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1.
Med ; 2024 May 19.
Article in English | MEDLINE | ID: mdl-38776915

ABSTRACT

BACKGROUND: Xenotransplantation of genetically engineered porcine organs has the potential to address the challenge of organ donor shortage. Two cases of porcine-to-human kidney xenotransplantation were performed, yet the physiological effects on the xenografts and the recipients' immune responses remain largely uncharacterized. METHODS: We performed single-cell RNA sequencing (scRNA-seq) and longitudinal RNA-seq analyses of the porcine kidneys to dissect xenotransplantation-associated cellular dynamics and xenograft-recipient interactions. We additionally performed longitudinal scRNA-seq of the peripheral blood mononuclear cells (PBMCs) to detect recipient immune responses across time. FINDINGS: Although no hyperacute rejection signals were detected, scRNA-seq analyses of the xenografts found evidence of endothelial cell and immune response activation, indicating early signs of antibody-mediated rejection. Tracing the cells' species origin, we found human immune cell infiltration in both xenografts. Human transcripts in the longitudinal bulk RNA-seq revealed that human immune cell infiltration and the activation of interferon-gamma-induced chemokine expression occurred by 12 and 48 h post-xenotransplantation, respectively. Concordantly, longitudinal scRNA-seq of PBMCs also revealed two phases of the recipients' immune responses at 12 and 48-53 h. Lastly, we observed global expression signatures of xenotransplantation-associated kidney tissue damage in the xenografts. Surprisingly, we detected a rapid increase of proliferative cells in both xenografts, indicating the activation of the porcine tissue repair program. CONCLUSIONS: Longitudinal and single-cell transcriptomic analyses of porcine kidneys and the recipient's PBMCs revealed time-resolved cellular dynamics of xenograft-recipient interactions during xenotransplantation. These cues can be leveraged for designing gene edits and immunosuppression regimens to optimize xenotransplantation outcomes. FUNDING: This work was supported by NIH RM1HG009491 and DP5OD033430.

2.
Nat Med ; 30(5): 1448-1460, 2024 May.
Article in English | MEDLINE | ID: mdl-38760586

ABSTRACT

In a previous study, heart xenografts from 10-gene-edited pigs transplanted into two human decedents did not show evidence of acute-onset cellular- or antibody-mediated rejection. Here, to better understand the detailed molecular landscape following xenotransplantation, we carried out bulk and single-cell transcriptomics, lipidomics, proteomics and metabolomics on blood samples obtained from the transplanted decedents every 6 h, as well as histological and transcriptomic tissue profiling. We observed substantial early immune responses in peripheral blood mononuclear cells and xenograft tissue obtained from decedent 1 (male), associated with downstream T cell and natural killer cell activity. Longitudinal analyses indicated the presence of ischemia reperfusion injury, exacerbated by inadequate immunosuppression of T cells, consistent with previous findings of perioperative cardiac xenograft dysfunction in pig-to-nonhuman primate studies. Moreover, at 42 h after transplantation, substantial alterations in cellular metabolism and liver-damage pathways occurred, correlating with profound organ-wide physiological dysfunction. By contrast, relatively minor changes in RNA, protein, lipid and metabolism profiles were observed in decedent 2 (female) as compared to decedent 1. Overall, these multi-omics analyses delineate distinct responses to cardiac xenotransplantation in the two human decedents and reveal new insights into early molecular and immune responses after xenotransplantation. These findings may aid in the development of targeted therapeutic approaches to limit ischemia reperfusion injury-related phenotypes and improve outcomes.


Subject(s)
Heart Transplantation , Heterografts , Transplantation, Heterologous , Humans , Animals , Swine , Male , Female , Graft Rejection/immunology , Graft Rejection/genetics , Proteomics , Metabolomics , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/immunology , Transcriptome , Gene Expression Profiling , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Lipidomics , Reperfusion Injury/immunology , Reperfusion Injury/genetics , Reperfusion Injury/metabolism , Multiomics
3.
Nature ; 628(8007): 373-380, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38448583

ABSTRACT

Pervasive transcriptional activity is observed across diverse species. The genomes of extant organisms have undergone billions of years of evolution, making it unclear whether these genomic activities represent effects of selection or 'noise'1-4. Characterizing default genome states could help understand whether pervasive transcriptional activity has biological meaning. Here we addressed this question by introducing a synthetic 101-kb locus into the genomes of Saccharomyces cerevisiae and Mus musculus and characterizing genomic activity. The locus was designed by reversing but not complementing human HPRT1, including its flanking regions, thus retaining basic features of the natural sequence but ablating evolved coding or regulatory information. We observed widespread activity of both reversed and native HPRT1 loci in yeast, despite the lack of evolved yeast promoters. By contrast, the reversed locus displayed no activity at all in mouse embryonic stem cells, and instead exhibited repressive chromatin signatures. The repressive signature was alleviated in a locus variant lacking CpG dinucleotides; nevertheless, this variant was also transcriptionally inactive. These results show that synthetic genomic sequences that lack coding information are active in yeast, but inactive in mouse embryonic stem cells, consistent with a major difference in 'default genomic states' between these two divergent eukaryotic cell types, with implications for understanding pervasive transcription, horizontal transfer of genetic information and the birth of new genes.


Subject(s)
Genes, Synthetic , Genome , Saccharomyces cerevisiae , Transcription, Genetic , Animals , Humans , Mice , Chromatin/genetics , CpG Islands , Genes, Synthetic/genetics , Genome/genetics , Mouse Embryonic Stem Cells/metabolism , Promoter Regions, Genetic/genetics , Saccharomyces cerevisiae/genetics , Hypoxanthine Phosphoribosyltransferase/genetics , Evolution, Molecular
4.
Cell ; 186(26): 5826-5839.e18, 2023 12 21.
Article in English | MEDLINE | ID: mdl-38101409

ABSTRACT

Super-enhancers are compound regulatory elements that control expression of key cell identity genes. They recruit high levels of tissue-specific transcription factors and co-activators such as the Mediator complex and contact target gene promoters with high frequency. Most super-enhancers contain multiple constituent regulatory elements, but it is unclear whether these elements have distinct roles in activating target gene expression. Here, by rebuilding the endogenous multipartite α-globin super-enhancer, we show that it contains bioinformatically equivalent but functionally distinct element types: classical enhancers and facilitator elements. Facilitators have no intrinsic enhancer activity, yet in their absence, classical enhancers are unable to fully upregulate their target genes. Without facilitators, classical enhancers exhibit reduced Mediator recruitment, enhancer RNA transcription, and enhancer-promoter interactions. Facilitators are interchangeable but display functional hierarchy based on their position within a multipartite enhancer. Facilitators thus play an important role in potentiating the activity of classical enhancers and ensuring robust activation of target genes.


Subject(s)
Gene Expression Regulation , Super Enhancers , Transcription, Genetic , alpha-Globins , Enhancer Elements, Genetic , Promoter Regions, Genetic , Transcription Factors/metabolism , alpha-Globins/genetics
5.
Mol Cell ; 83(23): 4424-4437.e5, 2023 Dec 07.
Article in English | MEDLINE | ID: mdl-37944526

ABSTRACT

Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes.


Subject(s)
Cell Nucleus , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genetics , Chromosomes/genetics , Genome, Fungal , Synthetic Biology/methods
6.
iScience ; 25(6): 104438, 2022 Jun 17.
Article in English | MEDLINE | ID: mdl-35692632

ABSTRACT

Overwriting counterselectable markers is an efficient strategy for removing wild-type DNA or replacing it with payload DNA of interest. Currently, one bottleneck of efficient genome engineering in mammals is the shortage of counterselectable (negative selection) markers that work robustly without affecting organismal developmental potential. Here, we report a conditional Piga knockout strategy that enables efficient proaerolysin-based counterselection in mouse embryonic stem cells. The conditional Piga knockout cells show similar proaerolysin resistance as full (non-conditional) Piga deletion cells, which enables the use of a PIGA transgene as a counterselectable marker for genome engineering purposes. Native Piga function is readily restored in conditional Piga knockout cells to facilitate subsequent mouse development. We also demonstrate the generality of our strategy by engineering a conditional knockout of endogenous Hprt. Taken together, our work provides a new tool for advanced mouse genome writing and mouse model establishment.

7.
Nat Commun ; 12(1): 349, 2021 01 13.
Article in English | MEDLINE | ID: mdl-33441553

ABSTRACT

The widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease derives its DNA targeting specificity from protein-DNA contacts with protospacer adjacent motif (PAM) sequences, in addition to base-pairing interactions between its guide RNA and target DNA. Previous reports have established that the PAM specificity of SpCas9 can be altered via positive selection procedures for directed evolution or other protein engineering strategies. Here we exploit in vivo directed evolution systems that incorporate simultaneous positive and negative selection to evolve SpCas9 variants with commensurate or improved activity on NAG PAMs relative to wild type and reduced activity on NGG PAMs, particularly YGG PAMs. We also show that the PAM preferences of available evolutionary intermediates effectively determine whether similar counterselection PAMs elicit different selection stringencies, and demonstrate that negative selection can be specifically increased in a yeast selection system through the fusion of compensatory zinc fingers to SpCas9.


Subject(s)
CRISPR-Associated Protein 9/metabolism , CRISPR-Cas Systems , DNA/metabolism , Gene Editing/methods , RNA, Guide, Kinetoplastida/metabolism , Streptococcus pyogenes/metabolism , Amino Acid Sequence , CRISPR-Associated Protein 9/genetics , Cell Line, Tumor , DNA/chemistry , DNA/genetics , Directed Molecular Evolution/methods , Humans , Mutation , Nucleic Acid Conformation , Nucleotide Motifs/genetics , Protein Engineering/methods , RNA, Guide, Kinetoplastida/genetics , Streptococcus pyogenes/genetics , Substrate Specificity
8.
Sci Rep ; 8(1): 17809, 2018 12 13.
Article in English | MEDLINE | ID: mdl-30546021

ABSTRACT

An imaging-integrated microfluidic cell volume sensor was used to evaluate the volumetric growth rate of single cells from a Saccharomyces cerevisiae population exhibiting two phenotypic expression states of the PDR5 gene. This gene grants multidrug resistance by transcribing a membrane transporter capable of pumping out cytotoxic compounds from the cell. Utilizing fluorescent markers, single cells were isolated and trapped, then their growth rates were measured in two on-chip environments: rich media and media dosed with the antibiotic cycloheximide. Approximating growth rates to first-order, we assessed the fitness of individual cells and found that those with low PDR5 expression had higher fitness in rich media whereas cells with high PDR5 expression had higher fitness in the presence of the drug. Moreover, the drug dramatically reduced the fitness of cells with low PDR5 expression but had comparatively minimal impact on the fitness of cells with high PDR5 expression. Our experiments show the utility of this imaging-integrated microfluidic cell volume sensor for high-resolution, single-cell analysis, as well as its potential application for studies that characterize and compare the fitness and morphology of individual cells from heterogeneous populations under different growth conditions.


Subject(s)
ATP-Binding Cassette Transporters/biosynthesis , Gene Expression Regulation, Fungal , Lab-On-A-Chip Devices , Microfluidic Analytical Techniques , Saccharomyces cerevisiae Proteins/biosynthesis , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/growth & development , Microfluidic Analytical Techniques/instrumentation , Microfluidic Analytical Techniques/methods
9.
J Clin Invest ; 126(4): 1555-65, 2016 Apr 01.
Article in English | MEDLINE | ID: mdl-26999603

ABSTRACT

The X chromosome-encoded histone demethylase UTX (also known as KDM6A) mediates removal of repressive trimethylation of histone H3 lysine 27 (H3K27me3) to establish transcriptionally permissive chromatin. Loss of UTX in female mice is embryonic lethal. Unexpectedly, male UTX-null mice escape embryonic lethality due to expression of UTY, a paralog that lacks H3K27 demethylase activity, suggesting an enzyme-independent role for UTX in development and thereby challenging the need for active H3K27 demethylation in vivo. However, the requirement for active H3K27 demethylation in stem cell-mediated tissue regeneration remains untested. Here, we employed an inducible mouse KO that specifically ablates Utx in satellite cells (SCs) and demonstrated that active H3K27 demethylation is necessary for muscle regeneration. Loss of UTX in SCs blocked myofiber regeneration in both male and female mice. Furthermore, we demonstrated that UTX mediates muscle regeneration through its H3K27 demethylase activity, as loss of demethylase activity either by chemical inhibition or knock-in of demethylase-dead UTX resulted in defective muscle repair. Mechanistically, dissection of the muscle regenerative process revealed that the demethylase activity of UTX is required for expression of the transcription factor myogenin, which in turn drives differentiation of muscle progenitors. Thus, we have identified a critical role for the enzymatic activity of UTX in activating muscle-specific gene expression during myofiber regeneration and have revealed a physiological role for active H3K27 demethylation in vivo.


Subject(s)
Gene Expression Regulation/physiology , Histone Demethylases/biosynthesis , Myofibrils/physiology , Myogenin/metabolism , Regeneration/physiology , Satellite Cells, Skeletal Muscle/enzymology , Animals , Female , Gene Knock-In Techniques , Histone Demethylases/genetics , Histones/genetics , Histones/metabolism , Male , Mice , Mice, Knockout , Myogenin/genetics , Satellite Cells, Skeletal Muscle/cytology
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