We performed the protein differential display approach in 2DGE with the H4-tail deleted mutant to identify the H4-tail-associated-proteins by MS and identified 22 candidate proteins, including the H4-tail binding proteins, Arp4p and Isw1p, and proteins with the SANT and WD40-repeat, which may be a histone tail-binding domain. We also provided in vivo evidence that one candidate, Pwp1p, associates with the rDNA chromatin in an H4 tail-dependent manner.
In previous reports, in order to identify protein complexes capable of binding specifically to histone H3 N-terminal tail, nuclear extracts were applied to affinity columns displaying either unmodified H3 tails or the same tails but differently methylated at specific residues. The NuRD complex binds specifically to unmodified H3 tails but not to K4 methylated tails (14
). Two proteins, INHAT complex subunits SET and pp32, bind specifically to unmodified H3 tails but not to phosphorylated tails at T3 (16
). hSNF2H and WDR5 were identified as H3 tail-binding proteins that associate with the methylated tail at K4 (15
). The 14-3-3 isoforms also bind to the phosphorylated H3-tail at S10 (18
). These protein identifications proved that the peptide affinity column is a powerful method to identify the tail-binding proteins. However, this approach is dependent on the preparation of a nuclear extract and the salt concentration of the binding buffer. Most importantly, the peptide conformation could be different from that in the chromatin context in vivo
. Thus, the screening may be restricted, and some proteins would not be identified. By a yeast two-hybrid screen employing yeast genomic libraries in combination with a bait plasmid expressing a fragment of histone H4 (amino acids 1–59), only two proteins, Bdf1p and Hif1p, were identified (54
). This indicates that the two-hybrid screen also has limitations. The authors suggested that a folding difference may exist between the full-length histone H4 and the portion of amino acids 1–59 (54
). Again, the tail in this assay may have a different conformation as compared to that in the chromatin context. In this regard, our approach seems to be more suitable than the others to identify tail-binding proteins. The nucleosome ladder was detected by MNase digestion in our H4-tail mutant, showing the maintenance of the nucleosome structure in the absence of H4-tails (50 and data not shown). Therefore, our screening reflects the H4-tail binding ability in vivo
We enriched chromatin proteins by the Mg2+-dependent oligomerization for an efficient identification of a histone-tail binding protein (). However, silver stained 2D gels were not sensitive enough to identify Sir3p, whose abundance was reduced in western blot (). Total mixture mass spectrometry is more sensitive than the silver stain. This method will help to identify less-abundant proteins.
Our screening is based on in vivo
binding activity. So a candidate protein may interact either directly or indirectly with an H4-tail. We identified Isw1p, whose fly homolog, ISWI, probably recognizes a DNA-bound H4-tail. ISWI does not interact stably with the GST–H4 tail fusion protein in the absence of DNA (32
). This type of a binding protein cannot be identified by a widely used approach such as the peptide affinity column.
Histone tails are involved in folding higher-order chromatin structures (2
). In the tail mutant strain, the chromatin structures may be altered at the higher-order folding level. The MNase digestion produced a nucleosomal ladder in the H4-tailΔ, and its MNase sensitivity was higher than that of the WT, suggesting a structural alteration beyond the nucleosomal level by the tail-deletion (50
). If there is a protein that recognizes the higher-order chromatin structure, then the amount of this protein would also be decreased in our screening. Such a protein may be included among our candidate proteins. We cannot examine this possibility at present, as there is no biochemical method to assess the folding of higher-order chromatin in vivo
The WD40-repeat is defined by a sequence repeat of ~40 amino acids, typically beginning with a glycine–histidine pair and ending with a tryptophan–aspartic acid pair. This motif is shared among over 30 functional families, which are involved in signal transduction, mRNA synthesis, RNA splicing, vascular trafficking, cytoskeletal assembly, control of transcription initiation complex assembly and a chromatin-regulated complex (56
). Among the chromatin-regulated proteins, several WD40-repeat proteins are known as histone-tail binding proteins. For example, the transcriptional repressor proteins Tup1p, Groucho and transducin beta-like protein (TBL1)/TBL1-related protein (TBLR1) associate with a histone-tail via a domain other than the WD40-repeat domain (57
). WDR5, a common component of three H3 K4 methyltransferase complexes (the mixed-lineage leukemia gene (MLL)1, MLL2 and hSet1), directly associates with a histone H3-tail di- and trimethylated at K4 via the WD40-repeat domain itself (17
). We plan to investigate whether the three candidates (Pwp1p, Tif34p, and YDL156Wp) interact with the H4-tail directly or indirectly.
rDNA transcription accounts for ~60% of the transcription in a rapidly growing yeast cell. However, only about half of the ~150 copies of the rDNA are active at any given time, whereas the remaining copies are maintained in a silenced state. This ratio of active to inactive genes is stably propagated throughout the cell cycle and is independent of the transcriptional activity of the cell. The regulatory mechanism that controls the ratio of active to inactive rDNA genes is poorly understood, but rDNA silencing is one of the factors that establish and maintain the transcriptionally inactive rDNA genes. We showed that Pwp1p associates with the rDNA chromatin, and its association is dependent on the H4-tail. The importance of the H4-tail for rDNA regulation was also suggested by studies of a mammalian nucleolar remodeling complex (NoRC) and a yeast regulator of nucleolar silencing and telophase exit (RENT) complex, which both regulate rDNA silencing. The NoRC induces nucleosome sliding in an ATP- and histone H4 tail-dependent fashion, and the NoRC-directed rDNA repression requires the histone H4-tail (60
). The RENT complex mediates the rDNA silencing and deacetylates the acetylated K16 of the H4-tail by Sir2p (52
). These studies raised the possibility that the H4-tail binding protein that associates with the rDNA chromatin performs a role similar to that of Sir3p in the silencing at telomeric and HM loci. Pwp1p may be the factor that links the RENT complex to the rDNA chromatin through the H4-tail.
The yTAP-C288 complex consists of eight subunits: Act1p, Eno2p, Fpr4p, Nan1p, Pol5p, Pwp1p, Smc1p and YPL207Wp (27
). Other subunits besides Pwp1p may also function in the rDNA regulation. For instance, Fpr4p binds to rDNA chromatin and regulates rDNA silencing (37
); Nan1p associates with the RENT complex (63
) and Pol5p is required for the synthesis of rRNA (65
). In addition, Smc1p is a member of a ubiquitous family of chromosome-associated ATPases and plays a role in chromosome dynamics (66
). Taken together, we propose that the yTAP-C228 complex associates with rDNA chromatin by the histone H4-tail and regulates rDNA transcription by modulating the chromatin structure.
We compared the Mg2+-dependent oligomerized chromatins isolated from H4 tail deletion cells and wild type cells by the protein differential display approach in 2DGE, and effectively identified histone-tail binding proteins. Other tail-deleted and point mutant cells are possible sources for the identification of tail-binding proteins for other core histones and specifically modified histones, respectively.