Dosage Compensation Complex Research Articles

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.

Two-step mechanism for selective incorporation of lncRNA into a chromatin modifier

The MLE DExH helicase and the roX lncRNAs are essential components of the chromatin modifying Dosage Compensation Complex (DCC) in Drosophila. To explore the mechanism of ribonucleoprotein complex assembly, we developed vitRIP, an unbiased, transcriptome-wide in vitro assay that reveals RNA binding specificity. We found that MLE has intrinsic specificity for U-/A-rich sequences and tandem stem-loop structures and binds many RNAs beyond roX in vitro. The selectivity of the helicase for physiological substrates is further enhanced by the core DCC. Unwinding of roX2 by MLE induces a highly selective RNA binding surface in the unstructured C-terminus of the MSL2 subunit and triggers-specific association of MLE and roX2 with the core DCC. The exquisite selectivity of roX2 incorporation into the DCC thus originates from intimate cooperation between the helicase and the core DCC involving two distinct RNA selection principles and their mutual refinement.

Faint gray bands in Drosophila melanogaster polytene chromosomes are formed by coding sequences of housekeeping genes.

In Drosophila melanogaster, the chromatin of interphase polytene chromosomes appears as alternating decondensed interbands and dense black or thin gray bands. Recently, we uncovered four principle chromatin states (4НММ model) in the fruit fly, and these were matched to the structures observed in polytene chromosomes. Ruby/malachite chromatin states form black bands containing developmental genes, whereas aquamarine chromatin corresponds to interbands enriched with 5' regions of ubiquitously expressed genes. Lazurite chromatin supposedly forms faint gray bands and encompasses the bodies of housekeeping genes. In this report, we test this idea using the X chromosome as the model and MSL1 as a protein marker of the lazurite chromatin. Our bioinformatic analysis indicates that in the X chromosome, it is only the lazurite chromatin that is simultaneously enriched for the proteins and histone marks associated with exons, transcription elongation, and dosage compensation. As a result of FISH and EM mapping of a dosage compensation complex subunit, MSL1, we for the first time provide direct evidence that lazurite chromatin forms faint gray bands. Our analysis proves that overall most of housekeeping genes typically span from the interbands (5' region of the gene) to the gray band (gene body). More rarely, active lazurite chromatin and inactive malachite/ruby chromatin may be found within a common band, where both the housekeeping and the developmental genes reside together.

Targeting of the Dosage-Compensated Male X-Chromosome during Early Drosophila Development

SUMMARYDosage compensation, which corrects for the imbalance in X-linked gene expression between XX females and XY males, represents a model for how genes are targeted for coordinated regulation. However, the mechanism by which dosage compensation complexes identify the X chromosome during early development remains unknown because of the difficulty of sexing embryos before zygotic transcription using X- or Y-linked reporter transgenes. We used meiotic drive to sex Drosophila embryos before zygotic transcription and ChIP-seq to measure the dynamics of dosage compensation factor targeting. The Drosophila male-specific lethal dosage compensation complex (MSLc) requires the ubiquitous zinc-finger protein chromatin-linked adaptor for MSL proteins (CLAMP) to identify the X chromosome. We observe a multi-stage process in which MSLc first identifies CLAMP binding sites throughout the genome, followed by concentration at the strongest X-linked MSLc sites. We provide insight into the dynamics of binding site recognition by a large transcription complex during early development.

Global chromatin conformation differences in the Drosophila dosage compensated chromosome X

In Drosophila melanogaster the single male chromosome X undergoes an average twofold transcriptional upregulation for balancing the transcriptional output between sexes. Previous literature hypothesised that a global change in chromosome structure may accompany this process. However, recent studies based on Hi-C failed to detect these differences. Here we show that global conformational differences are specifically present in the male chromosome X and detectable using Hi-C data on sex-sorted embryos, as well as male and female cell lines, by leveraging custom data analysis solutions. We find the male chromosome X has more mid-/long-range interactions. We also identify differences at structural domain boundaries containing BEAF-32 in conjunction with CP190 or Chromator. Weakening of these domain boundaries in male chromosome X co-localizes with the binding of the dosage compensation complex and its co-factor CLAMP, reported to enhance chromatin accessibility. Together, our data strongly indicate that chromosome X dosage compensation affects global chromosome structure.

Multilayered Tuning of Dosage Compensation and Z-Chromosome Masculinization in the Wood White (Leptidea sinapis) Butterfly.

In species with genetic sex determination, dosage compensation can evolve to equal expression levels of sex-linked and autosomal genes. Current knowledge about dosage compensation has mainly been derived from male-heterogametic (XX/XY) model organisms, whereas less is understood about the process in female-heterogametic systems (ZZ/ZW). In moths and butterflies, downregulation of Z-linked expression in males (ZZ) to match the expression level in females (ZW) is often observed. However, little is known about the underlying regulatory mechanisms, or if dosage compensation patterns vary across ontogenetic stages. In this study, we assessed dynamics of Z-linked and autosomal expression levels across developmental stages in the wood white (Leptidea sinapis). We found that although expression of Z-linked genes in general was reduced compared with autosomal genes, dosage compensation was actually complete for some categories of genes, in particular sex-biased genes, but equalization in females was constrained to a narrower gene set. We also observed a noticeable convergence in Z-linked expression between males and females after correcting for sex-biased genes. Sex-biased expression increased successively across developmental stages, and male-biased genes were enriched on the Z-chromosome. Finally, all five core genes associated with the ribonucleoprotein dosage compensation complex male-specific lethal were detected in adult females, in correspondence with a reduction in the expression difference between autosomes and the single Z-chromosome. We show that tuning of gene dosage is multilayered in Lepidoptera and argue that expression balance across chromosomal classes may predominantly be driven by enrichment of male-biased genes on the Z-chromosome and cooption of available dosage regulators.

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.

Structural insights reveal the specific recognition of roX RNA by the dsRNA-binding domains of the RNA helicase MLE and its indispensable role in dosage compensation in Drosophila.

In Drosophila, dosage compensation globally upregulates the expression of genes located on male single X-chromosome. Maleless (MLE) helicase plays an essential role to incorporate the roX lncRNA into the dosage compensation complex (MSL-DCC), and such function is essentially dependent on its dsRNA-binding domains (dsRBDs). Here, we report a 2.90Å crystal structure of tandem dsRBDs of MLE in complex with a 55mer stem-loop of roX2 (R2H1). MLE dsRBDs bind to R2H1 cooperatively and interact with two successive minor grooves and a major groove of R2H1, respectively. The recognition of R2H1 by MLE dsRBDs involves both shape- and sequence-specificity. Moreover, dsRBD2 displays a stronger RNA affinity than dsRBD1, and mutations of key residues in either MLE dsRBD remarkably reduce their affinities for roX2 both in vitro and in vivo. In Drosophila, the structure-based mle mutations generated using the CRISPR/Cas9 system, are partially male-lethal and indicate the inter-regulation among the components of the MSL-DCC at multiple levels. Hence, our research provides structural insights into the interactions between MLE dsRBDs and R2H1 and facilitates a deeper understanding of the mechanism by which MLE tandem dsRBDs play an indispensable role in specific recognition of roX and the assembly of the MSL-DCC in Drosophila dosage compensation.

Targeting of the Dosage-Compensated Male X-Chromosome During Early <i>Drosophila</i> Development

The essential process of dosage compensation, which corrects for the imbalance in X-linked gene expression between XX females and XY males, represents a key model for how genes are targeted for coordinated regulation. However, the mechanism by which dosage compensation complexes identify the X-chromosome during early development remained unknown because of the difficulty of sexing embryos prior to zygotic transcription. We used meiotic drive to sex Drosophila embryos prior to zygotic transcription and ChIP-seq to measure dynamics of dosage compensation factor targeting. The Drosophila Male-Specific Lethal dosage compensation complex (MSLc) requires the ubiquitous zinc-finger protein Chromatin-Linked Adaptor for MSL Proteins (CLAMP) to identify the X-chromosome. We observe a multi-stage process in which MSLc first identifies CLAMP binding sites throughout the genome followed by concentration at the strongest X-linked MSLc sites. We provide insight into the dynamic mechanism by which a large transcription complex identifies its binding sites during early development.

The simultaneous interaction of MSL2 with CLAMP and DNA provides redundancy in the initiation of dosage compensation in Drosophila males

The binding of the Drosophila male-specific lethal dosage compensation complex (DCC) exclusively to the male X chromosome provides an excellent model system to understand mechanisms of selective recruitment of protein complexes to chromatin. Previous studies showed that the male-specific organizer of the complex, MSL2, and the ubiquitous DNA-binding protein CLAMP are key players in the specificity of X chromosome binding. The CXC domain of MSL2 binds to genomic sites of DCC recruitment in vitro Another conserved domain of MSL2, named Clamp-binding domain (CBD) directly interacts with the N-terminal zinc-finger domain of CLAMP. Here, we found that inactivation of CBD or CXC individually only modestly affected recruitment of the DCC to the X chromosome in males. However, combination of these two genetic lesions within the same MSL2 mutant resulted in an increased loss of DCC recruitment to the X chromosome. Thus, proper MSL2 positioning requires an interaction with either CLAMP or DNA to initiate dosage compensation in Drosophila males.

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.

Sex-specific phenotypes of histone H4 point mutants establish dosage compensation as the critical function of H4K16 acetylation in Drosophila

Acetylation of histone H4 at lysine 16 (H4K16) modulates nucleosome-nucleosome interactions and directly affects nucleosome binding by certain proteins. In Drosophila, H4K16 acetylation by the dosage compensation complex subunit Mof is linked to increased transcription of genes on the single X chromosome in males. Here, we analyzed Drosophila containing different H4K16 mutations or lacking Mof protein. An H4K16A mutation causes embryonic lethality in both sexes, whereas an H4K16R mutation permits females to develop into adults but causes lethality in males. The acetyl-mimic mutation H4K16Q permits both females and males to develop into adults. Complementary analyses reveal that males lacking maternally deposited and zygotically expressed Mof protein arrest development during gastrulation, whereas females of the same genotype develop into adults. Together, this demonstrates the causative role of H4K16 acetylation by Mof for dosage compensation in Drosophila and uncovers a previously unrecognized requirement for this process already during the onset of zygotic gene transcription.

Sex and death: from cell fate specification to dynamic control of X-chromosome structure and gene expression.

Determining sex is a binary developmental decision that most metazoans must make. Like many organisms, Caenorhabditis elegans specifies sex (XO male or XX hermaphrodite) by tallying X-chromosome number. We dissected this precise counting mechanism to determine how tiny differences in concentrations of signals are translated into dramatically different developmental fates. Determining sex by counting chromosomes solved one problem but created another—an imbalance in X gene products. We found that nematodes compensate for the difference in X-chromosome dose between sexes by reducing transcription from both hermaphrodite X chromosomes. In a surprising feat of evolution, X-chromosome regulation is functionally related to a structural problem of all mitotic and meiotic chromosomes: achieving ordered compaction of chromosomes before segregation. We showed the dosage compensation complex is a condensin complex that imposes a specific three-­dimensional architecture onto hermaphrodite X chromosomes. It also triggers enrichment of histone modification H4K20me1. We discovered the machinery and mechanism underlying H4K20me1 enrichment and demonstrated its pivotal role in regulating higher-order X-chromosome structure and gene expression.

Non-canonical Drosophila X chromosome dosage compensation and repressive topologically associated domains

BackgroundIn animals with XY sex chromosomes, X-linked genes from a single X chromosome in males are imbalanced relative to autosomal genes. To minimize the impact of genic imbalance in male Drosophila, there is a dosage compensation complex (MSL) that equilibrates X-linked gene expression with the autosomes. There are other potential contributions to dosage compensation. Hemizygous autosomal genes located in repressive chromatin domains are often derepressed. If this homolog-dependent repression occurs on the X, which has no pairing partner, then derepression could contribute to male dosage compensation.ResultsWe asked whether different chromatin states or topological associations correlate with X chromosome dosage compensation, especially in regions with little MSL occupancy. Our analyses demonstrated that male X chromosome genes that are located in repressive chromatin states are depleted of MSL occupancy; however, they show dosage compensation. The genes in these repressive regions were also less sensitive to knockdown of MSL components.ConclusionsOur results suggest that this non-canonical dosage compensation is due to the same transacting derepression that occurs on autosomes. This mechanism would facilitate immediate compensation during the evolution of sex chromosomes from autosomes. This mechanism is similar to that of C. elegans, where enhanced recruitment of X chromosomes to the nuclear lamina dampens X chromosome expression as part of the dosage compensation response in XX individuals.

A Simple Method for Visualization of Locus-Specific H4K20me1 Modifications in Living Caenorhabditis elegans Single Cells.

Recently, advances in next-generation sequencing technologies have enabled genome-wide analyses of epigenetic modifications; however, it remains difficult to analyze the states of histone modifications at a single-cell resolution in living multicellular organisms because of the heterogeneity within cellular populations. Here we describe a simple method to visualize histone modifications on the specific sequence of target locus at a single-cell resolution in living Caenorhabditis elegans, by combining the LacO/LacI system and a genetically-encoded H4K20me1-specific probe, “mintbody”. We demonstrate that Venus-labeled mintbody and mTurquoise2-labeled LacI can co-localize on an artificial chromosome carrying both the target locus and LacO sequences, where H4K20me1 marks the target locus. We demonstrate that our visualization method can precisely detect H4K20me1 depositions on the her-1 gene sequences on the artificial chromosome, to which the dosage compensation complex binds to regulate sex determination. The degree of H4K20me1 deposition on the her-1 sequences on the artificial chromosome correlated strongly with sex, suggesting that, using the artificial chromosome, this method can reflect context-dependent changes of H4K20me1 on endogenous genomes. Furthermore, we demonstrate live imaging of H4K20me1 depositions on the artificial chromosome. Combined with ChIP assays, this mintbody-LacO/LacI visualization method will enable analysis of developmental and context-dependent alterations of locus-specific histone modifications in specific cells and elucidation of the underlying molecular mechanisms.

Transcriptional modulation of entire chromosomes: dosage compensation

Dosage compensation is a regulatory system designed to equalize the transcription output of the genes of the sex chromosomes that are present in different doses in the sexes (X or Z chromosome, depending on the animal species involved). Different mechanisms of dosage compensation have evolved in different animal groups. In Drosophila males, a complex (male-specific lethal) associates with the X chromosome and enhances the activity of most X-linked genes by increasing the rate of RNAPII elongation. In Caenorhabditis, a complex (dosage compensation complex) that contains a number of proteins involved in condensing chromosomes decreases the level of transcription of both X chromosomes in the XX hermaphrodite. In mammals, dosage compensation is achieved by the inactivation, early during development, of most X-linked genes on one of the two X chromosomes in females. The mechanism involves the synthesis of an RNA (Tsix) that protects one of the two Xs from inactivation, and of another RNA (Xist) that coats the other X chromosome and recruits histone and DNA modifying enzymes. This review will focus on the current progress in understanding the dosage compensation mechanisms in the three taxa where it has been best studied at the molecular level: flies, round worms and mammals.

Dosage Compensation of the X Chromosome: A Complex Epigenetic Assignment Involving Chromatin Regulators and Long Noncoding RNAs.

X chromosome regulation represents a prime example of an epigenetic phenomenon where coordinated regulation of a whole chromosome is required. In flies, this is achieved by transcriptional upregulation of X chromosomal genes in males to equalize the gene dosage differences in females. Chromatin-bound proteins and long noncoding RNAs (lncRNAs) constituting a ribonucleoprotein complex known as the male-specific lethal (MSL) complex or the dosage compensation complex mediate this process. MSL complex members decorate the male X chromosome, and their absence leads to male lethality. The male X chromosome is also enriched with histone H4 lysine 16 acetylation (H4K16ac), indicating that the chromatin compaction status of the X chromosome also plays an important role in transcriptional activation. How the X chromosome is specifically targeted and how dosage compensation is mechanistically achieved are central questions for the field. Here, we review recent advances, which reveal a complex interplay among lncRNAs, the chromatin landscape, transcription, and chromosome conformation that fine-tune X chromosome gene expression.

Differential Occupancy of Two GA-Binding Proteins Promotes Targeting of the Drosophila Dosage Compensation Complex to the Male X Chromosome

SUMMARYLittle is known about how variation in sequence composition alters transcription factor occupancy to precisely recruit large transcription complexes. A key model for understanding how transcription complexes are targeted is the Drosophila dosage compensation system in which the male-specific lethal (MSL) transcription complex specifically identifies and regulates the male X chromosome. The chromatin-linked adaptor for MSL proteins (CLAMP) zinc-finger protein targets MSL to the X chromosome but also binds to GA-rich sequence elements throughout the genome. Furthermore, the GAGA-associated factor (GAF) transcription factor also recognizes GA-rich sequences but does not associate with the MSL complex. Here, we demonstrate that MSL complex recruitment sites are optimal CLAMP targets. Specificity for CLAMP binding versus GAF binding is driven by variability in sequence composition within similar GA-rich motifs. Therefore, variation within seemingly similar cis elements drives the context-specific targeting of a large transcription complex.

The Drosophila CLAMP protein associates with diverse proteins on chromatin.

Gaining new insights into gene regulation involves an in-depth understanding of protein-protein interactions on chromatin. A powerful model for studying mechanisms of gene regulation is dosage compensation, a process that targets the X-chromosome to equalize gene expression between XY males and XX females. We previously identified a zinc finger protein in Drosophila melanogaster that plays a sex-specific role in targeting the Male-specific lethal (MSL) dosage compensation complex to the male X-chromosome, called the Chromatin-Linked Adapter for MSL Proteins (CLAMP). More recently, we established that CLAMP has non-sex-specific roles as an essential protein that regulates chromatin accessibility at promoters genome-wide. To identify associations between CLAMP and other factors in both male and female cells, we used two complementary mass spectrometry approaches. We demonstrate that CLAMP associates with the transcriptional regulator complex Negative Elongation Factor (NELF) in both sexes and determine that CLAMP reduces NELF recruitment to several target genes. In sum, we have identified many new CLAMP-associated factors and provide a resource for further study of this little understood essential protein.