trithorax

Protein Interactions

Studies of deletion mutants of trithorax and Polycomb demonstrate that trithorax-dependent activation requires the central zinc-binding domain, while Polycomb-dependent repression requires the intact chromodomain. In addition, trithorax-dependent activity can be abrogated by increasing the amount of Polycomb, suggesting a competitive interaction between the products of trithorax and Polycomb (Chang, 1995).

Trithorax products can be detected at 16 discrete sites on larval salivary gland polytene chromosomes, 12 of which colocalize with binding sites of several Polycomb group proteins. The intensity of Trithorax protein binding is strongly decreased in larvae carrying mutations in another trithorax group gene ash-1, and in the Polycomb group gene E(z). This work provides solid evidence that trithorax and Polycomb group proteins assemble together at regulatory sites to carry out alternative strategies of gene activation and repression (Kuzin, 1994).

Trithorax proteins are bound to 63 specific sites on the polytene chromosomes of the larval salivary gland. Trithorax binding is detected at the sites of its known targets, the bithorax and Antennapedia complexes, despite the transcriptionally repressed state of these loci in the salivary gland. A temperature-sensitive trithorax mutation greatly reduces the number of binding sites. Simultaneous localization of Trithorax and Polycomb indicates that many of their chromosomal binding sites coincide. One Trithorax binding site is located within a portion of the large 5' regulatory region of the Ubx gene, in an interval which also contains binding sites for Polycomb group proteins. These results suggest that Trithorax exerts its effects by binding directly or indirectly to specific DNA sequences in its target genes. Co-localization with Polycomb also suggests that interactions between these activators and repressors of the homeotic genes may be a significant feature of their mode of action (Chinwalla, 1995).

The ALL-1 gene was discovered by virtue of its involvement in human acute leukemia. Its Drosophila homolog trithorax (trx) is a member of the trx-Polycomb gene family, which maintains correct spatial expression of the Antennapedia and bithorax complexes during embryogenesis. The C-terminal SET domain of ALL-1 and Trithorax (Trx) is a 150-aa motif, highly conserved during evolution. Yeast two hybrid screening of a Drosophila cDNA library was performed and interaction was detected between a Trx polypeptide spanning SET and the Snr1 protein. Snr1 is a product of snr1, which is classified as a trx group gene. Parallel interaction is found in yeast between the SET domain of ALL-1 and the human homolog of Snr1, INI1 (hSNF5). These results were confirmed by in vitro binding studies and by demonstrating coimmunoprecipitation of the proteins from cultured cells and/or transgenic flies. Epitope-tagged SNR1 is detected at discrete sites on larval salivary gland polytene chromosomes, and these sites colocalize with approximately 50% of Trx binding sites. Because Snr1 and INI1 are constituents of the SWI/SNF complex, which acts to remodel chromatin and consequently to activate transcription, the observed interactions suggest a mechanism by which the SWI/SNF complex is recruited to ALL-1/trx targets through physical interactions of the C-terminal domains of ALL-1 and Trx with INI1/Snr1 (Rozenblatt-Rosen, 1998).

Trithorax (Trx) and Ash1 belong to the trithorax group (trxG) of transcriptional activator proteins -- this group of proteins maintains homeotic gene expression during Drosophila development. Trx and Ash1 are localized on chromosomes and share several homologous domains with other chromatin-associated proteins, including a highly conserved SET domain and PHD fingers. Based on genetic interactions between trx and ash1 and the observation that association of the Trx protein with polytene chromosomes is ash1 dependent, the possibility of a physical linkage between the two proteins was investigated. Endogenous Trx and Ash1 proteins coimmunoprecipitate from embryonic extracts and colocalize on salivary gland polytene chromosomes. Furthermore, Trx and Ash1 bind in vivo to a relatively small (4 kb) bxd subregion of the homeotic gene Ultrabithorax (Ubx), which contains several trx response elements. Analysis of the effects of ash1 mutations on the activity of this regulatory region indicates that it also contains ash1 response element(s). This suggests that Ash1 and Trx act on Ubx in relatively close proximity to each other. Finally, Trx and Ash1 appear to interact directly through their conserved SET domains, based on binding assays in vitro and in yeast and on coimmunoprecipitation assays with embryo extracts. Collectively, these results suggest that Trx and Ash1 are components that interact either within trxG protein complexes or between complexes that act in close proximity on regulatory DNA to maintain Ubx transcription (Rozovskaia, 1999).

Since genetic experiments suggest that both trx and ash1 are involved in regulation of homeotic gene expression, it was of particular interest to determine whether binding of the proteins to polytene chromosomes and/or genetic responsiveness is conferred by the same DNA sequences. To test this, an analysis was performed to see whether both proteins bind in vivo to a well-characterized TRE-PRE-containing bxd regulatory module located 25 kb upstream of the Ubx promoter. Indeed, on salivary gland polytene chromosomes, both proteins are found at the site of insertion of a transgene containing this 4-kb bxd subregion. This indicates that Trx and Ash1 DNA binding elements may be close to each other. In addition, it has been shown that Ash1 is required for full function of the same regulatory region in vivo. Since this 4-kb region contains three trx-responsive TREs, this leaves open the possibility that Trx and Ash1 may function through the same DNA elements. Experiments aimed at fine mapping of the ash1 response element(s) within this region of Ubx are currently in progress. Nonetheless, these results suggest that Trx and Ash1 may act in concert on one or more bxd TREs. Two interesting possibilities are that both Trx and Ash1 are components of the same protein complex or that they are interacting components of two separate protein complexes that form on closely situated TREs. The physical association between Trx and Ash1 (probably through interaction of their SET domains) is apparently required for Trx binding to chromosomes, since Trx is only weakly associated with chromosomes in ash1 mutant larvae. These close physical and functional associations on Ubx regulatory DNA provide a biochemical rationale for the genetic interactions between trx and ash1 mutants (Rozovskaia, 1999).

By applying yeast two-hybrid assays as well as other methodologies, it has been found that the SET domains of both Trx and Ash1 proteins can self-associate. The self-associating Trx fragment (aa 3540 to 3759) spans the ~130-aa SET domain and includes an additional ~90 aa of upstream sequence. The self-interacting Ash1 region includes the entire SET domain (residues 1318 to 1448) in addition to upstream sequence (aa 1160 to 1317). An alternative self-associating region of Ash1 (aa 1245 to 1525) also includes the entire SET domain. Mutations within the SET domain of both Trx and Ash1 prevent self-association. Whether those TRX and ASH1 regions can also undergo hetero-oligomerization was examined. Indeed, the two polypeptides interact strongly in yeast, as evidenced by activation of both the HIS and lacZ reporters. To confirm this result, GST pull-down methodology as well as coimmunoprecipitation analysis was performed. A C-terminal Trx polypeptide (Trx SET) was synthesized and radiolabeled in a coupled transcription-translation system and tested for binding to the relevant Ash1 polypeptide (Ash1 SET) linked to GST. The ASH1-linked resin binds 10- to 20-fold more Trx SET than does GST resin alone. For in vitro coimmunoprecipitation analysis, the same Trx polypeptide was radiolabeled and mixed with unlabeled epitope-tagged (T7) Ash1 SET. The labeled Trx SET coimmunoprecipitates with the T7-ASH1 SET but not with two unrelated T7-tagged proteins. Similar results were obtained in a reciprocal experiment. Finally, plasmids encoding the T7-tagged Ash1 SET and HA-tagged Trx SET were transiently cotransfected into COS cells. The epitope-tagged polypeptides produced in vivo were also found to coimmunoprecipitate (Rozovskaia, 1999).

To address the biological significance of this hetero-oligomerization, conserved residues within the SET domain were mutagenized and their effects on interaction in yeast were tested. Thirteen different mutations at either single amino acids or nearby pairs of amino acids were constructed, 10 at highly conserved residues and 3 controls at nonconserved residues within Trx SET. Each of the alterations of conserved amino acids resulted in the loss of most or all of the capacity of TRX SET to interact with Ash1 SET in yeast. In contrast, the three alterations of nonconserved residues, located within the SET domain or immediately upstream of it, did not affect the interaction. A more limited mutagenesis analysis of conserved residues within the Ash1 SET domain shows that conversion of GRG (residues 1310 to 1321) to VRV, PN (1391 and 1392) to AY, I (1414) to A, or DY (1423 and 1424) to AA results in the loss of most or all of the interaction in yeast. These results argue for the functional significance of the TRX SET-ASH1 SET interactions seen in yeast and in vitro and suggest that the association in embryos between full-length Trx and Ash1 is direct and involves binding between their SET domains (Rozovskaia, 1999).

The compaction of the eukaryotic genome in nucleosomes limits the access of DNA to regulatory proteins and the enzymes that process genetic information. To overcome this constraint, cells employ a variety of strategies to disrupt locally the organization of nucleosomes. Prominent among these are several distinct classes of enzymes that covalently modify specific residues of the N-terminal histone tails, thereby affecting nucleosome stability or higher order nucleosome interactions. A second group of multisubunit protein complexes uses the energy of ATP hydrolysis to alter chromatin structure and mobilize nucleosomes. NURF is an Imitation SWI (ISWI) containing complex of four proteins that uses the energy of ATP hydrolysis to catalyze nucleosome sliding. Three NURF components have been identified previously. cDNAs encoding the largest NURF subunit have been cloned, revealing a 301 kDa polypeptide (NURF301) that shares structural motifs with ACF1. Full and partial NURF complexes have been reconstructed from recombinant proteins and NURF301 and the ISWI ATPase have been shown to be necessary and sufficient for accurate and efficient nucleosome sliding. An HMGA/HMGI(Y)-like domain of NURF301 that facilitates nucleosome sliding indicates the importance of DNA conformational changes in the sliding mechanism. NURF301 also shows interactions with sequence-specific transcription factors, providing a basis for targeted recruitment of the NURF complex to specific genes (Xiao, 2001).

To explore additional functions for NURF and NURF301, an analysis of proteins that interact with NURF was undertaken by identifying coimmunoprecipitating polypeptides in embryo nuclear extract fractions. Peptide sequencing of a 70 kDa species among numerous polypeptides that were substoichiometric with respect to NURF subunits reveal an unambiguous match with the embryonically expressed isoform of the GAGA transcription factor, GAGA519. A control experiment using beads coated with a nonspecific antibody showed no coimmunoprecipitation of GAGA factor. The association of GAGA factor and NURF was confirmed in binding assays. Binding to recombinant NURF complex was observed for S³⁵-labeled GAGA519 and the alternatively spliced GAGA581 isoform, which is expressed at later stages in Drosophila development (Xiao, 2001).

By systematic deletions, the NURF-interacting sequences common to both GAGA519 and GAGA581 were determined to be in a conserved region containing the DNA binding zinc finger and flanking sequences. This finding, which is consistent with a study of the GAGA factor regions necessary for chromatin remodeling, is clearly distinguished from known interactions involving the multimerization (POZ) and transactivating (glutamine-rich) domains of GAGA factor and is also reminiscent of interactions between the zinc finger of mammalian ELKF and the SWI/SNF complex. In addition to the interactions with the GAGA factor, the NURF complex binds to S³⁵-labeled GAL4-HSF and GAL4-VP16 activators containing the GAL4 DNA binding domain fused to strong activation regions of HSF and VP16. Binding was specific for HSF and VP16, since little or no binding was detected for the GAL4(1-147) DNA binding domain (Xiao, 2001).

To define region(s) of NURF301 that interact with GAGA factor, HSF, and VP16, the binding of GST-NURF301 segments to S³⁵-labeled activators was analyzed in GST-pull-down assays. Two regions on NURF301 responsible for GAGA factor binding are also nucleosome binding regions. Whether the interaction of NURF301 with GAGA factor or the nucleosome is mutually exclusive is presently unknown. A separate region of NURF301 is responsible for binding GAL4-HSF and GAL4-VP16. Consistent with these results, the DeltaN301NURF complex binds to GAGA factor less efficiently (~26% of wild-type) but still binds to GAL4-HSF and GAL4-VP16. The binding of other NURF subunits to activators was also examined. ISWI, NURF55, and NURF38 show little or no binding to S³⁵-labeled HSF and VP16. Interestingly, ISWI, but not NURF55 and NURF38, shows binding to GAGA factor, albeit at a level lower than binding to NURF (Xiao, 2001).

Trithorax (Trx) is a Drosophila SET domain protein that is required for the correct expression of homeotic genes. The Trx SET domain efficiently binds to core histones and nucleosomes. The primary target for the SET domain is histone H3 and binding requires the N-terminal histone tails. The previously described trx^Z11 mutation changes a strictly conserved glycine in the SET domain to serine and causes homeotic transformations in the fly. This mutation selectively interferes with histone binding, suggesting that histones represent a critical target during developmental gene regulation by Trx (Katsani, 2001).

Unlike the heterochromatic silencing factor SUV39H1, the activator Trx appears to be unable to methylate histone tails. This failure, however, is not due to the inability to bind histones. Thus, rather than an active enzymatic core, the Trx SET domain might act as a histone binding module that anchors Trx to the chromatin template. Similar to anti-phosphatases, the Trx SET domain may bind the substrate (i.e., the histone tails) and block modification by related active enzymes such as SUV39H1, thus preventing HP1 recruitment. Such a mechanism may contribute to the antagonistic activities of the silencer SUV39H1 and the activator Trx. Modification of the histone tails can influence binding of the Trx SET domain and may contribute to directing Trx to active chromatin. Trx is a developmental regulator that is essential for the normal expression of multiple homeotic genes. This function is impaired by the trx^Z11 mutation in the SET domain resulting in homeotic transformations. An analogous mutation in the SET domain of yeast Set1p causes a defect in telomeric silencing, supporting the notion that this mutation interferes with a highly conserved function. Although other SET domain functions may also be affected, these results show that the trx^Z11 mutation incapacitates its ability to bind histones. This finding provides a molecular explanation for the aberrant development of trx^Z11 flies and implies that histone recognition by the Trx SET domain is essential for the in vivo functioning of Trx (Katsani, 2001).

Trithorax (Trx) is a member of the trithorax group (trxG) of epigenetic regulators; these proteins are required to maintain active states of Hox gene expression during development. A trithorax acetylation complex (TAC1) has been purified that contains Trx, dCBP, and Sbf1. Like CBP, TAC1 acetylates core histones in nucleosomes, suggesting that this activity may be important for epigenetic maintenance of gene activity. dCBP and Sbf1 associate with specific sites on salivary gland polytene chromosomes, colocalizing with many Trx binding sites. One of these is the site of the Hox gene Ultrabithorax (Ubx). Mutations in either trx or the gene encoding dCBP reduce expression of the endogenous Ubx gene as well as of transgenes driven by the bxd regulatory region of Ubx. Thus Trx, dCBP, and Sbf1 are closely linked, physically and functionally, in the maintenance of Hox gene expression (Petruk, 2001).

Trithorax interacts with type 1 serine/threonine protein phosphatase in Drosophila

The catalytic subunit of type 1 serine/threonine protein phosphatase (PP1c) binds Trithorax (Trx) in the yeast two-hybrid system. Interaction between PP1c and Trx was confirmed in vivo by co-immunoprecipitation from Drosophila extracts. An amino-terminal fragment of Trx, containing a putative PP1c-binding motif, is sufficient for binding to PP1c by in vitro glutathione S-transferase pull-down assays using recombinant protein and fly extracts expressing epitope tagged PP1c. Disruption of the PP1c-binding motif abolished binding, indicating that this motif is necessary for interaction with PP1. On polytene chromosomes, PP1c is found at many discrete bands, which are widely distributed along the chromosomes. Many of the sites that stain strongly for PP1c correspond to sites of Trx, consistent with a physical association of PP1c with chromatin-bound Trx. Homeotic transformations of haltere to wing in flies mutant for trx are dominantly suppressed by PP1c mutants, indicating that PP1c not only binds Trx, but is a physiologically relevant regulator of Trx function in vivo (Rudenko, 2003).

It is proposed that PP1 is part of one or more TRX chromatin-associated complexes, in which PP1 exerts an effect on TRX-dependent transcriptional control. One such complex might be TAC1, which is reported to contain about 90% of total Trx protein. However, PP1 might also be part of other complexes, containing dCBP or SNF1 for instance. PP1 is found at other discrete sites that do not contain Trx, indicating that there are other PP1c-binding proteins that can recruit PP1 to different chromosomal regions (Rudenko, 2003).

The exact role of Trx-bound PP1 is not yet known. Trx, which contains four serine-rich domains, could itself be a direct target of PP1. However, it is not known whether Trx is a phosphoprotein, and because the exact role of Trx is unclear it is difficult to predict what the effect of phosphorylation might be. It is possible that Trx is an anchor for other chromatin-associated proteins and that it targets PP1 to some other member of this complex, such as dCBP. Mammalian CBP has been found to be phosphorylated in both quiescent and dividing cells, but surprisingly few studies have addressed the role of phosphorylation in controlling CBP activity. Phosphorylation of the carboxy terminus of CBP has been reported to repress its histone acetylation activity, whereas phosphorylation at the N terminus by protein kinase C (at Ser 89) has the opposite effect. Further studies will need to examine the phosphorylation state of dCBP in TRX complexes, but one possibility is that PP1 dephosphorylates CBP to allow patterns of transcription to be reprogrammed (Rudenko, 2003).

A role for phosphorylation in the control of TAC1 activity is implied by the presence of Sbf1 in the TAC1 complex. Sbf1 belongs to a subset of myotubularin-related dual-specificity phosphatases that are catalytically inactive and have been proposed to act as 'anti-phosphatases' by binding to and protecting substrates from dephosphorylation. Myotubularin phosphatases seem primarily to dephosphorylate phosphatidylinositol 3-OH monophosphatase but can also dephosphorylate phosphoserine and phosphothreonine residues, indicating that Sbf1 might protect against the effects of PP1. Identification of targets of Sbf1 might therefore help to identify relevant PP1 substrates (Rudenko, 2003).

Species selectivity of mixed-lineage leukemia/trithorax and HCF proteolytic maturation pathways

Site-specific proteolytic processing plays important roles in the regulation of cellular activities. The histone modification activity of the human trithorax group mixed-lineage leukemia (MLL) protein and the cell cycle regulatory activity of the cell proliferation factor herpes simplex virus host cell factor 1 (HCF-1) are stimulated by cleavage of precursors that generates stable heterodimeric complexes. MLL is processed by a protease called taspase 1, whereas the precise mechanisms of HCF-1 maturation are unclear, although they are known to depend on a series of sequence repeats called HCF-1(PRO) repeats. The Drosophila homologs of MLL and HCF-1, called Trithorax and dHCF, are both cleaved by Drosophila Taspase 1. Although highly related, the human and Drosophila taspase 1 proteins display cognate species specificity. Thus, human taspase 1 preferentially cleaves MLL and Drosophila taspase 1 preferentially cleaves Trithorax, consistent with coevolution of taspase 1 and MLL/Trithorax proteins. HCF proteins display even greater species-specific divergence in processing: whereas dHCF is cleaved by the Drosophila taspase 1, human and mouse HCF-1 maturation is taspase 1 independent. Instead, human and Xenopus HCF-1PRO repeats are cleaved in vitro by a human proteolytic activity with novel properties. Thus, from insects to humans, HCF proteins have conserved proteolytic maturation but evolved different mechanisms (Capotosti, 2007).

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