Transcription through intergenic chromosomal memory elements of the Drosophila bithorax complex correlates with an epigenetic switch

Abstract

The proteins of the trithorax and Polycomb groups maintain the differential expression pattern of homeotic genes established by the early embryonic patterning system during development. These proteins generate stable and heritable chromatin structures by acting via particular chromosomal memory elements. We established a transgenic assay system showing that the Polycomb group response elements bxd and Mcp confer epigenetic inheritance throughout development. With previously published data for the Fab7 cellular memory module, we confirmed the cellular memory function of Polycomb group response elements. In Drosophila melanogaster, several of these memory elements are located in the large intergenic regulatory regions of the homeotic bithorax complex. Using a transgene assay, we showed that transcription through a memory element correlated with the relief of silencing imposed by the Polycomb group proteins and established an epigenetically heritable active chromatin mode. A memory element remodeled by the process of transcription was able to maintain active expression of a reporter gene throughout development. Thus, transcription appears to reset and change epigenetic marks at chromosomal memory elements regulated by the Polycomb and trithorax proteins. Interestingly, in the bithorax complex of D. melanogaster, the segment-specific expression of noncoding intergenic transcripts during embryogenesis seems to fulfill this switching role for memory elements regulating the homeotic genes.

FIG. 1.

CMM function of bxd and Mcp. GAL4 was expressed during embryogenesis (A and C) by transferring embryos to a 37°C water bath for 1 h. Afterwards, heat-shocked lines were raised at 18°C until adulthood, like their control siblings (B and D), which were kept at 18°C constantly. Activated flies showed derepression of the mini-white gene. (E) The bxd, Mcp, and Fab7 subfragments were cloned into the pUZ transformation vector in both orientations (d, distal; p, proximal). From these constructs, transgenic flies were generated. The bxd and Mcp strains inherit the activated state of the two reporter genes, whereas flies carrying the Fab7 subfragments do not, as illustrated. The boundary and the PRE region depicted in the Mcp element are based on information published by Karch et al. (18) and Strutt et al. (36). The separation between boundary and PRE is arbitrary and not based on functional analysis. Constructs which displayed pairing-sensitive silencing or showed CMM function are marked with +; otherwise, a − is indicated.

FIG. 2.

CMMs are transcribed upon ubiquitous GAL4 expression during embryogenesis: in situ hybridization of embryos carrying the bxd, Mcp, or Fab7 construct. The strand-specific probes detected transcripts in the proximal-distal (d, distal; p, proximal) orientation relative to the bithorax complex. Transcripts were mostly expressed in a homogenous manner. In some lines, silencing was not completely removed, and a variegated expression pattern was observed (see Mcp line). In the absence of GAL4, minimal background expression was observed. ssRNA, single-stranded RNA.

FIG. 3.

CMM of heat shock (hs)-induced FLW-1/Fab7¹ is transcribed two- to threefold more compared with non-heat-shocked controls. A digoxigenin-labeled 3.6-kb Fab7 RNA probe (distal-proximal [d-p] orientation) was hybridized to a slot blot of 5 μg of polyadenylated RNA of various lines. The endogenous Fab7 locus was transcribed weakly in the wild type (wt). Additional controls included 10 ng of in vitro-transcribed Fab7 RNA.

FIG. 4.

Transcription pattern of cis regulatory intergenic sequences in the bithorax complex. RNA in situ hybridization was performed with 1- to 12-h embryos. cis regulatory sequences are marked by red bars. Homeotic genes according to their most common splicing form are illustrated in black, and the in situ hybridization probes are marked in blue. The upper panels show embryos of the blastula stage, whereas the lower panels depict embryos around germ band extension. All the transcripts depicted are in distal-to-proximal orientation, detected by strand-specific probes. Transcripts remained through embryogenesis or appeared at later stages, as shown for probe D 1E1. The anterior expression domains of CMMs and intergenic cis regulatory sequences are transcribed in a linear pattern along the distal-proximal axis of the bithorax complex. Probe D 1E1 detects transcripts in the most posterior parasegment 14, although only after germ band retraction. Fab7 hybridized to transcripts in parasegments 12 to 14; Mcp to parasegments 10 to 14; iab4 to parasegments 8 to 14; and the bxd PRE to parasegments 6 to 12.

FIG. 5.

Upstream and downstream region of the Fab7¹ deletion are transcribed. Fab7¹ embryos were in situ hybridized to 1-kb probes flanking the Fab7¹ deletion. Distal (d) as well as proximal (p) probes generated the same staining pattern observed for the 3.6-kb Fab7 element. The probes hybridized in parasegments 12 to 14.

FIG. 6.

Transcriptional remodeling of CMMs. Initially, gap and segmentation genes set up the expression pattern of the homeotic genes. CMMs are subsequently required to maintain the preset homeotic gene expression patterns throughout development by distinguishing transcriptionally active and silenced chromatin. Transcription through a CMM switches the element from the silenced to activated state. We can envisage two possible, not mutually exclusive, functions to achieve this heritable switch. In model A, the RNA polymerase (Pol) II complex passes through CMMs in intergenic regions, displacing the repressive complexes. As a consequence, chromatin becomes accessible for further epigenetic modifications. In model B, RNA polymerase II “piggybacks” remodeling complexes which epigenetically mark histones. These modifications are stably sustained throughout development and inherit an open chromatin conformation (ovals, nucleosomes; half-circles, silencing complexes; Mod, epigenetic modification of histones like histone acetyltransferase [HAT], histone deacetylase [HDAC], or methyltransferase [MT]).