One Hundred Years of Gene Balance: How Stoichiometric Issues Affect Gene Expression, Genome Evolution, and Quantitative Traits

A century ago experiments with the flowering plant <i>Datura stramonium</i> and the fruit fly <i>Drosophila melanogaster</i> revealed that adding an extra chromosome to a karyotype was much more detrimental than adding a whole set of chromosomes. This phenomenon was referred to as gene balance and has been recapitulated across eukaryotic species. Here, we retrace some developments in this field. Molecular studies suggest that the basis of balance involves stoichiometric relationships of multi-component interactions. This concept has implication for the mechanisms controlling gene expression, genome evolution, sex chromosome evolution/dosage compensation, speciation mechanisms, and the underlying genetics of quantitative traits.

Interplay between Sublethal Aminoglycosides and Quorum Sensing: Consequences on Survival in V. cholerae

Antibiotics are well known drugs which, when present above certain concentrations, are able to inhibit the growth of certain bacteria. However, a growing body of evidence shows that even when present at lower doses (subMIC, for sub-minimal inhibitory concentration), unable to inhibit or affect microbial growth, antibiotics work as signaling molecules, affect gene expression and trigger important bacterial stress responses. However, how subMIC antibiotic signaling interplays with other well-known signaling networks in bacteria (and the consequences of such interplay) is not well understood. In this work, through transcriptomic and genetic approaches, we have explored how quorum-sensing (QS) proficiency of V. cholerae affects this pathogen’s response to subMIC doses of the aminoglycoside tobramycin (TOB). We show that the transcriptomic signature of V. cholerae in response to subMIC TOB depends highly on the presence of QS master regulator HapR. In parallel, we show that subMIC doses of TOB are able to negatively interfere with the AI-2/LuxS QS network of V. cholerae, which seems critical for survival to aminoglycoside treatment and TOB-mediated induction of SOS response in this species. This interplay between QS and aminoglycosides suggests that targeting QS signaling may be a strategy to enhance aminoglycoside efficacy in V. cholerae.

Comparison of CRISPR–Cas9 Tools for Transcriptional Repression and Gene Disruption in the BEVS

The generation of knock-out viruses using recombineering of bacmids has greatly accelerated scrutiny of baculovirus genes for a variety of applications. However, the CRISPR–Cas9 system is a powerful tool that simplifies sequence-specific genome editing and effective transcriptional regulation of genes compared to traditional recombineering and RNAi approaches. Here, the effectiveness of the CRISPR–Cas9 system for gene disruption and transcriptional repression in the BEVS was compared. Cell lines constitutively expressing the cas9 or dcas9 gene were developed, and recombinant baculoviruses delivering the sgRNA were evaluated for disruption or repression of a reporter green fluorescent protein gene. Finally, endogenous AcMNPV genes were targeted for disruption or downregulation to affect gene expression and baculovirus replication. This study provides a proof-of-concept that CRISPR–Cas9 technology may be an effective tool for efficient scrutiny of baculovirus genes through targeted gene disruption and transcriptional repression.

Comprehensive characterization of somatic variants associated with intronic polyadenylation in human cancers

Somatic single nucleotide variants (SNVs) in cancer genome affect gene expression through various mechanisms depending on their genomic location. While somatic SNVs near canonical splice sites have been reported to cause abnormal splicing of cancer-related genes, whether these SNVs can affect gene expression through other mechanisms remains an open question. Here, we analyzed RNA sequencing and exome data from 4,998 cancer patients covering ten cancer types and identified 152 somatic SNVs near splice sites that were associated with abnormal intronic polyadenylation (IPA). IPA-associated somatic variants favored the localization near the donor splice sites compared to the acceptor splice sites. A proportion of SNV-associated IPA events overlapped with premature cleavage and polyadenylation events triggered by U1 small nuclear ribonucleoproteins (snRNP) inhibition. GC content, intron length and polyadenylation signal were three genomic features that differentiated between SNV-associated IPA and intron retention. Notably, IPA-associated SNVs were enriched in tumor suppressor genes (TSGs), including the well-known TSGs such as PTEN and CDH1 with recurrent SNV-associated IPA events. Minigene assay confirmed that SNVs from PTEN, CDH1, VEGFA, GRHL2, CUL3 and WWC2 could lead to IPA. This work reveals that IPA acts as a novel mechanism explaining the functional consequence of somatic SNVs in human cancer.

Concerted genomic and epigenomic changes accompany stabilization of Arabidopsis allopolyploids

During evolution successful allopolyploids must overcome ‘genome shock’ between hybridizing species but the underlying process remains elusive. Here, we report concerted genomic and epigenomic changes in resynthesized and natural Arabidopsis suecica (TTAA) allotetraploids derived from Arabidopsisthaliana (TT) and Arabidopsisarenosa (AA). A. suecica shows conserved gene synteny and content with more gene family gain and loss in the A and T subgenomes than respective progenitors, although A. arenosa-derived subgenome has more structural variation and transposon distributions than A. thaliana-derived subgenome. These balanced genomic variations are accompanied by pervasive convergent and concerted changes in DNA methylation and gene expression among allotetraploids. The A subgenome is hypomethylated rapidly from F1 to resynthesized allotetraploids and convergently to the T-subgenome level in natural A. suecica, despite many other methylated loci being inherited from F1 to all allotetraploids. These changes in DNA methylation, including small RNAs, in allotetraploids may affect gene expression and phenotypic variation, including flowering, silencing of self-incompatibility and upregulation of meiosis- and mitosis-related genes. In conclusion, concerted genomic and epigenomic changes may improve stability and adaptation during polyploid evolution.

Cis-regulatory variants affect gene expression dynamics in yeast

Evolution of cis-regulatory sequences depends on how they affect gene expression and motivates both the identification and prediction of cis-regulatory variants responsible for expression differences within and between species. While much progress has been made in relating cis-regulatory variants to expression levels, the timing of gene activation and repression may also be important to the evolution of cis-regulatory sequences. We investigated allele-specific expression (ASE) dynamics within and between Saccharomyces species during the diauxic shift and found appreciable cis-acting variation in gene expression dynamics. Within species ASE is associated with intergenic variants, and ASE dynamics are more strongly associated with insertions and deletions than ASE levels. To refine these associations we used a high-throughput reporter assay to test promoter regions and individual variants. Within the subset of regions that recapitulated endogenous expression we identified and characterized cis-regulatory variants that affect expression dynamics. Between species, chimeric promoter regions generate novel patterns and indicate constraints on the evolution of gene expression dynamics. We conclude that changes in cis-regulatory sequences can tune gene expression dynamics and that the interplay between expression dynamics and other aspects expression are relevant to the evolution of cis-regulatory sequences.

Histone H2B Mutations in Cancer

Oncohistones have emerged as a new area in cancer epigenetics research. Recent efforts to catalogue histone mutations in cancer patients have revealed thousands of histone mutations across different types of cancer. In contrast to previously identified oncohistones (H3K27M, H3G34V/R, and H3K36M), where the mutations occur on the tail domain and affect histone post-translational modifications, the majority of the newly identified mutations are located within the histone fold domain and affect gene expression via distinct mechanisms. The recent characterization of the selected H2B has revealed previously unappreciated roles of oncohistones in nucleosome stability, chromatin accessibility, and chromatin remodeling. This review summarizes recent advances in the study of H2B oncohistones and other emerging oncohistones occurring on other types of histones, particularly those occurring on the histone fold domain.

Human embryoid bodies as a novel system for genomic studies of functionally diverse cell types

Most disease-associated loci, though located in putatively regulatory regions, have not yet been confirmed to affect gene expression. One reason for this could be that we have not examined gene expression in the most relevant cell types or conditions. Indeed, even large-scale efforts to study gene expression broadly across tissues are limited by the necessity of obtaining human samples post-mortem, and almost exclusively from adults. Thus, there is an acute need to expand gene regulatory studies in humans to the most relevant cell types, tissues, and states. We propose that embryoid bodies (EBs), which are organoids that contain a multitude of cell types in dynamic states, can provide an answer. Single cell RNA-sequencing now provides a way to interrogate developmental trajectories in EBs and enhance the potential to uncover dynamic regulatory processes that would be missed in studies of static adult tissue. Here, we examined the properties of the EB model for the purpose mapping inter-individual regulatory differences in a large variety of cell types.