Heterochromatin formation in Drosophila requires genome-wide histone deacetylation in cleavage chromatin before mid-blastula transition in early embryogenesis.

Su(var) mutations define epigenetic factors controlling heterochromatin formation and gene silencing in Drosophila. Here, we identify SU(VAR)2-1 as a novel chromatin regulator that directs global histone deacetylation during the transition of cleavage chromatin into somatic blastoderm chromatin in early embryogenesis. SU(VAR)2-1 is heterochromatin-associated in blastoderm nuclei but not in later stages of development. In larval polytene chromosomes, SU(VAR)2-1 is a band-specific protein. SU(VAR)2-1 directs global histone deacetylation by recruiting the histone deacetylase RPD3. In Su(var)2-1 mutants H3K9, H3K27, H4K8 and H4K16 acetylation shows elevated levels genome-wide and heterochromatin displays aberrant histone hyper-acetylation. Whereas H3K9me2- and HP1a-binding appears unaltered, the heterochromatin-specific H3K9me2S10ph composite mark is impaired in heterochromatic chromocenters of larval salivary polytene chromosomes. SU(VAR)2-1 contains an NRF1/EWG domain and a C2HC zinc-finger motif. Our study identifies SU(VAR)2-1 as a dosage-dependent, heterochromatin-initiating SU(VAR) factor, where the SU(VAR)2-1-mediated control of genome-wide histone deacetylation after cleavage and before mid-blastula transition (pre-MBT) is required to enable heterochromatin formation.

The SUUR protein is involved in binding of SU(VAR)3-9 and methylation of H3K9 and H3K27 in chromosomes of Drosophila melanogaster.

In the present work, we found that the SUUR protein is required for the SU(VAR)3-9 enzyme to bind to the salivary gland polytene chromosomes. The SuUR mutation results in loss of SU(VAR)3-9 on the chromosomes, whereas artificial expression of the SuUR gene restores its binding. The SUUR protein is also involved in methylation of the residues H3K9 and H3K27. However, mono-, di-, and tri-methylated forms of H3K9 and H3K27 behave differently in various chromosomal domains in response to the SuUR mutation. Euchromatin and chromosome 4 are almost completely deprived of mono-, di-, and tri-methylation of H3K9. In the chromocenter, mono-methylation is reduced, di-methylation shows no noticeable changes, and tri-methylation is lost. Furthermore, mono- and di-methylation of H3K27 are not influenced by the SuUR mutation, whereas tri-methylation is lost in the chromocenter. Artificial expression of the SuUR gene on the SuUR (-) background restores the pattern of methylated residues characteristic for the wild type.

Aurora1 phosphorylation activity on histone H3 and its cross-talk with other post-translational histone modifications in Arabidopsis.

The enzymological properties of AtAurora1, a kinase responsible for the cell cycle-dependent phosphorylation of histone H3 at S10, and its cross-talk with other post-translational histone modifications, were determined. In vitro phosphorylation of H3S10 by AtAurora1 is strongly increased by K9 acetylation, and decreased by K14 acetylation and T11 phosphorylation. However, S10 phosphorylation activity is unaltered by mono-, di- or trimethylation of K9. An interference of H3K9 dimethylation by SUVR4 occurs by a pre-existing phosphorylation at S10. Hence, cross-talk in plants exists between phosphorylation of H3S10 and methylation, acetylation or phosphorylation of neighbouring amino acid residues. AtAurora1 undergoes autophosphorylation in vivo regardless of the presence of substrate, and forms dimers in planta. Of the three ATP-competitive Aurora inhibitors tested, Hesperadin was most effective in reducing the in vivo kinase activity of AtAurora1. Hesperadin consistently inhibited histone H3S10 phosphorylation during mitosis in Arabidopsis cells, but did not affect other H3 post-translational modifications, suggesting a specific inhibition of AtAurora in vivo. Inactivation of AtAurora also caused lagging chromosomes in a number of anaphase cells, but, unlike the situation in mammalian cells, Hesperadin did not influence the microtubule dynamics in dividing cells.

Conserved introns reveal novel transcripts in Drosophila melanogaster.

Noncoding RNAs that are-like mRNAs-spliced, capped, and polyadenylated have important functions in cellular processes. The inventory of these mRNA-like noncoding RNAs (mlncRNAs), however, is incomplete even in well-studied organisms, and so far, no computational methods exist to predict such RNAs from genomic sequences only. The subclass of these transcripts that is evolutionarily conserved usually has conserved intron positions. We demonstrate here that a genome-wide comparative genomics approach searching for short conserved introns is capable of identifying conserved transcripts with a high specificity. Our approach requires neither an open reading frame nor substantial sequence or secondary structure conservation in the surrounding exons. Thus it identifies spliced transcripts in an unbiased way. After applying our approach to insect genomes, we predict 369 introns outside annotated coding transcripts, of which 131 are confirmed by expressed sequence tags (ESTs) and/or noncoding FlyBase transcripts. Of the remaining 238 novel introns, about half are associated with protein-coding genes-either extending coding or untranslated regions or likely belonging to unannotated coding genes. The remaining 129 introns belong to novel mlncRNAs that are largely unstructured. Using RT-PCR, we verified seven of 12 tested introns in novel mlncRNAs and 11 of 17 introns in novel coding genes. The expression level of all verified mlncRNA transcripts is low but varies during development, which suggests regulation. As conserved introns indicate both purifying selection on the exon-intron structure and conserved expression of the transcript in related species, the novel mlncRNAs are good candidates for functional transcripts.

Heterochromatin formation in Drosophila is initiated through active removal of H3K4 methylation by the LSD1 homolog SU(VAR)3-3.

Epigenetic indexing of chromatin domains by histone lysine methylation requires the balanced coordination of methyltransferase and demethylase activities. Here, we show that SU(VAR)3-3, the Drosophila homolog of the human LSD1 amine oxidase, demethylates H3K4me2 and H3K4me1 and facilitates subsequent H3K9 methylation by SU(VAR)3-9. Su(var)3-3 mutations suppress heterochromatic gene silencing, display elevated levels of H3K4me2, and prevent extension of H3K9me2 at pericentric heterochromatin. SU(VAR)3-3 colocalizes with H3K4me2 in interband regions and is abundant during embryogenesis and in syncytial blastoderm, where it appears concentrated at prospective heterochromatin during cycle 14. In embryos of Su(var)3-3/+ females, H3K4me2 accumulates in primordial germ cells, and the deregulated expansion of H3K4me2 antagonizes heterochromatic H3K9me2 in blastoderm cells. Our data indicate an early developmental function for the SU(VAR)3-3 demethylase in controlling euchromatic and heterochromatic domains and reveal a hierarchy in which SU(VAR)3-3-mediated removal of activating histone marks is a prerequisite for subsequent heterochromatin formation by H3K9 methylation.

Histone modification and the control of heterochromatic gene silencing in Drosophila.

Covalent modifications of histones index structurally and functionally distinct chromatin domains in eukaryotic nuclei. Drosophila with its polytene chromosomes and developed genetics allows detailed cytological as well as functional analysis of epigenetic histone modifications involved in the control of gene expression pattern during development. All H3K9 mono- and dimethylation together with all H3K27 methylation states and H4K20 trimethylation are predominant marks of pericentric heterochromatin. In euchromatin, bands and interbands are differentially indexed. H3K4 and H3K36 methylation together with H3S10 phosphorylation are predominant marks of interband regions whereas in bands different H3K27 and H4K20 methylation states are combined with acetylation of H3K9 and H3K14. Genetic dissection of heterochromatic gene silencing in position-effect variegation (PEV) by Su(var) and E(var) mutations allowed identification and functional analysis of key factors controlling the formation of heterochromatin. SU(VAR)3-9 association with heterochromatic sequences followed by H3K9 methylation initiates the establishment of repressive SU(VAR)3-9/HP1/SU(VAR)3-7 protein complexes. Differential enzymatic activities of novel point mutants demonstrate that the silencing potential of SU(VAR)3-9 is mainly determined by the kinetic properties of the HMTase reaction. In Su(var)3-9ptn a significantly enhanced enzymatic activity results in H3K9 hypermethylation, enhanced gene silencing and extensive chromatin compaction. Mutations in factors controlling active histone modification marks revealed the dynamic balance between euchromatin and heterochromatin. Further analysis and definition of Su(var) and E(var) genes in Drosophila will increase our understanding of the molecular hierarchy of processes controlling higher-order structures in chromatin.

Saturation transfer difference measurements with SU(VAR)3-9 and S-adenosyl-L-methionine.

Saturation transfer difference NMR measurements were performed to investigate the interaction of S-adenosyl-l-methionine (AdoMet) with SU(VAR)3-9 from Drosophila melanogaster. SU(VAR)3-9 has a SET domain and plays an important role in methylation of lysine-9 of histone H3 which results in gene silencing. We determined the binding epitope of AdoMet and compared it with a crystal structure of another SET protein.

Su(var) genes regulate the balance between euchromatin and heterochromatin in Drosophila.

Histone lysine methylation is an epigenetic mark to index chromosomal subdomains. In Drosophila, H3-K9 di- and trimethylation is mainly controlled by the heterochromatic SU(VAR)3-9 HMTase, a major regulator of position-effect variegation (PEV). In contrast, H3-K27 methylation states are independently mediated by the Pc-group enzyme E(Z). Isolation of 19 point mutants demonstrates that the silencing potential of Su(var)3-9 increases with its associated HMTase activity. A hyperactive Su(var)3-9 mutant, pitkin(D), displays extensive H3-K9 di- and trimethylation within but also outside pericentric heterochromatin. Notably, mutations in a novel Su(var) gene, Su(var)3-1, severely restrict Su(var)3-9-mediated gene silencing. Su(var)3-1 was identified as "antimorphic" mutants of the euchromatic H3-S10 kinase JIL-1. JIL-1(Su(var)3-1) mutants maintain kinase activity and do not detectably impair repressive histone lysine methylation marks. However, analyses with seven different PEV rearrangements demonstrate a general role of JIL-1(Su(var)3-1) in controlling heterochromatin compaction and expansion. Our data provide evidence for a dynamic balance between heterochromatin and euchromatin, and define two distinct mechanisms for Su(var) gene function. Whereas the majority of Su(var)s encode inherent components of heterochromatin that can establish repressive chromatin structures [intrinsic Su(var)s], Su(var)3-1 reflects gain-of-function mutants of a euchromatic component that antagonize the expansion of heterochromatic subdomains [acquired Su(var)s].