Chromatin, the physiological template of all eukaryotic genetic information, is subject to a diverse array of posttranslational modifications that largely impinge on histone amino termini, thereby regulating access to the underlying DNA. Distinct histone amino-terminal modifications can generate synergistic or antagonistic interaction affinities for chromatin-associated proteins, which in turn dictate dynamic transitions between transcriptionally active or transcriptionally silent chromatin states. The combinatorial nature of histone amino-terminal modifications thus reveals a “histone code” that considerably extends the information potential of the genetic code. We propose that this epigenetic marking system represents a fundamental regulatory mechanism that has an impact on most, if not all, chromatin-templated processes, with far-reaching consequences for cell fate decisions and both normal and pathological development.

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Translating the Histone Code

Histone proteins and the nucleosomes they form with DNA are the fundamental building blocks of eukaryotic chromatin. A diverse array of post-translational modifications that often occur on tail domains of these proteins has been well documented. Although the function of these highly conserved modifications has remained elusive, converging biochemical and genetic evidence suggests functions in several chromatin-based processes. We propose that distinct histone modifications, on one or more tails, act sequentially or in combination to form a 'histone code' that is, read by other proteins to bring about distinct downstream events.

The language of covalent histone modifications.

'The amino termini of histones extend from the nucleosomal core and are modified by acetyltransferases and deacetylases during the cell cycle These acetylation patterns may direct histone assembly and help regulate the unfolding and activity of genes

Histone acetylation in chromatin structure and transcription

More than 30 years ago, Vincent Allfrey proposed that histone acetylation was associated with transcriptional activity in eukaryotic cells (Allfrey et al. 1964; Pogo et al. 1966). Subsequently, acetylated core histones were shown to preferentially associate with transcriptionally active chromatin (Sealy and Chalkley 1978; Vidali et al. 1978; Hebbes et al. 1988). Acetylation occurs at lysine residues on the amino-terminal tails of the histones, thereby neutralizing the positive charge of the histone tails and decreasing their affinity for DNA (Hong et al. 1993). As a consequence, histone acetylation alters nucleosomal conformation (Norton et al. 1989), which can increase the accessibility of transcriptional regulatory proteins to chromatin templates (Lee et al. 1993; Vettese-Dadey et al. 1996). Taken together, these observations suggested how histone acetylation could result in increased transcriptional activity in vivo. However, there was essentially no information about the cause and effect relationship between histone acetylation and transcriptional activity or about the underlying molecular mechanisms. A mechanistic and physiologically relevant connection between histone acetylation and transcriptional regulation was initially provided by two independent lines of evidence. First, yeast cells unable to acetylate the histone H4 tail because of mutations of the target lysine residues show altered patterns of transcription (Durrin et al. 1991). However, these mutations broadly affect chromatin structure in vivo, and hence are likely to influence other molecular processes involving DNA (e.g., DNA replication and repair, recombination, chromosome segregation). Second, treatment of mammalian cells with potent inhibitors of histone deacetylase activity such as trapoxin or trichostatin A resulted in increased expression of a variety of genes (Yoshida et al. 1995). However, these drugs might inhibit other cellular targets, and they affect a variety of cellular processes, including cell proliferation, apoptosis, differentiation, and DNA synthesis. Although these observations were suggestive, understanding of the relationship between chromatin structure and transcription regulation was hampered significantly by a lack of knowledge about the enzymes that acetylate and deacetylate histones. In the past 2 years, our understanding of the causal relationship between histone acetylation and gene expression has been enhanced dramatically by the identification of proteins with intrinsic histone acetylase and deacetylase activity (Brownell et al. 1996; for recent reviews, see Grunstein 1997; Pazin and Kadonaga 1997; Wade et al. 1997). Of particular significance, some of these enzymes had been identified previously as components of the RNA polymerase II (Pol II) transcription machinery itself, proteins that associate with transcriptional regulatory factors, or proteins that positively or negatively affect transcription in vivo. These discoveries have led to a major paradigm shift. It is now clear that chromatin structure and modification can not be viewed as a process that is independent of transcriptional initiation, that is, chromatin is not simply a structure that serves to compact DNA in the nucleus and provide a relatively passive substrate for the action of transcription factors. Instead, histone acetylases and deacetylases provide a critical link between chromatin structure and transcriptional output, and this link is now accessible to experimental intervention. This review will focus on molecular mechanisms by which histone acetylation affects transcriptional activity in living cells.

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Histone acetylation and transcriptional regulatory mechanisms

The state of chromatin (the packaging of DNA in eukaryotes) has long been recognized to have major effects on levels of gene expression, and numerous chromatin-altering strategies-including ATP-dependent remodeling and histone modification-are employed in the cell to bring about transcriptional regulation. Of these, histone acetylation is one of the best characterized, as recent years have seen the identification and further study of many histone acetyltransferase (HAT) proteins and their associated complexes. Interestingly, most of these proteins were previously shown to have coactivator or other transcription-related functions. Confirmed and putative HAT proteins have been identified from various organisms from yeast to humans, and they include Gcn5-related N-acetyltransferase (GNAT) superfamily members Gcn5, PCAF, Elp3, Hpa2, and Hat1: MYST proteins Sas2, Sas3, Esa1, MOF, Tip60, MOZ, MORF, and HBO1; global coactivators p300 and CREB-binding protein; nuclear receptor coactivators SRC-1, ACTR, and TIF2; TATA-binding protein-associated factor TAF(II)250 and its homologs; and subunits of RNA polymerase III general factor TFIIIC. The acetylation and transcriptional functions of these HATs and the native complexes containing them (such as yeast SAGA, NuA4, and possibly analogous human complexes) are discussed. In addition, some of these HATs are also known to modify certain nonhistone transcription-related proteins, including high-mobility-group chromatin proteins, activators such as p53, coactivators, and general factors. Thus, we also detail these known factor acetyltransferase (FAT) substrates and the demonstrated or potential roles of their acetylation in transcriptional processes.

Acetylation of Histones and Transcription-Related Factors

Histone H4 isoforms acetylated at specific lysine residues define individual chromosomes and chromatin domains in Drosophila polytene nuclei.

Objective. To test the hypothesis that genetic characterization of patients at the time they present with inflammatory arthritis can predict subsequent destructive disease.



Methods. We evaluated 177 patients with early arthritis. Patients were serologically tested for rheumatoid factor (RF) and were DNA oligotyped for the presence of conserved base sequences in the third hypervariable region (HVR3) of the DRB1 gene, previously shown to be associated with severe rheumatoid arthritis (RA). Homozygosity in the patient's genotype was confirmed using restriction fragment length polymorphism analysis. The main outcome measure was radiologic erosions at 1 year.



Results. At presentation, 120 patients fulfilled the American College of Rheumatology 1987 criteria for RA, 64% of whom possessed the conserved base sequences, compared with 45% of 347 healthy controls (P < 0.001) and with 56% of 57 patients with other inflammatory arthritis (P not significant). Within the RA population, the frequency of Dw4/Dw14 compound heterozygotes was disproportionately increased. The presence of either HVR3 or RF had a relative risk of 13.49 for erosions, with a sensitivity of 95% (specificity 39%); the presence of both HVR3 and RF had a relative risk of 8.13, with a specificity of 88% (sensitivity 53%). All but 1 patient with the Dw4/Dw14 genotype developed erosions within 1 year.



Conclusion. Knowledge of a patient's HLA—DR type and RF status allows clinically useful prediction of erosive disease; patients possessing Dw4/Dw14 represent a particularly high-risk subgroup.

Genetic typing of patients with inflammatory arthritis at presentation can be used to predict outcome.

Two behavioral rhythm phenotypes, oviposition and locomotor activity, have been compared in the four period genotypes (per+, pers, per°, and per1) of Drosophila. Period, signal-to-noise ratio, and phase were all analyzed, and the genetic penetrance of the two characters was estimated. Significant rhythmicity of both oviposition and locomotor activity was evident in all four genotypes. The entrained and free-running periods of the activity rhythms of per+, pers, and per1 were within the range reported for these flies by previous workers, and rhythmic behavior was also shown by the per° flies. The free-running period of the oviposition rhythm varied similarly between the four genotypes and showed significant correlation with that of the locomotor activity rhythm. It is suggested that both rhythm phenotypes are determined by die period gene, and estimates of the genetic penetrance of rhydimicity in oviposition and locomotor activity, based on period and signal-to-noise ratios (SNRs) of die different strains,...

Oviposition in the Period Genotypes of Drosophila Melanogaster

In the fruit flyDrosophila, dosage compensation involves several proteins acting in concert to double the transcriptional activity of genes on the single male X chromosome. Three of these proteins, MLE, MSL-1 and histone H4 acetylated at lysine 16 (H4Ac16), have recently been shown to be located almost exclusively on the male X chromosome in interphase (polytene) cells. We show here that in neuroblasts from third instarDrosophila larvae antisera to H4Ac16, MLE and MSL-1 uniquely label the distal, euchromatic region of the male X chromosome through mitosis. The centromere-proximal, heterochromatic region of the male X is not labelled with these antisera, nor are male autosomes or any chromosomes in female cells. That the association of H4Ac16 with the male X chromosome persists, even when the chromosome is maximally compacted and transcriptionally quiescent, argues that this modified histone is an integral component of the dosage compensation pathway. In the nuclei of interphase neuroblasts from male (but never female) larvae, antibodies to H4Ac16 revealed a small, brightly labelled patch against a background of generally weak nuclear staining. In double-labelling experiments, this patch was also labelled, albeit comparatively weakly, with antibodies to MSL-1. These results strongly suggest that the distal, euchromatic region of the X chromosome in male cells occupies a limited and relatively compact nuclear domain.

Histone H4 acetylated at lysine 16 and proteins of theDrosophila dosage compensation pathway co-localize on the male X chromosome through mitosis

An intragenic elastin Hinf I polymorphism has been used to study the inheritance of elastin alleles in a family considered to show recessive inheritance of pseudoxanthoma elasticum (PXE). The marker has proved informative, excluding the elastin gene as a cause of PXE in this family. In addition, whole genomic human elastin clones were used in Southern analysis to screen the family for gross elastin gene rearrangements, but none were detected.

A. J. Birley

Papers

Histone H4 isoforms acetylated at specific lysine residues define individual chromosomes and chromatin domains in Drosophila polytene nuclei.

Genetic typing of patients with inflammatory arthritis at presentation can be used to predict outcome.

Oviposition in the Period Genotypes of Drosophila Melanogaster

Histone H4 acetylated at lysine 16 and proteins of theDrosophila dosage compensation pathway co-localize on the male X chromosome through mitosis

Exclusion of an elastin gene (ELN) mutation as the cause of pseudoxanthoma elasticum (PXE) in one family.