Targets of Activity: DNA-binding proteins required for pairing-sensitive silencing by a polycomb group response element from the Drosophila engrailed gene

Regulatory DNA from engrailed causes silencing of a linked reporter gene (mini-white) in transgenic Drosophila. This silencing is strengthened in flies homozygous for the transgene and has been called 'pairing-sensitive silencing'. The pairing-sensitive silencing activities of a large fragment (2.6 kb) and a small subfragment (181 bp) were explored. Since pairing-sensitive silencing is often associated with Polycomb group response elements (PREs), the activities of each of these engrailed fragments were tested in a construct designed to detect PRE activity in embryos. Both fragments behave as PREs in a bxd-Ubx-lacZ reporter construct, while the larger fragment shows additional silencing capabilities. Using the mini-white reporter gene, a 139-bp minimal pairing-sensitive element (PSE) was defined. DNA mobility-shift assays using Drosophila nuclear extracts suggest that there are eight protein-binding sites within this 139-bp element. Mutational analysis shows that at least five of these sites are important for pairing-sensitive silencing. One of the required sites is for the Polycomb group protein Pleiohomeotic and another is GAGAG, a sequence bound by the proteins GAGA factor and Pipsqueak. The identity of the other proteins is unknown. These data suggest a surprising degree of complexity in the DNA-binding proteins required for PSE function (Americo, 2002).

Targets of Activity: Psq functions within a Pc protein complex as a DNA binding activity specific to the (GA)n motif in a PRE from the bithoraxoid region of Ultrabithorax

Polycomb group proteins act through Polycomb group response elements (PREs) to maintain silencing at homeotic loci. The minimal 1.5-kb bithoraxoid (bxd) PRE contains a region required for pairing-sensitive repression and flanking regions required for maintenance of embryonic silencing. Little is known about the identity of specific sequences necessary for function of the flanking regions. Using gel mobility shift analysis, DNA binding activities have been identified that interact specifically with a multipartite 70-bp fragment (MHS-70) downstream of the pairing-sensitive sequence. Deletion of MHS-70 in the context of a 5.1-kb bxd Polycomb group response element derepresses maintenance of silencing in embryos. A partially purified binding activity requires multiple, nonoverlapping d(GA)(3) repeats for MHS-70 binding in vitro. Mutation of d(GA)(3) repeats within MHS-70 in the context of the 5.1-kb bxd PRE destabilizes maintenance of silencing in a subset of cells in vivo but gives weaker derepression than deletion of MHS-70. These results suggest that d(GA)(3) repeats are important for silencing but that other sequences within MHS-70 also contribute to silencing. Antibody supershift assays and Western analyses show that distinct isoforms of Polyhomeotic and two proteins that recognize d(GA)(3) repeats, the Trl/GAGA factor and Pipsqueak (Psq), are present in the MHS-70 binding activity. Mutations in Trl and psq enhance homeotic phenotypes of ph, indicating that Trl/GAGA factor and Psq are enhancers of Polycomb that have sequence-specific DNA binding activity. These studies demonstrate that site-specific recognition of the bxd PRE by d(GA)(n) repeat binding activities mediates PcG-dependent silencing (Hodgson, 2001).

The results of the sequence-specific analysis suggest that d(GA)n-specific binding factors are present in a complex defined by electrophoretic mobility, termed complex 2. Therefore, antibodies directed against two nuclear factors that bind d(GA)n sequences, Trl/GAF and Psq, were tested in binding reactions with a bxd fragment termed MHS-70. In the presence of increasing amounts of antibody to Trl/GAF, the mobility of complex 2 was significantly retarded, migrating close to the sample well. Antibodies to Psq caused a modest but detectable retardation of complex 2. Neither of these antibodies alters the mobility of a second complex, complex 1. These results show that complex 2 contains detectable levels of Trl/GAF and Psq. The significantly reduced mobility of complex 2 in the presence of anti-TRL/GAF antibody presumably results from the ability to induce multimeric aggregates of DNA-TRL/GAF complexes. To show that the complexes formed by MHS-70 and the competing oligomers are equivalent, the formation of complex 2 with synthetic oligomers was tested with antibodies to Trl/GAF, Ph, and Psq. Antibodies to Ph, Trl/GAF, and Psq supershift complex 2 in synthetic oligomer binding reactions (Hodgson, 2001).

Polyhomeotic proximal (Php), Trl/GAF, and Psq have multiple isoforms. To determine which of these isoforms are potential components of complex 2, Western analysis of the Separose AS, BR0.6 and Q0.15 fractions was undertaken. Q0.15 is enriched for isoforms of Trl/GAF P67 plus Trl/GAF P54, Php105 plus Php64, and Psq P70. Taken together with the antibody supershift analysis, these results show that the distinct isoforms of Trl/GAF, Php and Psq coelute with complex 2 and suggest that these isoforms constitute potential subunits of complex 2 binding activities. It has been shown that the full-length isoform of PhP, Php-170, coimmunoprecipitates with the PcG proteins Pc, Psc, Su(z)2, and Scm. Western analyses of the three fractions described above show that there are no detectable levels of Pc, Su(z)2, Psc, or Sex Combs on Midleg (Scm) in Q0.15, indicating that these PcG proteins do not coelute with complex 1 and 2 binding activities. These results suggest that the complex 2 activity is a novel PcG activity containing distinct isoforms of PHP, TRL/GAF, and Psq (Hodgson, 2001).

Trl null or hypomorphic alleles (Trl13C, Trl62, and Trl85) do not affect maintenance of silencing in vivo by bxd5.1 UbxlacZ. Similarly, embryos mutant for psqRF13 (deletion of psq) and Df(2R)psq-lolaDelta18 (deletion of psq, termed psqlola hereafter) show wild-type bxd5.1 UbxlacZ silencing. One potential reason for these results is that maternally deposited Trl or psq protein or mRNA rescue the effects of absence of zygotic proteins on embryonic silencing (Hodgson, 2001).

Genetic interactions between PcG genes are monitored by the enhancement of PcG mutations, providing a sensitive genetic assay for genes required in PcG-mediated silencing. Therefore, the ability of Trl and Psq mutations to enhance the extra sex combs phenotype of ph was tested. Trl enhances the extra sex combs phenotype of Pc. Similarly, Trl62 enhances the extra sex combs phenotype of ph2 and ph409. The effects of psqlola-Delta18 and psq2403 on enhancement of ph2 and ph409 were tested. There is strong enhancement of the expressivity of the extra sex combs phenotype. These results are consistent with a role for Trl/GAF and Psq in PcG-mediated silencing of homeotic loci and indicate that Trl/GAF and Psq are enhancers of PC that have sequence-specific DNA binding activity (Hodgson, 2001).

pipsqueak encodes a factor essential for sequence-specific targeting of a polycomb group protein complex

To facilitate the biochemical study of the Pc-G complex, a Drosophila S2 cell line PC-FH was established that can express Pc protein with both FLAG epitope and hexahistidine tags at its C terminus. These modifications do not appear to affect the activity of Pc, since similar constructs can repress the Ubx reporter gene in cultured cells and partially rescue Pc mutants in transgenic flies. The tagged PC is under the control of a metallothionein promoter, which allows protein induction by the copper ion in a range up to 0.7 mM. A suboptimal concentration of CuSO4 (i.e., 0.1 mM) was chosen for induction, since it appears to provide sufficient amounts of tagged protein complexes for purification. Nuclear extracts prepared from induced cells were fractionated by 10% to 40% (NH4)2SO4 precipitation to enrich for large protein complexes. The extracts were then passed through a FLAG antibody column (i.e., M2 resin) and eluted with the FLAG peptide. Approximately 200-fold purification was obtained by affinity chromatography compared with the crude extracts. Many proteins appeared to be specifically coeluted with the Pc protein. Although the region corresponding to the size of Pc proteins appears to be heavily stained, the relative abundance of Pc proteins has been exaggerated by the presence of several proteins of similar size that can be better separated in high-resolution gels. An earlier study has shown that a specific subset of Pc-G proteins, including Psc, Ph, and HDAC1 are copurified, indicating the presence of multimeric Pc-G complexes in these fractions. Since this is the first Drosophila Pc-G complex shown to contain both histone modification activity and DNA binding activity and since homeotic genes are its best characterized targets, this complex is referred to as CHRASCH (chromatin-associated silencing complex for homeotics) to distinguish it from commonly referred Pc-G complexes or complexes characterized by other workers (Huang, 2002).

Since a functional Pc-G complex must act specifically on its response element (i.e., PRE), whether CHRASCH can bind such sequences in vitro was examined. PRE from several homeotic genes have been mapped, including the one from the upstream bxd region of Ubx. An ~440-bp fragment (B-151) from this region recapitulates transcriptional regulation by either Pc or trx in cultured cells. DNA fragments encompassing this region can also confer pairing-sensitive repression and are enriched for Pc proteins in chromatin immunoprecipitation experiments. B-151 therefore contains physiologically relevant binding sites for the Pc-G complex. Using two subfragments from B-151 as probes for EMSAs, CHRASCH was found to bind strongly to the bxd-b fragment, resulting in several slow-migrating bands. These bands presumably reflect the binding to a reiterated motif in the bxd-b fragment. The binding of CHRASCH to the bxd-b fragment appears to be specific, since it can be completely competed out by the addition of bxd-b but not by bxd-a or nonspecific vector sequences. A much weaker but specific binding of CHRASCH to bxd-a was also detected. The observation that bxd-b can compete for binding to bxd-a but that bxd-a can not compete effectively for binding to bxd-b suggests that these fragments might contain similar binding sequences, albeit with a lower affinity in the bxd-a fragment. Due to the difficulty in studying a weak binding activity with certainty, subsequent studies focused on bxd-b (Huang, 2002).

Four partially overlapping fragments from B-151 were used in competition assays to further map the binding sites of CHRASCH. Only the bxd-3 and bxd-4 fragments compete effectively for the binding to CHRASCH. In addition, bxd-4 appears to compete better than bxd-3. Therefore, it was deduced that the right half of B-151 must contain the primary binding sites for CHRASCH. Interestingly, transgenes containing small deletions in this region, but not in the left half of B-151, fail to silence the reporter gene effectively and can no longer respond to mutations in several Pc-G, indicating that this region is indeed relevant for Pc-G-mediated silencing. Three major sequence motifs can be identified in B-151. The first motif (C/T)GAG(C/T)G is the consensus binding site of the Zeste protein. Both the left and right halves of B-151 contain one Zeste binding site. The second motif ATGGC represents the binding site of a newly characterized member of Pc-G, pho, which encodes the Drosophila homolog of YY1. bxd-3 and bxd-4 each contain one copy of an almost identical YY1 site. The third motif is a (GA)n repeat, which represents the consensus binding site of GAF encoded by Trithorax-like (Trl). Trl was originally identified as a member of trx-G; however, some recent studies suggest that it may also share some characteristics with Pc-G. While both bxd-3 and bxd-4 contain 2 separate clusters of this motif, one cluster in bxd-4 is much further extended (GAGAGAGGGAGAG versus GAGAG). Since bxd-4 has been shown to be more effective in competition assays, it is likely that the (GA)n motif is most critical for CHRASCH binding. This possibility is further supported by the observation that CHRASCH binding to bxd-b is completely competed out by a fragment containing multiple (GA)n repeats but not by the one containing multiple Zeste repeats or an oligonucleotide containing a YY1 binding site. Therefore, it is concluded that the binding sequences of CHRASCH consist primarily of the (GA)n motif (Huang, 2002).

The association of the (GA)n binding activity with CHRASCH is further confirmed by the following observations. When the DNA binding activity was examined in peptide-eluted fractions, it was found that fractions 6 and 8 had the strongest binding activities. These fractions also contained the highest amounts of PC and other associated proteins. Thus, the binding protein coelutes with CHRASCH in the immunoaffinity chromatography. In addition, it was found the DNA-protein complexes formed on bxd-b can be slightly supershifted by a preincubation with an affinity-purified PC antibody but not with a nonspecific IgG. The small supershift might be expected for a large complex in a gel composed of 3.5% polyacrylamide. Taken together, these results indicate that the (GA)n binding protein is physically associated with CHRASCH (Huang, 2002).

Since chromatin immunoprecipitation experiments have shown that Trithorax-like is enriched in the region encompassing B-151 and its vicinity, whether the binding protein of CHRASCH is related to Trithorax-like was examined. The results are not consistent with this notion. As expected, a purified recombinant Trithorax-like binds specifically to the bxd-b fragment. In addition, the binding activity of Trithorax-like is drastically stimulated by zinc ions over a wide range, resulting in a further retardation of the DNA-protein complexes in the EMSA. By contrast, the binding activity of CHRASCH is not significantly affected at intermediate concentrations of zinc ions and becomes completely inactivated at a high concentration (i.e., 0.5 mM). The differential effects of zinc ions on DNA binding properties argue that different DNA binding proteins are involved in the binding. Furthermore, a Trithorax-like-specific antiserum was used to examine the protein preparations. Trithorax-like antiserum detected multiple bands corresponding to two major classes of isoforms, Trithorax-like-519 and -581, from the column input, but no specific Trl immunoreactivity was found in the eluate (Huang, 2002).

To further characterize the DNA binding protein of CHRASCH, a cross-linking method was used to specifically label this protein. An oligonucleotide probe was designed that allowed specific incorporation of both radioactive dCTP and a photoactivated cross-linking dTTP analogue (i.e., AB-dUTP) into the nucleotide sequences that correspond to the extended (GA)n motif in bxd-4. Following DNA binding and UV irradiation, the cross-linked nucleoprotein complex was digested extensively with both DNase I and micrococcal nuclease to remove excessive DNA sequences. The indirectly labeled proteins were then resolved on an SDS-9% polyacrylamide gel. A major band of ~85 kDa was identified for the purified recombinant Trithorax-like, whereas a different pattern was observed for CHRASCH, which consists of a doublet of ~130 kDa and a doublet of ~70 kDa. Taken together, these results provide strong evidence that the DNA binding activity of CHRASCH is not contributed by Trithorax-like but by a novel factor(s) (Huang, 2002).

A (GA)n-binding protein has been identified in Apis mellifera by screening an expression library with (GA)n repeats (Lehmann, 1998). The Drosophila homolog of this protein was found to be encoded by pipsqueak (psq), identified originally by its grandchildless phenotype and subsequently by its effect on eye development. By differential transcriptional and translational initiation, psq produces two major mRNAs containing open reading frames for 1,065 (PSQ-A) and 630 to 646 amino acids (PSQ-B). These two isoforms share a common C-terminal PSQ domain capable of binding to the (GA)n motif (Lehmann, 1998). The size similarity between one of the cross-linked proteins and PSQ-A prompted a test of whether Psq proteins copurify with CHRASCH. Using an antibody that reacts with both PSQ-A and B (Horowitz, 1996), it was found that PSQ-A and much less PSQ-B are clearly detectable in CHRASCH sample. For a preparation of FLAG-tagged TATA-box binding protein (TBP), however, a large amount of PSQ-A was found in the input but not in the eluted fraction. These results demonstrate that PSQ-A and CHRASCH have indeed been copurified. To further examine the association between Pc and Psq in vivo, coimmunoprecipitation was performed with embryonic nuclear extracts. Pc was precipitated by the Psq antibody. Although it is not clear whether the smaller ~70-kDa proteins detected in the cross-linking experiments represent degradation products of Psq or other unrelated proteins, these results strongly suggest that Psq-A may play a major role in DNA binding (Huang, 2002).

Control of germline torso expression by the BTB/POZ domain protein pipsqueak is required for embryonic terminal patterning in Drosophila

Early embryogenesis in Drosophila is controlled by maternal gene products, which are deposited in the egg during oogenesis. It is not well understood how maternal gene expression is controlled during germline development. pipsqueak (psq) is a complex locus that encodes several nuclear protein variants containing a PSQ DNA-binding domain and a BTB/POZ domain. Psq proteins are thought to regulate germline gene expression through epigenetic silencing. While psq was originally identified as a posterior-group gene, this study shows a novel role of psq in embryonic terminal patterning. A new psq loss-of-function allele, psqrum, was identified that specifically affects signaling by the Torso (Tor) receptor tyrosine kinase (RTK). Using genetic epistasis, gene expression analyses, and rescue experiments, it was demonstrated that the sole function impaired by the psqrum mutation in the terminal system is an essential requirement for controlling transcription of the tor gene in the germline. In contrast, the expression of several other maternal genes, including those encoding Tor pathway components, is not affected by the mutation. Rescue of the psqrum terminal phenotype does not require the BTB/POZ domain, suggesting that the PSQ DNA-binding domain can function independently of the BTB/POZ domain. The finding that tor expression is subject to dedicated transcriptional regulation suggests that different maternal genes may be regulated by multiple distinct mechanisms, rather than by a general program controlling nurse-cell transcription (Grillo, 2001).

psq has functions in many developmental stages, and presumptive null mutants are lethal. This study reports that a set of psq mutations unveils a specific role in tor transcription. Why is this phenotype only observed associated with these particular psq mutations? Among the psq alleles that allow adult survival, strong mutations block oogenesis at early stages. Thus, in those cases, an early requirement in oogenesis would mask a later requirement for tor transcription. Psq proteins are present in multiple isoforms. Only a few psq alleles have been molecularly characterized and among those many are due to transposon insertions. Therefore, it is difficult on the basis of the molecular analysis of the psq mutations to assign distinct functions to the isoforms generated by the different transcripts (Grillo, 2001).

A number of reasons argue for the psqrum mutation unveiling a physiological function of psq in tor transcription, rather than the rum phenotype being a neomorphic effect caused by a special truncated Psq protein. First, the terminal phenotype of the psqrum mutation is observed in homozygosity, as well as in trans-heterozygous combinations with several other psq loss-of-function alleles. Second, the terminal phenotype of the psqrum mutation arises in association with a mild posterior phenotype, a well-known psq loss-of-function phenotype. Third, a transheterozygous combination of other psq loss-of-function alleles also produces embryos showing reduced tor expression and terminal defects. And finally, expression of psq rescues the psqrum terminal phenotype (Grillo, 2001).

The psqrum mutation causes a decrease in the wild-type splicing at one specific site. Nevertheless, since all known isoforms share this splicing, it cannot be inferred whether a particular isoform is responsible for tor transcriptional regulation. However, the rescue experiments indicate that both a long Psq isoform containing the BTB/POZ domain and a short isoform lacking this domain are capable of providing the psq function controlling tor transcription that is missing in psqrum mutants. Thus, on the one hand, the BTB/POZ domain appears to be dispensable for this psq function, but on the other hand, a long isoform can substitute for a short isoform, arguing against separate functions of these psq isoforms in the context of Tor signaling. Thus, an overall decrease of many psq isoforms in the psqrum mutant could be affecting tor transcription (Grillo, 2001).

In this regard, it is worth considering together the terminal and posterior defects associated with the psqrum mutation. tor is affected more strongly than vas in psqrum mutants, while the opposite is true for other psq mutants (e.g., psqHK38, psq2403, and psqfs1, in which vas is strongly affected, but not tor, according to their cuticle phenotype. These data argue against a simple model in which tor and vas transcription would be impaired below different thresholds of psq activity (Grillo, 2001).

As Psq is thought to repress gene expression through epigenetic silencing, Psq could activate tor expression indirectly, through the repression of a still unidentified tor repressor. Alternatively, Psq could activate tor expression directly. Indeed, genetic interaction studies suggest that psq and Trithorax-like (Trl) act together in transcriptional activation as well as in transcriptional silencing of homeotic genes (Grillo, 2001).

Not much is known about how transcription is regulated in Drosophila nurse cells. One possibility is that spatially and temporally coexpressed genes share a common mode of transcriptional regulation. Indeed, enrichment of specific core motifs in the promoters of genes with female germline expression is consistent with such a hypothesis. The multiple effects of psq mutations during oogenesis might argue for such a general role of psq. However, and in spite of psq's multiple requirements in the germline, tor transcription appears to be distinctly regulated. A similar case appears to apply to bcd transcription, which was found to be specifically controlled by Serendipity-δ (Sry-δ), a zinc finger protein. sry-δ null alleles block oogenesis, and only a particular genetic combination revealed the specific requirement of sry-δ for bcd transcription. Thus, both psq and sry-δ have a basic function in oogenesis, probably through the transcriptional control of other germline genes, and a specific function in the control of tor and bcd, respectively. This similarity is particularly intriguing considering the peculiarities of early Drosophila embryogenesis and the fact that anterior patterning by bcd seems to be restricted to Diptera and that tor-dependent terminal patterning appears in Diptera and Coleoptera, but not in Hymenoptera. Thus, the regulation of bcd and tor transcription by a specific function of more general germline transcription factors might be related to their particular recruitment to embryonic patterning. Interestingly, in the case of terminal patterning, tor transcription appears to be regulated independently from that of genes encoding the other elements of the signaling pathway (e.g., Ras and Raf), as induced expression of tor is sufficient to rescue the psqrum terminal phenotype. However, the only essential function of these other elements of the Tor pathway in the oocyte is to transmit the Tor signal, as indicated by the phenotype of mutant germline clones. Thus, in the absence of tor activity, these products appear to be silent in the Drosophila germline. Altogether, these data suggest a possible multiple-step way to acquire new regulatory mechanisms in a given set of cells. This possibility appears particularly suggestive in the light of recent results pointing to Tor as the receptor for prothoracicotropic hormone (PTTH), which stimulates the production of the molting hormone ecdysone. Could this be a more ancient role of tor that would subsequently have been recruited for embryonic terminal patterning in some insects? Such a scenario appears to apply to the Toll signaling pathway, which shares some similarities with the Tor pathway, and has a widely conserved function in immunity in many animals and whose components are also transcribed by the Drosophila female germline to specify the embryonic dorsoventral pattern (Grillo, 2001).

Ecdysone-induced 3D chromatin reorganization involves active enhancers bound by Pipsqueak and Polycomb

Evidence suggests that Polycomb (Pc) is present at chromatin loop anchors in Drosophila. Pc is recruited to DNA through interactions with the GAGA binding factors GAF and Pipsqueak (Psq). Using HiChIP in Drosophila cells, this study found that the psq gene, which has diverse roles in development and tumorigenesis, encodes distinct isoforms with unanticipated roles in genome 3D architecture. The BR-C, ttk, and bab domain (BTB)-containing Psq isoform (Psq(L)) colocalizes genome-wide with known architectural proteins. Conversely, Psq lacking the BTB domain (Psq(S)) is consistently found at Pc loop anchors and at active enhancers, including those that respond to the hormone ecdysone. After stimulation by this hormone, chromatin 3D organization is altered to connect promoters and ecdysone-responsive enhancers bound by Psq(S). These findings link Psq variants lacking the BTB domain to Pc-bound active enhancers, thus shedding light into their molecular function in chromatin changes underlying the response to hormone stimulus (Gutierrez-Perez, 2019).

The BTB domain has been shown to contribute to the oncogenic roles of these proteins. Most BTB-containing transcription factors also encode isoforms that lack the BTB domain and the role of these short isoforms is uncertain. This study shows that different isoforms of Psq appear to play different roles in nuclear function, which may explain their opposing roles in tumorigenesis ascribed to the gene. The BTB-containing PsqL isoform colocalizes with a specific class of architectural proteins that includes Su(Hw), CP190, and Mod(mdg4)2.2. In contrast, the PsqS isoform, which lacks the BTB domain, colocalizes with GAF and Pc at developmental enhancers (dCP) and is mainly associated with active chromatin states. Therefore, PsqS appears to contribute to enhancer function, whereas PsqL is an architectural protein that binds to sequences that have insulator function. How these two isoforms display different genomic distributions while sharing the same DNA binding domain is unclear. However, based on previous findings, it is speculated that the conformation adopted by the protein in the presence of the BTB-interaction domain might inhibit its direct binding to DNA. In addition, the two isoforms coincide in regions in which both Pc and architectural proteins are found. This may explain the reported involvement of PsqL in the recruitment of PcG proteins to chromatin, where it might act with the help of other architectural proteins. In addition to its canonical role, Pc is found, together with PsqS, ISWI, GAF, and CBP, in regions containing H3K27ac and previously characterized experimentally as housekeeping enhancers (hkCP) or dCP enhancers. These findings, suggesting an association of Pc with active enhancers, agree with previous observations showing that PRC1 can be recruited to active genes by the cohesin complex, where it affects phosphorylation of Pol II and Spt5 occupancy (Gutierrez-Perez, 2019).

H3K27me3 is present in the genome of Kc167 cells at very high levels in Pc-repressed domains such as Hox genes. The rest of the genome containing silenced genes in Kc167 cells has low but significant levels of H3K27me3 that represent B compartment sequences. Pc HiChIP analysis provides insights into the dual role of Pc in regulating chromatin organization. Classical Pc-repressed domains interact with each other and with other B compartments with a frequency that correlates with the amount of H3K27me3 present in these compartments. Distinct from these interactions, Pc also forms punctate point-to-point contacts. Two types of loops, defined as puncta of an intense signal in Hi-C heatmaps, have been identified when analyzing changes in 3D organization during Drosophila embryonic development. These loops were classified as active loops containing H3K27ac, Zelda, and Pol II at their anchors or as Pc loops bound by GAF. Zelda loops are absent from Kc167 cells. Like Pc loop anchors observed in embryos, loops represented by puncta in Hi-C heatmaps of Kc167 cells are located within regions enriched in H3K27me3. However, this study found that the center of these sites in Kc167 cells is depleted of H3K27me3 and enriched in H3K27ac. The exact roles of H3K27ac, Pc, PsqS, and GAF found at these loop anchors are unknown, but it is speculated that maintaining a localized active chromatin state may be important for the binding of these proteins and the establishment of these loops. These results suggest a dual and context-dependent function of regulatory elements and agree with previous studies showing that dCP enhancers can act as PREs, and vice versa, during Drosophila embryogenesis (Gutierrez-Perez, 2019).

Protein Interactions

The Drosophila GAGA factor Trithorax like controls transcription and other chromosome functions by altering chromatin structure. A second GAGA-binding protein of Drosophila, Pipsqueak (Psq), can directly bind to Trithorax like and is associated with Trl in vivo. Genetic interaction studies provide evidence that Psq and Trl act together in the transcriptional activation and silencing of homeotic genes. A complete colocalization of Psq and Trl on polytene interphase chromosomes and mitotic chromosomes suggests that the two proteins cooperate as general partners not only at homeotic loci, but also at hundreds of other chromosomal sites (Schwendemann, 2002).

To identify chromosomal loci that are targets of both Trl and Psq, salivary gland polytene chromosomes were doubly immunostained with anti-Psq and anti-Trl antibodies. Surprisingly, the staining patterns obtained with the two antibodies appeared to be identical. Like Trl, Psq binds to hundreds of loci on the polytene chromosomes. Analysis of the binding patterns by confocal microscopy confirmed that virtually every signal derived with Trl antibody coincides with a signal derived with Psq antibody and vice versa. Occasionally, single sites appear to be stained by only one of the two antibodies. However, these sites vary between different chromosome preparations, and it is therefore believed that they do not represent binding sites truly specific for only one of the two proteins. Western analyses show that the overlapping staining patterns are not caused by cross-reaction of Psq antibody with Trl protein or Trl antibody with Psq protein (Schwendemann, 2002).

One possibility to explain the colocalization of Trl and Psq is that GAGA-binding sites are recognized in vivo by a protein complex that contains both proteins. Therefore protein extracts were prepared from salivary gland nuclei and immunoprecipitation assays were performed with the Psq antibody. This antibody efficiently coimmunoprecipitates Psq and Trl from these extracts. To test whether this effect is specific, whether two other transcription factors, dAP-4 and BR-C, were precipitated by the antibody was also analyzed. Both proteins are known to be expressed in salivary glands, from which the nuclear extract was derived. In addition, like Psq and Trl, all isoforms encoded by the BR-C contain a BTB protein interaction domain. However, neither dAP-4 nor BR-C was found to be precipitated by the Psq antibody. It is concluded that the interaction between Psq and Trl is specific (Schwendemann, 2002).

It was next asked whether the association of Psq and Trl in vivo might be due to direct binding of the two proteins to one another. To address this question, histidine (his)-tagged full-length and truncated Psq proteins were expressed in bacteria, and the ability of these proteins to retain the Trl519 isoform on a Ni2-NTA column was tested. Full-length Psq (Psq1064), but not polypeptides including only the Psq domain (Psq221) or lacking the BTB domain (Psq942), were able to efficiently bind Trl519. When Psq1064 was successively truncated from the C terminus, all resulting polypeptides were able to bind Trl519, including Psq166, which essentially consists of the BTB domain. Because previous studies have shown that the BTB domain forms a protein-protein interaction interface, this result suggests that binding is mediated by the BTB domains of both proteins. Therefore a GST fusion of the Trl BTB domain was expressed in bacteria (GST-Trl116) and tested to see whether this polypeptide can be coimmunoprecipitated with Psq331, a polypeptide that consists of the N-terminal third of Psq, which includes the BTB domain. As a control, whether Trl116 can be coimmunoprecipitated with Psq221 was tested. Psq331, but not Psq221, is bound by Trl116. Taken together, these results suggest that Psq and Trl are associated with one another in vivo through direct binding mediated by their BTB domains. The BTB domain of Trl has been shown to also mediate self-oligomerization of Trl that leads to formation of large protein complexes in vitro. These results suggest that Psq is a partner of Trl in similar complexes formed in vivo. Because both Trl and Psq have been shown to bind GA-rich sequences, it is likely that both proteins contribute to the cooperative binding of multiple GAGA elements through these complexes. The complete colocalization of Trl and Psq at hundreds of chromosomal loci predicts that, in general, Psq and Trl act as partners. Consistent with this notion, the bxd PRE of Ubx (Hodgson, 2001) and the MCP silencer of Abdominal B (Abd-B) (S. Sakonju, personal communication to Schwendemann, 2002) emerge as first examples of specific loci where Psq and Trl appear to interact (Schwendemann, 2002).

To further test the model that Psq and Trl are partners, it was asked whether Psq is also bound to the GA-rich satellite DNA of the centromeric heterochromatin, which is occupied by Trl during mitosis. When mitotic chromosomes from larval brains are stained with Trl antibodies, strong fluorescent signals are observed in the pericentric regions. A similar staining of the pericentric regions is observed when chromosomes are stained with the Psq antibody. Double staining experiments show that this staining cannot be distinguished from the staining pattern obtained with the Trl antibody. As on the polytene chromosomes, there thus seems to be a complete overlap of the Trl and Psq binding sites. Taken together, the results of the polytene and mitotic chromosome staining strongly suggest that Psq and Trl are general partners that not only share common functions in the control of target genes at euchromatic sites, but also in heterochromatin organization and mitosis (Schwendemann, 2002).

The genetic, biochemical, and cytogenetic data presented in this study strongly suggest that Psq and Trl act together as partners in the control of homeotic and many other genes. Since both Trl and Psq are encoded by essential genes, the functions of these proteins are not redundant. Psq may even be an obligatory partner of Trl. Future studies on Trl function will therefore have to include this partner, and may thus provide novel insights into the mechanism of action of this important chromatin factor. The finding that Psq is a member of a larger family of DNA-binding proteins, which includes many Drosophila BTB proteins with previously unknown DNA-binding activity (Siegmund, 2002), may help to further elucidate the composition and function of Psq/Trl-containing protein complexes (Schwendemann, 2002)

pipsqueak: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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