Heterochromatin: On the ADAR Radar?

AbstractVigilin proteins, the absence of which is known to cause abnormalities in heterochromatin, have been found to bind edited RNAs. Molecular complexes including vigilin comprise proteins involved with RNA editing and with DNA repair, making connections between these processes and RNA-based silencing mechanisms

Interaction Study of the Male Specific Lethal (MSL) Complex and Trans-Acting Dosage Effects in Metafemales of Drosophila melanogaster

The effect of ectopic expression of male specific lethal 2 (msl2) on chromatin modification and gene expression was studied in Drosophila diploid females and metafemales (3X;2A). Results show that ectopic expression of MSL2 in transgenic msl2 females and metafemales sequesters the MOF histone acetylase to the X, which occurs concordantly with an increase of histone acetylation. Gene expression studies indicate that the X-linked genes are not affected by direct targeting of the MSL complex and the resulting increased H4Lys16 acetylation on the X chromosomes, suggesting one function of the MSL complex is to nullify the effect of a high level of histone acetylation. These results are not consistent with the hypothesis that the presence of the MSL complex conditions a two-fold upregulation. Autosomal gene expression is generally decreased in ectopically expressed MSL2 females, which correlates with the reduced autosomal histone acetylation. Metafemales show dosage compensation of X-linked genes with some autosomal reductions in expression. Interestingly, in metafemales with ectopically expressed MSL2, the autosomal expression is returned to a more normal level. There is a lower autosomal level of histone acetylation compared to the normal metafemales, suggesting a nullifying effect on the negative dosage effect of the X chromosome as previously hypothesized to occur in normal males

Studies on the short range spreading of the male specific lethal (MSL) complex on the X chromosome in Drosophila

A series of autosomal insertions of chromosomal fragments derived from around the X linked white eye color locus have been examined for male specific lethal (MSL) complex binding using both immunostaining and fluorescence in situ hybridization (FISH) techniques. The results show that the transposing elements (TEs) composed of several genes in the white region (3C2-3C5) do not recruit the MSL complex when inserted into an autosome. The same result is found for the Tp(1:3)wzh insertion, a fragment of the X chromosome inserted into the third chromosome. Two other insertions, Dp(1:2)w70h (3A7-3C2-3) and Dp(1:2)51b (3C2-3D6), which extend more distally or proximally beyond the TE insertion, respectively, display a binding pattern of the MSL complex at the autosomal location. These insertions were also examined in females ectopically expressing MSL-2 and show similar binding activity. In addition, the Tp(3:1)O5 transposition strain containing an autosomal segment in the X chromosome was examined for spreading of the MSL complex. Limited spreading of the MSL complex into autosomal regions was indicated by immunostaining and FISH. This spreading was further confirmed by chromatin immunoprecipitation of the MSL complex covering the autosomal sequences

Supplementary Material for: Accumulation of Multiple Copies of Maize Minichromosomes

Multiple copies of B chromosomes in maize <i>(Zea mays)</i> can accumulate in the genome using the B chromosome’s accumulation mechanism, specifically nondisjunction at the second pollen mitosis and preferential fertilization of the egg. Using this mechanism, we accumulated 4 different-sized minichromosomes derived from the B chromosome to test the chromosome limits of the cell. The accumulation of normal B chromosomes is associated with multiple phenotypes including white stripes and asymmetric leaf blades, but when minichromosomes are accumulated these symptoms are absent. We also found that multiple B chromosome-derived minichromosomes can coexist with A chromosome-derived minichromosomes. During the years that these experiments were conducted, we found many B chromosome rearrangements and fragments, 2 recoverable A chromosome fragments, and observed a minichromosome breakage-fusion-bridge cycle in roots

Heterochromatic Silencing and HP1 Localization in Drosophila Are Dependent on the RNAi Machinery

Genes normally resident in euchromatic domains are silenced when packaged into heterochromatin, as exemplified in Drosophila melanogaster by position effect variegation (PEV).Loss-of-function mutations resulting in suppression of PEV have identified critical components of heterochromatin, including proteins HP1, HP2, and histone H3 lysine 9 methyltransferase.Here, we demonstrate that this silencing is dependent on the RNA interference machinery, using tandem mini-white arrays and white transgenes in heterochromatin to show loss of silencing as a result of mutations in piwi, aubergine, or spindle-E (homeless), which encode RNAi components. These mutations result in reduction of H3 Lys9 methylation and delocalization of HP1 and HP2, most dramatically in spindle-E mutants

Whole-chromosome paints in maize reveal rearrangements, nuclear domains, and chromosomal relationships

Whole-chromosome painting probes were developed for each of the 10 chromosomes of maize by producing amplifiable libraries of unique sequences of oligonucleotides that can generate labeled probes through transcription reactions. These paints allow identification of individual homologous chromosomes for many applications as demonstrated in somatic root tip metaphase cells, in the pachytene stage of meiosis, and in interphase nuclei. Several chromosomal aberrations were examined as proof of concept for study of various rearrangements using probes that cover the entire chromosome and that label diverse varieties. The relationship of the supernumerary B chromosome and the normal chromosomes was examined with the finding that there is no detectable homology between any of the normal A chromosomes and the B chromosome. Combined with other chromosome-labeling techniques, a complete set of whole-chromosome oligonucleotide paints lays the foundation for future studies of the structure, organization, and evolution of genomes