Conserved chromosomal clustering of genes governed by chromatin regulators in Drosophila

Abstract

Background: The trithorax group (trxG) and Polycomb group (PcG) proteins are responsible for the maintenance of stable transcriptional patterns of many developmental regulators. They bind to specific regions of DNA and direct the post-translational modifications of histones, playing a role in the dynamics of chromatin structure.

Results: We have performed genome-wide expression studies of trx and ash2 mutants in Drosophila melanogaster. Using computational analysis of our microarray data, we have identified 25 clusters of genes potentially regulated by TRX. Most of these clusters consist of genes that encode structural proteins involved in cuticle formation. This organization appears to be a distinctive feature of the regulatory networks of TRX and other chromatin regulators, since we have observed the same arrangement in clusters after experiments performed with ASH2, as well as in experiments performed by others with NURF, dMyc, and ASH1. We have also found many of these clusters to be significantly conserved in D. simulans, D. yakuba, D. pseudoobscura and partially in Anopheles gambiae.

Conclusion: The analysis of genes governed by chromatin regulators has led to the identification of clusters of functionally related genes conserved in other insect species, suggesting this chromosomal organization is biologically important. Moreover, our results indicate that TRX and other chromatin regulators may act globally on chromatin domains that contain transcriptionally co-regulated genes.

Figure 1

Genomic map of clusters of genes deregulated in trx mutants. The location of each gene significantly deregulated in the microarray is indicated with a vertical line (up-regulated genes in red, down-regulated genes in green). Genes in the forward strand are displayed above the chromosome line; genes in the reverse strand are displayed below. Clusters of genes are indicated with a triangle in red or green according to their expression. The genome map was produced using the program GFF2PS [102].

Figure 2

Specificity controls in the clustering process. (a) Statistical significance of clusters. Bar plots representing the number of clusters observed in the set of genes regulated by TRX (up-regulated clusters in red, down-regulated clusters in green) and the number of clusters expected by chance (in white). The number of trx clusters observed in each chromosome was highly significant (Z-score >2). Error bars represent the standard deviation of the random samples. (b) Quantitative RT-PCR of target expression (clusters 4 and 20) in third instar wild-type (WT) and trx mutant larvae. Error bars represent variability between replicates.

Figure 3

Genomic map of clusters of genes on chromosome 3L that are regulated by several chromatin regulators. The location of each gene reported on every microarray is indicated with a vertical line (up-regulated genes in red, down-regulated genes in green). Genes in the forward strand are displayed above the chromosome line, genes in the reverse strand are displayed below. Clusters of genes in each experiment are indicated with a triangle in red or green according to their expression. Clusters present in two or more microarrays are highlighted by gray bands. Clusters of small genes identified along the fly genome are denoted with a triangle in gray.

Figure 4

Co-expression of genes in clusters. (a,b) Expression of genes in the same cluster in different microarrays. (a) Cluster of four down-regulated genes (in green) in trx microarrays. (b) Cluster of four up-regulated genes (in red) in ash1 microarrays. Notice the boundaries and the co-regulated genes of the cluster are not the same in both experiments. These images were produced using the program GFF2PS [102]. (c) Graphical comparison between co-expression of genes in trx and artificial clusters, according to the expression data provided in [30]. For each cluster, the co-expression level was computed as the mean of Pearson's correlation coefficient between all pairs of genes in the cluster. The distribution of co-expression values within the boundaries of the trx clusters (including all genes or only the deregulated ones) is clearly skewed to the right, indicating much stronger co-expression than expected at random.

Figure 5

Genomic map of 'hybrid' clusters of genes deregulated by TRX in D. melanogaster. Computational identification of clusters was performed on a set of up- and down-regulated genes in the microrray. The new hybrid clusters of genes are indicated with a blue triangle. The clusters detected before - using one of both sets - are indicated with a red triangle (up-regulated genes) or a green triangle (down-regulated genes). Some of them have been enriched using genes expressed in the opposite sense (displayed in light red or light green).

Figure 6

Genomic map in other species of clusters deregulated in trx mutants. The location in each species of the orthologous gene deregulated in D. melanogaster is indicated with a vertical line (up-regulated genes in red, down-regulated genes in green). Genes in the forward strand are displayed above the chromosome line, genes in the reverse strand are displayed below. Clusters of genes identified on each genome are indicated with a blue triangle.

Figure 7

Functional annotation of genes deregulated in trx mutants. (a) Classification of the microarray gene set, the deregulated genes and the genes that constitute the clusters according to the GO category 'molecular function', level 3. (b) Genomic map of clusters of functionally related genes. Clusters of genes annotated as structural constituents of cuticle (displayed as blue stars) and clusters of genes annotated as chitin binding (displayed as purple circles). Clusters of co-regulated genes in the trx mutant are indicated with a triangle in red or green according to their expression. Notice that most functional clusters match regulatory clusters despite the fact that both approaches are completely different.

Figure 8

Clusters are enriched in genes expressed in particular tissues. (a) From left to right: tissue distribution of genes expressed 18 h before larval to pupal metamorphosis according to Li et al. [63]; expression pattern of genes included in our trx microarray; genes deregulated by TRX; and genes in clusters deregulated by TRX. (b) Tissue distribution of clustered genes (at least one gene must be expressed in that tissue). Clusters that contain genes annotated as structural proteins in GO are displayed for comparison.

Figure 9

Genomic map of clusters, PcG ChIP-on-chip data and predicted PRE/TREs. The location of each gene reported on the trx microarray is indicated with a vertical line (up-regulated genes in red, down-regulated genes in green). Genes in the forward strand are displayed above the chromosome line, genes in the reverse strand are displayed below. Clusters of genes on each experiment are indicated with a triangle in red or green according to their expression. PcG binding domains reported by Schwartz et al. [68], Tolhuis et al. [69] and Negre et al. [70] are displayed in blue. PRE/TRE predictions obtained by Ringrose et al. [67] are displayed in black.