The Quantitative Nuclear Matrix Proteome as a Biochemical Snapshot

Article pubs.acs.org/jpr

The Quantitative Nuclear Matrix Proteome as a Biochemical Snapshot of Nuclear Organization Rudolf Engelke,†,# Julia Riede,‡,§,∇ Jan Hegermann,∥,○ Andreas Wuerch,† Stefan Eimer,∥,◆ Joern Dengjel,‡,§,¶ and Gerhard Mittler*,†,⊥ †

Max Planck Institute of Immunobiology and Epigenetics, Stübeweg 51, 79108 Freiburg, Germany Freiburg Institute for Advanced Studies, School of Life Sciences − LifeNet, University of Freiburg, Albertstrasse 19, 79104 Freiburg, Germany § Center for Biological Systems Analysis, University of Freiburg, Habsburgerstrasse 49, 79104 Freiburg, Germany ∥ European Neuroscience Institute and Center for Molecular Physiology of the Brain (CMPB), 37077 Göttingen, Germany ⊥ BIOSS, Center for Biological Signalling Studies, University of Freiburg, Schänzlestrasse 18, 79104 Freiburg, Germany ‡

S Supporting Information *

ABSTRACT: The nuclear matrix (NM) is an operationally defined structure of the mammalian cell nucleus that resists stringent biochemical extraction procedures applied subsequent to nuclease-mediated chromatin digestion of intact nuclei. This comprises removal of soluble biomolecules and chromatin by means of either detergent (LIS: lithium diiodosalicylate) or high salt (AS: ammonium sulfate, sodium chloride) treatment. So far, progress toward defining bona f ide NM proteins has been hindered by the problem of distinguishing them from copurifying abundant contaminants and extraction-method-intrinsic precipitation artifacts. Here, we present a highly improved NM purification strategy, adding a FACS sorting step for efficient isolation of morphologically homogeneous lamin B positive NM specimens. SILAC-based quantitative proteome profiling of LIS-, AS-, or NaCl-extracted matrices versus the nuclear proteome together with rigorous statistical filtering enables the compilation of a high-quality catalogue of NM proteins commonly enriched among the three different extraction methods. We refer to this set of 272 proteins as the NM central proteome. Quantitative NM retention profiles for 2381 proteins highlight elementary features of nuclear organization and correlate well with immunofluorescence staining patterns reported in the Human Protein Atlas, demonstrating that the NM central proteome is significantly enriched in proteins exhibiting a nuclear body as well as nuclear speckle-like morphology. KEYWORDS: Nuclear organization, nuclear matrix, nuclear compartment, organellar proteomics, SILAC quantitative proteomics, flow cytometry

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inner nuclear space.4−6 Apart from the nuclear intermediate filament proteins, named lamins (discovered in 1978), the constituents repeatedly identified in the insoluble nuclear material include a bewildering spectrum of proteins assigned to various functions, predominantly correlating with lamina formation, DNA repair, higher-order chromosome organization, genome replication, gene transcription, and RNA processing.7 Conversely, solid data explaining how these identified proteins could organize to form the aforementioned mesh-like filamentous NM structure in living cells are clearly lacking. Consequently, the concept of the biochemically defined NM has been a subject of considerable controversy.8−10 The description of so-called scaffold-associated regions or matrix

INTRODUCTION It is well-accepted that nuclear architecture provides a framework that is ultimately linked to the functional regulation of most, if not all, metabolic processes taking place in the mammalian cell nucleus.1,2 In the 1970s, subnuclear organization was proposed to be dependent on the presence of a rigid structural framework. This putative nucleoskeleton, derived from nuclei that were subjected to DNase I digestion followed by ammonium sulfate or NaCl salt extraction, was commonly termed the nuclear matrix (NM).3 Notably, the NM could be visualized only by negative staining electron microscopy (EM) after the removal of chromatin, which otherwise confined the ultrastructural characterization of the NM.3 Thus, several procedures that deplete nuclear chromatin while trying to preserve the NM architecture have been developed, revealing a filamentous meshwork spanning the © 2014 American Chemical Society

Received: March 8, 2014 Published: August 4, 2014 3940

dx.doi.org/10.1021/pr500218f | J. Proteome Res. 2014, 13, 3940−3956

Journal of Proteome Research

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proteins have been reproducibly shown to localize to nuclei of somatic cells or to play a role in nuclear organization. Here, in order to overcome these obstacles, we have decided to address this question by using a highly sophisticated combination of SILAC-based quantitative proteomics with a fluorescence activated cell sorting-assisted next generation procedure for isolating subcellular organellar structures. We employ SILAC to comprehensively measure the differential protein retention profiles in NM preparations (versus nuclei), comparing all three previously described extraction procedures (LIS, ammonium sulfate, and NaCl). Our synoptic view, together with stringent statistical filtering, permits the definition of a NM central proteome comprising 272 proteins, thereby establishing a valuable resource for nuclear organization research.

attachment sites (S/MARs), comprising A+T-rich DNA sequence elements that remain or can be bound to salt or detergent (lithium 3,5-diiodosalicylate; LIS) extracted nuclei, led to another overlapping but distinct area of research.5,11 It is commonly believed that S/MAR elements are important for the functional organization of chromatin into higher-order structures consisting of distinct loop and “matrix-attached” S/ MAR units. While ubiquitously expressed “classical” NM proteins like lamin B1 and NuMA (nuclear mitotic apparatus protein) exhibit basic S/MAR-binding capabilities,12,13 more cell-type restricted transcription factors like SATB114 and SATB215 that are highly expressed in lymphocytes specifically bind to a stretch of A+T-rich DNA with asymmetric distribution of G/C residues.16 The S/MAR sequences, which are bound by SATB2 and that are flanking the intronic μ enhancer of the immunoglobulin heavy chain locus, are retained in LIS-extracted nuclei in both B- and non-B-cells.17 This suggests that matrix attachment of S/MARs is likely dependent on ubiquitously expressed as well as cell-type specific proteins. An initially independent line of research has been fuelled by the development of methodological advancements in (confocal) immunofluorescence microscopy (IF) and live cell imaging of GFP-tagged nuclear proteins, leading to the discovery of a variety of nuclear suborganelles.18−20 Most prominent examples are the nucleolus, splicing speckles, paraspeckles, PML, and Cajal bodies. In contrast to cellular organelles, these functional compartments are not surrounded by lipid membranes, but still they appear as stable inert structures and can be observed for several hours by time-lapse microscopy or purified by biochemical methods (e.g., nucleolus, Cajal bodies). However, rapid exchange of proteins between compartments and the nucleoplasm has been observed for virtually all nuclear suborganelles, thereby further challenging the concept of a static NM or nucleoskeleton. Nevertheless, the complex spatial arrangements in the nucleus (nuclear neighborhood) are maintained, at least in part, by the biochemical NM purification procedures.3,21 Until now, the progress toward defining the molecular identity and physiological relevance of the NM has been mainly hampered by difficulties to define bona f ide protein constituents of the quite complex NM proteome.22 Modern state-of-the-art nanoliquid chromatography coupled ESI-mass spectrometry can easily overcome the protein identification challenge of complex samples. Unfortunately, proteomic studies of cellular organelles often identify copurifying contaminant proteins, which, in the worst case, adhere to the structure of interest.23−25 The latter represents a serious concern in the case of the NM preparation protocol that employs nonphysiological extraction procedures, which might possibly induce rearrangement and artificial formation of macromolecular complexes or aggregates. This also implies that a simple proteomic cataloguing cannot distinguish between genuine NM constituents and non-quantitatively extracted highly abundant nucleoplasmic and chromatinic proteins, which is reflected, as mentioned above, in the detection of a miscellaneous spectrum of nuclear proteins.26−28 Accordingly, our experiments reveal that the classical NM purification procedure3 results in the generation of matrices with varying degrees of homogeneity. Not surprisingly, diverse components known from the cytoskeleton and the extracellular matrix, such as actin, tubulin, and vimentin, have been identified in NM preparations.22,29−33 By now, none of these

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EXPERIMENTAL SECTION

Cloning and Expression of eGFP-LmnB1 and NLS-mCherry

LmnB1 was amplified from pYX-Asc-LmnB1 (clone IRAVp968B04159D; ImaGenes) by RT-PCR with Pfx DNA polymerase (Invitrogen) using a forward primer with a XhoI restriction site (5′-ACGTCTCGAGTTGGTGGAAGTGGAGGTAGTGCGACCGCGACCCCC-3′) and a reverse primer with a KpnI restriction site (5′-CATGGTACCGCTATGCGGTCGACTCACATAATGGCACAGCTTTTATTGGATGCT-3′). The PCR product was inserted into the pGEM-T vector using the pGEM-T Easy Vector System (Promega). The eGFP restriction fragment obtained by AatII and XhoI digestion from pEGFP-C1 (Clontech Laboratories) was inserted N-terminally to LmnB1 into pGEM-T. The eGFPLmnB1 fusion product was amplified by PCR with primers (5′PHO-TACCGGTCGCCACCATGGTGA-3′, 5′-PHOGCGGATCTGACGCGAATTCACTA-3′) and cloned into the SciI site of pMXs to create pMXs-eGFP-LmnB1. pMXs was previously created by religation of SalI and XhoI digested pMXs-IG (Cell Biolabs). All primers were acquired from Eurofins MWG Biotech. Complementary oligonucleotides coding for the SV40 nuclear localization signal (NLS) (5′-GGATCCCATGCCGCCGAAAAAAAAACGCAAAGTGGCGCCCGGG3′, 5′-CCCGG GCGCCACTTT GCGTT TTT TTT TCGGCGGCATGGGATCC-3′) were annealed and cloned via BamHI and SmaI into pBluescript II SK(−) (Fermentas) to create pBluescript-NLS. mCherry amplification product (5′CCCGGGGTGAGCAAGGGCGAGGAGGATA-3′, 5′GAATTCCTTGTACAGCTCGTCCATGCC-3′) from pcDNA3.1-N3-mCherry (provided by P. Heun, Max Planck Institute for Immunobiology and Epigenetics, Freiburg, Germany) was fused to NLS by SmaI and EcoRI subcloning into pBluescript-NLS. Subsequently, NLS-mCherry was cloned by BamHI and EcoRI restriction into pMXs to create the pMXs-NLS-mCherry vector. Chemical Transfection and Retroviral Transduction

The Moloney murine leukemia virus (MoMuLV)-based bicistronic vector system derived constructs pMXs-eGFPLmnB1 and pMXs-NLS-mCherry were used for transduction of the murine pre-B-cell line PD36. Virus particle production was performed with the packaging cell line Plat-E.34 Plat-E cells were transfected chemically with 20 μg of vector DNA in 500 μL of 0.125 M CaCl2 in HEPES buffered saline (HBS; 25 mM HEPES, pH 7.0, 140 mM NaCl, 5 mM KCl, 0.75 mM Na2HPO4). The Plat-E cell culture medium was changed 24 h 3941



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mCherry−. After FACS, NMs were reanalyzed to confirm the purity and harvested by centrifugation at 1200g for 5 min.

after transfection. 48 h later, the supernatant containing the viral particles was harvested. This supernatant was adjusted to 6 μg/mL polybrene (Sigma-Aldrich) and filtered through a 0.2 μm cellulose acetate filter (Whatman). The transduction was performed with 2 mL lymphoid cells at 6 × 105 cells/mL and 2 mL of retrovirus-containing supernatant in 6-well plates. Spinning at 500g for 45 min was performed to increase the transduction efficiency. Antibiotic selection with 1 μg/mL puromycin or FACS sorting was performed 24−48 h posttransduction.

Immunocytochemistry and Immunofluorescence Microscopy

Cells, as well as nuclear and subnuclear preparations, were fixed on slides with 4% paraformaldehyde (PFA; Sigma) for immunostaining. After blocking with 1% milk powder in PBS, slides were incubated with the primary antibody in PBS-Tx (PBS, 0.1% Triton X-100) supplemented with 1% milk powder. Primary antibodies were used at 1:150 dilutions: anti-hnRNP U goat polyclonal IgG, anti-Lamin B1 rabbit polyclonal IgG (Santa Cruz Biotechnology), anti-NPC (414) mouse monoclonal IgG1 (Abcam), anti-Fibrillarin goat polyclonal IgG (Santa Cruz Biotechnology), and anti-dimethyl Histone H4 K20me2 (kindly provided by E. Kremmer, Institute of Molecular Immunology, Munich, Germany). Secondary antibodies coupled to Alexa 488 and Alexa 568 fluorophores (Invitrogen) were used at 1:1000 dilutions. Finally, preparations were stained for chromatin with 1 μg/mL 4′,6-diamidino2-phenylindole (DAPI; Invitrogen), and slides were mounted in SlowFade Gold antifade reagent (Invitrogen). Immunofluorescence images at 100× magnification were taken on a Deltavision RT microscope system (Applied Precision). Images were acquired as z-axis stacks and were deconvolved using the softWoRx Explorer Suite (Applied Precision). Immunofluorescence images at 20× and 40× magnification were taken on an Axiovert 200 microscope (Carl Zeiss) equipped with the AxioCam HR (Carl Zeiss).

Cell Culture and SILAC Labeling

PD36 pre-B-cells were cultured in RPMI 1640 (PAA Laboratories), and Plat-E cells, in DMEM (PAA Laboratories). Media were supplemented with 10% FCS (Thermo Fisher Scientific), 50 μM 2-mercaptoethanol (Sigma-Aldrich), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/mL streptomycin (PAA Laboratories). SILAC medium was prepared accordingly from RPMI 1640 w/o L-lysine and L-arginine (Thermo Fisher Scientific) with 42 μg/mL 13C6-L-arginine and 40 μg/mL D4-Llysine (Sigma-Aldrich) or corresponding nonlabeled amino acids 12C6-L-arginine and H3-L-lysine (Sigma-Aldrich). Preparation of Nuclei and Nuclear Matrices

With several modifications, crude nuclei were isolated according to Gerner et al.22 and further purified to acquire clean nuclei according to Graham et al.35 All procedures were carried out at 4 °C if not specifically indicated. 109 murine lymphocytes were resuspended in 2 mL of buffer A (10 mM HEPES, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1 mM EGTA, 0.05% NP-40) and disrupted by shearing in a glass homogenizer. The cell homogenate was pelleted through 40 mL of 250 mM sucrose in buffer B (50 mM HEPES, pH 7.4, 50 mM NaCl, 5 mM MgCl2, 1 mM EGTA) at 400g for 8 min to obtain crude nuclei. The crude nuclear pellet was resuspended in 1 mL of 25% iodixanol and 250 mM sucrose in buffer B and placed on top of a iodixanol step gradient consisting of 1.5 mL of 25% iodixanol, 5 mL of 30% iodixanol, and 5 mL of 35% iodixanol in buffer B for ultracentrifugation at 10 000g for 20 min. The isopycnic band at the 30−35% iodixanol interface containing clean nuclei was collected, washed in 5 mL of buffer C (10 mM PIPES, pH 6.8, 250 mM sucrose, 50 mM NaCl, 3 mM MgCl2, 1 mM CaCl2, 1 mM EGTA, 0.5% Triton X-100, 2 mM VRC), and pelleted at 350g for 3 min. The nuclear pellet was digested in 2 mL of buffer C containing 100 U/mL DNase I (Roche) for 1 h at 24 °C. Three preparationally defined nuclear matrices were isolated using reported extraction strategies of digested nuclei with lithium 3,5-diiodosalicylate (LIS; Sigma-Aldrich), ammonium sulfate, or 2 M NaCl.4,5,36 Consequently, digested nuclei were extracted twice with 12 mL of (a) 25 mM LIS in buffer C, (b) 250 mM ammonium sulfate in buffer C, or (c) 2 M NaCl in buffer C w/o Triton X-100, each for maximal 5 min. So-called crude nuclear matrices were pelleted at 1000g for 5 min, resuspended at 107 mL−1 in buffer C, and FACS sorted.

Electron Microscopy

Purified nuclear matrices were transferred onto a 100 μm deep aluminum platelet (Microscopy Services Dähnhardt) and immediately frozen using a BalTec HPM 010 freezing machine (BalTec). Freeze substitution was carried out at −90 °C for 100 h in 0.1% tannic acid, 7 h in 2% OsO4, and at −20 °C for 16 h in 2% OsO4 (all solutions w/v in dry acetone),37 followed by embedding in EPON resin at RT. 40 nm EPON sections were stained with saturated uranyl acetate in 75% methanol and 4% lead citrate.38 Micrographs were taken with a 1024 × 1024 CCD detector (Proscan CCD HSS 512/1024; Proscan Electronic Systems) in a Zeiss EM 902A microscope (Carl Zeiss). For electron tomography, 250 nm EPON sections were poststained as described above. 10 nm gold beads were applied to both sides. A tilt series from −60 to +60° with 1° increments was recorded on a JEOL 2100 transmission electron microscope (JEOL) at 200 kV with an Orius SC1000W CCDCamera (Gatan) using 2-fold binning. Reconstruction and rendering was done using the IMOD software (http://bio3d. colorado.edu/imod). Western Immunoblotting

Protein samples were solubilized in SDT buffer (4% (w/v) SDS, 100 mM Tris-HCl, pH 7.6, 1 mM DTT), separated on 12% SDS-PAGE (18 × 16 × 0.1 cm) gels, and processed for semidry western blotting on a Trans-Blot SD electrophoretic transfer cell (Bio-Rad Laboratories) using standard protocols. Primary antibodies used were rabbit anti-LmnB1 (Santa Cruz Biotechnology) at 1:1500, goat anti-hnRNP U (Santa Cruz Biotechnology) at 1:1500, rabbit anti-MZB1 immunoserum39 at 1:100, mouse anti-PCNA (PC10; Santa Cruz Biotechnology) at 1:1000, and anti-PC4 (kindly provided by M. Meisterernst, WWU, Münster, Germany) at 1:1000. Secondary HRP-coupled

Nuclear Matrix Isolation via FACS

Crude NMs at a concentration of 107 mL−1 in buffer C (10 mM PIPES pH 6.8, 300 mM sucrose, 50 mM NaCl, 3 mM MgCl2, 1 mM CaCl2, 1 mM EGTA, 0.5% Triton X-100, 2 mM VRC) were filtered through a 40 μm Nitex mesh and sorted by FACS. Gate settings were based on the marker intensities of positive and negative NMs markers as follows: GFP+, DAPIlow, and 3942



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acetonitrile, 0.5% acetic acid) at a constant flow rate of 200 nL/ min for 165 min. LTQ Orbitrap XL was operated in positive ion mode, employing a data-dependent automatic switch between survey full-scans and MS/MS spectra acquisition. For each cycle, one full MS scan in the orbitrap at 1 000 000 AGC target was followed by 10 CID MS/MS in the LTQ at 5000 AGC target on the 10 most intense ions. Selected ions were excluded from repeated sequencing for 60 s, with a relative exclusion mass window of 100 ppm. Survey full-scans were acquired at a resolution of 60 000 at m/z 400 using the polydimethylcyclosiloxane ion [Si(CH3) 2 O]6 H + at m/z 445.120025 ion as lock mass for internal calibration of the orbitrap mass analyzer.

antibodies (Santa Cruz Biotechnology) were used for detection with a commercial ECL solution (GE Healthcare). Q-RT-PCR

Total RNA and DNA were isolated using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Nucleic acid concentration was measured at 260 nm with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). The SYBR Green PCR Master Mix (Applied Biosystems) was used to prepare a 20 μL PCR reaction mix with 200 nM forward and 200 nM reverse primers. The reaction was monitored with the 7500 Fast-Real-Time PCR System (Applied Biosystems). Protein Solubilization and Digestion

Proteomic Data Analysis

Protein samples were solubilized in SDT buffer w/o DTT. Chromatin was digested by adding 50 U/mL benzonase (Novagen) and incubation for 1 h at 24 °C. To increase solubilization, samples were bath sonicated and incubated for 3 min at 95 °C. Insoluble material was cleared by centrifugation at 20 000g for 15 min. Protein concentration was determined with the BCA protein estimation kit (Novagen) according to manufacturer’s instructions. Filter-aided digestion was performed in Microcon YM-10 (Millipore) centrifugal devices as described previously.40 The procedure applying sequential digestion with Lys-C (Wako) and trypsin (Promega) was applied. Obtained peptides were desalted with Empore C18-SD extraction disk cartridges (3M).

FT-MS raw data were analyzed using MaxQuant v. 1.2.0.18 with the integrated Andromeda database search engine.44,45 For peptide identification, enzyme specificity was set to trypsin with a maximum of 2 miscleavages, allowing for cleavage N-terminal to proline and between aspartic acid and proline. Carbamidomethyl cysteine was set as a fixed modification. Oxidized methionine, N-acetylation, and formation of N-pyro-glutamate were set as variable modifications. The MS and MS/MS tolerance were specified as 6 ppm and 0.5 Da, respectively. The required peptide false discovery rate (FDR) and the required protein FDR were set to 0.01, with the minimum required peptide length of 6 amino acids. The “match between runs” and “second peptide” features were enabled. Protein sequence searches were performed against the IPI mouse database v3.68 (56 729 protein sequence entries) appended with sequences of 248 common protein contaminants and decoy protein sequences. Proteins were identified with at least two peptides, wherein one of them should be unique to this protein. Protein quantification ratios were calculated only for proteins with ≥2 acquired SILAC pairs on lysine and arginine containing peptide doublets with lysine-d4 and arginine-13C6, respectively. Intensity-based relative and absolute quantifications were performed using the label-free quantification (LFQ) and iBAQ algorithms implemented in MaxQuant.46 The quantification of NM preparations was performed in minimum of two independent biological replicates, and quantification of nuclei was performed in four replicates.

OFFGEL Peptide Isoelectric Focusing

Fractionation of desalted peptides was performed on a 3100 OFFGEL fractionator (Agilent Technologies) as described previously.41 Briefly, fractionation was performed after assembly of a 24-well setup using the OFFGEL Kit (Agilent) equipment and 24 cm immobilized pH gradient (IPG) strips, pH 3−10 (GE Healthcare). Prior to sample loading, IPG strips were rehydrated with 20 μL of rehydration buffer (2% IPG ampholyte solution, 5% glycerol) per well for 20 min. A desalted peptide solution from 250 μg of protein was adjusted to a final volume of 3.6 mL in 2% IPG ampholyte solution and 5% glycerol. A 150 μL sample aliquot was then loaded into each of 24 wells. The sample was focused at a maximum current of 50 μA and power of 200 mW until 50 kV h was reached. Focused fractions were collected and mixed with 15 μL of 10% TFA, 30% acetonitrile, and 5% acetic acid prior to desalting. Modified peptide purification with StageTips42 was performed using two layers of 0.9 × 1.0 mm Empore C18 (3M) discs. StageTips were conditioned with 50 μL of methanol and equilibrated using 80 μL of buffer A (0.5% acetic acid, 0.05% HFBA). After peptide sample loading, StageTip columns were washed twice using 200 μL of buffer A with 5% acetonitrile. Peptides were eluted with 50 μL of buffer B (0.5% acetic acid, 80% acetonitrile) and vaporized in a vacuum concentrator.

Nuclear FDR and NM Classifier Calculation

The data was analyzed using R and standard statistical packages if not stated differently. Initially, the protein SILAC ratios from the MaxQuant output were logarithmized and quantile normalized. The median of the nuclear data set was adjusted to zero. The NM data sets were adjusted to a logarithmized LMNB1 ratio of zero, mimicking total nuclear retention of LMNB1. The nuclear protein FDR for the nuclear data set (filter criterion I) was calculated using acquired experimental and bioinformatic parameters (logarithmic SILAC ratio, ratio variance between biological replicates of nuclear preparations, iBAQ-derived protein abundance, and the WoLF PSORT47 nuclear localization prediction score). Thus, the FDR for proteins without nuclear annotation was estimated based on the distribution of annotated proteins within the parameter matrix. The FDR was calculated as a running sum ratio between false positive proteins (not nuclear) and the total number of proteins after sorting the protein list by the means of the parameter values. Sorting was performed based on the tested ability of the parameter to identify a true positive nuclear protein starting at the logarithmic SILAC ratio of zero at the lowest ratio variance

LC−MS/MS

Electrospray experiments were conducted on an Agilent 1200 nanoflow LC system (Agilent Technologies) coupled online to an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific) through a nanoelectrospray ion source (Proxeon). For chromatographic separation of peptides, a 15 cm in-house packed fused-silica emitter microcolumn (SilicaTip PicoTip; New Objective) filled with reverse-phase ReproSil-Pur C18-AQ beads (Dr. Maisch) was used.43 Peptide mixtures were loaded onto the column for 20 min at a flow rate of 500 nL/min and eluted with a segment gradient of 10−60% buffer B (80% 3943



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Figure 1. FACS strategy leads to microscopically pure and homogeneous nuclear matrices. (A) A schematic outline of flow cytometry-assisted purification of the NM. PD36 pre-B-cells stably transduced with EGFP-LMNB1 (lamin B1) and NLS-mCherry were used for the purification of clean nuclei. The clean nuclear fraction was subsequently digested with DNase I followed by extraction with 25 mM lithium 3,5-diiodosalicylate (LIS), 250 mM ammonium sulfate (AS), or 2 M NaCl. The crude NM fractions were used in fluorescence-activated sorting for the GFP+, mCherry−, and DAPIlow population. Here, EGFP-LMNB1 represents a positive NM marker, NLS-mCherry, a negative nucleoplasmic marker, and DAPI, a negative bulk chromatin marker. (B) The assigned panels show the fluorescence microscopy analysis of intact cells, purified nuclei, and three NM preparations from PD36 pre-B-cells expressing stably transfected EGFP-LMNB1 (lamin B1) and NLS-mCherry. The NM fractions were prepared by an improved purification strategy involving FACS sorting for GFP+, mCherry−, and DAPIlow populations. The NM preparations are homogeneous and virtually devoid of chromatin and nucleoplasm according to DAPI and mCherry signals (Scale bar: 50 μm).

and cytosolic proteins with a MW larger than 50 kDa as false positive contaminants. Due to their ability to pass the nuclear pore complex (NPC) by passive diffusion,49,50 proteins with a MW below 50 kDa are treated as true positives. The NM classifier I was calculated from standard z-scores indicating by how many standard deviations a ratio is above the mean of all measured ratios for a particular preparation.

between four nuclear analyses, at the highest iBAQ-derived protein abundance, and finally starting at the highest nuclear localization prediction score. Annotations for FDR estimation were taken from the UniProt-GO annotation database,48 while treating nuclear and cytosolic proteins with a molecular weight (MW) below 50 kDa as true positives and proteins from all other compartments (ER, Golgi apparatus, mitochondria, cytoplasmic vesicles, plasma membrane, extracellular region) 3944


Journal of Proteome Research z=

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r − rLMNB1 σ

staining patterns. The staining pattern descriptors were transformed into ordinal numbers in the rank order 1 for diffuse to 5 for compartment. Thus, differential medians between the staining pattern distributions within protein groups were tested using the Mann−Whitney U test.

where r is the measured protein SILAC ratio, rLMNB1 is the LMNB1 SILAC ratio, and σ is the standard deviation of the SILAC ratio within the dataset. These z-scores were inversed and used to derive p-values from the integral of the Gaussian distribution, using the complementary error function. p-values were concatenated using the Fisher’s combined probability test and adjusted using the Benjamini−Hochberg multiple testing correction. The NM classifier II is an abundance weighted modification of the NM classifier I. The assessment was performed using z-scores from the iBAQ distribution for the particular NM preparation in a weighting ratio 2:1 NM retention/NM abundance according to Stouffer’s z-score method z(NM classifier II) =

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RESULTS AND DISCUSSION

Proteomic Workflow and Purification of the Nuclear Matrix (NM)

The NM represents a biochemically generated subnuclear proteinaceous structure that resists extensive extraction with either high salt (2 M sodium chloride, 0.25 M ammonium sulfate) or detergent (25 mM LIS) after treatment of nuclei with DNase I.4−6 Here, we have chosen the PD36 pre-B-cell line as a model system based on earlier work demonstrating that the S/MARs flanking the immunoglobulin heavy chain intronic μ enhancer are efficiently retained in B-cell NMs prepared by the LIS extraction method,54,55 which additionally served as a positive control for our purification regimen. For a high-quality definition of a NM core proteome, it is of the outmost importance to be able to control for nonspecific extraction-induced protein precipitations. We also note that it is equally essential to obtain a highly homogeneous preparation of nuclear matrices since the applied classical NM purification procedures suffer from chromatin agglutination and the presence of nonextracted nuclei or nuclear debris, as assessed by differential interference microscopy (DIC) and DAPI staining (Figure S1). It was also obvious that the proteomic identification of bona fide NM proteins is further hampered by copurifying contaminants as well as the incomplete extraction of highly abundant nuclear proteins (introduction and data not shown). In order to address these issues, we have not only developed a proteomic approach to identify truly enriched NM proteins but also have drastically improved the classical NM purification protocol. First, to evaluate the generation of precipitation-borne artifacts, we compared NMs prepared by three physicochemically different extraction procedures (Figure 1A), namely, 0.25 M ammonium sulfate (AS), 2 M NaCl, or 25 mM lithium 3,5diiodosalicylate (LIS). LIS is a mild anionic protein solubilizing detergent,56 whereas AS represents a kosmotropic salt (saltingout). In contrast, NaCl is considered to be a weak chaotropic (salting-in) salt.57 These have been extensively used in the past and are considered as standard extraction agents for the operational definition of the NM.3 We put significant effort toward starting NM preparations from a two-gradient purified clean and homogeneous nuclear fraction that was virtually free of adherent ER and cytoskeletal remnants, as judged by microscopy (Figure S3) and immunoblotting (Figure S4C), for the abundant B-cell-specific ER marker protein MZB1.39 During the establishment of the extraction procedures, we put effort into maintaining overall nuclear morphology, as assessed by immunofluorescence staining with anti-SAF-A, antiH3, anti-LMNB1, and anti-NPC antibodies (Figure S2), thereby compromising on yield and efficiency of chromatin removal. This is reflected by the results of our EM ultrastructural analysis of the NaCl NM preparation (Figure S3), which reveals a fibrogranular structure, recapitulating earlier observations.4 Second, in order to improve the purity of NMs preparations, we generated a PD36 cell line expressing EGFP-LMNB1 (lamin B1) as a positive NM marker58 and NLS-mCherry as a negative

2z(NM classifier I)−1 + z(iBAQ) 22 + 12

Bioinformatic Analyses

For plotting in a heat map, missing log ratios in the NM data set (LIS NM, n = 282; AS NM, n = 258; NaCl NM, n = 269; total number of plotted nuclear proteins, n = 2892) were filled with random values between the 0.95 to 1.0 percentile of the distribution of measured values in each NM preparation. Logarithmic ratios were linkage clustered using the Manhattan distance metric and plotted in heat maps using the MultiExperiment Viewer v4.6.51 The enrichment analysis of gene ontology (GO) annotations for biological function and cellular compartment was performed for protein fractions of interest against the complete reference data set or the murine genome. The calculation of Fisher’s exact statistics for the enrichment of individual annotation categories was performed within the DAVID Bioinformatic resources v6.7.52 Statistical significance for domain and compositional bias enrichment in comparison to a reference data set was performed with the Fisher’s exact test. For hierarchical clustering, GO categories were filtered for significant enrichment (Benjamini−Hochberg corrected EASE score < 0.05) in at least one of the analyzed fractions and additionally checked for nonredundancy of category terms. The filtered p-values were logarithmized, inverted, and z-transformed. These scores were then clustered by one-way hierarchical average linkage clustering using the Manhattan distance function and plotted in a heat map. Text mining was performed with the MedScan Natural Language Processing technology module within the Pathway Studio software (Ariadne Genomics). Image Data Mining

Immunofluorescence images from the Human Protein Atlas53 were used for the evaluation of nuclear staining patterns. 516 proteins with images assigned to have a supportive IF and WB validation were found among significant nuclear proteins from our study. The evaluation of nuclear staining patterns was performed unbiased by two independent researchers. Following staining pattern descriptors were chosen: diffuse (diffuse, smooth pattern), granular (dense granular pattern), speckled (speckles), spotty (>20 separated dots), and compartment (nuclear bodies as few separated spots, nucleolus, nuclear membrane). Multiple assignments were possible when the staining could not be described with a single descriptor or when cell lines stained with the same antibody showed different 3945



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Figure 2. Workflow for proteomic analysis of the nuclear matrix (NM). (A) A schematic overview of the quantitative proteomic strategy combined with statistical filtering and classification criteria. The nuclear proteome of PD36 pre-B-cells was used to define filter criterion I (nuclear protein FDR < 0.05). Filtering criterion II required the protein to be quantified in the three differentially prepared (LIS, AS, and NaCl) nuclear matrices. The NM classifier I represents a p-value and is a function of protein retention in the NM. NM classifier II corresponds to a transformed NM classifier I value that is further weighted by protein abundance in the three differentially extracted nuclear matrices. (B) Shown are Pearson correlation coefficients of log2-transformed SILAC ratios for biological replicates of nuclear and NM preparations as well as in-between sample correlation coefficients that amount to 0.88, 0.94, and 0.84 for LIS NM, AS NM, and NaCl NM, respectively. The correlation coefficients for nuclei ranged from 0.71 to 0.95. (C) Principle component analysis of nuclear and NM preparations. Principal component 2 (PC2) demonstrates that NM preparations clearly differ from the nuclear proteome but are quite similar to each other.

enriched. The previous data are further corroborated by immunofluorescence microscopy studies (Figure 1B). Sorting for GFP+ specimens led to integral removal of LMNB1negative and enrichment of structurally preserved LMNB1containing GFP+ nuclear matrices following either AS or sodium chloride or LIS extraction. More importantly, sorting for DAPIlow and mCherry− particles completely removed chromatin clots and nonextracted nuclei. Differential interference contrast (DIC) microscopy further confirmed this remarkable purity, which is highlighted by the complete absence of granular and debris material. In line with this being most effective for the sodium chloride extraction method, DNA depletion is quite efficient, with an average depletion to less than 0.01% (compared to nuclei) as measured by photometry subsequent to total DNA isolation/purification (Figure S5A). Similarly, more than 99% of nuclear RNA is removed by both the sodium chloride and AS extraction procedure, whereas LIS treatment depleted slightly more than 90% of it. Next, we performed quantitative RT-PCR analysis of the Ig μ chain locus, employing two primer pairs specifically amplifying either the 5′- or 3′-S/MAR elements positioned juxtaposed to the Eμ enhancer. As reported previously,17 both S/MARs exhibit a substantial and specific enrichment in the LIS NM compared to the gene body (exon 3 amplicon),

(nucleoplasmic) marker, thereby enabling the inclusion of a novel FACS-based NM enrichment step. This extended NM purification workflow (Figure 1A) allows a marker-selective fractionation when sorted for GFP+ (LMNB1 positive), mCherry− (nucleoplasmic NLS-mCherry negative), and DAPIlow (chromatin depleted) specimens. On the basis of measuring the light scatter, FACS additionally excels in its ability to apply a filter on particle size, resulting in morphologically homogeneous preparations. The anticipated efficacy of flow cytometry-based enrichment of nuclear matrices was confirmed by reanalysis, which performed at as least as well as preparative sorting of other organelles like nuclei and mitochondria.24,59−61 The marker signal distributions after sorting were nearly normal and nonoverlapping, indicating homogeneity and high purity of the sample (Figure S4A,B). Averaged signal intensity medians of two independent experiments underscore the reproducibility and efficiency of the sorting procedure, resulting in selective enrichment of morphologically homogeneous EGFP-LMNB1 containing matrices and exhaustive removal of mCherry and DAPI marker signals (Figure S4B). The efficiency of the complete workflow was analyzed by western blotting (Figure S4C). During nuclear extraction, nucleoplasmic proteins PC4 and PCNA were quantitatively removed, whereas LMNB1 was strongly 3946



Article

Figure 3. Definition of the NM central proteome. (A) Number of proteins identified and quantified in the nuclear, LIS, AS and NaCl NM preparations. (B) Receiver operating characteristic (ROC) curves demonstrating the specificity of filtering criterion I. (C) Effect of filtering and classification on the quantitative nuclear proteome and (D) NM proteome. The statistical analysis of experimental parameters (ratio, ratio variance, iBAQ-based abundance) combined with the WoLF PSORT nuclear localization score define a high-quality (FDR < 0.05) nuclear proteome (filter criterion I). Filter criterion II demands a protein both to be present and quantified in all three NM preparations. Classifier I: degree of retention in the LIS, AS, and NaCl NM. Classifier II: abundance weighted NM classifier I. (E) The NM central proteome visualized by the quantitative profile analysis of the LIS, AS, and NaCl NM. Shown is a heat map dendrogram of quantified NM proteins. For completeness, all proteins that fulfilled filtering criteria I (n = 2892; panel C) were selected for plotting. The NM classifier I and II map highlights significant NM proteins that constitute the NM central proteome corresponding to the extraction-resilient structure of the nucleus. Overall, the NM protein extraction profile reflects a biochemical snapshot of the structural landscape of the nucleus remaining after nucleoplasm and chromatin removal.

Supportive to the relatively high overall similarity between NM preparations is the fact that they show no difference in the second principal component. Hence, we concluded that the biochemical purification of nuclear matrices by our approach is highly reproducible. Furthermore, the relatively high correlation between the three different extraction methods (low principle component variance) argues against the purification of a randomly generated structure.

whereas sodium chloride and AS extraction seem to remove these DNA elements (Figure S5B). This is in agreement with observations from others showing that S/MARs associated with active genes are selectively retained in LIS NM but not in sodium chloride NM preparations.62 Third, for high-confidence assignment of NM proteins, we devised a SILAC-based quantitative proteomic strategy to accurately describe the degree of retention of a nuclear protein in the NM. In this process, FACS-sorted NMs were compared against heavy labeled nuclei by nanoLC−MS (Figure 2A). Consequently, nucleoplasmic proteins that are efficiently extracted by our NM purification procedure exhibit a high heavy-to-light ratio. In contrast, proteins refractory to extraction (e.g., lamin B1) are characterized by a low ratio. The Pearson correlation coefficient of logarithmized SILAC ratios for intersample reproducibility of biochemical preparations was 0.88, 0.94 and 0.84 for the LIS NM, AS NM, and NaCl NM, respectively (Figure 2B). The quantitative analysis of nuclei showed a robust correlation with correlation coefficients from 0.71 to 0.95 and can be compared to results obtained in DNA microarray experiments showing correlations from 0.68 to 0.91.63 To evaluate the relationship between the different proteomic data sets, we performed an unbiased principle component analysis (PCA; Figure 2C). The relatedness of all sample-specific replicates is demonstrated, as they form distinct nuclear, LIS, AS, and NaCl NM clusters.

Proteomic Identification and Classification of NM Proteins: Definition of the NM Central Proteome

Assuming that a protein identified in the NM preparation should also appear in the nuclear preparation, we generated a high-quality nuclear proteome data set for filtering out nonnuclear copurifying exogenous (e.g., keratins) and endogenous (e.g., cytoskeletal proteins) contaminants from our NM protein catalogue (Figure 2A). To pursue this, we obtained a light labeled and a heavy labeled (lysine-2H4 and arginine 13C6) nuclear preparation of high purity from the murine pre-B-cell line PD36 by combining differential centrifugation on a 0.25 M sucrose cushion with iodixanol density gradient ultracentrifugation (Figure 1A). Under these conditions, nuclear integrity was virtually unaffected, as judged by phase contrast and immunofluorescence microscopy (Figure S2). However, electron microscopy revealed that a substantial amount of the nuclear envelope double membrane system was removed 3947


AATF, BCLAF1, BHLHE40, BOP1, BRIX1, CDKN2A, CIRH1A, DCAF13, DDX18, DDX51, DHX33, DHX9, DKC1, DNTTIP2, EBNA1BP2, ESF1, FBL, FBLL1, FCF1, FOP, FTSJ3, GNL3, GTPBP4, IMP3, IMP4, MAK16, MKI67IP, MPHOSPH10, MYBBP1A, NAT10, NHP2, NOC2L, NOC3L, NOL10, NOL11, NOL7, NOL8, NOL9, NOP10, NOP56, NOP58, NSA2, NVL, PES1, PINX1, PWP2, RBM28, RCL1, RPF1, RPF2, RPL23, RPL4, RPL6, RRP1, RRP12, RRP15, RRP1B, RRP8, RRS1, RSL24D1, TBL3, TCOF1, UBTF, URB1, USP36, UTP11L, UTP14A, UTP15, Utp18, UTP20, UTP3, UTP6, WDR12, WDR3, WDR43, WDR74, XRN2, ZFP106 CCNL1, CCNL2, CRNKL1, DDX3X, DDX42, EIF4A3, FYTTD1, LUC7L3, MAGOH, NOC3L, NSRP1, PIAS1, PNN, PPIG, PRPF40A, PRPF8, RBM25, RBM27, RBM8A, SRRM1, SRRM2, SRSF1, SRSF10, SRSF3, SRSF5, SUMO1, TFIP11, THOC1, U2AF1, U2AF2, ZC3H14, ZNF638 AAAS, AHCTF1, CCAR1, EIF6, GLE1, GTPBP4, LMNA, LMNB1, LMNB2, MAD1L1, NUP107, NUP133, NUP153, NUP160, NUP188, NUP210, NUP214, NUP43, NUP50, NUP62, NUP85, NUP88, NUP93, NUP98, NXF1, POM121, RANBP2, RANGAP1, RRP12, SUMO1, SUN1, SUN2, TMEM48, TPR, WDR3

BCLAF1, BHLHE40, BOP1, BRIX1, CIRH1A, DCAF13, DDX51, DHX33, DKC1, DNTTIP2, EBNA1BP2, ESF1, FBL, FOP, FTSJ3, GNL3, GTPBP4, HJURP, IMP3, IMP4, MAK16, MKI67IP, MPHOSPH10, NOL10, NOL11, NOL7, NOL8, NOP56, NOP58, NSA2, NUP153, NVL, PES1, PWP2, RCL1, RPF1, RRP1, RRP12, RSL24D1, SETX, TBL3, URB1, USP36, UTP11L, UTP14A, UTP15, UTP18, UTP3, UTP6, WDR12, WDR3, WDR43, ZFP106

1110037F02RIK, ACIN1, AIMP1, AQR, ARGLU1, BMS1, BUD13, CASC5, CASP8AP2, CBLL1, CDC40, CDK11B, CEBPZ, CENPQ, CHCHD3, CHD1, CHERP, CLK2, CLK3, CLP1, CWC22, D2WSU81E, DDX10, DDX21, DDX24, DDX27, DDX41, DDX54, DHX15, DHX8, DNAJA2, DNAJA3, DNAJB6, EEF1A1, EFTUD2, EHD4, ERH, ETS1, FIP1L1, FOXP1, GCFC1, GCFC2, GLTSCR2, GNL2, GPS2, GTL3, HEATR1, HNRNPF, HNRNPL, IK, IKZF5, KDM4B, KHDRBS1, KIF24, KRI1, LUC7L, LUC7L2, MACF1, MAML2, MCG_18410, MCM3AP, METTL16, MFAP1, MIER1, MIS18BP1, MKI67, MYEF2, NCOR1, NGDN, NIPBL, NKRF, NO66, NOLC1, NOP14, NUDT21, PAF1, PCID2, PHF3, PLEK, PPAN, PPHLN1, PPIL4, PRPF38A, PRPF38B, PRPF4B, Q3TTL0, Q4FZC9, Q80XR9, Q8BZR9, Q8R3Y5, Q8VDP2, RAE1, RALY, RBBP8, RBM10, RBM14, RBM15, RBM17, RBM26, RBM39, RBM5, RBMX2, RBMXL1, RIF1, RNPS1, RPL10A, RPL13, RPL14, RPL15, RPL18, RPL18A, RPL27A, RPL3, RPL32, RPL34, RPL35, RPL35A, RPL36, RPL7A, RPL7L1, RPRD2, RPS23, RSRC2, RUNX1, SAFB2, SAMM50, SAP18, SENP1, SF3B1, SFRS18, SFSWAP, SLTM, SNRNP200, SNRNP27, SNRNP70, SNRPD1, SNRPD2, SNRPD3, SNRPF, SNW1, SON, SREK1, SRRT, SRSF11, SRSF2, SRSF4, SRSF6, SRSF7, SRSF9, STAG3, SYMPK, TBL1X, THOC2, THOC5, THOC6, THRAP3, TINF2, TMEM209, TMEM43, TRA2A, TRA2B, TRIP13, TRP53, TXNL1, U2SURP, URB2, VIM, WDR36, WDR46, WDR75, WRNIP1, XAB2, YTHDC1, ZC3H13, ZC3H18, ZC3HAV1, ZCCHC10, ZFP397, ZFP42, ZMYM4, ZNF609, ZRANB2

1110037F02RIK, ACIN1, AIMP1, AKAP17B, ARGLU1, BC006779, BMS1, BUD13, CASC5, CASP8AP2, CBLL1, CCDC165, CCNL1, CCNL2, CDK11B, CENPC1, CENPL, CHCHD3, CHCHD6, CHERP, CLK2, CLK3, CLP1, CRNKL1, CWC22, DDX10, DDX24, DMD, DNAJA2, DNAJA3, DNAJB6, EG547091, EHD4, EIF6, ETS1, FIP1L1, FOXJ3, FOXP1, FOXP4, FYTTD1, GCFC1, GCFC2, GLE1, GM14420, GPS2, HEATR1, HNRNPL, IKZF5, KDM3A, KDM4B, KDM5C, KEAP1, KIAA1551, KIF24, LUC7L, LUC7L2, LUC7L3, MACF1, MAD1L1, MAGOH, MAML2, MCM3AP, METTL16, MIER1, MIS18BP1, MLL5, MYEF2, MYH7, NCOA2, NCOR1, NGDN, NKRF, NKTR, NO66, NOLC1, NUDT21, NUP107, NUP133, NUP160, NUP188, NUP214, NUP43, NUP88, NXF1, PAPD4, PAPD5, PCF11, PCID2, PIAS1, PNN, PPHLN1, PPIL4, PRPF38A, PRPF4B, PRPF8, PTPN23, Q3TTL0, Q4FZC9, RANBP2, RASSF1, RBBP8, RBM10, RBM14, RBM17, RBM25, RBM26, RBM39, RBM45, RBM8A, RBMX2, RBMXL1, REPIN1, RERE, RIF1, RNPS1, RPL7L1, RSRC1, SAMM50, SAP18, SECISBP2, SENP1, SFRS18, SFSWAP, SNRNP27, SNRNP48, SNRNP70, SPC25, SPEN, SREK1, SRSF1, SRSF10, SRSF2, SRSF3, SRSF5, SRSF6, SRSF7, SRSF9, STAG3, SYMPK, TDRD3, TFIP11, THOC2, THRAP3, TINF2, TMEM209, TMEM43, TPR, TRA2A, TRA2B, TRIM56, TRIM59, TRIP13, TRP53, TTN, TXNL1, U2AF1, U2AF2, U2SURP, UBR5, URB2, WDR36, WDR46, WDR75, WRNIP1, YTHDC1, ZC3H13, ZC3HAV1, ZCCHC10, ZFP26, ZFP397, ZFP42, ZFP462, ZFP748, ZFR, ZNF507, ZNF574, ZNF609, ZNF638, ZRANB2

EIF6, LMNB1

AHCTF1, HDAC3, OIP5 EIF3E GEMIN7, MAML2, NACC1, NCOR2, NUDT21 CENPC1, MAD1L1, NUP107, NUP133, NUP160, NUP43, SPC25

DDX42, DKC1, FBL, GAR1, NHP2, NOP10, NOP58, NPAT, OIP5, SART1, SRRM2 AHCTF1, HNRNPM, LMNA, LMNB1, MATR3, NONO, NUMA1, PPIG, PRPF40A, SFPQ, SRRM1, THOC1, ZNF326 AHCTF1, PPP1R10, OIP5 EIF3E, SUMO1 NACC1, SUMO1 AHCTF1, CENPA, CENPC1, MAD1L1, MAD2L1, NUP107, NUP133, NUP160, NUP43, NUP85, RANGAP1 EIF6, LMNA, LMNB1, LMNB2

DKC1, FBL, GAR1, GEMIN7, NOLC1, NOP58, NPAT, OIP5 AHCTF1, HNRNPM, LMNB1, MATR3, PPIG, PRPF40A, PSMA6

AKAP17B, CCNL1, CCNL2, CRNKL1, FYTTD1, LUC7L3, MAGOH, MLL5, NSRP1, NXF1, PIAS1, PNN, PPIG, PRPF40A, PRPF8, RBM25, RBM8A, SRSF1, SRSF10, SRSF3, SRSF5, TFIP11, U2AF1, U2AF2, ZNF638 AHCTF1, EIF6, GLE1, GTPBP4, LMNB1, MAD1L1, NUP107, NUP133, NUP153, NUP160, NUP188, NUP210, NUP214, NUP43, NUP62, NUP85, NUP88, NUP93, NUP98, NXF1, POM121, RANBP2, RANGAP1, RRP12, SUN1, TMEM48, TPR, WDR3

gene name (NM classifier II < 0.05)

gene name (NM classifier I < 0.05)

Table 1. NM Central Proteome

Nuclear lamina Other

Nuclear membrane, nuclear envelope, nuclear pore Cajal body Nuclear matrix Chromatin PML body Nuclear body Kinetochore

Nuclear speck

Nucleolus

nuclear compartment

Journal of Proteome Research Article

3948



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share a high probability to play an important role in nuclear organization. Hence, proteins passing filter criteria I and II were further assigned with a statistical value describing the chance of a protein being efficiently retained (exhibiting a low heavy-tolight ratio) in an individual NM preparation. We calculate this value based on an outlier analysis of the log2-transformed SILAC ratio distribution. Thus, we were able to precisely quantify the enrichment of proteins in NM preparations and assign 320, 346, and 344 significant (FDR < 0.05) proteins to the LIS, AS, and NaCl NM. To have an integrated view on the NM central proteome, the statistical values for each extraction method were concatenated to a combinatorial (multiple testing correction adjusted) statistical p-value, NM classifier I (Figure 3E and Tables 1 and S1). The latter represents a rather conservative classification for core NM proteins because even proteins that are substantially enriched in only two of the three extraction techniques will be filtered out. In essence, NM classifier I annotates a protein to be either highly extractable (pvalue close to 1) or very efficiently retained (p-value < 0.05) in the NM. The latter group (comprising 272 proteins) is listed in Table 1 and contains proteins characterized by a welldocumented scaffolding function like lamin B1,70 TPR,71 AHCTF1/Elys,72 FBL/fibrillarin,73 and SUN1.74 Surprisingly, the well-known and abundantly expressed lamina component lamin A/C, which forms a nuclear filament structure separated from the lamin B meshwork, was found with a NM classifier I value of only 0.27. Interestingly, FRAP experiments have shown that lamin A/C basically adopts two distinct fates in the nucleus, a very dynamic as well as a highly static one.58 In addition, the well-established S/MAR-binding NM protein SAF-A/hnRNPU75 was, to a large extent, extracted in the NM preparations (NM classifier I = 1.0), reflecting the fact that SAF-A has additional nucleoplasmic functions (transcription, splicing, mRNA stability and export). To cope with this, we have defined a second descriptive classifier, termed NM classifier II, which represents a protein abundance (iBAQ-based46) weighted transformation of NM classifier I. We propose to evaluate nuclear components based on the tendency of NM classifier II to increase or decrease compared to NM classifier I (Figure 3E and Table S1). This should reflect that abundant and enriched proteins play a more general structural role in the nucleus than enriched but low abundance proteins. For example, the NM classifier I for LMNB1 is 2.8 × 10−11. NM classifier II is more significant, 2.0 × 10−16, reflecting the global structural importance76,77 of this protein. Similarly, the NM classifier II for lamin A/C (p = 0.018) mirrors its well-documented role in nuclear organization. Other copious nuclear proteins like NONO and NUMA behave accordingly with an increase of the NM classifiers II significance from 4.6 × 10−2 and 5.3 × 10−2 to 2.8 × 10−4 and 2.1 × 10−3, respectively. Vice versa, low abundance proteins, as quantified in the nuclear proteome, show a loss of significance from NM classifier I to NM classifier II. For instance, MATR3 from 3.9 × 10−11 to 1.4 × 10−06, CPSF from 1.7 × 10−2 to 6.7 × 10−2, and PML (promyelocytic leukemia protein) from 6.9 × 10−2 to 0.5. The latter example reflects nicely that PML, which was suggested to form the core of PML bodies,78 is structurally important but plays only a localized role in the nucleus. Also our EM tomography (Figure S3) analysis of the NaCl NM reveals the presence of many local scaffolding platforms rather than a contiguous proteinaceous meshwork spanning and statically compartmentalizing the entire nuclear space. In conclusion, NM classifier I or II filtering leads to the

(Figure S3A, D). This is in accordance with previous observations demonstrating removal of the outer nuclear membrane in the presence of low amounts of nonionic detergent (Triton X-100 or NP-40) while preserving the integrity of nuclei.64 Following one-to-one mixing of the nuclei and in-solution digestion, quantitative LC−MS/MS data was used to calculate the FDR for a protein to be a true nuclear protein. In this step, we reasoned that nuclear proteins will be purified reproducibly and give a ratio of one when two nuclear preparations are equally mixed. Accordingly, the intersample variance of these preparations should be lower compared to stochastically copurifying contaminants. Furthermore, we expected to see a relationship between nuclear proteins and protein abundance, with more true nuclear proteins in the abundant fraction. Thus, the acquired experimental parameters (SILAC ratio, ratio variance between biological replicates, and protein abundance) from the analysis of one-to-one heavy to light mixed nuclei were used to calculate the FDR for each protein to be localized to the nucleus. More precisely, initial FDR estimation was based on the annotated proteins in these nuclear data set. The WoLF PSORT47 nuclear localization prediction score was additionally used to increase the sensitivity of this approach. Thus, the calculated nuclear FDR was sought to be used as a first filter criterion operating at the input proteome level (filter criterion I) to discriminate between true nuclear proteins and contaminants from other cellular compartments. Starting from a depth of 4367 quantified proteins (Figure 3A), this very conservative approach (Figure 3B) enabled the definition of 2892 nuclear proteins with a nuclear FDR < 0.05 (Figure 3C), of which 579 proteins were previously not annotated for nuclear localization. The second filter criterion (filter criterion II) was the requirement for a protein to be quantified in all three NM preparations using different extraction reagents (LIS, AS, NaCl). Accordingly, by applying both filter criteria on initially 3646, 3962, and 3950 quantified proteins, 2381 proteins were considered for further analysis in the LIS-, AS-, and NaCl-extracted NM proteome data sets, respectively (Figure 3D). As demonstrated in previous studies on organelles, state-ofthe-art proteomics has reached a sensitivity level65 that results in the repeated identification of cofractionating contaminants.66,67 Additionally, qualitative interpretation of proteomic data sets is complicated by stochastic undersampling of lower abundance proteins.68 Hence, in order to obtain a global picture of the NM protein inventory, the quantitative retention profile of nuclear proteins (passing filter criterion I) in the NM preparations was plotted in a heat map (Figure 3E) and illustrates, to our knowledge, the first comprehensive biochemical view on nuclear organization. As expected from the principal component analysis, the extraction profiles among the three different extraction techniques are largely similar. However, small clusters of differential extraction behavior are present and reflect the bias associated with each individual method. Notably, cluster II, which shows strong extraction of its components in the LIS NM, contains almost the complete set of the RNA polymerase II Mediator head module proteins (Med6, Med8, Med11, Med18, Med20, Med22, Med28, Med29, Med30).69 Likewise, compared to that of the AS and NaCl NM, histones (especially histone H1) are less efficiently extracted (cluster III) by LIS (Figure 3E). To facilitate the extraction of the NM core components from our large data set, we introduce the concept of the NM central proteome. We define the latter to contain those proteins that 3949



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Figure 4. Comparison of quantified NM proteins with previously published data. (A) Illustrated are the numbers of previously published NM proteins. Proteins from all species were collected by text mining and GO annotation. The LC−MS/MS proteomic analysis identifying 158 significant NM proteins by Albrethsen et al.32 (see text) was treated separately. Shown is the total number of compiled NM proteins, followed by the total number of proteins quantified in this study. The effect of applying stringent nuclear and NM filtering criteria is depicted. (B) Heat map demonstrating the nuclear extractability (based on our data set) of previously published NM proteins. In the zoomed view, the most enriched NM proteins are listed with an emphasis on NM classifier I and II. Nineteen proteins (colored in red) from the text and GO mining as well as three proteins from the Albrethsen et al. data set exhibit a NM classifier I FDR > 0.05. Similarly, 33 and 7 proteins display a NM classifier II FDR > 0.05, respectively. In the latter case, all 7 proteins from the Albrethsen et al. study are also part of the text/GO mining data set. (C) Intensity-based protein abundance profile (log iBAQ) of previously published NM proteins, as measured in the nuclear preparations described in this work. Almost 60% of the previously suggested NM components belong to the most abundant proteins in our unfiltered quantitative nuclear proteome data set.

mining, and GO annotations. We have found 390 mouse proteins or orthologues of previously published NM proteins. A recently published LC−MS/MS analysis of human colorectal cancer tissue NM preparation identified 158 proteins32 and was considered separately. We were able to quantify 211 out of the 390 reported NM proteins in our study. Likewise, we quantified 91 out of 158 proteins from the Albrethsen et al.32 study (Figure 4A). In turn, only 175 out of the 211 and, more strikingly, 54 of the 91 quantified proteins were considered to be nuclear based on filter criterion I. The union of these remaining proteins (n = 211) was plotted in a heat map to visualize the wide range of extractability (Figure 4B). It was apparent that most of the previously published NM proteins were false-positive identifications based on our filtering and classification criteria. On one side, many proteins, in particular from the Albrethsen et al. study, were coenriched contaminants from the extracellular matrix, cytosol, and cytoskeleton. On the other side, almost 60% of these proteins were previously misassigned to the NM based mainly on their shear abundance (Figure 4C) in the nucleus, not considering their degree of extraction.

assignment of 272 or 351 NM central proteome constituents, respectively (Figure 3D, Table 1, see Supporting Information Table S1 for the complete proteomic data set). Thus, in line with a recent study,79 SILAC protein enrichment quantification of biochemical purifications combined with additional experimental and bioinformatics classifiers allow a much more precise assignment of proteins to cellular compartments. Comparison with Previously Published Data

Since the establishment of the NM concept in 1974, 4232 publications have been listed on PubMed containing the keywords “nuclear matrix”. Many of the listed publications focus solely on the identification of single NM proteins. First proteome-wide studies on the NM from rodent cells performed in the group of G. Sauermann resolved up to 200 spots on 2D gels.22 A recent 2D gel-based proteomic study of the high-saltextracted NM from Drosophila melanogaster identified about 135 reproducible spots.80 GeLC−MS studies identified 174 proteins from Jurkat NMs and 158 proteins from human colorectal adenoma tissue derived NMs.28,32 To evaluate the concordance of our NM data set with previously published data, we curated a list of proteins identified in the NM from the NMPdb,81 MedScan text 3950



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Structural Hierarchy in Nuclear Organization

in the definition of nuclear architecture. Additionally, Ser-, Arg-, Glu-, and Pro-rich repeats are statistically overrepresented in the NM, whereas Gly-, Asp-, His-, Gln-, Thr-, and Leu-rich sequences are not. Notably, Pro-rich sequences were shown to be important protein−protein interaction modules in signaling pathway scaffolds and spliceosome complex formation.87,88 Given that proteins containing such intrinsically disordered regions are known for providing important scaffolding functions,89 the potential role of LC regions in nuclear organization should be further addressed in the future. Despite recent advances in fluorescence microscopy, particularly the use of fluorescent protein tagged reporters,90 the understanding of nuclear structure and function is notably limited. An important goal is to identify and characterize proteins that are involved in either global or local orchestration of nuclear architecture. To appraise the extent to which this proteomic study could provide valuable information on nuclear structure, we performed functional annotation of quantified proteins, subdivided into similarly sized bins based on their extractability as expressed by the NM classifier I. The analysis of GO terms describing the affiliation with a known subnuclear compartment revealed that nuclear body proteins from the nucleolus, nuclear speckle, nuclear pore, nuclear envelope, and Cajal body were enriched (Figure 5A) in our NM specimens. Additionally, PML (promyelocytic leukemia protein) body components significantly peak in the slightly depleted fraction with NM classifier I values ranging from 0.2−0.4. Because only isoform V of PML displays a profound scaffolding function as measured by FRAP and FCS,91,92 the failure of shotgun proteomics to accurately quantify highly related protein isoforms may account for the moderate NM classifier I value obtained for PML. Notably, other extraction-resilient components of nuclear bodies exhibit a much better correlation of protein extractability with nuclear protein dynamics. FRAP studies identified NuMA,93 coilin, and FBL/fibrillarin94,95 as kinetically slow components in the nucleus comprising immobile fractions of 10−40% and FRAP half-times of up to 300 s. Interestingly, EBNA1BP2 (NM classifier II = 0.0009) and RPF2/BXDC1 (NM classifier II = 0.0056), which have been identified as important scaffolding proteins for the nucleolus, demonstrate nucleolar FRAP-measured residence times around 120 s.96 In comparison, nuclear EGFP has a recovery half-time of ∼1 s95 and FITC-dextran in the range of 2.5−10 s (for 70 kDa to 2 MDa polymers).97 This is in line with our observation that the EGFP variant protein mCherry is very efficiently extracted during NM preparation (Figures 1B and S3A,B). In summary, we defined a large portion of proteins from well-known nuclear subcompartments belonging to a partially extraction-resistant nuclear structure. To evaluate the extent of structural integrity of nuclear compartments in the NM preparations, we analyzed the degree of extraction for known individual proteins within nuclear bodies (Figure 5B). The analysis revealed that about 20−40% of known components of the Cajal body, nuclear speckle, PML body, and NPC were retained in the NM. The degree of extraction using three different extraction methods was strongly comparable. More heterogeneity was observed for the nucleolus, where only 19% of nucleolar proteins were commonly retained by all methods. In contrast to that using LIS and AS, NaCl treatment extracts the vast majority of nucleolar proteins. The latter might reflect a more complex functional and structural organization of the nucleolus98 compared to that of other nuclear microdomains. Interestingly,

To obtain information on protein domains, which could be involved in the structural organization of the NM, we performed InterPro motif enrichment analysis by comparing significantly enriched NM proteins (NM classifier I < 0.05) against the nuclear proteome defined in this work. Interestingly, the NM protein fraction was enriched (Benjamini−Hochberg corrected Fisher’s exact test p < 0.05) for RNA-binding signatures like RNA-recognition motif (RNP-1), α/β-plait, DEAD and DEAH box helicases, Brix domain, helicase superfamily 1 and 2, and D111/G-patch. Consistent with the traditional NM concept,3 we indeed observed residual RNA in our NM preparations (Figure S5A) and an increased NM yield (data not shown) in the presence of the RNase inhibitor VRC.22 Conversely, RNase treatment results in morphological disintegration of our specimens (data not shown), suggesting a role for RNA in NM integrity. Not significantly enriched, but very abundant, were proteins with C2H2-type zinc finger domains. Recently, a couple of elegant studies demonstrated that certain ncRNAs in collaboration with RNA-interacting proteins are instrumental for the establishment of chromosome territories75 or de novo assembly of subnuclear organelles.82 In agreement with the observations of others,83 this indicates that RNA binders might constitute major components of the NM. Hence, the structural hierarchy, as revealed by the striking differences in extractability of nuclear proteins (Figure 3E), supports the “seeding model” of nuclear organization that has been proposed in the context of RNA-dependent nuclear body formation.19,84 Additionally, we analyzed whether structural motifs and low amino acid complexity (LC) regions (compositional biases) listed in the UniProt database are enriched. The analysis revealed that the NM central proteome is enriched for proteins harboring coiled-coil structural motifs and WD40 repeats. It was shown that both WD40 repeats and coiled-coil motifs are important for protein−protein interaction and higher-order structure formation.85,86 Thus, from the structural perspective, proteins containing such modules (Table 2) might play a role Table 2. Protein Domains Enriched in the NM Central Proteome α/β-plait MKI67IP, SR140, C130057N11RIK, KIAA0122, RBM14, RBM15, RBM25, RBM26, KIAA1311, RBM28, CAPER, LUCA15, RBM8, RBMXRT, RBMX2, MET, KIAA0929, U2AF2, ACIN1, CPSF6, HNRNPL, HNRNPM, RNPS1, MATR3, KIAA1341, NONO, MCG_17848, NOL8, PPIL4, U2AF1, HRS, HNRNPF, FUSIP1, RBM17, SFRS3, SAFB2, DRB1, SNRNP70, SFPQ, SFRS9, SFRS1, SFRS12, SFRS2, MCG_16265, MCG_1675, SFRS7, TRA2A, SFRS10, SR140 WD40 repeat PWP2, MRNP41, THOC6, UTP15, UTP18, MNCB-5414, WDR3, WDR36, KIAA0007, BING4, WDR74, MCG_116076, DCAF13, AAAS, BOP1, CDC40, CIRH1A, GM67, NUP133, KIAA0023, NUP43, TBL1, TBL3, H3A Pro-rich SR140, CHERP, DNJB6, CBLL1, RBM26, CPSF6, SFRS8, NCOR1, BUD13, SPEN, NCOR2, TTN, MLL5, KIAA1471, EDD, KIAA0802, SKIIP, POP101, ARS2, SFPQ, KHDRBS1, SRRM2, KIAA0460, KIAA1311 Coiled coil AKAP17B, BUD13, CHCHD6, DNTTIP2, EBNA1BP2, ESF1, GCFC2, GLE1, GNL3, LMNA, LMNB1, LMNB2, LUC7L, LUC7L2, LUC7L3, MAD1L1, MPHOSPH10, MYH7, NCBP1, NCOR2, NGDN, NOC3L, NOL10, NOL8, NONO, NSRP1, NUP62, NUP88, PAF1, PRPF38A, PRPF38B, PTPN23, RBBP8, RBM26, RBM27, RRP15, SART1, SFSWAP, SOGA2, SPC25, SPEN, SRRM2, SUN1, SUN2, THOC2, TRIM56, TTN, U2SURP, UPF0428, UTP14A, UTP20, VIM 3951



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Figure 5. Detailed biochemical view of the structural hierarchy of the nucleus. (A) Heat map showing GO cellular component enrichment in bins of similar size based on the NM classifier I. Bins were analyzed for enrichment of GO cellular component terms emphasizing annotations relevant for known nuclear compartments and cellular organelles. All categories were filtered with high stringency for nonredundant and significantly enriched terms (Benjamini method corrected Fisher’s exact test < 0.05). A positive control was performed by the analysis of the nuclear proteome data set defined here against the murine proteome. The GO term nuclear matrix is not enriched in any bin because previously suggested NM proteins do not pass our NM classifier I or II cutoff. (B) Heat maps of proteins from known nuclear compartments reveal differential extractability (log2 SILAC ratios) within a nuclear subcompartment. A substantial fraction of nuclear body components is retained in the NM following extraction of DNase Idigested nuclei with LIS, AS, and NaCl, respectively.

follow-up studies, we mined the immunofluorescence images provided by the Human Protein Atlas project (www. proteinatlas.org53). Altogether, 516 nuclear proteins were evaluated for their nuclear distribution pattern based on the associated immunocytochemical antibody staining in the depository. The staining pattern was classified into categories including nuclear

the NM-retained proteins GAR1 and FBL were both shown to be part of the dense fibrillar component of the nucleolus99,100 together with MPHOSPH10, being one of the marker proteins for prenucleolar bodies.101 To test whether identified NM proteins could comprise components of already well-described (e.g., speckles) or unknown nuclear subcompartments that would merit further 3952



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Figure 6. Nuclear matrix proteins preferentially show a localized nuclear staining pattern in the Human Protein Atlas IF database. (A) Distribution of nuclear staining patterns in dependence of NM classifier I. Immunofluorescence (IF) image data in the Human Protein Atlas depository were evaluated by two independent researchers and separated into five categories ranging from diffuse (no compartmentalization) to nuclear body (high degree of compartmentalization). The distributions of NM classifier I bins between the two experts are not significantly different, whereas a decreasing trend was observed from the fraction with NM classifier I < 0.05 to > 0.95 (Mann−Whitney U test: * < 0.05, ** < 0.01, *** < 0.001). The most common distribution containing the majority of proteins exhibiting compartmentalized staining was observed for proteins with NM classifier I < 0.2. (B) The NM central proteome contains a potentially large fraction of proteins with compartmentalized nuclear distribution. 30.5% of the proteins in our data set with a NM classifier I value < 0.2 are presently annotated in the Human Protein Atlas IF data repository. This fraction contains 71% of proteins with a compartmentalized staining, of which only 50% have been previously reported in literature. Iteratively, this data set is a promising resource for both the identification of novel nuclear substructures and the assignment of proteins to already known compartments.

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compartment, speckle, spotty, dense granular, and diffuse. The analysis revealed that, indeed, proteins found to be enriched in the NM have a different immunostaining pattern compared to that for depleted proteins (Figure 6A). The staining pattern showed a successive change from compartmental, spotty, and speckled for most enriched proteins to a granular and diffuse staining for NM-depleted proteins. The largest portion of nuclear compartment candidate proteins was found in the fraction with NM classifier I below 0.05 but also in the fraction with NM classifier I from 0.05 to 0.1. Hence, proteins belonging to the NM central proteome (NM classifier I < 0.05) demonstrate a strikingly different IF staining pattern (significance level < 0.01) compared to that of proteins with a NM classifier I value greater than 0.2. Interestingly, this ensemble of proteins (NM classifier < 0.02) is highly enriched for proteins that display a compartmentalized IF staining in the Human Protein Atlas database. Of importance, only 50% of them have been previously described in the literature to be part of a nuclear subcompartment (Figure 6B). Among them, SLTM, a general inhibitor of transcription, shows a localized punctate nuclear structure.102 Granular nuclear staining was also observed for the NM-enriched proteins WRNIP1, involved in DNA polymerase δ-mediated DNA synthesis, and RERE, involved in transcriptional repression and regulation of apoptosis.103,104 In summary, we observe that a significant fraction of proteins from known nuclear compartments is retained in our NM preparation, promoting the use of this data as a valuable resource for the identification of new proteins from known compartments and, more importantly, for the identification of novel nuclear substructures. The protein extraction profiles of proteins belonging to a certain compartment could also assist follow-up studies to understand the mechanisms of nuclear body formation.

ASSOCIATED CONTENT

S Supporting Information *

Figure S1: classical nuclear matrix (NM) preparation procedures applied to pre-B lymphocytes are inadequate for proteomic analysis. Figure S2: nuclear neighborhood integrity is maintained after extraction for the NM. Figure S3: electron microscopy of the NM. Figure S4: flow-cytometry-assisted purification of the NM efficiently removes nucleoplasmic proteins and chromatin. Figure S5: nucleic acid content of individual NM preparations. Table S1: identification and quantification of nuclear and nuclear matrix proteins including Nuclear Matrix Classifiers I and II. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +49 761 5108 712. Present Addresses # (R.E.) Weill Cornell Medical College in Qatar, Qatar Foundation Education City, Doha, State of Qatar. ∇ (J.R.) Novartis Institutes for BioMedical Research, Drug Metabolism and Pharmacokinetics, Fabrikstrasse 14, CH-4056 Basel, Switzerland. ○ (J.H.) Institute for Functional and Applied Anatomy, Hannover Medical School (MHH), Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. ◆ (S.E.) BIOSS, Center for Biological Signaling Studies, University of Freiburg, Schänzlestrasse 18, 79104 Freiburg, Germany. ¶ (J.D.) Department of Dermatology, Medical Center-University of Freiburg, Hauptstrasse 7, 79104 Freiburg, Germany.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. E. Kremmer and Prof. Dr. M. Meisterernst for the antibodies, Dr. P. Heun for the plasmid encoding mCherry and his support in fluorescence microscopy, and Prof. Dr. Rudolf Grosschedl for critical reading of the manuscript. This research was supported in part by the Max Planck Society.

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ABBREVATIONS AS, ammonium sulfate; BCA, bicinchoninic acid; DAPI, 4′,6diamidino-2-phenylindole; DIC, differential interference contrast; DNA, deoxyribonucleic acid; EGFP, enhanced green fluorescent protein; EM, electron microscopy; FCS, fluorescence correlation spectroscopy; FDR, false discovery rate; FRAP, fluorescence recovery after photobleaching; GO, gene ontology; HFBA, heptafluorobutyric acid; iBAQ, intensity based absolute quantification; IF, immunofluorescence; IPI, international protein index; LC, low complexity; LIS, lithium 3,5-diiodosalicylate; MW, molecular weight; NLS, nuclear localization signal; NM, nuclear matrix; NPC, nuclear pore complex; PFA, paraformaldehyde; RNA, ribonucleic acid; S/ MAR, scaffold/matrix attachment region; SILAC, stable isotope labeling by amino acids in cell culture; VRC, vanadyl ribonucleoside complex; WB, western blot

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