Barbara A. Hamkalo

A unified phylogeny-based nomenclature for histone variants

Histone variants are non-allelic protein isoforms that play key roles in diversifying chromatin structure. The known number of such variants has greatly increased in recent years, but the lack of naming conventions for them has led to a variety of naming styles, multiple synonyms and misleading homographs that obscure variant relationships and complicate database searches. We propose here a unified nomenclature for variants of all five classes of histones that uses consistent but flexible naming conventions to produce names that are informative and readily searchable. The nomenclature builds on historical usage and incorporates phylogenetic relationships, which are strong predictors of structure and function. A key feature is the consistent use of punctuation to represent phylogenetic divergence, making explicit the relationships among variant subtypes that have previously been implicit or unclear. We recommend that by default new histone variants be named with organism-specific paralog-number suffixes that lack phylogenetic implication, while letter suffixes be reserved for structurally distinct clades of variants. For clarity and searchability, we encourage the use of descriptors that are separate from the phylogeny-based variant name to indicate developmental and other properties of variants that may be independent of structure.

Curvature of mouse satellite DNA and condensation of heterochromatin

Cloned, sequenced mouse satellite DNA exhibits properties characteristic of molecules that possess a stable curvature. Circularly permuted fragments containing the region predicted to bend were used to map the curvature relative to DNA sequence. The altered mobility of these fragments in polyacrylamide gels is reversed when gels are run in the presence of distamycin A, a drug that binds preferentially to AT-rich DNA. Treatment of living mouse cells with this drug dramatically reduces the condensation of centromeric heterochromatin, the exclusive location of satellite sequences. In situ hybridization of satellite probes to extended chromosomes at the electron microscope level shows that satellite does not comprise a single block but is distributed throughout the centromere region. Based on these experiments, we hypothesize that the structure of mouse satellite DNA is an important feature of centromeric heterochromatin condensation.

Chromatin Condensing Functions of the Linker Histone C-terminal Domain are mediated by Specific Amino Acid Composition and Intrinsic Protein Disorder †

Linker histones bind to the nucleosomes and linker DNA of chromatin fibers, causing changes in linker DNA structure and stabilization of higher order folded and oligomeric chromatin structures. Linker histones affect chromatin structure acting primarily through their approximately 100-residue C-terminal domain (CTD). We have previously shown that the ability of the linker histone H1 degrees to alter chromatin structure was localized to two discontinuous 24-/25-residue CTD regions (Lu, X., and Hansen, J. C. (2004) J. Biol. Chem. 279, 8701-8707). To determine the biochemical basis for these results, we have characterized chromatin model systems assembled with endogenous mouse somatic H1 isoforms or recombinant H1 degrees CTD mutants in which the primary sequence has been scrambled, the amino acid composition mutated, or the location of various CTD regions swapped. Our results indicate that specific amino acid composition plays a fundamental role in molecular recognition and function by the H1 CTD. Additionally, these experiments support a new molecular model for CTD function and provide a biochemical basis for the redundancy observed in H1 isoform knockout experiments in vivo.

Electron microscopy of whole mount metaphase chromosomes

Whole mount metaphase chromosomes, from cultured L cells, have been centrifuged onto grids and examined by electron microscopy. Compact and dispersed chromosome forms provide extensive ultrastructural information. Condensed chromosome arms appear as packed fibers with centromeric heterochromatin identifiable because it stains more intensely than the rest of the chromosome. Kinetochores are readily visible in these preparations. Under appropriate isolation conditions, it is possible to obtain mitotic spindles in which bundles of microtubules remain attached to kinetochores, suggesting that the kinetochores retain basic structural integrity throughout the isolation procedure. Dispersal of metaphase chromosomes by treatment with formalin and distilled water shows that these chromosomes are composed of a basic fiber that is normally highly condensed. This fiber is made up of regularly repeating 70–90 Å diameter nucleoprotein granules separated from neighboring granules by a 20–40 Å diameter fiber whose continuity is maintained by DNA. This structural arrangement is totally analagous to that reported for interphase chromatin from a variety of sources.

Hoechst 33258, distamycin A, and high mobility group protein I (HMG-I) compete for binding to mouse satellite DNA

The experiments described were designed to test the hypothesis that the (A+T)-specific DNA binding ligands Hoechst 33258 and distamycin A affect the condensation of mouse centromeric heterochromatin by competing for binding to satellite DNA with one or more chromosomal proteins. The studies focused on the nonhistone chromosomal protein HMG-I since its binding properties predict it would be a target for competition. Gel mobility shift assays show that HMG-I forms specific complexes with satellite DNA and that the formation of these complexes is competed for by both Hoechst and distamycin. In addition, methidium propyl EDTA Fe(II) [MPE Fe(II)] footprints of ligand-satellite DNA complexes showed essentially the same protection pattern for both drugs and a similar, but not identical, HMG-I footprint. If these in vitro results reflect the in vivo situation then the incomplete condensation of centromeric heterochromatin observed when mouse cells are grown in the presence of either chemical ligand could be a consequence of competition for binding of HMG-I (and possibly other proteins) to satellite DNA.

Preparation of centromeric heterochromatin by restriction endonuclease digestion of mouse L929 cells

When L929 cells in metaphase are digested with either Eco RI or Alu I, chromatin containing about 85% of the DNA is released. DNA from the Alu I- and Eco RI-resistant chromatin is enriched 6.8- and 3.7-fold, respectively, in satellite sequences. Analysis by electron microscopy of these digests reveals the existence of structures containing condensed heterochromatin and kinetochores. When these preparations are incubated with anticentromere serum from a human CREST scleroderma patient and then with rhodamine-conjugated antihuman IgG, fluorescence appears in the form of paired dots, the same pattern found in whole metaphase chromosomes. The fluorescent staining pattern, the electron microscopy, and the enrichment of satellite DNA sequences together support the conclusion that the Eco RI- and Alu I-resistant structures contain centromeres. We anticipate that these preparations will be useful in studies of the interactions between centromeric heterochromatin, kinetochores, and microtubules.

Characterization of a highly repetitive sequence DNA family in rat

Abstract A 92 to 93 base-pair highly reiterated DNA from rat liver has been isolated by digestion with Eco RI and cloned in pBR322. Three recombinant plasmids have been studied in detail; these are p93-2 and p93-10, both of which contain two 93 base-pair inserts, and p93-15, which contains one 92 base-pair and two 93 base-pair inserts. Analysis of these seven cloned DNA fragments reveals that 93 base-pair highly repetitive DNA exhibits as much as 50% overall sequence heterogeneity although several families can be identified on the basis of a high degree of sequence homology among particular cloned inserts. Individual families have also been identified on the basis of specific restriction endonuclease sites. This property, coupled with a limited degree of cross-hybridization among the groups of inserts in filter hybridizations, has been utilized to demonstrate that the three 93(2) base-pair families identified here are repeated in a regularly interspersed, tandem manner with each family repeated no more frequently than once every fourth sequence. Similar conclusions have been reached by analysis of a specific fraction of 370 base-pair rat repeated DNA (Pech et al. , 1979 a ). The chromosomal location of these 93 base-pair repeated sequences has been determined by in situ hybridization. They are found primarily, though not exclusively, at centromeres and telomeres of metaphase chromosomes. Highly reiterated 93 base-pair DNA is transcribed in HTC cells. Large RNA transcripts containing sequences homologous to 93 base-pair DNA have been identified in HTC cell nuclei utilizing the RNA-DNA filter hybridization technology of Alwine et al. , (1977).

Distribution of somatic H1 subtypes is non‐random on active vs. inactive chromatin II: Distribution in human adult fibroblasts

For nearly twenty years researchers have observed changes in the histone H1 subtype content of tissues as an organism develops into an adult. To better understand the consequences of such changes, immunofractionation of chromatin using previously characterized antibodies specific for human H1 subtypes was employed in the analysis of a fibroblast cell strain derived from a 37‐year‐old individual. DNAs isolated from immunoprecipitates were probed for the existence of a variety of DNA sequences. The results presented lend further support to a previously‐proposed model (Parseghian et al. [ 2000 ] Chromosome Res 8:405‐424) in which transcription of a sequence is accompanied by the selective depletion of subtypes. The data also suggest that there is more total H1 on actively transcribed sequences in these cells as compared to fetal fibroblasts and that there is less difference in the subtype compositions of active genes vs. inactive sequences in this strain. Specifically, the consequences of these changes appear to correlate with the attenuation of the heat shock response in aging fibroblasts. In a broader context, these results could explain why there are reductions in transcription in cells from mature tissue that approach senescence. J. Cell. Biochem. 83: 643–659, 2001.

Chromatin organization during meiotic prophase ofBombyx mori

Chromatin organization during the early stages of male meiotic prophase inBombyx mori was investigated by electron microscopy. The analysis of nuclei prepared by the Miller spreading procedure, suggests that chromatin fibers which are 200–300 Å in diameter undergo an orderly folding coincident with the formation of the synaptonemal complex. In very early stages the chromatin is released in linear arrays typical of interphase chromatin material. With time loops containing 5–25 μ of B conformation DNA, initially visualized at the periphery of early meiotic prophase nuclei, aggregate into discrete foci. These foci coalesce to form the longitudinal axis of the chromosome in conjunction with the initial appearance of the axial elements of the synaptonemal complex. At pachytene, the loops are evenly distributed along the length of the chromosome and extend radially so that in well spread preparations the chromosome has a brush-like appearance. Throughout this period nascent RNP-fibers were visualized along some of the loops.

Fractionation of human H1 subtypes and characterization of a subtype-specific antibody exhibiting non-uniform nuclear staining

Four histone H1 subtypes and H1° were fractionated from human placental nuclei and purified to homogeneity by a combination of Bio-Rex 70 chromatography and reversephase high-performance liquid chromatography (RPHPLC). Polyclonal antibodies were generated in rabbits against one of these subtypes designated H1-3. Antibodies reacted only against this subtype in enzymelinked immunosorbent assays and Western assays; subtype specificity was documented further by Western blotting of cell and nuclear extracts. They crossreacted with monkey H1, but not with H1 from other vertebrates tested. The epitope(s) recognized were mapped by immunoblotting against peptides prepared by cleavage with N-bromosuccinimide (NBS) and α-chymotrypsin; it includes the variant amino-terminal tail of the protein as well as a portion of the globular domain. The antibody stains mitotic chromosomes weakly but uniformly and, unlike antibodies that recognize total H1 which show uniform nuclear staining after indirect immunofluoresence localization, anti-H1-3 exhibits preferential labelling of the nuclear periphery. This non-uniform staining suggests compartmentalization of this subtype which may have functional significance with respect to differential chromatin condensation.