Design and testing of a humanized porcine donor for xenotransplantation.

Recent human decedent model studies1,2 and compassionate xenograft use3 have explored the promise of porcine organs for human transplantation. To proceed to human studies, a clinically ready porcine donor must be engineered and its xenograft successfully tested in nonhuman primates. Here we describe the design, creation and long-term life-supporting function of kidney grafts from a genetically engineered porcine donor transplanted into a cynomolgus monkey model. The porcine donor was engineered to carry 69 genomic edits, eliminating glycan antigens, overexpressing human transgenes and inactivating porcine endogenous retroviruses. In vitro functional analyses showed that the edited kidney endothelial cells modulated inflammation to an extent that was indistinguishable from that of human endothelial cells, suggesting that these edited cells acquired a high level of human immune compatibility. When transplanted into cynomolgus monkeys, the kidneys with three glycan antigen knockouts alone experienced poor graft survival, whereas those with glycan antigen knockouts and human transgene expression demonstrated significantly longer survival time, suggesting the benefit of human transgene expression in vivo. These results show that preclinical studies of renal xenotransplantation could be successfully conducted in nonhuman primates and bring us closer to clinical trials of genetically engineered porcine renal grafts.

A multi-gene knockdown approach reveals a new role for Pax6 in controlling organ number in Drosophila.

Genetic screens are designed to target individual genes for the practical reason of establishing a clear association between a mutant phenotype and a single genetic locus. This allows for a developmental or physiological role to be assigned to the wild-type gene. We previously observed that the concurrent loss of Pax6 and Polycomb epigenetic repressors in Drosophila leads the eye to transform into a wing. This fate change is not seen when either factor is disrupted separately. An implication of this finding is that standard screens may miss the roles that combinations of genes play in development. Here, we show that this phenomenon is not limited to Pax6 and Polycomb but rather applies more generally. We demonstrate that in the Drosophila eye-antennal disc, the simultaneous downregulation of Pax6 with either the NURF nucleosome remodeling complex or the Pointed transcription factor transforms the head epidermis into an antenna. This is a previously unidentified fate change that is also not observed with the loss of individual genes. We propose that the use of multi-gene knockdowns is an essential tool for unraveling the complexity of development.

Simple and efficient profiling of transcription initiation and transcript levels with STRIPE-seq.

Accurate mapping of transcription start sites (TSSs) is key for understanding transcriptional regulation. However, current protocols for genome-wide TSS profiling are laborious and/or expensive. We present Survey of TRanscription Initiation at Promoter Elements with high-throughput sequencing (STRIPE-seq), a simple, rapid, and cost-effective protocol for sequencing capped RNA 5' ends from as little as 50 ng total RNA. Including depletion of uncapped RNA and reaction cleanups, a STRIPE-seq library can be constructed in about 5 h. We show application of STRIPE-seq to TSS profiling in yeast and human cells and show that it can also be effectively used for quantification of transcript levels and analysis of differential gene expression. In conjunction with our ready-to-use computational workflows, STRIPE-seq is a straightforward, efficient means by which to probe the landscape of transcriptional initiation.

A Cyclin A-Myb-MuvB-Aurora B network regulates the choice between mitotic cycles and polyploid endoreplication cycles.

Endoreplication is a cell cycle variant that entails cell growth and periodic genome duplication without cell division, and results in large, polyploid cells. Cells switch from mitotic cycles to endoreplication cycles during development, and also in response to conditional stimuli during wound healing, regeneration, aging, and cancer. In this study, we use integrated approaches in Drosophila to determine how mitotic cycles are remodeled into endoreplication cycles, and how similar this remodeling is between induced and developmental endoreplicating cells (iECs and devECs). Our evidence suggests that Cyclin A / CDK directly activates the Myb-MuvB (MMB) complex to induce transcription of a battery of genes required for mitosis, and that repression of CDK activity dampens this MMB mitotic transcriptome to promote endoreplication in both iECs and devECs. iECs and devECs differed, however, in that devECs had reduced expression of E2F1-dependent genes that function in S phase, whereas repression of the MMB transcriptome in iECs was sufficient to induce endoreplication without a reduction in S phase gene expression. Among the MMB regulated genes, knockdown of AurB protein and other subunits of the chromosomal passenger complex (CPC) induced endoreplication, as did knockdown of CPC-regulated cytokinetic, but not kinetochore, proteins. Together, our results indicate that the status of a CycA-Myb-MuvB-AurB network determines the decision to commit to mitosis or switch to endoreplication in both iECs and devECs, and suggest that regulation of different steps of this network may explain the known diversity of polyploid cycle types in development and disease.

Glycolytic Disruption Triggers Interorgan Signaling to Nonautonomously Restrict Drosophila Larval Growth.

Drosophila larval growth requires efficient conversion of dietary nutrients into biomass. Lactate Dehydrogenase (Ldh) and Glycerol-3-phosphate dehydrogenase (Gpdh1) support larval biosynthetic metabolism by maintaining NAD+/NADH redox balance and promoting glycolytic flux. Consistent with the cooperative functions of Ldh and Gpdh1, the loss of both enzymes, but neither single enzyme, induces a developmental arrest. However, Ldh and Gpdh1 exhibit complex and often mutually exclusive expression patterns, suggesting that the Gpdh1; Ldh double mutant lethal phenotype could be mediated nonautonomously. Here we find that the developmental arrest displayed by the double mutants extends beyond simple metabolic disruption and instead stems, in part, from changes in systemic growth factor signaling. Specifically, we demonstrate that this synthetic lethality is linked to the upregulation of Upd3, a cytokine involved in the Jak/Stat signaling pathway. Moreover, we demonstrate that either loss of the Upd3 or dietary administration of the steroid hormone 20-hydroxyecdysone (20E) rescue the synthetic lethal phenotype of Gpdh1; Ldh double mutants. Together, these findings demonstrate that metabolic disruptions within a single tissue can nonautonomously modulate interorgan signaling to ensure synchronous developmental growth.

A CUT&RUN protocol to determine patterns of epigenetic marks in imaginal discs of Drosophila.

Cleavage Under Targets & Release Using Nucleases (CUT&RUN) sequencing is a technique used to study gene regulation. The protocol presented here has been used successfully to identify the pattern of histone modifications within the genome of the eye-antennal disc of the fruit fly, Drosophila melanogaster. In its present form, it can be used to analyze genomic features of other imaginal discs. It can be modified for use with other tissues and applications including identifying the pattern of transcription factor occupancy.

Clinical and molecular correlation defines activity of physiological pathways in life-sustaining kidney xenotransplantation.

Porcine kidney xenotransplantation is accelerating towards clinical translation. However, despite the demonstrated ability of porcine kidneys to remove metabolic waste products, questions remain about their ability to faithfully recapitulate renal endocrine functions after transplantation. Here we analyze xenograft growth and function of two kidney dependent endocrine pathways in seventeen cynomolgus macaques after kidney xenotransplantation from gene edited Yucatan minipigs. Xenograft growth, the renin-angiotensinogen aldosterone-system, and the calcium-vitamin D-parathyroid hormone axis are assessed using clinical chemistries data, renin activity and beta-C-terminal-telopeptide assays, kidney graft RNA-sequencing and serial ultrasonography. We demonstrate that xenografts transplanted from minipigs show only modest growth and do not substantially contribute to recipient RAAS pathway activity. However, parathyroid hormone-independent hypercalcemia and hypophosphatemia are observed, suggesting a need for close monitoring and timely intervention during human testing. Further study of these phenotypes is warranted in designing prospective clinical trials.

Genome-Wide Profiling of Transcription Initiation with STRIPE-seq.

Transcription start site (TSS) usage is a critical factor in the regulation of gene expression. A number of methods for global TSS mapping have been developed, but barriers of expense, technical difficulty, time, and/or cost have limited their broader adoption. To address these issues, we developed Survey of TRanscription Initiation at Promoter Elements with high-throughput sequencing (STRIPE-seq). Requiring only three enzymatic steps with intervening bead cleanups, a STRIPE-seq library can be prepared from as little as 50 ng total RNA in ~5 h at a cost of ~$12 (US). In addition to profiling TSS usage, STRIPE-seq provides information on transcript levels that can be used for differential expression analysis. Thanks to its simplicity and low cost, we envision that STRIPE-seq could be employed by any molecular biology laboratory interested in profiling transcription initiation.

Global approaches for profiling transcription initiation.

Transcription start site (TSS) selection influences transcript stability and translation as well as protein sequence. Alternative TSS usage is pervasive in organismal development, is a major contributor to transcript isoform diversity in humans, and is frequently observed in human diseases including cancer. In this review, we discuss the breadth of techniques that have been used to globally profile TSSs and the resulting insights into gene regulation, as well as future prospects in this area of inquiry.

Flexible analysis of TSS mapping data and detection of TSS shifts with TSRexploreR.

Heterogeneity in transcription initiation has important consequences for transcript stability and translation, and shifts in transcription start site (TSS) usage are prevalent in various developmental, metabolic, and disease contexts. Accordingly, numerous methods for global TSS profiling have been developed, including most recently Survey of TRanscription Initiation at Promoter Elements with high-throughput sequencing (STRIPE-seq), a method to profile transcription start sites (TSSs) on a genome-wide scale with significant cost and time savings compared to previous methods. In anticipation of more widespread adoption of STRIPE-seq and related methods for construction of promoter atlases and studies of differential gene expression, we built TSRexploreR, an R package for end-to-end analysis of TSS mapping data. TSRexploreR provides functions for TSS and transcription start region (TSR) detection, normalization, correlation, visualization, and differential TSS/TSR analyses. TSRexploreR is highly interoperable, accepting the data structures of TSS and TSR sets generated by several existing tools for processing and alignment of TSS mapping data, such as CAGEr for Cap Analysis of Gene Expression (CAGE) data. Lastly, TSRexploreR implements a novel approach for the detection of shifts in TSS distribution.

LSD1 and Aberrant DNA Methylation Mediate Persistence of Enteroendocrine Progenitors That Support BRAF-Mutant Colorectal Cancer.

Despite the connection of secretory cells, including goblet and enteroendocrine (EEC) cells, to distinct mucus-containing colorectal cancer histologic subtypes, their role in colorectal cancer progression has been underexplored. Here, our analysis of The Cancer Genome Atlas (TCGA) and single-cell RNA-sequencing data demonstrates that EEC progenitor cells are enriched in BRAF-mutant colorectal cancer patient tumors, cell lines, and patient-derived organoids. In BRAF-mutant colorectal cancer, EEC progenitors were blocked from differentiating further by DNA methylation and silencing of NEUROD1, a key gene required for differentiation of intermediate EECs. Mechanistically, secretory cells and the factors they secrete, such as trefoil factor 3, promoted colony formation and activation of cell survival pathways in the entire cell population. Lysine-specific demethylase 1 (LSD1) was identified as a critical regulator of secretory cell specification in vitro and in a colon orthotopic xenograft model, where LSD1 loss blocks formation of EEC progenitors and reduces tumor growth and metastasis. These findings reveal an important role for EEC progenitors in supporting colorectal cancer. SIGNIFICANCE: This study establishes enteroendocrine progenitors as a targetable population that promotes BRAF-mutant colorectal cancer and can be blocked by LSD1 inhibition to suppress tumor growth.

Early satellite cell communication creates a permissive environment for long-term muscle growth.

Using in vivo muscle stem cell (satellite cell)-specific extracellular vesicle (EV) tracking, satellite cell depletion, in vitro cell culture, and single-cell RNA sequencing, we show satellite cells communicate with other cells in skeletal muscle during mechanical overload. Early satellite cell EV communication primes the muscle milieu for proper long-term extracellular matrix (ECM) deposition and is sufficient to support sustained hypertrophy in adult mice, even in the absence of fusion to muscle fibers. Satellite cells modulate chemokine gene expression across cell types within the first few days of loading, and EV delivery of miR-206 to fibrogenic cells represses Wisp1 expression required for appropriate ECM remodeling. Late-stage communication from myogenic cells during loading is widespread but may be targeted toward endothelial cells. Satellite cells coordinate adaptation by influencing the phenotype of recipient cells, which extends our understanding of their role in muscle adaptation beyond regeneration and myonuclear donation.

Lysine-Specific Demethylase 1 Mediates AKT Activity and Promotes Epithelial-to-Mesenchymal Transition in PIK3CA-Mutant Colorectal Cancer.

Activation of the epithelial-to-mesenchymal transition (EMT) program is a critical mechanism for initiating cancer progression and migration. Colorectal cancers contain many genetic and epigenetic alterations that can contribute to EMT. Mutations activating the PI3K/AKT signaling pathway are observed in >40% of patients with colorectal cancer contributing to increased invasion and metastasis. Little is known about how oncogenic signaling pathways such as PI3K/AKT synergize with chromatin modifiers to activate the EMT program. Lysine-specific demethylase 1 (LSD1) is a chromatin-modifying enzyme that is overexpressed in colorectal cancer and enhances cell migration. In this study, we determine that LSD1 expression is significantly elevated in patients with colorectal cancer with mutation of the catalytic subunit of PI3K, PIK3CA, compared with patients with colorectal cancer with WT PIK3CA. LSD1 enhances activation of the AKT kinase in colorectal cancer cells through a noncatalytic mechanism, acting as a scaffolding protein for the transcription-repressing CoREST complex. In addition, growth of PIK3CA-mutant colorectal cancer cells is uniquely dependent on LSD1. Knockdown or CRISPR knockout of LSD1 blocks AKT-mediated stabilization of the EMT-promoting transcription factor Snail and effectively blocks AKT-mediated EMT and migration. Overall, we uniquely demonstrate that LSD1 mediates AKT activation in response to growth factors and oxidative stress, and LSD1-regulated AKT activity promotes EMT-like characteristics in a subset of PIK3CA-mutant cells. IMPLICATIONS: Our data support the hypothesis that inhibitors targeting the CoREST complex may be clinically effective in patients with colorectal cancer harboring PIK3CA mutations.

Enzymatic methods for genome-wide profiling of protein binding sites.

Genome-wide mapping of protein-DNA interactions is a staple approach in many areas of modern molecular biology. Genome-wide profiles of protein-binding sites are most commonly generated by chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq). Although ChIP-seq has played a central role in studying genome-wide protein binding, recent work has highlighted systematic biases in the technique that warrant technical and interpretive caution and underscore the need for orthogonal techniques to both confirm the results of ChIP-seq studies and uncover new insights not accessible to ChIP. Several such techniques, based on genetic or immunological targeting of enzymatic activity to specific genomic loci, have been developed. Here, we review the development, applications and future prospects of these methods as complements to ChIP-based approaches and as powerful techniques in their own right.

Yeast Vps13 promotes mitochondrial function and is localized at membrane contact sites.

The Vps13 protein family is highly conserved in eukaryotic cells. Mutations in human VPS13 genes result in a variety of diseases, such as chorea acanthocytosis (ChAc), but the cellular functions of Vps13 proteins are not well defined. In yeast, there is a single VPS13 orthologue, which is required for at least two different processes: protein sorting to the vacuole and sporulation. This study demonstrates that VPS13 is also important for mitochondrial integrity. In addition to preventing transfer of DNA from the mitochondrion to the nucleus, VPS13 suppresses mitophagy and functions in parallel with the endoplasmic reticulum-mitochondrion encounter structure (ERMES). In different growth conditions, Vps13 localizes to endosome-mitochondrion contacts and to the nuclear-vacuole junctions, indicating that Vps13 may function at membrane contact sites. The ability of VPS13 to compensate for the absence of ERMES correlates with its intracellular distribution. We propose that Vps13 is present at multiple membrane contact sites and that separation-of-function mutants are due to loss of Vps13 at specific junctions. Introduction of VPS13A mutations identified in ChAc patients at cognate sites in yeast VPS13 are specifically defective in compensating for the lack of ERMES, suggesting that mitochondrial dysfunction might be the basis for ChAc.