Epigenetic regulation by histone methylation and histone variants

Epigenetics is the study of heritable changes in gene expression that are not mediated at the DNA sequence level. Molecular mechanisms that mediate epigenetic regulation include DNA methylation and chromatin/histone modifications. With the identification of key histone-modifying enzymes, the biological functions of many histone posttranslational modifications are now beginning to be elucidated. Histone methylation, in particular, plays critical roles in many epigenetic phenomena. In this review, we provide an overview of recent findings that shape the current paradigms regarding the roles of histone methylation and histone variants in heterochromatin assembly and the maintenance of the boundaries between heterochromatin and euchromatin. We also highlight some of the enzymes that mediate histone methylation and discuss the stability and inheritance of this modification.     

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.     

Human histone demethylase LSD1 reads the histone code

Human histone demethylase LSD1 is a flavin-dependent amine oxidase that catalyzes the specific removal of methyl groups from mono- and dimethylated Lys4 of histone H3. The N-terminal tail of H3 is subject to various covalent modifications, and a fundamental question in LSD1 biology is how these epigenetic marks affect the demethylase activity. We show that LSD1 does not have a strong preference for mono- or dimethylated Lys4 of H3. Substrate recognition is not confined to the residues neighboring Lys4, but it requires a sufficiently long peptide segment consisting of the N-terminal 20 amino acids of H3. Electrostatic interactions are an important factor in protein-substrate recognition, as indicated by the high sensitivity of Km to ionic strength. We have probed LSD1 for its ability to demethylate Lys4 in presence of a second modification on the same peptide substrate. Methylation of Lys9 does not affect enzyme catalysis. Conversely, Lys9 acetylation causes an almost 6-fold increase in the Km value, whereas phosphorylation of Ser10 totally abolishes activity. LSD1 is inhibited by a demethylated peptide with an inhibition constant of 1.8 microM, suggesting that LSD1 can bind to H3 independently of Lys4 methylation. LSD1 is a chromatin-modifying enzyme, which is able to read different epigenetic marks on the histone N-terminal tail and can serve as a docking module for the stabilization of the associated corepressor complex(es) on chromatin.     

Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification

We report the identification of 67 previously undescribed histone modifications, increasing the current number of known histone marks by about 70%. We further investigated one of the marks, lysine crotonylation (Kcr), confirming that it represents an evolutionarily-conserved histone posttranslational modification. The unique structure and genomic localization of histone Kcr suggest that it is mechanistically and functionally different from histone lysine acetylation (Kac). Specifically, in both human somatic and mouse male germ cell genomes, histone Kcr marks either active promoters or potential enhancers. In male germinal cells immediately following meiosis, Kcr is enriched on sex chromosomes and specifically marks testis-specific genes, including a significant proportion of X-linked genes that escape sex chromosome inactivation in haploid cells. These results therefore dramatically extend the repertoire of histone PTM sites and designate Kcr as a specific mark of active sex chromosome-linked genes in postmeiotic male germ cells.     

Interplay between histone deacetylase SIR-2, linker histone H1 and histone methyltransferases in heterochromatin formation

Maintenance of intact heterochromatin structure through epigenetic mechanisms is essential for cell survival. Defects in heterochromatin formation caused by loss of chromatin-modifying enzymes lead to genomic instability and cellular senescence. The NAD+-dependent histone deacetylase SIR-2 and the H1 linker histone are intriguing chromatin elements that are connected to chromatin regulation and cell viability in the single cellular eukaryotic organism yeast. However, it remains an open question how SIR-2 and H1 mediate heterochromatin formation in simple multi-cellular organisms such as C. elegans and in even more complex organisms such as mammals. Recently we have identified SIR-2.1 and the H1 histone subtype, HIS-24 as factors involved in heterochromatin regulation at subtelomeric regions in C. elegans. In addition we show that SIR-2.1, HIS-24, and MES-2, a ortholog to Enhancer of zeste E(Z) are functionally related in heterochromatin formation contributing to fertility and embryogenesis. Here we discuss the interplay between SIR-2, H1 histone and histone methyltransferases in modulation of chromatin structure in further detail.     

Copper induces histone hypoacetylation through directly inhibiting histone acetyltransferase activity

The abnormal accumulation of Cu2+ is closely correlated with the incidence of different diseases, such as Alzheimer's disease and Wilson disease. To study in vivo functions of Cu2+ will lead to a better understanding of the nature of these diseases. In the present study, effect of Cu2+ on histone acetylation was investigated in human hepatoma cells. Exposure of cells to Cu2+ resulted in a significant decrease of histone acetylation, as indicated by the decrease of the overall histone acetylation and the decrease of histone H3 and H4 acetylation. Since histone acetyltransferase (HAT) and histone deacetylase (HDAC) are the enzymes controlled the state of histone acetylation in vivo, we tested their contribution to the inhibition of Cu2+ on histone acetylation. One hundred nanomolar trichostatin A, the specific inhibitor of HDAC, did not attenuate the inhibitory effect of Cu2+ on histone acetylation. Combined with that Cu2+ showed no effect on the in vitro activity of HDAC, these results led to the conclusion that it is HAT, but not HDAC that is involved in Cu2+ -induced histone hypoacetylation. This conclusion was confirmed by the facts that (1) Cu2+ significantly inhibited the in vitro activity of HAT, (2) Cu2+ -treated cells possessed a lower HAT activity than control cells, and (3) 50 or 100 microM bathocuproine disulfonate, a chelator of Cu2+, significantly attenuated the inhibition of Cu2+ on HAT activity and histone acetylation in the similar pattern. Combined with that Cu2+ showed no or obvious cytotoxicity at 100 or 200 microM in human hepatoma cells, and the previous study that Cu2+ inhibits the histone H4 acetylation of yeast cells at nontoxic or toxic levels, the data presented here suggest that inhibiting histone acetylation is probably one general in vivo function of Cu2+, where HAT is its molecular target.     

Mycardial histone acetylation

Myocardial histone acetylation was investigated in an isolated perfused heart preparation. Radioactive acetate rapidly accumulated in the intracellular compartment which preceded the covalent modification of histones. The acetylation of nucleohistones was rapid and reached a maximum during the first 20 min of perfusion. After 60 min of continuous perfusion there was a three-fold decrease in the amount of acetate bound to histone proteins. Histone H3 appeared to be preferred substrate for acetylation and was most responsive to a change in the concentration of radioactive acetate as well as the perfusion time.     

Cross-talk between histone modifications in response to histone deacetylase inhibitors: MLL4 links histone H3 acetylation and histone H3K4 methylation

Histones are subject to a wide variety of post-translational modifications that play a central role in gene activation and silencing. We have used histone modification-specific antibodies to demonstrate that two histone modifications involved in gene activation, histone H3 acetylation and H3 lysine 4 methylation, are functionally linked. This interaction, in which the extent of histone H3 acetylation determines both the abundance and the "degree" of H3K4 methylation, plays a major role in the epigenetic response to histone deacetylase inhibitors. A combination of in vivo knockdown experiments and in vitro methyltransferase assays shows that the abundance of H3K4 methylation is regulated by the activities of two opposing enzyme activities, the methyltransferase MLL4, which is stimulated by acetylated substrates, and a novel and as yet unidentified H3K4me3 demethylase.     

Core histone acetylation is regulated by linker histone stoichiometry in vivo

We investigated the relationship between linker histone stoichiometry and the acetylation of core histones in vivo. Exponentially growing cell lines induced to overproduce either of two H1 variants, H1(0) or H1c, displayed significantly reduced rates of incorporation of [(3)H]acetate into all four core histones. Pulse-chase experiments indicated that the rates of histone deacetylation were similar in all cell lines. These effects were also observed in nuclei isolated from these cells upon labeling with [(3)H]acetyl-CoA. Nuclear extracts prepared from control and H1-overexpressing cell lines displayed similar levels of histone acetylation activity on chromatin templates prepared from control cells. In contrast, extracts prepared from control cells were significantly less active on chromatin templates prepared from H1-overexpressing cells than on templates prepared from control cells. Reduced levels of acetylation in H1-overproducing cell lines do not appear to depend on higher order chromatin structure, because it persists even after digestion of the chromatin with micrococcal nuclease. The results suggest that alterations in chromatin structure, resulting from changes in linker histone stoichiometry may modulate the levels or rates of core histone acetylation in vivo.