Biology subjects

Kocher, A. A.

Publications and source records attributed to Kocher, A. A..

6 recordsLinked to original sources

bioRxiv2026

Local chromatin, 3D genome folding and transposon cargo shape Sleeping Beauty transposition

The cut-and-paste DNA transposon Sleeping Beauty (SB) is widely used for genome engineering, mutagenesis and gene delivery. When mobilized from a genomic donor position ("launch site"), SB preferentially reintegrates locally (< 2 Mb), but factors governing its transposition from genomic sites remain incompletely understood. Here we analysed local transposition experiments in mouse embryonic stem cells to determine the influence of chromatin context and transposon cargo on SB transposition. Transposons in launch sites with transcriptionally active, accessible chromatin showed lower transposition efficiency. Large cargo and cargo with higher transcriptional activity also result in reduced transposition efficiency. After excision, local reintegration patterns closely resembled 3D contact frequencies of the launch site. This indicates that chromosome folding is a major determinant of the transposition pattern. Surprisingly, we did not detect any effect of the destination chromatin composition on reintegration efficiency. Cargo sequence modified the reintegration patterns: promoter-containing cargo increased both very proximal (<10kb) and interchromosomal transpositions, while CTCF binding sites in the cargo increased reintegration on the launch chromosome. Together, these results define key determinants of SB transposition in chromosomes and inform the design of SB-based genome engineering strategies.

genomics↗

bioRxiv2025

Sedative choice alters Klebsiella pneumoniae lung pathogenesis and dissemination

Klebsiella pneumoniae make up 85% of carbapenem-resistant Enterobacteriaceae (CRE), bacteria that have become an urgent threat to public health. K. pneumoniae is largely transmitted in healthcare settings, where inpatients and outpatients are often anesthetized with the widely-used anesthetic induction agent propofol. Recent evidence obtained from rodent infection models indicates that propofol exposure can dramatically increase host susceptibility to microbial infections. Given that intensive care patients who are at a greater risk for K. pneumoniae lung infections are often given propofol during their hospitalization, we investigated the outcome of K. pneumoniae infections in mice briefly sedated with either propofol or ketamine/xylazine as control. Propofol-sedated mice experienced more rapid dissemination from the lungs to secondary sites of infection and their lungs exhibited more severe pathology. Based on these observations, we investigated bacterial factors involved in infection and dissemination in mice with propofol or ketamine/xylazine sedation using a high throughput insertion sequencing (INSeq) approach. We identified numerous novel potential virulence factors together with previously identified gene products, confirming the validity of our screen. We further characterized a mutant lacking the phospholipid retrograde trafficking chaperone MlaC and found that the degree of mutant attenuation was dependent upon sedation method. These results highlight the importance of sedative choice when studying hospital-acquired microbial infections and suggest that sedation can influence outcome of K. pneumoniae infection and dissemination in animal models. IMPORTANCEHost sedation by either propofol or ketamine exposure differentially impacted the severity of K. pneumoniae lung infection following intranasal inoculation of mice. While propofol-sedated mice exhibited increased lung pathology, some bacterial mutants, such as those lacking the MlaC gene product associated with the maintenance of inner and outer membrane lipid asymmetry, exhibited more severe attenuation following propofol sedation versus ketamine/xylazine. Given the dominating use of propofol in health care settings for the induction and maintenance of anesthesia, procedural sedation, and sedation for intensive care patients, this work provides important perspective as to how the choice of an anesthetic agent may impact the outcome of healthcare-associated infections.

microbiology↗

bioRxiv2024

Evolutionary innovation in conserved regulatory elements across the mammalian tree of life

Transcriptional enhancers orchestrate cell type- and time point-specific gene expression programs. Evolution of enhancer sequences can alter target gene expression without causing detrimental misexpression in other contexts. It has long been thought that this modularity allows evolutionary changes in enhancers to escape pleiotropic constraints, which is especially important for evolutionary constrained developmental patterning genes. However, there is still little data supporting this hypothesis. Here we identified signatures of accelerated evolution in conserved enhancer elements across the mammalian phylogeny. We found that pleiotropic genes involved in gene regulatory and developmental processes were enriched for accelerated sequence evolution within their enhancer elements. These genes were associated with an excess number of enhancers compared to other genes, and due to this they exhibit a substantial degree of sequence acceleration over all their enhancers combined. We provide evidence that sequence acceleration is associated with turnover of regulatory function. We studied one acceleration event in depth and found that its sequence evolution led to the emergence of a new enhancer activity domain that may be involved in the evolution of digit reduction in hoofed mammals. Our results provide tangible evidence that enhancer evolution has been a frequent contributor to modifications involving constrained developmental signaling genes in mammals.

evolutionary biology↗

bioRxiv2023

CpG island turnover events predict evolutionary changes in enhancer activity

Genetic changes that modify the function of transcriptional enhancers have been linked to the evolution of biological diversity across species. Multiple studies have focused on the role of nucleotide substitutions, transposition, and insertions and deletions in altering enhancer function. Here we show that turnover of CpG islands (CGIs), which contribute to enhancer activation, is broadly associated with changes in enhancer activity across mammals, including humans. We integrated maps of CGIs and enhancer activity-associated histone modifications obtained from multiple tissues in nine mammalian species and found that CGI content in enhancers was strongly associated with increased histone modification levels. CGIs showed widespread turnover across species and species-specific CGIs were strongly enriched for enhancers exhibiting species-specific activity across all tissues and species we examined. Genes associated with enhancers with species-specific CGIs showed concordant biases in their expression, supporting that CGI turnover contributes to gene regulatory innovation. Our results also implicate CGI turnover in the evolution of Human Gain Enhancers (HGEs), which show increased activity in human embryonic development and may have contributed to the evolution of uniquely human traits. Using a humanized mouse model, we show that a highly conserved HGE with a large CGI absent from the mouse ortholog shows increased activity at the human CGI in the humanized mouse diencephalon. Collectively, our results point to CGI turnover as a mechanism driving gene regulatory changes potentially underlying trait evolution in mammals.

genetics↗

bioRxiv2019

The Human Accelerated Region HACNS1 modifies developmental gene expression in humanized mice

The evolution of uniquely human traits likely entailed changes in developmental gene regulation. Human Accelerated Regions (HARs), which include transcriptional enhancers harboring a significant excess of human-specific sequence changes, are leading candidates for driving gene regulatory modifications in human development. However, insight into whether HARs alter the level, distribution and timing of endogenous gene expression remains limited. We examined the role of the HAR HACNS1 (HAR2) in human evolution by interrogating its molecular functions in a humanized mouse model. We find that HACNS1 maintains its human-specific enhancer activity in humanized mice and that it modifies expression of Gbx2, which encodes a homeobox transcription factor, during limb development. Using single-cell RNA-sequencing, we demonstrate that Gbx2 is upregulated in the chondrogenic mesenchyme of humanized limbs, supporting that HACNS1 alters gene expression in cell types involved in skeletal patterning. Our findings illustrate that humanized mouse models provide mechanistic insight into how HARs modified gene expression in human evolution.

genetics↗

bioRxiv2019

Massively parallel discovery of human-specific substitutions that alter neurodevelopmental enhancer activity

Genetic changes that altered the function of gene regulatory elements have been implicated in the evolution of the human brain. However, identifying the particular changes that modified regulatory activity during neurodevelopment remains challenging. Here we used massively parallel enhancer assays in human neural stem cells to measure the impact of 32,776 human-specific substitutions on enhancer activity in 1,363 Human Accelerated Regions (HARs) and 3,027 Human Gain Enhancers (HGEs), which include enhancers with novel activities in humans. We found that 31.9% of active HARs and 36.4% of active HGEs exhibited differential activity between human and chimpanzee. This enabled us to isolate the effects of 401 human-specific substitutions from other types of genetic variation in HARs and HGEs. Substitutions acted in both an additive and non-additive manner to alter enhancer activity. Human-specific substitutions altered predicted binding sites for a specific set of human transcription factors (TFs) that were a subset of TF binding sites associated with enhancer activity in our assay. Substitutions within HARs, which are overall highly constrained compared to HGEs, showed smaller effects on enhancer activity, suggesting that the impact of human-specific substitutions may be buffered in enhancers with constrained ancestral functions. Our findings yield insight into the mechanisms by which human-specific genetic changes impact enhancer function and provide a rich set of candidates for experimental studies of regulatory evolution in humans.

genomics↗