The Differences Between Cis- and Trans-Gene Inactivation Caused by Heterochromatin in Drosophila

Abstract

Position-effect variegation (PEV) is the epigenetic disruption of gene expression near the de novo-formed euchromatin-heterochromatin border. Heterochromatic cis-inactivation may be accompanied by the trans-inactivation of genes on a normal homologous chromosome in trans-heterozygous combination with a PEV-inducing rearrangement. We characterize a new genetic system, inversion In(2)A4, demonstrating cis-acting PEV as well as trans-inactivation of the reporter transgenes on the homologous nonrearranged chromosome. The cis-effect of heterochromatin in the inversion results not only in repression but also in activation of genes, and it varies at different developmental stages. While cis-actions affect only a few juxtaposed genes, trans-inactivation is observed in a 500-kb region and demonstrates а nonuniform pattern of repression with intermingled regions where no transgene repression occurs. There is no repression around the histone gene cluster and in some other euchromatic sites. trans-Inactivation is accompanied by dragging of euchromatic regions into the heterochromatic compartment, but the histone gene cluster, located in the middle of the trans-inactivated region, was shown to be evicted from the heterochromatin. We demonstrate that trans-inactivation is followed by de novo HP1a accumulation in the affected transgene; trans-inactivation is specifically favored by the chromatin remodeler SAYP and prevented by Argonaute AGO2.

Keywords: Drosophila; PEV; heterochromatin; nuclear compartmentalization; trans-inactivation.

Figure 1

Structure of the A4 rearrangement and the manifestation of cis- and trans-inactivation of mini-white in reporter transgenes. (A) Structure of the A4 chromosome. Breakpoint positions (bp) in euchromatin and heterochromatin are located in the second exon of the Mcm10 gene and in the h37 heterochromatin block [according to Dimitri (1991)], respectively. C, centromere. The purple triangle designates the mini-white-containing P-element in the Hr39 gene. Dashed arrows show the spreading of inactivation caused by the main heterochromatic block and the detached small block of heterochromatin. (B) Localization of the heterochromatic breakpoint in A4. (Left) DAPI staining of mitotic chromosomes from A12/A4 larval brains. (Right) In situ hybridization using the (AATAACATAG)_n probe (red). The positions of the h37 block are marked on chromosome 2. (С) Eye color phenotypes resulting from the expression of mini-white in the P-element inserted into the Hr39 gene. The A4 inversion leads to mosaic repression of mini-white in A4/+, A4/A4, A12/A4, and A12/A4(ΔP) flies. Dosage effect is seen in A12/A12, A4/A4, and A12/A4 flies. (D) trans-Inactivation of the mini-white reporter (P(w)) on normal chromosome 2 in heterozygous P(w)/A4(ΔP) flies. The degree of trans-inactivation is ranked into four categories (+++, ++, +, −) according to the observed degree of mini-white repression. The examples of trans-inactivation phenotypes of four transgene insertions (20708, 20250, 18735, and 20102) are shown. P(w)/+ is a mini-white reporter over a wild-type chromosome; P(w)/A4(ΔP) is the same transgene over an A4(ΔP) chromosome.

Figure 2

cis-Effects of heterochromatin in the A4 inversion in larvae and adults. (A) Structure of the A4 inversion. C, centromere. Black, gray, and white blocks represent the organization of heterochromatin of chromosome 2 (Dimitri 1991). Exon-intron maps of the genes in the region are presented, and the genes with confirmed greater than twofold expression changes in either larvae or adults have captions. Euchromatic position of the A4 breakpoint is shown by the vertical dotted line. (B) Chromosome distributions of log₂-transformed ratios (A4/A4 to A12/A12) of normalized gene expression levels based on RNA-Seq (blue diamonds) and qPCR (horizontal red strips) data. cis-Repression corresponds to negative values. These data show that euchromatic genes respond to heterochromatin-induced cis-effects individually and differently in larvae and adults. (C) Changes in HP1a and H3K4me2 abundance (log₂-transformed A4/A4-to-A12/A12 ratio) for the genes Acon, CG8678, CG8679, Hr39, and crc at the larval stage. The positions of the bars correspond to the positions of genes on the chromosome. Genes Acon, CG8678, CG8679, Hr39, but not crc change their expression near the heterochromatin (B). Significant changes (greater than twofold) in HP1a are detected for the Hr39 and CG8678 genes, and no significant difference is observed for H3K4me2 enrichments.

Figure 3

Detailed map of mini-white reporter trans-inactivation in A4(ΔP)/P(w) flies. (Top) Schematic representation of paired A4(ΔP) and transgene-bearing P(w) chromosomes forming an inversion loop. The A4(ΔP) chromosome contains pericentromeric and detached smaller heterochromatin blocks that cause propagations of trans-inactivation (shown by thick red arrows). Colored vertical strokes represent the positions and degree of transgene repression: red, strong inactivation; orange, moderate inactivation; brown, weak inactivation; and blue, no inactivation. The whole area of trans-inactivation spreading is subdivided into regions of total repression (A and B with the single exception of 10662), moderate/interspersed repression (C and E), and no repression (D) of inserted transgenes. Dashed colored lines outline the approximate borders of these regions. (Bottom) Close-up views of regions A, B, C, D, and E showing the positions of endogenous genes and transgene insertions. Transgene names are constructed as “stock number_transgene type_insertion orientation”; the colors of transgenes correspond to the degree of repression, as in the top panel. Regions A and B, ∼40 kb each, are immediately adjacent to heterochromatin in the A4 chromosome. Region C is the region of interspersed inactivation (∼80 kb). Region D includes the histone gene cluster; no trans-inactivation is observed here. Region E is the “island” of trans-inactivation after the histone gene cluster. The furthermost transgene still repressed is 19883 (region E, 475 kb from the breakpoint position).

Figure 4

HP1a accumulates at the trans-inactivated transgene and on A4 at the site homologous to insertion of the trans-inactivated reporter. Primer pairs used for ChIP analysis of the transgenes (mini-white, LacZ) and the site on the A4(ΔP) chromosome corresponding to transgene insertion 11127 are marked by different colors. The histogram bars in B and C presents the log₂-transformed ratios of HP1a (blue) or H3K4me2 (red) abundance in P(w)/A4(∆P) individuals to P(w)/+ individuals; the bar positions correspond to the locations of the regions analyzed. The trans-inactivation phenotypes are shown for 11127 and 20102. (A) Fragments of A4(ΔP) inversion and normal P(w) chromosome carrying reporter transgenes. The direction of trans-inactivation spreading along the nonrearranged chromosome is indicated by the red dotted arrow. The positions of the 11127 (trans-inactivated) and 20102 (noninactivated) transgenes are indicated by pink and green triangles, respectively. (B) trans-Inactivated insertion 11127 in 11127/A4(ΔP) larvae. The scheme shows the arrangement of the 11127 and A4(ΔP) chromosomes, positions of insertion and primers, and the simplified structure of the P[lacW] transgene inserted (below the chromosome). The positions of histogram bars showing the HP1a and H3K4me2 enrichment levels correspond to the positions of primers. HP1a accumulates at the constituents of trans-inactivated transgene 11127 (histogram below the transgene image) and in the site of this transgene insertion on the opposite A4(∆P) chromosome (primers 11127). (C) Noninactivated insertion 20102 in 20102/A4(∆P) larvae (for designations, see B). No accumulation of HP1a in the mini-white of transgene 20102 was observed. There was also no accumulation of HP1a in the site of transgene 11127 insertion in the absence of the proper transgene. A significant enrichment in HP1a was observed in trans-inactivated but not in noninactivated transgenes. The comparison of chromatin state on A4(∆P) when heterozygous with the trans-inactivated transgene reveals an HP1a enrichment.

Figure 5

The 39AB region located in A12/A12 nuclei in euchromatin is moved to the heterochromatic compartment in A12/A4, while the histone gene cluster maintains its position at the euchromatin-heterochromatic border. (A) The positions of the in situ probes for 39AB (green), histone gene cluster (pink), and AACAC satellite (red) are shown on a schematic chromosome 2 map. The arrow points to the position of the A4 breakpoint. (B) Examples of confocal nuclei cross section and the schematic view (below) of paired homologous chromosomes of typical A12/A12 and A12/A4 nuclei after in situ staining. C, centromere. Colored dots on the scheme represent the positions of the in situ hybridization probes; their colors correspond to those of the FISH signals on the confocal images and on the chromosome scheme at the top. The yellow cloud is HP1a staining of the heterochromatin compartment. (C) Mean HP1a staining intensities calculated at the positions of in situ signals of the histone gene cluster and the 39AB region and normalized to the mean intensity of HP1a staining at the position of the AACAC satellite signal.

Figure 6

Effect of PEV modifiers and the e(y)3 transcriptional coactivator on trans-inactivation. trans-Inactivated transgene 11127 is the insertion of P[lacW] at a distance of 80 kb from the border between euchromatin and the pericentromeric block of heterochromatin in A4. Su(var)2-5, Su(var)3-7, Su(var)3-6, and Su(var)3-1 suppress trans-inactivation of mini-white in 11127/A4 flies. Su(var)3-9 does not suppress trans-inactivation, while e(y)3^u1 suppresses trans- but not cis-inactivation. AGO2 mutations (AGO2⁴¹⁴/AGO2^51B and AGO2⁴¹⁴/AGO2⁴¹⁴ have the same effect) enhance trans-inactivation but to a much lesser degree cis-inactivation of mini-white in A4.