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A Homeotic Mutation in the Trithorax SET Domain Impedes Histone Binding

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A Homeotic Mutation in the Trithorax SET Domain Impedes Histone Binding Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION al. 1998). The SET domain has been implicated in a mul- A homeotic mutation titude of different protein–protein interactions and func- in the trithorax SET domain tions. The SET domains of MLL, yeast Set1p, and E(z) bind to myotubularin-related dual-specificity phosphata- impedes histone binding ses and anti-phosphatases that modulate growth control 1 1 (Cui et al. 1998). The TRX and MLL SET domains bind to Katerina R. Katsani, Juan J. Arredondo, the SNF5 component of the ATP-dependent remodeler Arnoud J. Kal,and C. Peter Verrijzer 2 SWI/SNF (Rozenblatt-Rosen et al. 1998) and mediate self-association (Rozovskaia et al. 2000). Furthermore, Department of Molecular and Cell Biology, MGC Center the SETdomain of yeast Set1p binds the Mec3p check- for Biomedical Genetics, Leiden University Medical Center, point protein and has been implicated in DNA repair and 2300 RA Leiden, The Netherlands telomere function (Corda et al. 1999). The Set1p SET domain alone suffices to mediate telomeric silencing, Drosophila Trithorax (TRX) is a SET domain protein suggesting that it forms a functional unit (Nislow et al. that is required for the correct expression of homeotic 1997). Recently, it was shown that SUV39H1, the mam- genes. Here,we show that the TRX SET domain effi- malian homolog of Su(var)3–9, selectively methylates ly- ciently binds to core histones and nucleosomes. The pri- sine 9 of histone H3 (Rea et al. 2000; Jenuwein 2001). mary target for the SET domain is histone H3 and bind- This modification creates a binding site for HP1 and thus ing requires the N-terminal histone tails. The previously can contribute to the propagation of a heterochromatin described trxZ11 mutation changes a strictly conserved domain (Bannister et al. 2001; Lachner et al. 2001; Na- glycine in the SET domain to serine and causes homeotic kayama et al. 2001). Whereas the histone–methylase ac- transformations in the fly. We found that this mutation tivity of SUV39H1 was critically dependent on the SET domain, additional protein domains were also required. selectively interferes with histone binding,suggesting In contrast to SUV39H1, the SETdomains of TRXand that histones represent a critical target during develop- E(z) do not appear to mediate histone methylation (Rea mental gene regulation by TRX. et al. 2000; Jenuwein 2001). Thus, the TRX SET domain Received March 1, 2001; revised version accepted July 11, may form part of a methylase with a substrate other than 2001. histones or, alternatively, this SETdomain may present a histone-recognizing module that is not a methylase. Here, we report that the TRX SET domain efficiently The Polycomb group (PcG) of repressors and trithorax binds to core histones and to nucleosomes. We found group (trxG) of activators target chromatin in order to that binding depends on the N-terminal histone tails and “freeze” a mitotically stable pattern of gene expression investigated the role of their covalent modifications. The and determined cell fate (Pirrotta 1998; Lyko and Paro effect of a homeotic mutation in the SETdomain ( trxZ11) 1999; Mahmoudi and Verrijzer 2001). The founding on histone binding suggests that histone recognition member of the trxG, the Drosophila trx gene, is required constitutes an essential step during the in vivo control of throughout development and controls the expression of gene expression by TRX. several developmental regulators, including the homeo- tic genes (Ingham and Whittle 1980; Ingham 1985; Breen Results and Discussion 1999). trx is related to the human Mixed Lineage Leuke- mia (MLL) gene, which is involved in translocations as- sociated with the majority of cases of infant leukemias The TRX SET domain binds core histones (Waring and Cleary 1997). TRX and MLL are part of a and nucleosomes highly conserved regulatory network that is required for the correct expression of the homeotic selector genes and To assess whether the SET domain is a histone-recogniz- determination of segment identity in both mammals and ing module, we performed GSTpull-down experiments Drosophila Drosophila. They are very large proteins that contain using purified core histones (Fig. 1B). Bound structural motifs common to chromatin-associated fac- proteins were analyzed by SDS-PAGE followed by Coo- tors such as PHD fingers and a C-terminal SETdomain massie staining. We found that the GST–SET domain (Fig. 1A; Mazo et al. 1990; Stassen et al. 1995). fusion protein selectively retained histones H3 and H4, The SET domain is a highly conserved 130–150 amino whereas binding to H2A and H2B was significantly acids motif initially recognized as a common element in weaker. Binding is efficient since almost all H3 and H4 is chromatin regulators with opposing activities: the sup- depleted from the unbound fraction (Fig. 1B, cf. lanes 5 pressor of position affect variegation Su(var)3-9, the PcG and 7). The SET domain–histone interaction survived protein Enhancer of Zeste [E(z)], and TRX (Jenuwein et stringent washing conditions with a buffer containing 600 mM NaCl and 0.2% NP-40. Moreover, both the SET domain (isoelectric point of 8.6) and histones have a net positive charge at pH 7, arguing against a nonspecific [KeyWords: Trithorax; SET domain; chromatin; histones; homeotic genes] electrostatic interaction. It should be noted that when 1These authors contributed equally to this work and should both be higher amounts of histones were added, binding to all considered first author. four core histones could be detected (see Fig. 2B, below). 2 Corresponding author. Next, we prepared an affinity-matrix by covalent cou- E-MAIL [email protected]; FAX 31-71-527-6284. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ pling of either purified core histones or BSA to CNBr gad.201901. activated-Sepharose beads. We found that radiolabeled GENES & DEVELOPMENT 15:2197–2202 © 2001 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/01 $5.00; www.genesdev.org 2197 Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Katsani et al. beads but not by the control matrix. Next, we examined binding of the SETdomain to radiolabeled mononucleo- somes in a mobility shift assay. Figure 1E reveals that the SETdomain efficiently binds to mononucleosomes. The further retardation of SET–nucleosome complexes in the presence of higher amounts of the SETdomain indicates binding of more than one SETmolecule per nucleosome. Glycerol gradient sedimentation studies using nucleosomal arrays also revealed chromatin bind- ing of the SETdomain (data not shown). We conclude that the TRX SET domain is a histone-binding module that mediates association with chromatin. The TRX SET domain recognizes the N-terminal histone tails Histone tail domains are the protruding, flexible parts of the histones that mediate contacts between adjacent nucleosomes and interact with chromatin-associated proteins (Kingston and Narlikar 1999; Strahl and Allis 2000; Turner 2000; Jenuwein 2001). The unstructured histone tails are much more sensitive to digestion by trypsin than the globular domains and can be selectively removed by limited trypsinization (Fig. 2A). The fraction of trypsinized histones shown in lane 4 was used in a GSTpull-down experiment to test the role of the tail Figure 1. The TRX SET domain binds histones and nucleo- domains in SETdomain binding (Fig. 2B). In contrast to somes. (A) Schematic representation of the domain structure of intact histones, the SETdomain failed to recognize the TRX. Ignoring other conserved regions, only the PHD fingers, tailless histones. Thus, the histone tails appear to be and the C-terminal SETdomain are indicated. ( B) The TRX SET critical for binding by the TRX SET domain. To investi- domain interacts preferentially with the H3–H4 tetramer. GST gate whether they might also be sufficient, we assayed Drosophila and GST–SET domain fusion proteins were immobilized on glu- SETdomain binding to histone tail GST-fu- tathione-Sepharose and incubated with Drosophila core his- sion proteins (Georgel et al. 1997). Figure 2C shows mod- tones. Unbound proteins and first wash fractions were TCA est binding to all the histone tails with a preference for precipitated. Protein complexes were resolved by 15% SDS- the H3 tail domain, whereas binding to the H4 tail was PAGE and visualized by Coomassie staining. Input corresponds very weak. Similar results were obtained using GST– to 30% of the material used in the binding reactions. (C)The yeast histone tail fusion proteins (data not shown). TRX SET domain binds to a histone-affinity matrix. BSA–Seph- As an additional approach to identify its target(s), we arose control beads (lane 2) and histone–Sepharose beads (lane 3) performed a far-Western analysis of SETdomain binding Drosophila were incubated with radiolabeled SETdomain. Protein com- to purified endogenous histones and recom- Xenopus plexes were resolved by 15% SDS-PAGE and bound proteins binant, bacterially expressed histones (Luger et were detected by autoradiography. Lane 1 represents 5% of the al. 1999). Histones were separated by SDS-PAGE and input. (D) TRX protein interacts with immobilized histones. transferred to a nitrocellulose membrane that was BSA–Sepharose control beads (lane 2) and histone–Sepharose probed with radiolabeled TRX SET domain. Autoradiog- beads (lane 3) were incubated with Drosophila nuclear extracts. raphy revealed strong binding to the endogenous histone Bound proteins were resolved by 7.5% SDS-PAGE, transferred H3 but only weak binding to the other endogenous his- to nitrocellulose, and probed with an ␣-TRX antibody. Lane 1 tones (Fig. 2D). The recombinant histone H3 (rH3) is also represents 5% of the input. (E) The TRX SET domain binds recognized efficiently; but in addition, rH2A is also mononucleosomes. In vitro assembled mononucleosomes were bound and weaker interactions with rH2B and rH4 were incubated with either no protein (lane 1) or with increasing detected.
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