N terminus of Swr1 binds to histone H2AZ and provides a platform for subunit assembly in the chromatin remodeling complex - PubMed (original) (raw)

N terminus of Swr1 binds to histone H2AZ and provides a platform for subunit assembly in the chromatin remodeling complex

Wei-Hua Wu et al. J Biol Chem. 2009.

Abstract

Variant histone H2AZ-containing nucleosomes are involved in the regulation of gene expression. In Saccharomyces cerevisiae, chromatin deposition of histone H2AZ is mediated by the fourteen-subunit SWR1 complex, which catalyzes ATP-dependent exchange of nucleosomal histone H2A for H2AZ. Previous work defined the role of seven SWR1 subunits (Swr1 ATPase, Swc2, Swc3, Arp6, Swc5, Yaf9, and Swc6) in maintaining complex integrity and H2AZ histone replacement activity. Here we examined the function of three additional SWR1 subunits, bromodomain containing Bdf1, actin-related protein Arp4 and Swc7, by analyzing affinity-purified mutant SWR1 complexes. We observed that depletion of Arp4 (arp4-td) substantially impaired the association of Bdf1, Yaf9, and Swc4. In contrast, loss of either Bdf1 or Swc7 had minimal effects on overall complex integrity. Furthermore, the basic H2AZ histone replacement activity of SWR1 in vitro required Arp4, but not Bdf1 or Swc7. Thus, three out of fourteen SWR1 subunits, Bdf1, Swc7, and previously noted Swc3, appear to have roles auxiliary to the basic histone replacement activity. The N-terminal region of the Swr1 ATPase subunit is necessary and sufficient to direct association of Bdf1 and Swc7, as well as Arp4, Act1, Yaf9 and Swc4. This same region contains an additional H2AZ-H2B specific binding site, distinct from the previously identified Swc2 subunit. These findings suggest that one SWR1 enzyme might be capable of binding two H2AZ-H2B dimers, and provide further insight on the hierarchy and interdependency of molecular interactions within the SWR1 complex.

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Figures

FIGURE 1.

FIGURE 1.

Association of Bdf1 and Swc7 is dependent on the N terminus of Swr1. A, SDS-PAGE and Western blotting analysis of Bdf1 and Swc7 in partial SWR1 complexes purified from wild-type and mutant swr1 strains as indicated. PVDF membrane strips corresponding to subunit(s) were probed with antibodies against Bdf1 and Swc7. The schematic diagram of Swr1 indicates subunit interaction domains of Swr1 and fragments used in this study:N1 (residues 1–210), N2 (residues 278–681),Insert (residues 1002–1221), Swr1 (residues 1–681). SWR1(ΔN1), SWR1(ΔN2), and SWR1(ΔInsert) are mutant SWR1 complexes isolated from cells lacking the N1, N2, and Insert regions of the Swr1 ATPase subunit, respectively (32). B, SDS-PAGE and Western blotting analysis of Swr1, Bdf1, Arp4, Swc4, Yaf9, and Swc7 in SWR1 complexes isolated from wild-type, _bdf1_Δ and_swc7_Δ strains. PVDF membranes were probed with anti-Flag (to reveal Swr1), and antibodies against individual SWR1 subunits as indicated.C, SDS-PAGE (4–20% gel) and Western blotting analysis of Yaf9, Bdf1, and Swc7 in SWR1 complexes isolated from wild-type and_yaf9_Δ cells. PVDF membranes were probed with antibodies against Yaf9, Bdf1, and Swc7, respectively.

FIGURE 2.

FIGURE 2.

Arp4 is required for association of several components of the SWR1 complex. A, SDS-PAGE and Western blotting analysis of SWR1 subunits in SWR1 complexes purified from wild-type and arp4-td strains as indicated. Numbers in parentheses indicate subunit abundance in arp4-td strains relative to WT, normalized to the levels of the Swr1-Flag subunit. One asterisk indicates a low level of 10–30% abundance. Two asterisks indicate a moderate level of 70–80% abundance. The open circle indicates a nonspecific signal by anti-Arp4. Level of arp4-td was also compared at 25 and 37 °C in the arp4-td strain using anti-Arp4. A trace amount of arp4-td was detectable at 37 °C when overexposed. B, SDS-PAGE and Western blotting analysis of Yaf9 and Arp4 in SWR1 complexes purified from wild type,_yaf9_Δ, and arp4-td strains as indicated.

FIGURE 3.

FIGURE 3.

Arp4 but not Bdf1 or Swc7 is required for basic H2AZ replacement in vitro. SDS-PAGE and Western blotting showing H2AZ-Flag transfer from solution to immobilized, conventional nucleosome arrays (18). Equivalent amounts of SWR1 complexes measured by the Swr1 subunit were used in the reaction. The relative H2AZ histone replacement activity is determined by dividing the ratio of H2AZ signal from ATP (+) lane to the signal from ATP (–) lane from mutant SWR1 complexes by the same ratio obtained from wild-type SWR1 complexes. A, Bdf1 and Swc7 are not essential for functional H2AZ replacement in vitro. The relative H2AZ replacement activity is an average of three independent experiments. B, Arp4 is required for functional H2AZ replacement.

FIGURE 4.

FIGURE 4.

Subunit association in the SWR1(1–681) complex. Complexes were normalized to the Flag-tagged Swr1 subunit by Western blotting. A, N-terminal region of Swr1 (Swr1(1–681)) is sufficient for association of Bdf1, Arp4, Swc4, Act1, Yaf9, and Swc7 but not Swc5. SDS-PAGE and Western blotting analysis of Bdf1, Arp4, Swc4, Act1, Swc5, Yaf9, and Swc7 abundance in the wild-type SWR1 and SWR1(1–681) complexes. Association of Swc5 was also examined in SWR1(1–681) complex purified using a less stringent condition (0.3

m

KCl). Little or no Swc5 was detected (data not shown). B, subunit composition of SWR1(1–681) complexes purified from wild-type, _swc7_Δ, and _yaf9_Δ strains. The numbers (%) are determined by measuring protein level of each subunit in mutant relative to the level in wild type. C, association map of SWR1(1–681) components.

FIGURE 5.

FIGURE 5.

H2AZ binding to the SWR1(1–681) or SWR1(ΔN2) complexes. Complexes were normalized to the Swr1 subunit by Western blotting using anti-Swr1. The presence of H2AZ was detected using anti-H2AZ. A, H2AZ binding of SWR1(1–681) and SWR1(ΔN2) complexes. SDS-PAGE (14% gel) and Western blotting analysis of H2AZ pull-down by SWR1(1–681) or SWR1(ΔN2) complexes at the 0.2 or 0.3

m

KCl condition.B, subunits required for H2AZ binding in partial SWR1(1–681) and SWR1(ΔN2) complexes. SDS-PAGE (14% gel) and Western blotting analysis of H2AZ pull-down by SWR1(1–681) and SWR1(ΔN2) complexes isolated from wild-type and subunit deletion strains at the 0.2 or 0.3

m

KCl condition.

FIGURE 6.

FIGURE 6.

Bacterially expressed Swr1(1–681) binds to H2AZ-H2B in vitro. A, binding of recombinant H2AZ-H2B or H2A-H2B dimers to immobilized, His6-tagged Swr1(1–681) was assayed by Western blotting using anti-H2B. B, binding of recombinant H2AZ-H2B dimers to immobilized, bacterially expressed Swr1(1–681), Swr1(371–678), and Swc7 was determined by Western blotting using anti-H2B.

FIGURE 7.

FIGURE 7.

SWR1 subunit association map. One-headed arrows indicate that the association of one subunit with the complex requires the subunit to which the arrow points. Two-headed arrows indicate that the associations are interdependent. Note: Arp4 and Act1 have been shown to bind to the HSA domain (residues 340–411) of Swr1 (42). In the NuA4 complex, with which SWR1 shares the Swc4, Yaf9, Arp4, and Act1 subunits, the association of Yaf9 requires the C-terminal region of Swc4 (51).

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References

    1. Kornberg, R. D., and Lorch, Y. (1999) Cell 98 285–294 - PubMed
    1. Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., and Richmond, T. J. (1997) Nature 389 251–260 - PubMed
    1. Fischle, W., Wang, Y., and Allis, C. D. (2003) Curr. Opin. Cell Biol. 15 172–183 - PubMed
    1. Gangaraju, V. K., and Bartholomew, B. (2007) Mutat. Res. 618 3–17 - PMC - PubMed
    1. Redon, C., Pilch, D., Rogakou, E., Sedelnikova, O., Newrock, K., and Bonner, W. (2002) Curr. Opin. Genet. Dev. 12 162–169 - PubMed

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