The effects of two mutations connected with chromatin functions on female germ-line cells of Drosophila

Summary We have studied the developmental effects of two dominant suppressor mutations of position-effect variegation mutations on female germ-line cells. Su-var (2) 1⁰¹, which has been shown to affect chromatin structure though altering histone deacetylation, and Su-var (3) 3⁰³are recessive female steriles and zygotic lethals in the presence of butyrate or an additional Y chromosome. We have analysed mosaic females with mutant germ-line and normal soma and concluded that intact functions of the Su-var (2) 1 and the Su-var (3) 3 genes are required for development of both the soma and the germ-line and that as indirect evidence suggest, their maternally provided products are needed for normal embryonic development. It is suggested that there is possibly a common control of chromatin structure and gene expression in the soma, female germ-line and embryonic cells of Drosophila.     

Carnitine suppression of position-effect variegation in Drosophila melanogaster

Carnitine is a well-known naturally occurring compound, very similar to butyrate, with an essential role in intermediary metabolism mainly at the mitochondrial level. Since butyrate inhibits the enzyme histone deacetylase and is capable of suppressing position-effect variegation in Drosophila melanogaster, we tested a further possible function of carnitine in the nucleus, using an assay for the suppression of position-effect variegation. We tested three physiological forms of carnitine (l-carnitine, l-propionylcarnitine, l-acetylcarnitine) for the ability to suppress two different chromosomal rearrangements, inducing variegation of the white ⁺ and brown ⁺ genes. The results show that the carnitine derivatives are capable of suppressing the position-effect variegation, albeit with different efficiencies. The carnitine derivatives interact lethally with Su-var(2)1 ⁰¹, a mutation that induces hyperacetylation of histones, whilst hyperacetylated histories accumulated in both the nuclei of HeLa cells and Drosophila polytene chromosomes treated with the same compounds. These results strongly suggest that the carnitine derivatives suppress position-effect variegation by a mechanism similar to that of butyrate. It is suggested that carnitines may have a functional role in the nucleus, probably at the chromatin level.     

Carnitine suppression of position-effect variegation in Drosophila melanogaster

Carnitine is a well-known naturally occurring compound, very similar to butyrate, with an essential role in intermediary metabolism mainly at the mitochondrial level. Since butyrate inhibits the enzyme histone deacetylase and is capable of suppressing position-effect variegation in Drosophila melanogaster, we tested a further possible function of carnitine in the nucleus, using an assay for the suppression of position-effect variegation. We tested three physiological forms of carnitine (l-carnitine, l-propionylcarnitine, l-acetylcarnitine) for the ability to suppress two different chromosomal rearrangements, inducing variegation of the white ⁺ and brown ⁺ genes. The results show that the carnitine derivatives are capable of suppressing the position-effect variegation, albeit with different efficiencies. The carnitine derivatives interact lethally with Su-var(2)1 ⁰¹, a mutation that induces hyperacetylation of histones, whilst hyperacetylated histories accumulated in both the nuclei of HeLa cells and Drosophila polytene chromosomes treated with the same compounds. These results strongly suggest that the carnitine derivatives suppress position-effect variegation by a mechanism similar to that of butyrate. It is suggested that carnitines may have a functional role in the nucleus, probably at the chromatin level.     

Modifiers of position-effect variegation in the region from 86C to 88B of the Drosophila melanogaster third chromosome

Summary Four dominant suppressor and one enhancer of variegation loci were mapped in the polytene chromosome region extending from section 86C to section 88B of the Drosophila melanogaster third chromosome using a set of deficiencies. The suppressor locus Su-var(3) 14 maps in 86CD, Su-var(3) 13 in 86F4-7, Su-var(3)6 in 87B4-7 and Su-var(3)7 in 87E4-5. The enhancer locus E-var(3)3 maps in 87E12-F11. Su-var(3)13, Su-var(3)6 and Su-var(3)7 are also defined by point mutant alleles originally identified by other criteria (Reuter et al. 1986). Duplications covering the suppressor loci Su-var(3)14, Su-var(3)13, Su-var(3)6 and Su-var(3)7 were found to reduce considerably the haplo-abnormal effect of heterozygous point mutants of the corresponding loci. One suppressor locus, Su-var(3)7, maps within a region which has previously been cloned. The positions of deficiency breakpoints delimiting the suppressor locus indicate that all the necessary sequences for its function are located within 10 kb of cloned DNA.     

Inhibition of histone H3-H4 chaperone pathways rescues C. elegans sterility by H2B loss

Oncohistone mutations are crucial drivers for tumorigenesis, but how a living organism governs the loss-of-function oncohistone remains unclear. We generated a histone H2B triple knockout (3KO) strain in Caenorhabditis elegans, which decreased the embryonic H2B, disrupted cell divisions, and caused animal sterility. By performing genetic suppressor screens, we uncovered that mutations defective in the histone H3-H4 chaperone UNC-85 restored H2B 3KO fertility by decreasing chromatin H3-H4 levels. RNA interference of other H3-H4 chaperones or H3 or H4 histones also rescued H2B 3KO sterility. We showed that blocking H3-H4 chaperones recovered cell division in C. elegans carrying the oncohistone H2B^E74K mutation that distorts the H2B-H4 interface and induces nucleosome instability. Our results indicate that reducing chromatin H3-H4 rescues the dysfunctional H2B in vivo and suggest that inhibiting H3-H4 chaperones may provide an effective therapeutic strategy for treating cancers resulting from loss-of-function H2B oncohistone.     

The multi zinc-finger protein Trps1 acts as a regulator of histone deacetylation during mitosis

TRPS1, the gene mutated in human "Tricho-Rhino-Phalangeal syndrome," encodes a multi zinc-finger nuclear regulator of chondrocyte proliferation and differentiation. Here, we have identified a new function of Trps1 in controlling mitotic progression in chondrocytes. Loss of Trps1 in mice leads to an increased proportion of cells arrested in mitosis and, subsequently, to chromosome segregation defects. Searching for the molecular basis of the defect, we found that Trps1 acts as regulator of histone deacetylation. Trps1 interacts with two histone deacetylases, Hdac1 and Hdac4, thereby increasing their activity. Loss of Trps1 results in histone H3 hyperacetylation, which is maintained during mitosis. Consequently, chromatin condensation and binding of HP1 is impaired, and Trps1-deficient chondrocytes accumulate in prometaphase. Overexpression of Hdac4 rescues the mitotic defect of Trps1-deficient chondrocytes, identifying Trps1 as an important regulator of chromatin deacetylation during mitosis in chondrocytes. Our data provide the first evidence that the control of mitosis can be linked to the regulation of chondrocyte differentiation by epigenetic consequences of altered Hdac activity.     

Hyperacetylation of histone H4 promotes chromatin decondensation prior to histone replacement by protamines during spermatogenesis in rainbow trout

During the final stages of spermatogenesis in rainbow trout a dramatic increase in the level of histone H4 hyperacetylation is observed which is closely correlated with the replacement of histones by protamines. In order to understand further how H4 hyperacetylation might assist in protamine replacement of the histones, we have investigated the effect of H4 hyperacetylation on chromatin structure in trout testes actively undergoing the replacement process. Long chromatin fragments enriched in hyperacetylated H4 have been isolated and characterized. Evidence is presented that hyperacetylated H4 is clustered in certain regions (domains) of late stage testis chromatin and within these domains the chromatin exhibits an altered, highly relaxed structure which is believed to be the result of the extensive hyperacetylation. These domains, which are nearly devoid of protamine, are postulated to represent an initial structural transition which is necessary for the proper histone removal and protamine replacement process to take place.     

Budding yeast CENP-ACse4 interacts with the N-terminus of Sgo1 and regulates its association with centromeric chromatin

Shugoshin is an evolutionarily conserved protein, which is involved in tension sensing on mitotic chromosomes, kinetochore biorientation, and protection of centromeric (CEN) cohesin for faithful chromosome segregation. Interaction of the C-terminus of Sgo1 with phosphorylated histone H2A regulates its association with CEN and pericentromeric (peri-CEN) chromatin, whereas mutations in histone H3 selectively compromise the association of Sgo1 with peri-CEN but not CEN chromatin. Given that histone H3 is absent from CEN and is replaced by a histone H3 variant CENP-A^Cse4, we investigated if CENP-A^Cse4 interacts with Sgo1 and promotes its association with the CEN chromatin. In this study, we found that Sgo1 interacts with CENP-A^Cse4 in vivo and in vitro. The N-terminus coiled-coil domain of Sgo1 without the C-terminus (sgo1-NT) is sufficient for its interaction with CENP-A^Cse4, association with CEN but not the peri-CEN, and this CEN association is cell cycle dependent with maximum enrichment in mitosis. In agreement with the role of CENP-A^Cse4 in CEN maintenance of Sgo1, depletion of CENP-A^Cse4 results in the loss of Sgo1 and sgo1-NT from the CEN chromatin. The N-terminus of Sgo1 is required for genome stability as a mutant lacking the N-terminus (sgo1-CT) exhibits increased chromosome missegregation when compared to a sgo1-NT mutant. In summary, our results define a novel role for the N-terminus of Sgo1 in CENP-A^Cse4 mediated recruitment of Sgo1 to CEN chromatin for faithful chromosome segregation.     

A lesson learned from the H3.3K27M mutation found in pediatric glioma

Glioblastoma (GBM) is the most aggressive primary brain tumor in human. Recent studies on high-grade pediatric GBM have identified two recurrent mutations (K27M and G34R/V) in genes encoding histone H3 (H3F3A for H3.3 and HIST1H3B for H3.1).^1,2 The two histone H3 mutations are mutually exclusive and give rise to tumors in different brain compartments.³ Recently, we⁴ and others⁵ have shown that the histone H3 K27M mutation specifically altered the di- and tri-methylation of endogenous histone H3 at Lys27. Genome-wide studies using ChIP-seq on H3.3K27M patient samples indicate a global reduction of H3K27me3 on chromatin. Remarkably, we also found a dramatic enrichment of H3K27me3 and EZH2 (the catalytic subunit H3K27 methyltransferase) at hundreds of gene loci in H3.3K27M patient cells. Here, we discuss potential mechanisms whereby H3K27me3 is enriched at chromatin loci in cells expressing the H3.3K27M mutation and report effects of Lys-to-Met mutations of other well-studied lysine residues of histone H3.1/H3.3 and H4 on the corresponding endogenous lysine methylation. We suggest that mutation(s) on histones may be found in a variety of human diseases, and the expression of mutant histones may help to address the function of histone lysine methylation and possibly other modifications in mammalian cells.     

Distinct histone modifications define initiation and repair of meiotic recombination in the mouse

Little is known about the factors determining the location and activity of the rapidly evolving meiotic crossover hotspots that shape genome diversity. Here, we show that several histone modifications are enriched at the active mouse Psmb9 hotspot, and we distinguish those marks that precede from those that follow hotspot recombinational activity. H3K4Me3, H3K4Me2 and H3K9Ac are specifically enriched in the chromatids that carry an active initiation site, and in the absence of DNA double-strand breaks (DSBs) in Spo11^−/− mice. We thus propose that these marks are part of the substrate for recombination initiation at the Psmb9 hotspot. In contrast, hyperacetylation of H4 is increased as a consequence of DSB formation, as shown by its dependency on Spo11 and by the enrichment detected on both recombining chromatids. In addition, the comparison with another hotspot, Hlx1, strongly suggests that H3K4Me3 and H4 hyperacetylation are common features of DSB formation and repair, respectively. Altogether, the chromatin signatures of the Psmb9 and Hlx1 hotspots provide a basis for understanding the distribution of meiotic recombination.     

A negatively charged residue in place of histone H3K56 supports chromatin assembly factor association but not genotoxic stress resistance

In fungal species, lysine 56 of newly synthesized histone H3 molecules is modified by the acetyltransferase Rtt109, which promotes resistance to genotoxic agents. To further explore how H3 K56ac contributes to genome stability, we conducted screens for suppressors of the DNA damage sensitivity of budding yeast rtt109Δ mutants. We recovered a single extragenic suppressor mutation that efficiently restored damage resistance. The suppressor is a point mutation in the histone H3 gene HHT2, and converts lysine 56 to glutamic acid. In some ways, K56E mimics K56ac, because it suppresses other mutations that interfere with the production of H3 K56ac and restores histone binding to chromatin assembly proteins CAF-1 and Rtt106. Therefore, we demonstrate that enhanced association with chromatin assembly factors can be accomplished not only by acetylation-mediated charge neutralization of H3K56 but also by the replacement of the positively charged lysine with an acidic residue. These data suggest that removal of the positive charge on lysine 56 is the functionally important consequence of H3K56 acetylation. Additionally, the suppressive function of K56E requires the presence of a second H3 allele, because K56E impairs growth when it is the sole source of histones, even more so than does constitutive H3K56 acetylation. Our studies therefore emphasize how H3 K56ac not only promotes chromatin assembly but also leads to chromosomal malfunction if not removed following histone deposition.     

Inhibition of histone deacetylases by Trichostatin A leads to a HoxB4-independent increase of hematopoietic progenitor/stem cell frequencies as a result of selective survival

BackgroundDNA and chromatin modifications are critical mediators in the establishment and maintenance of cell type-specific gene expression patterns that constitute cellular identities. One type of modification, the acetylation and deacetylation of histones, occurs reversibly on lysine ε-NH₃⁺ groups of core histones via histone acetyl transferases (HAT) and histone deacetylases (HDAC). Hyperacetylated histones are associated with active chromatin domains, whereas hypoacetylated histones are enriched in non-transcribed loci.MethodsWe analyzed global histone H4 acetylation and HDAC activity levels in mature lineage marker-positive (Lin⁺) and progenitor lineage marker-negative (Lin^?) hematopoietic cells from murine bone marrow (BM). In addition, we studied the effects of HDAC inhibition on hematopoietic progenitor/stem cell (HPSC) frequencies, cell survival, differentiation and HoxB4 dependence.ResultsWe observed that Lin^? and Lin⁺ cells do not differ in global histone H4 acetylation but in HDAC activity levels. Further, we saw that augmented histone acetylation achieved by transient Trichostatin A (TSA) treatment increased the frequency of cells with HPSC immunophenotype and function in the heterogeneous pool of BM cells. Induction of histone hyperacetylation in differentiated BM cells was detrimental, as evidenced by preferential death of mature BM cells upon HDAC inhibition. Finally, TSA treatment of BM cells from HoxB4^?/? mice revealed that the HDAC inhibitor-mediated increase in HPSC frequencies was independent of HoxB4.ConclusionsOverall, these data indicate the potential of chromatin modifications for the regulation of HPSC. Chromatin-modifying agents may provide potential strategies for ex vivo expansion of HPSC.     

Secretion of MIP-1β and MIP-1α by CD8(+) T-lymphocytes correlates with HIV-1 inhibition independent of coreceptor usage

CD8⁺ T-lymphocytes can utilize noncytolytic mechanisms to suppress HIV-1 replication through the secretion of soluble factors. The secretion of MIP-1β, MIP-1α, IP-10, MIG, IL-1α, and interferon gamma correlated most strongly with soluble noncytolytic suppression (p < 0.0001). Since the noncytolytic response is impaired by histone hyperacetylation, we examined the ability of histone hyperacetylation to alter the expression of immune-related genes. MIP-1α and IP-10 were also among the genes that were down-regulated by histone hyperacetylation. We define a multifactorial cytokine profile of CD8⁺ T-lymphocytes capable of mediating noncytolytic suppression of CXCR4-tropic HIV-1 replication.     

Influence of distamycin A on the methylation,extraction, and formation of UV-inducible DNA-protein cross links of histone H1 in the interphase nuclei of rat liver cells

The incubation in vitro of rat liver nuclei in the presence of S-adenosyl[methyl-³H]methionine ([³H] SAM) leads to the incorporation of a radioactive label not only into core histones H3 and H4, but also into linker histone H1. The addition of distamycin A to the incubation medium stimulates label incorporation into histone H1 by approximately six times and into histone H3 by around two times. The presence of distamycin facilitates histone H1 extraction by polyglutamic acid (poly(Glu)) and decreases UV-induced DNA—histone cross-link formation. These effects give evidence that the weakening H1—chromatin interaction by distamycin may be the result of a histone H1 position change relative toward the nucleosome and (or) a disturbance of the histone H1–H3 interactions, as these histones are exposed to additional methylation.