Concentration and dosage sensitivity of proteins driving liquid-liquid phase separation

Liquid-liquid phase separation (LLPS) is a molecular process that leads to the formation of membraneless organelles (MLOs), i.e. functionally specialized liquid-like cellular condensates formed by proteins and nucleic acids. Integration of data on LLPS-associated proteins from dedicated databases revealed only modest overlap between them and resulted in a confident set of 89 human LLPS driver proteins. Since LLPS is highly concentration-sensitive, the underlying experiments are often criticized for applying higher-than-physiological protein concentrations. To clarify this issue, we performed a naive comparison of in vitro applied and quantitative proteomics-derived protein concentrations and discuss a number of considerations that rationalize the choice of apparently high in vitro concentrations in most LLPS studies. The validity of in vitro LLPS experiments is further supported by in vivo phase-separation experiments and by the observation that the corresponding genes show a strong propensity for dosage sensitivity. This observation implies that the availability of the respective proteins is tightly regulated in cells to avoid erroneous condensate formation. In all, we propose that although local protein concentrations are practically impossible to determine in cells, proteomics-derived cellular concentrations should rather be considered as lower limits of protein concentrations, than strict upper bounds, to be respected by in vitro experiments.

A cross-disorder dosage sensitivity map of the human genome

SUMMARYRare deletions and duplications of genomic segments, collectively known as rare copy number variants (rCNVs), contribute to a broad spectrum of human diseases. To date, most disease-association studies of rCNVs have focused on recognized genomic disorders or on the impact of haploinsufficiency caused by deletions. By comparison, our understanding of duplications in disease remains rudimentary as very few individual genes are known to be triplosensitive (i.e., duplication intolerant). In this study, we meta-analyzed rCNVs from 753,994 individuals across 30 primarily neurological disease phenotypes to create a genome-wide catalog of rCNV association statistics across disorders. We discovered 114 rCNV-disease associations at 52 distinct loci surpassing genome-wide significance (P=3.72×10−6), 42% of which involve duplications. Using Bayesian fine-mapping methods, we further prioritized 38 novel triplosensitive disease genes (e.g., GMEB2 in brain abnormalities), including three known haploinsufficient genes that we now reveal as bidirectionally dosage sensitive (e.g., ANKRD11 in growth abnormalities). By integrating our results with prior literature, we found that disease-associated rCNV segments were enriched for genes constrained against damaging coding variation and identified likely dominant driver genes for about one-third (32%) of rCNV segments based on de novo mutations from exome sequencing studies of developmental disorders. However, while the presence of constrained driver genes was a common feature of many pathogenic large rCNVs across disorders, most of the rCNVs showing genome-wide significant association were incompletely penetrant (mean odds ratio=11.6) and we also identified two examples of noncoding disease-associated rCNVs (e.g., intronic CADM2 deletions in behavioral disorders). Finally, we developed a statistical model to predict dosage sensitivity for all genes, which defined 3,006 haploinsufficient and 295 triplosensitive genes where the effect sizes of rCNVs were comparable to deletions of genes constrained against truncating mutations. These dosage sensitivity scores classified disease genes across molecular mechanisms, prioritized pathogenic de novo rCNVs in children with autism, and revealed features that distinguished haploinsufficient and triplosensitive genes, such as insulation from other genes and local cis-regulatory complexity. Collectively, the cross-disorder rCNV maps and metrics derived in this study provide the most comprehensive assessment of dosage sensitive genomic segments and genes in disease to date and set the foundation for future studies of dosage sensitivity throughout the human genome.

Sequence Transpositions Restore Genes on the Highly Degenerated W Chromosomes of Songbirds

The female-specific W chromosomes of most Neognathae birds are highly degenerated and gene-poor. Previous studies have demonstrated that the gene repertoires of the Neognathae bird W chromosomes, despite being in small numbers, are conserved across bird species, likely due to purifying selection maintaining the regulatory and dosage-sensitive genes. Here we report the discovery of DNA-based sequence duplications from the Z to the W chromosome in birds-of-paradise (Paradisaeidae, Passeriformes), through sequence transposition. The original transposition involved nine genes, but only two of them (ANXA1 and ALDH1A1) survived on the W chromosomes. Both ANXA1 and ALDH1A1 are predicted to be dosage-sensitive, and the expression of ANXA1 is restricted to ovaries in all the investigated birds. These analyses suggest the newly transposed gene onto the W chromosomes can be favored for their role in restoring dosage imbalance or through female-specific selection. After examining seven additional songbird genomes, we further identified five other transposed genes on the W chromosomes of Darwin’s finches and one in the great tit, expanding the observation of the Z-to-W transpositions to a larger range of bird species, but not all transposed genes exhibit dosage-sensitivity or ovary-biased expression We demonstrate a new mechanism by which the highly degenerated W chromosomes of songbirds can acquire genes from the homologous Z chromosomes, but further functional investigations are needed to validate the evolutionary forces underlying the transpositions.

Dosage-sensitivity of human transcription factor genes

Changes in the copy number of protein-coding genes would lead to detrimental effects if the consequent changes in protein concentration disrupt essential cellular functions. Large-scale genomic studies have identified thousands of dosage-sensitive genes in human genome. We are interested in the dosage-sensitivity of transcription factor (TF) genes whose products are essential for the growth, division and differentiation of cells by regulating the expression of the genetic information encoded in the genome. We first surveyed the enrichment of human TF genes in four recently curated datasets of dosage-sensitive genes, including the haploinsufficient genes identified by a large-scale genomic study, the haploinsufficient genes predicted by a machine learning approach, the genes with conserved copy number across mammals, and the ohnologs. Then we selected the dosage-sensitive genes that are present in all the four dataset and regarded them as the most reliable dosage-sensitive genes, and the genes that are absent from any one of the four datasets as the most reliable dosage-insensitive genes, and surveyed the enrichments of TFs genes in these two datasets. A large number of TF genes were found to be dosage-insensitive, which is beyond the expectation based on the role of TFs. In spite of this, the likeness of TF genes to be dosage-sensitive were supported by five datasets, with the conserved-copy-number genes as the exception. The nuclear receptors are the only one family of TFs whose dosage-sensitivity was consistently supported by all the six datasets. In addition, we found that TF families with very few members are also more likely to be dosage-sensitive while the largest TF family, C2H2-ZF, are most likely dosage-insensitive. The most extensively studied TFs, p53, are not special in dosage-sensitivity. They are significantly enriched in only three datasets. We also confirmed that dosage-sensitive genes generally have long coding sequences, high expression levels and experienced stronger selective pressure. Our results indicate some TFs function in a dose-dependent manner while some other not. Gene dosage changes in some TF families like nuclear receptor would result in disease phenotypes while the effects of such changes in some TFs like C2H2-ZF would be mild.