Diverse Molecular Mechanisms Contribute to Differential Expression of Human Duplicated Genes.

Emerging evidence links genes within human-specific segmental duplications (HSDs) to traits and diseases unique to our species. Strikingly, despite being nearly identical by sequence (>98.5%), paralogous HSD genes are differentially expressed across human cell and tissue types, though the underlying mechanisms have not been examined. We compared cross-tissue mRNA levels of 75 HSD genes from 30 families between humans and chimpanzees and found expression patterns consistent with relaxed selection on or neofunctionalization of derived paralogs. In general, ancestral paralogs exhibited greatest expression conservation with chimpanzee orthologs, though exceptions suggest certain derived paralogs may retain or supplant ancestral functions. Concordantly, analysis of long-read isoform sequencing data sets from diverse human tissues and cell lines found that about half of derived paralogs exhibited globally lower expression. To understand mechanisms underlying these differences, we leveraged data from human lymphoblastoid cell lines (LCLs) and found no relationship between paralogous expression divergence and post-transcriptional regulation, sequence divergence, or copy-number variation. Considering cis-regulation, we reanalyzed ENCODE data and recovered hundreds of previously unidentified candidate CREs in HSDs. We also generated large-insert ChIP-sequencing data for active chromatin features in an LCL to better distinguish paralogous regions. Some duplicated CREs were sufficient to drive differential reporter activity, suggesting they may contribute to divergent cis-regulation of paralogous genes. This work provides evidence that cis-regulatory divergence contributes to novel expression patterns of recent gene duplicates in humans.

Why Antibiotic Treatment Is Not Enough for Sepsis Resolution: an Evaluation in an Experimental Animal Model.

Sepsis remains a major health problem at the levels of mortality, morbidity, and economic burden to the health care system, a condition that is aggravated by the development of secondary conditions such as septic shock and multiple-organ failure. Our current understanding of the etiology of human sepsis has advanced, at least in part, due to the use of experimental animal models, particularly the model of cecum ligation and puncture (CLP). Antibiotic treatment has been commonly used in this model to closely mirror the treatment of human septic patients. However, whether their use may obscure the elucidation of the cellular and molecular mechanisms involved in the septic response is questionable. The objective of the present study was to determine the effect of antibiotic treatment in the outcome of a fulminant model of CLP. Various dosing strategies were used for the administration of imipenem, which has broad-spectrum coverage of enteric bacteria. No statistically significant differences in the survival of mice were observed between the different antibiotic dosing strategies and no treatment, suggesting that live bacteria may not be the only factor inducing septic shock. To further investigate this hypothesis, mice were challenged with sterilized or unsterilized cecal contents. We found that exposure of mice to sterilized cecal contents also resulted in a high mortality rate. Therefore, it is possible that bacterial debris, apart from bacterial proliferation, triggers a septic response and contributes to mortality in this model, suggesting that additional factors are involved in the development of septic shock.

Zebrafish models of human-duplicated gene SRGAP2 reveal novel functions in microglia and visual system development.

Recent expansion of duplicated genes unique in the Homo lineage likely contributed to brain evolution and other human-specific traits. One hallmark example is the expansion of the human SRGAP2 family, resulting in a human-specific paralog SRGAP2C . Introduction of SRGAP2C in mouse models is associated with altering cortical neuronal migration, axon guidance, synaptogenesis, and sensory-task performance. Truncated, human-specific SRGAP2C heterodimerizes with the full-length ancestral gene product SRGAP2A and antagonizes its functions. However, the significance of SRGAP2 duplication beyond neocortex development has not been elucidated due to the embryonic lethality of complete Srgap2 knockout in mice. Using zebrafish, we showed that srgap2 knockout results in viable offspring that phenocopy "humanized" SRGAP2C larvae. Specifically, human SRGAP2C protein interacts with zebrafish Srgap2, demonstrating similar Srgap2 functional antagonism observed in mice. Shared traits between knockout and humanized zebrafish larvae include altered morphometric features (i.e., reduced body length and inter-eye distance) and differential expression of synapse-, axogenesis-, vision-related genes. Through single-cell transcriptome analysis, we further observed a skewed balance of excitatory and inhibitory neurons that likely contributes to increased susceptibility to seizures displayed by Srgap2 mutant larvae, a phenotype resembling SRGAP2 loss-of-function in a child with early infantile epileptic encephalopathy. Single-cell data also pointed to strong microglia expression of srgap2 with mutants exhibiting altered membrane dynamics and likely delayed maturation of microglial cells. srgap2 -expressing microglia cells were also detected in the developing eye together with altered expression of genes related to axogenesis and synaptogenesis in mutant retinal cells. Consistent with the perturbed gene expression in the retina, we found that SRGAP2 mutant larvae exhibited increased sensitivity to broad and fine visual cues. Finally, comparing the transcriptomes of relevant cell types between human (+ SRGAP2C ) and non-human primates (- SRGAP2C ) revealed significant overlaps of gene alterations with mutant cells in our zebrafish models; this suggests that SRGAP2C plays similar roles altering microglia and the visual system in modern humans. Together, our functional characterization of zebrafish Srgap2 and human SRGAP2C in zebrafish uncovered novel gene functions and highlights the strength of cross-species analysis in understanding the development of human-specific features.