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Hematopoietic stem cells (HSCs) have a number of unique physiologic adaptations that enable lifelong maintenance of blood cell production, including a highly regulated rate of protein synthesis. Yet, the precise vulnerabilities that arise from such adaptations have not been fully characterized. Here, inspired by a bone marrow failure disorder due to the loss of the histone deubiquitinase MYSM1, characterized by selectively disadvantaged HSCs, we show how reduced protein synthesis in HSCs results in increased ferroptosis. HSC maintenance can be fully rescued by blocking ferroptosis, despite no alteration in protein synthesis rates. Importantly, this selective vulnerability to ferroptosis not only underlies HSC loss in MYSM1 deficiency but also characterizes a broader liability of human HSCs. Increasing protein synthesis rates via MYSM1 overexpression makes HSCs less susceptible to ferroptosis, more broadly illustrating the selective vulnerabilities that arise in somatic stem cell populations as a result of physiologic adaptations. [Display omitted] • MYSM1 deficiency causes human HSC loss by ferroptosis • MYSM1-deficient HSCs have reduced translation of ferroptosis-protective mRNAs • Human HSCs, but not progenitors, are vulnerable to ferroptosis • Human HSC vulnerability to ferroptosis arises due to low rates of protein synthesis Hematopoietic stem cells have a low rate of protein synthesis that makes them selectively vulnerable to ferroptosis. [ABSTRACT FROM AUTHOR]

Somatic mutations in nonmalignant tissues accumulate with age and injury, but whether these mutations are adaptive on the cellular or organismal levels is unclear. To interrogate genes in human metabolic disease, we performed lineage tracing in mice harboring somatic mosaicism subjected to nonalcoholic steatohepatitis (NASH). Proof-of-concept studies with mosaic loss of Mboat7 , a membrane lipid acyltransferase, showed that increased steatosis accelerated clonal disappearance. Next, we induced pooled mosaicism in 63 known NASH genes, allowing us to trace mutant clones side by side. This in vivo tracing platform, which we coined MOSAICS, selected for mutations that ameliorate lipotoxicity, including mutant genes identified in human NASH. To prioritize new genes, additional screening of 472 candidates identified 23 somatic perturbations that promoted clonal expansion. In validation studies, liver-wide deletion of Tbx3 , Bcl6 , or Smyd2 resulted in protection against hepatic steatosis. Selection for clonal fitness in mouse and human livers identifies pathways that regulate metabolic disease. [Display omitted] • Clones with Mboat7 loss-of-function mutations are selected against in NASH livers • Clones with Gpam loss-of-function mutations are positively selected for in NASH • In vivo screening was built to identify genes that alter cell fitness and steatosis • Screening of transcription and epi-factors identified unexpected NASH target genes An in vivo lineage tracing platform was developed to identify somatically mutated clones that are positively selected in chronic liver disease, revealing genes that modify hepatocyte fitness in the context of fatty liver disease in a high-throughput manner. [ABSTRACT FROM AUTHOR]

Normal tissues accumulate genetic changes with age, but it is unknown if somatic mutations promote clonal expansion of non-malignant cells in the setting of chronic degenerative diseases. Exome sequencing of diseased liver samples from 82 patients revealed a complex mutational landscape in cirrhosis. Additional ultra-deep sequencing identified recurrent mutations in PKD1 , PPARGC1B, KMT2D , and ARID1A. The number and size of mutant clones increased as a function of fibrosis stage and tissue damage. To interrogate the functional impact of mutated genes, a pooled in vivo CRISPR screening approach was established. In agreement with sequencing results, examination of 147 genes again revealed that loss of Pkd1 , Kmt2d , and Arid1a promoted clonal expansion. Conditional heterozygous deletion of these genes in mice was also hepatoprotective in injury assays. Pre-malignant somatic alterations are often viewed through the lens of cancer, but we show that mutations can promote regeneration, likely independent of carcinogenesis. • Deep seq reveals an accumulation of mutations in chronic liver disease tissues • PKD1 , PPARGC1B , KMT2D , and ARID1A are recurrently mutated • In vivo CRISPR screens validate functional relevance of Pkd1 , Kmt2d , and Arid1a • Mutations seen in liver tissues but not in cancer promote hepatocyte fitness Deep sequencing reveals the mutational landscape of human cirrhosis and in vivo CRISPR screening identifies functional roles for recurrently mutated genes in hepatocyte fitness and liver regeneration. [ABSTRACT FROM AUTHOR]

Despite advances in defining diverse somatic mutations that cause myeloid malignancies, a significant heritable component for these cancers remains largely unexplained. Here, we perform rare variant association studies in a large population cohort to identify inherited predisposition genes for these blood cancers. CTR9 , which encodes a key component of the PAF1 transcription elongation complex, is among the significant genes identified. The risk variants found in the cases cause loss of function and result in a ∼10-fold increased odds of acquiring a myeloid malignancy. Partial CTR9 loss of function expands human hematopoietic stem cells (HSCs) by increased super elongation complex-mediated transcriptional activity, which thereby increases the expression of key regulators of HSC self-renewal. By following up on insights from a human genetic study examining inherited predisposition to the myeloid malignancies, we define a previously unknown antagonistic interaction between the PAF1 and super elongation complexes. These insights could enable targeted approaches for blood cancer prevention. [Display omitted] • Inherited CTR9 loss-of-function variants predispose to the myeloid malignancies • Partial, but not complete, loss of CTR9 expands human HSCs • Partial CTR9 loss expands HSCs through increased transcription elongation • Select PAF1 complex subunits interact with and activate the super elongation complex Rare inherited CTR9 loss-of-function variants predispose to myeloid malignancies by altering the balance between the PAF1 and super elongation complexes. Specific subunits of the PAF1 complex then act in concert with the super elongation complex to promote transcription elongation of genes that can drive the self-renewal of hematopoietic stem cells. [ABSTRACT FROM AUTHOR]

Cellular lineage histories and their molecular states encode fundamental principles of tissue development and homeostasis. Current lineage-recording mouse models have insufficient barcode diversity and single-cell lineage coverage for profiling tissues composed of millions of cells. Here, we developed DARLIN, an inducible Cas9 barcoding mouse line that utilizes terminal deoxynucleotidyl transferase (TdT) and 30 CRISPR target sites. DARLIN is inducible, generates massive lineage barcodes across tissues, and enables the detection of edited barcodes in ∼70% of profiled single cells. Using DARLIN, we examined fate bias within developing hematopoietic stem cells (HSCs) and revealed unique features of HSC migration. Additionally, we established a protocol for joint transcriptomic and epigenomic single-cell measurements with DARLIN and found that cellular clonal memory is associated with genome-wide DNA methylation rather than gene expression or chromatin accessibility. DARLIN will enable the high-resolution study of lineage relationships and their molecular signatures in diverse tissues and physiological contexts. [Display omitted] • DARLIN generates massive barcode diversity and labels ∼70% of profiled cells • DARLIN identifies early fate bias among HSCs and their transcriptomic signatures • DARLIN reveals low-level HSC circulation between bone-marrow niches in adulthood • Strong clonal memory in DNA methylation rather than mRNA or chromatin accessibility DARLIN is an inducible barcoding system that allows for lineage tracing and analysis across mouse tissues as well as combined transcriptional and epigenomic single-cell measurements. [ABSTRACT FROM AUTHOR]

The dynamic regulation of β-cell abundance is poorly understood. Since chromatin remodeling plays critical roles in liver regeneration, these mechanisms could be generally important for regeneration in other tissues. Here, we show that the ARID1A mammalian SWI/SNF complex subunit is a critical regulator of β-cell regeneration. Arid1a is highly expressed in quiescent β-cells but is physiologically suppressed when β-cells proliferate during pregnancy or after pancreas resection. Whole-body Arid1a knockout mice are protected against streptozotocin-induced diabetes. Cell-type and temporally specific genetic dissection show that β-cell-specific Arid1a deletion can potentiate β-cell regeneration in multiple contexts. Transcriptomic and epigenomic profiling of mutant islets reveal increased neuregulin-ERBB-NR4A signaling. Chemical inhibition of ERBB or NR4A1 blocks increased regeneration associated with Arid1a loss. Mammalian SWI/SNF (mSWI/SNF) complex activity is a barrier to β-cell regeneration in physiologic and disease states., Competing Interests: Declaration of interests H.Z. has a sponsored research agreement with Alnylam Pharmaceuticals, consults for Flagship Pioneering and Chroma Medicines, and serves on the SAB of Ubiquitix. These interests are not related to the contents of this paper., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Identifying new pathways that regulate mammalian regeneration is challenging due to the paucity of in vivo screening approaches. We employed pooled CRISPR knockout and activation screening in the regenerating liver to evaluate 165 chromatin regulatory proteins. Both screens identified the imitation-SWI chromatin remodeling components Baz2a and Baz2b, not previously implicated in regeneration. In vivo sgRNA, siRNA, and knockout strategies against either paralog confirmed increased regeneration. Distinct BAZ2-specific bromodomain inhibitors, GSK2801 and BAZ2-ICR, resulted in accelerated liver healing after diverse injuries. Inhibitor-treated mice also exhibited improved healing in an inflammatory bowel disease model, suggesting multi-tissue applicability. Transcriptomics on regenerating livers showed increases in ribosomal and cell cycle mRNAs. Surprisingly, CRISPRa screening to define mechanisms showed that overproducing Rpl10a or Rpl24 was sufficient to drive regeneration, whereas Rpl24 haploinsufficiency was rate limiting for BAZ2 inhibition-mediated regeneration. The discovery of regenerative roles for imitation-SWI components provides immediate strategies to enhance tissue repair., Competing Interests: Declaration of interests H.Z. has a sponsored research agreement with Alnylam Pharmaceuticals, consults for Flagship Pioneering, and serves on the SAB of Ubiquitix. These interests are not directly related to the contents of this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

ARID1A, an SWI/SNF chromatin-remodeling gene, is commonly mutated in cancer and hypothesized to be tumor suppressive. In some hepatocellular carcinoma patients, ARID1A was highly expressed in primary tumors but not in metastatic lesions, suggesting that ARID1A can be lost after initiation. Mice with liver-specific homozygous or heterozygous Arid1a loss were resistant to tumor initiation while ARID1A overexpression accelerated initiation. In contrast, homozygous or heterozygous Arid1a loss in established tumors accelerated progression and metastasis. Mechanistically, gain of Arid1a function promoted initiation by increasing CYP450-mediated oxidative stress, while loss of Arid1a within tumors decreased chromatin accessibility and reduced transcription of genes associated with migration, invasion, and metastasis. In summary, ARID1A has context-dependent tumor-suppressive and oncogenic roles in cancer., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Gene fusions and their encoded products (fusion RNAs and proteins) are viewed as one of the hallmarks of cancer. Traditionally, they were thought to be generated solely by chromosomal rearrangements. However, recent discoveries of trans-splicing and cis-splicing events between neighboring genes, suggest that there are other mechanisms to generate chimeric fusion RNAs without corresponding changes in DNA. In addition, chimeric RNAs have been detected in normal physiology, complicating the use of fusions in cancer detection and therapy. On the other hand, "intergenically spliced" fusion RNAs represent a new repertoire of biomarkers and therapeutic targets. Here, we review current knowledge on chimeric RNAs and implications for cancer detection and treatment, and discuss outstanding questions for the advancement of the field.