Isolation and Proteomics Analysis of Barley Centromeric Chromatin

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Isolation and proteomics analysis of barley centromeric chromatin using PICh Zixian Zeng, and Jiming Jiang J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00063 • Publication Date (Web): 04 May 2016 Downloaded from http://pubs.acs.org on May 4, 2016

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Journal of Proteome Research

Isolation

and

proteomics

analysis

of

barley

centromeric chromatin using PICh Zixian Zeng and Jiming Jiang* Department of Horticulture, University of Wisconsin-Madison, Madison, WI 53706, USA

ABSTRACT: Identification of proteins that are directly or indirectly associated with a specific DNA sequence is often an important goal in molecular biology research. Proteomics of isolated chromatin fragments (PICh) is a technique used to isolate chromatin that contains homologous DNA sequence to a specific nucleic acid probe. All proteins directly and indirectly associated with the DNA sequences that hybridize to the probe are then identified by proteomics.1 We used the PICh technique to isolate chromatin associated with the centromeres of barley (Hordeum valgare), by using a 2’-deoxy-2’fluoro-ribonucleotides (2’-F RNA) probe that is homologous to the AGGGAG satellite DNA specific to barely centromeres. Proteins associated with the barley centromeric chromatin were then isolated and identified by mass spectrometry. Both alphacenH3 and beta-cenH3, the two centromeric histone H3 variants associated with barley centromeres, were positively identified. Interestingly, several different H2A and H2B variants were recovered in the PIChed chromatin. The limitations and future potential of PICh in plant chromatin research are discussed.

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KEYWORDS: Proteomics of isolated chromatin fragments (PICh), centromere, cenH3, barley.

INTRODUCTION Characterization of specific DNA-protein and protein-protein interactions is an essential step for understanding the regulation of gene expression, as well as for studying the structure and function of eukaryotic chromatin. Several traditional techniques have been developed to isolate and enrich target chromatin regions for locus-specific protein characterization.2-5 However, these early techniques can only be used to study highly abundant proteins or proteins associated with highly repetitive DNA sequences. Numerous modern techniques have been developed to allow the detection of DNA-protein interactions. Chromatin immunoprecipitation (ChIP) is one of the most powerful and widely adopted methods used to identify DNA sequences associated with a specific protein.6-7 ChIP is a chromatin enrichment technique that utilizes antibodies to purify chromatin fragments and the underlying DNA sequences associated with a specific protein of interest, such as a transcription factor or covalently modified histone. This technique, however, is limited to analysis of proteins that are already known and relies on antibody availability for the protein of interest. In contrast to ChIP, DNA-centered methods have also been developed to identify proteins associated with specific genomic regions, such as nucleic acid affinity capture,8 electrophoretic mobility shift assays,9 and yeast one hybrid assays (Y1H).10 However, none of these methods can be used to capture the complete protein composition associated with a specific DNA sequence. For instance, Y1H assays use a specific DNA sequence as a “bait” to capture DNA sequencespecific bound proteins in the yeast nucleus. Y1H assays are limited by their singular mode of


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detection, which only allows for the identification of proteins that directly interact with the bait sequence. Additionally, Y1H assays are often prone to undesirable levels of false positive detection.11 A new technique, termed Proteomics of Isolated Chromatin segments (PICh), was developed to identify all proteins both directly and indirectly associated with a specific DNA sequence.1 PICh employs a locked nucleic acid (LNA) oligo probe to hybridize with the underlying DNA sequence in formaldehyde-crosslinked chromatin. The hybridized chromatin is captured on magnet beads and proteins associated with these chromatin fragments are then isolated and identified by mass spectrometry (MS). The first PICh was employed to isolate telomereassociated proteins in human by taking advantage of a LNA oligo probe specific to mammalian telomeric repeats (TTAGGG)n.1 We wanted to adapt this method in order to identifying centromere-associated proteins in barley, since barely centromeres contain a unique short satellite repeat (AGGGAG)n.12 The simplicity and abundance of this centromere-specific repeat allowed us to design a 2’-F-RNA oligo probe to target centromeric nucleosomes. Using this probe, we were able to specifically purify two centromeric histone H3 variants (alpha-cenH3 and beta-cenH3) from the barley centromere using a modified PICh protocol specific to plants. In addition, we discuss the adaptation and optimization of the PICh technique for use in plant systems, specifically for targeting barley centromeres.

EXPERIMENTAL PROCEDURES Nuclear isolation and chromatin fragmentation


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Two-week-old barley seedlings were grown in the greenhouse. The whole seedlings including roots were collected and ground into fine powder in liquid nitrogen. The resulting powder was suspended in nuclear extraction buffer (10 mM Potassium Phosphate pH 7.0, 100 mM NaCl, 0.1% mercaptoethanol, 12% Hexylene glycol) and followed the standard protocol for plant nuclei extraction. Extracted nuclei were washed 2 times with wash buffer (10 mM Potassium Phosphate pH 7.0, 100 mM NaCl, 10 mM MgCl2, 0.1% mercaptoethanol, 12% Hexylene glycol, 0.4% Triton X-100) and then cross-linked by suspension in 3% formaldehyde for 30 minutes at room temperature (RT) with vacuum infiltration at 15 mmHg. Excessive formaldehyde was removed by washing 3 times with distilled water and centrifuging at 1200g for 10 minutes at 4ºC between each wash. RNA was removed by incubating nuclei with 50 U RNase A (Invitrogen AM2274) in 1×PBS for 1 hour at RT. Chromatin was re-suspended in digestion buffer (10 mM Tris-HCl pH 7.5, 4mM MgCl2, 1mM CaCl2) and fragmented using Micrococcal Nuclease (Sigma N5386) digestion with final the concentration of 0.15U per 1 µg chromatin for 10 minutes at 37°C. Then 30 mM NaCl, 2 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 0.2% SDS and 0.1% Sodium Sarkosyl were added into the digested chromatin sample. Chromatin was further fragmented by moderate sonication with a total of 8 cycles of continuous pulse-on for 15 seconds at 10% power output and 45 seconds pulse-off. Samples were always kept in ice during sonication. An average chromatin fragment length between 500 bp and 1 kb was obtained for downstream chromatin capture. Capture probe synthesis Modified RNA, with fluorine instead of oxygen at the 2nd position of nucleoside, has the feature of increased thermodynamic affinity for complementary bases due to the high electronegativity of fluorine. A 2’-fluoro-RNA (2’-F-RNA) oligo for barley centromere capture was designed


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according to the sequence of barley centromeric satellite repeat (AGGGAG)n, containing 4 units of the repeat. A scrambled 2’-F-RNA probe with the same number of bases as the centromere probe but without homology to any region within the barley genome, was synthesized in random order as a negative control. Desthiobiotin, a biotin derivative, was labeled at the 5’ end of both probes for affinity purification purposes. A carbon chain, 108 atoms in length, was synthesized to join the desthiobiotin residue with the 2’-F-RNA oligo, in order to prevent steric hindrance between desthiobiotin and the oligo binding complex. Both the 2’-F-RNA centromere probe and scramble probe were synthesized by Fidelity Systems Inc. Capture probes are as follows: Centromere: Desthiobiotin-108Carbons-5’-AgGgAgAgGgAgAgGgAgAgGgAga-3’. Scramble: Desthiobiotin-108Carbons-5’-CuUcCcAcCcUaAaUaCgCaAaUaa-3’. Capitalized letters represent 2’-F-RNA residues and small letters represent regular RNA residues. PICh The soluble chromatin sample from fragmentation was collected by centrifugation at 16000g for 10 min at RT and the supernatant was subsequently incubated at 58°C for 5 min to unmask endogenous biotin. Streptavidin resin (Pierce 53113), which was pre-equilibrated by wash buffer 2 (10 mM Tris-HCl pH 7.5, 30 mM NaCl, 2 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 0.2% SDS, 0.1% Sodium Sarkosyl), was added to the chromatin sample and incubated for 2 hours at RT with nutation. By centrifugation at 3200g for 10 minute at RT, the supernatant was preserved for the next step. Total amount of pre-cleaned chromatin was estimated by measuring the amount of crosslink-reversed DNA. Approximately 45 µg of pre-cleaned chromatin sample for each PICh was centrifuged an additional 15 min at 16000g, RT and then 1% of final volume of SDS was added together with the 2’-F RNA probe (1 µM final concentration), followed by mixing


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and splitting into 150 µL aliquots for the hybridization process. Hybridization was conducted using a thermal cycler with the following parameters: 25°C for 3 min, 73°C for 7 min, 38°C for 60 min, 60°C for 2 min, 38°C for 60 min, 60°C for 2 min, 38°C for 180 min and 25°C final hold temperature. Aliquots after hybridization were pooled together and the pooled sample was then centrifuged at 16000g for 15 min at RT. The supernatant was diluted twice with milliQ water (1:1 dilution), and then 0.6 mL of wash buffer 2 pre-equilibrated MyONETM Streptavidin C1 magnet beads (Invitrogen 65002) was added for capturing hybridized chromatin. After incubation for 12 hours at RT with nutation, magnet beads were immobilized on a magnetic stand and washed with 10 mL of wash buffer 1 (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 2 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 0.2% SDS, 0.1% Sodium Sarkosyl) five times at RT and additional 2 washes with 1mL wash buffer 1 at 42°C for 5 minutes. The magnet beads were resuspended with 1mL of elution buffer (12.5 mM biotin, 7.5 mM Tris-HCl pH 7.5, 75 mM NaCl, 1.5 mM EDTA pH 8.0, 0.75 mM EGTA pH 8.0, 0.15% SDS, 0.075% Na-lauroylsarcosine) and incubated at room temperature for 2 hours with orbit shaking, followed by incubation at 65°C for 10 min. The elution was collected by immobilizing the magnetic beads and then precipitated using 100% tri-chloroacetic acid (final concentration 20%) followed by incubation in ice for 10 min. The pellet was collected by centrifugation for 15 min at room temperature and the supernatant was carefully removed. The pellet was then washed two times with cold acetone by vortexing for 10 seconds and centrifugation between washes. The pellet was finally air dried and resuspended with 15 µL of cross-link reversal buffer (0.25 M Tris-HCl pH 8.8, 2% SDS, 0.5 M β-mercaptoethanol). The sample was incubated at 99°C for 25 min and immediately soaked in ice for 5 min. The sample was separated in a 12% SDS-PAGE gel (Bio-Rad) and visualized using SilverQuest staining kit (Invitrogen) according to the manufacturer’s instruction. The


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separated proteins that were subject to in-gel digestion and mass spectrometry detection were excised from a Coomassie Blue-stained SDS-PAGE gel. The total proteins eluted from three biological PICh replicates were pool together and directly submitted for Orbitrap-Mass Spectrometry analysis. Enzymatic “in liquid” digestion “In Liquid” digestion and mass spectrometric analysis was done at the Mass Spectrometry Facility [Biotechnology Center, University of Wisconsin-Madison]. In short, TCA/acetone precipitated protein samples were re-solubilized and denatured in 7.5 µl of 8M Urea / 50mM NH4HCO3 (pH8.5) for 5 minutes then diluted for reduction with: 1.25 µl of 25mM DTT, 2.5 µl MeOH, 0.1 µl 1mM Tris-HCl (pH7.5) and 18.74 µl 25mM NH4HCO3 (pH8.5). Samples were reduced for 15 minutes at 55°C, cooled, and then alkylated with 1.5 µl of 55mM IAA for 15 minutes at room temperature. Reaction was terminated by adding 3.95 µl of 25mM DTT and digestion commenced by adding 15 µl Trypsin [10ng/µl Trypsin Gold from PROMEGA Corp. in 25mM NH4HCO3] and 25mM NH4HCO3 (pH8.5) to 50 µl final volume. Digestion was conducted for 1 hour at 42°C then additional 10 µl of trypsin was added and digestion proceeded overnight at 37°C. Reaction was terminated by acidification with 2.5% TFA [Trifluoroacetic Acid] to 0.3% final. NanoLC-MS/MS Peptides were analyzed by nanoLC-MS/MS using the Agilent 1100 nanoflow system (Agilent Technologies, Palo Alto, CA) connected to a hybrid linear ion trap-orbitrap mass spectrometer (LTQ-Orbitrap XL, Thermo Fisher Scientific, Bremen, Germany) equipped with a nanoelectrospray ion source. Chromatography of peptides prior to mass spectral analysis was


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accomplished using C18 reverse phase HPLC trap column (Zorbax 300SB-C18, 5 µM, 5x0.3mm, Agilent Technologies) and capillary cm emitter column (in-house packed with MAGIC C18, 3 µM, 200x0.075mm, Michrom Bioresources, Inc.) onto which 8 µl of digest was automatically loaded. NanoHPLC system delivered solvents A: 0.1% (v/v) formic acid in water, and B: 95% (v/v) acetonitrile, 0.1% (v/v) formic acid at either 10 µL/min, to load sample, or 0.20 µL/min, to elute peptides directly into the nano-electrospray over a 195 minutes 1% (v/v) B to 40% (v/v) B followed by 20 minute 40% (v/v) B to 60% (v/v) B and 5 minute 60% (v/v) B to 100% (v/v) B gradient. As peptides eluted from the HPLC-column/electrospray source survey MS scans were acquired in the Orbitrap with a resolution of 100,000 and up to 5 most intense peptides per scan were fragmented and detected in the ion trap over a mass range of 300-2000 m/z; redundancy was limited by dynamic exclusion with settings of repeat count of 1 with 60 sec repeat duration, exclusion list size 500 with exclusion duration for 40sec, and exclusion mass width low: 0.55 and high: 1.05. MS1 signals only exceeding 500 counts were allowed to trigger MS2 scans. Ions with unassigned charge state and singly charged ions were excluded from triggering MS2. Data analysis Raw data files were converted to the mascot generic format (mgf) through initial open source mzXML format conversion using the Trans-Proteomic Pipeline (TPP) software suite version 4.4 (Seattle Proteome Center, Seattle, WA). Resulting mgf files were used to search against Uniprot Hordeum vulgare database (46,230 protein entries) using in-house Mascot search engine 2.2.07 [Matrix Science] with variable Methionine oxidation with Asparagine and Glutamine deamidation. Peptide mass tolerance was set at 20 ppm and fragment mass at 0.8 Da. Protein annotations, significance of identification and spectral based quantification was done with help


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of Scaffold software (version 4.3.2, Proteome Software Inc., Portland, OR). Peptide identifications were accepted if they could be established at greater than 80.0% probability by the Peptide Prophet algorithm13 with Scaffold delta-mass correction. Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm.14 Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Western blot hybridization Centromere histone H3 detection was performed according to the standard protocol for Western blotting. Proteins were separated in a 12% SDS-PAGE gel and then transferred to a PVDF membrane (Bio-Rad 162-0174) for immuno-hybridization. An antibody against rice cenH3 (antiOscenH3) was used for detecting the presence of barley centromere histone H3. HRP-based chemiluminescent exposed X-ray film was developed to visualize the presence of the signal.

RESULTS Optimization of the PICh protocol in barley PICh has been demonstrated as a feasible approach for studying proteins associated with a defined genomic region.1,15 The first PICh experiment was designed using human telomeric repeats, (TTAGGG)n, as a capture probe.1 Since the human genome contains a large number of the TTAGGG repeats and many of these repeats are associated with the same telomeric proteins, application of a repeat-based DNA probe increases the likelihood of isolating the target telomeric


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proteins. We intended to use a similar strategy to isolate the centromeric proteins in barley. Barley centromeres contain long arrays of a short satellite repeat (AGGGAG)n. This centromerespecific repeat represents the most dominant DNA component in each of the seven barley centromeres, whereas it is absent in non-centromere regions of all chromosomes according to the FISH detection.12 Long arrays of AGGGAG repeats would ensure that these repeats wrap many barley centromeric nucleosomes, including the linker sequences. Therefore, we designed a probe consisting of several AGGGAG units, which is expected to hybridize multiple positions of the barley centromeric nucleosomes. We first tried to optimize the PICh technique1 for applications in plants (protocol outlined in Figure 1). The presence of the resilient cell wall and the large amount of organelles in plant cells often generate technical problems for direct applications of chromatin/protein-related techniques developed in model animal species.16-17 As such, we employed nuclei extraction and chromatin crosslinking protocols developed for plant species, which is adapted to cicumvent these issues.18 To limit contamination by organelle and cell wall proteins, we isolated nuclei prior to formaldehyde crosslinking, rather than crosslinking frozen and homogenized tissue directly. We also implemented two additional nuclei washes with 0.4% Triton X-100 to minimize contamination from chloroplast proteins. An initially sonication for 40 cycles with 10% power output, and 15 seconds of constant pulse and 45 seconds of pause for each cycle was used to fragment barley nuclei cross-linked with 3% formaldehyde. This process resulted 5-kilobase pair (kb) fragments on average, which is too large for targeting proteins associated with specific nucleosomes. We were not able to further reduce the size by increasing the number of cycles of sonication. Experiments with more intensive sonication (15% and 20% power output) yielded slightly shorter fragments,


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approximately 4 kb in length. However, physical vibrations that accompany higher power output by the sonicator can break the DNA-protein and protein-protein interactions, and in our case, resulted in substantially reduced protein recovery compared to the initial trials using 10% power output. In order to achieve an optimal tradeoff between desired fragment size and protein recovery, a combination of fragmentation methods were employed. We first digested crosslinked nuclei with Micrococcal nuclease to reduce whole chromatin fragment size down to ~1 kb without violent physical vibration, and subsequently sonicated these smaller fragments for 8 cycles at 10% power output. This combination released most of sheared chromatin fragments from the nuclei and yielded fragments between 500 bp and 1 kb, which is suitable for hybridization. Identification of centromeric histone alpah-cenH3 A 2’-F-RNA probe was used in place of the LNA probes employed in the original PICh protocol.1 The 2’-F-RNA probes offer increased thermal stability and affinity than regular oligonucleotide probes. Such probes have increased Tm up to 1ºC per 2’-F-RNA/nucleotide substitution. While LNA probes form more stable duplexes with the target DNA than 2’-F-RNA probes, LNA probes are substantially more expensive. We designed a 2’-F-RNA capture probe (Cen-probe) 25 nucleotides (nt) in length complementary to the barley centromeric satellite repeat (AGGGAG)n and a scramble 2’-F-RNA probe (Scr-probe) to serve as a negative control (probe details see Methods). The Scr-probe is the same length in nucleotides as the Cen-probe but is composed of randomly assembled nucleotides. Since the number of input nuclei cannot be accurately counted in PICh experiments for plant species, we were not able to assess the appropriate amount of input from barley seedling tissue based on the mammalian protocol. Instead, we estimated the total amount of pre-cleaned digested chromatin by measuring the


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amount of crosslink-reversed DNA. We started with 12 g of finely-ground tissue (relevant to ~15 µg DNA after chromatin pre-clean) from barley whole seedlings and evaluated whether this amount of tissue was adequate to isolate enough proteins for subsequent MS analysis. Using 12 g of initial tissue gave inadequate PICh-purified protein yields, since only a few faint bands were detected on a silver-stained SDS-PAGE gel. These results suggested either low efficiency of probe hybridization or an insufficient amount of input material. This prompted us to attempt again with a substantially larger amount of input materials. PICh experiments using 36 g of tissue (relevant to ~45 µg DNA after chromatin pre-clean) yielded differential bands with intensive density between the Cen-probe and the Scr-probe isolations (Figure 2). After optimization of various adaptive modifications, our PICh experiments resulted in stable and repeatable band patterns in the SDS-PAGE gels. Since cenH3 is the most abundant centromeric protein, it should be purified by the Cen-probe in a successful PICh experiment. Two differential regions below 20 kD were only detected in the Cen-probe isolation lane (Arrow marked in Figure 2). The corresponding regions were excised from a Coomassie Blue stained SDS-PAGE gel and treated with trypsin for MS analysis. A total of 6 unique peptides were detected in the region marked as “Alpha-cenH3” (Figure 2). These peptides correspond to four unique proteins, including barley centromeric histone alpha-cenH3 (one of two barley cenH3 variants)19, histone H2A, RUBISCO and an unknown protein. Additionally, two and three unique peptides were found in the region marked as “Histone H4” (Figure 2), corresponding to RUBISCO and histone H4, respectively. Regardless of the contamination of the RUBISCO protein and normal histones, the presence of the alpha-cenH3 in the Cen-probe isolation was a good indicator of proof-ofconcept in our optimized PICh protocol for barley.


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To further confirm the presence of alpha-cenH3, we performed Western blot hybridization using the antibody against rice cenH320. This antibody showed high specificity to barely centromeres in immunofluorescence staining21. Approximately equal amount of proteins purified from Cen-probe and Scr-probe, respectively, were loaded in a 12% SDS-PAGE gel and the separated proteins were then transferred onto PVDF membrane for immuno-hybridization. A cenH3 hybridization signal was detected between 12 kDa and 20 kDa (Figure 3), at approximately 18 kDa, which is consistent with previous report19. Furthermore, this band appeared in the Cen-probe isolation as well as in the input lane (total protein extraction from barley chromatin), but was absent in Scr-probe isolation (Figure 3). Recovery of barley centromeric proteins Our goal was to identify all potential barley centromeric proteins by MS of PICh-isolated samples. Since a considerable amount of total protein is required for MS analysis, our first goal was to increase protein yield by improving the hybridization efficiency with the Cen-probe, which prompted us to experiment with modifying the hybridization temperature and buffer system. However, these modifications did not result in significant improvements. Increasing the denaturation temperature (higher than 75°C) caused loss of cross-linked proteins. In contrast, denaturation temperatures less than 70°C resulted in substantially reduced protein yields. Eventually, we adopted an optimal hybridization program with the denaturation temperature set at 73°C. Total proteins were purified using both the Cen-probe and Scr-probe from three biological replicates of independent experiments and were pooled together for subsequent Orbitrap-MS detection.


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We identified a total of 94 proteins using the Cen-probe and 73 proteins using the Scr-probe after removing contaminations such as RuBisCo and proteins with low probability. Scaffold (version 4.3.0; Proteome Software) was used with relatively loose stringency to retain as many likely proteins as possible. A threshold of at least one unique validated peptide with greater than 80% possibility was required to retain an identified protein, resulting in greater than 80% probability at the protein level. A total of 22 proteins were shared in both Cen-probe and Scrprobe isolations (Figure 4) and 72 proteins were specific to the Cen-probe. In comparison, 210 and 190 unique proteins were recovered from the HeLa 1.2.11 telomeres and ALT telomeres of the WI38-VA13 cells, respectively, using the human telomere LNA probe with 25-nt in length.1 The alpha-cenH3 was identified specifically by the Cen-probe. In addition, beta-cenH3, another centromeric cenH3 variant of barley19, was also detected only in the Cen-probe isolation. We ranked the list of proteins identified by the Cen-probe based on the abundance of each protein, which is calculated using the ratio of peptide number to protein size. Twelve of the top 20 scored proteins were identified as histones and their variants, including alpha-cenH3 (ranked 20th), beta-cenH3 (ranked 12th), and the canonical H3, H4, H2A, H2B and H1. Four proteins were predicted with either putative or unknown function, and three proteins, non-specific lipidtransfer protein, beta-amylase and cystatin were also identified, though they were not previously known to be associated with centromeres (Table 1). One of the top 20 proteins (ranked 14th) was identified as a High Mobility Group1/2-like protein (HMG1/2-like). HMG proteins are known as abundant non-histone components of chromatin involved in the organization of chromatin structure.22-24 Immunofluorescence staining using anti-HMG1 antibody showed that the HMG proteins are predominantly associated with heterochromatin, including the centromeres of human and mouse chromosomes.25 In addition,


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HMG proteins were predicted to play a role in positioning of the centromere DNA.26 HMG1binding sites were significantly enriched in the CENP-A (cenH3 in humans)-binding domain in a human neocentromere.27 Interestingly, the HMG2 protein was one of the 86 Drosophila melanogaster centromeric proteins detected by proteomics analysis using a cell line expressing GFP-tagged cenH3 (CENP-ACID in Drosophila).28 Nevertheless, the association of the HMG proteins with centromere function needs to be experimentally confirmed in both animal and plant species. Among the four unknown but predicted proteins, two of them (7th and 15th) have not been characterized thus far, although homologues to these two proteins can be identified in related grass species. The functions for the other two predicted proteins (17th and 20th) were speculated as ion binding and RNA binding, respectively. It seems unlikely that an ion binding protein and a RNA binding protein would be associated with centromeres, however without further confirmation, the function and localization of these predicted proteins remains to be elucidated. We further extended our search and tried to identify more possible proteins by loosening stringency of minimum peptide possibility to 50%. However, no other proteins previously shown to associate with centromere were found in both Cen-probe and Scr-probe isolation.

Discussion A number of centromeric proteins have been identified and characterized in model animal species.29 Homologues of several of these centromeric proteins were identified in plants30, including cenH331-32, CENP-C33-34, CENP-E35, MAD236-37, and MIS1238-39. Besides the cenH3 variants, we did not detect other previously characterized centromeric proteins in our PICh


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experiments. Failure to identify known proteins was also encountered in PICh-based telomere proteomics experiments in both human and Drosophila.1,15 Seven previously known telomeric proteins were not recovered in the human telomere PICh experiments, including SMC5, SIRT6, Tankyrase1, WRN, Rad51D, TERT and DKC, while the Drosophila study failed to recover PcG proteins, which were reported to be localized at telomeric repeats. There are several possible reasons that may lead to failure of detection for previously known proteins in our PICh experiments: (i) Depletion of some centromeric proteins, such as CENP-C, could be due to the application of MNase digestion in our PICh procedure. A previous study showed that MNase digestion releases the CENP-A/B/C chromatin complex into the soluble fraction first, and later removes CENP-C from the complex.40 Our PICh protocol implemented a MNase digestion step in order to fragment cross-linked chromatin into the desired fragment size. Therefore, CENP-C and possibly other centromeric proteins could be depleted by the MNase treatment of the PICh samples. In addition, a previous study showed that CENP-C was only detectable in meristematic cells in A. thaliana.41 Thus, young seedling, which was used in the present study, may not be enriched with CENP-C and possibly other centromeric proteins. (ii) Low efficiency of PICh hybridization caused by low binding affinity of the 2’F-RNA probe. The thermal stability of the 2’-F-RNA probes is lower than that of LNA probes. Thus, PICh using 2’F-RNA probes likely has lower hybridization efficiency than PICh using LNA probes. (iii) Inefficient crosslinking during our PICh experiments. CENP-C binds both centromeric DNA and CENP-A in humans42-44. However, the association between CENP-C and DNA is substantially looser compared with the binding affinity of the nucleosome core histones to centromeric DNA. Deeper crosslinking, however, would require higher concentrations of MNase to break the chromatin into the desired size. (iv) The depth of proteomic coverage of the target loci by PICh.


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Our PICh-isolated sample contained limited amount of proteins required for Orbitrap-MS detection, which resulted in only 111 unique peptides specific to the Cen-probe isolation. Of the 111, 70 unique peptides (~63%) were assigned to regular histones and predicted but unknown proteins. Thus we were only able to recover the most abundant proteins in the sample. Less abundant proteins, however, may still be recovered by using a larger amount of starting input. (v) Tissue type. Dividing cells provide opportunity to capture centromeric and kinetochore proteins that only bind to centromere regions during mitosis. In this study, we used 2-week-old whole seedlings for chromatin isolation. The whole seedlings harbor dividing cells in tissues such as root and shoot. However, the proportion of dividing cells to all tissues that processed was limited. In the future, callus or young spike tissue might be considered. It is worthy to note that only a single variant of histones H1, H3 and H4 was identified by the Cen-probe or by both the Cen-probe and Scr-probe in different PICh experiments. In contrast, eight H2A variants were identified by the Cen-probe, and only one of these eight variants was identified by the Scr-probe. In addition, two H2B variants were recovered by the Cen-probe, but only one of these two variants was also detected by Scr-probe. In Arabidopsis, the H2A family consists of 13 variants that are encoded by 13 different HTA genes45, while H2B family consists of 11 variants46. The Arabidopsis genome also contains 15 histone H3 variants47. The barley genome contains at least 18 H2A variants and 13 H2B variants that were identified according to the Uniprot database (http://www.uniprot.org). The noticeable difference in the number of detected histone variants captured by the Cen-probe and Scr-probe lead us to speculate that barley centromeric chromatin is potentially associated with a diverse set of histone H2A variants in contrast to other genomic regions. However, the Scr-probe is not an ideal control probe to investigate whether the histone variants captured by the Cen-probe are specific to the


17


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Page 18 of 30

centromeres. The Scr-probe did not appear to capture most of the nucleosomal proteins (Figure 2). Thus, control probes designed from non-centromeric satellite repeats can be used to test if the histone variants captured by the Cen-probe are derived from the centromeric nucleosomes.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Phone: +1(608)-262-1878 Funding Sources This work was partially supported by the National Science Foundation (IOS-1237969) and a Vilas Associate Fellowship from the University of Wisconsin-Madison to J.J.

ABBREVIATIONS PICh, proteomics of isolated chromatin fragments; cenH3, centromeric histone H3.

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Figure legends Figure 1. Workflow of the PICh procedure. Figure 2. Silver-staining of the barley centromeric proteins obtained from PICh purification. PICh and protein purifications were performed using Cen-probe and Scr-probe, respectively. Several bands specific to the Cen-probe (arrows) were labeled. The corresponding regions were visualized in a Coomassie Blue stained SDS-PAGE gel and sliced out for the enzymatic ‘in liquid’ digestion. Figure 3. Detection of cenH3 from PICh purification by Western blot hybridization. The presence of barley cenH3 proteins from PICh purification was detected using antibody against rice cenH3. Input: total proteins isolated from barley chromatin as positive control. Scr-probe: proteins obtained from PICh purification using the Scr-probe. Cen-probe: proteins obtained from PICh purification with the Cen-probe. Equal amounts of total proteins were loaded in SDSPAGE gel for detection. The rice anti-cenH3 antibody was developed from the N-terminus of rice cenH3, which shares 16/19 amino acids with alpha-cenH3 of barley, but only 4/16 amino acids with beta-cenH3 of barley. Figure 4. Comparison of proteins identified from PICh purification. The numbers of proteins identified by Orbitrap-MS from both Cen-probe and Scr-probe isolation were shown with the threshold of 80% of minimum unique peptide and protein identity probability.


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Table 1. Proteins only detected in Cen-probe isolation

Rank

Accession Molecular Identified Number Weight Proteins

Unique Total Protein Putative Coverage Peptides Spectra Length Function

1

F2CQJ8

15 kDa

Histone H3

3

22

20%

136 AA

2

F2EJW9

15 kDa

Histone H2B

1

6

28%

141 AA

3

F2DNY2

17 kDa

Histone H2A

2

6

37%

159 AA

4

F2E067

16 kDa

Histone H2A

2

5

39%

155 AA

5

F2CQ71

16 kDa

Histone H2A

2

4

47%

145 AA

6

F2EJV3

18 kDa

Histone H2A

3

4

35%

165 AA

7

F2D6K8

5 kDa

Predicted protein

1

1

28%

43 AA

8

F2E8Q3

14 kDa

Histone H2A

2

3

45%

145 AA

9

F2CT96

17 kDa

Histone H2A

2

3

41%

166 AA

Unknown


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10

F2CSG7

11 kDa

Nonspecific lipidtransfer protein

11

F2DXK0

27 kDa

Histone H1

2

4

10%

269 AA

12

G1APU3

16 kDa

Beta cenH3

2

2

24%

139 AA

13

F2DKK8

16 kDa

Histone H2A

1

2

46%

148 AA

1

2

8%

160 AA

1

2

14%

115 AA

14

Q43481

17 kDa

HMG1/2like protein

15

F2EAD5

12 kDa

Predicted protein

1

1

7%

109 AA

16

A8CFR3

60 kDa

Betaamylase

3

4

10%

269 AA

17

F2E395

15 kDa

Predicted protein

1

1

4%

137 AA

18

Q1ENF6

16 kDa

Cystatin Hv-CPI2

1

1

9%

140 AA

19

G1APU2

18 kDa

Alpha cenH3

1

1

14%

158 AA

20

F2CR90

17 kDa

Predicted protein

1

1

8%

175 AA

Unknown

Ion binding

RNA binding


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For TOC only


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Isolation and Proteomics Analysis of Barley Centromeric Chromatin

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