Dosage Compensation Complex Binding Research Articles

X-chromosome target specificity diverged between dosage compensation mechanisms of two closely related Caenorhabditis species.

An evolutionary perspective enhances our understanding of biological mechanisms. Comparison of sex determination and X-chromosome dosage compensation mechanisms between the closely related nematode species Caenorhabditis briggsae (Cbr) and Caenorhabditis elegans (Cel) revealed that the genetic regulatory hierarchy controlling both processes is conserved, but the X-chromosome target specificity and mode of binding for the specialized condensin dosage compensation complex (DCC) controlling X expression have diverged. We identified two motifs within Cbr DCC recruitment sites that are highly enriched on X: 13 bp MEX and 30 bp MEX II. Mutating either MEX or MEX II in an endogenous recruitment site with multiple copies of one or both motifs reduced binding, but only removing all motifs eliminated binding in vivo. Hence, DCC binding to Cbr recruitment sites appears additive. In contrast, DCC binding to Cel recruitment sites is synergistic: mutating even one motif in vivo eliminated binding. Although all X-chromosome motifs share the sequence CAGGG, they have otherwise diverged so that a motif from one species cannot function in the other. Functional divergence was demonstrated in vivo and in vitro. A single nucleotide position in Cbr MEX can determine whether Cel DCC binds. This rapid divergence of DCC target specificity could have been an important factor in establishing reproductive isolation between nematode species and contrasts dramatically with the conservation of target specificity for X-chromosome dosage compensation across Drosophila species and for transcription factors controlling developmental processes such as body-plan specification from fruit flies to mice.

Combinatorial clustering of distinct DNA motifs directs synergistic binding of Caenorhabditis elegans dosage compensation complex to X chromosomes

Organisms that count X-chromosome number to determine sex utilize dosage compensation mechanisms to balance X-gene expression between sexes. Typically, a regulatory complex is recruited to X chromosomes of one sex to modulate gene expression. A major challenge is to determine the mechanisms that target regulatory complexes specifically to X. Here, we identify critical X-sequence motifs in Caenorhabditis elegans that act synergistically in hermaphrodites to direct X-specific recruitment of the dosage compensation complex (DCC), a condensin complex. We find two DNA motifs that collaborate with a previously defined 12-bp motif called MEX (motif enriched on X) to mediate binding: MEX II, a 26-bp X-enriched motif and Motif C, a 9-bp motif that lacks X enrichment. Inserting both MEX and MEX II into a new location on X creates a DCC binding site equivalent to an endogenous recruitment site, but inserting only MEX or MEX II alone does not. Moreover, mutating MEX, MEX II, or Motif C in endogenous recruitment sites with multiple different motifs dramatically reduces DCC binding invivo to nearly the same extent as mutating all motifs. Changing the orientation or spacing of motifs also reduces DCC binding. Hence, synergy in DCC binding via combinatorial clustering of motifs triggers DCC assembly specifically on X chromosomes. Using an invitro DNA binding assay, we refine the features of motifs and flanking sequences that are critical for DCC binding. Our work reveals general principles by which regulatory complexes can be recruited across an entire chromosome to control its gene expression.

Induction and inhibition of Drosophila X chromosome gene expression are both impeded by the dosage compensation complex.

Sex chromosomes frequently differ from the autosomes in the frequencies of genes with sexually dimorphic or tissue-specific expression. Multiple hypotheses have been put forth to explain the unique gene content of the X chromosome, including selection against male-beneficial X-linked alleles, expression limits imposed by the haploid dosage of the X in males, and interference by the dosage compensation complex on expression in males. Here, we investigate these hypotheses by examining differential gene expression in Drosophila melanogaster following several treatments that have widespread transcriptomic effects: bacterial infection, viral infection, and abiotic stress. We found that genes that are induced (upregulated) by these biotic and abiotic treatments are frequently under-represented on the X chromosome, but so are those that are repressed (downregulated) following treatment. We further show that whether a gene is bound by the dosage compensation complex in males can largely explain the paucity of both up- and downregulated genes on the X chromosome. Specifically, genes that are bound by the dosage compensation complex, or close to a dosage compensation complex high-affinity site, are unlikely to be up- or downregulated after treatment. This relationship, however, could partially be explained by a correlation between differential expression and breadth of expression across tissues. Nonetheless, our results suggest that dosage compensation complex binding, or the associated chromatin modifications, inhibit both up- and downregulation of X chromosome gene expression within specific contexts, including tissue-specific expression. We propose multiple possible mechanisms of action for the effect, including a role of Males absent on the first, a component of the dosage compensation complex, as a dampener of gene expression variance in both males and females. This effect could explain why the Drosophila X chromosome is depauperate in genes with tissue-specific or induced expression, while the mammalian X has an excess of genes with tissue-specific expression.

Divergent evolution toward sex chromosome-specific gene regulation in Drosophila.

The dosage compensation complex (DCC) of Drosophila identifies its X-chromosomal binding sites with exquisite selectivity. The principles that assure this vital targeting are known from the D. melanogaster model: DCC-intrinsic specificity of DNA binding, cooperativity with the CLAMP protein, and noncoding roX2 RNA transcribed from the X chromosome. We found that in D. virilis, a species separated from melanogaster by 40 million years of evolution, all principles are active but contribute differently to X specificity. In melanogaster, the DCC subunit MSL2 evolved intrinsic DNA-binding selectivity for rare PionX sites, which mark the X chromosome. In virilis, PionX motifs are abundant and not X-enriched. Accordingly, MSL2 lacks specific recognition. Here, roX2 RNA plays a more instructive role, counteracting a nonproductive interaction of CLAMP and modulating DCC binding selectivity. Remarkably, roX2 triggers a stable chromatin binding mode characteristic of DCC. Evidently, X-specific regulation is achieved by divergent evolution of protein, DNA, and RNA components.

The Influence of Chromosomal Environment on X-Linked Gene Expression in Drosophila melanogaster.

Sex chromosomes often differ from autosomes with respect to their gene expression and regulation. In Drosophila melanogaster, X-linked genes are dosage compensated by having their expression upregulated in the male soma, a process mediated by the X-chromosome-specific binding of the dosage compensation complex (DCC). Previous studies of X-linked gene expression found a negative correlation between a gene’s male-to-female expression ratio and its distance to the nearest DCC binding site in somatic tissues, including head and brain, which suggests that dosage compensation influences sex-biased gene expression. A limitation of the previous studies, however, was that they focused on endogenous X-linked genes and, thus, could not disentangle the effects of chromosomal position from those of gene-specific regulation. To overcome this limitation, we examined the expression of an exogenous reporter gene inserted at many locations spanning the X chromosome. We observed a negative correlation between the male-to-female expression ratio of the reporter gene and its distance to the nearest DCC binding site in somatic tissues, but not in gonads. A reporter gene’s location relative to a DCC binding site had greater influence on its expression than the local regulatory elements of neighboring endogenous genes, suggesting that intra-chromosomal variation in the strength of dosage compensation is a major determinant of sex-biased gene expression. Average levels of sex-biased expression did not differ between head and brain, but there was greater positional effect variation in the brain, which may explain the observed excess of endogenous sex-biased genes located on the X chromosome in this tissue.

Progressive dosage compensation during Drosophila embryogenesis is reflected by gene arrangement.

In Drosophila melanogaster males, X-chromosome monosomy is compensated by chromosome-wide transcription activation. We found that complete dosage compensation during embryogenesis takes surprisingly long and is incomplete even after 10h of development. Although the activating dosage compensation complex (DCC) associates with the X-chromosome and MOF acetylates histone H4 early, many genes are not compensated. Acetylation levels on gene bodies continue to increase for several hours after gastrulation in parallel with progressive compensation. Constitutive genes are compensated earlier than developmental genes. Remarkably, later compensation correlates with longer distances to DCC binding sites. This time-space relationship suggests that DCC action on target genes requires maturation of the active chromosome compartment.

Binding of an X-Specific Condensin Correlates with a Reduction in Active Histone Modifications at Gene Regulatory Elements.

Condensins are evolutionarily conserved protein complexes that are required for chromosome segregation during cell division and genome organization during interphase. In Caenorhabditis elegans, a specialized condensin, which forms the core of the dosage compensation complex (DCC), binds to and represses X chromosome transcription. Here, we analyzed DCC localization and the effect of DCC depletion on histone modifications, transcription factor binding, and gene expression using chromatin immunoprecipitation sequencing and mRNA sequencing. Across the X, the DCC accumulates at accessible gene regulatory sites in active chromatin and not heterochromatin. The DCC is required for reducing the levels of activating histone modifications, including H3K4me3 and H3K27ac, but not repressive modification H3K9me3. In X-to-autosome fusion chromosomes, DCC spreading into the autosomal sequences locally reduces gene expression, thus establishing a direct link between DCC binding and repression. Together, our results indicate that DCC-mediated transcription repression is associated with a reduction in the activity of X chromosomal gene regulatory elements.

Factor cooperation for chromosome discrimination in Drosophila.

Transcription regulators select their genomic binding sites from a large pool of similar, non-functional sequences. Although general principles that allow such discrimination are known, the complexity of DNA elements often precludes a prediction of functional sites. The process of dosage compensation in Drosophila allows exploring the rules underlying binding site selectivity. The male-specific-lethal (MSL) Dosage Compensation Complex (DCC) selectively binds to some 300 X chromosomal ‘High Affinity Sites’ (HAS) containing GA-rich ‘MSL recognition elements’ (MREs), but disregards thousands of other MRE sequences in the genome. The DNA-binding subunit MSL2 alone identifies a subset of MREs, but fails to recognize most MREs within HAS. The ‘Chromatin-linked adaptor for MSL proteins’ (CLAMP) also interacts with many MREs genome-wide and promotes DCC binding to HAS. Using genome-wide DNA-immunoprecipitation we describe extensive cooperativity between both factors, depending on the nature of the binding sites. These are explained by physical interaction between MSL2 and CLAMP. In vivo, both factors cooperate to compete with nucleosome formation at HAS. The male-specific MSL2 thus synergises with a ubiquitous GA-repeat binding protein for refined X/autosome discrimination.

Chromosome topology guides the Drosophila Dosage Compensation Complex for target gene activation.

X chromosome dosage compensation in Drosophila requires chromosome-wide coordination of gene activation. The male-specific lethal dosage compensation complex (DCC) identifies and binds to X-chromosomal high-affinity sites (HAS) from which it boosts transcription. A sub-class of HAS, PionX sites, represent first contacts on the X. Here, we explored the chromosomal interactions of representative PionX sites by high-resolution 4C and determined the global chromosome conformation by Hi-C in sex-sorted embryos. Male and female X chromosomes display similar nuclear architecture, concordant with clustered, constitutively active genes. PionX sites, like HAS, are evenly distributed in the active compartment and engage in short- and long-range interactions beyond compartment boundaries. Long-range, inter-domain interactions between DCC binding sites are stronger in males, suggesting that the complex refines chromatin organization. By de novo induction of DCC in female cells, we monitored the extent of activation surrounding PionX sites. This revealed a remarkable range of DCC action not only in linear proximity, but also at megabase distance if close in space, suggesting that DCC profits from pre-existing chromosome folding to activate genes.

Cooperation between a hierarchical set of recruitment sites targets the X chromosome for dosage compensation.

In many organisms, it remains unclear how X chromosomes are specified for dosage compensation, since DNA sequence motifs shown to be important for dosage compensation complex (DCC) recruitment are themselves not X-specific. Here, we addressed this problem in C. elegans. We found that the DCC recruiter, SDC-2, is required to maintain open chromatin at a small number of primary DCC recruitment sites, whose sequence and genomic context are X-specific. Along the X, primary recruitment sites are interspersed with secondary sites, whose function is X-dependent. A secondary site can ectopically recruit the DCC when additional recruitment sites are inserted either in tandem or at a distance (>30 kb). Deletion of a recruitment site on the X results in reduced DCC binding across several megabases surrounded by topologically associating domain (TAD) boundaries. Our work elucidates that hierarchy and long-distance cooperativity between gene-regulatory elements target a single chromosome for regulation.

Faster-X evolution of gene expression in Drosophila.

DNA sequences on X chromosomes often have a faster rate of evolution when compared to similar loci on the autosomes, and well articulated models provide reasons why the X-linked mode of inheritance may be responsible for the faster evolution of X-linked genes. We analyzed microarray and RNA–seq data collected from females and males of six Drosophila species and found that the expression levels of X-linked genes also diverge faster than autosomal gene expression, similar to the “faster-X” effect often observed in DNA sequence evolution. Faster-X evolution of gene expression was recently described in mammals, but it was limited to the evolutionary lineages shortly following the creation of the therian X chromosome. In contrast, we detect a faster-X effect along both deep lineages and those on the tips of the Drosophila phylogeny. In Drosophila males, the dosage compensation complex (DCC) binds the X chromosome, creating a unique chromatin environment that promotes the hyper-expression of X-linked genes. We find that DCC binding, chromatin environment, and breadth of expression are all predictive of the rate of gene expression evolution. In addition, estimates of the intraspecific genetic polymorphism underlying gene expression variation suggest that X-linked expression levels are not under relaxed selective constraints. We therefore hypothesize that the faster-X evolution of gene expression is the result of the adaptive fixation of beneficial mutations at X-linked loci that change expression level in cis. This adaptive faster-X evolution of gene expression is limited to genes that are narrowly expressed in a single tissue, suggesting that relaxed pleiotropic constraints permit a faster response to selection. Finally, we present a conceptional framework to explain faster-X expression evolution, and we use this framework to examine differences in the faster-X effect between Drosophila and mammals.

Regulation of DCC Localization by HTZ-1/H2A.Z and DPY-30 Does not Correlate with H3K4 Methylation Levels

Dosage compensation is a specialized form of gene regulation that balances sex-chromosome linked gene expression between the sexes. In C. elegans, dosage compensation is achieved by the activity of the dosage compensation complex (DCC). The DCC binds along both X chromosomes in hermaphrodites to down-regulate gene expression by half, limiting X-linked gene products to levels produced in XO males. Sequence motifs enriched on the X chromosome play an important role in targeting the DCC to the X. However, these motifs are not strictly X-specific and therefore other factors, such as the chromatin environment of the X chromosome, are likely to aid in DCC targeting. Previously, we found that loss of HTZ-1 results in partial disruption of dosage compensation localization to the X chromosomes. We wanted to know whether other chromatin components coordinated with HTZ-1 to regulate DCC localization. One candidate is DPY-30, a protein known to play a role in DCC localization. DPY-30 homologs in yeast, flies, and mammals are highly conserved members of histone H3 lysine 4 (H3K4) methyltransferase Set1/MLL complexes. Therefore, we investigated the hypothesis that the dosage compensation function of DPY-30 involves H3K4 methylation. We found that in dpy-30 animals the DCC fails to stably bind chromatin. Interestingly, of all the C. elegans homologs of Set1/MLL complex subunits, only DPY-30 is required for stable DCC binding to chromatin. Additionally, loss of H3K4 methylation does not enhance DCC mislocalization in htz-1 animals. We conclude that DPY-30 and HTZ-1 have unique functions in DCC localization, both of which are largely independent of H3K4 methylation.

An MLL/COMPASS subunit functions in the C. elegans dosage compensation complex to target X chromosomes for transcriptional regulation of gene expression.

Here we analyze the essential process of X-chromosome dosage compensation (DC) to elucidate mechanisms that control the assembly, genome-wide binding, and function of gene regulatory complexes that act over large chromosomal territories. We demonstrate that a subunit of Caenorhabditis elegans MLL/COMPASS, a gene activation complex, acts within the DC complex (DCC), a condensin complex, to target the DCC to both X chromosomes of hermaphrodites for chromosome-wide reduction of gene expression. The DCC binds to two categories of sites on X: rex (recruitment element on X) sites that recruit the DCC in an autonomous, sequence-dependent manner, and dox (dependent on X) sites that reside primarily in promoters of expressed genes and bind the DCC robustly only when attached to X. We find that DC mutations that abolish rex site binding greatly reduce dox site binding but do not eliminate it. Instead, binding is diminished to the low level observed at autosomal sites in wild-type animals. Changes in DCC binding to these non-rex sites occur throughout development and correlate directly with transcriptional activity of adjacent genes. Moreover, autosomal DCC binding is enhanced by rex site binding in cis in X-autosome fusion chromosomes. Thus, dox and autosomal sites have similar binding potential but are distinguished by linkage to rex sites. We propose a model for DCC binding in which low-level DCC binding at dox sites is dictated by intrinsic properties correlated with high transcriptional activity. Sex-specific DCC recruitment to rex sites then enhances the magnitude of DCC binding to dox sites in cis, which lack high affinity for the DCC on their own. We also show that the DCC balances X-chromosome gene expression between sexes by controlling transcription.

A sequence motif enriched in regions bound by the Drosophila dosage compensation complex

BackgroundIn Drosophila melanogaster, dosage compensation is mediated by the action of the dosage compensation complex (DCC). How the DCC recognizes the fly X chromosome is still poorly understood. Characteristic sequence signatures at all DCC binding sites have not hitherto been found.ResultsIn this study, we compare the known binding sites of the DCC with oligonucleotide profiles that measure the specificity of the sequences of the D. melanogaster X chromosome. We show that the X chromosome regions bound by the DCC are enriched for a particular type of short, repetitive sequences. Their distribution suggests that these sequences contribute to chromosome recognition, the generation of DCC binding sites and/or the local spreading of the complex. Comparative data indicate that the same sequences may be involved in dosage compensation in other Drosophila species.ConclusionsThese results offer an explanation for the wild-type binding of the DCC along the Drosophila X chromosome, contribute to delineate the forces leading to the establishment of dosage compensation and suggest new experimental approaches to understand the precise biochemical features of the dosage compensation system.

The C. elegans Dosage Compensation Complex Propagates Dynamically and Independently of X Chromosome Sequence

The C. elegans dosage compensation complex (DCC) associates with both X chromosomes of XX animals to reduce X-linked transcript levels. Five DCC members are homologous to subunits of the evolutionarily conserved condensin complex, and two noncondensin subunits are required for DCC recruitment to X. We investigated the molecular mechanism of DCC recruitment and spreading along X by examining gene expression and the binding patterns of DCC subunits in different stages of development, and in strains harboring X;autosome (X;A) fusions. We show that DCC binding is dynamically specified according to gene activity during development and that the mechanism of DCC spreading is independent of X chromosome DNA sequence. Accordingly, in X;A fusion strains, DCC binding propagates from X-linked recruitment sites onto autosomal promoters as a function of distance. Quantitative analysis of spreading suggests that the condensin-like subunits spread from recruitment sites to promoters more readily than subunits involved in initial X targeting. A highly conserved chromatin complex is appropriated to accomplish domain-scale transcriptional regulation during C. elegans development. Unlike X recognition, which is specified partly by DNA sequence, spreading is sequence independent and coupled to transcriptional activity. Similarities to the X recognition and spreading strategies used by the Drosophila DCC suggest mechanisms fundamental to chromosome-scale gene regulation.

The worm solution: a chromosome-full of condensin helps gene expression go down

Dosage compensation in the nematode Caenorhabditis elegans is achieved by the binding of a condensin-like dosage compensation complex (DCC) to both X chromosomes in hermaphrodites to downregulate gene expression two-fold. Condensin I(DC), a sub-part of the DCC, differs from the mitotic condensin I complex by a single subunit, strengthening the connection between dosage compensation and mitotic chromosome condensation. The DCC is targeted to X chromosomes by initial binding to a number of recruiting elements, followed by dispersal or spreading to secondary sites. While the complex is greatly enriched on the X chromosomes, many sites on autosomes also bind the complex. DCC binding does not correlate with DCC-mediated repression, suggesting that the complex acts in a chromosome-wide manner, rather than on a gene-by-gene basis. Worm dosage compensation represents an excellent model system to study how condensin-mediated changes in higher order chromatin organization affect gene expression.

Two classes of dosage compensation complex binding elements along Caenorhabditis elegans X chromosomes.

Dosage compensation equalizes X-linked gene products between the sexes. In Caenorhabditis elegans, the dosage compensation complex (DCC) binds both X chromosomes in XX animals and halves the transcription from each. The DCC is recruited to the X chromosomes by a number of loci, rex sites, and is thought to spread from these sites by an unknown mechanism to cover the rest of the chromosome. Here we describe a novel class of DCC-binding elements that we propose serve as "way stations" for DCC binding and spreading. Both rex sites and way stations comprise strong foci of DCC binding on the native X chromosome. However, rex sites maintain their ability to bind large amounts of DCC even on X duplications detached from the native X, while way stations do not. These results suggest that two distinct classes of DCC-binding elements facilitate recruitment and spreading of the DCC along the X chromosome.

A condensin-like dosage compensation complex acts at a distance to control expression throughout the genome.

In many species, a dosage compensation complex (DCC) is targeted to X chromosomes of one sex to equalize levels of X-gene products between males (1X) and females (2X). Here we identify cis-acting regulatory elements that target the Caenorhabditis elegans X chromosome for repression by the DCC. The DCC binds to discrete, dispersed sites on X of two types. rex sites (recruitment elements on X) recruit the DCC in an autonomous, DNA sequence-dependent manner using a 12-base-pair (bp) consensus motif that is enriched on X. This motif is critical for DCC binding, is clustered in rex sites, and confers much of X-chromosome specificity. Motif variants enriched on X by 3.8-fold or more are highly predictive (95%) for rex sites. In contrast, dox sites (dependent on X) lack the X-enriched variants and cannot bind the DCC when detached from X. dox sites are more prevalent than rex sites and, unlike rex sites, reside preferentially in promoters of some expressed genes. These findings fulfill predictions for a targeting model in which the DCC binds to recruitment sites on X and disperses to discrete sites lacking autonomous recruitment ability. To relate DCC binding to function, we identified dosage-compensated and noncompensated genes on X. Unexpectedly, many genes of both types have bound DCC, but many do not, suggesting the DCC acts over long distances to repress X-gene expression. Remarkably, the DCC binds to autosomes, but at far fewer sites and rarely at consensus motifs. DCC disruption causes opposite effects on expression of X and autosomal genes. The DCC thus acts at a distance to impact expression throughout the genome.

Dual sex-specific functions ofDrosophilaUpstream of N-ras in the control of X chromosome dosage compensation

Dosage compensation in Drosophila melanogaster involves the assembly of the MSL-2-containing dosage compensation complex (DCC) on the single X chromosome of male flies. Translational repression of msl-2 mRNA blocks this process in females. Previous work indicated that the ubiquitous protein Upstream of N-ras (UNR) is a necessary co-factor for msl-2 repression in vitro. Here, we explore the function of UNR in vivo. Hypomorphic Unr mutant flies showed DCC assembly on high-affinity sites in the female X chromosomes, confirming that UNR inhibits dosage compensation in female flies. Unexpectedly, male mutant flies and UNR-depleted SL2 cells showed decreased DCC binding to the X chromosome, suggesting a role for UNR in DCC assembly or targeting. Consistent with this possibility, UNR overexpression resulted in moderate loss of DCC from the male X chromosome and predominant male lethality. Immunoprecipitation experiments revealed that UNR binds to roX1 and roX2, the non-coding RNA components of the DCC, providing possible targets for UNR function in males. These results uncover dual sex-specific functions of UNR in dosage compensation: to repress DCC formation in female flies and to promote DCC assembly on the male X chromosome.

The Chromosomal High-Affinity Binding Sites for the Drosophila Dosage Compensation Complex

Dosage compensation in male Drosophila relies on the X chromosome–specific recruitment of a chromatin-modifying machinery, the dosage compensation complex (DCC). The principles that assure selective targeting of the DCC are unknown. According to a prevalent model, X chromosome targeting is initiated by recruitment of the DCC core components, MSL1 and MSL2, to a limited number of so-called “high-affinity sites” (HAS). Only very few such sites are known at the DNA sequence level, which has precluded the definition of DCC targeting principles. Combining RNA interference against DCC subunits, limited crosslinking, and chromatin immunoprecipitation coupled to probing high-resolution DNA microarrays, we identified a set of 131 HAS for MSL1 and MSL2 and confirmed their properties by various means. The HAS sites are distributed all over the X chromosome and are functionally important, since the extent of dosage compensation of a given gene and its proximity to a HAS are positively correlated. The sites are mainly located on non-coding parts of genes and predominantly map to regions that are devoid of nucleosomes. In contrast, the bulk of DCC binding is in coding regions and is marked by histone H3K36 methylation. Within the HAS, repetitive DNA sequences mainly based on GA and CA dinucleotides are enriched. Interestingly, DCC subcomplexes bind a small number of autosomal locations with similar features.