Roles of common subunits within distinctmultisubunit complexesYu Nakabayashia, Satoshi Kawashimab, Takemi Enomotoc, Masayuki Sekia,1, and Masami Horikoshid,1

aDepartment of Biochemistry, Tohoku Pharmaceutical University, Sendai, Miyagi 981-8558, Japan; bMolecular Cell Biology Laboratory, Graduate School ofPharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan; cResearch Institute of Pharmaceutical Sciences, Faculty of Pharmacy, MusashinoUniversity, Nishitokyo, Tokyo 202-8585, Japan; and dLaboratory of Developmental Biology, Institute of Molecular and Cellular Biosciences, The Universityof 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)

Currently, there is no method to distinguish between the roles ofa subunit in one multisubunit protein complex from its roles inother complexes in vivo. This is because a mutation in a commonsubunit will affect all complexes containing that subunit. Here, wedescribe a unique method to discriminate between the functionsof a common subunit in different multisubunit protein complexes.In this method, a common subunit in a multisubunit protein com-plex is genetically fused to a subunit that is specific to that com-plex and point mutated. The resulting phenotype(s) identify thespecific function(s) of the subunit in that complex only. HistoneH2B is a common subunit in nucleosomes containing H2A/H2B orHtz1/H2B dimers. The H2B was fused to H2A or Htz1 and pointmutated. This strategy revealed that H2B has common and distinctfunctions in different nucleosomes. This method could be used tostudy common subunits in other multisubunit protein complexes.

FALC | histone variant | modification | chromatin | epigenetics

More than half a century has passed since molecular biologyrevolutionized the life sciences. However, there remain no

general strategies to differentiate between the in vivo role ofa particular subunit within one multisubunit protein complexfrom its roles in other complexes. Such subunits are referred toas common subunits.A protein can function as a single component or as a compo-

nent of a multisubunit protein complex (Fig. 1A). The functionalrole of a protein as a single component or in a multisubunitprotein complex in the cell can be ascertained via gene knockoutor knockdown of the corresponding mRNA. However, someproteins participate in several complexes and processes; there-fore, although such studies would indicate the net phenotypeinduced by removal of the protein, they would not discern thespecific role(s) of the protein in each complex. Thus, if a proteinfunctions in more than one biological process (i.e., it is a commonsubunit in several multisubunit protein complexes), its role ina 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 proteincomplexes that contain a common subunit include (i) eukaryoticRNA polymerases I, II, and III, which all share several Rpbsubunits (2); (ii) general transcription initiation factor com-plexes, SL1, TFIID, and TFIIIB, which share the TBP (TATAbox-binding protein) subunit (3); (iii) the histone acetyl-transferase NuA4 complex and the histone deacetylase Rpd3complex, which share the Eaf3 subunit (4); and (iv) the NuA4complex and the chromatin remodeling complexes SWR1 andINO80, which share the Act1 and Arp4 subunits (5) (Fig. 1B).The specific roles of Rpb, TBP, Eaf3, Act1, and Arp4 in eachmultisubunit protein complex remain elusive largely because in vivoanalyses of common subunits inmultisubunit protein complexes havebeen 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 distin-guished from its function(s) in other complexes. The physiolog-ical role of a common subunit in a specific multisubunit protein

complex can be elucidated if the subunit can be made specific toone complex. To accomplish this, the common subunit couldbe covalently linked to another subunit that is specific to onecomplex (Fig. 1C), followed by the introduction of a mutation intothe linked common subunit (Fig. 1 D and E). We call this strategyFunctional Analysis of Linker-mediated Complex (FALC).A nucleosome, the basic structural unit of chromatin (6), is

comprised of eight histones, typically two histone H2A/H2Bdimers and a histone (H3/H4)2 tetramer, and wrapped DNA(7, 8), and is the most abundant and evolutionarily conservedmultisubunit protein complex in eukaryotic cell nuclei. Nucleo-somes regulate a variety of DNA-mediated processes such astranscription and DNA replication (8, 9). Thus, the regulation ofnucleosome structure and function in individual DNA-mediatedreactions is a central subject in modern biology (7, 9, 10). Al-though the H2B subunit within nucleosomes remains constant,the H2A subtype changes. The H2A subtype (variant) H2A.Z isthe only isoform that is conserved among all eukaryotes. Each oftwo H2A proteins in a nucleosome (called the A/A nucleosome)is replaced with the H2A variant Htz1 (budding yeast H2A.Z) ina stepwise manner to first yield an A/Z nucleosome containingone H2A and one Htz1 molecule and then a Z/Z nucleosomecontaining two Htz1 proteins (11). Thus, H2B is a commonsubunit 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 nucleosomesrevealed that there are no marked structural differences com-pared with canonical nucleosomes (7, 12, 13). Budding yeastHtz1 is involved in gene regulation, protection against gene si-lencing 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 ofH2B that is paired with H2A.Z.

Significance

The same protein is often a subunit of more than one multi-subunit protein complex, each of which has a distinct functionwithin cells. Mutating such a protein would cause multiplecellular defects; therefore, it is difficult to distinguish betweenthe function of a protein in one complex from its functions inother complexes. Here, we developed a unique strategy toovercome this problem, which will help to analyze a variety ofbiological processes by revealing the specific roles played bysuch proteins within multisubunit protein complexes.

Author contributions: Y.N., M.S., and M.H. designed research; Y.N. and S.K. performedresearch; Y.N., S.K., M.S., and M.H. analyzed data; and Y.N., T.E., M.S., and M.H. wrote thepaper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence may be addressed. E-mail: seki@tohoku-pharm.ac.jp orhorikosh@iam.u-tokyo.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1316433111/-/DCSupplemental.

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ResultsH2B-H2A and H2B-Htz1 Fusions Are Functional in Vivo. To elucidatethe individual roles of the common H2B subunit in H2A- andHtz1-containing nucleosomes (Figs. 1E and 2A), we created fu-sion proteins consisting of a C-terminal portion of H2B fused inframe to the N terminus of full-length H2A or Htz1 (12, 13) (Fig.2 B and C). Although these fusions cannot be used to elucidatethe potential function(s) of junctional regions, the H2A N-ter-minal tail is reportedly dispensable (15).The H2B-H2A and H2B-Htz1 fusions were extensively char-

acterized to determine whether they were functional in yeast. Totest whether the H2B-H2A fusion was functional, this fusion wasexpressed 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 plasmidcontaining the URA3 marker, and cell viability was assessed.Because the URA3 plasmid carrying the HTA1 and HTB1 genesis lost from cells grown in the presence of 5-fluoroorotic acid (5-FOA), htaΔ/htbΔ cells harboring nonfused HTA1 and HTB1genes on the non-URA3 plasmid were viable, whereas cellsharboring an empty plasmid were not (Fig. 2D, rows 1 and 2).Under the same experimental conditions, the H2B-H2A fusionrescued htaΔ/htbΔ cells from lethality, indicating that the H2B-H2A fusion was functional (Fig. 2D, row 3). The slightly reducedgrowth 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 mimickedby 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 methylmethanesulfonate (MMS)-sensitive phenotypes of htz1Δ cells(19) (Fig. 2F, row 2). Whereas htz1Δ cells (Fig. 2F, row 2) andhtaΔ/htbΔ/htz1Δ cells expressing only the H2B-H2A fusion (row3) were sensitive to HU and MMS, htaΔ/htbΔ/htz1Δ cells ex-pressing both H2B-H2A and H2B-Htz1 fusions were not, similarto WT cells (rows 1 and 4). The ability of the H2B-H2A andH2B-Htz1 fusions to rescue the drug-sensitive phenotype ofhtaΔ/htbΔ/htz1Δ cells indicates that both fusions are functional.

Both nonfused H2A and Htz1 (Fig. 2G, Top and Middle, lane1) and only nonfused H2A (lane 2) were detected via immuno-blot analysis at a molecular weight less than 20 kDa in samplesfrom 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 wasdetected at a molecular weight greater than 30 kDa in samplesfrom cells expressing the H2B-H2A fusion (Fig. 2G, Top, lanes 3and 4) and the H2B-Htz1 fusion (Middle, lane 4), suggesting thatmonoubiquitination had occurred on the H2B-K123 residue ofboth fusions (20) (see Monoubiquitination Occurs on Both theH2B-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 MNaseassay 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 promotersand pericentromeric regions of chromosome III (CEN3) atwhich Htz1 is known to localize (11, 22) (Fig. 2I and Fig. S2 Band C). Nonfused Htz1 was detected at promoters and near tothe CEN3-left and -right regions in WT cells (Fig. 2J and Fig. S2D and E, lane 1). As expected, the Htz1 signal was abolished inhtz1Δ 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 Htz1were much lower around the ORFs of the LSB5 and GID7 genesthan at their corresponding promoters (Fig. S2F), as previouslyreported (11). Therefore, the H2B-Htz1 fusion seems to specifi-cally localize to chromatin, similar to nonfused Htz1. Importantly,the H2B-Htz1 fusion localized at promoters and near to CEN3-left and -right regions (Fig. 2J and Fig. S2 D and E, lane 4), albeitat a much lower level than nonfused Htz1 in WT cells. Although

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, intwo different multisubunit complexes, each with a specific subunit (X or Y). (B) Examples of common subunits in RNA polymerases, general transcriptioninitiation 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 witha 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 commonsubunit (Z) fused with a specific subunit (X or Y) in a complex can allow complex-specific phenotypes to be evaluated. (−) indicates that a mutation thatspecifically affects one complex cannot be identified.

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this difference could be explained by a partial replacement of thefunction of H2B-H2A by the H2B-Htz1 fusion on chromatin, itcould also be due to a technical artifact. Namely, the immuno-precipitation of chromatin containing the H2B-Htz1 fusion by theanti-Htz1 antibody could be less efficient than that of chromatincontaining nonfused Htz1. Irrespective of this, these observationsindicate that both H2B-H2A and H2B-Htz1 fusions are incor-porated into chromatin. Taking all of the data together (cell via-bility, drug sensitivity, protein expression, MNase assay, and ChIPassay), the H2B-H2A and H2B-Htz1 fusions seem to be functionalin cells.

Contribution of the H2B-L109 Residue in Fused H2B-H2A and H2B-Htz1to Cell Viability. To determine the functional roles of H2B inH2A/H2B and Htz1/H2B dimers, we point-mutated H2B in theH2B-H2A and H2B-Htz1 fusions (Fig. 3A and Fig. S3A). Thirty-eight phenotypic residues of H2B have been identified by pre-vious screening of the histone-GLibrary (nonfused H2B pointmutant library) (23–25). We individually mutated 3 (H2B-D71,-L109, or -K123) of 38 residues in the H2B-H2A and H2B-Htz1fusions. Expression of H2B-L109A in nonfused H2B-expressingcells caused cell lethality, whereas expression of H2B-D71A or-K123A did not (23, 24). However, the introduction of the lattertwo mutants into nonfused H2B-expressing cells led to cellsbecoming strongly sensitive to HU, MMS, benomyl (a microtu-bule-depolymerizing drug), and/or 6-azauracil (6AU) (23–25).We analyzed the effect of introducing the H2B-L109A muta-

tion into the H2B-H2A and H2B-Htz1 fusions on cell viability.Nonfused HTA1 and HTB1 genes on the URA3 plasmid, whichenabled htaΔ/htbΔ/htz1Δ cells to survive, would be lost duringculture 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 plas-mids, were viable (Fig. 3B, rows 1 and 3, and Fig. S3B, lanes 1 and3). By contrast, cells harboring nonfused H2A, H2B(L109A), andHtz1 were not viable (Fig. 3B, row 2, and Fig. S3B, lane 2), asexpected. Lethality was also observed under 5-FOA conditions incells expressing both mutant fusions [i.e., H2B(L109A)-H2A andH2B(L109A)-Htz1; Fig. 3B, row 4, and Fig. S3B, lane 4].H2A and Htz1 are indispensable and dispensable for cell vi-

ability, respectively (17, 18). Therefore, cells expressing bothH2B(L109A)-H2A and H2B(WT)-Htz1 fusions or both H2B(WT)-H2A and H2B(L109A)-Htz1 fusions should die and growproficiently, respectively. As expected, cells expressing both H2B(WT)-H2A and H2B(L109A)-Htz1 fusions grew well in thepresence of 5-FOA (Fig. 3B, row 6, and Fig. S3B, lane 6), similarto cells expressing both H2B(WT)-H2A and H2B(WT)-Htz1fusions (Fig. 3B, row 3, and Fig. S3B, lane 3). Surprisingly, cellsFig. 2. Functional analysis of H2B-H2A and H2B-Htz1 fusions. (A) Schematic

representation of nonfused and fused H2B-H2A– and H2B-Htz1–containingnucleosomes. (B) Schematic representation of the constructs used to gen-erate fused H2B-H2A (Upper) and H2B-Htz1 (Lower). A C-terminal portion ofH2B was fused in frame to the N terminus of full-length H2A or Htz1. PHTB1,HTB1 promoter. PHTZ1, HTZ1 promoter. (C) Nucleosome structure and en-larged 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 grewslightly poorer on (+) 5-FOA plates than on (−) 5-FOA plates because 5-FOAcan kill cells with the URA3 gene. (E) Schematic representation of nucleo-somes in the strains (htaΔ/htbΔ/htz1Δ background) used for experimentsF–H and J. (1) Cells expressing nonfused H2A, H2B, and Htz1 (WT); (2) cellsexpressing nonfused H2A and H2B, but not Htz1 (htz1-deleted cells); (3) cellsexpressing only the H2B-H2A fusion; and (4) cells expressing both the H2B-H2A 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 asa loading control. (H) MNase analysis of chromatin with digestion for increasingamounts of time. M, 100-bp marker. (I) Schematic representation of the peri-centromeric region of CEN3 and the locations of primer sets used for ChIPanalysis. (J) ChIP analysis of Htz1 and the H2B-Htz1 fusion. Total Htz1 occupancyin WT cells was used for normalization. Error bars represent SD (n = 3).

Fig. 3. Functional analysis of H2B-L109 in H2B-H2A and H2B-Htz1 fusions.(A) Schematic representation of nucleosomes containing nonfused or fusedH2B point mutants. The cell strains examined were as follows: (1) nonfusedWT 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 ofcells expressing fused H2B-H2A and/or H2B-Htz1 with the H2B-L109A pointmutation. (C) Interacting region between H2B-L109, H2A-Y58 (or Htz1-Y65),and H2A-E62 (or Htz1-E69) residues in the nucleosome structure.

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expressing both H2B(L109A)-H2A and H2B(WT)-Htz1 fusionsgrew in the presence of 5-FOA (Fig. 3B, row 5, and Fig. S3B, lane5), although to a lesser extent than cells expressing both H2B(WT)-H2A and H2B(L109A)-Htz1 fusions (Fig. 3B, row 6, andFig. S3B, lane 6). We confirmed that two independent clones iso-lated from cells expressing both H2B(L109A)-H2A and H2B(WT)-Htz1 fusions in the presence of 5-FOA grew extremelyslowly (Fig. S3 C–E). These results suggest that H2B-L109promotes cell viability not only through H2A- but also viaHtz1-containing nucleosomes (Table 1).H2B-L109 physically interacts with the H2A-Y58, H2A-E62,

Htz1-Y65, and Htz1-E69 residues, which are exposed to thenucleosome surface (23, 24, 26). Mutation of the H2A-Y58 and-E62 residues in nonfused H2A- and H2B-expressing cellscaused cell death (23, 24), whereas mutation of the Htz1-Y65and -E69 residues in nonfused Htz1- and H2B-expressing cellsresulted in HU/MMS/benomyl sensitivities (26). Thus, the prox-imity and functional relationship of H2B-L109 to H2A-Y58,H2A-E62, Htz1-Y65, and Htz1-E69 might explain why H2B-L109 is functionally important in both the H2B-H2A and H2B-Htz1 fusions.

H2B-D71 Residue Plays a Specific Role in the H2B-Htz1 Fusion. Wenext mutated another H2B residue, H2B-D71 (Fig. 4A and Figs.S4A and S5A), to investigate its effects on cell growth and sen-sitivity to HU, MMS, and benomyl (24, 25). Cells expressingnonfused H2B-D71A grew slower than WT cells (Fig. 4B, rows 1and 2). Cells expressing both H2B(D71A)-H2A and H2B(WT)-Htz1 fusions grew well (Fig. 4B, row 5), whereas cells expressingboth H2B(WT)-H2A and H2B(D71A)-Htz1 fusions (row 6)grew poorer than cells expressing both WT fusions (row 3). Cellsexpressing both H2B-H2A and H2B-Htz1 fusions in which theH2B-D71 residue was mutated exhibited different HU, MMS,and benomyl sensitivities (Fig. 4B, rows 3–6). Quantitativeanalysis of benomyl sensitivity confirmed that the H2B-D71Amutation in the H2B-Htz1 fusion conferred benomyl sensitivity,whereas the H2B-D71A mutation in the H2B-H2A fusion didnot (Fig. 4C and Table 1).

H2B-D71A Mutation Abolishes the Localization of Htz1 on ChromatinNear to CEN3 and at Promoters. H2B-D71 is located on the H2Bα-helix 2 region and interacts with the H4-L97 and -Y98 residuesin the H4 C-terminal tail region (7, 13, 14, 25). The H4-L97 and

-Y98 residues are located adjacent to a short β-strand of theH2A and Htz1 C-terminal tail regions (Fig. 4D). H4-L97, whichfaces H2B-D71, confers benomyl sensitivity when mutated and isnecessary for the localization of Htz1 at pericentromeric regionsof CEN3 (25), raising the possibility that H2B-D71 is also re-quired 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 in-dicated that the Htz1 level of the fusion near to the CEN3 locuswas the same in cells expressing the H2B(D71A)-H2A fusion asin cells expressing the H2B(WT)-H2A fusion (Fig. 4E, lanes 3and 5). By contrast, the level of the H2B(D71A)-Htz1 fusionnear to the CEN3 locus differed between cells expressing H2B(D71A)-H2A and cells expressing H2B(WT)-H2A (lanes 4 and6). Consistent with this, the introduction of the H2B-D71A pointmutation into nonfused H2B dramatically reduced the Htz1ChIP signal near to the CEN3 locus compared with the signal inWT cells (Fig. 4E, lanes 1 and 2). The same results were obtained

Table 1. Effects of H2B mutation on the H2B-H2A and H2B-Htz1fusions

H2B mutation Analyzed phenotype

Effects of mutationon

H2B-H2A H2B-Htz1

D71A Growth − ++HU sensitivity + +MMS sensitivity + +Benomyl sensitivity − ++H2A chromatin localization + −Htz1 chromatin localization − ++

L109A Cell viability* +* +*Growth ++ −

K123A 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 inspot assay and as lethal (−) by the absence of those cells, respectively.

Fig. 4. Functional analysis of H2B-D71 in H2B-H2A and H2B-Htz1 fusions.(A) Schematic representation of nucleosomes containing nonfused or fusedH2B-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 sensitivityanalysis of fused H2B-H2A and/or H2B-Htz1 with the H2B-D71A point mu-tation. (C) Yeast survival analysis after exposure to benomyl for a shortamount of time. The survival rate of each strain was calculated by dividingthe total number of colonies at each time point by the number of colonies att = 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 nucleosomestructure. (E and F) ChIP analysis of Htz1 or the H2B-Htz1 fusion (E) and H2A orthe H2B-H2A fusion (F) in the indicated cells. Total Htz1 and H2A occupancies inWT cells were used for normalization. Error bars represent SD (n = 3).

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at the promoters of the LSB5 and GID7 genes (Fig. S4B). Thesedata indicate that the H2B-D71A mutation dramatically reducesthe localization of the H2B-Htz1 fusion and nonfused Htz1 atchromatin. On the other hand, the levels of nonfused Htz1 (Fig.S5B, lane 2) and H2B(D71A)-Htz1 fusion proteins (lanes 4 and6), and their corresponding mRNAs (Fig. S5C, lanes 2, 4, and 6),were not substantially lower than the levels of the WT proteinsand transcripts (Fig. S5 B and C, lanes 1 and 3). Thus, the H2B-D71A mutation likely affects some functions of the dimer, suchas incorporation into chromatin, rather than the protein stabilityof 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 CEN3and at these two promoters (Fig. 4F and Fig. S4C, lanes 3) wasnot significantly altered by the introduction of the H2B-D71Amutation into the H2B-H2A fusion and/or the H2B-Htz1 fusion(Fig. 4F and Fig. S4C, lanes 4–6). These results suggested thatthe H2B-D71A mutation markedly affected Htz1-containingnucleosomes but not H2A-containing nucleosomes (Table 1).Furthermore, the introduction of a different point mutation intoH2B, H2B-K123A, did not affect the localization of Htz1 lo-calization at the CEN3 locus (Fig. S6). Thus, it is suggested thatthe proper localization of H2B-Htz1 at the CEN3 locus requiresthe H2B-D71 residue and that this residue is important formaintaining the function of Htz1-containing nucleosomes (Table1). 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 thelocalization of Htz1 around CEN3 (25). Therefore, we prefera model in which the H2B-D71A mutant cannot bind the (H3-H4)2 tetramer or can be easily released from the nucleosome.

H2B-K123 Predominantly Functions in the H2B-H2A Fusion. We nextexamined the effect of the introduction of the H2B-K123Amutation into the H2B-H2A and H2B-Htz1 fusions on the sen-sitivity of cells to HU, MMS, and/or 6AU (Fig. 5 A and B). 6AUdisturbs the balance of intracellular nucleotide concentrationsand blocks transcription elongation. Cells expressing nonfusedH2B-K123A were sensitive to HU, MMS, and 6AU (Fig. 5B,rows 1 and 2), as were cells expressing both H2B(K123A)-H2Aand H2B(K123A)-Htz1 fusions (Fig. 5B, rows 3 and 4). Underthe same conditions, cells expressing both H2B(K123A)-H2Aand 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 beimportant in the H2B-H2A fusion, but not in the H2B-Htz1fusion, the function of H2B-K123 in the H2A/H2B dimer seemsto be different from its function in the Htz1/H2B dimer.

Monoubiquitination Occurs on Both the H2B-H2A and H2B-Htz1Fusions. Among the three H2B residues (H2B-D71, -L109, and-K123) analyzed in this study, only H2B-K123 is chemically modi-fiable. Specifically, H2B-K123 can be monoubiquitinated (20), amodification that is important in transcription elongation (27, 28),DNA replication (29), DNA repair (30), and di-/trimethylation oflysine 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 comparedthe state of H2B monoubiquitination in cells expressing non-mutated fusions to that in cells expressing the H2B(K123A)-H2A and/or H2B(K123A)-Htz1 fusions (Fig. 5C). The status ofH2B-K123 monoubiquitination was indirectly evaluated by theappearance of a band migrating slightly higher than 30 kDa, thesize at which the H2B-H2A and H2B-Htz1 fusions were detectedusing anti-H2A and anti-Htz1 antibodies, respectively (Fig. 2G).

Higher molecular weight bands of ∼37 kDa were detected incells expressing the H2B-H2A fusion (Fig. 5C, Top, lanes 3 and6) and the H2B-Htz1 fusion (Middle, lanes 3 and 5). These bandswere 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 ismonoubiquitinated at K123, irrespective of whether H2B is pairedwith 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 fu-sion (lane 3), suggesting that the lack of monoubiquitination inthe H2B-H2A fusion (Fig. 5C, Top, lane 5) leads to accumula-tion of the monoubiquitinated H2B-Htz1 fusion (Middle, lane 5)in cells. The FALC strategy successfully determines whetherH2B-K123 is monoubiquitinated in the H2B-H2A fusion and/orthe H2B-Htz1 fusion; therefore, a variety of chemical mod-ifications of residues (such as S10, K11, and K16) (33–35) in thetail region of H2B could be tested to discern other roles playedby H2B depending on whether it is paired with H2A or Htz1.

DiscussionIn this study, we developed the FALC strategy to overcome thedifficulties associated with functional analyses of proteins thatare subunits of more than one multisubunit complex (Fig. 1). Weused H2B as a common subunit, and H2A- and Htz1-containingnucleosomes as multisubunit complexes (Figs. 2–5). To imple-ment the FALC strategy, we made functional H2B-H2A andH2B-Htz1 fusions (Fig. 2) and analyzed the phenotypes follow-ing point mutations of three H2B residues (H2B-D71, -L109,and -K123) in nonfused H2B, the H2B-H2A fusion, and theH2B-Htz1 fusion (Figs. 3–5 and Table 1). This innovative FALCstrategy led to unique functional information about H2A, Htz1,and H2B, which could not have been revealed by analysis ofcombinations 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 H2A-containing nucleosomes predominantly contributes to support cellviability, as expected (Fig. 3). Unexpectedly, it was revealed that

Fig. 5. Functional analysis of H2B-K123 in H2B-H2A and H2B-Htz1 fusions.(A) Schematic representation of nucleosomes containing nonfused or fusedH2B-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 sensitivityanalysis of fused H2B-H2A and/or H2B-Htz1 with the H2B-K123A point mu-tation. (C) Immunoblot analysis of monoubiquitination of H2B-K123 in H2B-H2A and H2B-Htz1. Lanes labeled 1 show a series of fourfold dilutions ofsamples from WT cells. Histone H4 was used as a loading control.

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H2B-L109 in Htz1-containing nucleosomes also supports cell vi-ability, although with a lesser contribution than in H2A-containingnucleosomes (Fig. 3). By contrast, cell growth and resistance tobenomyl largely relied on the H2B-D71 residue of fused H2B-Htz1 (Fig. 4). The localization of Htz1 at promoters and aroundCEN3 was abolished with the H2B-D71A mutant, but not with theH2B-K123A mutant, suggesting that the H2B-D71 and H2B-K123residues have different structural and functional roles (Fig. 4).Although the structures of H2A- and Htz1-containing nucleo-somes have been determined (7, 12, 13), the current study re-vealed the role of H2B-D71 in these nucleosomes by using a FALCstrategy. H2B-K123 is most likely to be monoubiquitinated inboth H2B-H2A and H2B-Htz1 fusions (Fig. 5). On the otherhand, H2B-K123 was functional within H2A- but not withinHtz1-containing nucleosomes, as shown by drug sensitivityexperiments (Fig. 5). Thus, in drug sensitivity-related reactions,monoubiquitinated H2B-K123 seems to have different functionsdepending on whether it is paired with H2A or Htz1. It will beinteresting to reveal the roles of monoubiquitination of H2B-K123 in the H2B-Htz1 fusion.Although some common phenotypes resulted from the pres-

ence of the H2B(mut)-H2A fusion or the H2B(mut)-Htz1 fusion(Table 1), distinct phenotypes were also detected. The FALCstrategy led to the successful determination of distinct roles foran individual residue in a common subunit, H2B, depending onwhether it was part of the H2B-H2A or H2B-Htz1 fusion.Generalizing the FALC strategy to other multisubunit com-

plexes (Fig. 1) is an interesting challenge. It is not known how toselect a specific subunit that is linked to the common subunit, es-pecially when the structural information of a multisubunit complex

is unavailable. To overcome this challenge, experimental pre-dictions could be made by gaining topological information oncommon and specific subunits in multisubunit complexes via theFRET assay, electron microscopy, and protein cross-linking. Us-ing the latter technique, information about the topology betweencommon subunits (such as Act1 and Arp4) and specific subunits ofthe INO80 complex (Fig. 1B) has recently been gained (36). In-troducing mutations into a protein fusion is another critical stepof the FALC strategy. Comprehensive mutagenesis analysis(GLibrary strategy = GLASP + GLAMP) (23–26) of the targetcommon subunit(s) would greatly facilitate mutagenic analysesof fused complexes, as shown in this study (Figs. 3–5 and Table 1).Thus, combining the FALC strategy with mutational analysesopens a new avenue to address the functional roles of a commonsubunit(s) in multiple multisubunit complexes in cells.

Materials and MethodsDetailed information on materials and methods in this study is provided in SIMaterials and Methods. Information on materials includes the characteristicsof yeast strains and construction of plasmids. Technical information onmethods contains drug sensitivity assays, immunoblot analysis, Northern blotanalysis, 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 foradministrative assistance. We also thank all members of our laboratoriesfor fruitful discussion of this study. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports,Science and Technology of Japan and a Grant-in-Aid for a Japan Society forthe Promotion of Science Research Fellow.

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