The testis is a peculiar tissue in many respects. It shows patterns of rapid gene evolution and provides a hotspot for the origination of genetic novelties such as de novo genes, duplications and mutations. To investigate the expression patterns of genetic novelties across cell types, we performed single-cell RNA-sequencing of adult Drosophila testis. We found that new genes were expressed in various cell types, the patterns of which may be influenced by their mode of origination. In particular, lineage-specific de novo genes are commonly expressed in early spermatocytes, while young duplicated genes are often bimodally expressed. Analysis of germline substitutions suggests that spermatogenesis is a highly reparative process, with the mutational load of germ cells decreasing as spermatogenesis progresses. By elucidating the distribution of genetic novelties across spermatogenesis, this study provides a deeper understanding of how the testis maintains its core reproductive function while being a hotbed of evolutionary innovation.

8The testis is a peculiar tissue in many respects. It shows patterns of rapid gene evolution 9 and provides a hotspot for the origination of genetic novelties such as de novo genes, 10 duplications and mutations. To investigate the expression patterns of genetic novelties across cell 11 types, we performed single-cell RNA-sequencing of adult Drosophila testis. We found that new 12 genes were expressed in various cell types, the patterns of which may be influenced by their 13 mode of origination. In particular, lineage-specific de novo genes are commonly expressed in 14 early spermatocytes, while young duplicated genes are often bimodally expressed. Analysis of 15 germline substitutions suggests that spermatogenesis is a highly reparative process, with the 16 mutational load of germ cells decreasing as spermatogenesis progresses. By elucidating the 17 distribution of genetic novelties across spermatogenesis, this study provides a deeper 18 understanding of how the testis maintains its core reproductive function while being a hotbed of 19 evolutionary innovation.

Dosage compensation equalizes X-linked expression between XY males and XX females. In male fruit flies, expression levels of the X-chromosome are increased approximately two-fold to compensate for their single X chromosome. In testis, dosage compensation is thought to cease during meiosis; however, the timing and degree of the resulting transcriptional suppression is difficult to separate from global meiotic downregulation of each chromosome. To address this, we analyzed testis single-cell RNA-sequencing (scRNA-seq) data from two Drosophila melanogaster strains. We found evidence that the X chromosome is equally transcriptionally active as autosomes in somatic and pre-meiotic cells, and less transcriptionally active than autosomes in meiotic and post-meiotic cells. In cells experiencing dosage compensation, close proximity to MSL (male-specific lethal) chromatin entry sites (CES) correlates with increased X chromosome transcription. We found low or undetectable levels of germline expression of most msl genes, mle, roX1 and roX2 via scRNA-seq and RNA-FISH, and no evidence of germline nuclear roX1/2 localization. Our results suggest that, although dosage compensation occurs in somatic and pre-meiotic germ cells in Drosophila testis, there might be non-canonical factors involved in the dosage compensation mechanism. The single-cell expression patterns and enrichment statistics of detected genes can be explored interactively in our database: https://zhao.labapps.rockefeller.edu/gene-expr/.

No abstract

The production of fuels or chemicals from lignocellulose currently requires thermochemical pretreatment to release fermentable sugars. These harsh conditions also generate numerous small-molecule inhibitors of microbial growth and fermentation, limiting production. We applied small-insert functional metagenomic selections to discover genes that confer microbial tolerance to these inhibitors, identifying both individual genes and general biological processes associated with tolerance to multiple inhibitory compounds. Having screened over 248 Gb of DNA cloned from 16 diverse soil metagenomes, we describe gain-of-function tolerance against acid, alcohol, and aldehyde inhibitors derived from hemicellulose and lignin, demonstrating that uncultured soil microbial communities hold tremendous genetic potential to address the toxicity of pretreated lignocellulose. We recovered genes previously known to confer tolerance to lignocellulosic inhibitors as well as novel genes that confer tolerance via unknown functions. For instance, we implicated galactose metabolism in overcoming the toxicity of lignin monomers and identified a decarboxylase that confers tolerance to ferulic acid; this enzyme has been shown to catalyze the production of 4-vinyl guaiacol, a valuable precursor to vanillin production. These metagenomic tolerance genes can enable the flexible design of hardy microbial catalysts, customized to withstand inhibitors abundant in specific bioprocessing applications. Many lignocellulosic feedstocks (e.g., switchgrass) are preferred to maize, sugarcane, and other traditional food crops for the production of fuels and chemicals because they are able to grow on marginal land, often require little attention or energy input, and do not compete directly with the food supply (1-6). However, lignocellulose requires harsh thermochemical pretreatment methods to liberate fermentable monosaccharides (7-9), producing an additional compendium of compounds inhibitory to microbial growth that ultimately reduce production efficiencies (10-12). These small-molecule inhibitors derived from lignocellulose pretreatment (here called lignocellulosic inhibitors) are typically aldehydes, organic acids, furans, or phenolics and can originate from the cellulosic, hemicellulosic, and lignified fractions of the feedstock (11-15).Engineering hardier microbial production hosts with elevated tolerance to these inhibitors offers potential to ameliorate the toxic effects of these compounds without incurring the high process costs associated with detoxifying the lignocellulosic hydrolysate (16, 17). Unfortunately, the modes of toxicity of many of these toxins are poorly described, and genes conferring tolerance to many of these compounds have not been identified (18,19). An expanded catalog of tolerance-conferring genotypes may shed light on mechanisms of toxicity and enable synthetic biology approaches for the design of diverse production hosts with broadspectrum tolerance.Soil microorganisms, including white-rot fungi (20) and many bacteria (21), ar...

Evolutionarily young genes are usually preferentially expressed in the testis across species. Although it is known that older genes are generally more broadly expressed than younger genes, the properties that shaped this pattern are unknown. Older genes may gain expression across other tissues uniformly, or faster in certain tissues than others. Using Drosophila gene expression data, we confirmed previous findings that younger genes are disproportionately testis biased and older genes are disproportionately ovary biased. We found that the relationship between gene age and expression is stronger in the ovary than any other tissue and weakest in testis. We performed ATAC-seq on Drosophila testis and found that although genes of all ages are more likely to have open promoter chromatin in testis than in ovary, promoter chromatin alone does not explain the ovary bias of older genes. Instead, we found that upstream transcription factor (TF) expression is highly predictive of gene expression in ovary but not in testis. In the ovary, TF expression is more predictive of gene expression than open promoter chromatin, whereas testis gene expression is similarly influenced by both TF expression and open promoter chromatin. We propose that the testis is uniquely able to express younger genes controlled by relatively few TFs, whereas older genes with more TF partners are broadly expressed with peak expression most likely in the ovary. The testis allows widespread baseline expression that is relatively unresponsive to regulatory changes, whereas the ovary transcriptome is more responsive to trans-regulation and has a higher ceiling for gene expression.

Dosage compensation (DC) is a mechanism by which X chromosome transcription is equalized in the somatic cells of both males and females. In male fruit flies, expression levels of the X-chromosome are increased two-fold to compensate for their single X chromosome. In testis, dosage compensation is thought to cease during meiosis, however, the timing and degree of the resulting transcriptional suppression is difficult to separate from global meiotic downregulation of each chromosome. To address this, we analyzed testis single-cell RNA-sequencing (scRNA-seq) data from two Drosophila melanogaster strains. We found evidence that the X chromosome is equally transcriptionally active as autosomes in somatic and pre-meiotic cells, and less transcriptionally active than autosomes in meiotic and post-meiotic cells. In cells experiencing dosage compensation, close proximity to MSL (male-specific lethal) chromatin entry sites (CES) correlates with increased X chromosome transcription. We found low or undetectable level of germline expression of most msl genes, mle, roX1 and roX2 via sequencing or RNA-FISH, and no evidence of germline nuclear roX1/2 localization. Our results suggest that, although DC occurs in somatic and premeiotic germ cells in Drosophila testis, there might be non-canonical factors involved in the dosage compensation. The single-cell expression patterns and enrichment statistics of detected genes can be explored interactively in our database: https://zhao.labapps.rockefeller.edu/gene-expr/.

Evolutionarily young genes are usually preferentially expressed in the testis across species. While it is known that older genes are generally more broadly expressed than younger genes, the properties that shaped this pattern are unknown. Older genes may gain expression across other tissues uniformly, or faster in certain tissues than others. Using Drosophila gene expression data, we confirmed previous findings that younger genes are disproportionately testis-biased and older genes are disproportionately ovary-biased. We found that the relationship between gene age and expression is stronger in the ovary than any other tissue, and weakest in testis. We performed ATAC-seq on Drosophila testis and found that while genes of all ages are more likely to have open promoter chromatin in testis than in ovary, promoter chromatin alone does not explain the ovary-bias of older genes. Instead, we found that upstream transcription factor (TF) expression is highly predictive of gene expression in ovary, but not in testis. In ovary, TF expression is more predictive of gene expression than open promoter chromatin, whereas testis gene expression is similarly influenced by both TF expression and open promoter chromatin. We propose that the testis is uniquely able to expresses younger genes controlled by relatively few TFs, while older genes with more TF partners are broadly expressed with peak expression most likely in ovary. The testis allows widespread baseline expression that is relatively unresponsive to regulatory changes, whereas the ovary transcriptome is more responsive to trans-regulation and has a higher ceiling for gene expression.