suggest that HP1 regulates both positively and negatively several genes of euchromatin (Shidlovskii, 2005).

The influence of the mutation was tested on expression of the same P-element construct located in 2L, 2R, and 3R telomeres, where SAYP binding was also detectable. No significant changes were found in the level of white expression. The results obtained demonstrate that SAYP participates in repression of transcription in heterochromatin of the fourth chromosome and in pericentric heterochromatin, but not in telomeres (Shidlovskii, 2005).

In previous genetic experiments, the e(y)3 gene was shown to be required for activation of several genes (Georgiev, 1989). This study whether individual domains of SAYP would have transcriptional activation functions. To this end, several domains of SAYP were fused to the C terminus of LexA. In yeast cells, the fusion peptide containing the conserved region of SAYP (amino acids 1273-1629) efficiently activated the HIS3 and LacZ reporter genes containing LexA-binding sites upstream of their promoter regions, while other LexA-SAYP fusions, in particular those containing the PHDs, did not. Cells expressing the LexA-SAYP(1273-1629) fusion grew efficiently on selective medium lacking histidine, as well as those expressing the LexA-GAL4 activation domain fusion used as the positive control. The rate of LacZ reporter gene activation by LexA-SAYP(1273-1629) was 10 times lower than that provided by LexA-GAL4. However, it was about 80 times higher than that provided by LexA-SAYP(1366-1629) that lacked the first 26 amino acids of the SAY domain. This fusion also resulted in a weaker growth. Deletion of 80 amino acids from the C terminus of the SAY domain in LexA-SAYP(1273-1493) completely abolished the activity of the fusion peptide. Thus, the whole conserved domain of SAYP (about 350 amino acids) is required for transcription activation in the yeast two-hybrid system, while the other domains do not possess this activity. These results testify that the SAY domain is a potent transcriptional activator that is responsible for the coactivator function of SAYP. However, in vivo, the other domains of SAYP may also be important for transcription activation (Shidlovskii, 2005).

The function of the SAY domain and PHDs was tested using the obtained mutations. The influence of the e(y)3^EMSl mutation on fly viability is much more severe than that of e(y)3^u1, suggesting that the SAY domain is indispensable for development of flies. To ascertain this, a transgene P{e(y)3^ΔPHD} was constructed that expressed a protein truncated shortly after the SAY domain, retaining the Ser-rich stretch but having no PHDs. The expression of P{e(y)3^ΔPHD} was confirmed by Western blot. The P{e(y)3⁺} construct producing the full-length SAYP was used as a control (Shidlovskii, 2005).

Both constructs were first tested for the ability to rescue the lethal e(y)3^EMSl allele. Eight independent insertions of P{e(y)3⁺} and five independent insertions of P{e(y)3^ΔPHD} were tested in rescue experiments. Just as P{e(y)3⁺}, the P{e(y)3^ΔPHD} transgene restored the visible wild-type phenotype of the e(y)3^EMSl mutants (Shidlovskii, 2005).

The influence of the P{e(y)3^ΔPHD} transgene was tested on the phenotype of the e(y)3^u1 mutation. This mutation results in synthesis of the protein lacking PHDs. In addition, the amount of SAYP is decreased in the e(y)3^u1 strain. Introduction of P{e(y)3^ΔPHD} would increase the amount of truncated protein, making it possible to detect the consequences of mutation caused by the lack of PHDs. Unexpectedly, like the construct expressing the wild-type SAYP, P{e(y)3^ΔPHD} was able to complement the main manifestations of the e(y)3^u1 mutation, restoring female fertility and increasing the viability of the e(y)3^u1 strain. Thus, a lower content of the SAY domain resulting from decreased e(y)3 expression, rather than the lack of PHDs, is the main cause of disturbances in e(y)3^u1 flies (Shidlovskii, 2005).

The influence was assessed of the SAY domain on gene expression in vivo. A significant manifestation of the e(y)3^u1 mutation described previously was its ability to interfere with the expression of several genes (Georgiev, 1994). In particular, it affects the yellow gene, decreasing expression of the y² allele in bristles (Georgiev, 1989). The y² allele is generated by insertion of the retrotransposon gypsy in the yellow regulatory region. To exclude the role of gypsy in the e(y)3-mediated regulation of the yellow gene, the y^Inr allele was used that was generated by mutation in the Initiator element of the yellow promoter. While the y^Inr allele displayed a wild-type phenotype, the e(y)3^u1 mutation strongly reduced its expression in bristles (Shidlovskii, 2005).

Whether introduction of the P{e(y)3^ΔPHD} construct in y²e(y)3^u1 or y^Inre(y)3^u1 flies would influence the expression of yellow was also checked, and complete restitution of the original y² or y^Inr phenotype was observed in transgenic y²e(y)3^u1/Y; P{e(y)3^ΔPHD} males (Shidlovskii, 2005).

Altogether, these results show the SAY domain to be crucial for the functioning of SAYP. A drop in its content to one-fourth in e(y)3^u1 flies leads to disturbances in fly development, while deletion of the SAY domain appears to be lethal. It is involved in activation of transcription of the yellow gene in vivo, which confirms the results obtained in vitro in yeasts. At the same time, removal of PHD fingers seems to be not essential for these functions (Shidlovskii, 2005).

As SAYP is involved in repression of transcription in heterochromatin, whether the P{e(y)3^ΔPHD} transgene would interfere with the influence of the e(y)3^u1 mutation on PEV was investigated. In the e(y)3^u1 strain, SAYP mutation does not prevent the binding of mutated SAYP to polytene chromosomes. Thus, either the weaker transcription of e(y)3 or the lack of PHDs, or both, suppresses PEV (Shidlovskii, 2005).

To discriminate between these possibilities, the P{e(y)3^ΔPHD} and P{e(y)3⁺} constructs were introduced in e(y)3^u1; P[hsp26-pt, hsp70-w] flies. Unlike the construct expressing the full-length protein, P{e(y)3^ΔPHD} producing the truncated PHD-finger-less version failed to suppress the influence of e(y)3^u1 on the expression of P[hsp26-pt, hsp70-w] transgenes in three tested lines. The transgenic females heterozygous for e(y)3^u1 did not increase the expression of the reporter white gene after introduction of either one or two copies of P{e(y)3^ΔPHD}. The same applied to males hemizygous for e(y)3^u1. Hence, it is the PHD fingers of SAYP that are instrumental in repressing transcription in heterochromatin in vivo (Shidlovskii, 2005).

These results demonstrate that SAYP is a chromatin-binding protein with a dual function that depends on chromatin surroundings. It operates positively or negatively in transcription regulation via different domains, which may interact with various transcription factors or protein complexes (Shidlovskii, 2005).

Previously, strong genetic interaction was observed between e(y)3 and e(y)1/taf9. This result suggests that SAYP may coactivate transcription by Pol II via interaction with TAF9-containing complexes, like TFIID or TFTC. This interaction may involve the SAY domain that was shown to possess an activator function; the high evolutionary conservation of SAY points to its possible interaction with general factors of transcription, while the variable N terminus may interact with some factors specific for particular promoters (Shidlovskii, 2005).

These data demonstrate that the PHD domains are not important for SAYP functions in euchromatin. At the same time, PHD fingers are required for repression of the euchromatic genes inserted into the heterochromatin region. Thus, SAYP, and particularly its PHD fingers, may perform dissimilar functions in euchromatic and heterochromatic regions. The presence of PHD fingers in many chromatin-associated proteins suggests that PHD has chromatin-related function. Several PHDs were shown to participate in protein-protein interactions. However, the PHD fingers are very diverse in sequence, suggesting that their molecular function related to chromatin is also diverse. Recent studies have demonstrated that the bromodomain and PHD of transcriptional cofactor p300 cooperate in binding nucleosomes that have a high degree of histone acetylation, pointing to the possible function of PHD in histone code recognition. Deletion of the PHD domain from SAYP does not influence its ability to bind to polytene chromosome in euchromatin and heterochromatin regions. Thus, the PHD domains mediate some specific protein-protein interactions rather than recruit SAYP to chromatin (Shidlovskii, 2005).

The results do not yet disclose the mechanisms of action of SAYP domains. Several models can be proposed to explain the dual activity of SAYP. It is possible that SAYP mutation suppresses PEV indirectly, decreasing the transcription level of genes responsible for transcription repression in heterochromatin. As the increase in the SAY domain content in transgenic flies does not influence PEV, this model implicates PHDs in transcription activation. This study did not reveal the involvement of PHDs in transcription activation in yeast two-hybrid or in rescue experiments on Drosophila. The high concentration of SAYP in heterochromatin regions also suggests that SAYP is directly involved in repression (Shidlovskii, 2005).

The attractive possibility is that the SAYP-dependent silencing is realized via recruiting by the PHD domains of a protein or a protein complex involved in formation of pericentric heterochromatin. No interaction was found between SAYP and HP1 in additional genetic experiments. Also, no interaction was found between SAYP and Drosophila Mi-2 ATPase, a component of the NuRD complex that represses transcription through its remodeling and deacetylation activities. However, these results do not exclude that the PHDs of SAYP may recruit to heterochromatin another complex responsible for transcription repression (Shidlovskii, 2005).

To explain the opposite activities of SAYP, it is speculated that the SAY domain, once bound to euchromatin proteins, alters the PHD finger structure, thus blocking their interaction with a hypothetical transcription repressor (or repression complex). Conversely, in heterochromatin, there is no target for the SAY domain, and it is free or is blocked by heterochromatin proteins and thus does not prevent PHDs from binding with the repressor. It is also conceivable that SAYP enters into the composition of different multiprotein complexes having either coactivator or corepressor functions. Further studies should clarify the mechanism of action of the SAY domain and the PHD fingers of this versatile regulator protein (Shidlovskii, 2005).

Search PubMed for articles about Drosophila Sayp

Banga, S. S., Peng, L., Dasgupta, T., Palejwala, V. and Ozer, H. L. (2009). PHF10 is required for cell proliferation in normal and SV40-immortalized human fibroblast cells. Cytogenet Genome Res 126: 227-242. PubMed ID: 20068294

Chalkley, G. E., et al. (2008). The transcriptional coactivator SAYP is a trithorax group signature subunit of the PBAP chromatin remodeling complex. Mol Cell Biol. 28: 2920-2929. PubMed ID: 18299390

Georgiev, P. G. and Gerasimova, T. I. (1989). Novel genes influencing the expression of the yellow locus and mdg4 (gypsy) in Drosophila melanogaster. Mol. Gen. Genet. 220(1): 121-6. PubMed ID: 2558282

Georgiev, P. G. (1994). Identification of mutations in three genes that interact with zeste in the control of white gene expression in Drosophila melanogaster. Genetics 138(3): 733-9. PubMed ID: 7851770

Kal, A. J., Mahmoudi, T., Zak, N. B. and Verrijzer, C. P. (2000). The Drosophila brahma complex is an essential coactivator for the trithorax group protein zeste. Genes Dev. 14: 1058-1071. PubMed ID: 10809665

Lall, S. (2007). Primers on chromatin. Nat. Struct. Mol. Biol. 1110-1115. PubMed ID: 17984971

Lemon, B., Inouye, C., King, D. S. and Tjian, R. (2001). Selectivity of chromatin-remodelling cofactors for ligand-activated transcription. Nature 414: 924-928. PubMed ID: 11780067

Lessard, J., Wu, J. I., Ranish, J. A., Wan, M., Winslow, M. M., Staahl, B. T., Wu, H., Aebersold, R., Graef, I. A. and Crabtree, G. R. (2007). An essential switch in subunit composition of a chromatin remodeling complex during neural development. Neuron 55: 201-215. PubMed ID: 17640523

Melnikova, L., Kulikov, A. and Georgiev, P. (1996). Interactions between cut wing mutations and mutations in zeste, and the enhancer of yellow and Polycomb group genes of Drosophila melanogaster. Mol. Gen. Genet. 252(3): 230-6. PubMed ID: 8842142

Mohrmann, L., et al. (2004). Differential targeting of two distinct SWI/SNF-related Drosophila chromatin-remodeling complexes. Mol. Cell. Biol. 24: 3077-3088. PubMed ID: 15060132

Mohrmann, L., and Verrijzer, C. P. (2005). Composition and functional specificity of SWI2/SNF2 class chromatin remodeling complexes. Biochim. Biophys. Acta 168: 59-73. PubMed ID: 15627498

Moshkin, Y. M., et al. (2007). Functional differentiation of SWI/SNF remodelers in transcription and cell cycle control. Mol. Cell. Biol. 27: 651-661. PubMed ID: 17101803

Panov, V. V., Kuzmina, J. L., Doronin, S. A., Kopantseva, M. R., Nabirochkina, E. N., Georgieva, S. G., Vorobyeva, N. E. and Shidlovskii, Y. V. (2012). Transcription co-activator SAYP mediates the action of STAT activator. Nucleic Acids Res 40: 2445-2453. PubMed ID: 22123744

Shidlovskii, Y. V., et al. (2005). A novel multidomain transcription coactivator SAYP can also repress transcription in heterochromatin. EMBO J. 24: 97-107. PubMed ID: 15616585

Vorobyeva, N. E., et al. (2009a). Transcription coactivator SAYP combines chromatin remodeler Brahma and transcription initiation factor TFIID into a single supercomplex. Proc. Natl. Acad. Sci. 106(27): 11049-54. PubMed ID: 19541607

Vorobyeva, N. E., et al. (2009b). The novel regulator of metazoan development SAYP organizes a nuclear coactivator supercomplex. Cell Cycle 8(14): 2152-6. PubMed ID: 19556896

Vorobyeva, N. E., et al. (2011). SAYP interacts with DHR3 nuclear receptor and participates in ecdysone-dependent transcription regulation. Cell Cycle 10(11): 1821-7. PubMed ID: 21519192

Vorobyeva, N. E., Nikolenko, J. V., Nabirochkina, E. N., Krasnov, A. N., Shidlovskii, Y. V. and Georgieva, S. G. (2012). SAYP and Brahma are important for 'repressive' and 'transient' Pol II pausing. Nucleic Acids Res 40: 7319-7331. PubMed ID: 22638575

date revised: 5 August 2021

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