EvoprintHD of ash1

BIOLOGICAL OVERVIEW

The establishment and maintenance of mitotic and meiotic stable (epigenetic) transcription patterns is fundamental for cell determination and function. Epigenetic regulation of transcription is mediated by epigenetic activators and repressors, and may require the establishment, 'spreading' and maintenance of epigenetic signals. Although these signals remain unclear, it has been proposed that chromatin structure and consequently post-translational modification of histones may have an important role in epigenetic gene expression. The epigenetic activator Ash1 (Tripoulas, 1994) is a multi-catalytic histone methyl-transferase (HMTase) that methylates lysine residues 4 and 9 in Histone H3 and 20 in Histone H4. Transcriptional activation by Ash1 coincides with methylation of these three lysine residues at the promoter of Ash1 target genes. The methylation pattern placed by Ash1 may serve as a binding surface for a chromatin remodelling complex containing the epigenetic activator Brahma (Brm), an ATPase, and inhibits the interaction of epigenetic repressors with chromatin. Chromatin immunoprecipitation indicates that epigenetic activation of Ultrabithorax transcription in Drosophila coincides with trivalent methylation by Ash1 and recruitment of Brm. Thus, histone methylation by Ash1 may provide a specific signal for the establishment of epigenetic, active transcription patterns (Beisel, 2002).

Acetylation/de-acetylation, ubiquitination and methylation of histones (H1, H2A, H2B, H3, H4) have been correlated with the activation and silencing of transcription. Histone methylation occurs predominantly at arginine and lysine residues in the amino-terminal tails of H3 and H4. Arginine methylation mediates transcriptional activation by hormone receptors and probably other chromatin-dependent processes. By contrast, methylation of K9 and K4 in H3 and K20 in H4 has been linked to transcriptionally inactive chromatin, and corresponding HMTases have been identified. Methylation of H3 K4 has also been detected in transcription-competent chromatin, but the functional link between histone methylation and activation has not been dissected (Beisel, 2002).

To identify HMTases that establish activation-specific methylation patterns, a biochemical screen was used that identified Ash1, a member of the trithorax group of epigenetic activators as an HMTase. Ash1 contains a SET domain -- the 'signature motif' of lysine-specific HMTases -- flanked by cysteine-rich regions (pre-SET and post-SET domains). To confirm that Ash1 has HMTase activity, the ability to methylate histones was assessed in recombinant Ash1 derivatives Ash1DeltaN (deleted N terminus) and Ash1(SET) (containing the pre-SET, post-SET and SET domains only). The Ash1 derivatives methylate H3 and, to a lesser extent, H4 in polynucleosomes and histone core octamers. By contrast, 'free' H3 and H4 were methylated to a lesser extent compared with nucleosomes, even though free histones were present at a fivefold excess over polynucleosomal histones or when supplemented with DNA. These results suggest that Ash1 methylates H3 and H4. Since Ash1 used in the described HMTase assays was purified from eukaryotic cells, the HMTase activity of Ash1 could result from an associated rather than intrinsic activity. To test this, Ash1DeltaN was subjected to protein transfer membrane assays that detect intrinsic enzymatic activities in proteins. Ash1(SET) was separated by SDS-polyacrylamide gel electrophoresis (PAGE), transferred electrophoretically onto polyvinylidene fluoride (PVDF) membrane, and denatured/re-natured. Reconstituted Ash1(SET) methylates H3 and H4, suggesting that Ash1 has intrinsic HMTase activity (Beisel, 2002).

To identify the target amino acid residue(s) of Ash1, radiolabelled H3 was subjected to Edman-degradation. Scintillation counting of the released amino acid fractions detected radiolabelling of H3 K4 and K9. To support this, the ability of Ash1DeltaN to methylate peptides consisting of amino acids 1-20 of H3 [H3(1-20)] was tested. Ash1DeltaN methylates the peptides H3(1-20), H3(1-20)K4 (which contains H3 K4 but leucine residues instead of lysine residues at positions 9, 14 and 18) and H3(1-20)K9 (which contains H3 K9 but leucine residues instead of lysine residues at positions 4, 14 and 18). By contrast, H3(1-20)L4/L9 peptides, which contain leucine residues at position 4 and 9 of H3, are not significantly methylated, indicating further that Ash1 methylates H3 K4 and K9. Owing to the weak radiolabelling, the target(s) of Ash1 in H4 could not be identified by Edman-degradation. Since H4 K20 is the only H4 residue being methylated in vivo, a monoclonal antibody was generated against dimethylated H4 K20 [anti-dim(H4-K20)] to investigate whether Ash1 methylates H4 K20. H4 that was free, in histone core octamers or polynucleosomes, was methylated by Ash1 and analysed by Western blot analysis. Anti-dim(H4-K20) antibody recognizes Ash1-methylated H4, but not un-methylated H4, indicating that Ash1 methylates H4 K20 (Beisel, 2002).

Single amino acid point mutations ash1¹⁰ and ash1²¹ abolish Ash1 activator function in Drosophila (Tripoulas, 1994). The mutation in ash1¹⁰ (N1458I) resides within the SET domain, and in ash1²¹ (E1357K) in the pre-SET domain (Tripoulas, 1994). To assess whether these mutations affect HMTase activity, recombinant proteins were expressed and purified containing one of these mutations (Ash1DeltaN¹⁰, Ash1DeltaN²¹) and a third mutant (Ash1DeltaN¹¹⁴²) whose mutation (H1459K) resides in the SET domain and abolishes HMTase activity of SUV39H1. HMTase assays revealed that the mutants do not significantly methylate H3 and H4, indicating that the mutations abolish HMTase activity and that both the pre-SET and SET domains of Ash1 contribute to HMTase activity and transcriptional activation by Ash1 (Beisel, 2002).

To assess whether the mutations in Ash1 specifically inactivate HMTase activity or cause a general functional inactivation, the ability of mutant Ash1DeltaN to bind the known interaction partner Trx was investigated. Ash1DeltaN and the three mutants can interacte with Trx in vitro, suggesting that the inability of mutant Ash1 proteins to methylate histones is based on a specific inactivation of HMTase activity (Beisel, 2002).

To investigate the effect of ash1¹⁰ and ash1²¹ on transcriptional activation by Ash1 in Drosophila, transgenic flies were used carrying the Ash1-dependent reporter gene N18/15, which contains a 4-kilobase (kb) regulatory element of the bxd region from the Ash1 target gene Ubx fused to the mini-white gene. Ash1 supports activation of N18/15 transcription in the Drosophila eye. By contrast, N18/15 expression is significantly reduced in ash1¹⁰/ + or ash1²¹/+ heterozygous flies. Since Ash1²¹ and Ash1¹⁰ lack HMTase activity in vitro, these results imply that HMTase activity contributes to transcriptional activation by Ash1 in vivo (Beisel, 2002).

To dissect the functional relationship between transcriptional activation and histone methylation by Ash1, transcriptional activation by Ash1 was reconstituted in Drosophila S2 cells. To monitor transcription in chromatin, S2 (BCAT5) cells were generated that carry the stable integrated reporter gene BCAT5, which contains five DNA-binding sites for the yeast activator Gal4, a core promoter and the bacterial cat gene. To recruit Ash1 to chromatin, Ash1 derivatives were fused to the Gal4 DNA-binding domain (amino acids 1-147) [Gal4(DBD)]. BCAT5 cells were transfected with plasmids expressing fusion proteins comprising Gal4(DBD) and either wild type or mutant Ash1DeltaN. Gal4(DBD)-Ash1DeltaN activates BCAT5 expression 20-fold, whereas HMTase-inactive Ash1DeltaN derivatives did not. These results support the hypothesis that HMTase activity of Ash1 mediates activation of transcription (Beisel, 2002).

To link transcriptional activation by Ash1 to histone methylation, crosslinked chromatin immunoprecipitation (XChIP), which detects protein-DNA interactions in vivo, was used. Crosslinked chromatin was isolated from BCAT5 cells expressing Gal4(DBD)-Ash1DeltaN, Gal4(DBD)-Ash1DeltaN¹⁰ or Gal4(DBD)-Ash1DeltaN²¹, and immunoprecipitated by antibodies recognizing dimethylated H3 K4, H3 K9 or H4 K20. Precipitated DNA was purified and the enhancer/promoter of target genes was detected by polymerase chain reaction. All three antibodies precipitate chromatin containing the BCAT5 enhancer/promoter from cells in which Gal4(DBD)-Ash1DeltaN activates transcription. Methylation of these lysine residues was detectable 500 bp upstream of the enhancer/promoter and at the 3'-end of the cat gene. In cells expressing Gal4(DBD)-Ash1DeltaN¹⁰, methylation of H3 K4 was undetectable, but weak methylation of H3 K9 and H4 K20 could be observed. This finding supports current models proposing that transcriptional repression correlates with methylation of H3 K9 and H4 K20. As, however, H3 K9 and H4 K20 methylation is enhanced at the transcriptionally active (active) reporter, transcriptional activation by Ash1 correlates with de novo methylation of not only H3 K4 but also H3 K9 and H4 K20 (Beisel, 2002).

Methylation of H3 K9 at the transcriptionally silent (silent) enhancer/promoter implies that BCAT5 might be associated with HP1, which binds methylated H3 K9. This was tested by XChIP using anti-HP1 polyclonal antibody. The antibody precipitated the BCAT5 enhancer/promoter from cells expressing HMTase-inactive Gal4(DBD)-Ash1DeltaN¹⁰. By contrast, the enhancer/promoter was only weakly precipitated from cells in which Gal4(DBD)-Ash1DeltaN activates reporter expression. These results suggest that HP1 binds the silent enhancer/promoter and is removed/relocated from the reporter by Ash1-mediated histone methylation (Beisel, 2002).

To investigate whether Ash1 methylates histones to activate transcription of a natural target gene, methylation of Ubx was monitored in BCAT5 cells. Ubx is not expressed in S2-cells but PCR with reverse transcription (RT-PCR) indicates that transiently expressed Gal4(DBD)-Ash1DeltaN activates expression of this gene in BCAT5 cells. XChIP experiments indicate that H3 K4 is not methylated and that H3 K9 and H4 K20 are only weakly methylated at the silent Ubx promoter. By contrast, methylation of all three lysine residues is significantly enhanced when Ash1 activates BCAT5 expression, indicating that transcriptional activation of Ubx by Ash1 coincides with methylation of H3 K4, K9 and H4 K20 (Beisel, 2002).

Genetic data indicate that Ash1 activates Ubx expression in imaginal discs of the third leg (LaJeunesse, 1995). Therefore, to investigate histone methylation by Ash1 in the natural context of the activator, the methylation pattern of Ubx was examined in third leg discs by XChIP. Crosslinked chromatin was prepared from third leg discs dissected from third instar larvae. Chromatin immunoprecipitations indicated methylation of H3 K4, K9 and H4 K20 at the Ubx promoter, suggesting that Ash1-mediated methylation of all three lysine residues coincides with epigenetic activation of Ubx transcription in Drosophila (Beisel, 2002).

On the basis of the result that methylated lysine residues facilitate or inhibit the binding of proteins, an investigation was carried out to determine whether the trivalent (H3 K4, K9 and H4 K20) methylation pattern placed by Ash1 attracts or repels proteins to establish epigenetic activation. The XChIP experiments in indicate that the trivalent methylation pattern removes/relocates HP1 from chromatin. To support this finding, the interaction was investigated of HP1 with methylated H3 peptides and histone core octamers that had been methylated by Ash1 or Drosophila SU(VAR)3-9, which methylates H3 K9. HP1 binds H3 K9-methylated peptides and histone core octamers, as well as H3 K4/K9-methylated peptides. By contrast, HP1 does not bind Ash1-methylated core octamers, suggesting that the trivalent methylation pattern inhibits the interaction of HP1 with chromatin (Beisel, 2002).

Protein-protein interaction assays using H3(1-20) peptides methylated at H3 K4, K9 or H3 K4 and K9 (H3 K4/K9), and Drosophila embryonic nuclear extract or recombinant proteins, resulted in the identification of three proteins that exhibit differential binding to methylated peptides. Two of these proteins -- the epigenetic repressor Polycomb (Pc) and Caf-1 p55, a subunit of different protein complexes involved in, for example, epigenetic repression -- bind H3 K9-methylated peptides and histone core octamers, but show significantly reduced binding to H3 K4- or H3 K4/K9-methylated peptides and trivalently methylated histone core octamers. Furthermore, protein-binding assays indicate that Brm and Moira (Mor) interact with H3 K4/K9-methylated peptides. In contrast, both proteins were not recruited to peptides methylated at H3 K4 or H3 K9. Brm and Mor are subunits of a SWI/SNF-like chromatin remodelling complex, suggesting that this complex, rather than individual proteins, is recruited to K4/K9-methylated H3. These results imply that the trivalent methylation pattern established by Ash1 facilitates or prevents the interaction of proteins with methylated H3 during epigenetic activation. To support this finding, XChIP was used to investigate the interaction of Brm and repressors with Ash1 target genes. These analyses indicate that Brm and Mor are present at the active but not at silent promoters of Ash1 target genes in cells or third leg imaginal discs. By contrast, the repressors were only detected at silent promoters. Thus, transcriptional activation by Ash1 may coincide with the recruitment of Brm and Mor and the extinction of repressor binding at the promoter of Ash1 target genes (Beisel, 2002).

Collectively, these data indicate that the epigenetic activator Ash1 activates transcription by methylation of H3 K4, K9 and H4 K20 at the promoter of target genes. This suggests that epigenetic activation and silencing, which has been linked to methylated H3 K9, may correlate with different histone methylation patterns. Each of the three lysine residues targeted by Ash1 can be individually methylated by specific HMTases, resulting in transcriptional repression and probably activation (H3 K4). Combining these three modifications results in a novel biological readout: epigenetic activation. Why does the trivalent modification pattern generated by Ash1 mediate epigenetic activation? The results indicate that each modification of the pattern fulfils a specific function. Methylation of H3 K4 prevents the interaction of repressors (Pc, p55) with Ash1 target genes. Methylation of H4 K20 in addition to H3 K4 and H3 K9 prevents the interaction of HP1 with chromatin. Inhibition of repressor binding is an important mechanism, as epigenetic activators and repressors are expressed together during Drosophila development. Finally, methylation of H3 K4 and H3 K9 generates an interaction surface for a chromatin-remodelling complex. These results imply that a specific functional interplay between the epigenetic activators Ash1 and Brm mediates epigenetic activation of transcription. Ash1 initially binds target genes and generates the trivalent histone methylation pattern, which subsequently recruits a Brm-containing chromatin-remodelling complex. The activity of this complex may contribute to the establishment of epigenetic active chromatin structures (Beisel, 2002).

Noncoding RNAs of trithorax response elements recruit Drosophila Ash1 to Ultrabithorax

Homeotic genes contain cis-regulatory trithorax response elements (TREs) that are targeted by epigenetic activators and transcribed in a tissue-specific manner. The transcripts of three TREs located in the Drosophila homeotic gene Ultrabithorax mediate transcription activation by recruiting the epigenetic regulator Ash1 to the template TREs. TRE transcription coincides with Ubx transcription and recruitment of Ash1 to TREs in Drosophila. The SET domain of Ash1 binds all three TRE transcripts, with each TRE transcript hybridizing with and recruiting Ash1 only to the corresponding TRE in chromatin. Transgenic transcription of TRE transcripts restores recruitment of Ash1 to Ubx TREs and restores Ubx expression in Drosophila cells and tissues that lack endogenous TRE transcripts. Small interfering RNA-induced degradation of TRE transcripts attenuates Ash1 recruitment to TREs and Ubx expression, which suggests that noncoding TRE transcripts play an important role in epigenetic activation of gene expression (Sanchez-Elsner, 2006).

The identity of cells in metazoan organisms is established during development and mitotically propagated throughout the entire life cycle. Phylogenetically highly conserved protein families of epigenetic regulators determine the fate of developing cells by establishing and maintaining mitotically stable gene expression programs. In Drosophila, members of the trithorax group (trxG) of epigenetic regulators maintain active transcription states, whereas members of the Polycomb group (PcG) maintain repressed transcription states. Many epigenetic regulators control gene expression by establishing transcriptional competent or silent chromatin structures. Several epigenetic activators [Trx, trithorax-related (Trr)] and repressors (Enhancer of zeste) are lysine-specific histone methyltransferases (HMTs) and contain a SET domain, the catalytic hallmark motif of HMTs. Methylation of lysine residues in histones H3 and H4 has been correlated with epigenetic activation [Lys⁴ in H3 (H3-K4)] and repression [Lys⁹ and Lys²⁷ in H3 (H3-K9)] (Sanchez-Elsner, 2006).

The epigenetic activator Absent small and homeotic discs(Ash1) promotes transcriptional activation by trimethylating H3-K4, H3-K9, and Lys²⁰ in H4 (H4-K20). Ash1 maintains activated transcription states in larval imaginal discs that give rise to the appendages in the adult fly. For example, Ash1 is essential for the expression of the homeotic gene Ultrabithorax (Ubx) in third-leg and haltere imaginal discs, and Ubx expression coincides with Ash1-mediated histone methylation (Sanchez-Elsner, 2006).

PcG and trxG regulators are recruited to specific chromosomal elements that are present in the cis-regulatory region of target genes. The same element can act as an activating or a silencing module. In the repressed state, the elements represent Polycomb response elements (PREs) and facilitate the recruitment of PcG proteins. In the activated state, the DNA-elements function as trithorax response elements (TREs) and recruit trxG proteins. Transcription of noncoding RNAs (ncRNAs) from TRE/PRE elements switches silent PREs into TREs, which indicates that TRE/PRE transcription plays an important role in epigenetic activation. How transcription of TREs culminates in the recruitment of trxG regulators is unknown. This study addressd the question of how epigenetic regulators without known DNA binding capabilities, such as Ash1, recognize and bind target genes in chromatin (Sanchez-Elsner, 2006).

The coincidence of the tissue-specific transcription and trans-regulatory activity patterns of TREs and trxG proteins, respectively, suggests that not only TRE/PRE transcription but also the resulting ncRNAs might play a role in epigenetic activation. This study analyzed the role of ncRNAs transcribed from three Ubx TRE/PREs. The Ubx locus contains a cluster of three characterized TRE/PREs (TRE1 to TRE3; see TRE1 to TRE3 in the UCSC genome browser) within the boundaries of the chromosomal memory element (CME) bxd that is located 22 kb upstream of the Ubx promoter (Sanchez-Elsner, 2006).

To correlate the transcriptional activity of Ubx with bxd transcription in Drosophila, RACE was used to detect bxd transcripts in third-leg discs. Three capped, polyadenylated bxd transcripts transcribed by RNA polymerase II (tre1, tre2, tre3) were detected in third-leg and haltere discs (Sanchez-Elsner, 2006).

The RT-PCR was used to determine whether the presence of the three TRE transcripts coincides with Ubx transcription. RNA was isolated from third-leg discs and haltere imaginal discs (haltere discs), which both transcribe Ubx, and from wing imaginal discs (wing discs) and embryonic Drosophila Schneider 2 (S2) cells that do not transcribe Ubx. Transcripts from Ubx and all three TREs were detected in third-leg and haltere discs, whereas Ubx and TRE transcripts were not detected in S2 cells and wing discs (Sanchez-Elsner, 2006).

To investigate whether Ash1 is recruited to transcriptionally active Ubx TREs, in vivo cross-linked chromatin immunoprecipitation (XChIP) was used to detect Ash1 at the Ubx TREs in third-leg, haltere, and wing discs and in S2 cells, all of which express ash1. Ash1 was detected at all three TREs in third-leg and haltere discs. In addition, the characteristic Ash1 histone methylation pattern was detectable in all three TREs and the transcriptionally active Ubx promoter in third-leg discs. Ash1 was not detected at the TREs of the transcriptionally inactive Ubx locus in wing discs and S2 cells, which do not transcribe TREs (Sanchez-Elsner, 2006).

The recruitment of Ash1 to Ubx was compared in wild-type and homozygous mutant ash1²² third-leg discs by XChIP. The ash1²² mutant is recessive lethal and expresses a truncated protein that lacks the SET domain and trans-activation activity. Ash1 and the characteristic Ash1 histone methylation pattern were detected at the transcriptionally active Ubx locus in wild-type discs but not in ash1²² mutant discs; this finding indicates that recruitment of Ash1 and Ash1-mediated histone methylation coincides with activation of Ubx expression in third-leg discs. TRE transcription was monitored in the wild-type and ash1²² mutant third-leg discs by RT-PCR. TRE transcripts were detected at comparable levels in wild-type and mutant discs, which indicates that Ash1 is not a major regulator of TRE transcription in imaginal discs (Sanchez-Elsner, 2006).

The association of Ash1 with TREs in cells producing TRE transcripts suggests that TRE transcription or TRE transcripts nucleate recruitment of Ash1 to Ubx TREs. SET-domain proteins can bind single-stranded RNA and DNA in vitro, and ncRNA has been implicated in protein recruitment in gene dosage compensation. In vitro protein-RNA binding assays were used to assess whether Ash1 associates with TRE transcripts. Ash1SET, which consists of amino acids 1001 to 1619, retained TRE1(+), TRE2(+), and TRE3(+) but not the H3-K9-specific HMT Medusa (Mdu). In contrast, Ash1, Ash1DeltaN, and Mdu did not bind the antisense RNA of the Ubx TREs. Ash1DeltaN did not interact with the N-element in tre2, which corresponds to the DNA spacer separating TRE-2 and TRE-3 (Sanchez-Elsner, 2006).

In competition experiments, unlabeled TRE transcripts could outcompete the interaction of Ash1 with the corresponding TRE transcript. In contrast, double-stranded TRE transcripts, double-stranded DNA TRE sequences, and DNA-RNA hybrids consisting of TRE transcripts and TREs failed to disrupt the interaction; these findings suggest that Ash1 associates with single-stranded TRE transcripts (Sanchez-Elsner, 2006).

To delineate the RNA-binding motif of Ash1, the interaction of truncated ash1 proteins with TRE transcripts was investigated. In addition to Ash1SET, Ash1DeltaN (amino acids 1001 to 2218), which contains the Ash1 SET module, and Ash1N (amino acids 1 to 1001) and Ash1C (amino acids 1619 to 2218), which both lack the SET domain and cysteine-rich regions, were tested. Ash1DeltaN and Ash1SET, but not Ash1N and Ash1C, retained TRE transcripts, indicating that the SET domain of Ash1 binds TRE transcripts in vitro (Sanchez-Elsner, 2006).

XChIP was used to investigate whether Ash1 associates with TRE transcripts in vivo. Ash1 coprecipitated with TRE transcripts but not control transcripts from mock-treated chromatin. Ash1 binds TRE transcripts in ribonuclease (RNase) III-treated chromatin, indicating that double-stranded RNA (dsRNA) motifs within TRE transcripts do not mediate the association of TRE transcripts with Ash1. In contrast, Ash1 did not interact with TRE transcripts from RNase A- and RNase H-treated chromatin, indicating that single-stranded RNA (ssRNA) is important for the association of Ash1 with TRE transcripts. The disruption of the association between Ash1 and TRE transcripts by RNase H (which degrades DNA-RNA hybrids) in chromatin suggests that TRE transcripts hybridize with DNA in chromatin (Sanchez-Elsner, 2006).

Is the association of Ash1 with TREs dependent on RNA? XChIP to compare the interaction of Ash1 and TRE in mock- and RNase-treated chromatin. Antibodies to Ash1 precipitated all three TREs, but not the spacer DNAs, from mock-treated and RNase III-treated chromatin, indicating that dsRNA does not contribute to the interaction of Ash1 with TREs. In contrast, treating chromatin with RNase H or RNase A attenuated the association of Ash1 with TREs, indicating that the association of Ash1 with the Ubx TREs is RNA-dependent. The disruption of the interaction of Ash1 with TREs in chromatin by RNase H and RNase A raises the possibility that ssRNA motifs in RNA-DNA hybrids play a role in the recruitment of Ash1 to TREs (Sanchez-Elsner, 2006).

To verify that the observed attenuation of Ash1-TRE interactions is based on specific rather than general disruption of protein-DNA interactions in RNase-treated chromatin, the recruitment of the general transcription factor TFIID to target genes was investigated in mock- and RNase-treated chromatin. The TATA-binding protein (TBP) subunit of TFIID interacts with the TATA box in eukaryotic promoters. PCR detected the interaction of TBP with the promoter of Ubx and string, whose transcription requires TFIID activity. TBP interacted with both promoters in mock-treated and RNase A-, RNase H-, and RNase III-treated chromatin, indicating that RNase treatment did not attenuate TBP-promoter interactions and protein-gene interactions in general (Sanchez-Elsner, 2006).

To test whether the detected association of Ash1 with TREs and TRE transcripts occurs in chromatin or is the result of fortuitous interactions generated in chemically cross-linked chromatin, the association of Ash1 with TRE transcripts and TREs in native chromatin was investigated with the use of native chromatin immunoprecipitation (NChIP). Ash1 bound all three TREs and TRE transcripts in mock- and RNase III-treated chromatin but not in RNase H- or RNase A-treated chromatin, indicating that Ash1 coimmunoprecipitates with TREs and TRE transcripts in native chromatin. An association of Ash1 with the N portion of the TRE2(+) transcript, as observed in cross-linked chromatin, was not detectable in native chromatin; this result indicates that, as in vitro, Ash1 binds the RNA corresponding to TRE-2 but not the N region of the TRE2(+) transcript (Sanchez-Elsner, 2006).

Collectively, these data indicate that the recruitment of Ash1 to the TREs of Ubx is mediated by RNA and suggests the existence of a trimeric protein-nucleic acid complex in chromatin, consisting of Ash1, TREs, and TRE transcripts (Sanchez-Elsner, 2006).

Ash1 is detectable at about 150 loci on Drosophila polytene chromosomes. To assess whether RNA facilitates Ash1 recruitment to target loci other than Ubx, the interaction was compared of Ash1 with target loci on mock- and RNase-treated chromosome squashes. Compared to mock-treated chromosomes, RNase treatment attenuated the association of Ash1 with the majority of the target loci. This result suggests that RNA plays an important role in the recruitment of Ash1 to target genes in chromatin (Sanchez-Elsner, 2006).

To assess whether TRE transcripts associate with chromatin, whether Ash1 coprecipitates TRE transcripts from chromatin-free nuclear extract was investigated. Ash1 bound TRE transcripts in chromatin but not chromatin-free nuclear extract, indicating that TRE transcripts are preferentially associated with chromatin in the cell (Sanchez-Elsner, 2006).

XChIP was used to determine whether the association of Ash1 with TRE transcripts precedes the recruitment of Ash1 to TREs in chromatin, or vice versa. In vivo cross-linked chromatin was isolated from wild-type and ash1²² mutant third-leg discs, sheared, and immunoprecipitated with antibodies to dimethylated H3-K9 present at the TREs of the transcriptionally active and inactive Ubx locus in third-leg discs. The antibody to dimethylated H3-K9 coprecipitated with TREs and TRE transcripts from the chromatin of wild-type and ash1²² third-leg discs, indicating that TRE transcripts are retained at Ubx TREs before recruitment of Ash1 (Sanchez-Elsner, 2006).

To dissect the role of TRE transcripts in Ubx transcription, it was asked whether transiently transcribed TRE transcripts could restore the recruitment of Ash1 to Ubx TREs and Ubx expression in S2 cells, which express Ash1 but lack endogenous TRE transcripts. S2 cells were transiently transfected with plasmids transcribing sense or antisense TRE transcripts. In PCR assays, Ubx transcription was undetectable in S2 cells transiently transcribing antisense TRE transcripts or mdu. In contrast, Ubx transcription was activated by one TRE transcript and cooperatively activated by multiple TRE transcripts (Sanchez-Elsner, 2006).

XChIP was used to determine whether activation of Ubx transcription by transient TRE transcripts coincides with the recruitment of Ash1 to TREs. In vivo cross-linked chromatin was isolated from wild-type S2 cells and cells transiently transcribing one or multiple TRE transcripts and control RNAs, and it was then immunoprecipitated with antibodies to Ash1 and the Ash1 histone methylation pattern. Ash1 was not detected at the TREs of transcriptionally silent Ubx in cells transcribing mdu or antisense TRE RNAs. In contrast, Ash1 and the Ash1 histone methylation pattern were detected at the Ubx TREs in cells transcribing TRE1(+), TRE2(+), and/or TRE3(+). Each of the three TRE transcripts facilitated the association of Ash1 only with the corresponding template TRE but not with other TREs (Sanchez-Elsner, 2006).

To verify the specificity of the described recruitment, whether TRE transcripts facilitate recruitment of Ash1 to CMEs containing TREs/PREs and genes other than Ubx was investigated. In XChIP assays, Ash1 was not detected at Drosophila genes and the CMEs MCP and Fab7 in S2 cells transcribing TRE1(+), TRE2(+), or TRE3(+). Thus, TRE transcripts facilitate Ash1 recruitment specifically to the corresponding TRE template DNA (Sanchez-Elsner, 2006).

NChIP and XChIP were used to assess whether transiently transcribed TRE transcripts associate with TREs and Ash1 in chromatin. Native chromatin was isolated from wild-type S2 cells and S2 cells transiently cotranscribing all three sense or antisense TRE transcripts. Ash1 did not associate with TRE transcripts and TREs in cross-linked and native chromatin from S2 cells transcribing mdu. In contrast, Ash1 interacted with TREs and TRE transcripts in S2 cells cotranscribing TRE1(+), TRE2(+), and TRE3(+) (Sanchez-Elsner, 2006).

The association of Ash1 with TREs and TRE transcripts was attenuated by RNase A and RNase H but not RNase III. RNase treatment did not abolish the association of TBP with the Ubx promoter. These results indicate that Ash1 associates with TRE transcripts and TREs in vivo and that TRE transcripts mediate the association of Ash1 with TREs in trans (Sanchez-Elsner, 2006).

To test this hypothesis, RNA interference (RNAi) was used to assess whether degradation of TRE transcripts attenuates recruitment of Ash1 to Ubx TREs and Ubx expression in third-leg discs. In vitro cultivated third-leg discs were incubated with small interfering RNAs (siRNAs) targeting all three TRE transcripts or with control siRNA. RT-PCR and XChIP assays indicated that siRNA-mediated degradation of TRE transcripts attenuates Ubx transcription and the interaction of Ash1 with TREs (Sanchez-Elsner, 2006).

Next, The binary Gal4/UAS system was used to determine whether ectopic transcription of TRE transcripts restores recruitment of Ash1 to Ubx TREs and Ubx transcription. Effector flies carrying a heat-inducible driver (hsp70Gal4) were crossed with reporter flies carrying Gal4-dependent reporter genes (UAS-TRE) consisting of Gal4-responsive UAS DNA sites and a promoter driving the transcription of sense and antisense TRE transcripts. Heat treatment of second-instar larvae resulted in ectopic transcription of TRE transcripts in all imaginal discs of third-instar larvae. Ectopic transcription of each TRE transcript nucleated ectopic transcription of Ubx in wing imaginal discs and facilitated the recruitment of Ash1 to the corresponding Ubx TREs. It is noteworthy that ectopic TRE transcription in second-instar larvae caused lethality in pupae. In contrast, ectopic Ubx expression was not observed in discs prepared from heat-treated parental strains and discs transcribing antisense TRE transcripts (Sanchez-Elsner, 2006).

Transcription of antisense TRE transcripts attenuated endogenous transcription of Ubx in wing discs isolated from young third-instar larvae, which suggests that ectopic transcription of antisense RNA interferes with the TRE transcript-mediated recruitment of Ash1 to Ubx TREs. In summary, these data provide evidence that noncoding TRE transcripts facilitate activation of Ubx expression by recruiting Ash1 to the Ubx TREs in the fly (Sanchez-Elsner, 2006).

Recent studies have shown that noncoding RNAs play an important role in the recruitment of proteins in several epigenetic phenomena. siRNAs have been lined to heterochromatin formation and transcriptional silencing of transgenes and transposons. siRNAs facilitate the recruitment of HMTs and DNA methyltransferases to chromatin. In Schizosaccharomyces pombe, heterochromatic silencing involves the RNA-induced initiator of transcriptional gene silencing complex (RITS), which contains an siRNA component that is essential for the recruitment of RITS to heterochromatic loci. The inability of RNase III, the key enzyme of the RNAi machinery, to degrade TRE transcripts into siRNAs and the interaction of Ash1 with full-length TRE transcripts in chromatin strongly argues against the involvement of siRNAs in the described RNA-dependent recruitment of Ash1 to chromatin (Sanchez-Elsner, 2006 and references therein).

Long ncRNAs are key players in imprinting and gene dosage compensation. In Drosophila, gene dosage compensation is achieved by a global twofold up-regulation of transcription from the male X chromosome and depends on the activity of the dosage compensation complex (DCC) that contains male-specific proteins and two ncRNAs, RNA on X 1 (rox1) and RNA on X 2 (rox2). Both RNAs are transcribed by single-copy genes that, as well as several other X chromosome regions, serve as chromatin entry sites for the DCC on paternal X chromosomes. Rox1 and Rox2 facilitate the assembly and recruitment of the DCC to chromatin entry sites. In mammals, spreading of Xist RNA culminates in X chromosome inactivation. Current models propose that the association between ncRNAs and chromatin involves their interaction with proteins, nascent transcripts at template DNA, or the template DNA. The observed attenuation of the association between TRE transcripts and TREs by RNase H suggests that TRE transcripts are retained at TREs through hybridization with the corresponding template DNA. Because none of the known DNA repair systems targets DNA-RNA hybrids, RNA-DNA hybrids represent stable molecular entities that, in general, may anchor ncRNAs at corresponding DNA templates in chromatin (Sanchez-Elsner, 2006 and references therein).

The three TRE transcripts of Ubx do not share common sequence motifs. This is not surprising, because the functionally redundant rox RNAs and functionally identical regions in Xist, which are required for chromatin localization and protein recruitment, lack identifiable sequence motifs. Because many RNA-protein interactions are facilitated by RNA secondary structures, the interaction of Ash1 with TRE transcripts might be mediated by secondary RNA structures rather than sequence motifs. In addition, the specificity of RNA-protein interactions is often generated by induced-fit mechanisms that involve complex, extensive conformational changes in both proteins and the target RNA generating a specific interaction surface (Sanchez-Elsner, 2006).

rox1 and rox2 RNAs transcribed from autosomes can localize to and mediate gene dosage compensation on the male X chromosome, indicating that the chromatin entry of rox RNAs does not depend on transcription of chromatin entry sites in cis. Thus, the association of transiently transcribed TRE transcripts with TREs in S2 cells suggests that TREs function as chromatin entry sites for the corresponding TRE transcripts in trans and cis, and that the transcription and chromatin entry site activities of TREs are functionally separated. Cumulatively, these results support a model in which RNAs transcribed from the TREs of Ubx are retained at TREs through DNA-RNA interactions and provide a RNA scaffold that is bound by Ash1 (Sanchez-Elsner, 2006).

GENE STRUCTURE

cDNA clone length - 7053

Bases in 5' UTR - 548

Exons - 6

Bases in 3' UTR - 525

PROTEIN STRUCTURE

Amino Acids - 2144

Structural Domains

The primary translation product of the 7.5-kb ash1 transcript is predicted to be a basic protein of 2144 amino acids. The ASH1 protein contains a SET domain, a PHD finger and a BAH (Bromo adjacent homology) domain (see NCBI Conserved Domain Summary. These motifs are found in the products of some trithorax group and Polycomb group genes (Tripoulas, 1996).

date revised: 16 February 2003

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