A basic domain in the histone H2B N-terminal tail is important for nucleosome assembly by FACT.

Nucleosome assembly in vivo requires assembly factors, such as histone chaperones, to bind to histones and mediate their deposition onto DNA. In yeast, the essential histone chaperone FACT (FAcilitates Chromatin Transcription) functions in nucleosome assembly and H2A-H2B deposition during transcription elongation and DNA replication. Recent studies have identified candidate histone residues that mediate FACT binding to histones, but it is not known which histone residues are important for FACT to deposit histones onto DNA during nucleosome assembly. In this study, we report that the histone H2B repression (HBR) domain within the H2B N-terminal tail is important for histone deposition by FACT. Deletion of the HBR domain causes significant defects in histone occupancy in the yeast genome, particularly at HBR-repressed genes, and a pronounced increase in H2A-H2B dimers that remain bound to FACT in vivo Moreover, the HBR domain is required for purified FACT to efficiently assemble recombinant nucleosomes in vitro We propose that the interaction between the highly basic HBR domain and DNA plays an important role in stabilizing the nascent nucleosome during the process of histone H2A-H2B deposition by FACT.

Ascending the nucleosome face: recognition and function of structured domains in the histone H2A-H2B dimer.

Research over the past decade has greatly expanded our understanding of the nucleosome's role as a dynamic hub that is specifically recognized by many regulatory proteins involved in transcription, silencing, replication, repair, and chromosome segregation. While many of these nucleosome interactions are mediated by post-translational modifications in the disordered histone tails, it is becoming increasingly apparent that structured regions of the nucleosome, including the histone fold domains, are also recognized by numerous regulatory proteins. This review will focus on the recognition of structured domains in the histone H2A-H2B dimer, including the acidic patch, the H2A docking domain, the H2B α3-αC helices, and the HAR/HBR domains, and will survey the known biological functions of histone residues within these domains. Novel post-translational modifications and trans-histone regulatory pathways involving structured regions of the H2A-H2B dimer will be highlighted, along with the role of intrinsic disorder in the recognition of structured nucleosome regions.

Association of microinjected myosin and its subfragments with myofibrils in living muscle cells.

Purified skeletal muscle myosin was labeled with iodoacetamidofluorescein and microinjected into cultured chick myotubes. The fluorescent myosin analogue became incorporated within 10-15 min after injection, into either periodic (mean periodicity = 2.23 +/- 0.02 micron) bands or apparently continuous fibrillar structures. Comparison of rhodamine-labeled alpha-actinin with coinjected fluorescein-labeled myosin suggested that myosin fluorescence was localized at the A-bands of myofibrils. In addition, close examination of the fluorescent myosin bands indicated that they were composed of two fluorescent bars separated by a nonfluorescent line that corresponded to the H-zone. Once incorporated, the myosin underwent a relatively slow exchange along myofibrils as indicated by fluorescence recovery after photobleaching. Glycerinated myofibrils were able to bind fluorescent myosin in a similar pattern in the presence or absence of MgATP, indicating that actin-myosin interactions had little effect on this process. Fluorescent heavy meromyosin did not incorporate into myofibrillar structures after injection. Light meromyosin, however, associated with A-bands as did whole myosin. These results suggest that microinjected myosin, even with its relatively low solubility under the cytoplasmic ionic condition, is capable of association with physiological structures in living muscle cells. Additionally, the light meromyosin portion of the molecule appears to be mainly responsible for the incorporation.

Formation and alignment of Z lines in living chick myotubes microinjected with rhodamine-labeled alpha-actinin.

We have used fluorescence analogue cytochemistry in conjunction with time lapse recording to study the dynamics of alpha-actinin, a major component of the Z line, during myofibrillogenesis. Rhodamine-labeled alpha-actinin microinjected into living cultured chick skeletal myotubes became localized in discrete cellular structures within 1 h and remained specifically associated with structures for up to 4 d, allowing individual identified structures to be followed during development. In the most immature cells used, alpha-actinin was found in diffuse aggregates, some of which displayed sarcomeric periodicity. Aggregates were observed to coalesce into better defined structures (Z bands) that were approximately 1.0-micron wide. Z bands condensed into narrow, more intensely fluorescent Z lines in 4-48 h. During this period, Z lines grew laterally, primarily by the addition of small beads of alpha-actinin to existing Z lines or by the merging of small Z lines. In more mature cells, alpha-actinin added to Z lines without going through a visible intermediary structure. Mean sarcomere length did not change significantly during the stages examined, although the variability of sarcomere length did decrease markedly over time for identified sets of sarcomeres. At early stages, myofibrils frequently shifted position in both the longitudinal and lateral directions. Neighboring myofibrils were frequently associated for one or more sarcomeres sporadically along their length, such that the intervening sarcomeres were often misaligned. Associations between myofibrils were often transitory. Shifts in myofibril location in conjunction with the formation, breaking, and reformation of lateral associations between myofibrils facilitated the alignment of Z lines through a trial and error process.

Formation and movement of myosin-containing structures in living fibroblasts.

Gizzard myosin, fluorescently labeled with tetramethylrhodamine iodoacetamide, was microinjected into living 3T3 fibroblasts to label myosin-containing structures. The fluorophore was located predominantly on the heavy chain near the COOH terminus of the S1 head and on the 17-kD light chain. After microinjection of a tracer amount into living 3T3 cells, the fluorescent myosin showed a distribution identical to that revealed by immunofluorescence with antimyosin antibodies. Injected myosin became localized in small beads, which were found along large stress fibers, along fine fibers, and in a poorly organized form near the lamellipodia. De novo assembly of beads was observed continuously within or near the lamellipodia, suggesting that myosin molecules may undergo a constant cycling between polymerized and unpolymerized states. The nascent structures then moved away from lamellipodia and became organized into linear arrays. Similar movement was also observed for beads already associated with linear structures, and may represent a continuous flux of myosin structures. The dynamic reorganization of myosin may play an important role in cell movement and polarity.

Exchangeability of alpha-actinin in living cardiac fibroblasts and muscle cells.

We have investigated the exchangeability of alpha-actinin in various structures of cultured chick cardiac fibroblasts and muscle cells using fluorescent analogue cytochemistry in combination with fluorescence recovery after photobleaching. Living cells were microinjected with tetramethylrhodamine-labeled alpha-actinin, which became localized in cellular structures. Small areas of labeled structures were then photobleached with a laser pulse, and the subsequent recovery of fluorescence was monitored with an image intensifier coupled to an image-processing system. In fibroblasts, fluorescence recovery was studied in stress fibers and in adhesion plaques. Bleached spots in adhesion plaques generally attained complete recovery within 20 min; whereas complete recovery in stress fibers occurred within 30 to 60 min. In muscle cells, alpha-actinin became localized in the Z-lines of sarcomeres, in punctate structures, and in apparently continuous bundle-like structures. Fluorescence recovery in Z-lines, punctate structures, and some bundle-like structures was extremely slow. Complete recovery did not occur within the 6- to 7-h observation period. However, some bundle-like structures recovered completely within 60 min, a rate similar to that of stress fibers in fibroblasts. These results indicate that fluorescently labeled alpha-actinin is more stably associated with structures in muscle cells than in fibroblasts. In addition, different structures within the same cell can display different alpha-actinin exchangeabilities which, in muscle cells, could be developmentally related.

Novel functional residues in the core domain of histone H2B regulate yeast gene expression and silencing and affect the response to DNA damage.

Previous studies have identified novel modifications in the core fold domain of histone H2B, but relatively little is known about the function of these putative histone modification sites. We have mutated core modifiable residues that are conserved in Saccharomyces cerevisiae histone H2B and characterized the effects of the mutants on yeast silencing, gene expression, and the DNA damage response. We identified three histone H2B core modifiable residues as functionally important. We find that mutating H2B K49 in yeast confers a UV sensitivity phenotype, and we confirm that the homologous residue in human histone H2B is acetylated and methylated in human cells. Our results also indicate that mutating H2B K111 impairs the response to methyl methanesulfonate (MMS)-induced DNA lesions and disrupts telomeric silencing and Sir4 binding. In contrast, mutating H2B R102 enhances silencing at yeast telomeres and the HML silent mating loci and increases Sir4 binding to these regions. The H2B R102A mutant also represses the expression of endogenous genes adjacent to yeast telomeres, which is likely due to the ectopic spreading of the Sir complex in this mutant strain. We propose a structural model by which H2B R102 and K111 regulate the binding of the Sir complex to the nucleosome.

Possible translocation of actin and alpha-actinin along stress fibers.

We have employed fluorescent analogue cytochemistry and fluorescence photobleaching to study the mobility of actin and alpha-actin along stress fibers. Rhodamine-labeled actin or alpha-actinin microinjected into embryonic chick cardiac fibroblasts soon became incorporated into stress fibers. A pulse of a laser microbeam was used to photobleach small spots on the fluorescent stress fibers. Images of the bleached fiber were recorded with an intensified image processing system at 2-3 min intervals. The distance between the bleached spot and the terminus of the stress fiber, which remained stationary throughout the experiment, was then measured in the successive images. Movement of bleached spots was detected along stress fibers located in the apparently trailing processes of polygonal fibroblasts, and only occurred in one direction: away from the distal tip of the stress fiber. The rate of movement calculated for alpha-actinin-injected cells was 0.24 +/- 0.12 micron/min, for actin-injected cells, 0.29 +/- 0.11 micron/min. The rate did not seem to be affected by the location of the spot relative to the distal end of the stress fiber unless the spot was located within the most distal 5 microns of the stress fiber. Anti-myosin antibody staining indicated that stress fibers which demonstrated translocation were relatively depleted of myosin. The apparent translocation of proteins along stress fibers, possibly generated by stretching, may be related to the retraction of cell processes during locomotion.

Alternatively spliced sodium channel transcripts in brain and muscle.

Sodium (Na) channel cDNAs were synthesized from RNA isolated from rat brain, cardiac muscle, and skeletal muscle. Partial cDNAs coding for the largest cytoplasmic loop of the Na channel were amplified with PCR. Sequence analysis of these cDNAs revealed that Na channel cDNAs originally described as brain genes were also expressed in both cardiac and skeletal muscle. Some of these cDNAs were isoforms that differed by insertions or deletions and can be explained by alternative choices of a 5' splice site. Southern blot analysis of genomic DNA confirmed the presence of introns in this region of the gene. Transcripts of multiple isoforms were detected with RNase protection in brain, heart, and skeletal muscle. Several conclusions can be drawn from the data. (1) Some rat sodium channel genes are transcribed in all excitable tissues studied here: brain, cardiac muscle, and skeletal muscle. (2) Each of these three tissues expresses multiple sodium channel genes. (3) Alternative splicing of sodium channel transcripts occurs in these tissues. (4) Expression of multiple genes and alternative splicing of the transcripts is responsible for at least seven different sodium channel mRNAs in skeletal muscle.