YY1 knockout in pro-B cells impairs lineage commitment, enabling unusual hematopoietic lineage plasticity.

During B-cell development, cells progress through multiple developmental stages, with the pro-B-cell stage defining commitment to the B-cell lineage. YY1 is a ubiquitous transcription factor that is capable of both activation and repression functions. We found here that knockout of YY1 at the pro-B-cell stage eliminates B lineage commitment. YY1 knockout pro-B cells can generate T lineage cells in vitro using the OP9-DL4 feeder system and in vivo after injection into sublethally irradiated Rag1-/- mice. These T lineage-like cells lose their B lineage transcript profile and gain a T-cell lineage profile. Single-cell RNA-seq experiments showed that as YY1 knockout pro-B cells transition into T lineage cells in vitro, various cell clusters adopt transcript profiles representing a multiplicity of hematopoietic lineages, indicating unusual lineage plasticity. In addition, YY1 KO pro-B cells in vivo can give rise to other hematopoietic lineages in vivo. Evaluation of RNA-seq, scRNA-seq, ChIP-seq, and scATAC-seq data indicates that YY1 controls numerous chromatin-modifying proteins leading to increased accessibility of alternative lineage genes in YY1 knockout pro-B cells. Given the ubiquitous nature of YY1 and its dual activation and repression functions, YY1 may regulate commitment in multiple cell lineages.

APC and P53 mutations synergise to create a therapeutic vulnerability to NOTUM inhibition in advanced colorectal cancer.

OBJECTIVE: Colorectal cancer (CRC) is a leading cause of cancer-related deaths, with the majority of cases initiated by inactivation of the APC tumour suppressor. This results in the constitutive activation of canonical WNT pathway transcriptional effector ß-catenin, along with induction of WNT feedback inhibitors, including the extracellular palmitoleoyl-protein carboxylesterase NOTUM which antagonises WNT-FZD receptor-ligand interactions. Here, we sought to evaluate the effects of NOTUM activity on CRC as a function of driver mutation landscape. DESIGN: Mouse and human colon organoids engineered with combinations of CRC driver mutations were used for Notum genetic gain-of-function and loss-of-function studies. In vitro assays, in vivo endoscope-guided orthotopic organoid implantation assays and transcriptomic profiling were employed to characterise the effects of Notum activity. Small molecule inhibitors of Notum activity were used in preclinical therapeutic proof-of-principle studies targeting oncogenic Notum activity. RESULTS: NOTUM retains tumour suppressive activity in APC-null adenomas despite constitutive ß-catenin activity. Strikingly, on progression to adenocarcinoma with P53 loss, NOTUM becomes an obligate oncogene. These phenotypes are Wnt-independent, resulting from differential activity of NOTUM on glypican 1 and 4 in early-stage versus late-stage disease, respectively. Ultimately, preclinical mouse models and human organoid cultures demonstrate that pharmacological inhibition of NOTUM is highly effective in arresting primary adenocarcinoma growth and inhibiting metastatic colonisation of distal organs. CONCLUSIONS: Our findings that a single agent targeting the extracellular enzyme NOTUM is effective in treating highly aggressive, metastatic adenocarcinomas in preclinical mouse models and human organoids make NOTUM and its glypican targets therapeutic vulnerabilities in advanced CRC.

Lineage-specific reorganization of nuclear peripheral heterochromatin and H3K9me2 domains.

Dynamic organization of chromatin within the three-dimensional nuclear space has been postulated to regulate gene expression and cell fate. Here, we define the genome-wide distribution of nuclear peripheral heterochromatin as a multipotent P19 cell adopts either a neural or a cardiac fate. We demonstrate that H3K9me2-marked nuclear peripheral heterochromatin undergoes lineage-specific reorganization during cell-fate determination. This is associated with spatial repositioning of genomic loci away from the nuclear periphery as shown by 3D immuno-FISH. Locus repositioning is not always associated with transcriptional changes, but a subset of genes is upregulated. Mef2c is specifically repositioned away from the nuclear periphery during early neurogenic differentiation, but not during early cardiogenic differentiation, with associated transcript upregulation. Myocd is specifically repositioned during early cardiogenic differentiation, but not during early neurogenic differentiation, and is transcriptionally upregulated at later stages of cardiac differentiation. We provide experimental evidence for lineage-specific regulation of nuclear architecture during cell-fate determination in a mouse cell line.

A Balance Between Intermediate Filaments and Microtubules Maintains Nuclear Architecture in the Cardiomyocyte.

Rationale: Mechanical forces are transduced to nuclear responses via the linkers of the nucleoskeleton and cytoskeleton (LINC) complex, which couples the cytoskeleton to the nuclear lamina and associated chromatin. While disruption of the LINC complex can cause cardiomyopathy, the relevant interactions that bridge the nucleoskeleton to cytoskeleton are poorly understood in the cardiomyocyte, where cytoskeletal organization is unique. Furthermore, while microtubules and desmin intermediate filaments associate closely with cardiomyocyte nuclei, the importance of these interactions is unknown. Objective: Here, we sought to determine how cytoskeletal interactions with the LINC complex regulate nuclear homeostasis in the cardiomyocyte. Methods and Results: To this end, we acutely disrupted the LINC complex, microtubules, actin, and intermediate filaments and assessed the consequences on nuclear morphology and genome organization in rat ventricular cardiomyocytes via a combination of super-resolution imaging, biophysical, and genomic approaches. We find that a balance of dynamic microtubules and desmin intermediate filaments is required to maintain nuclear shape and the fidelity of the nuclear envelope and lamina. Upon depletion of desmin (or nesprin [nuclear envelope spectrin repeat protein]-3, its binding partner in the LINC complex), polymerizing microtubules collapse the nucleus and drive infolding of the nuclear membrane. This results in DNA damage, a loss of genome organization, and broad transcriptional changes. The collapse in nuclear integrity is concomitant with compromised contractile function and may contribute to the pathophysiological changes observed in desmin-related myopathies. Conclusions: Disrupting the tethering of desmin to the nucleus results in a loss of nuclear homeostasis and rapid alterations to cardiomyocyte function. Our data suggest that a balance of forces imposed by intermediate filaments and microtubules is required to maintain nuclear structure and genome organization in the cardiomyocyte.

Brown marmorated stink bug, Halyomorpha halys (Stål), genome: putative underpinnings of polyphagy, insecticide resistance potential and biology of a top worldwide pest.

BACKGROUND: Halyomorpha halys (Stål), the brown marmorated stink bug, is a highly invasive insect species due in part to its exceptionally high levels of polyphagy. This species is also a nuisance due to overwintering in human-made structures. It has caused significant agricultural losses in recent years along the Atlantic seaboard of North America and in continental Europe. Genomic resources will assist with determining the molecular basis for this species' feeding and habitat traits, defining potential targets for pest management strategies. RESULTS: Analysis of the 1.15-Gb draft genome assembly has identified a wide variety of genetic elements underpinning the biological characteristics of this formidable pest species, encompassing the roles of sensory functions, digestion, immunity, detoxification and development, all of which likely support H. halys' capacity for invasiveness. Many of the genes identified herein have potential for biomolecular pesticide applications. CONCLUSIONS: Availability of the H. halys genome sequence will be useful for the development of environmentally friendly biomolecular pesticides to be applied in concert with more traditional, synthetic chemical-based controls.

Unusual lineage plasticity revealed by YY1 knockout in pro-B cells.

During B cell development, cells progress through multiple developmental stages with the pro-B cell stage defining commitment to the B cell lineage. YY1 is a ubiquitous transcription factor that is capable of both activation and repression functions. We find here that knockout of YY1 at the pro-B cell stage eliminates B lineage commitment. YY1 knockout pro-B cells can generate T lineage cells in vitro using the OP9- DL4 feeder system, as well as in vivo after injection into sub-lethally irradiated Rag1 -/- mice. These T lineage-like cells lose their B lineage transcript profile and gain a T cell lineage profile. Single cell-RNA-seq experiments showed that as YY1 knockout pro-B cells transition into T lineage cells, various cell clusters adopt transcript profiles representing a multiplicity of hematopoietic lineages indicating unusual lineage plasticity. Given the ubiquitous nature of YY1 and its dual activation and repression functions, YY1 likely regulates commitment in multiple cell lineages.

Real-time transcription polymerase chain reaction cycle threshold values as criteria for utilization of incidental COVID-19 positive lung donors.

Shortage of organ donors is an ongoing limiting factor in lung transplantation (LT). Despite increasing prevalence of asymptomatic COVID-19 infection, positive COVID-19 testing from a potential donor remains a contraindication at many LT centers. In this report, we present the outcomes of LT utilizing an algorithm based on donor clinical presentation, and COVID-19 real-time reverse transcription polymerase chain reaction (RT-PCR) with cycle threshold (Ct) values evaluation. The Ct value threshold for organ acceptance was >35. A total of 8 COVID-positive donors were included. No donor-to-recipient transmissions of COVID-19 were observed. Short-term outcomes were comparable to those reported in pre-COVID literature. Survival-to-date is 100% with median POD of 161 days. Our findings support the safety and efficacy of utilizing our algorithm including Ct value threshold for selection of donors with incidental COVID-19 positive testing.

Patient-Induced Pluripotent Stem Cell-Derived Hepatostellate Organoids Establish a Basis for Liver Pathologies in Telomeropathies.

BACKGROUND & AIMS: Dyskeratosis congenita (DC) is a telomere biology disorder caused primarily by mutations in the DKC1 gene. Patients with DC and related telomeropathies resulting from premature telomere dysfunction experience multiorgan failure. In the liver, DC patients present with nodular hyperplasia, steatosis, inflammation, and cirrhosis. However, the mechanism responsible for telomere dysfunction-induced liver disease remains unclear. METHODS: We used isogenic human induced pluripotent stem cells (iPSCs) harboring a causal DC mutation in DKC1 or a CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9)-corrected control allele to model DC liver pathologies. We differentiated these iPSCs into hepatocytes (HEPs) or hepatic stellate cells (HSCs) followed by generation of genotype-admixed hepatostellate organoids. Single-cell transcriptomics were applied to hepatostellate organoids to understand cell type-specific genotype-phenotype relationships. RESULTS: Directed differentiation of iPSCs into HEPs and stellate cells and subsequent hepatostellate organoid formation revealed a dominant phenotype in the parenchyma, with DC HEPs becoming hyperplastic and also eliciting a pathogenic hyperplastic, proinflammatory response in stellate cells independent of stellate cell genotype. Pathogenic phenotypes in DKC1-mutant HEPs and hepatostellate organoids could be rescued via suppression of serine/threonine kinase AKT (protein kinase B) activity, a central regulator of MYC-driven hyperplasia downstream of DKC1 mutation. CONCLUSIONS: Isogenic iPSC-derived admixed hepatostellate organoids offer insight into the liver pathologies in telomeropathies and provide a framework for evaluating emerging therapies.

Inhibition of nonalcoholic fatty liver disease in mice by selective inhibition of mTORC1.

Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) remain without effective therapies. The mechanistic target of rapamycin complex 1 (mTORC1) pathway is a potential therapeutic target, but conflicting interpretations have been proposed for how mTORC1 controls lipid homeostasis. We show that selective inhibition of mTORC1 signaling in mice, through deletion of the RagC/D guanosine triphosphatase-activating protein folliculin (FLCN), promotes activation of transcription factor E3 (TFE3) in the liver without affecting other mTORC1 targets and protects against NAFLD and NASH. Disease protection is mediated by TFE3, which both induces lipid consumption and suppresses anabolic lipogenesis. TFE3 inhibits lipogenesis by suppressing proteolytic processing and activation of sterol regulatory element-binding protein-1c (SREBP-1c) and by interacting with SREBP-1c on chromatin. Our data reconcile previously conflicting studies and identify selective inhibition of mTORC1 as a potential approach to treat NASH and NAFLD.

Pathogenic LMNA variants disrupt cardiac lamina-chromatin interactions and de-repress alternative fate genes.

Pathogenic mutations in LAMIN A/C (LMNA) cause abnormal nuclear structure and laminopathies. These diseases have myriad tissue-specific phenotypes, including dilated cardiomyopathy (DCM), but how LMNA mutations result in tissue-restricted disease phenotypes remains unclear. We introduced LMNA mutations from individuals with DCM into human induced pluripotent stem cells (hiPSCs) and found that hiPSC-derived cardiomyocytes, in contrast to hepatocytes or adipocytes, exhibit aberrant nuclear morphology and specific disruptions in peripheral chromatin. Disrupted regions were enriched for transcriptionally active genes and regions with lower LAMIN B1 contact frequency. The lamina-chromatin interactions disrupted in mutant cardiomyocytes were enriched for genes associated with non-myocyte lineages and correlated with higher expression of those genes. Myocardium from individuals with LMNA variants similarly showed aberrant expression of non-myocyte pathways. We propose that the lamina network safeguards cellular identity and that pathogenic LMNA variants disrupt peripheral chromatin with specific epigenetic and molecular characteristics, causing misexpression of genes normally expressed in other cell types.

Cardiac endothelial cells maintain open chromatin and expression of cardiomyocyte myofibrillar genes.

Endothelial cells (ECs) are widely heterogenous depending on tissue and vascular localization. Jambusaria et al. recently demonstrated that ECs in various tissues surprisingly possess mRNA signatures of their underlying parenchyma. The mechanism underlying this observation remains unexplained, and could include mRNA contamination during cell isolation, in vivo mRNA paracrine transfer from parenchymal cells to ECs, or cell-autonomous expression of these mRNAs in ECs. Here, we use a combination of bulk RNASeq, single-cell RNASeq datasets, in situ mRNA hybridization, and most importantly ATAC-Seq of FACS-isolated nuclei, to show that cardiac ECs actively express cardiomyocyte myofibril (CMF) genes and have open chromatin at CMF gene promoters. These open chromatin sites are enriched for sites targeted by cardiac transcription factors, and closed upon expansion of ECs in culture. Together, these data demonstrate unambiguously that the expression of CMF genes in ECs is cell-autonomous, and not simply a result of technical contamination or paracrine transfers of mRNAs, and indicate that local cues in the heart in vivo unexpectedly maintain fully open chromatin in ECs at genes previously thought limited to cardiomyocytes.

Differential Gene Expression in Red Imported Fire Ant (Solenopsis invicta) (Hymenoptera: Formicidae) Larval and Pupal Stages.

Solenopsis invicta Buren is an invasive ant species that has been introduced to multiple continents. One such area, the southern United States, has a history of multiple control projects using chemical pesticides over varying ranges, often resulting in non-target effects across trophic levels. With the advent of next generation sequencing and RNAi technology, novel investigations and new control methods are possible. A robust genome-guided transcriptome assembly was used to investigate gene expression differences between S. invicta larvae and pupae. These life stages differ in many physiological processes; of special importance is the vital role of S. invicta larvae as the colonies' "communal gut". Differentially expressed transcripts were identified related to many important physiological processes, including digestion, development, cell regulation and hormone signaling. This dataset provides essential developmental knowledge that reveals the dramatic changes in gene expression associated with social insect life stage roles, and can be leveraged using RNAi to develop effective control methods.

A Transcriptome Survey Spanning Life Stages and Sexes of the Harlequin Bug, Murgantia histrionica.

The harlequin bug, Murgantia histrionica (Hahn), is an agricultural pest in the continental United States, particularly in southern states. Reliable gene sequence data are especially useful to the development of species-specific, environmentally friendly molecular biopesticides and effective biolures for this insect. Here, mRNAs were sampled from whole insects at the 2nd and 4th nymphal instars, as well as sexed adults, and sequenced using Illumina RNA-Seq technology. A global assembly of these data identified 72,540 putative unique transcripts bearing high levels of similarity to transcripts identified in other taxa, with over 99% of conserved single-copy orthologs among insects being detected. Gene ontology and protein family analyses were conducted to explore the functional potential of the harlequin bug's gene repertoire, and phylogenetic analyses were conducted on gene families germane to xenobiotic detoxification, including glutathione S-transferases, carboxylesterases and cytochrome P450s. Genic content in harlequin bug was compared with that of the closely related invasive pest, the brown marmorated stink bug, Halyomorpha halys (Stål). Quantitative analyses of harlequin bug gene expression levels, experimentally validated using quantitative real-time PCR, identified genes differentially expressed between life stages and/or sexes.