Regulated chromatin domain comprising cluster of co-expressed genes in Drosophila melanogaster

Abstract

Recently, the phenomenon of clustering of co-expressed genes on chromosomes was discovered in eukaryotes. To explore the hypothesis that genes within clusters occupy shared chromatin domains, we performed a detailed analysis of transcription pattern and chromatin structure of a cluster of co-expressed genes. We found that five non-homologous genes (Crtp, Yu, CK2betates, Pros28.1B and CG13581) are expressed exclusively in Drosophila melanogaster male germ-line and form a non-interrupted cluster in the 15 kb region of chromosome 2. The cluster is surrounded by genes with broader transcription patterns. Analysis of DNase I sensitivity revealed 'open' chromatin conformation in the cluster and adjacent regions in the male germ-line cells, where all studied genes are transcribed. In contrast, in somatic tissues where the cluster genes are silent, the domain of repressed chromatin encompassed four out of five cluster genes and an adjacent non-cluster gene CG13589 that is also silent in analyzed somatic tissues. The fifth cluster gene (CG13581) appears to be excluded from the chromatin domain occupied by the other four genes. Our results suggest that extensive clustering of co-expressed genes in eukaryotic genomes does in general reflect the domain organization of chromatin, although domain borders may not exactly correspond to the margins of gene clusters.

Figure 1

Structure of the 60D1-2 region including the cluster of five testes-specific genes, and surrounding genes with broader expression pattern. Exon–intron structure and the location of genes from the region is according to the GadFly (release 3.1) with addition of the Yu transcription unit, according to our data. Testis-specific genes are in black. Gene CG13590 (dashed) is predicted in Drosophila genome annotation, but its existence is not supported by our data.The horizontal bars (labeled a through d) indicate the probes used for testes cDNA library screening and for in situ hybridizations.

Figure 2

Transcriptional analysis of genes from the 60D1-2 region. (A) Developmental northern analysis shows testis specificity of expression of genes from the 60D cluster. Total RNA sources are indicated on top; culture corresponds to the Schneider-2 cells; m.carc. and f.carc. represent gonadectomized males and females, respectively; ad.testes and lar.testes represent adult and larvae testes, respectively. ³²P-labeled antisense RNA probes were used. Hybridization with the antisense RNA probe for the constitutively expressed gene Rp49 served as the loading control. RNA in situ hybridization of DIG-labeled CG13581 (B) or Crtp (C) probes with whole-mount adult testes show onset of transcription in spermatocytes; transcripts persist until late post-meiotic stages of spermatogenesis. Arrow marks tip of testis occupied by stem cells and spermatogonia, where no hybridization was detected. (D) Northern analysis of the transcription of genes from the 60D cluster in the bam⁸⁶ and aly⁵ mutants shows universal requirement for the bam function, but diverse response to the aly deficiency. Total RNA was isolated from testes of males with the genotype indicated on top; the Df(1)w ^67c23(2), y males with normally proceeding spermatogenesis were used as positive control.

Figure 3

Profiles of chromatin DNase I-resistance across the 60D cluster region and surrounding sequences. Chromatin resistance to DNase I [as normalized relative yield (NRY) for each amplicon] is plotted on the vertical axis; the length of 60D1-2 genomic region (in kb) is plotted on the horizontal. Positions of the genes in the region are shown at bottom. Circles (black for the regulated chromatin domain, and white for the rest of the region) indicate average NRY for each amplicon, the grey area corresponds to the calculated 95% confidence interval. Upper panel: in larval testes, the entire region shows nearly uniformal low resistance to DNase I typical for the ‘open’ chromatin. In contrast, in larval brains (middle panel) and in embryos (lower panel) regulated chromatin domain that contains the genes CG13589 through Pros28.1B shows significantly higher resistance to DNase I, indicative of ‘closed’ chromatin configuration.