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Originally published In Press as doi:10.1074/mcp.M600255-MCP200 on October 15, 2006.
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Molecular & Cellular Proteomics 6:72-87, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Mass Spectrometric Mapping of Linker Histone H1 Variants Reveals Multiple Acetylations, Methylations, and Phosphorylation as Well as Differences between Cell Culture and Tissue*,S

Jacek R. Wisniewski{ddagger}, Alexandre Zougman, Sonja Krüger and Matthias Mann

From the Department of Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, D-82152 Martinsried, Germany


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Posttranslational modifications of histones are involved in regulation of chromatin structure and gene activity. Whereas the modifications of the core histones H2A, H2B, H3, and H4 have been extensively studied, our knowledge of H1 modifications remained mainly limited to its phosphorylation. Here we analyzed the composition of histone H1 variants and their modifications in two human cell lines and nine mouse tissues. Use of a hybrid linear ion trap-orbitrap mass spectrometer facilitated assignment of modifications by high resolution and low ppm mass accuracy for both the precursor and product mass spectra. Across different tissues we identified a range of phosphorylation, acetylation, and methylation sites. We also mapped sites of ubiquitination and report identification of formylated lysine residues. Interestingly many of the mapped modifications are located within the globular domain of the histones at sites that are thought to be involved in binding to nucleosomal DNA. Investigation of mouse tissue in addition to cell lines uncovered a number of interesting differences. For example, whereas methylation sites are frequent in tissues, this type of modification was much less abundant in cultured cells and escaped detection. Our study significantly extends the known spectrum of linker histone variability.

In eukaryotic cells, genomic DNA is primarily packaged into nucleosomes through sequentially ordered binding of the core histones (H2A, H2B, H3, and H4) (for a review, see Ref. 1). Another protein, histone H1, binds between the nucleosomes and forms a macromolecular structure of 30-nm chromatin fiber. Whereas the core histones are highly conserved, the linker histones are much variable, and in the vast majority of organisms, H1 occurs in multiple sequence variants (2). Somatic mammalian cells contain seven major variants of histone H1: H1.0, H1.1, H1.2, H1.3, H1.4, H1.5 (3–6), and H1X (7, 8). Four of them, H1.2, H1.3, H1.4, and H1.5, have been found in all cells, whereas the H1.1 is considered a specific variant for thymus, testis, spleen, lymphocytic, and neuronal cells. The H1.0 variant is thought to be mainly restricted to non-proliferating and terminally differentiated cells (9). Expression of the H1.X variant gene has been observed across a majority of tissues, but so far the protein was identified solely in cultured cells (8, 10). In testis, oocytes, and spermatids, the specific variants H1t (11, 12) and H1T2 (13), H1oo (14), and HILS1 (15), respectively, were identified.

The histone H1 consists of a well conserved hydrophobic GH1 and less conserved N- and C-terminal parts. GH1 (16, 17) consists of three helices and is primarily involved in DNA binding (18). The terminal parts are thought to be involved in binding of non-histone chromatin proteins regulating transcriptional activity (for reviews, see Refs. 19 and 20). The C-terminal tail of the linker histone is involved in chromatin condensation. The determinants for both the DNA binding and stabilization of chromatin fiber were mapped to two distinct regions of the tail (21). The C-terminal part of the linker histone also was shown to mediate protein-protein interactions and is involved in H1-dependent activation of the apoptotic nuclease DFF40/CAD (22). The role of the N-terminal portion of the linker histone is not understood. The individual variants of the linker histone are highly similar between human and mouse with at least 90% sequence identity within the GH1 portion.

The mode of binding of histone H1 to the linker DNA of nucleosomal fiber is unclear, and there are several competing models for this interaction. According to the first model, H1 binds symmetrically to DNA, entering and leaving nucleosomes and therefore protecting 10 bp from each site against digestion with nuclease (23). The second model suggests that the GH1 domain of the linker histone is asymmetrically located inside the gyres of DNA, which are also wrapped around the core histones. This domain extends the conformation of the protein superhelix to one side of the core particle (24). Similarly to the core histones, the linker histones are components of both condensed and transcriptionally active chromatin (25); however, the H1 binding to chromatin in its different states is not understood. Diverse posttranslational modifications such as acetylation, methylation, and phosphorylation are considered to be regulators of histone H1 function in chromatin condensation and attraction of chromatin-specific proteins. Mass spectrometry-based proteomics facilitates mapping of posttranslational modifications (26–32) as well as studying primary structure of core histones (30–32). Recently published work on linker histones has mainly been aimed at the mapping of phosphorylation sites (10, 33). These are usually mapped to either the N- or C-terminal tail of the histones. In addition, acetylation of the N-terminal residues and methylation of a single lysyl residue in the N-terminal tail of the H1.4 variant in HeLa cells has been reported (10). Recently lysine acetylation also was reported for the Tetrahymena linker histone H1 (34).

Identification of posttranslational modifications of histones has been done nearly exclusively using cultured cells. In these artificial systems of rapidly proliferating cells, the modifications of the proteins do not necessarily reflect the situation in vivo. In this work, we analyzed and compared posttranslational modifications of the histone H1 in two human cell lines and nine mouse tissues. We describe novel posttranslational modification sites including phosphorylation, acetylation, methylation, and ubiquitination. Moreover we identified formylated lysine residues. We used the high mass accuracy and resolution for both precursor and product mass spectra for all modification assignments reported here, greatly improving their confidence.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Tissue—
10–14-week-old mice of the inbred strain C57BL/6 were sacrificed by decapitation. Tissues were dissected, frozen, and stored at –80 °C. Human MCF7 and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were washed with PBS, pH 7.5, and stored at –80 °C.

Histone H1 Extraction and Purification—
Frozen cells and tissues were extracted with HClO4 as described previously (35). Briefly ~200–400 mg of each tissue was blended in 0.6–1.2 ml of 5% (v/v) HClO4 using an Ultra Turbax blender (IKA, Staufen, Germany) at a maximum speed of approximately 25,000 rpm for 30 s. The suspension was subjected to three freezing (–20 °C)/thawing/vortexing (10 s) cycles. The MCF7 and HeLa cells were extracted in the same way but without initial homogenization. The final suspension was centrifuged at 15,000 x g for 10 min, and to the clear supernatants ice-cold 100% (v/v) CCl3COOH was added to a final concentration of 33% (v/v). The proteins were precipitated for 30 min on ice and collected by centrifugation at 15,000 x g for 10 min. The pellets were washed with 0.2% HCl in acetone, then washed twice with pure acetone, and vacuum-dried. The protein pellets were solubilized in 0.1% (v/v) CF3COOH in water and were chromatographed on a C18 reverse phase column as described previously (36). Fractions containing histone H1 were collected, and the proteins were vacuum-dried.

Digestion and LC-MS/MS Analysis—
Dried protein pellets were reconstituted in 100 mM ammonium bicarbonate and digested with trypsin overnight at 37 °C. The resulting peptide mixtures were desalted using in-house made C18 STAGE tips (37), vacuum-dried, and reconstituted in 0.1% trifluoroacetic acid prior to analysis.

The LC-MS/MS setup was similar to that described before (38). Briefly the samples were injected into an in-house made 15-cm reverse phase spraying fused silica capillary column (inner diameter, 75 µm; packed with 3-µm ReproSil-Pur C18-AQ medium (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) using the Agilent 1100 nanoflow system (Agilent Technologies, Palo Alto, CA). The LC setup was connected to an LTQ-Orbitrap mass spectrometer (Thermo Electron, Bremen, Germany) equipped with a nanoelectrospray ion source (Proxeon Biosystems, Odense, Denmark). The peptides were separated with either 30- or 80-min gradients from 5 to 40% acetonitrile in 0.5% acetic acid. The mass spectrometer was operated in the data-dependent mode. Survey MS scans were acquired in the orbitrap with the resolution set to a value of 60,000. Up to five of the most intense ions per scan were fragmented in the linear trap, and the corresponding MS/MS spectra were acquired in the orbitrap with the resolution value of 15,000. Target values were 1,000,000 for the survey scan and 100,000 for the MS/MS scan. This acquisition cycle requires about 2 s, excluding fill time of the ion trap. Target ions already selected for the MS/MS were dynamically excluded for 45 s. For accurate mass measurements the lock-mass option was used as described previously (38).

The data were initially searched against the full International Protein Index (IPI) database with the aid of the Mascot (Matrix Science, London, UK) search engine (39). Searches for posttranslational modifications were done against a subset of the database containing mouse and human histone proteins. Mascot does not increase the probability score based on high accuracy MS/MS information. Therefore, we used an initial mass filter of 0.05 Da for both precursor and fragment mass spectra. In a second step, all relevant hits were checked manually for mass deviation of the fragments in the Mascot result file. MS/MS spectra with fragments deviating by more than 10 ppm were rejected. All spectra showing unique posttranslational modifications of human and mouse H1 are presented in Supplemental Files 1 and 2, respectively.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
In this work we sought to characterize posttranslational modifications of the linker histone H1 in both cell lines and tissue. To identify modifications with high confidence, we used a high accuracy mass spectrometer. Both precursor and product mass spectra were analyzed in the orbitrap. This lowered the duty cycle by a factor of 2 compared with parallel acquisition in the orbitrap and the LTQ part of the instruments. Furthermore about 30 times more ions were accumulated to acquire the MS/MS spectra compared with LTQ analysis. However, the high mass accuracy in both MS and MS/MS modes results in an exceptional level of confidence for pinpointing modification sites as it allows discriminating between modifications with the same nominal masses both at the intact peptide and fragment level. This turned out to be crucial, as in the case of formylation reported below, in discovering putative modifications.

Occurrence of H1 Variants in Mouse Organs and Human Cells—
Diluted HClO4 is a powerful tool for extraction of histone H1, high mobility group proteins, and more than a hundred other proteins (40). Extraction of whole cells and tissues, without nuclei isolation, preserves the proteins and their posttranslational modifications (40). This is also important because fractions of H1 can be present outside nuclei as it has been demonstrated for H1.X variant of Caenorhabditis elegans (41). For H1 identification the extracts can be directly subjected to digestion and LC-MS/MS analysis (42), but protein fractionation is necessary to achieve higher sequence coverage and enable identification of posttranslational modifications of lower abundance. This enrichment allowed characterization of individual histone variants with a sequence coverage of typically 70–85%. One exception was H1t in spleen, which was only identified with two peptides, probably because it occurs with very low abundance in this tissue. Because histones are small and solubilize well in acids they can be efficiently separated by means of reverse phase chromatography. With this method, human linker histones from the cells were separated into four fractions containing H1.0, H1.5 and H1.X, H1.1, and the variants H1.2, H1.3, and H1.4 (Fig. 1). Chromatography of HClO4-extracted proteins from different mouse tissues resulted in separation of histone H1 variants into five fractions containing H1.0, H1.5, H1.1, a mixture of H1.3 and H1.4, and H1.2, respectively (Fig. 2). Due to their posttranslational modifications linker histones can change their retention on the column. Therefore, in the protein fractions eluting closely together, such as H1.1 (fraction C), H1.3 and H1.4 (fraction D), and H1.2 (fraction E) (Fig. 1), we usually found modified versions of other histones. In testis, another late eluting fraction contained H1t. According to the classical view, somatic mammalian cells contain five main class subtypes, H1.2, H1.3, H1.4, H1.5, and the replacement subtype H1.0. These variants were prominent in all human and mouse fractions with the exception of cultured cells, testis, and spleen where the content of H1.0 variant was lower (Fig. 1). In contrast, in spleen and testis the levels of H1.1 variant were elevated; this is in accord with the fact that histone H1.1 is considered to be restricted to testis, thymus, and spleen. Against this view, we found that the H1.1 variant was present in all analyzed tissues and cells. In testis the H1t variant occurs at high levels, but we identified it also in spleen extracts (Fig. 2). The presence of H1.1 and H1t in more tissues than expected until now demonstrates advantages of modern proteomics, which makes possible the identification of proteins present in minute amounts that remained undetected in earlier analyses using conventional biochemical methods. Histones H1 are proteins with low turnover times with the potential to accumulate over many cell generations (42). Therefore, in tissue some variants may originate from early developmental stages as well as from gene leaking, a nonspecific transcription at very low rates. It remains to be determined whether H1.1 and H1t variants have any specific functions in these cells.


Figure 1
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  		FIG. 1. Chromatography of human (A) and mouse (B) histone H1 variants. Proteins were extracted from frozen HeLa cells and chromatographed on a reverse phase C18 column. The proteins were eluted with an acetonitrile gradient in 0.1% trifluoroacetic acid. Au, absorbance unit. In B, A–E, fractions containing histone H1 variants.

 

Figure 2
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  		FIG. 2. Summary of identified posttranslational modifications of human (A) and mouse (B) linker histones H1. In A and B, the helices (boxes) of GH1 of the avian erythrocyte H5 variant are aligned to the proteins sequences. The putative DNA binding residues (50) are indicated with arrows. Residues are highlighted as follows: blue, phosphorylation; red, acetylation; green, monomethylation; pale green, dimethylation; orange, acetylation or/and monomethylation; pale green outlined, ubiquitination; yellow outlined, N{alpha}-terminal acetylation; gray outlined, formylation. Underlined sequence, peptides unique to H1t identified in spleen: LIPEALSTSQER (mass calculated, 1342.709912; mass measured, 1342.709213; mass accuracy, 0.000699 Da; Mascot score, 54) and AGMSLAALK (mass calculated, 860.479042; mass measured, 860.478928; mass accuracy, 0.000114 Da; Mascot score, 60).

 
General Differences in Posttranslational Modifications of Histone Variants—
Analysis of the proteins in mouse tissues and human cell lines revealed the presence of different types of posttranslational modifications including phosphorylation, acetylation, methylation, and ubiquitination. All mapped sites of posttranslational modifications of human and mouse H1 histones are given in Tables I and II, respectively. A summary of the all identified modifications is shown in Fig. 2. Some of the identified modifications are common to most of the H1 variants, whereas others are restricted to single variants. In rapidly proliferating human cells and in spleen, the site of lymphopoiesis, most of the acetylation or/and methylation sites were observed, respectively (Table II). In contrast, in tissues containing mainly terminally differentiated cells, the overall extent of modifications was lower. In particular, only a few sites of modification were identified in liver. A most intriguing observation is that methylation sites are frequent in tissues, whereas we could not unequivocally demonstrate this type of modification in cultured cells.


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  		TABLE I List of identified tryptic peptides with posttranslational modifications from HeLa and MCF-7 cells

fK, formylated lysine; pS, phosphoserine; aK, acetylated lysine; pT, phosphothreonine; ubK, ubiquitinated lysine; mK, methylated lysine; a,mK, acetylated, methylated lysine; a, acetyl.

 

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  		TABLE II List of identified tryptic peptides with posttranslational modifications from mouse tissues

aK, acetylated lysine; pS, phosphoserine; mK, methylated lysine; dmK, dimethylated lysine; pT, phosphothreonine; mR, methylated arginine; fK, formylated lysine; ubK, ubiquitinated lysine; s, seminal; a, acetyl.

 
In human cells and mouse spleen, which contained the highest number of modifications, the somatic variants H1.2–H1.5 were found to be modified at multiple sites, whereas only few modifications were identified in H1.0, H1.1, and H1X variants. In particular, modifications within GH1 are missing in these variants. This may reflect a fact that histone variants with restricted occurrence and probably also specific functions are not regulated by posttranslational modifications to the same degree as the more ubiquitous histones.

HPLC fractions from tissues eluting before H1.0 and between H1.5 and H1.1 (Fig. 1) contained other abundant proteins, the high mobility box proteins HMGB1 and HMGB2, and ubiquitin. Only a few modifications were found in HMGB1 and ubiquitin. These are phosphorylation of Ser-35 of the HMGB1 and two ubiquitination sites (Arg-54 and Arg-72) in ubiquitin (not shown). In brain extracts the brain acid-soluble protein (BACS) co-eluted with histones in fractions D and E (Fig. 1). Up to five phosphorylation sites (depending on the experiment) and an N-terminal myristoylation were identified in this protein (not shown). In contrast to the linker histones, we did not observe sites of acetylation, methylation, and formylation in these abundant proteins; this probably reflects specificity of the modifications found in H1.

Heterogeneity of the N{alpha} Termini—
Cleavage of the N-terminal methionine and N{alpha}-terminal acetylation are the most common modifications of the majority of eukaryotic proteins (for a review, see Ref. 43). Both processes take place co-translationally on the nascent polypeptide. Although it is known that 95% of N{alpha}-terminal acetylation in eukaryotic proteins occurs on serine, alanine, methionine, glycine, and threonine, it seems there is no simple consensus sequence for N{alpha}-terminal acetylation. Attempts to sequence the N-terminal peptides of histone H1 by Edman degradation technique were unsuccessful and led to the assumption that the N{alpha} termini are acetylated (5). Recent proteomics studies confirmed these results showing that histones H1 have cleaved off N-terminal methionine and are acetylated at their N-terminal residues (10). In our study, we also identified the canonical termini, but in addition, we found that the N{alpha} termini of the histone H1 also occur in non-acetylated forms (Supplemental Table S1). Moreover we observed that in liver a portion of the histone H1.0 molecules carries a non-cleaved off methionyl residue 1 that is acetylated (Supplemental Table S1; Fig. 3). It appears that this type of heterogeneity is a widespread phenomenon because we observed it in cell lines and tissues of different origin. Recently we have reported that the variant C of the hematological and neurological expressed protein occurs in two forms with N{alpha}-terminally acetylated either methionine or alanine (40). In previous studies on linker histones (44, 45) it was shown that only the variant H1.0 and its avian counterpart H5 occur in N-terminally acetylated and non-acetylated forms. Whereas the ratio of both forms of H5 is unchanged in the erythrocytes from newly hatched and adult chickens, the extent of acetylation of the N{alpha} terminus of H1.0 increases in rat tissues during aging (44). Due to the fact that the non-acetylated forms of human histone H1 were not previously found (the H1.0 variant being an exception), it seems they are much less abundant than the acetylated ones and therefore escaped detection. Because we found the N{alpha}-terminally non-acetylated forms of all linker histone variants in both tissues and cultured cells, it appears to be a widespread phenomenon, although its function remains obscure.


Figure 3
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  		FIG. 3. Heterogeneity of the N{alpha} terminus of linker histone H1.0. Fragmentation spectra of the tryptic peptides of N termini of the histone H1.0 from mouse liver that contain acetylated methionine (A), acetylated threonine (B), and non-acetylated threonine (C) are shown. oM, oxidized methionine; a, acetyl.

 
Novel Phosphorylation Sites on Linker Histone Variants—
Phosphorylation has been the most extensively studied posttranslational modification in linker histones. Phosphorylation of H1 is complex and cell cycle-dependent. Already in the early work Gurley and co-workers (46–48) observed that H1 from Chinese hamster ovary cells is singly phosphorylated in late G1, triply phosphorylated in S and G2, and carries up to six phosphates per molecule in M phase. All of the phosphates in H1 are lost during telophase, thus resetting these histones to a phosphate-free state at the beginning of the next cell cycle (48). In human cells, H1 is phosphorylated at up to five sites with the consensus motif of cyclin-dependent kinase 1 (CDK1)1 ((S/T)PXK) (33). Up to four of these sites are located in the C-terminal tails of the H1, and one is located in the N-terminal portion of the histone. In addition to these sites, phosphorylation of non-CDK1 sites within the N-terminal tails was reported. These sites are Ser-1 and Thr-3 of H1.4 (10, 49), Thr-10 of H1.5 (33), Ser-26 of H1.4 (10), and Ser-30 of H1.X (10). Moreover phosphorylation of Ser-35, which is located N-terminally to the GH1 of the H1.4, was reported (10). In this study we identified nearly all these sites in tissues and human cells and have mapped several previously only predicted CDK1 sites (33) (Table II). In human cells these are H1.2 (Thr(P)-146), H1.3 (Thr(P)-18; Thr(P)-147), and H1.4 (Thr(P)-18). We also found novel non-CDK1 consensus sites in all analyzed histones except for H1t. Apart from the Thr(P)-165 site in H1.2, other non-CDK1 sites are located in the N-terminal tails and GH1 of the variants (Fig. 2). Fig. 4 shows the fragmentation spectrum of the N-terminal peptide derived from histone H1.5 that is phosphorylated at the acetylated Ser-1 residue. In histones H1.0 and H1.1 only non-CDK1 phosphorylation sites were identified (Fig. 2). In mouse histone variants H1.1–H1.4 we found a non-CDK1 site, Ser(P)-41 (H1.1 residue numbering), that is located within the helix 1 of GH1. This modification together with the other modification sites (see below) within GH1 potentially may regulate interaction of linker histones with DNA.


Figure 4
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  		FIG. 4. Fragmentation spectrum of the N{alpha}-terminally acetylated and Ser-1 phosphorylated peptide SETAPAETATPAPVEK derived from H1.5 variant isolated from MCF7 cells. a, acetyl; pS, phosphoserine.

 
Formylation of Histone H1 Variants, a Novel Type of Modification—
With a routine low ppm accuracy, the LTQ-Orbitrap used in this study is not only a powerful device for identifying proteins and posttranslational modifications but also an excellent tool for differentiating between the modifications whose nominal masses are the same. Dimethylation (28.031300 Da) and formylation (27.994915 Da) are of the same nominal mass, 28 Da, and differ by 0.036385 Da. "Formylation" was not included in the Mascot search query originally. However, some of the reported dimethylated peptides had unusually low reported mass accuracies. This was the case for the modified peptide SGVSLAALKK from H1.2 (Fig. 5C), which was reported by Mascot as a dimethylated peptide with a mass accuracy of –0.035665 Da (–36 ppm). Further enquiry using the MS/MS fragmentation information showed that the masses for the ions starting from y2 and up and for the b9 ion were also shifted to the left, whereas the masses of the b5 and b7 ions corresponded to the predicted values. We assumed that the formylation of lysine 63 is the most likely candidate for the modification, yielding the peptide mass accuracy of 0.00072 Da (0.7 ppm) and MS/MS root mean square (r.m.s.) error of 1 ppm.


Figure 5
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  		FIG. 5. Fragmentation spectra of the monomethylated (A) and dimethylated (B) at lysine 63 peptide SGVSLAALK isolated from mouse spleen are shown. This peptide is shared among H1.1, H1.2, H1.3, and H1.4 variants. C, fragmentation spectrum of the formylated at lysine 63 peptide SGVSLAALKK isolated from mouse spleen. The peptide was originally reported by Mascot as dimethylated with unusually low mass accuracy (–36 ppm). Further enquiry using the MS/MS fragmentation information showed that the masses for the ions starting from y2 and up and for the b9 ion were also shifted to the left, whereas the masses of the b5 and b7 ions corresponded to the predicted values. We assumed that the formylation of lysine- 63 is the most likely candidate for the modification, yielding the peptide mass accuracy of 0.00072 Da (0.7 ppm). This peptide is shared among H1.1, H1.2, H1.3, and H1.4 variants. mK, methylated lysine; dmK, dimethylated lysine; fK, formylated lysine.

 
Besides dimethylation and formylation, lysine to arginine mutation also causes a nominal mass increase of 28 Da (28.00615-Da delta mass). The difference between this modification and formylation is 0.01123 Da. For a typical formylated tryptic peptide this will result in a mass shift of about 10 ppm for the precursor ion (formylation compared with mutation) and mass differences much higher than 10 ppm for MS/MS fragmentation products. For example, if caused by lysine to arginine substitution, the reported formylated peptide KASGPPVSELITK would have a mass inaccuracy of 9 ppm. The r.m.s. error for the characteristic b ion series would be 24 ppm with the b ions in the low m/z range having mass errors up to 46 ppm (b2 ion) (Fig. 6). The correct assignment of formylation, however, yields 0.6 ppm accuracy for the peptide mass and 3 ppm for the MS/MS r.m.s. error.


Figure 6
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  		FIG. 6. Fragmentation spectrum of the formylated at lysine 34 peptide KASGPPVSELITK isolated from mouse spleen. This peptide is shared between H1.2 and H1.3 variants. fK, formylated lysine.

 
Few lysine residues from fractions originating from human cells, spleen, seminal vesicles, brain, and lung, were found to be modified with the moiety of a mass that corresponds to a formyl group. The origin and potential function of this modification remain obscure. In the biosynthetic single carbon pathway formylation can be catalyzed from formyltetrafolate. Alternatively formaldehyde liberated during demethylation of lysine (50) could be used in a formylation reaction by a specific formylating enzyme or the LSD1 demethylase in the reverse reaction. Because the observed modification is restricted to a few sites both in human and mouse and one of them is in the same location in human and mouse, we believe that it reflects a specific posttranslational modification and is not an artifact resulting from applied extraction and analysis procedures. More examples of fragmentation spectra of peptides containing a formylated lysine residue are in Supplemental Data File 3.

Acetylation and Methylation Sites—
Acetylation and methylation of [cepsilon]-amino group of lysine and guanidinyl group of arginine residues are the most frequent modifications of the core histones. Acetylation of histones is usually linked to transcriptional activation, whereas methylation can result in either transcriptional activation or repression depending on the modified residue and the simultaneous occurrence of other modifications on the histone (for a review, see Ref. 51).

In our study we identified different sites of acetylation and methylation of lysine residues (Tables I and II and Fig. 2). Up to nine acetylation sites were mapped to a single histone variant (human H1.4). The observed acetylation sites are located most frequently in GH1 on residues that have been considered to be directly involved in DNA binding (52). These residues are Lys-52, Lys-64, Lys-85, and Lys-97 (H1.2 residue numbering). As acetylation can interfere with DNA binding, it is possible that these modifications can significantly change the binding properties of H1 conferring specific properties in chromatin to some H1 variants. A second group of acetylation sites constitutes those identified in the N- and C-terminal regions of the H1 histones. Up to two and three acetylation sites were identified for a single variant in its N- and C-terminal tails.

Whereas we were unable to unequivocally identify methylation sites in the cultured cells, we identified different sites of lysine methylation in tissues including mono-, di-, and trimethylated residues (Table II). We cannot exclude that the inability to identify in cultured cells the methylation sites found in mouse tissues may reflect a lower extent of this modification in the cultured cells rather than complete absence of H1 methylation. In some cases the same lysine residues were found with different modifications. For example, lysine 63 was identified in mono- and dimethylated and also in formylated forms (Fig. 5).

Our data on H1 resemble the situation in core histones where individual residues can be modified either by acetylases or methyltransferases. The extent of methylation, from mono- to trimethylation, determines whether transcription of certain genes is activated or repressed (51, 53).

The observed differences in posttranslational modifications, in particular methylation of lysine residues, between culture cells and mouse tissues do not reflect differences between mouse and human because we recently found a number of methylated lysine residues in H1 isolated from human cancers and normal tissue.2 These include Lys-34, Lys-46, Lys-63, and Lys-106 of H1.2–H1.4 (H1.2 residue numbering) sites that were found in mouse H1. In depth analyses of human tissues and cancer samples, ongoing in our laboratory, may provide insights into acetylation and methylation of linker histones and their possible variation between normal and diseased tissues as well as during the cell cycle.

Ubiquitination—
Physiological observations suggest that ubiquitinated histones may have multiple functions and structural effects. Ubiquitination of core histones is generally associated with increased gene expression, implying that it may prevent chromatin folding or help maintain an open conformation (for a review, see Ref. 54). Until now ubiquitination of H1 has been reported by Pham and Sauer (55) for Drosophila melanogaster, but the modification site has not been revealed. In this study we mapped ubiquitination sites in human cells and mouse tissues. Interestingly the Lys-46 is present only in the ubiquitous H1 variants, and this residue is absent in the replacement variants H1.0 and H1.X as well as in tissue-specific H1.1 and H1.t. Ubiquitinated lysine residues were found in histones H1.2, H1.3, and H1.4 at Lys-46, whereas in H1.1 they were found at Lys-116. Fig. 7 shows the MS/MS spectrum of the ubiquitinated KASGPPVSELITubKAVAASK (where ubK is ubiquitinated lysine) peptide with Lys-46 carrying the characteristic Gly-Gly ubiquitination signature of 114.042927 Da, reflected in the ion series starting from y7 and up and from b13 and up.


Figure 7
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  		FIG. 7. Fragmentation spectrum of the ubiquitinated at lysine 46 peptide KASGPPVSELITKAVAASK isolated from MCF7 cells. This peptide is shared among H1.2, H1.3, and H1.4 variants. ubK, ubiquitinated lysine.

 
Conclusions—
Histone H1 variants were isolated from different cell lines and analyzed by LC-MS/MS. The proteomics analysis of purified linker histone variants from two human cell lines and nine mouse tissues revealed previously undiscovered types of histone H1 posttranslational modifications. This work demonstrates for the first time that linker histones are extensively modified not only by phosphorylation but also by acetylation, methylation, and ubiquitination. Furthermore we found that some lysine residues are formylated. We identified far more posttranslational modifications than all the previously published work on H1 over the last 3 decades. The extent and diversity of H1 modifications seems to be similar to those described for core histones suggesting that functions of linker histones are regulated with a complexity similar to those of core histones.

Against the established view that linker histones are mainly modified within the C-terminal domain, we show that many of these modifications are located within GH1. We demonstrate that phosphorylation of linker histones is not restricted to the cell cycle-dependent action of CDK1. Other types of phosphorylation at residues in the N-terminal part of the proteins and at the serine residue in the helix 1 of the GH1 are more frequent particularly in tissues. The identified acetylation sites are at lysine residues that are thought to be involved in interaction with nucleosomal DNA (56, 57), and therefore, it is possible that these modifications significantly change the binding properties of H1, conferring regulatory functions in chromatin to the linker histones.

Understanding the biological function of posttranslational modifications of core histones, the "histone code" is already advanced. Distinct modifications of core histones can act sequentially or in combination to regulate chromatin structure, activating or repressing genes. Potential involvement of linker histone H1 in the code has largely been neglected until recently. Kuzmichev et al. (58) demonstrated that histone H1 can be methylated in vivo at a lysine residue that is important for transcriptional repression. In this view, our data should be helpful in the future studies aimed at deciphering the histone code for H1.


   ACKNOWLEDGMENTS
 
We thank Christian Luber for help with dissection of tissues and Dr. Matthias Selbach for providing the MCF7 cells. We are also grateful to Dr. Yong Zhang for technical advice.


  FOOTNOTES
 
Received, July 12, 2006, and in revised form, September 22, 2006.

Published, MCP Papers in Press, October 15, 2006, DOI 10.1074/mcp.M600255-MCP200

1 The abbreviations used are: CDK1, cyclin-dependent kinase 1; r.m.s., root mean square; LTQ, linear trap quadrupole. Back

2 J. R. Wisniewski, A. Zougman, and M. Mann, unpublished results. Back

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

{ddagger} To whom correspondence should be addressed: Dept. of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried bei München, Germany. Tel.: 49-89-8578-2205; Fax: 49-89-8578-2219; E-mail: jwisniew{at}biochem.mpg.de


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