Dosage Compensation Complex Subunits Research Articles

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.

Anchoring of Heterochromatin to the Nuclear Lamina Reinforces Dosage Compensation-Mediated Gene Repression.

Higher order chromosome structure and nuclear architecture can have profound effects on gene regulation. We analyzed how compartmentalizing the genome by tethering heterochromatic regions to the nuclear lamina affects dosage compensation in the nematode C. elegans. In this organism, the dosage compensation complex (DCC) binds both X chromosomes of hermaphrodites to repress transcription two-fold, thus balancing gene expression between XX hermaphrodites and XO males. X chromosome structure is disrupted by mutations in DCC subunits. Using X chromosome paint fluorescence microscopy, we found that X chromosome structure and subnuclear localization are also disrupted when the mechanisms that anchor heterochromatin to the nuclear lamina are defective. Strikingly, the heterochromatic left end of the X chromosome is less affected than the gene-rich middle region, which lacks heterochromatic anchors. These changes in X chromosome structure and subnuclear localization are accompanied by small, but significant levels of derepression of X-linked genes as measured by RNA-seq, without any observable defects in DCC localization and DCC-mediated changes in histone modifications. We propose a model in which heterochromatic tethers on the left arm of the X cooperate with the DCC to compact and peripherally relocate the X chromosomes, contributing to gene repression.

SUMOylation is essential for sex-specific assembly and function of the Caenorhabditis elegans dosage compensation complex on X chromosomes

The essential process of dosage compensation equalizes X-chromosome gene expression between Caenorhabditis elegans XO males and XX hermaphrodites through a dosage compensation complex (DCC) that is homologous to condensin. The DCC binds to both X chromosomes of hermaphrodites to repress transcription by half. Here, we show that posttranslational modification by the SUMO (small ubiquitin-like modifier) conjugation pathway is essential for sex-specific assembly and function of the DCC on X. Depletion of SUMO in vivo severely disrupts binding of particular DCC subunits and causes changes in X-linked gene expression similar to those caused by deleting genes encoding DCC subunits. Three DCC subunits are SUMOylated, and SUMO depletion preferentially reduces their binding to X, suggesting that SUMOylation of DCC subunits is essential for robust association with X. DCC SUMOylation is triggered by the signal that initiates DCC assembly onto X. The initial step of assembly-binding of X-targeting factors to recruitment sites on X-is independent of SUMOylation, but robust binding of the complete complex requires SUMOylation. SUMOylated DCC subunits are enriched at recruitment sites, and SUMOylation likely enhances interactions between X-targeting factors and condensin subunits that facilitate DCC binding beyond the low level achieved without SUMOylation. DCC subunits also participate in condensin complexes essential for chromosome segregation, but their SUMOylation occurs only in the context of the DCC. Our results reinforce a newly emerging theme in which multiple proteins of a complex are collectively SUMOylated in response to a specific stimulus, leading to accelerated complex formation and enhanced function.

The DNA binding CXC domain of MSL2 is required for faithful targeting the Dosage Compensation Complex to the X chromosome

Dosage compensation in Drosophila melanogaster involves the selective targeting of the male X chromosome by the dosage compensation complex (DCC) and the coordinate, ∼2-fold activation of most genes. The principles that allow the DCC to distinguish the X chromosome from the autosomes are not understood. Targeting presumably involves DNA sequence elements whose combination or enrichment mark the X chromosome. DNA sequences that characterize ‘chromosomal entry sites’ or ‘high-affinity sites’ may serve such a function. However, to date no DNA binding domain that could interpret sequence information has been identified within the subunits of the DCC. Early genetic studies suggested that MSL1 and MSL2 serve to recognize high-affinity sites (HAS) in vivo, but a direct interaction of these DCC subunits with DNA has not been studied. We now show that recombinant MSL2, through its CXC domain, directly binds DNA with low nanomolar affinity. The DNA binding of MSL2 or of an MSL2–MSL1 complex does not discriminate between different sequences in vitro, but in a reporter gene assay in vivo, suggesting the existence of an unknown selectivity cofactor. Reporter gene assays and localization of GFP-fusion proteins confirm the important contribution of the CXC domain for DCC targeting in vivo.

The dosage compensation complex shapes the conformation of the X chromosome in Drosophila

The dosage compensation complex (DCC) in Drosophila globally increases transcription from the X chromosome in males to compensate for its monosomy. We discovered a male-specific conformation of the X chromosome that depends on the associations of high-affinity binding sites (HAS) of the DCC. The core DCC subunits MSL1-MSL2 are responsible for this male-specific organization. Contrary to emerging concepts, we found that neither DCC assembly nor the conformation of the male X chromosome are influenced by nuclear pore components. We propose that nuclear organization of HAS is central to the faithful distribution of the DCC along the X chromosome.

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.

C. elegans dosage compensation: A window into mechanisms of domain-scale gene regulation

The C. elegans dosage compensation complex (DCC) reduces transcript levels from each of the two hermaphrodite X chromosomes to equalize X-linked gene expression to that of XO males. Several of the proteins that comprise the DCC are homologous to subunits of the evolutionarily conserved condensin complexes, which in most organisms function in mitotic and meiotic chromosome condensation. These include the DCC subunits MIX-1 and DPY-27, which belong to the structural maintenance of chromosomes (SMC) family of proteins. Several of the C. elegans DCC subunits also perform double duty as members of the canonical meiotic and mitotic condensin complexes. Here, we review what is known about the C. elegans DCC and how study of this model might shed light on general mechanisms of domain-scale transcriptional regulation. We discuss how condensin-like complexes may be targeted to specific chromosomal locations for performance of their functions.

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.

Cumulative contributions of weak DNA determinants to targeting the Drosophila dosage compensation complex

Fine-tuning of X chromosomal gene expression in Drosophila melanogaster involves the selective interaction of the Dosage Compensation Complex (DCC) with the male X chromosome, in order to increase the transcription of many genes. However, the X chromosomal DNA sequences determining DCC binding remain elusive. By adapting a ‘one-hybrid’ assay, we identified minimal DNA elements that direct the interaction of the key DCC subunit, MSL2, in cells. Strikingly, several such novel MSL2 recruitment modules have very different DNA sequences. The assay revealed a novel, 40 bp DNA element that is necessary for recruitment of DCC to an autosomal binding site in flies in the context of a longer sequence and sufficient by itself to direct recruitment if trimerized. Accordingly, recruitment of MSL2 to the single 40 bp element in cells was weak, but as a trimer approached the power of the strongest DCC recruitment site known to date, the roX1 DH site. This element is the shortest MSL2 recruitment sequence known to date. The results support a model for MSL2 recruitment according to which several different, degenerate sequence motifs of variable affinity cluster and synergise to form a high affinity site.

The Drosophila dosage compensation complex binds to polytene chromosomes independently of developmental changes in transcription.

In Drosophila, the dosage compensation complex (DCC) mediates upregulation of transcription from the single male X chromosome. Despite coating the polytene male X, the DCC pattern looks discontinuous and probably reflects DCC dynamic associations with genes active at a given moment of development in a salivary gland. To test this hypothesis, we compared binding patterns of the DCC and of the elongating form of RNA polymerase II (PolIIo). We found that, unlike PolIIo, the DCC demonstrates a stable banded pattern throughout larval development and escapes binding to a subset of transcriptionally active areas, including developmental puffs. Moreover, these proteins are not completely colocalized at the electron microscopy level. These data combined imply that simple recognition of PolII machinery or of general features of active chromatin is either insufficient or not involved in DCC recruitment to its targets. We propose that DCC-mediated site-specific upregulation of transcription is not the fate of all active X-linked genes in males. Additionally, we found that DCC subunit MLE associates dynamically with developmental and heat-shock-induced puffs and, surprisingly, with those developing within DCC-devoid regions of the male X, thus resembling the PolIIo pattern. These data imply that, independently of other MSL proteins, the RNA-helicase MLE might participate in general transcriptional regulation or RNA processing.