Roles of common subunits within distinct multisubunit complexes Yu Nakabayashia, Satoshi Kawashimab, Takemi Enomotoc, Masayuki Sekia,1, and Masami Horikoshid,1 a Department of Biochemistry, Tohoku Pharmaceutical University, Sendai, Miyagi 981-8558, Japan; bMolecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan; cResearch Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, Nishitokyo, Tokyo 202-8585, Japan; and dLaboratory of Developmental Biology, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan
Edited by Fred M. Winston, Harvard Medical School, Boston, MA, and approved November 27, 2013 (received for review August 31, 2013)
FALC
|
histone variant
|
modification
|
chromatin
M
|
epigenetics
ore than half a century has passed since molecular biology revolutionized the life sciences. However, there remain no general strategies to differentiate between the in vivo role of a particular subunit within one multisubunit protein complex from its roles in other complexes. Such subunits are referred to as common subunits. A protein can function as a single component or as a component of a multisubunit protein complex (Fig. 1A). The functional role of a protein as a single component or in a multisubunit protein complex in the cell can be ascertained via gene knockout or knockdown of the corresponding mRNA. However, some proteins participate in several complexes and processes; therefore, although such studies would indicate the net phenotype induced by removal of the protein, they would not discern the specific role(s) of the protein in each complex. Thus, if a protein functions in more than one biological process (i.e., it is a common subunit in several multisubunit protein complexes), its role in a specific biological process or complex cannot be determined easily. Such common subunits are found in a variety of multisubunit protein complexes (1). Four examples of multisubunit protein complexes that contain a common subunit include (i) eukaryotic RNA polymerases I, II, and III, which all share several Rpb subunits (2); (ii) general transcription initiation factor complexes, SL1, TFIID, and TFIIIB, which share the TBP (TATA box-binding protein) subunit (3); (iii) the histone acetyltransferase NuA4 complex and the histone deacetylase Rpd3 complex, which share the Eaf3 subunit (4); and (iv) the NuA4 complex and the chromatin remodeling complexes SWR1 and INO80, which share the Act1 and Arp4 subunits (5) (Fig. 1B). The specific roles of Rpb, TBP, Eaf3, Act1, and Arp4 in each multisubunit protein complex remain elusive largely because in vivo analyses of common subunits in multisubunit protein complexes have been hampered by weaknesses in current experimental approaches. Here, we developed a strategy that permits the in vivo function of a subunit in one multisubunit protein complex to be distinguished from its function(s) in other complexes. The physiological role of a common subunit in a specific multisubunit protein www.pnas.org/cgi/doi/10.1073/pnas.1316433111
complex can be elucidated if the subunit can be made specific to one complex. To accomplish this, the common subunit could be covalently linked to another subunit that is specific to one complex (Fig. 1C), followed by the introduction of a mutation into the linked common subunit (Fig. 1 D and E). We call this strategy Functional Analysis of Linker-mediated Complex (FALC). A nucleosome, the basic structural unit of chromatin (6), is comprised of eight histones, typically two histone H2A/H2B dimers and a histone (H3/H4)2 tetramer, and wrapped DNA (7, 8), and is the most abundant and evolutionarily conserved multisubunit protein complex in eukaryotic cell nuclei. Nucleosomes regulate a variety of DNA-mediated processes such as transcription and DNA replication (8, 9). Thus, the regulation of nucleosome structure and function in individual DNA-mediated reactions is a central subject in modern biology (7, 9, 10). Although the H2B subunit within nucleosomes remains constant, the H2A subtype changes. The H2A subtype (variant) H2A.Z is the only isoform that is conserved among all eukaryotes. Each of two H2A proteins in a nucleosome (called the A/A nucleosome) is replaced with the H2A variant Htz1 (budding yeast H2A.Z) in a stepwise manner to first yield an A/Z nucleosome containing one H2A and one Htz1 molecule and then a Z/Z nucleosome containing two Htz1 proteins (11). Thus, H2B is a common subunit in H2A-containing (A/A- and A/Z-) and Htz1-containing (A/Z- and Z/Z-) multisubunit protein complexes of nucleosomes (Fig. 2A). Analysis of crystals of H2A.Z-containing nucleosomes revealed that there are no marked structural differences compared with canonical nucleosomes (7, 12, 13). Budding yeast Htz1 is involved in gene regulation, protection against gene silencing around boundaries, DNA repair, cell cycle progression, and chromosome stability (14). Although H2A.Z is widely studied, there is no experimental approach to reveal the specific roles of H2B that is paired with H2A.Z. Significance The same protein is often a subunit of more than one multisubunit protein complex, each of which has a distinct function within cells. Mutating such a protein would cause multiple cellular defects; therefore, it is difficult to distinguish between the function of a protein in one complex from its functions in other complexes. Here, we developed a unique strategy to overcome this problem, which will help to analyze a variety of biological processes by revealing the specific roles played by such proteins within multisubunit protein complexes. Author contributions: Y.N., M.S., and M.H. designed research; Y.N. and S.K. performed research; Y.N., S.K., M.S., and M.H. analyzed data; and Y.N., T.E., M.S., and M.H. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1
To whom correspondence may be addressed. E-mail: [email protected] or [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1316433111/-/DCSupplemental.
PNAS | January 14, 2014 | vol. 111 | no. 2 | 699–704
CELL BIOLOGY
Currently, there is no method to distinguish between the roles of a subunit in one multisubunit protein complex from its roles in other complexes in vivo. This is because a mutation in a common subunit will affect all complexes containing that subunit. Here, we describe a unique method to discriminate between the functions of a common subunit in different multisubunit protein complexes. In this method, a common subunit in a multisubunit protein complex is genetically fused to a subunit that is specific to that complex and point mutated. The resulting phenotype(s) identify the specific function(s) of the subunit in that complex only. Histone H2B is a common subunit in nucleosomes containing H2A/H2B or Htz1/H2B dimers. The H2B was fused to H2A or Htz1 and point mutated. This strategy revealed that H2B has common and distinct functions in different nucleosomes. This method could be used to study common subunits in other multisubunit protein complexes.
Fig. 1. Study concept. (A) Schematic representation of a protein as a single component (W) and as a common subunit (Z), with and without a mutation, in two different multisubunit complexes, each with a specific subunit (X or Y). (B) Examples of common subunits in RNA polymerases, general transcription initiation factor complexes, the NuA4 histone acetyltransferase and the Rpd3 deacetylase, and the NuA4, SWR1, and INO80 chromatin remodeling complexes. The red zone depicts common subunits and the other colored zones depict specific subunits. (C) Schematic representation of a common subunit (Z) fused with a specific subunit (X or Y). (D) Introduction of a mutation into a common subunit (Z) of each fusion. (E) Schematic representation of how mutating a common subunit (Z) fused with a specific subunit (X or Y) in a complex can allow complex-specific phenotypes to be evaluated. (−) indicates that a mutation that specifically affects one complex cannot be identified.
Results H2B-H2A and H2B-Htz1 Fusions Are Functional in Vivo. To elucidate
the individual roles of the common H2B subunit in H2A- and Htz1-containing nucleosomes (Figs. 1E and 2A), we created fusion proteins consisting of a C-terminal portion of H2B fused in frame to the N terminus of full-length H2A or Htz1 (12, 13) (Fig. 2 B and C). Although these fusions cannot be used to elucidate the potential function(s) of junctional regions, the H2A N-terminal tail is reportedly dispensable (15). The H2B-H2A and H2B-Htz1 fusions were extensively characterized to determine whether they were functional in yeast. To test whether the H2B-H2A fusion was functional, this fusion was expressed in hta and htb double KO (htaΔ/htbΔ) cells (16, 17), which lack genomic H2A (HTA1/HTA2) and H2B (HTB1/HTB2) genes but carry nonfused HTA1 and HTB1 genes on a plasmid containing the URA3 marker, and cell viability was assessed. Because the URA3 plasmid carrying the HTA1 and HTB1 genes is lost from cells grown in the presence of 5-fluoroorotic acid (5FOA), htaΔ/htbΔ cells harboring nonfused HTA1 and HTB1 genes on the non-URA3 plasmid were viable, whereas cells harboring an empty plasmid were not (Fig. 2D, rows 1 and 2). Under the same experimental conditions, the H2B-H2A fusion rescued htaΔ/htbΔ cells from lethality, indicating that the H2BH2A fusion was functional (Fig. 2D, row 3). The slightly reduced growth of cells expressing the H2B-H2A fusion (Fig. 2D, row 3) seemed to be due to degradation of monomeric Htz1 (Fig. S1). htz1Δ cells grow slowly (18), and this phenotype was mimicked by cells expressing the H2B-H2A fusion. To evaluate the functionality of the H2B-Htz1 fusion (Fig. 2E and Fig. S2A), we used the hydroxyurea (HU)- and methyl methanesulfonate (MMS)-sensitive phenotypes of htz1Δ cells (19) (Fig. 2F, row 2). Whereas htz1Δ cells (Fig. 2F, row 2) and htaΔ/htbΔ/htz1Δ cells expressing only the H2B-H2A fusion (row 3) were sensitive to HU and MMS, htaΔ/htbΔ/htz1Δ cells expressing both H2B-H2A and H2B-Htz1 fusions were not, similar to WT cells (rows 1 and 4). The ability of the H2B-H2A and H2B-Htz1 fusions to rescue the drug-sensitive phenotype of htaΔ/htbΔ/htz1Δ cells indicates that both fusions are functional. 700 | www.pnas.org/cgi/doi/10.1073/pnas.1316433111
Both nonfused H2A and Htz1 (Fig. 2G, Top and Middle, lane 1) and only nonfused H2A (lane 2) were detected via immunoblot analysis at a molecular weight less than 20 kDa in samples from WT and htz1Δ cells, respectively. H2B-H2A in H2B-H2A– expressing cells, and H2B-H2A and H2B-Htz1 in H2B-H2A– and H2B-Htz1–expressing cells, were detected at ∼30 kDa (Fig. 2G, Top and Middle, lanes 3 and 4). Notably, another band was detected at a molecular weight greater than 30 kDa in samples from cells expressing the H2B-H2A fusion (Fig. 2G, Top, lanes 3 and 4) and the H2B-Htz1 fusion (Middle, lane 4), suggesting that monoubiquitination had occurred on the H2B-K123 residue of both fusions (20) (see Monoubiquitination Occurs on Both the H2B-H2A and H2B-Htz1 Fusions). An examination of nucleosome ladders produced following digestion with MNase for increasing amounts of time (6, 21) provides information about chromatin structure. This MNase assay indicated that the chromatin of htz1Δ cells, htaΔ/htbΔ/ htz1Δ cells expressing only the H2B-H2A fusion, and htaΔ/htbΔ/ htz1Δ cells expressing both H2B-H2A and H2B-Htz1 fusions (Fig. 2H, columns 2–4) are similar to the chromatin of WT cells (column 1). ChIP was performed to determine whether the H2B-Htz1 fusion could replace the H2B-H2A fusion at several promoters and pericentromeric regions of chromosome III (CEN3) at which Htz1 is known to localize (11, 22) (Fig. 2I and Fig. S2 B and C). Nonfused Htz1 was detected at promoters and near to the CEN3-left and -right regions in WT cells (Fig. 2J and Fig. S2 D and E, lane 1). As expected, the Htz1 signal was abolished in htz1Δ cells (Fig. 2J and Fig. S2 D and E, lane 2) and in htaΔ/ htbΔ/htz1Δ cells expressing only the H2B-H2A fusion (lane 3). The ChIP signals of the H2B-Htz1 fusion and nonfused Htz1 were much lower around the ORFs of the LSB5 and GID7 genes than at their corresponding promoters (Fig. S2F), as previously reported (11). Therefore, the H2B-Htz1 fusion seems to specifically localize to chromatin, similar to nonfused Htz1. Importantly, the H2B-Htz1 fusion localized at promoters and near to CEN3left and -right regions (Fig. 2J and Fig. S2 D and E, lane 4), albeit at a much lower level than nonfused Htz1 in WT cells. Although Nakabayashi et al.
Contribution of the H2B-L109 Residue in Fused H2B-H2A and H2B-Htz1 to Cell Viability. To determine the functional roles of H2B in
this difference could be explained by a partial replacement of the function of H2B-H2A by the H2B-Htz1 fusion on chromatin, it could also be due to a technical artifact. Namely, the immunoprecipitation of chromatin containing the H2B-Htz1 fusion by the anti-Htz1 antibody could be less efficient than that of chromatin containing nonfused Htz1. Irrespective of this, these observations indicate that both H2B-H2A and H2B-Htz1 fusions are incorporated into chromatin. Taking all of the data together (cell viability, drug sensitivity, protein expression, MNase assay, and ChIP assay), the H2B-H2A and H2B-Htz1 fusions seem to be functional in cells. Nakabayashi et al.
CELL BIOLOGY
Fig. 2. Functional analysis of H2B-H2A and H2B-Htz1 fusions. (A) Schematic representation of nonfused and fused H2B-H2A– and H2B-Htz1–containing nucleosomes. (B) Schematic representation of the constructs used to generate fused H2B-H2A (Upper) and H2B-Htz1 (Lower). A C-terminal portion of H2B was fused in frame to the N terminus of full-length H2A or Htz1. PHTB1, HTB1 promoter. PHTZ1, HTZ1 promoter. (C) Nucleosome structure and enlarged view of the C terminus of H2B and the N terminus of H2A or Htz1 (PDB ID: 1ID3). (D) Viability of cells expressing fused H2B-H2A. Cells grew slightly poorer on (+) 5-FOA plates than on (−) 5-FOA plates because 5-FOA can kill cells with the URA3 gene. (E) Schematic representation of nucleosomes in the strains (htaΔ/htbΔ/htz1Δ background) used for experiments F–H and J. (1) Cells expressing nonfused H2A, H2B, and Htz1 (WT); (2) cells expressing nonfused H2A and H2B, but not Htz1 (htz1-deleted cells); (3) cells expressing only the H2B-H2A fusion; and (4) cells expressing both the H2BH2A and H2B-Htz1 fusions. (F) Drug sensitivity analysis using HU or MMS. (G) Immunoblot analysis of H2B-H2A and H2B-Htz1 fusions. Histone H4 was used as a loading control. (H) MNase analysis of chromatin with digestion for increasing amounts of time. M, 100-bp marker. (I) Schematic representation of the pericentromeric region of CEN3 and the locations of primer sets used for ChIP analysis. (J) ChIP analysis of Htz1 and the H2B-Htz1 fusion. Total Htz1 occupancy in WT cells was used for normalization. Error bars represent SD (n = 3).
H2A/H2B and Htz1/H2B dimers, we point-mutated H2B in the H2B-H2A and H2B-Htz1 fusions (Fig. 3A and Fig. S3A). Thirtyeight phenotypic residues of H2B have been identified by previous screening of the histone-GLibrary (nonfused H2B point mutant library) (23–25). We individually mutated 3 (H2B-D71, -L109, or -K123) of 38 residues in the H2B-H2A and H2B-Htz1 fusions. Expression of H2B-L109A in nonfused H2B-expressing cells caused cell lethality, whereas expression of H2B-D71A or -K123A did not (23, 24). However, the introduction of the latter two mutants into nonfused H2B-expressing cells led to cells becoming strongly sensitive to HU, MMS, benomyl (a microtubule-depolymerizing drug), and/or 6-azauracil (6AU) (23–25). We analyzed the effect of introducing the H2B-L109A mutation into the H2B-H2A and H2B-Htz1 fusions on cell viability. Nonfused HTA1 and HTB1 genes on the URA3 plasmid, which enabled htaΔ/htbΔ/htz1Δ cells to survive, would be lost during culture on plates containing 5-FOA. In the presence of 5-FOA, htaΔ/htbΔ/htz1Δ cells expressing nonfused H2A, H2B, and Htz1, or both H2B-H2A and H2B-Htz1 fusions from non-URA3 plasmids, were viable (Fig. 3B, rows 1 and 3, and Fig. S3B, lanes 1 and 3). By contrast, cells harboring nonfused H2A, H2B(L109A), and Htz1 were not viable (Fig. 3B, row 2, and Fig. S3B, lane 2), as expected. Lethality was also observed under 5-FOA conditions in cells expressing both mutant fusions [i.e., H2B(L109A)-H2A and H2B(L109A)-Htz1; Fig. 3B, row 4, and Fig. S3B, lane 4]. H2A and Htz1 are indispensable and dispensable for cell viability, respectively (17, 18). Therefore, cells expressing both H2B(L109A)-H2A and H2B(WT)-Htz1 fusions or both H2B (WT)-H2A and H2B(L109A)-Htz1 fusions should die and grow proficiently, respectively. As expected, cells expressing both H2B (WT)-H2A and H2B(L109A)-Htz1 fusions grew well in the presence of 5-FOA (Fig. 3B, row 6, and Fig. S3B, lane 6), similar to cells expressing both H2B(WT)-H2A and H2B(WT)-Htz1 fusions (Fig. 3B, row 3, and Fig. S3B, lane 3). Surprisingly, cells
Fig. 3. Functional analysis of H2B-L109 in H2B-H2A and H2B-Htz1 fusions. (A) Schematic representation of nucleosomes containing nonfused or fused H2B point mutants. The cell strains examined were as follows: (1) nonfused WT H2B; (2) nonfused mutant (mut) H2B; (3) fused H2B-H2A/H2B-Htz1; (4) fused H2B(mut)-H2A/H2B(mut)-Htz1; (5) fused H2B(mut)-H2A/H2B(WT)Htz1; and (6) fused H2B(WT)-H2A/H2B(mut)-Htz1. (B) Lethality analysis of cells expressing fused H2B-H2A and/or H2B-Htz1 with the H2B-L109A point mutation. (C) Interacting region between H2B-L109, H2A-Y58 (or Htz1-Y65), and H2A-E62 (or Htz1-E69) residues in the nucleosome structure.
PNAS | January 14, 2014 | vol. 111 | no. 2 | 701
expressing both H2B(L109A)-H2A and H2B(WT)-Htz1 fusions grew in the presence of 5-FOA (Fig. 3B, row 5, and Fig. S3B, lane 5), although to a lesser extent than cells expressing both H2B (WT)-H2A and H2B(L109A)-Htz1 fusions (Fig. 3B, row 6, and Fig. S3B, lane 6). We confirmed that two independent clones isolated from cells expressing both H2B(L109A)-H2A and H2B (WT)-Htz1 fusions in the presence of 5-FOA grew extremely slowly (Fig. S3 C–E). These results suggest that H2B-L109 promotes cell viability not only through H2A- but also via Htz1-containing nucleosomes (Table 1). H2B-L109 physically interacts with the H2A-Y58, H2A-E62, Htz1-Y65, and Htz1-E69 residues, which are exposed to the nucleosome surface (23, 24, 26). Mutation of the H2A-Y58 and -E62 residues in nonfused H2A- and H2B-expressing cells caused cell death (23, 24), whereas mutation of the Htz1-Y65 and -E69 residues in nonfused Htz1- and H2B-expressing cells resulted in HU/MMS/benomyl sensitivities (26). Thus, the proximity and functional relationship of H2B-L109 to H2A-Y58, H2A-E62, Htz1-Y65, and Htz1-E69 might explain why H2BL109 is functionally important in both the H2B-H2A and H2BHtz1 fusions. H2B-D71 Residue Plays a Specific Role in the H2B-Htz1 Fusion. We next mutated another H2B residue, H2B-D71 (Fig. 4A and Figs. S4A and S5A), to investigate its effects on cell growth and sensitivity to HU, MMS, and benomyl (24, 25). Cells expressing nonfused H2B-D71A grew slower than WT cells (Fig. 4B, rows 1 and 2). Cells expressing both H2B(D71A)-H2A and H2B(WT)Htz1 fusions grew well (Fig. 4B, row 5), whereas cells expressing both H2B(WT)-H2A and H2B(D71A)-Htz1 fusions (row 6) grew poorer than cells expressing both WT fusions (row 3). Cells expressing both H2B-H2A and H2B-Htz1 fusions in which the H2B-D71 residue was mutated exhibited different HU, MMS, and benomyl sensitivities (Fig. 4B, rows 3–6). Quantitative analysis of benomyl sensitivity confirmed that the H2B-D71A mutation in the H2B-Htz1 fusion conferred benomyl sensitivity, whereas the H2B-D71A mutation in the H2B-H2A fusion did not (Fig. 4C and Table 1). H2B-D71A Mutation Abolishes the Localization of Htz1 on Chromatin Near to CEN3 and at Promoters. H2B-D71 is located on the H2B
α-helix 2 region and interacts with the H4-L97 and -Y98 residues in the H4 C-terminal tail region (7, 13, 14, 25). The H4-L97 and Table 1. Effects of H2B mutation on the H2B-H2A and H2B-Htz1 fusions Effects of mutation on H2B mutation D71A
L109A K123A
Analyzed phenotype
H2B-H2A
H2B-Htz1
Growth HU sensitivity MMS sensitivity Benomyl sensitivity H2A chromatin localization Htz1 chromatin localization Cell viability* Growth Growth HU sensitivity MMS sensitivity 6AU sensitivity H2B monoubiquitination
− + + − + − +* ++ − ++ ++ ++ ++
++ + + ++ − ++ +* − − − − − ++
++, significantly affected; +, slightly affected; −, not affected. *Cell viability is defined as viable (+) by the presence of any growing cells in spot assay and as lethal (−) by the absence of those cells, respectively.
702 | www.pnas.org/cgi/doi/10.1073/pnas.1316433111
Fig. 4. Functional analysis of H2B-D71 in H2B-H2A and H2B-Htz1 fusions. (A) Schematic representation of nucleosomes containing nonfused or fused H2B-D71A point mutants. The cell strains examined were as follows: (1) nonfused WT H2B; (2) nonfused H2B(D71A); (3) fused H2B-H2A/H2B-Htz1; (4) fused H2B(D71A)-H2A/H2B(D71A)-Htz1; (5) fused H2B(D71A)-H2A/H2B(WT)Htz1; and (6) fused H2B(WT)-H2A/H2B(D71A)-Htz1. (B) Drug sensitivity analysis of fused H2B-H2A and/or H2B-Htz1 with the H2B-D71A point mutation. (C) Yeast survival analysis after exposure to benomyl for a short amount of time. The survival rate of each strain was calculated by dividing the total number of colonies at each time point by the number of colonies at t = 0. (D) Enlarged view of the interacting region between histone H2B α2, the H4 C-terminal tail, and the H2A or Htz1 C-terminal tail in the nucleosome structure. (E and F) ChIP analysis of Htz1 or the H2B-Htz1 fusion (E) and H2A or the H2B-H2A fusion (F) in the indicated cells. Total Htz1 and H2A occupancies in WT cells were used for normalization. Error bars represent SD (n = 3).
-Y98 residues are located adjacent to a short β-strand of the H2A and Htz1 C-terminal tail regions (Fig. 4D). H4-L97, which faces H2B-D71, confers benomyl sensitivity when mutated and is necessary for the localization of Htz1 at pericentromeric regions of CEN3 (25), raising the possibility that H2B-D71 is also required for the localization of Htz1 around CEN3. To test this possibility, we examined the localization of Htz1 around CEN3 in cells expressing H2B(D71A)-H2A and/or H2B (D71A)-Htz1 fusions (Fig. 4E and Table 1). ChIP analysis indicated that the Htz1 level of the fusion near to the CEN3 locus was the same in cells expressing the H2B(D71A)-H2A fusion as in cells expressing the H2B(WT)-H2A fusion (Fig. 4E, lanes 3 and 5). By contrast, the level of the H2B(D71A)-Htz1 fusion near to the CEN3 locus differed between cells expressing H2B (D71A)-H2A and cells expressing H2B(WT)-H2A (lanes 4 and 6). Consistent with this, the introduction of the H2B-D71A point mutation into nonfused H2B dramatically reduced the Htz1 ChIP signal near to the CEN3 locus compared with the signal in WT cells (Fig. 4E, lanes 1 and 2). The same results were obtained Nakabayashi et al.
H2B-K123 Predominantly Functions in the H2B-H2A Fusion. We next examined the effect of the introduction of the H2B-K123A mutation into the H2B-H2A and H2B-Htz1 fusions on the sensitivity of cells to HU, MMS, and/or 6AU (Fig. 5 A and B). 6AU disturbs the balance of intracellular nucleotide concentrations and blocks transcription elongation. Cells expressing nonfused H2B-K123A were sensitive to HU, MMS, and 6AU (Fig. 5B, rows 1 and 2), as were cells expressing both H2B(K123A)-H2A and H2B(K123A)-Htz1 fusions (Fig. 5B, rows 3 and 4). Under the same conditions, cells expressing both H2B(K123A)-H2A and H2B(WT)-Htz1 fusions, but not H2B(WT)-H2A and H2B (K123A)-Htz1 fusions, were sensitive to HU, MMS, and 6AU (Fig. 5B, rows 5 and 6). Because the H2B-K123 residue could be important in the H2B-H2A fusion, but not in the H2B-Htz1 fusion, the function of H2B-K123 in the H2A/H2B dimer seems to be different from its function in the Htz1/H2B dimer. Monoubiquitination Occurs on Both the H2B-H2A and H2B-Htz1 Fusions. Among the three H2B residues (H2B-D71, -L109, and
-K123) analyzed in this study, only H2B-K123 is chemically modifiable. Specifically, H2B-K123 can be monoubiquitinated (20), a modification that is important in transcription elongation (27, 28), DNA replication (29), DNA repair (30), and di-/trimethylation of lysine 4 of histone H3 (H3-K4me2/3) in the nucleosome (31, 32). To investigate whether monoubiquitination of H2B-K123 occurs in H2A- and Htz1-containing nucleosomes, we compared the state of H2B monoubiquitination in cells expressing nonmutated fusions to that in cells expressing the H2B(K123A)H2A and/or H2B(K123A)-Htz1 fusions (Fig. 5C). The status of H2B-K123 monoubiquitination was indirectly evaluated by the appearance of a band migrating slightly higher than 30 kDa, the size at which the H2B-H2A and H2B-Htz1 fusions were detected using anti-H2A and anti-Htz1 antibodies, respectively (Fig. 2G). Nakabayashi et al.
Fig. 5. Functional analysis of H2B-K123 in H2B-H2A and H2B-Htz1 fusions. (A) Schematic representation of nucleosomes containing nonfused or fused H2B-K123A point mutants. The cell strains examined were as follows: (1) nonfused WT H2B; (2) nonfused H2B(K123A); (3) fused H2B-H2A/H2B-Htz1; (4) fused H2B(K123A)-H2A/H2B(K123A)-Htz1; (5) fused H2B(K123A)-H2A/H2B (WT)-Htz1; and (6) fused H2B(WT)-H2A/H2B(K123A)-Htz1. (B) Drug sensitivity analysis of fused H2B-H2A and/or H2B-Htz1 with the H2B-K123A point mutation. (C) Immunoblot analysis of monoubiquitination of H2B-K123 in H2BH2A and H2B-Htz1. Lanes labeled 1 show a series of fourfold dilutions of samples from WT cells. Histone H4 was used as a loading control.
Higher molecular weight bands of ∼37 kDa were detected in cells expressing the H2B-H2A fusion (Fig. 5C, Top, lanes 3 and 6) and the H2B-Htz1 fusion (Middle, lanes 3 and 5). These bands were not detected in cells expressing the H2B(K123A)-H2A fusion (Fig. 5C, Top, lanes 4 and 5) and the H2B(K123A)-Htz1 fusion (Middle, lanes 4 and 6). These observations suggest that H2B is monoubiquitinated at K123, irrespective of whether H2B is paired with H2A or Htz1. The level of monoubiquitinated H2B-Htz1 fusion was much higher in cells expressing the H2B(K123A)-H2A fusion (Fig. 5C, Middle, lane 5) than in cells expressing the H2B(WT)-H2A fusion (lane 3), suggesting that the lack of monoubiquitination in the H2B-H2A fusion (Fig. 5C, Top, lane 5) leads to accumulation of the monoubiquitinated H2B-Htz1 fusion (Middle, lane 5) in cells. The FALC strategy successfully determines whether H2B-K123 is monoubiquitinated in the H2B-H2A fusion and/or the H2B-Htz1 fusion; therefore, a variety of chemical modifications of residues (such as S10, K11, and K16) (33–35) in the tail region of H2B could be tested to discern other roles played by H2B depending on whether it is paired with H2A or Htz1. Discussion In this study, we developed the FALC strategy to overcome the difficulties associated with functional analyses of proteins that are subunits of more than one multisubunit complex (Fig. 1). We used H2B as a common subunit, and H2A- and Htz1-containing nucleosomes as multisubunit complexes (Figs. 2–5). To implement the FALC strategy, we made functional H2B-H2A and H2B-Htz1 fusions (Fig. 2) and analyzed the phenotypes following point mutations of three H2B residues (H2B-D71, -L109, and -K123) in nonfused H2B, the H2B-H2A fusion, and the H2B-Htz1 fusion (Figs. 3–5 and Table 1). This innovative FALC strategy led to unique functional information about H2A, Htz1, and H2B, which could not have been revealed by analysis of combinations of nonfused proteins. The FALC strategy revealed that each residue has a distinct role depending on which fusion it was part of. H2B-L109 in H2Acontaining nucleosomes predominantly contributes to support cell viability, as expected (Fig. 3). Unexpectedly, it was revealed that PNAS | January 14, 2014 | vol. 111 | no. 2 | 703
CELL BIOLOGY
at the promoters of the LSB5 and GID7 genes (Fig. S4B). These data indicate that the H2B-D71A mutation dramatically reduces the localization of the H2B-Htz1 fusion and nonfused Htz1 at chromatin. On the other hand, the levels of nonfused Htz1 (Fig. S5B, lane 2) and H2B(D71A)-Htz1 fusion proteins (lanes 4 and 6), and their corresponding mRNAs (Fig. S5C, lanes 2, 4, and 6), were not substantially lower than the levels of the WT proteins and transcripts (Fig. S5 B and C, lanes 1 and 3). Thus, the H2BD71A mutation likely affects some functions of the dimer, such as incorporation into chromatin, rather than the protein stability of the dimer. The ChIP signal of nonfused H2A near to the CEN3 locus and at the promoters of the LSB5 and GID7 genes in WT cells (Fig. 4F and Fig. S4C, lane 1) was slightly reduced, but not abolished, by introduction of the H2B-D71A mutation (Fig. 4F and Fig. S4C, lane 2). The level of the H2B-H2A fusion around CEN3 and at these two promoters (Fig. 4F and Fig. S4C, lanes 3) was not significantly altered by the introduction of the H2B-D71A mutation into the H2B-H2A fusion and/or the H2B-Htz1 fusion (Fig. 4F and Fig. S4C, lanes 4–6). These results suggested that the H2B-D71A mutation markedly affected Htz1-containing nucleosomes but not H2A-containing nucleosomes (Table 1). Furthermore, the introduction of a different point mutation into H2B, H2B-K123A, did not affect the localization of Htz1 localization at the CEN3 locus (Fig. S6). Thus, it is suggested that the proper localization of H2B-Htz1 at the CEN3 locus requires the H2B-D71 residue and that this residue is important for maintaining the function of Htz1-containing nucleosomes (Table 1). H2B is likely to be involved in the transport, processing, deposition, unloading, and/or recycling of Htz1/H2B dimers. H2B-D71 faces the H4-L97 residue, which is involved in the localization of Htz1 around CEN3 (25). Therefore, we prefer a model in which the H2B-D71A mutant cannot bind the (H3H4)2 tetramer or can be easily released from the nucleosome.
H2B-L109 in Htz1-containing nucleosomes also supports cell viability, although with a lesser contribution than in H2A-containing nucleosomes (Fig. 3). By contrast, cell growth and resistance to benomyl largely relied on the H2B-D71 residue of fused H2BHtz1 (Fig. 4). The localization of Htz1 at promoters and around CEN3 was abolished with the H2B-D71A mutant, but not with the H2B-K123A mutant, suggesting that the H2B-D71 and H2B-K123 residues have different structural and functional roles (Fig. 4). Although the structures of H2A- and Htz1-containing nucleosomes have been determined (7, 12, 13), the current study revealed the role of H2B-D71 in these nucleosomes by using a FALC strategy. H2B-K123 is most likely to be monoubiquitinated in both H2B-H2A and H2B-Htz1 fusions (Fig. 5). On the other hand, H2B-K123 was functional within H2A- but not within Htz1-containing nucleosomes, as shown by drug sensitivity experiments (Fig. 5). Thus, in drug sensitivity-related reactions, monoubiquitinated H2B-K123 seems to have different functions depending on whether it is paired with H2A or Htz1. It will be interesting to reveal the roles of monoubiquitination of H2BK123 in the H2B-Htz1 fusion. Although some common phenotypes resulted from the presence of the H2B(mut)-H2A fusion or the H2B(mut)-Htz1 fusion (Table 1), distinct phenotypes were also detected. The FALC strategy led to the successful determination of distinct roles for an individual residue in a common subunit, H2B, depending on whether it was part of the H2B-H2A or H2B-Htz1 fusion. Generalizing the FALC strategy to other multisubunit complexes (Fig. 1) is an interesting challenge. It is not known how to select a specific subunit that is linked to the common subunit, especially when the structural information of a multisubunit complex 1. Havugimana PC, et al. (2012) A census of human soluble protein complexes. Cell 150(5):1068–1081. 2. Vannini A, Cramer P (2012) Conservation between the RNA polymerase I, II, and III transcription initiation machineries. Mol Cell 45(4):439–446. 3. Burley SK, Roeder RG (1996) Biochemistry and structural biology of transcription factor IID (TFIID). Annu Rev Biochem 65:769–799. 4. Smith E, Shilatifard A (2010) The chromatin signaling pathway: Diverse mechanisms of recruitment of histone-modifying enzymes and varied biological outcomes. Mol Cell 40(5):689–701. 5. van Attikum H, Gasser SM (2005) The histone code at DNA breaks: A guide to repair? Nat Rev Mol Cell Biol 6(10):757–765. 6. Kornberg RD (1974) Chromatin structure: A repeating unit of histones and DNA. Science 184(4139):868–871. 7. Luger K, Mäder AW, Richmond RK, Sargent DF, Richmond TJ (1997) Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389(6648):251–260. 8. Natsume R, et al. (2007) Structure and function of the histone chaperone CIA/ASF1 complexed with histones H3 and H4. Nature 446(7133):338–341. 9. Allis CD, Jenuwein T, Reinberg D, Caparros ML (2007) Epigenetics (Cold Spring Harbor Laboratory Press, New York). 10. Akai Y, et al. (2010) Structure of the histone chaperone CIA/ASF1-double bromodomain complex linking histone modifications and site-specific histone eviction. Proc Natl Acad Sci USA 107(18):8153–8158. 11. Luk E, et al. (2010) Stepwise histone replacement by SWR1 requires dual activation with histone H2A.Z and canonical nucleosome. Cell 143(5):725–736. 12. White CL, Suto RK, Luger K (2001) Structure of the yeast nucleosome core particle reveals fundamental changes in internucleosome interactions. EMBO J 20(18): 5207–5218. 13. Suto RK, Clarkson MJ, Tremethick DJ, Luger K (2000) Crystal structure of a nucleosome core particle containing the variant histone H2A.Z. Nat Struct Biol 7(12):1121–1124. 14. Zlatanova J, Thakar A (2008) H2A.Z: View from the top. Structure 16(2):166–179. 15. Schuster T, Han M, Grunstein M (1986) Yeast histone H2A and H2B amino termini have interchangeable functions. Cell 45(3):445–451. 16. Rykowski MC, Wallis JW, Choe J, Grunstein M (1981) Histone H2B subtypes are dispensable during the yeast cell cycle. Cell 25(2):477–487. 17. Kolodrubetz D, Rykowski MC, Grunstein M (1982) Histone H2A subtypes associate interchangeably in vivo with histone H2B subtypes. Proc Natl Acad Sci USA 79(24): 7814–7818. 18. Jackson JD, Gorovsky MA (2000) Histone H2A.Z has a conserved function that is distinct from that of the major H2A sequence variants. Nucleic Acids Res 28(19):3811–3816.
704 | www.pnas.org/cgi/doi/10.1073/pnas.1316433111
is unavailable. To overcome this challenge, experimental predictions could be made by gaining topological information on common and specific subunits in multisubunit complexes via the FRET assay, electron microscopy, and protein cross-linking. Using the latter technique, information about the topology between common subunits (such as Act1 and Arp4) and specific subunits of the INO80 complex (Fig. 1B) has recently been gained (36). Introducing mutations into a protein fusion is another critical step of the FALC strategy. Comprehensive mutagenesis analysis (GLibrary strategy = GLASP + GLAMP) (23–26) of the target common subunit(s) would greatly facilitate mutagenic analyses of fused complexes, as shown in this study (Figs. 3–5 and Table 1). Thus, combining the FALC strategy with mutational analyses opens a new avenue to address the functional roles of a common subunit(s) in multiple multisubunit complexes in cells. Materials and Methods Detailed information on materials and methods in this study is provided in SI Materials and Methods. Information on materials includes the characteristics of yeast strains and construction of plasmids. Technical information on methods contains drug sensitivity assays, immunoblot analysis, Northern blot analysis, ChIP assay, chromatin digestion assay with micrococcal nuclease, survival analysis following benomyl treatment, and molecular graphics. ACKNOWLEDGMENTS. We thank L. Sato for initial ideas on linker analysis, G. Ueno for creating some of the constructs, and K. Hasegawa for administrative assistance. We also thank all members of our laboratories for fruitful discussion of this study. This work was supported by a Grant-inAid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and a Grant-in-Aid for a Japan Society for the Promotion of Science Research Fellow.
19. Mizuguchi G, et al. (2004) ATP-driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin remodeling complex. Science 303(5656):343–348. 20. Robzyk K, Recht J, Osley MA (2000) Rad6-dependent ubiquitination of histone H2B in yeast. Science 287(5452):501–504. 21. Kent NA, Mellor J (1995) Chromatin structure snap-shots: Rapid nuclease digestion of chromatin in yeast. Nucleic Acids Res 23(18):3786–3787. 22. Krogan NJ, et al. (2004) Regulation of chromosome stability by the histone H2A variant Htz1, the Swr1 chromatin remodeling complex, and the histone acetyltransferase NuA4. Proc Natl Acad Sci USA 101(37):13513–13518. 23. Matsubara K, Sano N, Umehara T, Horikoshi M (2007) Global analysis of functional surfaces of core histones with comprehensive point mutants. Genes Cells 12(1):13–33. 24. Sakamoto M, et al. (2009) Global analysis of mutual interaction surfaces of nucleosomes with comprehensive point mutants. Genes Cells 14(11):1271–1330. 25. Kawashima S, et al. (2011) Global analysis of core histones reveals nucleosomal surfaces required for chromosome bi-orientation. EMBO J 30(16):3353–3367. 26. Kawano A, et al. (2011) Global analysis for functional residues of histone variant Htz1 using the comprehensive point mutant library. Genes Cells 16(5):590–607. 27. Xiao T, et al. (2005) Histone H2B ubiquitylation is associated with elongating RNA polymerase II. Mol Cell Biol 25(2):637–651. 28. Pavri R, et al. (2006) Histone H2B monoubiquitination functions cooperatively with FACT to regulate elongation by RNA polymerase II. Cell 125(4):703–717. 29. Trujillo KM, Osley MA (2012) A role for H2B ubiquitylation in DNA replication. Mol Cell 48(5):734–746. 30. Giannattasio M, Lazzaro F, Plevani P, Muzi-Falconi M (2005) The DNA damage checkpoint response requires histone H2B ubiquitination by Rad6-Bre1 and H3 methylation by Dot1. J Biol Chem 280(11):9879–9886. 31. Sun ZW, Allis CD (2002) Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast. Nature 418(6893):104–108. 32. Dover J, et al. (2002) Methylation of histone H3 by COMPASS requires ubiquitination of histone H2B by Rad6. J Biol Chem 277(32):28368–28371. 33. Hayashi Y, Senda T, Sano N, Horikoshi M (2009) Theoretical framework for the histone modification network: Modifications in the unstructured histone tails form a robust scale-free network. Genes Cells 14(7):789–806. 34. Sato L, et al. (2010) Global analysis of functional relationships between histone point mutations and the effects of histone deacetylase inhibitors. Genes Cells 15(6): 553–594. 35. Horikoshi M (2013) Histone acetylation: From code to web and router via intrinsically disordered regions. Curr Pharm Des 19(28):5019–5042. 36. Tosi A, et al. (2013) Structure and subunit topology of the INO80 chromatin remodeler and its nucleosome complex. Cell 154(6):1207–1219.
Nakabayashi et al.
Edited by Fred M. Winston, Harvard Medical School, Boston, MA, and approved November 27, 2013 (received for review August 31, 2013)
FALC
|
histone variant
|
modification
|
chromatin
M
|
epigenetics
ore than half a century has passed since molecular biology revolutionized the life sciences. However, there remain no general strategies to differentiate between the in vivo role of a particular subunit within one multisubunit protein complex from its roles in other complexes. Such subunits are referred to as common subunits. A protein can function as a single component or as a component of a multisubunit protein complex (Fig. 1A). The functional role of a protein as a single component or in a multisubunit protein complex in the cell can be ascertained via gene knockout or knockdown of the corresponding mRNA. However, some proteins participate in several complexes and processes; therefore, although such studies would indicate the net phenotype induced by removal of the protein, they would not discern the specific role(s) of the protein in each complex. Thus, if a protein functions in more than one biological process (i.e., it is a common subunit in several multisubunit protein complexes), its role in a specific biological process or complex cannot be determined easily. Such common subunits are found in a variety of multisubunit protein complexes (1). Four examples of multisubunit protein complexes that contain a common subunit include (i) eukaryotic RNA polymerases I, II, and III, which all share several Rpb subunits (2); (ii) general transcription initiation factor complexes, SL1, TFIID, and TFIIIB, which share the TBP (TATA box-binding protein) subunit (3); (iii) the histone acetyltransferase NuA4 complex and the histone deacetylase Rpd3 complex, which share the Eaf3 subunit (4); and (iv) the NuA4 complex and the chromatin remodeling complexes SWR1 and INO80, which share the Act1 and Arp4 subunits (5) (Fig. 1B). The specific roles of Rpb, TBP, Eaf3, Act1, and Arp4 in each multisubunit protein complex remain elusive largely because in vivo analyses of common subunits in multisubunit protein complexes have been hampered by weaknesses in current experimental approaches. Here, we developed a strategy that permits the in vivo function of a subunit in one multisubunit protein complex to be distinguished from its function(s) in other complexes. The physiological role of a common subunit in a specific multisubunit protein www.pnas.org/cgi/doi/10.1073/pnas.1316433111
complex can be elucidated if the subunit can be made specific to one complex. To accomplish this, the common subunit could be covalently linked to another subunit that is specific to one complex (Fig. 1C), followed by the introduction of a mutation into the linked common subunit (Fig. 1 D and E). We call this strategy Functional Analysis of Linker-mediated Complex (FALC). A nucleosome, the basic structural unit of chromatin (6), is comprised of eight histones, typically two histone H2A/H2B dimers and a histone (H3/H4)2 tetramer, and wrapped DNA (7, 8), and is the most abundant and evolutionarily conserved multisubunit protein complex in eukaryotic cell nuclei. Nucleosomes regulate a variety of DNA-mediated processes such as transcription and DNA replication (8, 9). Thus, the regulation of nucleosome structure and function in individual DNA-mediated reactions is a central subject in modern biology (7, 9, 10). Although the H2B subunit within nucleosomes remains constant, the H2A subtype changes. The H2A subtype (variant) H2A.Z is the only isoform that is conserved among all eukaryotes. Each of two H2A proteins in a nucleosome (called the A/A nucleosome) is replaced with the H2A variant Htz1 (budding yeast H2A.Z) in a stepwise manner to first yield an A/Z nucleosome containing one H2A and one Htz1 molecule and then a Z/Z nucleosome containing two Htz1 proteins (11). Thus, H2B is a common subunit in H2A-containing (A/A- and A/Z-) and Htz1-containing (A/Z- and Z/Z-) multisubunit protein complexes of nucleosomes (Fig. 2A). Analysis of crystals of H2A.Z-containing nucleosomes revealed that there are no marked structural differences compared with canonical nucleosomes (7, 12, 13). Budding yeast Htz1 is involved in gene regulation, protection against gene silencing around boundaries, DNA repair, cell cycle progression, and chromosome stability (14). Although H2A.Z is widely studied, there is no experimental approach to reveal the specific roles of H2B that is paired with H2A.Z. Significance The same protein is often a subunit of more than one multisubunit protein complex, each of which has a distinct function within cells. Mutating such a protein would cause multiple cellular defects; therefore, it is difficult to distinguish between the function of a protein in one complex from its functions in other complexes. Here, we developed a unique strategy to overcome this problem, which will help to analyze a variety of biological processes by revealing the specific roles played by such proteins within multisubunit protein complexes. Author contributions: Y.N., M.S., and M.H. designed research; Y.N. and S.K. performed research; Y.N., S.K., M.S., and M.H. analyzed data; and Y.N., T.E., M.S., and M.H. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1
To whom correspondence may be addressed. E-mail: [email protected] or [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1316433111/-/DCSupplemental.
PNAS | January 14, 2014 | vol. 111 | no. 2 | 699–704
CELL BIOLOGY
Currently, there is no method to distinguish between the roles of a subunit in one multisubunit protein complex from its roles in other complexes in vivo. This is because a mutation in a common subunit will affect all complexes containing that subunit. Here, we describe a unique method to discriminate between the functions of a common subunit in different multisubunit protein complexes. In this method, a common subunit in a multisubunit protein complex is genetically fused to a subunit that is specific to that complex and point mutated. The resulting phenotype(s) identify the specific function(s) of the subunit in that complex only. Histone H2B is a common subunit in nucleosomes containing H2A/H2B or Htz1/H2B dimers. The H2B was fused to H2A or Htz1 and point mutated. This strategy revealed that H2B has common and distinct functions in different nucleosomes. This method could be used to study common subunits in other multisubunit protein complexes.
Fig. 1. Study concept. (A) Schematic representation of a protein as a single component (W) and as a common subunit (Z), with and without a mutation, in two different multisubunit complexes, each with a specific subunit (X or Y). (B) Examples of common subunits in RNA polymerases, general transcription initiation factor complexes, the NuA4 histone acetyltransferase and the Rpd3 deacetylase, and the NuA4, SWR1, and INO80 chromatin remodeling complexes. The red zone depicts common subunits and the other colored zones depict specific subunits. (C) Schematic representation of a common subunit (Z) fused with a specific subunit (X or Y). (D) Introduction of a mutation into a common subunit (Z) of each fusion. (E) Schematic representation of how mutating a common subunit (Z) fused with a specific subunit (X or Y) in a complex can allow complex-specific phenotypes to be evaluated. (−) indicates that a mutation that specifically affects one complex cannot be identified.
Results H2B-H2A and H2B-Htz1 Fusions Are Functional in Vivo. To elucidate
the individual roles of the common H2B subunit in H2A- and Htz1-containing nucleosomes (Figs. 1E and 2A), we created fusion proteins consisting of a C-terminal portion of H2B fused in frame to the N terminus of full-length H2A or Htz1 (12, 13) (Fig. 2 B and C). Although these fusions cannot be used to elucidate the potential function(s) of junctional regions, the H2A N-terminal tail is reportedly dispensable (15). The H2B-H2A and H2B-Htz1 fusions were extensively characterized to determine whether they were functional in yeast. To test whether the H2B-H2A fusion was functional, this fusion was expressed in hta and htb double KO (htaΔ/htbΔ) cells (16, 17), which lack genomic H2A (HTA1/HTA2) and H2B (HTB1/HTB2) genes but carry nonfused HTA1 and HTB1 genes on a plasmid containing the URA3 marker, and cell viability was assessed. Because the URA3 plasmid carrying the HTA1 and HTB1 genes is lost from cells grown in the presence of 5-fluoroorotic acid (5FOA), htaΔ/htbΔ cells harboring nonfused HTA1 and HTB1 genes on the non-URA3 plasmid were viable, whereas cells harboring an empty plasmid were not (Fig. 2D, rows 1 and 2). Under the same experimental conditions, the H2B-H2A fusion rescued htaΔ/htbΔ cells from lethality, indicating that the H2BH2A fusion was functional (Fig. 2D, row 3). The slightly reduced growth of cells expressing the H2B-H2A fusion (Fig. 2D, row 3) seemed to be due to degradation of monomeric Htz1 (Fig. S1). htz1Δ cells grow slowly (18), and this phenotype was mimicked by cells expressing the H2B-H2A fusion. To evaluate the functionality of the H2B-Htz1 fusion (Fig. 2E and Fig. S2A), we used the hydroxyurea (HU)- and methyl methanesulfonate (MMS)-sensitive phenotypes of htz1Δ cells (19) (Fig. 2F, row 2). Whereas htz1Δ cells (Fig. 2F, row 2) and htaΔ/htbΔ/htz1Δ cells expressing only the H2B-H2A fusion (row 3) were sensitive to HU and MMS, htaΔ/htbΔ/htz1Δ cells expressing both H2B-H2A and H2B-Htz1 fusions were not, similar to WT cells (rows 1 and 4). The ability of the H2B-H2A and H2B-Htz1 fusions to rescue the drug-sensitive phenotype of htaΔ/htbΔ/htz1Δ cells indicates that both fusions are functional. 700 | www.pnas.org/cgi/doi/10.1073/pnas.1316433111
Both nonfused H2A and Htz1 (Fig. 2G, Top and Middle, lane 1) and only nonfused H2A (lane 2) were detected via immunoblot analysis at a molecular weight less than 20 kDa in samples from WT and htz1Δ cells, respectively. H2B-H2A in H2B-H2A– expressing cells, and H2B-H2A and H2B-Htz1 in H2B-H2A– and H2B-Htz1–expressing cells, were detected at ∼30 kDa (Fig. 2G, Top and Middle, lanes 3 and 4). Notably, another band was detected at a molecular weight greater than 30 kDa in samples from cells expressing the H2B-H2A fusion (Fig. 2G, Top, lanes 3 and 4) and the H2B-Htz1 fusion (Middle, lane 4), suggesting that monoubiquitination had occurred on the H2B-K123 residue of both fusions (20) (see Monoubiquitination Occurs on Both the H2B-H2A and H2B-Htz1 Fusions). An examination of nucleosome ladders produced following digestion with MNase for increasing amounts of time (6, 21) provides information about chromatin structure. This MNase assay indicated that the chromatin of htz1Δ cells, htaΔ/htbΔ/ htz1Δ cells expressing only the H2B-H2A fusion, and htaΔ/htbΔ/ htz1Δ cells expressing both H2B-H2A and H2B-Htz1 fusions (Fig. 2H, columns 2–4) are similar to the chromatin of WT cells (column 1). ChIP was performed to determine whether the H2B-Htz1 fusion could replace the H2B-H2A fusion at several promoters and pericentromeric regions of chromosome III (CEN3) at which Htz1 is known to localize (11, 22) (Fig. 2I and Fig. S2 B and C). Nonfused Htz1 was detected at promoters and near to the CEN3-left and -right regions in WT cells (Fig. 2J and Fig. S2 D and E, lane 1). As expected, the Htz1 signal was abolished in htz1Δ cells (Fig. 2J and Fig. S2 D and E, lane 2) and in htaΔ/ htbΔ/htz1Δ cells expressing only the H2B-H2A fusion (lane 3). The ChIP signals of the H2B-Htz1 fusion and nonfused Htz1 were much lower around the ORFs of the LSB5 and GID7 genes than at their corresponding promoters (Fig. S2F), as previously reported (11). Therefore, the H2B-Htz1 fusion seems to specifically localize to chromatin, similar to nonfused Htz1. Importantly, the H2B-Htz1 fusion localized at promoters and near to CEN3left and -right regions (Fig. 2J and Fig. S2 D and E, lane 4), albeit at a much lower level than nonfused Htz1 in WT cells. Although Nakabayashi et al.
Contribution of the H2B-L109 Residue in Fused H2B-H2A and H2B-Htz1 to Cell Viability. To determine the functional roles of H2B in
this difference could be explained by a partial replacement of the function of H2B-H2A by the H2B-Htz1 fusion on chromatin, it could also be due to a technical artifact. Namely, the immunoprecipitation of chromatin containing the H2B-Htz1 fusion by the anti-Htz1 antibody could be less efficient than that of chromatin containing nonfused Htz1. Irrespective of this, these observations indicate that both H2B-H2A and H2B-Htz1 fusions are incorporated into chromatin. Taking all of the data together (cell viability, drug sensitivity, protein expression, MNase assay, and ChIP assay), the H2B-H2A and H2B-Htz1 fusions seem to be functional in cells. Nakabayashi et al.
CELL BIOLOGY
Fig. 2. Functional analysis of H2B-H2A and H2B-Htz1 fusions. (A) Schematic representation of nonfused and fused H2B-H2A– and H2B-Htz1–containing nucleosomes. (B) Schematic representation of the constructs used to generate fused H2B-H2A (Upper) and H2B-Htz1 (Lower). A C-terminal portion of H2B was fused in frame to the N terminus of full-length H2A or Htz1. PHTB1, HTB1 promoter. PHTZ1, HTZ1 promoter. (C) Nucleosome structure and enlarged view of the C terminus of H2B and the N terminus of H2A or Htz1 (PDB ID: 1ID3). (D) Viability of cells expressing fused H2B-H2A. Cells grew slightly poorer on (+) 5-FOA plates than on (−) 5-FOA plates because 5-FOA can kill cells with the URA3 gene. (E) Schematic representation of nucleosomes in the strains (htaΔ/htbΔ/htz1Δ background) used for experiments F–H and J. (1) Cells expressing nonfused H2A, H2B, and Htz1 (WT); (2) cells expressing nonfused H2A and H2B, but not Htz1 (htz1-deleted cells); (3) cells expressing only the H2B-H2A fusion; and (4) cells expressing both the H2BH2A and H2B-Htz1 fusions. (F) Drug sensitivity analysis using HU or MMS. (G) Immunoblot analysis of H2B-H2A and H2B-Htz1 fusions. Histone H4 was used as a loading control. (H) MNase analysis of chromatin with digestion for increasing amounts of time. M, 100-bp marker. (I) Schematic representation of the pericentromeric region of CEN3 and the locations of primer sets used for ChIP analysis. (J) ChIP analysis of Htz1 and the H2B-Htz1 fusion. Total Htz1 occupancy in WT cells was used for normalization. Error bars represent SD (n = 3).
H2A/H2B and Htz1/H2B dimers, we point-mutated H2B in the H2B-H2A and H2B-Htz1 fusions (Fig. 3A and Fig. S3A). Thirtyeight phenotypic residues of H2B have been identified by previous screening of the histone-GLibrary (nonfused H2B point mutant library) (23–25). We individually mutated 3 (H2B-D71, -L109, or -K123) of 38 residues in the H2B-H2A and H2B-Htz1 fusions. Expression of H2B-L109A in nonfused H2B-expressing cells caused cell lethality, whereas expression of H2B-D71A or -K123A did not (23, 24). However, the introduction of the latter two mutants into nonfused H2B-expressing cells led to cells becoming strongly sensitive to HU, MMS, benomyl (a microtubule-depolymerizing drug), and/or 6-azauracil (6AU) (23–25). We analyzed the effect of introducing the H2B-L109A mutation into the H2B-H2A and H2B-Htz1 fusions on cell viability. Nonfused HTA1 and HTB1 genes on the URA3 plasmid, which enabled htaΔ/htbΔ/htz1Δ cells to survive, would be lost during culture on plates containing 5-FOA. In the presence of 5-FOA, htaΔ/htbΔ/htz1Δ cells expressing nonfused H2A, H2B, and Htz1, or both H2B-H2A and H2B-Htz1 fusions from non-URA3 plasmids, were viable (Fig. 3B, rows 1 and 3, and Fig. S3B, lanes 1 and 3). By contrast, cells harboring nonfused H2A, H2B(L109A), and Htz1 were not viable (Fig. 3B, row 2, and Fig. S3B, lane 2), as expected. Lethality was also observed under 5-FOA conditions in cells expressing both mutant fusions [i.e., H2B(L109A)-H2A and H2B(L109A)-Htz1; Fig. 3B, row 4, and Fig. S3B, lane 4]. H2A and Htz1 are indispensable and dispensable for cell viability, respectively (17, 18). Therefore, cells expressing both H2B(L109A)-H2A and H2B(WT)-Htz1 fusions or both H2B (WT)-H2A and H2B(L109A)-Htz1 fusions should die and grow proficiently, respectively. As expected, cells expressing both H2B (WT)-H2A and H2B(L109A)-Htz1 fusions grew well in the presence of 5-FOA (Fig. 3B, row 6, and Fig. S3B, lane 6), similar to cells expressing both H2B(WT)-H2A and H2B(WT)-Htz1 fusions (Fig. 3B, row 3, and Fig. S3B, lane 3). Surprisingly, cells
Fig. 3. Functional analysis of H2B-L109 in H2B-H2A and H2B-Htz1 fusions. (A) Schematic representation of nucleosomes containing nonfused or fused H2B point mutants. The cell strains examined were as follows: (1) nonfused WT H2B; (2) nonfused mutant (mut) H2B; (3) fused H2B-H2A/H2B-Htz1; (4) fused H2B(mut)-H2A/H2B(mut)-Htz1; (5) fused H2B(mut)-H2A/H2B(WT)Htz1; and (6) fused H2B(WT)-H2A/H2B(mut)-Htz1. (B) Lethality analysis of cells expressing fused H2B-H2A and/or H2B-Htz1 with the H2B-L109A point mutation. (C) Interacting region between H2B-L109, H2A-Y58 (or Htz1-Y65), and H2A-E62 (or Htz1-E69) residues in the nucleosome structure.
PNAS | January 14, 2014 | vol. 111 | no. 2 | 701
expressing both H2B(L109A)-H2A and H2B(WT)-Htz1 fusions grew in the presence of 5-FOA (Fig. 3B, row 5, and Fig. S3B, lane 5), although to a lesser extent than cells expressing both H2B (WT)-H2A and H2B(L109A)-Htz1 fusions (Fig. 3B, row 6, and Fig. S3B, lane 6). We confirmed that two independent clones isolated from cells expressing both H2B(L109A)-H2A and H2B (WT)-Htz1 fusions in the presence of 5-FOA grew extremely slowly (Fig. S3 C–E). These results suggest that H2B-L109 promotes cell viability not only through H2A- but also via Htz1-containing nucleosomes (Table 1). H2B-L109 physically interacts with the H2A-Y58, H2A-E62, Htz1-Y65, and Htz1-E69 residues, which are exposed to the nucleosome surface (23, 24, 26). Mutation of the H2A-Y58 and -E62 residues in nonfused H2A- and H2B-expressing cells caused cell death (23, 24), whereas mutation of the Htz1-Y65 and -E69 residues in nonfused Htz1- and H2B-expressing cells resulted in HU/MMS/benomyl sensitivities (26). Thus, the proximity and functional relationship of H2B-L109 to H2A-Y58, H2A-E62, Htz1-Y65, and Htz1-E69 might explain why H2BL109 is functionally important in both the H2B-H2A and H2BHtz1 fusions. H2B-D71 Residue Plays a Specific Role in the H2B-Htz1 Fusion. We next mutated another H2B residue, H2B-D71 (Fig. 4A and Figs. S4A and S5A), to investigate its effects on cell growth and sensitivity to HU, MMS, and benomyl (24, 25). Cells expressing nonfused H2B-D71A grew slower than WT cells (Fig. 4B, rows 1 and 2). Cells expressing both H2B(D71A)-H2A and H2B(WT)Htz1 fusions grew well (Fig. 4B, row 5), whereas cells expressing both H2B(WT)-H2A and H2B(D71A)-Htz1 fusions (row 6) grew poorer than cells expressing both WT fusions (row 3). Cells expressing both H2B-H2A and H2B-Htz1 fusions in which the H2B-D71 residue was mutated exhibited different HU, MMS, and benomyl sensitivities (Fig. 4B, rows 3–6). Quantitative analysis of benomyl sensitivity confirmed that the H2B-D71A mutation in the H2B-Htz1 fusion conferred benomyl sensitivity, whereas the H2B-D71A mutation in the H2B-H2A fusion did not (Fig. 4C and Table 1). H2B-D71A Mutation Abolishes the Localization of Htz1 on Chromatin Near to CEN3 and at Promoters. H2B-D71 is located on the H2B
α-helix 2 region and interacts with the H4-L97 and -Y98 residues in the H4 C-terminal tail region (7, 13, 14, 25). The H4-L97 and Table 1. Effects of H2B mutation on the H2B-H2A and H2B-Htz1 fusions Effects of mutation on H2B mutation D71A
L109A K123A
Analyzed phenotype
H2B-H2A
H2B-Htz1
Growth HU sensitivity MMS sensitivity Benomyl sensitivity H2A chromatin localization Htz1 chromatin localization Cell viability* Growth Growth HU sensitivity MMS sensitivity 6AU sensitivity H2B monoubiquitination
− + + − + − +* ++ − ++ ++ ++ ++
++ + + ++ − ++ +* − − − − − ++
++, significantly affected; +, slightly affected; −, not affected. *Cell viability is defined as viable (+) by the presence of any growing cells in spot assay and as lethal (−) by the absence of those cells, respectively.
702 | www.pnas.org/cgi/doi/10.1073/pnas.1316433111
Fig. 4. Functional analysis of H2B-D71 in H2B-H2A and H2B-Htz1 fusions. (A) Schematic representation of nucleosomes containing nonfused or fused H2B-D71A point mutants. The cell strains examined were as follows: (1) nonfused WT H2B; (2) nonfused H2B(D71A); (3) fused H2B-H2A/H2B-Htz1; (4) fused H2B(D71A)-H2A/H2B(D71A)-Htz1; (5) fused H2B(D71A)-H2A/H2B(WT)Htz1; and (6) fused H2B(WT)-H2A/H2B(D71A)-Htz1. (B) Drug sensitivity analysis of fused H2B-H2A and/or H2B-Htz1 with the H2B-D71A point mutation. (C) Yeast survival analysis after exposure to benomyl for a short amount of time. The survival rate of each strain was calculated by dividing the total number of colonies at each time point by the number of colonies at t = 0. (D) Enlarged view of the interacting region between histone H2B α2, the H4 C-terminal tail, and the H2A or Htz1 C-terminal tail in the nucleosome structure. (E and F) ChIP analysis of Htz1 or the H2B-Htz1 fusion (E) and H2A or the H2B-H2A fusion (F) in the indicated cells. Total Htz1 and H2A occupancies in WT cells were used for normalization. Error bars represent SD (n = 3).
-Y98 residues are located adjacent to a short β-strand of the H2A and Htz1 C-terminal tail regions (Fig. 4D). H4-L97, which faces H2B-D71, confers benomyl sensitivity when mutated and is necessary for the localization of Htz1 at pericentromeric regions of CEN3 (25), raising the possibility that H2B-D71 is also required for the localization of Htz1 around CEN3. To test this possibility, we examined the localization of Htz1 around CEN3 in cells expressing H2B(D71A)-H2A and/or H2B (D71A)-Htz1 fusions (Fig. 4E and Table 1). ChIP analysis indicated that the Htz1 level of the fusion near to the CEN3 locus was the same in cells expressing the H2B(D71A)-H2A fusion as in cells expressing the H2B(WT)-H2A fusion (Fig. 4E, lanes 3 and 5). By contrast, the level of the H2B(D71A)-Htz1 fusion near to the CEN3 locus differed between cells expressing H2B (D71A)-H2A and cells expressing H2B(WT)-H2A (lanes 4 and 6). Consistent with this, the introduction of the H2B-D71A point mutation into nonfused H2B dramatically reduced the Htz1 ChIP signal near to the CEN3 locus compared with the signal in WT cells (Fig. 4E, lanes 1 and 2). The same results were obtained Nakabayashi et al.
H2B-K123 Predominantly Functions in the H2B-H2A Fusion. We next examined the effect of the introduction of the H2B-K123A mutation into the H2B-H2A and H2B-Htz1 fusions on the sensitivity of cells to HU, MMS, and/or 6AU (Fig. 5 A and B). 6AU disturbs the balance of intracellular nucleotide concentrations and blocks transcription elongation. Cells expressing nonfused H2B-K123A were sensitive to HU, MMS, and 6AU (Fig. 5B, rows 1 and 2), as were cells expressing both H2B(K123A)-H2A and H2B(K123A)-Htz1 fusions (Fig. 5B, rows 3 and 4). Under the same conditions, cells expressing both H2B(K123A)-H2A and H2B(WT)-Htz1 fusions, but not H2B(WT)-H2A and H2B (K123A)-Htz1 fusions, were sensitive to HU, MMS, and 6AU (Fig. 5B, rows 5 and 6). Because the H2B-K123 residue could be important in the H2B-H2A fusion, but not in the H2B-Htz1 fusion, the function of H2B-K123 in the H2A/H2B dimer seems to be different from its function in the Htz1/H2B dimer. Monoubiquitination Occurs on Both the H2B-H2A and H2B-Htz1 Fusions. Among the three H2B residues (H2B-D71, -L109, and
-K123) analyzed in this study, only H2B-K123 is chemically modifiable. Specifically, H2B-K123 can be monoubiquitinated (20), a modification that is important in transcription elongation (27, 28), DNA replication (29), DNA repair (30), and di-/trimethylation of lysine 4 of histone H3 (H3-K4me2/3) in the nucleosome (31, 32). To investigate whether monoubiquitination of H2B-K123 occurs in H2A- and Htz1-containing nucleosomes, we compared the state of H2B monoubiquitination in cells expressing nonmutated fusions to that in cells expressing the H2B(K123A)H2A and/or H2B(K123A)-Htz1 fusions (Fig. 5C). The status of H2B-K123 monoubiquitination was indirectly evaluated by the appearance of a band migrating slightly higher than 30 kDa, the size at which the H2B-H2A and H2B-Htz1 fusions were detected using anti-H2A and anti-Htz1 antibodies, respectively (Fig. 2G). Nakabayashi et al.
Fig. 5. Functional analysis of H2B-K123 in H2B-H2A and H2B-Htz1 fusions. (A) Schematic representation of nucleosomes containing nonfused or fused H2B-K123A point mutants. The cell strains examined were as follows: (1) nonfused WT H2B; (2) nonfused H2B(K123A); (3) fused H2B-H2A/H2B-Htz1; (4) fused H2B(K123A)-H2A/H2B(K123A)-Htz1; (5) fused H2B(K123A)-H2A/H2B (WT)-Htz1; and (6) fused H2B(WT)-H2A/H2B(K123A)-Htz1. (B) Drug sensitivity analysis of fused H2B-H2A and/or H2B-Htz1 with the H2B-K123A point mutation. (C) Immunoblot analysis of monoubiquitination of H2B-K123 in H2BH2A and H2B-Htz1. Lanes labeled 1 show a series of fourfold dilutions of samples from WT cells. Histone H4 was used as a loading control.
Higher molecular weight bands of ∼37 kDa were detected in cells expressing the H2B-H2A fusion (Fig. 5C, Top, lanes 3 and 6) and the H2B-Htz1 fusion (Middle, lanes 3 and 5). These bands were not detected in cells expressing the H2B(K123A)-H2A fusion (Fig. 5C, Top, lanes 4 and 5) and the H2B(K123A)-Htz1 fusion (Middle, lanes 4 and 6). These observations suggest that H2B is monoubiquitinated at K123, irrespective of whether H2B is paired with H2A or Htz1. The level of monoubiquitinated H2B-Htz1 fusion was much higher in cells expressing the H2B(K123A)-H2A fusion (Fig. 5C, Middle, lane 5) than in cells expressing the H2B(WT)-H2A fusion (lane 3), suggesting that the lack of monoubiquitination in the H2B-H2A fusion (Fig. 5C, Top, lane 5) leads to accumulation of the monoubiquitinated H2B-Htz1 fusion (Middle, lane 5) in cells. The FALC strategy successfully determines whether H2B-K123 is monoubiquitinated in the H2B-H2A fusion and/or the H2B-Htz1 fusion; therefore, a variety of chemical modifications of residues (such as S10, K11, and K16) (33–35) in the tail region of H2B could be tested to discern other roles played by H2B depending on whether it is paired with H2A or Htz1. Discussion In this study, we developed the FALC strategy to overcome the difficulties associated with functional analyses of proteins that are subunits of more than one multisubunit complex (Fig. 1). We used H2B as a common subunit, and H2A- and Htz1-containing nucleosomes as multisubunit complexes (Figs. 2–5). To implement the FALC strategy, we made functional H2B-H2A and H2B-Htz1 fusions (Fig. 2) and analyzed the phenotypes following point mutations of three H2B residues (H2B-D71, -L109, and -K123) in nonfused H2B, the H2B-H2A fusion, and the H2B-Htz1 fusion (Figs. 3–5 and Table 1). This innovative FALC strategy led to unique functional information about H2A, Htz1, and H2B, which could not have been revealed by analysis of combinations of nonfused proteins. The FALC strategy revealed that each residue has a distinct role depending on which fusion it was part of. H2B-L109 in H2Acontaining nucleosomes predominantly contributes to support cell viability, as expected (Fig. 3). Unexpectedly, it was revealed that PNAS | January 14, 2014 | vol. 111 | no. 2 | 703
CELL BIOLOGY
at the promoters of the LSB5 and GID7 genes (Fig. S4B). These data indicate that the H2B-D71A mutation dramatically reduces the localization of the H2B-Htz1 fusion and nonfused Htz1 at chromatin. On the other hand, the levels of nonfused Htz1 (Fig. S5B, lane 2) and H2B(D71A)-Htz1 fusion proteins (lanes 4 and 6), and their corresponding mRNAs (Fig. S5C, lanes 2, 4, and 6), were not substantially lower than the levels of the WT proteins and transcripts (Fig. S5 B and C, lanes 1 and 3). Thus, the H2BD71A mutation likely affects some functions of the dimer, such as incorporation into chromatin, rather than the protein stability of the dimer. The ChIP signal of nonfused H2A near to the CEN3 locus and at the promoters of the LSB5 and GID7 genes in WT cells (Fig. 4F and Fig. S4C, lane 1) was slightly reduced, but not abolished, by introduction of the H2B-D71A mutation (Fig. 4F and Fig. S4C, lane 2). The level of the H2B-H2A fusion around CEN3 and at these two promoters (Fig. 4F and Fig. S4C, lanes 3) was not significantly altered by the introduction of the H2B-D71A mutation into the H2B-H2A fusion and/or the H2B-Htz1 fusion (Fig. 4F and Fig. S4C, lanes 4–6). These results suggested that the H2B-D71A mutation markedly affected Htz1-containing nucleosomes but not H2A-containing nucleosomes (Table 1). Furthermore, the introduction of a different point mutation into H2B, H2B-K123A, did not affect the localization of Htz1 localization at the CEN3 locus (Fig. S6). Thus, it is suggested that the proper localization of H2B-Htz1 at the CEN3 locus requires the H2B-D71 residue and that this residue is important for maintaining the function of Htz1-containing nucleosomes (Table 1). H2B is likely to be involved in the transport, processing, deposition, unloading, and/or recycling of Htz1/H2B dimers. H2B-D71 faces the H4-L97 residue, which is involved in the localization of Htz1 around CEN3 (25). Therefore, we prefer a model in which the H2B-D71A mutant cannot bind the (H3H4)2 tetramer or can be easily released from the nucleosome.
H2B-L109 in Htz1-containing nucleosomes also supports cell viability, although with a lesser contribution than in H2A-containing nucleosomes (Fig. 3). By contrast, cell growth and resistance to benomyl largely relied on the H2B-D71 residue of fused H2BHtz1 (Fig. 4). The localization of Htz1 at promoters and around CEN3 was abolished with the H2B-D71A mutant, but not with the H2B-K123A mutant, suggesting that the H2B-D71 and H2B-K123 residues have different structural and functional roles (Fig. 4). Although the structures of H2A- and Htz1-containing nucleosomes have been determined (7, 12, 13), the current study revealed the role of H2B-D71 in these nucleosomes by using a FALC strategy. H2B-K123 is most likely to be monoubiquitinated in both H2B-H2A and H2B-Htz1 fusions (Fig. 5). On the other hand, H2B-K123 was functional within H2A- but not within Htz1-containing nucleosomes, as shown by drug sensitivity experiments (Fig. 5). Thus, in drug sensitivity-related reactions, monoubiquitinated H2B-K123 seems to have different functions depending on whether it is paired with H2A or Htz1. It will be interesting to reveal the roles of monoubiquitination of H2BK123 in the H2B-Htz1 fusion. Although some common phenotypes resulted from the presence of the H2B(mut)-H2A fusion or the H2B(mut)-Htz1 fusion (Table 1), distinct phenotypes were also detected. The FALC strategy led to the successful determination of distinct roles for an individual residue in a common subunit, H2B, depending on whether it was part of the H2B-H2A or H2B-Htz1 fusion. Generalizing the FALC strategy to other multisubunit complexes (Fig. 1) is an interesting challenge. It is not known how to select a specific subunit that is linked to the common subunit, especially when the structural information of a multisubunit complex 1. Havugimana PC, et al. (2012) A census of human soluble protein complexes. Cell 150(5):1068–1081. 2. Vannini A, Cramer P (2012) Conservation between the RNA polymerase I, II, and III transcription initiation machineries. Mol Cell 45(4):439–446. 3. Burley SK, Roeder RG (1996) Biochemistry and structural biology of transcription factor IID (TFIID). Annu Rev Biochem 65:769–799. 4. Smith E, Shilatifard A (2010) The chromatin signaling pathway: Diverse mechanisms of recruitment of histone-modifying enzymes and varied biological outcomes. Mol Cell 40(5):689–701. 5. van Attikum H, Gasser SM (2005) The histone code at DNA breaks: A guide to repair? Nat Rev Mol Cell Biol 6(10):757–765. 6. Kornberg RD (1974) Chromatin structure: A repeating unit of histones and DNA. Science 184(4139):868–871. 7. Luger K, Mäder AW, Richmond RK, Sargent DF, Richmond TJ (1997) Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389(6648):251–260. 8. Natsume R, et al. (2007) Structure and function of the histone chaperone CIA/ASF1 complexed with histones H3 and H4. Nature 446(7133):338–341. 9. Allis CD, Jenuwein T, Reinberg D, Caparros ML (2007) Epigenetics (Cold Spring Harbor Laboratory Press, New York). 10. Akai Y, et al. (2010) Structure of the histone chaperone CIA/ASF1-double bromodomain complex linking histone modifications and site-specific histone eviction. Proc Natl Acad Sci USA 107(18):8153–8158. 11. Luk E, et al. (2010) Stepwise histone replacement by SWR1 requires dual activation with histone H2A.Z and canonical nucleosome. Cell 143(5):725–736. 12. White CL, Suto RK, Luger K (2001) Structure of the yeast nucleosome core particle reveals fundamental changes in internucleosome interactions. EMBO J 20(18): 5207–5218. 13. Suto RK, Clarkson MJ, Tremethick DJ, Luger K (2000) Crystal structure of a nucleosome core particle containing the variant histone H2A.Z. Nat Struct Biol 7(12):1121–1124. 14. Zlatanova J, Thakar A (2008) H2A.Z: View from the top. Structure 16(2):166–179. 15. Schuster T, Han M, Grunstein M (1986) Yeast histone H2A and H2B amino termini have interchangeable functions. Cell 45(3):445–451. 16. Rykowski MC, Wallis JW, Choe J, Grunstein M (1981) Histone H2B subtypes are dispensable during the yeast cell cycle. Cell 25(2):477–487. 17. Kolodrubetz D, Rykowski MC, Grunstein M (1982) Histone H2A subtypes associate interchangeably in vivo with histone H2B subtypes. Proc Natl Acad Sci USA 79(24): 7814–7818. 18. Jackson JD, Gorovsky MA (2000) Histone H2A.Z has a conserved function that is distinct from that of the major H2A sequence variants. Nucleic Acids Res 28(19):3811–3816.
704 | www.pnas.org/cgi/doi/10.1073/pnas.1316433111
is unavailable. To overcome this challenge, experimental predictions could be made by gaining topological information on common and specific subunits in multisubunit complexes via the FRET assay, electron microscopy, and protein cross-linking. Using the latter technique, information about the topology between common subunits (such as Act1 and Arp4) and specific subunits of the INO80 complex (Fig. 1B) has recently been gained (36). Introducing mutations into a protein fusion is another critical step of the FALC strategy. Comprehensive mutagenesis analysis (GLibrary strategy = GLASP + GLAMP) (23–26) of the target common subunit(s) would greatly facilitate mutagenic analyses of fused complexes, as shown in this study (Figs. 3–5 and Table 1). Thus, combining the FALC strategy with mutational analyses opens a new avenue to address the functional roles of a common subunit(s) in multiple multisubunit complexes in cells. Materials and Methods Detailed information on materials and methods in this study is provided in SI Materials and Methods. Information on materials includes the characteristics of yeast strains and construction of plasmids. Technical information on methods contains drug sensitivity assays, immunoblot analysis, Northern blot analysis, ChIP assay, chromatin digestion assay with micrococcal nuclease, survival analysis following benomyl treatment, and molecular graphics. ACKNOWLEDGMENTS. We thank L. Sato for initial ideas on linker analysis, G. Ueno for creating some of the constructs, and K. Hasegawa for administrative assistance. We also thank all members of our laboratories for fruitful discussion of this study. This work was supported by a Grant-inAid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and a Grant-in-Aid for a Japan Society for the Promotion of Science Research Fellow.
19. Mizuguchi G, et al. (2004) ATP-driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin remodeling complex. Science 303(5656):343–348. 20. Robzyk K, Recht J, Osley MA (2000) Rad6-dependent ubiquitination of histone H2B in yeast. Science 287(5452):501–504. 21. Kent NA, Mellor J (1995) Chromatin structure snap-shots: Rapid nuclease digestion of chromatin in yeast. Nucleic Acids Res 23(18):3786–3787. 22. Krogan NJ, et al. (2004) Regulation of chromosome stability by the histone H2A variant Htz1, the Swr1 chromatin remodeling complex, and the histone acetyltransferase NuA4. Proc Natl Acad Sci USA 101(37):13513–13518. 23. Matsubara K, Sano N, Umehara T, Horikoshi M (2007) Global analysis of functional surfaces of core histones with comprehensive point mutants. Genes Cells 12(1):13–33. 24. Sakamoto M, et al. (2009) Global analysis of mutual interaction surfaces of nucleosomes with comprehensive point mutants. Genes Cells 14(11):1271–1330. 25. Kawashima S, et al. (2011) Global analysis of core histones reveals nucleosomal surfaces required for chromosome bi-orientation. EMBO J 30(16):3353–3367. 26. Kawano A, et al. (2011) Global analysis for functional residues of histone variant Htz1 using the comprehensive point mutant library. Genes Cells 16(5):590–607. 27. Xiao T, et al. (2005) Histone H2B ubiquitylation is associated with elongating RNA polymerase II. Mol Cell Biol 25(2):637–651. 28. Pavri R, et al. (2006) Histone H2B monoubiquitination functions cooperatively with FACT to regulate elongation by RNA polymerase II. Cell 125(4):703–717. 29. Trujillo KM, Osley MA (2012) A role for H2B ubiquitylation in DNA replication. Mol Cell 48(5):734–746. 30. Giannattasio M, Lazzaro F, Plevani P, Muzi-Falconi M (2005) The DNA damage checkpoint response requires histone H2B ubiquitination by Rad6-Bre1 and H3 methylation by Dot1. J Biol Chem 280(11):9879–9886. 31. Sun ZW, Allis CD (2002) Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast. Nature 418(6893):104–108. 32. Dover J, et al. (2002) Methylation of histone H3 by COMPASS requires ubiquitination of histone H2B by Rad6. J Biol Chem 277(32):28368–28371. 33. Hayashi Y, Senda T, Sano N, Horikoshi M (2009) Theoretical framework for the histone modification network: Modifications in the unstructured histone tails form a robust scale-free network. Genes Cells 14(7):789–806. 34. Sato L, et al. (2010) Global analysis of functional relationships between histone point mutations and the effects of histone deacetylase inhibitors. Genes Cells 15(6): 553–594. 35. Horikoshi M (2013) Histone acetylation: From code to web and router via intrinsically disordered regions. Curr Pharm Des 19(28):5019–5042. 36. Tosi A, et al. (2013) Structure and subunit topology of the INO80 chromatin remodeler and its nucleosome complex. Cell 154(6):1207–1219.
Nakabayashi et al.
