Targeting X chromosomes for repression

Abstract

Dosage compensation is a chromosome-wide regulatory process that balances X-chromosome gene expression between males and females that have different complements. Recent advances have clarified the molecular nature of the Caenorhabditis elegans sex-determination signal, which tallies X-chromosome number relative to the ploidy and controls both the choice of sexual fate and the process of dosage compensation. Dissecting the sex signal has revealed molecular mechanisms by which small quantitative differences in intracellular signals are translated into dramatically different developmental fates. Recent experiments have also revealed fundamental principles by which C. elegans dosage compensation proteins recognize and bind X chromosomes of XX embryos to reduce gene expression. Dosage compensation proteins function not only in a condensin complex specialized for regulating X-chromosome gene expression, but also in distinct condensin complexes that control other chromosome-wide processes: chromosome segregation and meiotic crossover recombination. The reshuffling of interchangeable molecular parts creates independent machines with similar architecture but distinct biological functions.

Figure 1. Primary sex determination: the X:A signal model

(a) In C. elegans, sex is determined by the number of X chromosomes relative to the ploidy, the number of sets of autosomes. This X:A signal is composed of X signal elements (XSEs) that activate the hermaphrodite program of development and Autosomal signal elements (ASEs) that oppose the XSEs to promote male development. The direct target of the X:A signal is the sex determination switch gene xol-1, which controls both the choice of sexual fate and the level of X-linked gene expression achieved through the process of dosage compensation. (b) X and autosomal signals antagonize each other directly at xol-1 to determine C. elegans sex. Direct rivalry at the xol-1 promoter between XSE transcriptional repressors (the ONECUT homeodomain protein CEH-39 and the nuclear hormone receptor SEX-1) and ASE transcriptional activators (the T-box transcription factor SEA-1 and the zinc finger protein SEA-2) leads to high xol-1 transcript levels in XO embryos and low levels in XX embryos. FOX-1, an RNA binding protein that acts as an XSE, then enhances the fidelity of signaling process by creating an inactive xol-1 mRNA splice variant in a dose-dependent manner. High XOL-1 protein levels in XO animals cause male development, and low XOL-1 levels in XX animals cause hermaphrodite development, including loading of the DCC onto X. Decreasing XSE dose causes XX-specific lethality, while increasing it causes XO-specific lethality. The reciprocal is true for ASEs. Increasing ASE dose causes XX-specific lethality; decreasing it causes XO-specific lethality. (xol-1 gene: promoter, gray; exons, blue; introns, orange and yellow).

Figure 2. Regulation of Dosage Compensation in C. elegans

(a) Partial genetic pathway for sex determination and dosage compensation. In XX embryos, xol-1 is repressed by the double dose of XSEs, permitting the XX-specific protein SDC-2 to activate dosage compensation and to repress her-1, a male-specific sex determination gene. SDC-2 acts with SDC-3 and DPY-30 to recruit the DCC condensin subunits to X. SDC-2 acts with SDC-1 and SDC-3 to repress her-1. SDC-2 plays the lead role in recognizing X sequences, while SDC-3 predominates in recognizing the SDC binding sites at her-1. In XO embryos, ASEs overcome XSEs to activate xol-1, resulting in sdc-2 repression and her-1 activation, thereby setting the male sexual fate. The DCC is not loaded onto X. (b) The DCC consists of five condensin-like components (DPY-27, MIX-1, DPY-26, DPY-28, and CAPG-1) that are homologous to canonical condensin subunits SMC2, SMC4, CAP-H, CAP-D2, and CAP-G1, respectively The DCC also contains at least five additional factors that confer X- and sex-specificity (SDC-2, SDC-3, and DPY-30) or assist in repression (DPY-21 and SDC-1). (c) DCC binding sites have been mapped by ChIP chip experiments and classified into two categories based on their ability to bind the complex when detached from the X chromosome. rex sites (recruitment elements on X) bind the complex robustly when they are detached from X and present either in multiple copies on extrachromosomal arrays (see Figure 3a) or in low copy number integrated onto an autosome. dox sites (dependent on X) fail to bind the DCC when detached; they depend on the broader X chromosomal context for their ability to associate with the DCC. (d) Motif searches identified a twelve base pair consensus motif that is enriched at rex sites relative to dox sites and on X chromosomes relative to autosomes. Mutations within the motif disrupt the ability of rex sites to recruit the DCC (see Figure 3a).

Figure 3. Three condensin complexes carry out distinct functions in C. elegans

(a) The dosage compensation complex (the DCC, also called condensin I^DC) (top) resembles condensin I. It equalizes X-linked expression between the sexes (XX hermaphrodites and XO males) by reducing transcript levels by half in hermaphrodites. Shown is an XX embryo stained with the DCC component SDC-2 (red) and DAPI (blue) (middle). The DCC binds to both X chromosomes. Shown also are images of gut cell nuclei carrying extrachromosomal arrays with multiple copies of rex-1 derivatives stained with DAPI (blue), SDC-3 antibodies (red), and array FISH (green) (bottom). Arrays have rex-1 fragments with different numbers of MEX motifs. Left, a 33 bp rex-1 fragment in which its single MEX motif was mutated, thus abrogating DCC binding. X staining is apparent (red). Middle, a wild-type 33 bp rex-1 fragment with 1 MEX motif. SDC-3 colocalizes with the array and X. Right, a 60 bp rex-1 fragment with 2 MEX motifs. SDC-3 binds robustly to rex-1 and is titrated from X, showing that MEX motifs collaborate to recruit the DCC. An array carrying a rex-1 fragment that titrates the DCC from X can suppress the male lethality caused by mutation of xol-1 in XO embryos. (b) Condensin I. This complex differs from DCC condensin by a single subunit, SMC-4 (top). This complex controls meiotic DSB distribution through effects on chromosome structure. It also plays minor roles in chromosome segregation in mitosis and meiosis. Shown is a high resolution image of pachytene chromosomes in wild-type animals labeled with the axis protein HTP-3 and FISH probes (blue, red) to X (middle). X chromosomes from wild-type and dpy-28 mutant animals were traced (yellow) in three dimensions and straightened computationally (bottom). Straightened chromosomes are shown horizontally. Genotypes, average total axis length, and standard error of the mean are below each axis. Disruption of dpy-28 dramatically increases the X-chromosome axis length in a manner independent of DSBs made by SPO-11. In response, DSBs are increased in number and redistributed in dpy-28 mutations, causing an increase in crossovers and their redistribution. (c) Condensin II. This complex shares one subunit (MIX-1) with the DCC (condensin I^DC) and two subunits (MIX-1 and SMC-4) with condensin I (top). This complex is the prime condensin complex responsible for mitotic and meiotic chromosome compaction and resolution. Condensin II binds to the centromeres on the holocentric mitotic chromosomes. Shown is a two cell embryo (middle) with one cell in metaphase (left) and one in prometaphase (right). Centromeric proteins (yellow) and condensin II bind along the outer edge of each chromosome, adjacent to where the mitotic spindle (tubulin, blue) attaches. Condensin II also binds to meiotic chromosomes at pachytene exit to create the compact shape (diakinesis bivalents) required for chromosome segregation. Shown are the four sister chromatids of meiotic diakinesis bivalents (bottom) stained with HCP-6 antibodies (green) and DAPI (red). Merge is yellow.

Figure 4. Model for loading of the DCC onto X

This model integrates current data. The DCC binds to rex sites in a sequence-dependent manner requiring SDC-2, SDC-3, and DPY-30. rex sites confer X-specificity to DCC binding and recruit additional complexes to bind along X at dox sites, which are unable to recruit the DCC when detached from X. Many dox sites on X have a small, intrinsic DCC binding capability; this binding is enhanced in response to DCC loading onto rex sites. dox sites are located preferentially in promoters of active genes, and positions of dox sites change as gene expression changes, implying that dox-site binding is transcription dependent.