E-GEOD-3055 - RNAi knock down of heterochromatin protein 1 in male and female Drosophila flies
Submitted on 5 August 2005, released on 1 August 2007, last updated on 27 March 2012
Drosophila heterochromatin protein 1- HP1 is believed to be involved in active transcription, transcriptional gene silencing, and the formation of heterochromatin2-7. However, little is known about the function of HP1 during development. Using a Gal4-induced RNA interference system, we show that conditional depletion of HP1 in transgenic flies results in preferential lethality in male flies. Cytological analysis of mitotic chromosomes reveals that HP1 depletion causes sex-biased chromosomal defects, including telomere fusions. The global levels of specific histone modifications, particularly the hallmarks of active chromatin, are preferentially increased in males as well. Expression analysis revealed that approximately twice as many genes are specifically regulated by HP1 in males compared to females. Furthermore, HP1-regulated genes showed greater enrichment for HP1 binding in males. Taken together, these results reveal that HP1 modulates chromosomal integrity, histone modifications, and transcription in a sex-specific manner. Overall design. The extraction of total RNA was performed following a standard protocol (www.erin.utoronto.ca/~w3flyma/protocol.htm). Total RNA was isolated from two independent populations of male and female 3rd-instar larva of HP1-21/act-Gal4 and, as controls, larval progenies from line HP1-21 with the genotype y w; +/+; HP1-21/+, and larva with the genotype y w; + /+; +/act-Gal4. In brief, larvaa frozen in liquid nitrogen were homogenized and then resuspended in Trizol reagent by pipetting. The precipitated RNA was washed, and then dissolved in RNase-free water. Five micrograms of total RNA from each experimental sample were reverse-transcribed using the SuperScript Choice cDNA synthesis kit from Stratagene. One microgram of double-stranded cDNA was in vitro-transcribed using the Affymetrix IVT kit and labeled by the incorporation of biotinylated-UTP. Fifteen micrograms of cRNA were then fragmented and hybridized to Affymetrix DGv2 GeneChips as per the manufacturer’s instructions (Affymetrix, Santa Clara CA, USA).
RNAi profiling by array, cellular modification, individual genetic characteristics, sex
Sex-specific role of Drosophila melanogaster HP1 in regulating chromatin structure and gene transcription. Lu-Ping Liu,Jian-Quan Ni,Yan-Dong Shi,Edward J Oakeley,Fang-Lin Sun. Nature genetics :1361-6 (2005)