Open Access

Identification of replication-dependent and replication-independent linker histone complexes: Tpr specifically promotes replication-dependent linker histone stability

  • Pei Zhang1,
  • Owen E. Branson2,
  • Michael A. Freitas2 and
  • Mark R. Parthun1Email author
BMC BiochemistryBMC series – open, inclusive and trusted201617:18

DOI: 10.1186/s12858-016-0074-9

Received: 11 August 2016

Accepted: 20 September 2016

Published: 1 October 2016

Abstract

Background

There are 11 variants of linker histone H1 in mammalian cells. Beyond their shared abilities to stabilize and condense chromatin, the H1 variants have been found to have non-redundant functions, the mechanisms of which are not fully understood. Like core histones, there are both replication-dependent and replication-independent linker histone variants. The histone chaperones and other factors that regulate linker histone dynamics in the cell are largely unknown. In particular, it is not known whether replication-dependent and replication-independent linker histones interact with distinct or common sets of proteins. To better understand linker histone dynamics and assembly, we used chromatography and mass spectrometry approaches to identify proteins that are associated with replication-dependent and replication-independent H1 variants. We then used a variety of in vivo analyses to validate the functional relevance of identified interactions.

Results

We identified proteins that bind to all linker histone variants and proteins that are specific for only one class of variant. The factors identified include histone chaperones, transcriptional regulators, RNA binding proteins and ribosomal proteins. The nuclear pore complex protein Tpr, which was found to associate with only replication-dependent linker histones, specifically promoted their stability.

Conclusion

Replication-dependent and replication-independent linker histone variants can interact with both common and distinct sets of proteins. Some of these factors are likely to function as histone chaperones while others may suggest novel links between linker histones and RNA metabolism. The nuclear pore complex protein Tpr specifically interacts with histone H1.1 and H1.2 but not H1x and can regulate the stability of these replication-dependent linker histones.

Keywords

Chromatin Histone Histone chaperone Linker histone H1 Tpr

Background

The basic repeating structural unit of eukaryotic chromatin is the nucleosome core particle (NCP), which is a structured package of 147 bps of DNA and a histone octamer consisting of 2 copies of each core histone: H2A, H2B, H3, and H4. There are also linker histones, H1, flanking NCPs and sealing the structure with about 20 bps of additional DNA. The involvement of H1 is crucial for the formation and stabilization of chromatin structures and for the regulation of gene expression [1, 2].

There are 11 variants of linker histone H1 in mammalian cells: somatic replication-dependent variants (H1.1 to H1.5), somatic replication-independent variants (H1.0, H1x), and germ cell specific variants (H1t, H1T2m and HILS1 for testicular cells, and H1oo for oocytes) [3]. The replication-dependent variants are typically expressed during S phase and are incorporated during into chromatin during DNA replication, while the replication-independent variants are expressed throughout the cell cycle and can be incorporated into chromatin outside of S phase. The sequences of these H1 variants vary greatly in their C-terminal domains. The individual functions of H1 variants are not fully understood. It was believed that H1 served as a global gene regulator by binding to chromatin non-specifically. However, although partially redundant in function, there is evidence indicating H1 variants have distinct roles in gene regulation and development. While deleting a single H1 variant did not lead to any observable phenotype, the H1.2/H1.3/H1.4 triple knockout mouse exhibited developmental defects and embryonic lethality [4]. Microarray experiments showed that knockdown of each H1 variant altered a different subset of genes [5]. The expression levels and activities of the H1 variants were also found to be highly regulated during cell differentiation and tumorigenesis [6]. These observations suggest the hypothesis that each H1 variant has its individual function in the cells in addition to their roles as global chromatin modifiers.

Studies on core histones indicate that replication-dependent and replication-independent histone variants can be involved in distinct protein complexes and assembly pathways. For example, histone H3.1 is assembled into chromatin in coordination with DNA replication during S phage, while another variant, H3.3, is exchanged throughout the cell cycle. The dynamics of H3.1 and H3.3 are mediated by distinct protein complexes that contain different chaperones for each of the H3 variants. While H3.1 and H3.3 complexes have some shared histone associated proteins, such as NASP, ASF1A, ASF1B, HAT1, and importin 4, there are also exclusive histone binding partners in each complex. All three subunits of histone chaperone CAF-1 are only found in the H3.1 complex, while another histone chaperone, HIRA, is only found in the H3.3 complex [7, 8]. CAF-1 and HIRA then dictate the replication-coupled and replication-independent assembly of these H3 variants.

Currently it is not known whether the dynamics of replication-dependent and -independent variants of H1 are also regulated by distinct chaperones in a manner similar to H3.1 and H3.3 complexes. Previous studies proposed several linker histone chaperone candidates: Nucleosome Assembly Protein 1(NAP1) [9], Nuclear Autoantigenic Sperm Protein (NASP) [10], Nucleophosmin (NPM1) [11], Prothymosin α (ProTα) [12], and Template Activating Factor-I (TAF-1, also known as protein SET) [13]. The roles of these interactions between histone H1 and linker histone chaperones in H1 storage, transport, chromatin assembly and disassembly are not fully understood. How the linker histone chaperones interact with each H1 variant also remains to be determined. To better understand H1 variant - protein interactions, we purified non-chromatin associated protein complexes containing 6 × His-tagged H1 variants expressed in Tetracycline-inducible U2OS cell lines using column chromatography. Proteins associated with each H1 variant were identified by mass spectrometry analysis. We found replication-dependent and replication-independent H1 variants had distinct binding partners. For example, one protein bound to replication-dependent H1.1 and H1.2, nucleoprotein Tpr, was not found in the protein complex containing replication-independent H1x. Tpr knockdown leads to decreased levels of H1.1 and H1.2, but does not affect H1x levels. These findings suggest that association with variant-specific binding partners may regulate linker histone dynamics.

Results

Generation of inducible U2OS cell lines expressing 6 × His-tagged H1 variants

To purify H1 variants and their associated proteins for identification, we generated tetracycline-inducible U2OS cell lines over-expressing 6 × His-tagged H1 variants H1.1, H1.2 or H1x by transfecting U2OS cells engineered for tetracycline-inducible expression with pT-Rex-DEST31 plasmids carrying the corresponding H1 variant sequences (Fig. 1a). Cellular fractionation revealed that the vast majority of 6 × His-tagged linker histones were in the nuclear fraction (Fig. 1b), indicating the exogenous H1 proteins were properly localized. Since histone proteins are highly basic, excess accumulation of histones could disrupt normal chromatin structures and cause cytotoxicity. To verify that our over-expression of H1 variants did not disrupt normal cell functions, we isolated total histones from uninduced and doxycycline-induced U2OS cells. Figure 1c shows a Coomassie stained gel of uninduced and induced cells expressing histone H1.1, H1.2 and H1x. It is clear that H1 induction does not result in an overall increase in linker histone abundance. MNase digestion assays further confirmed that over-expression of 6 × His-tagged H1 variants did not significantly alter nucleosome organization or repeat length (compare time points from uninduced samples to the Dox-induced samples in Fig. 1df). Thus we conclude that these cell lines are suitable as the source for H1-containing protein complex purification.
Fig. 1

Over-expression of 6 × His-tagged H1 Variants Does Not Alter Global Chromatin Structure. a U2OS Tet/On cells were transfected with plasmids encoding 6 × His-tagged human histone H1 variants. These cells were incubated 72 h with or without doxycycline before extracted. Whole cell extracts were resolved by SDS-PAGE gel, and immunoblotted with anti-His antibodies. b Untransfected (UN) U2OS Tet/On cells or U2OS Tet/On cells expressing H1 variants (H1.1, H1.2 and H1x) were incubated with doxycycline for 72 h. Whole cell, cytosolic and nuclear extracts of these cells were resolved by SDS-PAGE gels and visualized by western blotting with antibodies targeting proteins indicated on the right. c Total histones were extracted from untransfected (UN) U2OS Tet/On cells or Doxycycline-induced U2OS cells expressing 6 × His-tagged H1 variants (H1.1, H1.2 and H1x) using acid precipitation method. 30 μg of each sample was resolved by SDS-PAGE gel, and visualized by coomassie staining or immunoblotting with anti-His antibodies. df Nuclei from uninduced (UN) or Doxycycline induced (Dox) U2OS cells transfected with vectors expressing H1.1 (d) , H1.2 (e) or H1x (f) were digested with 0.2 U/ml (Sigma units) of MNase at 37 °C for various lengths of time, then quenched with EDTA. Digested DNA samples were purified using phenol extraction and ethanol precipitation, and resolved on 1 % agarose gel with EtBr staining

Purification and identification of H1 complexes

To analyze proteins associating with the soluble pool of the H1 variants, we purified H1.1, H1.2 and H1x from U2OS whole cell extracts by sequential chromatography with an anion exchange column (Mono Q) and an affinity column (nickel column). The specific order of the chromatography was used to optimize the concentration of proteins prior to mass spec analysis. Interestingly, H1.1 eluted in two separate peaks on the Mono Q column, while H1.2 and H1x both eluted in only one peak (Fig. 2ac). Each peak eluting from the Mono Q column was collected and pooled separately, and chromatographed on a nickel chelate column (Fig. 2df, data not shown for the second peak of H1.1). We performed mock purification from whole cell extracts of untransfected U2OS Tet/On cells, which did not express 6 × His-tagged proteins, to serve as negative controls. For the mock purifications, extracts were resolved on a Mono Q column and the fractions containing an equivalent salt concentration as the peak of the H1-containing fractions was then resolved on a nickel chelating column.
Fig. 2

Purification of H1.1, H1.2 and H1x Complexes. ac Soluble whole cell extracts of U2OS cells expressing 6 × His - tagged H1.1 (a), H1.2 (b) or H1x (c) were purified on a Mono Q column. Top panel: 1 ml fractions were collected, resolved by SDS-PAGE gel, and immunoblotted with anti-His antibodies. Bottom panel: chromatogram showing chromatography conditions used. df Peaks containing 6 × His – tagged H1.1 (d), H1.2 (e) or H1x (f) from Mono Q column were pooled and applied to a nickel column in loading buffer containing 20 mM imidazole, washed with 50 mM imidazole and then eluted with 500 mM imidazole. Top panel: 1 ml fractions were collected, resolved by SDS-PAGE gel, and visualized by immunoblotting with anti-His antibodies. Bottom panel: chromatogram showing chromatography conditions used

Peak samples eluting from the nickel chelate column containing each histone variant, as well as the comparable negative control fraction, were analyzed in duplicate by mass spectrometry. A complete list of the proteins identified, as well as the number of unique peptides observed in each analysis, is listed in Additional file 1: Table S1. The distribution of specificities of the identified linker histone binding proteins for H1.1, H1.2 and H1x is represented by a Venn diagram in Fig. 3a. Approximately half of the proteins identified interact with more than one linker histone variant while half are specific for a single H1 species.
Fig. 3

Graphical representation of identified linker histone binding proteins. a Venn diagram of the distribution of proteins co-purifying with histones H1.1, H1.2 and H1x (as indicated). b Venn diagram of the distribution of linker histones binding proteins identified in the current study (H1.1, H1.2, H1x and the studies of Kalashnikova et al. 2013 and Szerlong et al. 2015 (as indicated) [14, 15]. c Venn diagram of the overlap of liner histone binding proteins identified from U2OS cells in the current study and the study of Kalashnikove et al. 2013 (as indicated) [15]

The protein list contains a large number of ribosomal proteins. Several recent proteomic studies have also found that linker histones bind to ribosomes and this interaction has been implicated in transcriptional repression [1417]. Recent proteomic studies of linker histone binding proteins have used a fundamentally different approach where a histone H1 variant was immobilized and incubated with nuclear or nucleolar extracts [14, 15]. In addition to these differences in experimental approach, these studies used a different linker histone variant than those used in the current study. Despite these differences, there is significant overlap in the H1 interacting proteins identified. As seen in Fig. 3b, there is approximately 30 % overlap in the linker histone binding proteins identified in the current study and in the previous studies. In addition, if only the linker histone binding proteins identified from the same cell line are considered (U2OS), the current study identified more than half of the linker histone binding proteins found by Kalashnikova and colleagues (Fig. 3c).

Table 1 lists all of the non-ribosomal proteins found to co-purify with the linker histones that were detected by at least 5 peptides in both of the duplicate samples (average number of peptides listed). These proteins are divided into 6 groups based on their pattern of interaction. Group 1 consists of the proteins that co-purified with all three linker histones. Group 2 proteins co-purified with the replication-dependent H1.1 and H1.2 but not with the replication-independent H1x. Group 3, group 4 and group 5 proteins were specifically associated with a single histone H1 variant (H1.1, H1.2 and H1x, respectively) and, as expected, each of these groups contained the corresponding H1 variant (proteins specifically identified in H1.1 peak 2 are indicated by an asterisk). Group 6 proteins were associated with both histone H1.1 and H1x.
Table 1

Linker histone binding proteins

Table lists the selected proteins identified by mass spectrometry associated with the linker histones H1.1, H1.2 and H1.X (color coded as indicated). Table lists the average number of peptides identified from each linker histone purification. The averages are derived from 2 to 4 replicates. * denotes proteins identified in histone H1.1 Mono-Q peak 2. = ALL H1’s (GROUP 1), = H1.1 AND H1.2 (GROUP 2), = H1.1 (GROUP 3), = H1.2 (GROUP 4), = H1.X (GROUP 5), = H1.1 AND H1.X (GROUP 6)

The group 1 proteins included two proteins that had previously been identified as histone chaperones, nucleolin and nucleophosmin (NPM1) [18]. Nucleolin and nucleophosmin are broad specificity histone binding proteins and have been found to associate with both core and linker histones [11, 1922]. Group 1 also included the SSRP subunit of the FACT complex, which also has been shown to interact with both core and linker histones [15, 23]. The second subunit of FACT, Spt16, was identified as specifically interacting with histone H1.1. However, this classification was due to a high level of Spt16 peptides in the negative control sample. Therefore, it is likely that both subunits of FACT are bound to all three of the linker histone variants tested.

Group 1 also included SRP68 and SRP72. Components of the signal recognition particle, SRP68 and SRP72 have also been shown to form a heterodimeric complex distinct from the signal recognition particle. In addition to its role in translation, the SRP68/72 complex was also found to bind histone H4 molecules that contain arginine 3 methylation [24].

YBX1 and YBX3 also co-purified with all three linker histones. Y box-binding proteins bind to Y box consensus promoter elements and are involved in mRNA processing [25]. YBX1 was found to be recruited together with PURα and histone H1.2 to the p53 target gene Bax in order to repress p53-induced transcription [26]. Our data indicates that YBX1 not only associates with H1 variant H1.2, but with H1.1 and H1x as well. Interestingly, these Y box proteins were only detected in the H1.1 complex that corresponded to the second peak of H1.1 on the Mono Q column.

Five of the group 1 proteins are linked to N6-methyladenosine modification of mRNA. VIR (virilizer homolog), WTAP (Wilms Tumor Associated Protein), ZC3H13 and Hakai are all components of the WTAP complex that serves to target the METTL3 and METTL14 methyltransferases to their substrate [2730]. In addition, YTDC1 is a YTH domain protein that can function as a reader of N6-methyladenosine [3134].

The remaining group 1 proteins include a cyclin/cdk complex; CCNL1 and CDK11b. Linker histones are highly phosphorylated and often used as non-specific substrates in kinase assays. In fact, CDK11b has been shown to be able to phosphorylate histone H1 in vitro [35]. The observation that the CCNL1/CDK11b complex can be purified in association with linker histones suggests that H1s may be a specific substrate of this kinase complex. Finally, all three H1 variants associate with the ubiquitin hydrolase UBP34.

The group 2 proteins bind specifically to the replication-dependent H1 variants H1.1 and H1.2 but don’t form a complex with the replication-independent variant H1x. The group 2 proteins include 4 subunits of the PAF1 complex, PAF1, CTR9, CDC73 and LEO1 [36]. The specificity of the interaction between the PAF1 complex and H1.1 and H1.2 is consistent with a recent study that showed that PAF1 co-purified with epitope tagged H1.1 and H1.2 but not with the other replication-dependent H1 variants H1.3, H1.4, H1.5 or with the replication-independent variant H1.0. The association of the PAF1 complex with H1.1 and H1.2 was shown to function with Cul4A in transcription-associated ubiquitylation [37].

CHD8 has previously been shown to function in transcriptional repression of p53 and β-catenin target genes through the recruitment of histone H1 [38, 39]. The proteomic data presented here suggests that the interaction between CHD8 and linker histones is variant specific.

The nuclear pore complex protein Tpr was also found to be replication-coupled H1 variant specific. Tpr (translocated promoter region) is a component of the nuclear pore complex (NPC), forming fibrous structures that extend into the nuclear interior [40]. Tpr is required for establishing heterochromatin exclusion zones in the vicinity of NPCs [41]. In addition to its roles in NPC architecture, Tpr is also involved in mRNA, unspliced RNA and nuclear protein export [4244]. Depletion of Tpr induces nuclear accumulation of p53, and facilitates autophagy [45].

Proteins that specifically co-purified with histone H1.1 (group 3) included several known histone chaperones. NapP1L1 and SET were recently shown to bind histone H1.0 in vitro [16, 46]. NAP1L4 has not previously been demonstrated to interact with linker histones.

Group 3 contained several proteins involved in RNA metabolism. These included three poly A binding proteins, PABP1, PABP3 and PABP4, as well as the RNaseP subunit POP1 [47, 48]. In addition, proteins involved in transcriptional regulation, NDE1, UBF1 and S30BP were also found to be specific for histone H1.1.

H1.2 specific binding partners (group 4) included DNA damage-binding protein 1 (DDB1) and protein VPRBP (also known as DDB1-CUL4-associated factor 1, DCAF1). DDB1 and VPRBP are both members of an E3 ubiquitin-protein ligase complex, named CUL4A-RBX1-DDB1-DCAF1/VPRBP complex, which is responsible for methylation-dependent ubiquitylation [49]. VPRBP and DDB1 were recently shown to co-purify with both H1.1 and H1.2 along with Cul4A and the PAF1 complex [37]. The distinct properties of the PAF1 and Cul4A complex subunits in the U2OS cells suggest that the specificity of the interaction between the Cul4A complex and linker histones is subject to cell type specific regulation.

Group 4 contained two RNA helicases, DDX21 and DDX50 and the nucleolar RNA binding protein EBP2. Group 4 also contained the transcriptional regulators TAF5 and TIF1B, as well as CHD7. While CHD7 has not been shown to be associated with linker histones, these results suggest that it may function in a manner analogous to CHD8.

Only two proteins were found to be exclusive binding partners of H1x (group 6). These were ubiquitin carboxyl-terminal hydrolase 15 (USP15) and insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3). USP15 is a deubiquitinating enzyme that can bind to ubiquitinated H2A/H2B dimers, and removes ubiquitin from ubiquitinated H2B that are not in nucleosomes [50]. IGFBP3 belongs to a family of three IGF-II mRNA-binding proteins that can bind to the 5′ UTR of the insulin-like growth factor II leader 3 mRNA and regulate the translation of insulin-like growth factor II during late mammalian development [51]. Interactions between USP15 and IGFBP3 and linker histones have not been reported previously.

Group 6 is an eclectic collection of proteins that co-purified with both H1.1 and H1x. This group includes the RNA binding proteins SRSF3 and SRSF7, the zinc finger protein ZNF16 and the anti-microbial protein dermcidin.

Interactions between nucleoprotein Tpr and H1 variants

The proteomics data clearly demonstrate that linker histones participate in a complex set of interactions that display varying degrees of specificity for the H1 variants. To explore in more detail the distinction between replication-dependent (H1.1 and H1.2) and replication-independent (H1x) H1 variants, we examined whether the specificity observed for replication-dependent H1.1 and H1.2 displayed by Tpr was a reflection of a specific in vivo connection between Tpr and replication-dependent linker histones. Unlike the PAF-1 complex and CHD8, which were also identified in the H1.1 and H1.2 complexes but not in the H1x complex (Table 1), Tpr has not been previously shown to interact with linker histones. To confirm the potential for interactions between Tpr and H1 variants in vivo, we resolved soluble whole cell extracts of U2OS cells expressing H1.1, H1.2 or H1x by size exclusion chromatography (Fig. 4). In all three cases, the peaks of H1 variants overlapped with the Tpr peak in a very high molecular weight complex (Fraction 9–10 in Fig. 4a, Fraction 9 in Fig. 4b, and Fraction 8–10 in Fig. 4c). These large complexes are likely due to other proteins associated with Tpr and/or H1 variants through direct or indirect interactions. The overlapping elution profiles of the linker histones and Tpr is consistent with the possibility that Tpr can form complexes with the linker histones in vivo but does not provide biochemical evidence for specific association of Tpr with the replication-dependent linker histones. Recprical co-immunopreciptations were also attempted to obtain additional evidence for for the specific interactions between Tpr and linker histones. However, currently available anti-Tpr antibodies were efficient for the immunopreciptation of Tpr and its associated proteins.
Fig. 4

Tpr Co-Elutes with H1 Variants on Size Exclusion Column. Soluble whole cell extracts of U2OS cells expressing 6 × His - tagged H1.1 (a), H1.2 (b) or H1x (c) were resolved by gel filtration chromatography (Superose 6). Fractions (indicated by numbers at top of gels) were precipitated with 20 % w/v TCA, then resolved by SDS-PAGE gel, and visualized by immunoblotting with anti-Tpr or anti-His antibodies

Tpr knockdown specifically reduces replication-dependent H1 variant levels via protein degradation

To determine whether the observed interactions between Tpr and replication-dependent linker histones are functionally relevant, we treated U2OS cells expressing 6 × His-tagged H1.1, H1.2 or H1x with Tpr-targeting siRNA in order to knockdown the expression of Tpr proteins. Surprisingly, we found that siTpr treated cells, which had significantly reduced Tpr immunofluorescence signals, also showed reduced immunofluorescence signals of 6 × His-tagged replication-dependent variants H1.1 and H1.2, but not replication-independent variant H1x (Fig. 5a). In both control siRNA and Tpr siRNA treated cells, all three H1 variants were localized within the nucleus, indicating that the loss of Tpr did not disrupt the nuclear distribution of histone H1. The fluorescence intensities of the signals for Tpr and the H1 variants, relative to the signal for DAPI, were quantitated and plotted (Fig. 5b). Based on a linear regression analysis of the data there was a clear positive correlation between the level of Tpr and the levels of histone H1.1 and H1.2 (r = 0.83 and 0.89, respectively) while there was no correlation between the level of Tpr and the level of H1x (r = 0.17). Consistent with the immunofluorescence data, we also observed reduced H1.1 and H1.2 protein levels in Tpr siRNA treated cells on western blots; however H1x protein levels were not affected (Fig. 5c).
Fig. 5

Tpr Knockdown Specifically Reduces Replication-dependent H1 Variant H1.1 and H1.2 Levels. a Untransfected (UN) U2OS Tet/On cells or U2OS Tet/On cells expressing H1 variants (H1.1, H1.2 and H1x) were treated with either control siRNA or siRNA targeting protein Tpr for 72 h. The cells were simultaneously stained with DAPI (blue), anti-Tpr (Red), and anti-His (Green). Overlay of anti-Tpr and anti-His staining was shown for comparison. b Scatter plot of quantified fluorescence intensities from Fig. 5a. Quantification and regression analysis was performed as described in Experimental Procedures. c Untransfected (UN) U2OS Tet/On cells or U2OS Tet/On cells expressing H1 variants (H1.1, H1.2 or H1x) were treated with either control siRNA or siRNA targeting protein Tpr for 72 h. 20 μg whole cell extracts of each sample were resolved on SDS-PAGE gel, and visualized by immunoblotting with antibodies targeting proteins indicated on the right

The reduction in H1.1 and H1.2 levels in Tpr knockdown cells could have been a result of a decrease in mRNA abundance or by protein destabilization. In order to investigate the effect of Tpr depletion on histone H1 gene expression, we performed quantitative real-time PCR analysis on the mRNA levels of H1.1, H1.2, and H1x in U2OS cells treated with either control siRNA or Tpr siRNA. Tpr knockdown did not cause any significant decrease in the mRNA levels of any of the three H1 variants (Fig. 6a and b).
Fig. 6

Tpr Stabilizes Replication-dependent H1 Variant H1.1 and H1.2 by Preventing Protein Degradation. ab mRNA levels of H1.1, H1.2 or H1x were quantified by quantitative Real-Time PCR in untransfected U2OS Tet/On cells (a) or U2OS Tet/On cells expressing H1 variants H1.1, H1.2 or H1x (b) treated with either control siRNA or siRNA targeting Tpr for 48 h. Expression levels of histone H1 genes were normalized relative to levels of GAPDH. Each experiment was completed on three biological replicates where each biological replicate was obtained in triplicate, and the mean of these values was used for further analysis. Statistical analysis was carried out using unpaired Student’s t-test: p ≤ 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.001 (***). The data are presented as means ± standard deviations (n = 3). c Untransfected (UN) U2OS Tet/On cells or U2OS Tet/On cells expressing H1 variants (H1.1, H1.2 or H1x) were treated with either control siRNA or siRNA targeting protein Tpr for 72 h. For the last 3 h of siRNA transfection, 20 μM of MG132 was added to the media. 20 μg whole cell extracts of each sample were resolved on SDS-PAGE gel, and visualized by immunoblotting with antibodies targeting proteins indicated on the right. d Untransfected (UN) U2OS Tet/On cells or U2OS Tet/On cells expressing H1 variants (H1.1, H1.2 or H1x) were treated with either control siRNA or siRNA targeting protein Tpr for 72 h. For the last 3 h of siRNA transfection, 20 μM of MG132 was added to the media. Treated cells were then simultaneously stained with DAPI (blue), Tpr (Red), and 6 × His-tag (Green). Overlay of anti-Tpr and anti-His staining was shown for comparison. e Scatter plot of quantified fluorescence intensities from Fig. 6d. Quantification and regression analysis was performed as described in Experimental Procedures

To test whether Tpr is required for histone H1.1 and H1.2 protein stability, we treated U2OS cells with MG132, which is a cell permeable proteasome inhibitor. In the presence of MG132, H1.1 and H1.2 protein levels were restored in Tpr siRNA treated cells to levels comparable to those of control siRNA treated cells (Fig. 6c). Immunofluorescence microscopy of MG132 treated cells also confirmed this rescue of H1.1 and H1.2 protein levels (Fig. 6d). Quantitation of the immunofluorescence data indicated a clear decrease in the correlation of the levels of Tpr and histones H1.1 and H1.2 with an increase in the number of cells with a low level of Tpr that maintain a high level of the replication-dependent linker histones (Fig. 6e). It is important to note that in MG132 treated cells all H1 variants were still localized within the nucleus, indicating that loss of Tpr did not cause replication-dependent H1 proteins to accumulate in the cytosol suggesting that Tpr is not required for the nuclear import of replication-dependent linker histones. These results suggest that the specific interaction between Tpr and replication-dependent histone H1 variants detected biochemically is a reflection of a specific in vivo interaction. Further, the interaction between Tpr and histones H1.1 and H1.2 is critical to maintain the proper abundance of these linker histones.

Discussion

There is accumulated evidence indicating functional differentiation among linker histone variants. The abundance of H1 variants fluctuates in diverse cell types and at development stages [6, 52]. Their ability to condense chromatin also varies (reviewed in [53]). In addition, the H1 variants can engage in a wide range of different protein-protein interactions (reviewed in [16, 54]). Several previous proteomic studies focused on the characterization of the linker histone variants H1.0 and H1.2 interaction networks, however the comparison of replication-dependent and replication-independent H1 variants is still lacking [14, 15, 37]. In order to understand how replication-dependent and replication-independent H1 variants carry out differentiated functions through protein-protein interactions, we purified protein complexes containing 6 × His-tagged replication-dependent H1 variants H1.1 and H1.2, or replication-independent variant H1x, and identified their binding partners using mass spectrometric analysis.

Broadly speaking, our proteomic characterization of linker histones binding proteins identifies four groups of proteins that interact with linker histones. We used soluble whole cell extracts as the starting material for the H1 purifications to facilitate the identification of histone chaperones that would regulate the transport and assembly of the linker histones. Indeed, a significant fraction of the H1 interacting proteins are previously known histone-binding proteins. While many of these proteins have primarily been characterized in the context their functions as core histone chaperones, it will be interesting to determine how they also impact linker histone dynamics.

The well-characterized function of linker histones in the regulation of transcription is reflected in the abundance of transcription factors identified as linker histone binding proteins. The observation that some transcription factors are highly specific for a single histone H1 variant suggests that some of the unique functions of the linker histones may be mediated by protein-protein interactions that regulate specific subsets of genes.

The other two groups of proteins were related to RNA biology. The largest groups of interacting factors were ribosomal proteins. The interaction of linker histones with ribosomes has been observed both in vitro and in vivo and has been implicated in transcriptional repression [14, 15, 17]. We also identified a number of proteins involved in RNA metabolism including RNA binding proteins and RNA helicases. Surprisingly, multiple members of the WTAP complex, which is required for a significant fraction of the N6-methyladenosine modification in the cell, as well as the N6-methyladenosine reader protein YTDC1 were associated with all three H1 variants [28]. Together, these results suggest the intriguing possibility that linker histones may provide an important link between chromatin structure and RNA metabolism.

Surprisingly, NASP (nuclear antigenic sperm protein) was not found to associate with any of the histone H1 variants in the present study and has not been identified in the recent proteomic analyses of in vivo linker histone complexes [16, 26, 37]. NASP was one of the first proteins proposed to be a linker histone chaperone and, in fact, can bind linker histones with nM affinity in vitro [5557]. The inability to identify NASP as linker histone-associated protein in multiple studies suggests a number of possibilities. NASP may not interact with linker histones to a significant extent despite the demonstrated in vitro affinity. Alternatively, NASP may interact in vivo with only a subset of the linker histone variants that have not yet been examined by proteomic methods.

In comparing the proteins that co-purified with each of the H1 variants, we found that the three H1 variants had shared binding partners that were identified in earlier studies, such as nucleophosmin, nucleolin and FACT complex subunits SPT16 and SSRP1, nucleolin, nucleophosmin (NPM1) and Y box-binding proteins YB1, YB2 and YB3. These H1-associating proteins may contribute to the common functions of H1 variants. Importantly, we identified shared components found in the replication-dependent H1 variant complexes that were not associated replication-independent H1 variants. These factors include the PAF1 complex, CHD8 and nucleoprotein Tpr. The recent study from Kim and colleagues demonstrated that the PAF1 complex is not associated with histone H1.0 consistent with the specificity of this complex for replication-dependent linker histones [37]. These exclusive protein-protein interactions may be crucial for mediating cell cycle-dependent functions of H1 variants.

The interactions of the PAF-1 complex and CHD8 with linker histones have demonstrated functions in transcriptional regulation [3739]. However, a link between Tpr and linker histones has not previously been identified. Our analysis of Tpr siRNA knockdown cells supports the proteomic data that indicated that Tpr engages in specific interactions as Tpr knockdown reduced the levels of replication-dependent H1 variants H1.1 and H1.2, but not replication-dependent H1 variant H1x. Future studies will determine whether Tpr also interacts with the remaining replication-dependent linker histones, H1.3, H1.4 and H1.5 or, whether, like the PAF1 complex, it is specific for H1.1 and H1.2.

While Tpr is required for the stability of histones H1.1 and H1.2, the functional significance of their interaction is not clear. As Tpr is a component of the nuclear pore complex, the most straightforward model is that Tpr is specifically involved in the nuclear import of replication-dependent linker histones. This model predicts that if the levels of H1.1 and H1.2 were restored in cells lacking Tpr that the histones would accumulate in the cytoplasm. However, when the Tpr siRNA knockdown was performed in the presence of the proteasome inhibitor MG132, we observed no cytoplasmic accumulation of H1.1 and H1.2.

A second possibility is that Tpr can function as a histone chaperone that participates in the targeting of replication-dependent linker histones to sites of chromatin assembly. Indeed, Tpr contains a region nears the COOH-terminus that is highly enriched in aspartic acid and glutamic acid as is observed in many histone chaperones. In addition, a recent proteomic analysis of nascent chromatin structure identified Tpr as a chromatin-associated protein [58].

Another potential function for the interaction of Tpr with histones H1.1 and H1.2 may be related to involvement of Tpr in the formation of heterochromatin exclusion zones (HEZs) around nuclear pores. While heterochromatin localizes near the nuclear envelope, regions near the nuclear pores are devoid of heterochromatin through the formation of Tpr-dependent HEZs [41]. The interaction between Tpr and replication-dependent H1 variants may be important for the maintenance of HEZs. Tpr may stabilize and preserve H1 variants with minimal DNA compaction abilities, such as H1.1 and H1.2, which establishes a basal level of chromatin packaging around HEZs, while keeping out H1 variants that promote strong condensation, such as H1x. To test this hypothesis, it would be interesting to investigate the interactions between Tpr and other H1 variants with higher affinity and/or higher capacity for the compaction of nucleosomes, such as H1.3 and H1.4 (Reviewed in [53]).

Conclusions

We have isolated and analyzed the soluble forms of the replication-dependent linker histones H1.1 and H1.2 and the replication-independent linker histone H1x. These linker histones interact with both a common group of proteins and with variant-specific interacting proteins. One protein that is specifically associated with H1.1 and H1.2 is nuclear pore complex protein Tpr. Tpr was not required for the nuclear import of histones H1.1 and H1.2. However, knockdown of Tpr expression resulted in a specific decrease in protein stability of the replication-dependent linker histones.

Methods

Antibodies and other materials

Sources of antibody employed are as follows: anti-His.M8 from Thermo Fisher Scientific was used for western blots; anti-His6 from Roche was used for immunofluorescence microscopy; anti-Tpr from Abcam. Control siRNA (sc-37007) and siRNA targeting Tpr (sc-45343) was purchased from Santa Cruz Biotech.

Plasmid construction

The human Ultimate ORF of histone H1.1 (IOH35288), H1.2 (IOH5275), and H1x (IOH3417) (Thermo Fisher Scientific) were inserted into mammalian expression vector pT-Rex-DEST31 (Thermo Fisher Scientific), which adds a 6 × His-tag at the NH2-terminus. The resulting constructs were transferred into DH10B E. coli strain for production.

Cell culture and transfections

Tetracycline-inducible human osteosarcoma cell line U2OS (kind gift from Dr. Dan Schoenberg) was grown in McCoy’s 5A media supplemented with 10 % (v/v) fetal bovine serum, 50 U/ml penicillin, 50 mg/ml streptomycin, and 1 % L-glutamate (Sigma) at 37 °C in 5 % CO2 supply. The U2OS cells were transfected with pT-Rex-DEST31 plasmids carrying H1.1, H1.2 or H1x using X-tremeGENE HP DNA transfection reagent (Roche) following the manufacturer’s instruction. Transfected clones were selected against 200 mg/ml G418 and 10 mg/ml blasticidin (Gibco, Thermo Fisher Scientific). Stable clones were induced by 1 μg/ml doxycycline (Sigma) for 48 h. The expression of 6 × His-tagged H1 variants was confirmed by western blot.

Cell fractionation

Cytosolic and nuclear extracts were prepared from U2OS cells as previously described [59]. The resulting extracts were resolved on SDS-PAGE gels and visualized on western blots.

Histone extractions

Total histones were prepared from U2OS cells as previously described [60]. The resulting histone pellets were resuspended in DN (300) buffer (300 mM NaCl, 25 mM Tris pH 7.0, 0.1 mM EDTA, 10 % glycerol, and 0.5 mM phenyl methyl sulfonate fluoride), and resolved on 18 % SDS PAGE gels. Linker histones isolated from chicken erythrocyte nuclei were also loaded in the gels as controls [56].

Micrococcal nuclease digestion assays

1 × 107 U2OS cells were collected and washed with cold PBS, and incubated in lysis buffer (300 mM HEPES pH7.5, 60 mM KCl, 300 mM sucrose, 5 mM K2HPO4, 5 mM MgCl2, 2 mM EDTA, and 0.5 % Triton X-100) for 5 min on ice. The cells were then broken with 7 gentle strokes in a type B Dounce homogenizer. Nuclei were pelleted by centrifuging at 120 × g for 10 min. The pelleted nuclei were washed with 1 ml MNase digestion buffer (10 mM Tris pH7.5, 15 mM NaCl, 1 mM CaCl2, 60 mM KCl, and 0.2 mM phenyl methyl sulfonate fluoride), resuspended in 1 ml MNase digestion buffer, and digested with 0.2 U/ml (Sigma units) of MNase at 37 °C for various lengths of time. Digestions were stopped with 10 mM EDTA followed by incubation with 0.1 mg/ml proteinase K at 37 °C for 5 min. The resulting DNA samples were isolated using phenol extraction and ethanol precipitation, and resolved on 1 % agarose gel [61].

Protein expression and purification

U2OS cells with 6 × His-tagged H1 inserts were induced by 1 μg/ml doxycycline (Sigma). After 48 h, cells were harvested, washed with PBS, and lysed with NP40 lysis buffer (150 mM NaCl, 50 mM Tris pH7.5, 10 % Glycerol, 0.75 % NP40) containing EDTA-free protease inhibitor cocktail (Roche) and 0.5 mM phenyl methyl sulfonate fluoride. The resulting whole cell extracts were applied to a Mono Q 5/50 GL column (GE Healthcare Life Sciences). Proteins were eluted with a NaCl gradient from 50 mM to 500 mM in 15 column volumes, then 1 M for 5 column volumes. Fractions containing eluted 6 × His-tagged H1 variants were pooled, desalted on desalting columns (GE Healthcare Life Sciences), and then applied to an HiTrap Chelating HP Column (1 ml, GE Healthcare Life Sciences) charged with Ni2+. The column was then washed extensively with NP40 lysis buffer containing 50 mM imidazole. Bound proteins were eluted with NP40 lysis buffer containing 500 mM imidazole. Fractions were collected and resolved on western blots.

Mass spectrometry

Tryptic peptides were desalted online using a μ-precolumn (PepMap100, C18, 5 μm, 100 Å, 0.3 x 50 mm, ThermoFisher, Waltham, MA, USA) and separated based on hydrophobicity at a flow rate of 2 μL/min (C18Aq, 5 μm, 300 Å 0.2 × 150 mm, Michrom Bioresources Inc., Auburn, CA, USA). Peptides were introduced into a ThermoFisher LTQ Orbitrap XL mass spectrometer (ThermoFisher, Waltham, MA, USA) with the aid of micro/nanospray ionization source (Michrom Bioresources Inc., Auburn, CA, USA). Mobile phase A (MPA) consisted of HPLC water with 0.1 % v/v formic acid (J.T. Baker, Center Valley, PA, USA) and mobile phase B (MPB) consisted of ACN (EMD Millipore, Billerica, MA, USA) with 0.1 % v/v formic acid. The HPLC gradient was ramped from 2 to 40 % MPB from 5 to 155 min, followed by a high organic wash and column equilibration period. The heated capillary temperature and electrospray voltage were set to 175 °C and 2.0 kV. The top 5 molecular ions (2+/3+ charges) were selected for CID fragmentation in a data-dependent fashion. Molecular ions were analyzed in the Orbitrap (AGC:1000 K ions; 60 K mass resolution; 300 ms max injection time, 1 microscan, precursor scan enabled). Molecular ions were selected and subjected to CID fragmentation in the linear ion trap (NCE: 35 %, AGC: 10 K ions; 300 ms max injection time). Dynamic exclusion parameters were enabled (30s ± 25 ppm, repeat count:1). Protein identifications were determined using the MassMatrix search engine (v 2.4.2) and the complete, reviewed Swiss-Prot entries from UniProt (taxon:9606, 20,258 entries, July 2013) [6265]. Search parameters included a maximum of two trypsin missed cleavages, molecular ion tolerance of ±20 ppm and a fragment ion tolerance of ±0.8 Da. The false discovery rate (FDR) was estimated using a target-decoy strategy. Final protein identifications and spectral counts were harmonized using an in-house python application [66]. Protein identification from this harmonized protein list, were retained based on an FDR threshold of 5 % where each protein consisted of at least two unique peptide matches.

Size-exclusion chromatography of H1 complexes

Whole cell extracts of U2OS cells expressing 6 × His-tagged H1 variants were applied to a Superose 6 10/300 GL column (GE Healthcare Life Sciences). The column was washed with 1.5 column volumes of NP40 lysis buffer. Proteins in each collected fraction were precipitated with 20 % w/v TCA, then resolved and visualized on western blots.

Immunofluorescence microscopy

U2OS cells seeded on cover slips were fixed in 4 % paraformaldehyde for 20 min at room temperature and then washed with PBS. The washed cells were permeabilized with 0.5 % Triton X-100 in PBS for 15 min, then washed with PBS three times, and blocked with blocking buffer (1 % bovine serum albumin and 0.3 % Triton X-100 in PBS) for 1 h. After blocking, the cells were incubated with primary antibodies diluted in blocking buffer overnight followed by three washes in PBST, and incubated with secondary antibodies for 1 h. Then the cover slips with cells were washed three times in PBS, and mounted onto microscope slides with Vectashield mounting serum containing DAPI (Vector Laboratories). The slides were examined using Axioskop 40 microscope. Immunofluorescence pictures were taken with an Axiocam HRC camera and processed in Zen 2 Pro software.

Quantification and analysis of immunofluorescence

Corrected total cell fluorescence (CTCF) was quantified using ImageJ as previously described [67]. CTCF of anti-Tpr and anti-His6 staining were standardized against CTCF of DAPI staining. Regression analysis was carried out using Microsoft Excel built-in statistical functions.

Quantitative real-time PCR analysis

RNAs were extracted from U2OS cells using Trizol reagent (Thermo Fisher Scientific) following the manufacturer’s instructions. The resulting RNAs were used to prepare cDNAs using High Capacity cDNA Reverse Transcription Kits (Applied Biosystems) following the manufacturer's instruction. Levels of gene expression were measured using TaqMan Gene Expression Assays (Applied Biosystems, Assay IDs as follows: H1.1 – Hs00271225_s1, H1.2 – Hs00271185_s1, H1x – Hs00366688_s1, and GAPDH – Hs02758991_g1), TagMan Universal PCR Master Mix (Applied Biosystems) and the ABI 7300 sequence detector as previously described [68]. Expression levels of histone H1 genes were normalized relative to levels of GAPDH. Each experiment was completed on three biological replicates where each biological replicate was obtained in triplicate, and the mean of these values was used for further analysis. Statistical analysis was carried out using unpaired Student’s t-test: p ≤ 0.05 (*), p ≤ 0.01(**), and p ≤ 0.001(***).

Declarations

Acknowledgements

We would like to thank Dr. Dan Schoenberg for the gift of Tet-inducible U2OS cells.

Funding

This work was supported by a grant from the National Institutes of Health (GM062970 to M.R.P.).

Availability of data and materials

All data generated or analysed during this study are included in this published article [and its Additional file].

Authors’ contributions

PZ, OEB, MAF and MRP designed experiments. PZ and OEB performed experiments. PZ, OEB, MAF and MRP analyzed data. PZ, OEB, MAF and MRP wrote the manuscript. All authors read and approved the final manuscript.

Competing interest

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

No human subjects or animals were used in this study.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Biological Chemistry and Pharmacology, The Ohio State University
(2)
Department of Cancer Biology and Genetics, The Ohio State University

References

  1. Tremethick DJ. Higher-order structures of chromatin: the elusive 30 nm fiber. Cell. 2007;128:651–4.View ArticlePubMedGoogle Scholar
  2. Thomas JO, Stott K. H1 and HMGB1: modulators of chromatin structure. Biochem Soc Trans. 2012;40:341–6.View ArticlePubMedGoogle Scholar
  3. Happel N, Doenecke D. Histone H1 and its isoforms: contribution to chromatin structure and function. Gene. 2009;431:1–12.View ArticlePubMedGoogle Scholar
  4. Fan Y, Nikitina T, Morin-Kensicki EM, Zhao J, Magnuson TR, Woodcock CL, Skoultchi AI. H1 linker histones are essential for mouse development and affect nucleosome spacing in vivo. Mol Cell Biol. 2003;23:4559–72.View ArticlePubMedPubMed CentralGoogle Scholar
  5. Sancho M, Diani E, Beato M, Jordan A. Depletion of human histone H1 variants uncovers specific roles in gene expression and cell growth. PLoS Genet. 2008;4:e1000227.View ArticlePubMedPubMed CentralGoogle Scholar
  6. Terme JM, Sese B, Millan-Arino L, Mayor R, Izpisua Belmonte JC, Barrero MJ, Jordan A. Histone H1 variants are differentially expressed and incorporated into chromatin during differentiation and reprogramming to pluripotency. J Biol Chem. 2011;286:35347–57.View ArticlePubMedPubMed CentralGoogle Scholar
  7. Campos EI, Fillingham J, Li G, Zheng H, Voigt P, Kuo WH, Seepany H, Gao Z, Day LA, Greenblatt JF, Reinberg D. The program for processing newly synthesized histones H3.1 and H4. Nat Struct Mol Biol. 2010;17:1343–51.View ArticlePubMedPubMed CentralGoogle Scholar
  8. Tagami H, Ray-Gallet D, Almouzni G, Nakatani Y. Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis. Cell. 2004;116:51–61.View ArticlePubMedGoogle Scholar
  9. Shintomi K, Iwabuchi M, Saeki H, Ura K, Kishimoto T, Ohsumi K. Nucleosome assembly protein-1 is a linker histone chaperone in Xenopus eggs. Proc Natl Acad Sci U S A. 2005;102:8210–5.View ArticlePubMedPubMed CentralGoogle Scholar
  10. Richardson RT, Alekseev OM, Grossman G, Widgren EE, Thresher R, Wagner EJ, Sullivan KD, Marzluff WF, O’Rand MG. Nuclear autoantigenic sperm protein (NASP), a linker histone chaperone that is required for cell proliferation. J Biol Chem. 2006;281:21526–34.View ArticlePubMedGoogle Scholar
  11. Gadad SS, Senapati P, Syed SH, Rajan RE, Shandilya J, Swaminathan V, Chatterjee S, Colombo E, Dimitrov S, Pelicci PG, Ranga U, Kundu TK. The multifunctional protein nucleophosmin (NPM1) is a human linker histone H1 chaperone. Biochemistry. 2011;50:2780–9.View ArticlePubMedGoogle Scholar
  12. George EM, Brown DT. Prothymosin alpha is a component of a linker histone chaperone. FEBS Lett. 2010;584:2833–6.View ArticlePubMedPubMed CentralGoogle Scholar
  13. Kato K, Okuwaki M, Nagata K. Role of template activating factor-I as a chaperone in linker histone dynamics. J Cell Sci. 2011;124:3254–65.View ArticlePubMedGoogle Scholar
  14. Szerlong HJ, Herman JA, Krause CM, DeLuca JG, Skoultchi A, Winger QA, Prenni JE, Hansen JC. Proteomic characterization of the nucleolar linker histone H1 interaction network. J Mol Biol. 2015;427:2056–71.View ArticlePubMedPubMed CentralGoogle Scholar
  15. Kalashnikova AA, Winkler DD, McBryant SJ, Henderson RK, Herman JA, DeLuca JG, Luger K, Prenni JE, Hansen JC. Linker histone H1.0 interacts with an extensive network of proteins found in the nucleolus. Nucleic Acids Res. 2013;41:4026–35.View ArticlePubMedPubMed CentralGoogle Scholar
  16. Kalashnikova AA, Rogge RA, Hansen JC. Linker histone H1 and protein-protein interactions. Biochim Biophys Acta. 2016;1859:455–61.View ArticlePubMedGoogle Scholar
  17. Ni JQ, Liu LP, Hess D, Rietdorf J, Sun FL. Drosophila ribosomal proteins are associated with linker histone H1 and suppress gene transcription. Genes Dev. 2006;20:1959–73.View ArticlePubMedPubMed CentralGoogle Scholar
  18. Das C, Tyler JK, Churchill ME. The histone shuffle: histone chaperones in an energetic dance. Trends Biochem Sci. 2010;35:476–89.View ArticlePubMedPubMed CentralGoogle Scholar
  19. Okuwaki M, Matsumoto K, Tsujimoto M, Nagata K. Function of nucleophosmin/B23, a nucleolar acidic protein, as a histone chaperone. FEBS Lett. 2001;506:272–6.View ArticlePubMedGoogle Scholar
  20. Swaminathan V, Kishore AH, Febitha KK, Kundu TK. Human histone chaperone nucleophosmin enhances acetylation-dependent chromatin transcription. Mol Cell Biol. 2005;25:7534–45.View ArticlePubMedPubMed CentralGoogle Scholar
  21. Angelov D, Bondarenko VA, Almagro S, Menoni H, Mongelard F, Hans F, Mietton F, Studitsky VM, Hamiche A, Dimitrov S, Bouvet P. Nucleolin is a histone chaperone with FACT-like activity and assists remodeling of nucleosomes. EMBO J. 2006;25:1669–79.View ArticlePubMedPubMed CentralGoogle Scholar
  22. Holmberg Olausson K, Elsir T, Moazemi Goudarzi K, Nister M, Lindstrom MS. NPM1 histone chaperone is upregulated in glioblastoma to promote cell survival and maintain nucleolar shape. Sci Rep. 2015;5:16495.View ArticlePubMedPubMed CentralGoogle Scholar
  23. Belotserkovskaya R, Oh S, Bondarenko VA, Orphanides G, Studitsky VM, Reinberg D. FACT facilitates transcription-dependent nucleosome alteration. Science. 2003;301:1090–3.View ArticlePubMedGoogle Scholar
  24. Li J, Zhou F, Zhan D, Gao Q, Cui N, Li J, Iakhiaeva E, Zwieb C, Lin B, Wong J. A novel histone H4 arginine 3 methylation-sensitive histone H4 binding activity and transcriptional regulatory function for signal recognition particle subunits SRP68 and SRP72. J Biol Chem. 2012;287:40641–51.View ArticlePubMedPubMed CentralGoogle Scholar
  25. Eliseeva IA, Kim ER, Guryanov SG, Ovchinnikov LP, Lyabin DN. Y-box-binding protein 1 (YB-1) and its functions. Biochemistry (Mosc). 2011;76:1402–33.View ArticleGoogle Scholar
  26. Kim K, Choi J, Heo K, Kim H, Levens D, Kohno K, Johnson EM, Brock HW, An W. Isolation and characterization of a novel H1.2 complex that acts as a repressor of p53-mediated transcription. J Biol Chem. 2008;283:9113–26.View ArticlePubMedPubMed CentralGoogle Scholar
  27. Horiuchi K, Kawamura T, Iwanari H, Ohashi R, Naito M, Kodama T, Hamakubo T. Identification of Wilms’ tumor 1-associating protein complex and its role in alternative splicing and the cell cycle. J Biol Chem. 2013;288:33292–302.View ArticlePubMedPubMed CentralGoogle Scholar
  28. Schwartz S, Mumbach MR, Jovanovic M, Wang T, Maciag K, Bushkin GG, Mertins P, Ter-Ovanesyan D, Habib N, Cacchiarelli D, Sanjana NE, Freinkman E, Pacold ME, Satija R, Mikkelsen TS, Hacohen N, Zhang F, Carr SA, Lander ES, Regev A. Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5′ sites. Cell Rep. 2014;8:284–96.View ArticlePubMedPubMed CentralGoogle Scholar
  29. Ping XL, Sun BF, Wang L, Xiao W, Yang X, Wang WJ, Adhikari S, Shi Y, Lv Y, Chen YS, Zhao X, Li A, Yang Y, Dahal U, Lou XM, Liu X, Huang J, Yuan WP, Zhu XF, Cheng T, Zhao YL, Wang X, Rendtlew Danielsen JM, Liu F, Yang YG. Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase. Cell Res. 2014;24:177–89.View ArticlePubMedPubMed CentralGoogle Scholar
  30. Liu J, Yue Y, Han D, Wang X, Fu Y, Zhang L, Jia G, Yu M, Lu Z, Deng X, Dai Q, Chen W, He C. A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat Chem Biol. 2014;10:93–5.View ArticlePubMedGoogle Scholar
  31. Xiao W, Adhikari S, Dahal U, Chen YS, Hao YJ, Sun BF, Sun HY, Li A, Ping XL, Lai WY, Wang X, Ma HL, Huang CM, Yang Y, Huang N, Jiang GB, Wang HL, Zhou Q, Wang XJ, Zhao YL, Yang YG. Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing. Mol Cell. 2016;61:507–19.View ArticlePubMedGoogle Scholar
  32. Xu C, Liu K, Ahmed H, Loppnau P, Schapira M, Min J. Structural basis for the discriminative recognition of N6-methyladenosine RNA by the human YT521-B homology domain family of proteins. J Biol Chem. 2015;290:24902–13.View ArticlePubMedGoogle Scholar
  33. Xu C, Wang X, Liu K, Roundtree IA, Tempel W, Li Y, Lu Z, He C, Min J. Structural basis for selective binding of m6A RNA by the YTHDC1 YTH domain. Nat Chem Biol. 2014;10:927–9.View ArticlePubMedGoogle Scholar
  34. Zhang Z, Theler D, Kaminska KH, Hiller M, de la Grange P, Pudimat R, Rafalska I, Heinrich B, Bujnicki JM, Allain FH, Stamm S. The YTH domain is a novel RNA binding domain. J Biol Chem. 2010;285:14701–10.View ArticlePubMedPubMed CentralGoogle Scholar
  35. Zhang S, Cai M, Zhang S, Xu S, Chen S, Chen X, Chen C, Gu J. Interaction of p58(PITSLRE), a G2/M-specific protein kinase, with cyclin D3. J Biol Chem. 2002;277:35314–22.View ArticlePubMedGoogle Scholar
  36. Mueller CL, Jaehning JA. Ctr9, Rtf1, and Leo1 are components of the Paf1/RNA polymerase II complex. Mol Cell Biol. 2002;22:1971–80.View ArticlePubMedPubMed CentralGoogle Scholar
  37. Kim K, Lee B, Kim J, Choi J, Kim JM, Xiong Y, Roeder RG, An W. Linker Histone H1.2 cooperates with Cul4A and PAF1 to drive H4K31 ubiquitylation-mediated transactivation. Cell Rep. 2013;5:1690–703.View ArticlePubMedPubMed CentralGoogle Scholar
  38. Nishiyama M, Skoultchi AI, Nakayama KI. Histone H1 recruitment by CHD8 is essential for suppression of the Wnt-beta-catenin signaling pathway. Mol Cell Biol. 2012;32:501–12.View ArticlePubMedPubMed CentralGoogle Scholar
  39. Nishiyama M, Oshikawa K, Tsukada Y, Nakagawa T, Iemura S, Natsume T, Fan Y, Kikuchi A, Skoultchi AI, Nakayama KI. CHD8 suppresses p53-mediated apoptosis through histone H1 recruitment during early embryogenesis. Nat Cell Biol. 2009;11:172–82.View ArticlePubMedPubMed CentralGoogle Scholar
  40. Krull S, Thyberg J, Bjorkroth B, Rackwitz HR, Cordes VC. Nucleoporins as components of the nuclear pore complex core structure and Tpr as the architectural element of the nuclear basket. Mol Biol Cell. 2004;15:4261–77.View ArticlePubMedPubMed CentralGoogle Scholar
  41. Krull S, Dorries J, Boysen B, Reidenbach S, Magnius L, Norder H, Thyberg J, Cordes VC. Protein Tpr is required for establishing nuclear pore-associated zones of heterochromatin exclusion. EMBO J. 2010;29:1659–73.View ArticlePubMedPubMed CentralGoogle Scholar
  42. Dieppois G, Iglesias N, Stutz F. Cotranscriptional recruitment to the mRNA export receptor Mex67p contributes to nuclear pore anchoring of activated genes. Mol Cell Biol. 2006;26:7858–70.View ArticlePubMedPubMed CentralGoogle Scholar
  43. Galy V, Gadal O, Fromont-Racine M, Romano A, Jacquier A, Nehrbass U. Nuclear retention of unspliced mRNAs in yeast is mediated by perinuclear Mlp1. Cell. 2004;116:63–73.View ArticlePubMedGoogle Scholar
  44. Frosst P, Guan T, Subauste C, Hahn K, Gerace L. Tpr is localized within the nuclear basket of the pore complex and has a role in nuclear protein export. J Cell Biol. 2002;156:617–30.View ArticlePubMedPubMed CentralGoogle Scholar
  45. Funasaka T, Tsuka E, Wong RW. Regulation of autophagy by nucleoporin Tpr. Sci Rep. 2012;2:878.View ArticlePubMedPubMed CentralGoogle Scholar
  46. Herz HM, Garruss A, Shilatifard A. SET for life: biochemical activities and biological functions of SET domain-containing proteins. Trends Biochem Sci. 2013;38:621–39.View ArticlePubMedPubMed CentralGoogle Scholar
  47. Smith RW, Blee TK, Gray NK. Poly(A)-binding proteins are required for diverse biological processes in metazoans. Biochem Soc Trans. 2014;42:1229–37.View ArticlePubMedPubMed CentralGoogle Scholar
  48. Brook M, Gray NK. The role of mammalian poly(A)-binding proteins in co-ordinating mRNA turnover. Biochem Soc Trans. 2012;40:856–64.View ArticlePubMedPubMed CentralGoogle Scholar
  49. Lee JM, Lee JS, Kim H, Kim K, Park H, Kim JY, Lee SH, Kim IS, Kim J, Lee M, Chung CH, Seo SB, Yoon JB, Ko E, Noh DY, Kim KI, Kim KK, Baek SH. EZH2 generates a methyl degron that is recognized by the DCAF1/DDB1/CUL4 E3 ubiquitin ligase complex. Mol Cell. 2012;48:572–86.View ArticlePubMedGoogle Scholar
  50. Long L, Thelen JP, Furgason M, Haj-Yahya M, Brik A, Cheng D, Peng J, Yao T. The U4/U6 recycling factor SART3 has histone chaperone activity and associates with USP15 to regulate H2B deubiquitination. J Biol Chem. 2014;289:8916–30.View ArticlePubMedPubMed CentralGoogle Scholar
  51. Nielsen J, Christiansen J, Lykke-Andersen J, Johnsen AH, Wewer UM, Nielsen FC. A family of insulin-like growth factor II mRNA-binding proteins represses translation in late development. Mol Cell Biol. 1999;19:1262–70.View ArticlePubMedPubMed CentralGoogle Scholar
  52. Meergans T, Albig W, Doenecke D. Varied expression patterns of human H1 histone genes in different cell lines. DNA Cell Biol. 1997;16:1041–9.View ArticlePubMedGoogle Scholar
  53. Parseghian H. What is the role of histone H1 heterogeneity? A functional model emerges from a 50 year mystery. AIMS Biophysics. 2015;2:724–72.View ArticleGoogle Scholar
  54. McBryant SJ, Lu X, Hansen JC. Multifunctionality of the linker histones: an emerging role for protein-protein interactions. Cell Res. 2010;20:519–28.View ArticlePubMedPubMed CentralGoogle Scholar
  55. Richardson RT, Batova IN, Widgren EE, Zheng LX, Whitfield M, Marzluff WF, O’Rand MG. Characterization of the histone H1-binding protein, NASP, as a cell cycle-regulated somatic protein. J Biol Chem. 2000;275:30378–86.View ArticlePubMedGoogle Scholar
  56. Wang H, Walsh ST, Parthun MR. Expanded binding specificity of the human histone chaperone NASP. Nucleic Acids Res. 2008;36:5763–72.View ArticlePubMedPubMed CentralGoogle Scholar
  57. Wang H, Ge Z, Walsh ST, Parthun MR. The human histone chaperone sNASP interacts with linker and core histones through distinct mechanisms. Nucleic Acids Res. 2012;40(2):660-9. Epub 2011 Sep 29.Google Scholar
  58. Alabert C, Bukowski-Wills JC, Lee SB, Kustatscher G, Nakamura K, de Lima Alves F, Menard P, Mejlvang J, Rappsilber J, Groth A. Nascent chromatin capture proteomics determines chromatin dynamics during DNA replication and identifies unknown fork components. Nat Cell Biol. 2014;16:281–93.View ArticlePubMedPubMed CentralGoogle Scholar
  59. Suzuki K, Bose P, Leong-Quong RY, Fujita DJ, Riabowol K. REAP: A two minute cell fractionation method. BMC Res Notes. 2010;3:294.View ArticlePubMedPubMed CentralGoogle Scholar
  60. Knapp AR, Ren C, Su X, Lucas DM, Byrd JC, Freitas MA, Parthun MR. Quantitative profiling of histone post-translational modifications by stable isotope labeling. Methods. 2007;41:312–9.View ArticlePubMedPubMed CentralGoogle Scholar
  61. Moore, D., and Dowhan, D. Purification and concentration of DNA from aqueous solutions. Curr Protoc Mol Biol. 2002. Chapter 2, Unit 2 1AGoogle Scholar
  62. Xu H, Freitas MA. A mass accuracy sensitive probability based scoring algorithm for database searching of tandem mass spectrometry data. BMC Bioinformatics. 2007;8:133.View ArticlePubMedPubMed CentralGoogle Scholar
  63. Xu H, Yang L, Freitas MA. A robust linear regression based algorithm for automated evaluation of peptide identifications from shotgun proteomics by use of reversed-phase liquid chromatography retention time. BMC Bioinformatics. 2008;9:347.View ArticlePubMedPubMed CentralGoogle Scholar
  64. Xu H, Zhang L, Freitas MA. Identification and characterization of disulfide bonds in proteins and peptides from tandem MS data by use of the MassMatrix MS/MS search engine. J Proteome Res. 2008;7:138–44.View ArticlePubMedGoogle Scholar
  65. Xu H, Freitas MA. Monte carlo simulation-based algorithms for analysis of shotgun proteomic data. J Proteome Res. 2008;7:2605–15.View ArticlePubMedPubMed CentralGoogle Scholar
  66. Zhang B, Chambers MC, Tabb DL. Proteomic parsimony through bipartite graph analysis improves accuracy and transparency. J Proteome Res. 2007;6:3549–57.View ArticlePubMedPubMed CentralGoogle Scholar
  67. McCloy RA, Rogers S, Caldon CE, Lorca T, Castro A, Burgess A. Partial inhibition of Cdk1 in G 2 phase overrides the SAC and decouples mitotic events. Cell Cycle. 2014;13:1400–12.View ArticlePubMedPubMed CentralGoogle Scholar
  68. Ge Z, Wang H, Parthun MR. Nuclear Hat1p complex (NuB4) components participate in DNA repair-linked chromatin reassembly. J Biol Chem. 2011;286:16790–9.View ArticlePubMedPubMed CentralGoogle Scholar

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