The DNA-binding region of RAG 1 is not a homeodomain

Functional annotation is used to catalog information that would be of value in experimental design and analysis but annotations in public databases are often incorrect. Here, one such case is discussed.     

H 1 histone and histone variants in Trypanosoma cruzi

Trypanosoma cruzi chromatin is not condensed in chromosomes during mitosis. In previous studies a characteristic H 1 was not found in SDS or in acid-urea-PAGE. Consequently, it was proposed that the particular behavior of T. cruzi chromatin in dividing cells was due to the absence of an H 1 histone. In the present work, histones from this parasite were systematically characterized by spectrofluorometric analysis, amino acid composition, PAGE in one and in two dimensions, differential extraction with PCA and TCA, immunological cross-reactivity with antisera, and immunoblotting. We conclude that T. cruzi contains all five histones, H 1 presenting solubility and immunological properties similar to those in other species, but with a particular electrophoretic mobility in Triton-PAGE. Thus an explanation other than the absence of H 1 should be offered in order to understand the behavior of T. cruzi chromatin during mitosis. Moreover, histone variants were described by two-dimensional PAGE. The presence of histone variants suggests that they may participate in the regulation of cell proliferation and differentiation of this parasite, as it has been postulated for higher eukaryotes.     

DNA-binding specificity of the S8 homeodomain.

The murine S8 homeobox gene is expressed in a mesenchyme-specific pattern in embryos, as well as in mesodermal cell lines. The S8 homeodomain is overall similar to paired type homeodomains, but at position 50, which is crucial for specific DNA recognition, it contains a Gln, as is found in Antennapedia (Antp)-type homeodomains. We determined the DNA-binding specificity of the purified S8 homeodomain by in vitro selection of random oligonucleotides. The resulting 11-bp consensus binding site, ANC/TC/TAATTAA/GC resembles, but subtly differs from, the recognition sequences of Antp-type homeodomains. Equilibrium binding constants of down to 6.0 x 10(-10) M were found for binding of the S8 homeodomain to selected oligonucleotides. Using specific antibodies and an oligonucleotide containing an S8-site, we detected by band-shift two abundant DNA binding activities in mesodermal cell lines that correspond to S8 and two more that correspond to its close relative MHox. These S8 protein forms are differentially expressed in retinoic acid-treated P19 EC cells.     

Organization and expression of H1 histone and H1 replacement histone genes

The H1 family is the most divergent subgroup of the highly conserved class of histone proteins [Cole: Int J Pept Protein Res 30:433–449, 1987]. In several vertebrate species, the H1 complement comprises five or more subtypes, and tissue specific patterns of H1 histones have been described. The diversity of the H1 histone family raises questions about the functions of different H1 subtypes and about the differential control of expression of their genes. The expression of main type H1 genes is coordinated with DNA replication, whereas the regulation of synthesis of replacement H1 subtypes, such as H1° and H5, and the testis specific H1t appears to be more complex. The differential control of H1 gene expression is reflected in the chromosomal organization of the genes and in different promoter structures. This review concentrates on a comparison of the chromosomal organization of main type and replacement H1 histone genes and on the differential regulation of their expression. General structural and functional data, which apply to both H1 and core histone genes and which are covered by recent reviews, will not be discussed in detail.     

The Yng1p plant homeodomain finger is a methyl-histone binding module that recognizes lysine 4-methylated histone H3

The ING (inhibitor of growth) protein family includes a group of homologous nuclear proteins that share a highly conserved plant homeodomain (PHD) finger domain at their carboxyl termini. Members of this family are found in multiprotein complexes that posttranslationally modify histones, suggesting that these proteins serve a general role in permitting various enzymatic activities to interact with nucleosomes. There are three members of the ING family in Saccharomyces cerevisiae: Yng1p, Yng2p, and Pho23p. Yng1p is a component of the NuA3 histone acetyltransferase complex and is required for the interaction of NuA3 with chromatin. To gain insight into the function of the ING proteins, we made use of a genetic strategy to identify genes required for the binding of Yng1p to histones. Using the toxicity of YNG1 overexpression as a tool, we showed that Yng1p interacts with the amino-terminal tail of histone H3 and that this interaction can be disrupted by loss of lysine 4 methylation within this tail. Additionally, we mapped the region of Yng1p required for overexpression of toxicity to the PHD finger, showed that this region capable of binding lysine 4-methylated histone H3 in vitro, and demonstrated that mutations of the PHD finger that abolish binding in vitro are no longer toxic in vivo. These results identify a novel function for the Yng1p PHD finger in promoting stabilization of the NuA3 complex at chromatin through recognition of histone H3 lysine 4 methylation.     

H1 histone variants in Xenopus laevis

The H1 histones from erythrocytes, livers, intestines, testes, and embryos of Xenopus laevis have been examined electrophoretically. This species has been found to contain at least five electrophoretically resolvable lysine-rich histones in addition to the presumptive H5 histone of erythrocytes. Quantitative and qualitative distinctions between the H1 histones from each source were readily observed. Three H1 histones (H1A, H1B, and H1C) were found in both embryos and adult tissues, although in varying amounts. Two other H1 histones (H1D and H1E) were found only in adult tissues. Comparative SDS gel V8 protease cleavage maps of the lysine-rich histones from testes and erythrocytes have demonstrated that the “adult-specific” H1D and H1E are not artifacts of proteolysis and may be closely related to the presumptive H5 histone. Spermatogenic cells were found to be similar to embryonic cells in being deficient in H1D and H1E. These observations suggest that H1D and H1E are enriched in cell types with low rates of cell division similar to the mammalian H1° histone. The results presented here demonstrate a previously unrecognized degree of developmental and cell-specific variance in the H1 histones of Xenopus laevis.     

Human histone acetyltransferase 1 (Hat1) acetylates lysine 5 of histone H2A in vivo

The primary structure of Histone Acetyltransferase 1 (Hat1) has been conserved throughout evolution; however, despite its ubiquity, its cellular function is not well characterized. To study its in vivo acetylation pattern and function, we utilized shRNAmir against Hat1 expressed in the well-substantiated HeLa (human cervical cancer) cell line. To reduce the interference by enzymes with similar HAT specificity, we used HeLa cells expressing histone acetyltransferase Tip60 with mutated acetyl-CoA binding site that abrogates its enzyme activity (mutant HeLa-tip60). Two shRNAmir were identified that reduced the expression of the cytoplasmic and nuclear forms of Hat1. Cytosolic protein preparations from these two clones showed decreased levels of acetylation of lysine 5 (K5) and K12 on histone H4, with the concomitant loss of the acetylation of histone H2A at K5. This pattern of decreased acetylation of H2AK5 was well defined in preparations of histone protein and insoluble nuclear-protein (INP) fractions as well. Abrogating the Hat1 expression caused a 74 % decrease in colony-forming efficiency of mutant HeLa-tip60 cells, reduced the size of the colonies by 50 %, and decreased the amounts of proteins with molecular weights below 35 kDa in the INP fractions.     

A helix-turn motif in the C-terminal domain of histone H1

The structural study of peptides belonging to the terminal domains of histone H1 can be considered as a step toward the understanding of the function of H1 in chromatin. The conformational properties of the peptide Ac-EPKRSVAFKKTKKEVKKVATPKK (CH-1), which belongs to the C-terminal domain of histone H1(o) (residues 99-121) and is adjacent to the central globular domain of the protein, were examined by means of 1H-NMR and circular dichroism. In aqueous solution, CH-1 behaved as a mainly unstructured peptide, although turn-like conformations in rapid equilibrium with the unfolded state could be present. Addition of trifluoroethanol resulted in a substantial increase of the helical content. The helical limits, as indicated by (i,i + 3) nuclear Overhauser effect (NOE) cross correlations and significant up-field conformational shifts of the C(alpha) protons, span from Pro100 to Val116, with Glu99 and Ala117 as N- and C-caps. A structure calculation performed on the basis of distance constraints derived from NOE cross peaks in 90% trifluoroethanol confirmed the helical structure of this region. The helical region has a marked amphipathic character, due to the location of all positively charged residues on one face of the helix and all the hydrophobic residues on the opposite face. The peptide has a TPKK motif at the C-terminus, following the alpha-helical region. The observed NOE connectivities suggest that the TPKK sequence adopts a type (I) beta-turn conformation, a sigma-turn conformation or a combination of both, in fast equilibrium with unfolded states. Sequences of the kind (S/T)P(K/R)(K/R) have been proposed as DNA binding motifs. The CH-1 peptide, thus, combines a positively charged amphipathic helix and a turn as potential DNA-binding motifs.     

Identification of two DNA-binding sites on the globular domain of histone H5.

The nature of the complexes of histones H1 and H5 and their globular domains (GH1 and GH5) with DNA suggested two DNA-binding sites which are likely to be the basis of the preference of H1 and H5 for the nucleosome, compared with free DNA. More recently the X-ray and NMR structures of GH5 and GH1, respectively, have identified two basic clusters on opposite sides of the domains as candidates for these sites. Removal of the positive charge at either location by mutagenesis impairs or abolishes the ability of GH5 to assemble cooperatively in ''tramline'' complexes containing two DNA duplexes, suggesting impairment or loss of its ability to bind two DNA duplexes. The mutant forms of GH5 also fail to protect the additional 20 bp of nucleosomal DNA that are characteristically protected by H1, H5 and wild-type recombinant GH5. They still bind to H1/H5-depleted chromatin, but evidently inappropriately. These results confirm the existence of, and identify the major components of, two DNA-binding sites on the globular domain of histone H5, and they strongly suggest that both binding sites are required to position the globular domain correctly on the nucleosome.     

A solitary human H3 histone gene on chromosome 1

A solitary histone H3 gene encoding a novel H3 protein sequence has been isolated. This H3 gene maps to chromosome 1 (1g42), whereas we have shown previously that the majority of the human histone genes form a large cluster on chromosome 6 (6p21.3). In addition, a small cluster has been described at 1q21. The clustered histone genes are expressed during the S-phase of the cell cycle, hence their definition as replication-dependent histone genes. In contrast, expression of replacement histone genes is essentially cell-cycle independent; they are solitary genes and map outside the major clusters. The newly described H3 gene maps outside all known histone gene clusters and varies by four amino acid residues from the consensus mammalian H3 structure. In contrast to other solitary histone genes, this human H3 gene shows the consensus promoter and 3 flanking portions that are typical for replication-dependent genes.