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The Y chromosome harbors nine multi-copy ampliconic gene families expressed exclusively in testis. The gene copies within each family are >99% identical to each other, which poses a major challenge in evaluating their copy number. Recent studies demonstrated high variation in Y ampliconic gene copy number among humans. However, how this variation affects expression levels in human testis remains understudied. Here we developed a novel computational tool Ampliconic Copy Number Estimator (AmpliCoNE) that utilizes read sequencing depth information to estimate Y ampliconic gene copy number per family. We applied this tool to whole-genome sequencing data of 149 men with matched testis expression data whose samples are part of the Genotype-Tissue Expression (GTEx) project. We found that the Y ampliconic gene families with low copy number in humans were deleted or pseudogenized in non-human great apes, suggesting relaxation of functional constraints. Among the Y ampliconic gene families, higher copy number leads to higher expression. Within the Y ampliconic gene families, copy number does not influence gene expression, rather a high tolerance for variation in gene expression was observed in testis of presumably healthy men. No differences in gene expression levels were found among major Y haplogroups. Age positively correlated with expression levels of the HSFY and PRY gene families in the African subhaplogroup E1b, but not in the European subhaplogroups R1b and I1. We also found that expression of five Y ampliconic gene families is coordinated with that of their non-Y (i.e. X or autosomal) homologs. Indeed, five ampliconic gene families had consistently lower expression levels when compared to their non-Y homologs suggesting dosage regulation, while the HSFY family had higher expression levels than its X homolog and thus lacked dosage regulation.

In mammals, dosage compensation of sex chromosomal genes between females (XX) and males (XY) is achieved through X-chromosome inactivation (XCI). The X-linked X-inactive-specific transcript (Xist) long noncoding RNA is indispensable for XCI and initiates the process early during development by spreading in cis across the X chromosome from which it is transcribed. During XCI, Xist RNA triggers gene silencing, recruits a plethora of chromatin modifying factors, and drives a major structural reorganization of the X chromosome. Here, we review our knowledge of the multitude of epigenetic events orchestrated by Xist RNA to allow female mammals to survive through embryonic development by establishing and maintaining proper dosage compensation. In particular, we focus on recent studies characterizing the interaction partners of Xist RNA, and we discuss how they have affected the field by addressing long-standing controversies or by giving rise to new research perspectives that are currently being explored. This review is dedicated to the memory of Denise Barlow, pioneer of genomic imprinting and functional long noncoding RNAs (lncRNAs), whose work has revolutionized the epigenetics field and continues to inspire generations of scientists.

Key Points Genomes of multicellular organisms produce thousands of different long non-coding RNA (lncRNA) species. lncRNAs have crucial roles in gene expression control during developmental and differentiation processes. lncRNAs can regulate gene expression by several mechanisms in both the nucleus and the cytoplasm. lncRNAs drive the formation of ribonucleoprotein complexes and guide them to specific targets to regulate gene expression. Different in vitro and in vivo systems have shown the importance of lncRNAs in developmental processes, such as in dosage compensation, genomic imprinting, cell differentiation and organogenesis. lncRNAs can form regulative networks with other RNA species, such as microRNAs and mRNAs. Our knowledge of the diverse types and roles of long non-coding RNAs (lncRNAs) is rapidly increasing. This Review discusses our latest understanding of lncRNAs that have validated functional roles in various differentiation and developmental processes. Genomes of multicellular organisms are characterized by the pervasive expression of different types of non-coding RNAs (ncRNAs). Long ncRNAs (lncRNAs) belong to a novel heterogeneous class of ncRNAs that includes thousands of different species. lncRNAs have crucial roles in gene expression control during both developmental and differentiation processes, and the number of lncRNA species increases in genomes of developmentally complex organisms, which highlights the importance of RNA-based levels of control in the evolution of multicellular organisms. In this Review, we describe the function of lncRNAs in developmental processes, such as in dosage compensation, genomic imprinting, cell differentiation and organogenesis, with a particular emphasis on mammalian development.

Genome-wide chromosome conformation capture analysis in C. elegans reveals that the dosage compensation complex, a condensin complex, remodels the X chromosomes of hermaphrodites into a sex-specific topology distinct from autosomes while regulating gene expression chromosome-wide. X-chromosome takes shapes Dosage compensation in the roundworm Caenorhabditis elegans is a good model for understanding the role of three-dimensional chromosome organization in regulating gene expression. Here, Barbara Meyer and colleagues use genome-wide chromosome conformation capture techniques in wild-type XX hermaphrodite embryos and those lacking the dosage compensation complex (DCC), to obtain three-dimensional maps of the C. elegans genome. The DCC remodels hermaphrodite X chromosomes into a spatial conformation of topologically associating domains that is distinct from that on autosomes. The three-dimensional organization of a genome plays a critical role in regulating gene expression, yet little is known about the machinery and mechanisms that determine higher-order chromosome structure 1 , 2 . Here we perform genome-wide chromosome conformation capture analysis, fluorescent in situ hybridization (FISH), and RNA-seq to obtain comprehensive three-dimensional (3D) maps of the Caenorhabditis elegans genome and to dissect X chromosome dosage compensation, which balances gene expression between XX hermaphrodites and XO males. The dosage compensation complex (DCC), a condensin complex, binds to both hermaphrodite X chromosomes via sequence-specific recruitment elements on X ( rex sites) to reduce chromosome-wide gene expression by half 3 , 4 , 5 , 6 , 7 . Most DCC condensin subunits also act in other condensin complexes to control the compaction and resolution of all mitotic and meiotic chromosomes 5 , 6 . By comparing chromosome structure in wild-type and DCC-defective embryos, we show that the DCC remodels hermaphrodite X chromosomes into a sex-specific spatial conformation distinct from autosomes. Dosage-compensated X chromosomes consist of self-interacting domains (∼1 Mb) resembling mammalian topologically associating domains (TADs) 8 , 9 . TADs on X chromosomes have stronger boundaries and more regular spacing than on autosomes. Many TAD boundaries on X chromosomes coincide with the highest-affinity rex sites and become diminished or lost in DCC-defective mutants, thereby converting the topology of X to a conformation resembling autosomes. rex sites engage in DCC-dependent long-range interactions, with the most frequent interactions occurring between rex sites at DCC-dependent TAD boundaries. These results imply that the DCC reshapes the topology of X chromosomes by forming new TAD boundaries and reinforcing weak boundaries through interactions between its highest-affinity binding sites. As this model predicts, deletion of an endogenous rex site at a DCC-dependent TAD boundary using CRISPR/Cas9 greatly diminished the boundary. Thus, the DCC imposes a distinct higher-order structure onto X chromosomes while regulating gene expression chromosome-wide.

Sex chromosomes impact chromatin organization and histone modification dynamics differently between males and females, particularly those involved in dosage compensation (DC). The evolutionary diversity, as well as the tissue- and age-dependent variations of DC mechanisms are incompletely understood. Here, we investigate the occurrence of histone H4 lysine 16 acetylation (H4K16ac), previously known for its role in sex chromosome DC in the male-heterogametic fruit fly Drosophila melanogaster and the green anole lizard Anolis carolinensis . By sampling multiple arthropods, we find the convergent evolution of H4K16ac for DC in a female-heterogametic (ZW) species, the crustacean Artemia franciscana . CUT&Tag analysis demonstrates that H4K16ac is confined to the non-recombining stratum of the Z chromosome in females. H4K16ac-mediated DC is established during embryogenesis. In aged individuals, we observe an overall decline in nuclear organization, disrupted H4K16ac territories and increased variability in local acetylation levels on the female Z chromosome. Our findings shed light on the evolutionary diversity of DC across species and raise the possibility of sex-specific histone acetylation contributing to male-female differences in lifespan.

Proper control of gene expression levels upon various perturbations is a fundamental aspect of cellular robustness. Protein-level dosage compensation is one mechanism buffering perturbations to stoichiometry of multiprotein complexes through accelerated proteolysis of unassembled subunits. Although N-terminal acetylation- and ubiquitin-mediated proteasomal degradation by the Ac/N-end rule pathway enables selective compensation of excess subunits, it is unclear how widespread this pathway contributes to stoichiometry control. Here we report that dosage compensation depends only partially on the Ac/N-end rule pathway. Our analysis of genetic interactions between 18 subunits and 12 quality control factors in budding yeast demonstrated that multiple E3 ubiquitin ligases and N-acetyltransferases are involved in dosage compensation. We find that N-acetyltransferases-mediated compensation is not simply predictable from N-terminal sequence despite their sequence specificity for N-acetylation. We also find that the compensation of Pop3 and Bet4 is due in large part to a minor N-acetyltransferase NatD. Furthermore, canonical NatD substrates histone H2A/H4 were compensated even in its absence, suggesting N-acetylation-independent stoichiometry control. Our study reveals the complexity and robustness of the stoichiometry control system.

Background Mammalian sex chromosomes provide dosage compensation, but avian lack a global mechanism of dose compensation. Herein, we employed nanopore sequencing to investigate the genetic basis of gene expression and gene dosage effects in avian Z chromosomes at the posttranscriptional level. Results In this study, the gonad and head skin of female and male duck samples ( n  = 4) were collected at 16 weeks of age for Oxford nanopore sequencing. Our results revealed a dosage effect and local regulation of duck Z chromosome gene expression. Additionally, AS and APA achieve tissue-specific gene expression, and male-biased lncRNA regulates its Z-linked target genes, with a positive regulatory role for gene dosage effects on the duck Z chromosome. In addition, GO enrichment and KEGG pathway analysis showed that the dosage effects of Z-linked genes were mainly associated with the cellular response to hormone stimulus, melanin biosynthetic, metabolic pathways, and melanogenesis, resulting in sex differences. Conclusions Our data suggested that post transcriptional regulation (AS, APA and lncRNA) has a potential impact on the gene expression effects of avian Z chromosomes. Our study provides a new view of gene regulation underlying the dose effects in avian Z chromosomes at the RNA post transcriptional level.

Condensins are molecular motors that compact DNA via linear translocation. In Caenorhabditis elegans , the X-chromosome harbors a specialized condensin that participates in dosage compensation (DC). Condensin DC is recruited to and spreads from a small number of r ecruitment e lements on the X -chromosome ( rex ) and is required for the formation of topologically associating domains (TADs). We take advantage of autosomes that are largely devoid of condensin DC and TADs to address how rex sites and condensin DC give rise to the formation of TADs. When an autosome and X-chromosome are physically fused, despite the spreading of condensin DC into the autosome, no TAD was created. Insertion of a strong rex on the X-chromosome results in the TAD boundary formation regardless of sequence orientation. When the same rex is inserted on an autosome, despite condensin DC recruitment, there was no spreading or features of a TAD. On the other hand, when a ‘ super rex ’ composed of six rex sites or three separate rex sites are inserted on an autosome, recruitment and spreading of condensin DC led to the formation of TADs. Therefore, recruitment to and spreading from rex sites are necessary and sufficient for recapitulating loop-anchored TADs observed on the X-chromosome. Together our data suggest a model in which rex sites are both loading sites and bidirectional barriers for condensin DC, a one-sided loop-extruder with movable inactive anchor.

Dosage compensation, the equalized X chromosome gene expression between males and females in Drosophila , has also been found in triple X metafemales. Inverse dosage effects, produced by genomic imbalance, are believed to account for this modulated expression, but they have not been studied on a global level. Here, we show a global expression comparison of metafemales (XXX; AA) with normal females (XX; AA) with high-throughput RNA-sequencing. We found that the majority of the X-linked genes in metafemales exhibit dosage compensation with an expression level similar to that of normal diploid females. In parallel, most of the autosomal genes were expressed at about two-thirds the level of normal females, the ratio of inverse dosage effects produced by the extra X chromosome. Both compensation and inverse effects were further confirmed by combination of X-linked and autosomally located miniwhite reporter genes in metafemales and relative quantitative PCR of selected genes. These data provide evidence for an inverse dosage component to X chromosome compensation.

The repeatability or predictability of evolution is a central question in evolutionary biology and most often addressed in experimental evolution studies. Here, we infer how genetically heterogeneous natural systems acquire the same molecular changes to address how genomic background affects adaptation in natural populations. In particular, we take advantage of independently formed neo-sex chromosomes in Drosophila species that have evolved dosage compensation by co-opting the dosage-compensation male-specific lethal (MSL) complex to study the mutational paths that have led to the acquisition of hundreds of novel binding sites for the MSL complex in different species. This complex recognizes a conserved 21-bp GA-rich sequence motif that is enriched on the X chromosome, and newly formed X chromosomes recruit the MSL complex by de novo acquisition of this binding motif. We identify recently formed sex chromosomes in the D. melanica and D. robusta species groups by genome sequencing and generate genomic occupancy maps of the MSL complex to infer the location of novel binding sites. We find that diverse mutational paths were utilized in each species to evolve hundreds of de novo binding motifs along the neo-X, including expansions of microsatellites and transposable element (TE) insertions. However, the propensity to utilize a particular mutational path differs between independently formed X chromosomes and appears to be contingent on genomic properties of that species, such as simple repeat or TE density. This establishes the \"genomic environment\" as an important determinant in predicting the outcome of evolutionary adaptations.