Relaxed Chromatin Formation and Weak ... - ACS Publications

Subscriber access provided by NEW MEXICO STATE UNIV

Article

Relaxed chromatin formation and weak suppression of homologous pairing by the testis-specific linker histone H1T Shinichi Machida, Ryota Hayashida, Motoki Takaku, Atsuhiko Fukuto, Jiying Sun, Aiko Kinomura, Satoshi Tashiro, and Hitoshi Kurumizaka Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b01126 • Publication Date (Web): 12 Jan 2016 Downloaded from http://pubs.acs.org on January 13, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Relaxed chromatin formation and weak suppression of homologous pairing by the testis-specific linker histone H1T Shinichi Machida,† Ryota Hayashida,† Motoki Takaku,† Atsuhiko Fukuto,‡ Jiying Sun,‡ Aiko Kinomura,‡ Satoshi Tashiro,‡ and Hitoshi Kurumizaka†§,* †

Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan

‡

Department of Cellular Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan

§

Institute for Medical-oriented Structural Biology, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan

KEYWORDS nucleosome, chromatosome, chromatin, linker histone, H1T, homologous recombination, RAD51, RAD54

ACS Paragon Plus Environment

1

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 33

ABSTRACT

Linker histones bind to nucleosomes and compact poly-nucleosomes into a higher-order chromatin configuration. Somatic and germ cell-specific linker histone subtypes have been identified, and may have distinct functions. In the present study, we reconstituted polynucleosomes containing human histones H1.2 and H1T, as representative somatic and germ cellspecific linker histones, respectively, and found that H1T forms less compacted chromatin, as compared to H1.2. An in vitro homologous-pairing assay revealed that H1T weakly inhibited RAD51/RAD54-mediated homologous pairing in chromatin, although the somatic H1 subtypes, H1.0, H1.1, H1.2, H1.3, H1.4, and H1.5, substantially suppressed it. An in vivo recombination assay revealed that H1T overproduction minimally affected the recombination frequency, but significant suppression was observed when H1.2 was overproduced in human cells. These results suggested that the testis-specific linker histone, H1T, possesses a specific function to produce the chromatin architecture required for proper chromosome regulation, such as homologous recombination.


2

Page 3 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

INTRODUCTION In the nucleus, genomic DNA is organized and packaged in chromatin. The fundamental repeating unit of chromatin is the nucleosome. In the nucleosome, a histone octamer is formed by two copies each of the core histones, H2A, H2B, H3, and H4, and 145-147 base pairs of DNA are left-handedly twisted around the histone octamer1-3. Nucleosomes are connected by linker DNAs and form poly-nucleosomes, which are further compacted as higher-order chromatin4. In addition to the core histones, comparable amounts of linker histones (H1 and its subtypes) also exist in higher eukaryotes5,6. Linker histones exhibit high affinity binding to nucleosomes containing linker DNAs and form chromatosomes7-10, and are considered as key regulatory factors for the formation of higher-order chromatin5,6. The linker histone family proteins have common structural features, with flexible N- and C-terminal regions and a central globular domain11,12. Cryo-electron microscopic and crystallographic studies revealed that H1 and the globular domain of H5 (a chicken erythrocyte subtype) bind near the nucleosomal dyad, and the DNA ends at the entry/exit sites of the nucleosome are compacted by direct interactions with the linker histones13,14. Seven human somatic H1 subtypes, H1.0, H1.1, H1.2, H1.3, H1.4, H1.5, and H1x, have been identified5,6. In addition, four H1 subtypes, H1T, H1T2, HILS1, and H1foo, are expressed in germ cell-specific manners5,6. A previous in vivo study with exogenously produced H1 proteins revealed that the somatic H1 subtypes, H1.0, H1.1, H1.2, H1.3, H1.4, and H1.5, have distinct mobilities in the nucleus and different localization preferences for heterochromatin and euchromatin15. An atomic force microscopic analysis of the somatic H1 subtypes demonstrated that H1.1, H1.2, and H1.3 have weak/intermediate chromatin condensation abilities, whereas H1.0, H1.4, H1.5, and H1x are strong chromatin condensers16. Genomic localization analyses


3

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 33

with H1.1, H1.2, H1.3, H1.4, and H1.5 revealed the subtype-specific localization in human cells17, suggesting that the H1 subtypes function to mark genomic domains by forming specific types of higher-order chromatin organization. A linker histone, which was probably H1T, prepared from rat testis reportedly exhibited weak chromatin condensation ability18,19. This suggested that the testis-specific H1 subtype may have a unique function for achieving the chromatin organization required for meiosis and/or spermatogenesis. Interestingly, in rat primary spermatocytes, about 60% of the total linker histone is replaced by H1T20-22. Homologous recombination occurs in this cellular stage, and the recombined paternal and maternal chromosomes are accurately segregated into daughter cells23,24. RAD51 is a eukaryotic recombinase25-28, and it requires a chromatin remodeler, RAD54, to promote homologous pairing in chromatin29-33. We previously found that the somatic linker histone H1.2 significantly inhibits the RAD51/RAD54-mediated homologous pairing in chromatin34. However, the homologous pairing in chromatin containing testis-specific linker histones has not been characterized. In the present study, we found that the human linker histone H1T binds to nucleosomes as efficiently as H1.2, but forms less compacted chromatin in vitro. Interestingly, the homologouspairing suppression by H1T in chromatin is quite weak, as compared to the suppression by the somatic H1 subtypes, H1.0, H1.1, H1.2, H1.3, H1.4, and H1.5, in vitro. Consistently, H1T overproduction in human cells minimally affected the in vivo recombination frequency, although H1.2 overproduction substantially reduced it. Therefore, H1T may form a specific chromatin structure that is suitable for homologous recombination in primary spermatocytes.

EXPERIMENTAL PROCEDURES


4

Page 5 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Preparation of Recombinant Proteins. The DNAs encoding human histone H1 subtypes (H1.2, H1.3, H1.4, H1.5, H1.1, H1.0, and H1T) were inserted between the NdeI and XhoI sites of the pET21a vector, and the proteins were expressed in Escherichia coli BL21 (DE3) cells, as the C-terminally SUMO-fused proteins. A minor tRNA expression vector (Codon(+)RIL; Stratagene) was co-expressed in the E. coli cells.

Recombinant histone H1 proteins were

purified by the method described previously34. During the purification procedure, the SUMOHis6 portion was removed by PreScission protease. Finally, the purified recombinant H1 proteins contained six amino acid residues, Leu-Glu-Val-Leu-Phe-Gln, at their C-terminal ends. The H1T mutants were expressed in E. coli BL21 (DE3) cells, and were purified by the same method used for the H1 subtypes. In the H1T-N mutant, the H1T N-terminal tail (amino acid residues 1-40) was replaced by the H1.2 N-terminal tail (amino acid residues 1-36). In the H1T-G mutant, the H1T central globular domain (amino acid residues 41-111) was replaced by the H1.2 central globular domain (amino acid residues 37-107). In the H1T-C protein, the H1T C-terminal tail (amino acid residues 112-207) was replaced by the H1.2 C-terminal tail (amino acid residues 108-213). Human RAD5135, RAD5434, Nap136, and histones37 (H2A, H2B, H3.1, and H4) were purified by the method described previously (Supplementary Figure 1B and C). Preparation of DNAs. The 5S 90-mer single-stranded oligonucleotide (ssDNA) used for the D-loop formation assay was purchased from Nihon Gene Research Laboratory, as an HPLCpurified oligonucleotide (5’- CCG GTA TAT TCA GCA TGG TAT GGT CGT AGG CTC TTG CTT GAT GAA AGT TAA GCT ATT TAA AGG GTC AGG GAT GTT ATG ACG TCA TCG GCT-3’). The superhelical double-stranded DNA (dsDNA) was prepared by the method without alkaline treatment of cells, as described previously38. The 193 base-pair dsDNA containing the Widom601 DNA sequence39 was prepared by the method described previously40. The dsDNA


5

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 33

containing twelve 208 base-pair Widom601 DNAs was prepared by the method described previously41. The DNA concentrations are expressed as moles of nucleotides. Preparation of Chromatin. Nucleosomes were reconstituted with the 193 base-pair dsDNA by the salt dialysis method, as described previously40. The poly-nucleosomes for the D-loop formation assay were reconstituted with the purified histone octamer and the superhelical dsDNA containing 5S DNA repeats, in a histone octamer : DNA (200 base pairs) ratio = 133,34. The polynucleosomes for the sedimentation assay were reconstituted with the purified histone octamer and the dsDNA containing the Widom601 repeat, at a histone octamer : DNA (208 base pairs) ratio = 1.8, and were further purified by nondenaturing agarose-acrylamide composite gel (0.7% agarose and 2% acrylamide) electrophoresis, using a Prep cell apparatus (Bio-Rad). Assay for Histone H1 Binding to Nucleosomes. Histone H1.2 or H1T (0.2, 0.3, 0.4, and 0.5 µM) was mixed with one-half the amount of Nap1, and the samples were kept on ice for 10 min. Each H1-Nap1 mixture was added to nucleosomes (30 µM) reconstituted with the 193 base-pair DNA in the reaction solution (10 µl), containing 22 mM HEPES-NaOH (pH 7.5), 9 mM TrisHCl (pH 7.5), 43 mM NaCl, 60 mM KCl, 0.2 mM EDTA, 1 mM DTT, 0.6 mM 2mercaptoethanol, 15 µM phenylmethylsulfonyl fluoride, 7.5% glycerol, 1 mM MgCl2, 1 mM CaCl2, and 100 µg/ml BSA. After a 15 min incubation at 37°C, the reaction mixtures were analyzed by 5% native polyacrylamide gel electrophoresis in 1x TBE buffer with EtBr staining. Analytical Ultracentrifugation. Histones H1.2 and H1T (1.8 µM) were each mixed with Nap1 (0.9 µM), and the H1/Nap1 mixtures were incubated with the poly-nucleosomes (120 µM) reconstituted on the twelve Widom601 repeats, in a reaction solution containing 30 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.1 mM EDTA, 2 mM DTT, 0.4 mM 2-mercaptoethanol, and 4% glycerol. The poly-nucleosomes with H1.2 and H1T were dialyzed against 10 mM Tris-HCl (pH


6

Page 7 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

7.5) buffer. Sedimentation velocity analyses were performed using a ProteomeLab XL-I analytical centrifuge (Beckman Coulter). The initial absorbance of the samples at 260 nm was between 0.5 and 0.8. The samples in 12 mm double-sector cells were equilibrated for 2 h at 20ºC under a vacuum, and were centrifuged at 22,000 rpm. Scans at 260 nm were collected in the continuous scanning mode with a 0.03 cm radial step size. Collected data were analyzed by the enhanced van Holde-Weischet method using UltraScanII 9.9 revision 1927, as reported previously42. Sedimentation coefficients (S20,W) were calculated with a partial specific volume of 0.65 ml/g. ScaI Analysis. The ScaI analysis was performed by the method reported previously41, with minor modifications. ScaI (14 units) was added to the poly-nucleosomes (30 µM) reconstituted on the twelve Widom601 repeats with or without a linker histone, in a reaction solution containing 15 mM Tris-HCl (pH 7.5), 55 mM NaCl, 1 mM DTT, 100 µg/ml BSA, 5% glycerol, and 0.5 mM MgCl2, and the samples were incubated at 22°C for 12 h. After complete digestion, products were analyzed by 5% native polyacrylamide gel electrophoresis in 1x TBE buffer with EtBr staining. Homologous Pairing Assay. The homologous pairing assay with the reconstituted polynucleosomes was performed according to the previously described method, with minor modifications34. RAD51 (400 nM) was mixed with the

32

P-labeled 5S 90-mer ssDNA, and the

RAD51-ssDNA complex was formed by an incubation at 37°C for 10 min, in the presence of 1 mM MgCl2, 1 mM CaCl2, and 1 mM ATP. The 5S 90-mer ssDNA sequence is homologous to the 5S rDNA. Histones H1.2 and H1T were incubated with the poly-nucleosomes (30 µM in nucleotides) in the presence of Nap1 (one-half the amount of H1) at 37°C for 10 min. The RAD51-ssDNA complex and RAD54 (400 nM) were mixed with the poly-nucleosomes with


7

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 33

H1.2 or H1T in the reaction solution (10 µl), containing 22 mM HEPES-NaOH (pH 7.5), 9 mM Tris-HCl (pH 7.5), 43 mM NaCl, 80 mM KCl, 0.2 mM EDTA, 1 mM DTT, 0.6 mM 2mercaptoethanol, 15 µM phenylmethylsulfonyl fluoride, 7.5% glycerol, 1 mM MgCl2, 1 mM CaCl2, 1 mM ATP, 20 mM creatine phosphate, 75 µg/ml creatine kinase, and 100 µg/ml BSA. After an incubation at 37°C for 10 min, the stop solution, containing SDS (0.2%) and proteinase K (1.4 mg/ml, Roche Applied Science), was added, and the products were separated by 1% agarose gel electrophoresis. The gels were dried and exposed to an imaging plate. The gel images were obtained using an FLA-7000 imaging analyzer (GE Healthcare). Homologous Recombination Assay. The homologous recombination assay was performed as reported previously43,44. Human U2OS-DR-GFP cells were transfected with the 3xFLAG-H1.2 or -H1T plasmid using GeneJuice (Novagen), and selected with G418 to obtain stably expressing cells. To induce DSBs, the I-SceI expression vector (pCBASce, 5 µg) was introduced into U2OSDR-GFP cells by transfection, using Lipofectamine 2000 (Thermo Fisher Scientific). To increase the protein expression level, 3 µg of 3xFLAG-H1.2 or -H1T was transfected together with pCBASce, respectively. Two days after transfection, GFP-positive cells were quantified by flow cytometry (FACSCanto II flow cytometer, Becton Dickinson). The expression levels of FLAG(3x)-H1.2 and -H1T in U2OS-DR-GFP cells were analyzed by western blotting. The 3xFLAG-H1.2 and -H1T proteins were detected by an anti-FLAG monoclonal antibody (SIGMA), and the 3xFLAG-H1.2 and endogenous H1.2 proteins were detected by an anti-H1.2 polyclonal antibody.

RESULTS


8

Page 9 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Linker Histone H1T Binds to Nucleosomes as Efficiently as Canonical H1.2. In chromatin, the linker histone H1 binds to nucleosomes and forms compacted higher-order chromatin at the poly-nucleosome level. H1T is considered to have a distinct character among the H1 isoforms, for the following reasons. Firstly, H1T is known as a testis-specific linker histone isoform, suggesting its function in chromatin reorganization during spermatogenesis5,6,21,22. Secondly, the amino acid sequence of the C-terminal half of H1T is significantly different from those of the somatic H1 subtypes5. The linker and core histones were bacterially expressed and purified to near homogeneity (Supplementary Figure 1A and B). Poly-nucleosomes containing twelve nucleosomes with the Widom601 sequence (208 base pairs) were used as the substrate (Figure 1A). The polynucleosomes were reconstituted by the salt-dialysis method, and purified by nondenaturing agarose-acrylamide composite gel electrophoresis. The purified poly-nucleosomes were then analyzed by native agarose gel electrophoresis (Figure 1B, lane 1). The linker histone H1.2 or H1T was then loaded onto the poly-nucleosomes in the presence of the histone chaperone Nap1, which is known to mediate proper H1 loading onto poly-nucleosomes9,34,45,46. The gel electrophoretic mobility shift assay (EMSA) revealed that both H1.2 and H1T efficiently bound to the poly-nucleosomes (Figure 1B, lanes 2 and 3). As a control experiment, mock linker histone loading without H1 was also performed, in the presence of Nap1 (Figure 1B, lane 4). To confirm the H1 binding to each nucleosome of the poly-nucleosome, the linker DNAs were cleaved by ScaI, and the resulting mono-nucleosomes containing H1 were analyzed by EMSA (Figure 1C). In this assay, nucleosome-free DNAs and H1-free nucleosomes were separately detected from the H1-nucleosome complex. After ScaI treatment of the reconstituted polynucleosome sample, only a trace amount of the free DNA was detected, although the mono-


9

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 33

nucleosome band was clearly observed (Figure 1C, lane 2). This indicated that the nucleosomes were efficiently formed on every Widom601 sequence in the poly-nucleosome. When the polynucleosomes complexed with H1.2 or H1T were treated with ScaI, the bands corresponding to the mono-nucleosomes bound to H1.2 or H1T were clearly observed (Figure 1C, lanes 3 and 4). Notably, no H1-free nucleosome bands were detected in these samples (Figure 1C, lanes 3 and 4). These results indicated that both H1.2 and H1T efficiently bound to every mono-nucleosome in the poly-nucleosomes. To confirm the efficient H1T binding to the nucleosome, we performed EMSA with the mono-nucleosome reconstituted with a 193 base-pair DNA. The results revealed that H1T bound to the nucleosome with linker DNAs as efficiently as H1.2 (Figure 1D and E).

Linker Histone H1T Forms a Relaxed Configuration of Chromatin. We next tested the effect of H1T binding on the poly-nucleosome configuration by an analytical ultracentrifugation sedimentation assay42. The reconstituted poly-nucleosomes (Figure 1B, lane 1) exhibited a sedimentation value of -30S (the 50% boundary value) under the experimental conditions used in this analysis (Figure 2A). Nap1 did not affect the sedimentation values of the poly-nucleosomes (Figure 2A). When H1.2 was loaded onto the poly-nucleosomes in the presence of Nap1, the sedimentation value of the poly-nucleosomes drastically increased to -43S (50% boundary value) (Figure 2B). This indicated that H1.2 causes the poly-nucleosomes to adopt a more compacted configuration. Interestingly, when H1T was loaded onto the poly-nucleosomes, the increment of the sedimentation value was clearly smaller (Figure 2B, -38S, 50% boundary value), indicating that H1T compacted the poly-nucleosomes less densely than H1.2. Since the H1T binding to the poly-nucleosomes was quite similar to the H1.2 binding (Figure 1B and C), these sedimentation-


10

Page 11 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

assay data indicate that H1T forms a more relaxed chromatin configuration upon binding to the poly-nucleosomes, as compared to H1.2.

Weak Suppression of in vitro Homologous Pairing in Chromatin by H1T. Homologous recombination is a major event occurring at an early stage of meiotic cell division in the testis. Homologous pairing is a central step in the homologous-recombination process, and inter- or intra-chromosomal homologous DNA sequences are paired by this reaction23,24,47-49. In chromatin, RAD51 promotes the homologous pairing reaction with the aid of RAD54, a chromatin remodeling factor29-34. Previously, we reported that a canonical H1 subtype, H1.2, substantially suppresses the RAD51/RAD54-mediated homologous pairing in chromatin34. We then tested the effect of the testis-specific H1T on homologous pairing in chromatin with purified proteins (Supplementary Figure 1C). As the chromatin template, poly-nucleosomes were prepared by the salt dialysis method with the superhelical dsDNA. This poly-nucleosome substrate contains 12 positioned nucleosomes, and the G5E4 promoter DNA segment for two nucleosomes is located at the center of the twelve positioned nucleosomes. Five tandem repeats of 5S rDNA flank both sides of the G5E4 promoter segment32-34 (Figure 3A). H1.2 or H1T was assembled onto the poly-nucleosomes in the presence of Nap1. We then performed an in vitro RAD51/RAD54-mediated homologous pairing assay (Figure 3B). In this assay, a

32

P-labeled

ssDNA 90-mer containing the homologous sequence to the 5S rDNA was used as the ssDNA substrate, and homologous pairing between the ssDNA and the poly-nucleosomes was detected by the D-loop formation assay (Figure 3B). Consistent with previous results, H1.2 efficiently inhibited the RAD51/RAD54-mediated Dloop formation (homologous pairing) in chromatin (Figure 3C, lanes 3-6). Intriguingly, we found


11

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 33

that H1T exhibited very weak H1-mediated suppression of homologous pairing, as compared to the canonical H1.2 (Figure 3C and D).

The N-terminal and C-terminal Tails of H1T Are Responsible for the Weak Suppression of Homologous Pairing in Chromatin. To study the H1T domains involved in the weak suppression of homologous pairing in chromatin, we purified three swapping mutants, H1T-N, H1T-G, and H1T-C, in which each H1T N-terminal tail, central globular domain, and C-terminal tail was replaced by the corresponding region of H1.2 (Figure 4A, and Supplementary Figure 1D). We then performed the D-loop formation assay with the reconstituted chromatin template, and tested the inhibitory effects of H1T-N, H1T-G, and H1T-C. We found that the H1T-G mutant exhibited low inhibition ability for the RAD51/RAD54-mediated homologous pairing, similar to H1T, indicating that the central globular domain of H1.2 may not be responsible for the strong suppression ability of H1.2 (Figure 4B and C). In contrast, the H1T-N and H1T-C mutants exhibited marked inhibition activity for homologous pairing (Figure 4B and C). These results suggested that the N-terminal and C-terminal tails of H1.2 induce the strong suppression of the RAD51/RAD54-mediated homologous pairing in chromatin.

Weak Suppression of Homologous Pairing in Chromatin is a Unique Characteristic of H1T. We next tested the homologous pairing suppression by other H1 subtypes. To do so, we purified human histones H1.0, H1.1, H1.3, H1.4, and H1.5, as recombinant proteins (Supplementary Figure 1A and B), and performed the RAD51/RAD54-mediated homologous pairing reaction in chromatin. Reproducibly, H1.2 significantly suppressed the homologous pairing in chromatin (Figure 5A and B, lanes 3-5), and the H1T-dependent suppression was very


12

Page 13 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

weak (Figure 5B, lanes 12-14). Interestingly, we found that H1.0, H1.1, H1.3, H1.4, and H1.5 robustly suppressed the RAD51/RAD54-mediated homologous pairing reaction in chromatin, as well as H1.2 (Figure 5A and B). Therefore, the chromatin containing H1T has a unique property that allows homologous recombination to occur more efficiently.

H1T Does Not Suppress Homologous Recombination in Human Cells. We finally tested whether H1T production is less suppressive for homologous recombination in cells. To do so, we performed the homologous recombination assay in U2OS DR-GFP cells43,44 (Figure 6A). In this assay system, the inactive GFP mutant gene containing an I-SceI cleavage site is repaired by homologous recombination with an 812 base-pair DNA fragment encoding the GFP sequence (GFP∆), as the donor (Figure 6A). As a result, the inactive GFP mutant gene is replaced by the intact GFP gene sequence, and wild type GFP is expressed in cells. The frequency of homologous recombination was then estimated, as the number of GFP-positive cells. We established stable cell lines expressing FLAG-tagged H1.2 and H1T. We confirmed that FLAGH1.2 was overproduced, as compared to endogenous H1.2 in the cells (Figure 6B). The expression levels of FLAG-H1.2 and FLAG-H1T were nearly equal (Figure 6B). As shown in Figure 6C, the homologous recombination frequency in the cells producing H1.2 was reduced by approximately 35%. In contrast, the cells producing H1T displayed only a 7% reduction. These results are perfectly consistent with the in vitro homologous pairing data. Therefore, we conclude that the testis-specific H1T is a unique linker histone that forms a relaxed configuration of chromatin for meiotic events, such as homologous recombination.

DISCUSSION


13

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 33

In rat primary spermatocytes, H1T reportedly represents up to ~60% of the total amount of linker histones20-22. Robust expression of H1T has also been reported in human primary spermatocytes50. These facts suggest that H1T probably has an important function in this cellular stage during spermatogenesis. Interestingly, the first meiotic cell division occurs in primary spermatocytes, and meiotic homologous recombination is essential for accurate chromosome segregation at this stage23,24. Therefore, the chromatin architecture containing H1T may have a unique character that facilitates homologous recombination in primary spermatocytes. In the present study, we analyzed the effect of H1T on the primary and secondary chromatin structure. We found that H1T and the canonical H1.2 bind to mono- and poly-nucleosomes with indistinguishable affinities (Figure 1). As expected, canonical H1.2 drastically compacted the poly-nucleosomes (Figure 2). However, the chromatin compaction by H1T was significantly low (Figure 2). H1T only weakly inhibited the in vitro homologous pairing in chromatin, although other somatic subtypes, H1.0, H1.1, H1.3, H1.4, and H1.5, significantly suppressed it as well as H1.2 (Figure 3 and Figure 5). Importantly, the in vivo homologous recombination assay consistently revealed that H1T is less suppressive for homologous recombination in cells (Figure 6). These results are the first demonstration that a testis-specific linker histone subtype actually has a specific character for the promotion of a meiotic event. In contrast, H1.1 reportedly exhibits a unique distribution profile in the human lung fibroblast genome17. Our homologouspairing assay revealed that the suppression function of H1.1 (and H1.0) may be stronger than that of the other somatic subtypes, H1.2, H1.3, H1.4, and H1.5 (Figure 5). These in vivo and in vitro distinctions suggest that H1.1 may have a unique function. Spermatogenesis occurs normally in the H1T knockout mice51-53, suggesting that the other testis-specific H1 subtype may complement the H1T function. Actually, in humans, two more


14

Page 15 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

testis-specific H1 subtypes, H1T2 and HILS1, and one oocyte-specific 1 subtype, H1foo, have been identified5,54-56. Interestingly, H1T-C, which contains the H1.2 C-terminal tail, suppressed the RAD51/RAD54-mediated homologous pairing in chromatin, to a level similar to H1.2 suppression (Figure 4). The amino acid sequence of the H1.2 C-terminal tail (amino acids 108213) is highly conserved among the somatic H1.0 (25%, amino acids 96-194), H1.1 (33%, amino acids 111-215), H1.3 (32%, amino acids 109-221), H1.4 (28%, amino acids 108-219), and H1.5 (35%, amino acids 111-226) subtypes, but not in the germ-cell specific H1T (16%, amino acids 112-207), H1T2 (11%, amino acids 123-255), HILS1 (9%, amino acids 123-231), and H1foo (7%, amino acids 128-346). The diversity among the less-conserved C-terminal tail sequences of the germ-cell H1 subtypes may be responsible for their specific functions during spermatogenesis and oogenesis. To understand the structural and functional transitions of chromatin during spermatogenesis and oogenesis, it is intriguing to study the biochemical and biophysical features of these germ cell-specific H1 subtypes, to elucidate their structural and functional roles in chromatin transitions during spermatogenesis. Specific core histone variants have also been identified in the testis57. In humans, the testisspecific histone H3T forms the nucleosome with local distortion, and the H3T nucleosome exhibits extreme instability in vitro and in vivo37. In addition, the histone H2A and H2B variants produced in the testis also reportedly have distinctive structures and physical properties in the nucleosomes58-64. Further analyses of these testis-specific core histones combined with testisspecific linker histones will be important to understand the organization of functional chromatin and its structural transitions during spermatogenesis.


15

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 33

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Preparation of recombinant human histone H1 subtypes, H1T mutants, core histones, a histone octamer, RAD51, RAD54, and Nap1. (PDF)

AUTHOR INFORMATION Corresponding Author *Tel +81-3-5369-7315; Fax +81-3-5367-2820; e-mail [email protected]. Present Addresses Motoki Takaku: Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, 27709, USA Author Contributions S.M., R.H., and M.T. purified proteins, prepared mono- and poly-nucleosomes, and performed biochemical analyses. A.F., J.S., A.K., and S.T. performed cell biological experiments. H.K. conceived, designed, and supervised all of the work. S.M. and H.K. wrote the paper. All of the authors discussed the results and commented on the manuscript. Funding Sources This work was supported in part by MEXT KAKENHI Grant Number 25116002 [to H.K.], JSPS KAKENHI Grant Number 25250023 [to H.K.] and Grant Number 26890023 [to S.M.], and was also partially supported by grants from the Platform Project for Supporting Drug Discovery and


16

Page 17 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science), from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and the Japan Agency for Medical Research and Development (AMED) [to H.K.]. H.K. was also supported by the Waseda Research Institute for Science and Engineering. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We thank Dr. Craig L. Peterson (University of Massachusetts) for providing the plasmid DNA for nucleosome arrays. We are grateful to Dr. Maria Jasin (Memorial Sloan Kettering Cancer Center) and Dr. Makoto Nakanishi (Nagoya City University) for providing the I-SceI expression system and the DR-GFP cells. ABBREVIATIONS ATP, adenosine 5'-triphosphate; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; EMSA,

electrophoretic

mobility

shift

assay;

HEPES,

4-(2-hydroxyethyl)-1-

piperazineethanesulfonic acid; NAP1, Nucleosome assembly protein 1; PAGE, poly-acrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; TBE, Tris-borate-EDTA; Tris, tris(hydroxymethyl)aminomethane.

REFERENCES (1) Luger, K., Mäder, A. W., Richmond, R. K., Sargent, D. F., and Richmond, T. J. (1997) Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature 389, 251−260.


17

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 33

(2) Davey, C. A., Sargent, D. F., Luger, K., Maeder, A. W., and Richmond, T. J. (2002) Solvent Mediated Interactions in the Structure of the Nucleosome Core Particle at 1.9Å Resolution. J. Mol. Biol. 319, 1097–1113. (3) Makde, R. D., England, J. R., Yennawar, H. P., and Tan, S. (2010) Structure of RCC1 chromatin factor bound to the nucleosome core particle. Nature 467, 562–566. (4) Luger, K., Dechassa, M. L., and Tremethick, D. J. (2012) New insights into nucleosome and chromatin structure: an ordered state or a disordered affair? Nat. Rev. Mol. Cell Biol. 13, 436–447. (5) Happel, N., and Doenecke, D. (2009) Histone H1 and its isoforms: Contribution to chromatin structure and function. Gene 431, 1–12. (6) Harshman, S. W., Young, N. L., Parthun, M. R., and Freitas, M. A. (2013) H1 histones: current perspectives and challenges. Nucleic Acids Res. 41, 9593–9609. (7) Whitlock, J. P., Jr., and Simpson, R. T. (1976) Removal of histone H1 exposes a fifty base pair DNA segment between nucleosomes. Biochemistry 15, 3307-3314. (8) Simpson, R. T. (1978) Structure of the chromatosome, a chromatin particle containing 160 base pairs of DNA and all the histones. Biochemistry 17, 5524–5531. (9) Syed, S. H., Goutte-Gattat, D., Becker, N., Meyer, S., Shukla, M. S., Hayes, J. J., Everaers, R., Angelov, D., Bednar, J., and Dimitrov, S. (2010) Single-base resolution mapping of H1nucleosome interactions and 3D organization of the nucleosome. Proc. Natl. Acad. Sci. U.S.A. 107, 9620–9625.


18

Page 19 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

(10) Caterino, T. L., Fang, H., and Hayes, J. J. (2011) Nucleosome linker DNA contacts and induces specific folding of the intrinsically disordered H1 carboxyl-terminal domain. Mol. Cell. Biol. 31, 2341–2348. (11) Hartman, P. G., Chapman, G. E., Moss, T., and Bradbury, E. M. (1977) Studies on the role and mode of operation of the very-lysine-rich histone H1 in eukaryote chromatin. The three structural regions of the histone H1 molecule. Eur. J. Biochem. 77, 45–51. (12) Allan, J., Hartman, P. G., Crane-Robinson, C., and Aviles, F. X. (1980) The structure of histone H1 and its location in chromatin. Nature 288, 675–679. (13) Song, F., Chen, P., Sun, D., Wang, M., Dong, L., Liang, D., Xu, R. M., Zhu, P., and Li, G. (2014) Cryo-EM study of the chromatin fiber reveals a double helix twisted by tetranucleosomal units. Science 344, 376–380. (14) Zhou, B.-R., Jiang, J., Feng, H., Ghirlando, R., Xiao, T. S., and Bai, Y. (2015) Structural mechanisms of nucleosome recognition by linker histones. Mol. Cell 59, 1–34. (15) Th'ng, J. P. H., Sung, R., Ye, M., and Hendzel, M. J. (2005) H1 family histones in the nucleus. Control of binding and localization by the C-terminal domain. J. Biol. Chem. 280, 27809–27814. (16) Clausell, J., Happel, N., Hale, T. K., Doenecke, D., and Beato, M. (2009) Histone H1 subtypes differentially modulate chromatin condensation without preventing ATP-dependent remodeling by SWI/SNF or NURF. PloS One 4, e0007243.


19

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 33

(17) Izzo, A., Kamieniarz-Gdula, K., Ramírez, F., Noureen, N., Kind, J., Manke, T., van Steensel, B., and Schneider, R. (2013) The genomic landscape of the somatic linker histone subtypes H1.1 to H1.5 in human cells. Cell Rep. 3, 2142–2154. (18) DeLucia, F., Faraone-Mennella, M. R., D’Erme, M., Quesada, P., Caiafa, P., and Farina, B. (1994) Histone-induced condensation of rat testis chromatin: testis-specific H1t versus somatic H1 variants. Biochem. Biophys. Res. Commun. 198, 32–39. (19) Khadake, J. R., and Rao, M. R. (1995) DNA- and chromatin-condensing properties of rat testes H1a and H1t compared to those of rat liver H1bdec; H1t is a poor condenser of chromatin. Biochemistry 34, 15792–15801. (20) Bucci, L. R., Brock, W. A., and Meistrich, M. L. (1982) Distribution and synthesis of histone 1 subfractions during spermatogenesis in the rat. Exp. Cell Res. 140, 111–118. (21) Grimes, S. R., Wilkersona, D. C., Noss, K. R., and Wolfe, S. A. (2003) Transcriptional control of the testis-specific histone H1t gene. Gene 304, 13-21. (22) Govin, J., Caron, C., Lestrat, C., Rousseaux, S., and Khochbin, S. (2004) The role of histones in chromatin remodelling during mammalian spermiogenesis. Eur, J. Biochem. 271, 3459–3469. (23) Petronczki, M., Siomos, M. F., and Nasmyth, K. (2003) Un ménage à quatre: the molecular biology of chromosome segregation in meiosis. Cell 112, 423–440. (24) Neale, M.J., and Keeney, S. (2006) Clarifying the mechanics of DNA strand exchange in meiotic recombination. Nature 442, 153–158.


20

Page 21 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

(25) Sung, P. (1994) Catalysis of ATP-dependent homologous DNA pairing and strand exchange by yeast RAD51 protein. Science 265, 1241–1243. (26) Baumann P., Benson F. E., and West, S. C. (1996) Human Rad51 protein promotes ATPdependent homologous pairing and strand transfer reactions in vitro. Cell 87, 757–766. (27) Gupta R. C., Bazemore L. R., Golub E. I., and Radding, C. M. (1997) Activities of human recombination protein Rad51. Proc. Natl. Acad. Sci. U.S.A. 94, 463–468. (28) Maeshima K., Morimatsu K., and Horii T. (1996) Purification and characterization of XRad51.1 protein, Xenopus RAD51 homologue: recombinant XRad51.1 promotes strand exchange reaction. Genes Cells 1, 1057–1068. (29) Alexiadis, V., and Kadonaga, J. T. (2002) Strand pairing by Rad54 and Rad51 is enhanced by chromatin. Genes Dev. 16, 2767–2771. (30) Jaskelioff, M., Van, Komen, S., Krebs, J. E., Sung, P., and Peterson, C. L. (2003) Rad54p is a chromatin remodeling enzyme required for heteroduplex DNA joint formation with chromatin. J. Biol. Chem. 278, 9212–9218. (31) Alexeev, A., Mazin, A., and Kowalczykowski, S. C. (2003) Rad54 protein possesses chromatin-remodeling activity stimulated by the Rad51–ssDNA nucleoprotein filament. Nat. Struct. Biol. 10, 182–186. (32) Zhang, Z., Fan, H. -Y., Goldman, J. A., and Kingston, R. E. (2007) Homology-driven chromatin remodeling by human RAD54. Nat. Struct. Mol. Biol. 14, 397–405.


21

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 33

(33) Sinha, M., and Peterson, C. L. (2008) A Rad51 presynaptic filament is sufficient to capture nucleosomal homology during recombinational repair of a DNA double-strand break. Mol. Cell 30, 803–810. (34) Machida, S., Takaku, M., Ikura, M., Sun, J., Suzuki, H., Kobayashi, W., Kinomura, A., Osakabe, A., Tachiwana, H., Horikoshi, Y., Fukuto, A., Matsuda, R., Ura, K., Tashiro, S., Ikura, T., and Kurumizaka, H. (2014) Nap1 stimulates homologous recombination by RAD51 and RAD54 in higher-ordered chromatin containing histone H1. Sci. Rep. 4, 4863. (35) Ishida, T., Takizawa, Y., Sakane, I., and Kurumizaka, H. (2008) The Lys313 residue of the human Rad51 protein negatively regulates the strand-exchange activity. Genes Cells 13, 91–103. (36) Tachiwana, H., Osakabe, A., Kimura, H., and Kurumizaka, H. (2008) Nucleosome formation with the testis-specific histone H3 variant, H3t, by human nucleosome assembly proteins in vitro. Nucleic Acids Res. 36, 2208–2218. (37) Tachiwana, H., Kagawa, W., Osakabe, A., Kawaguchi, K., Shiga, T., Hayashi-Takanaka, Y., Kimura, H., and Kurumizaka, H. (2010) Structural basis of instability of the nucleosome containing a testis-specific histone variant, human H3T. Proc. Natl. Acad. Sci. U.S.A. 107, 10454–10459. (38) Kagawa, W., Kurumizaka, H., Ikawa, S., Yokoyama, S., and Shibata, T. (2001) Homologous pairing promoted by the human Rad52 protein. J. Biol. Chem. 276, 35201–35208. (39) Lowary, P. T., and Widom, J. (1998) New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning. J. Mol. Biol. 276, 19–42.


22

Page 23 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

(40) Arimura, Y., Tachiwana, H., Oda, T., Sato, M., and Kurumizaka, H. (2012) Structural analysis of the hexasome, lacking one histone H2A/H2B dimer from the conventional nucleosome. Biochemistry 51, 3302–3309. (41) Dorigo, B., Schalch, T., Bystricky, K., and Richmond, T. J. (2003) Chromatin fiber folding: requirement for the histone H4 N-terminal tail. J. Mol. Biol. 327, 85–96. (42) Demeler, B., and van Holde, K. E. (2004) Sedimentation velocity analysis of highly heterogeneous systems. Anal. Biochem. 335, 279–288. (43) Pierce, A. J., Johnson, R. D., Thompson, L. H., and Jasin, M. (1999) XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. Genes Dev. 13, 2633–2638. (44) Kobayashi, J., Kato, A., Ota, Y., Ohba, R., and Komatsu, K. (2010) Bisbenzamidine derivative, pentamidine represses DNA damage response through inhibition of histone H2A acetylation. Mol. Cancer 9, e34. (45) Saeki, H., Ohsumi, K., Aihara, H., Ito, T., Hirose, S., Ura, K., and Kaneda, Y. (2005) Linker histone variants control chromatin dynamics during early embryogenesis. Proc. Natl. Acad. Sci. U.S.A. 102, 5697–5702. (46) Shintomi, K., Iwabuchi, M., Saeki, H., Ura, K., Kishimoto, T., and Ohsumi, K. (2005) Nucleosome assembly protein-1 is a linker histone chaperone in Xenopus eggs. Proc. Natl. Acad. Sci. U.S.A. 102, 8210–8215. (47) San Filippo, J., Sung, P., and Klein, H. (2008) Mechanism of eukaryotic homologous recombination. Annu. Rev. Biochem. 77, 229–257.


23

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 33

(48) Heyer, W. D., Ehmsen, K. T., and Liu, J. (2010) Regulation of homologous recombination in eukaryotes. Annu. Rev. Genet. 44, 113–139. (49) Krejci, L., Altmannova, V., Spirek, M., and Zhao, X. (2012) Homologous recombination and its regulation. Nucleic Acids Res. 40, 5795–5818. (50) Steger, K., Klonisch, T., Gavenis, K., Drabent, B., Doenecke, D., and Bergmann, M. (1998) Expression of mRNA and protein of nucleoproteins during human spermiogenesis. Mol. Hum. Reprod. 4, 939–945. (51) Drabent, B., Saftig, P., Bode, C., and Doenecke, D. (2000) Spermatogenesis proceeds normally in mice without linker histone H1t. Histochem. Cell Biol. 113, 433–442. (52) Lin, Q., Sirotkin, A., and Skoultchi, A. I. (2000) Normal spermatogenesis in mice lacking the testis-specific linker histone H1t. Mol. Cell. Biol. 20, 2122–2128. (53) Fantz, D. A., Hatfield, W. R., Horvath, G., Kistler, M. K., and Kistler, W. S. (2001) Mice with a targeted disruption of the H1t gene are fertile and undergo normal changes in structural chromosomal proteins during spermiogenesis. Biol. Reprod. 64, 425–431. (54) Tanaka, H., Matsuoka, Y., Onishi, M., Kitamura, K., Miyagawa Y., Nishimura, H., Tsujimura, A, Okuyama, A, and Nishimune, Y. (2006) Expression profiles and single-nucleotide polymorphism analysis of human HANP1/H1T2 encoding a histone H1-like protein. Int. J. Androl. 29, 353–359. (55) Yan, W., Ma, L., and Burns, K. H. (2003) HILS1 is a spermatid-specific linker histone H1like protein implicated in chromatin remodeling during mammalian spermiogenesis. Proc. Natl. Acad. Sci. U.S.A. 100, 10546–10551.


24

Page 25 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

(56) Tanaka, Y., Kato, S., Tanaka, M., Kuji, N., and Yoshimura, Y. (2003) Structure and expression of the human oocyte-specific histone H1 gene elucidated by direct RT-nested PCR of a single oocyte. Biochem. Biophys. Res. Commun. 304, 351–357. (57) Maze, I., Noh, K.-M., Soshnev, A. A., and Allis, C. D. (2014) Every amino acid matters: essential contributions of histone variants to mammalian development and disease. Nat. Rev. Genet. 15, 259–271. (58) Bao, Y., Konesky, K., Park, Y. J., Rosu, S., Dyer, P. N., Rangasamy, D., Tremethick, D. J., Laybourn, P. J., and Luger, K. (2004) Nucleosomes containing the histone variant H2A.Bbd organize only 118 base pairs of DNA. EMBO J. 23, 3314−3324. (59) Gautier, T., Abbott, D. W., Molla, A., Verdel, A., Ausio, J., and Dimitrov, S. (2004) Histone variant H2ABbd confers lower stability to the nucleosome. EMBO Rep. 5, 715–720. (60) Urahama, T., Horikoshi, N., Osakabe, A., Tachiwana, H., and Kurumizaka, H. (2014) Structure of human nucleosome containing the testis-specific histone variant TSH2B. Acta Cryst. F70, 444-449. (61) Arimura, Y., Kimura, H., Oda, T., Sato, K., Osakabe, A., Tachiwana, H., Sato, Y., Kinugasa, Y., Ikura, T., Sugiyama, M., Sato, M., and Kurumizaka, H. (2013) Structural basis of a nucleosome containing histone H2A.B/H2A.Bbd that transiently associates with reorganized chromatin. Sci. Rep. 3, 3510. (62) Sansoni, V., Casas-Delucchi, C. S., Rajan, M., Schmidt, A., Bonisch, C., Thomae, A. W., Staege, M.S., Hake, S.B., Cardoso, and M.C., and Imhof, A. (2014) The histone variant H2A.Bbd is enriched at sites of DNA synthesis. Nucleic Acids Res. 42, 6405–6420.


25

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 33

(63) Ishibashi, T., Li, A., Eirin-Lopez, J. M., Zhao, M., Missiaen, K., Abbott, D. W., Meistrich, M., Hendzel, M.J., and Ausió, J. (2010) H2A.Bbd: an X-chromosome-encoded histone involved in mammalian spermiogenesis. Nucleic Acids Res. 38, 1780–1789. (64) Padavattan, S., Shinagawa, T., Hasegawa, K., Kumasaka, T., Ishii, S., and Kumarevel, T. (2015) Structural and functional analyses of nucleosome complexes with mouse histone variants TH2a and TH2b, involved in reprogramming. Biochem. Biophys. Res. Commun. 464, 929-935.

Graphic for the Table of Contents (ToC Graphic) For Table of Contents Use Only


26

Page 27 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 1. Histone H1T binds to chromatin. (A) Schematic representation of H1 loading on the polynucleosome. (B) EMSA analysis of the poly-nucleosomes. The poly-nucleosomes with or without H1.2 or H1T were analyzed by 0.7 % agarose gel electrophoresis with EtBr staining. Lane 1 indicates the reconstituted poly-nucleosomes. Lanes 2 and 3 indicate the poly-nucleosomes complexed with H1.2 and H1T, respectively, in the presence of Nap1. Lane 4 indicates the poly-nucleosomes without linker histones, in the presence of Nap1. (C) ScaI analysis of the poly-nucleosomes. The poly-nucleosomes with or without linker histones were digested with ScaI, and the resulting mono-nucleosomes were analyzed by 5% native-PAGE with EtBr staining. Lane 1 indicates a control experiment with the naked DNA. Lane 2 indicates the experiment with the poly-nucleosomes, in the absence of Nap1. Lanes 3 and 4 indicate the experiments with the poly-nucleosomes complexed with H1.2 and H1T, respectively, in the presence of Nap1. Lane 5 indicates the experiment with the poly-nucleosomes without linker histones, in the presence of Nap1. (D) EMSA analysis of the reconstituted mono-nucleosomes. The mono-nucleosomes (30 µM) reconstituted with the 193-base-pair DNA were incubated with H1.2 or H1T in the presence of Nap1 (one-half the amount of the linker histone). The linker histone concentrations were 0.2 µM (lanes 3 and 7), 0.3 µM (lanes 4 and 8), 0.4 µM (lanes 5 and 9), and 0.5 µM (lanes 6 and 10). Lane 1 indicates naked DNA. Lane 2 indicates a control experiment without linker histones. The products were analyzed by 5% native-PAGE with EtBr stain. (E) Graphic representation of the experiments shown in panel (D). The H1-nucleosome complex formation ratios (%) were plotted against the linker histone concentrations. The experiments were independently performed three times, and the average values are shown with standard deviations. 141x122mm (300 x 300 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. Analytical ultracentrifugation sedimentation velocity analyses of poly-nucleosomes. (A) Sedimentation velocity analyses of poly-nucleosomes in the presence (closed diamonds) or absence (closed triangles) of Nap1 were performed in 10 mM Tris-HCl (pH 7.5). (B) Sedimentation velocity analyses of polynucleosomes with H1T (closed squares) or H1.2 (closed circles) were performed in 10 mM Tris-HCl (pH 7.5), in the presence of Nap1. The sedimentation coefficient (S20,w) distributions were determined by the method of van Holde and Weischet42. 81x40mm (300 x 300 DPI)


Page 28 of 33

Page 29 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 3. Homologous pairing assays with H1T and H1.2 in chromatin. (A) Schematic diagram of the polynucleosome template used in the homologous pairing assay. The arrow indicates the homologous region of the 5S ssDNA 90-mer. (B) Scheme for the homologous pairing assay with the poly-nucleosome containing histone H1. (C) The homologous pairing assay with the poly-nucleosomes containing histone H1. The polynucleosomes were incubated with either H1.2 or H1T (0.2, 0.3, 0.4, and 0.5 µM), in the presence of Nap1 (one-half the amount of histone H1). The reactions were initiated by the addition of the RAD51-ssDNA complex and RAD54. After deproteinization, the homologous pairing products (D-loops) were separated by 1% agarose gel electrophoresis, and were visualized with an FLA-7000 image analyzer (GE Healthcare). (D) Graphic representation of the experiment shown in panel (C). The amounts of ssDNA in the D-loops were quantitated, and the average values of three independent experiments are plotted with standard deviation values. 122x87mm (300 x 300 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 33

Figure 4. Homologous pairing assays with the swapping H1T-N, H1T-G, and H1T-C mutants in chromatin. (A) Schematic diagram of the swapping H1T-N, H1T-G, and H1T-C mutants. (B) Homologous pairing assays with the poly-nucleosomes containing H1T-N, H1T-G, and H1T-C. The poly-nucleosomes were incubated with either H1T-N, H1T-G, or H1T-C (0.3, 0.4, and 0.5 µM), in the presence of Nap1 (one-half the amount of histone H1). The reactions were initiated by the addition of the RAD51-ssDNA complex and RAD54. After deproteinization, the homologous pairing products (D-loops) were separated by 1% agarose gel electrophoresis, and were visualized with an FLA-7000 image analyzer (GE Healthcare). (C) Graphic representation of the experiment shown in panel (B). The amounts of ssDNA in the D-loops were quantitated, and the average values of three independent experiments are plotted with standard deviation values. 111x72mm (300 x 300 DPI)


Page 31 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 5. Homologous pairing assays with H1.0, H1.1, H1.2, H1.3, H1.4, H1.5, and H1T in chromatin. (A) Homologous pairing assays with poly-nucleosomes containing H1.2, H1.3, H1.4, and H1.5. The polynucleosomes were incubated with each linker histone subtype (0.3, 0.4, and 0.5 µM), in the presence of Nap1 (one-half the amount of histone H1). The reactions were initiated by the addition of the RAD51-ssDNA complex and RAD54. After deproteinization, the homologous pairing products (D-loops) were separated by 1% agarose gel electrophoresis, and were visualized with an FLA-7000 image analyzer (GE Healthcare). The D-loop formation rates relative to the experiment without the linker histone were estimated, and the average values of three independent experiments are plotted with the standard deviation values in the bottom panel. (B) The homologous pairing assays with the poly-nucleosomes containing H1.2, H1.1, H1.0, and H1T. Experiments were performed as described in panel (A). The D-loop formation rates relative to the experiment without the linker histone were estimated, and the average values of three independent experiments are plotted with the standard deviation values in the bottom panel. 99x55mm (300 x 300 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 6. Endogenously expressed H1T minimally affected homologous recombination in human cells. (A) Scheme of the homologous recombination assay in human cells. The reporter contained the SceGFP gene (mutant GFP) and the truncated GFP gene (GFP∆) lacking 5’- and 3’-sequences, and was introduced into human U2OS-DR-GFP cells. The SceGFP gene is inactive before the homologous recombination repair, because it contains the I-SceI site within the coding region. A DSB was induced at the I-SceI site by exogenously expressed I-SceI, and homologous recombination occurred between SceGFP and the downstream GFP∆. The rate of homologous recombination was estimated as the GFP signal, which is derived from the repaired GFP gene by homologous recombination. (B) Western blot analyses of FLAG-H1.2 and FLAG-H1T in human cells. FLAG-H1.2 and FLAG-H1T, which were exogenously produced in human cells, were detected by an anti-FLAG antibody (upper panel). Exogenously expressed FLAG-H1.2 and endogenous H1.2 were detected by an anti-H1.2 antibody (middle panel). (C) Graphic representation of the homologous recombination assay. GFP-positive cells expressing FLAG-H1.2 or FLAG-H1T were quantified by flow cytometry, and the rates relative to a mock experiment without exogenous linker histone production were


Page 32 of 33

Page 33 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

estimated. The average values of three independent experiments are shown with the standard deviation values. 111x133mm (300 x 300 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ToC Graphic 67x30mm (300 x 300 DPI)


Page 34 of 33

Relaxed Chromatin Formation and Weak ... - ACS Publications

Recommend Documents