A Cluster of Evolutionarily Recent KRAB Zinc Finger Proteins Protects Cancer Cells from Replicative Stress-Induced Inflammation.

Heterochromatin loss and genetic instability enhance cancer progression by favoring clonal diversity, yet uncontrolled replicative stress leads to mitotic catastrophe and inflammatory responses that promote immune rejection. KRAB domain-containing zinc finger proteins (KZFP) contribute to heterochromatin maintenance at transposable elements (TE). Here, we identified an association of upregulation of a cluster of primate-specific KZFPs with poor prognosis, increased copy-number alterations, and changes in the tumor microenvironment in diffuse large B-cell lymphoma (DLBCL). Depleting two of these KZFPs targeting evolutionarily recent TEs, ZNF587 and ZNF417, impaired the proliferation of cells derived from DLBCL and several other tumor types. ZNF587 and ZNF417 depletion led to heterochromatin redistribution, replicative stress, and cGAS-STING-mediated induction of an interferon/inflammatory response, which enhanced susceptibility to macrophage-mediated phagocytosis and increased surface expression of HLA-I, together with presentation of a neoimmunopeptidome. Thus, cancer cells can exploit KZFPs to dampen TE-originating surveillance mechanisms, which likely facilitates clonal expansion, diversification, and immune evasion. SIGNIFICANCE: Upregulation of a cluster of primate-specific KRAB zinc finger proteins in cancer cells prevents replicative stress and inflammation by regulating heterochromatin maintenance, which could facilitate the development of improved biomarkers and treatments.

Transposable elements mediate genetic effects altering the expression of nearby genes in colorectal cancer.

Transposable elements (TEs) are prevalent repeats in the human genome, play a significant role in the regulome, and their disruption can contribute to tumorigenesis. However, TE influence on gene expression in cancer remains unclear. Here, we analyze 275 normal colon and 276 colorectal cancer samples from the SYSCOL cohort, discovering 10,231 and 5,199 TE-expression quantitative trait loci (eQTLs) in normal and tumor tissues, respectively, of which 376 are colorectal cancer specific eQTLs, likely due to methylation changes. Tumor-specific TE-eQTLs show greater enrichment of transcription factors, compared to shared TE-eQTLs suggesting specific regulation of their expression in tumor. Bayesian networks reveal 1,766 TEs as mediators of genetic effects, altering the expression of 1,558 genes, including 55 known cancer driver genes and show that tumor-specific TE-eQTLs trigger the driver capability of TEs. These insights expand our knowledge of cancer drivers, deepening our understanding of tumorigenesis and presenting potential avenues for therapeutic interventions.

Ancestral genome reconstruction enhances transposable element annotation by identifying degenerate integrants.

Growing evidence indicates that transposable elements (TEs) play important roles in evolution by providing genomes with coding and non-coding sequences. Identification of TE-derived functional elements, however, has relied on TE annotations in individual species, which limits its scope to relatively intact TE sequences. Here, we report a novel approach to uncover previously unannotated degenerate TEs (degTEs) by probing multiple ancestral genomes reconstructed from hundreds of species. We applied this method to the human genome and achieved a 10.8% increase in coverage over the most recent annotation. Further, we discovered that degTEs contribute to various cis-regulatory elements and transcription factor binding sites, including those of a known TE-controlling family, the KRAB zinc-finger proteins. We also report unannotated chimeric transcripts between degTEs and human genes expressed in embryos. This study provides a novel methodology and a freely available resource that will facilitate the investigation of TE co-option events on a full scale.

Statistical learning quantifies transposable element-mediated cis-regulation.

BACKGROUND: Transposable elements (TEs) have colonized the genomes of most metazoans, and many TE-embedded sequences function as cis-regulatory elements (CREs) for genes involved in a wide range of biological processes from early embryogenesis to innate immune responses. Because of their repetitive nature, TEs have the potential to form CRE platforms enabling the coordinated and genome-wide regulation of protein-coding genes by only a handful of trans-acting transcription factors (TFs). RESULTS: Here, we directly test this hypothesis through mathematical modeling and demonstrate that differences in expression at protein-coding genes alone are sufficient to estimate the magnitude and significance of TE-contributed cis-regulatory activities, even in contexts where TE-derived transcription fails to do so. We leverage hundreds of overexpression experiments and estimate that, overall, gene expression is influenced by TE-embedded CREs situated within approximately 500 kb of promoters. Focusing on the cis-regulatory potential of TEs within the gene regulatory network of human embryonic stem cells, we find that pluripotency-specific and evolutionarily young TE subfamilies can be reactivated by TFs involved in post-implantation embryogenesis. Finally, we show that TE subfamilies can be split into truly regulatorily active versus inactive fractions based on additional information such as matched epigenomic data, observing that TF binding may better predict TE cis-regulatory activity than differences in histone marks. CONCLUSION: Our results suggest that TE-embedded CREs contribute to gene regulation during and beyond gastrulation. On a methodological level, we provide a statistical tool that infers TE-dependent cis-regulation from RNA-seq data alone, thus facilitating the study of TEs in the next-generation sequencing era.

Genetic features and genomic targets of human KRAB-zinc finger proteins.

Krüppel-associated box (KRAB) domain-containing zinc finger proteins (KZFPs) are one of the largest groups of transcription factors encoded by tetrapods, with 378 members in human alone. KZFP genes are often grouped in clusters reflecting amplification by gene and segment duplication since the gene family first emerged more than 400 million years ago. Previous work has revealed that many KZFPs recognize transposable element (TE)-embedded sequences as genomic targets, and that KZFPs facilitate the co-option of the regulatory potential of TEs for the benefit of the host. Here, we present a comprehensive survey of the genetic features and genomic targets of human KZFPs, notably completing past analyses by adding data on close to a hundred family members. General principles emerge from our study of the TE-KZFP regulatory system, which point to multipronged evolutionary mechanisms underlaid by highly complex and combinatorial modes of action with strong influences on human speciation.

The immunopeptidome landscape associated with T cell infiltration, inflammation and immune editing in lung cancer.

One key barrier to improving efficacy of personalized cancer immunotherapies that are dependent on the tumor antigenic landscape remains patient stratification. Although patients with CD3+CD8+ T cell-inflamed tumors typically show better response to immune checkpoint inhibitors, it is still unknown whether the immunopeptidome repertoire presented in highly inflamed and noninflamed tumors is substantially different. We surveyed 61 tumor regions and adjacent nonmalignant lung tissues from 8 patients with lung cancer and performed deep antigen discovery combining immunopeptidomics, genomics, bulk and spatial transcriptomics, and explored the heterogeneous expression and presentation of tumor (neo)antigens. In the present study, we associated diverse immune cell populations with the immunopeptidome and found a relatively higher frequency of predicted neoantigens located within HLA-I presentation hotspots in CD3+CD8+ T cell-excluded tumors. We associated such neoantigens with immune recognition, supporting their involvement in immune editing. This could have implications for the choice of combination therapies tailored to the patient's mutanome and immune microenvironment.

Decreased expression of mitochondrial aminoacyl-tRNA synthetases causes downregulation of OXPHOS subunits in type 2 diabetic muscle.

Type 2 diabetes mellitus (T2D) affects millions of people worldwide and is one of the leading causes of morbidity and mortality. The skeletal muscle (SKM) is one of the most important tissues involved in maintaining glucose homeostasis and substrate oxidation, and it undergoes insulin resistance in T2D. In this study, we identify the existence of alterations in the expression of mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) in skeletal muscle from two different forms of T2D: early-onset type 2 diabetes (YT2) (onset of the disease before 30 years of age) and the classical form of the disease (OT2). GSEA analysis from microarray studies revealed the repression of mitochondrial mt-aaRSs independently of age, which was validated by real-time PCR assays. In agreement with this, a reduced expression of several encoding mt-aaRSs was also detected in skeletal muscle from diabetic (db/db) mice but not in obese ob/ob mice. In addition, the expression of the mt-aaRSs proteins most relevant in the synthesis of mitochondrial proteins, threonyl-tRNA, and leucyl-tRNA synthetases (TARS2 and LARS2) were also repressed in muscle from db/db mice. It is likely that these alterations participate in the reduced expression of proteins synthesized in the mitochondria detected in db/db mice. We also document an increased iNOS abundance in mitochondrial-enriched muscle fractions from diabetic mice that may inhibit aminoacylation of TARS2 and LARS2 by nitrosative stress. Our results indicate a reduced expression of mt-aaRSs in skeletal muscle from T2D patients, which may participate in the reduced expression of proteins synthesized in mitochondria. An enhanced mitochondrial iNOS could play a regulatory role in diabetes.

Primate-specific transposable elements shape transcriptional networks during human development.

The human genome contains more than 4.5 million inserts derived from transposable elements (TEs), the result of recurrent waves of invasion and internal propagation throughout evolution. For new TE copies to be inherited, they must become integrated in the genome of the germline or pre-implantation embryo, which requires that their source TE be expressed at these stages. Accordingly, many TEs harbor DNA binding sites for the pluripotency factors OCT4, NANOG, SOX2, and KLFs and are transiently expressed during embryonic genome activation. Here, we describe how many primate-restricted TEs have additional binding sites for lineage-specific transcription factors driving their expression during human gastrulation and later steps of fetal development. These TE integrants serve as lineage-specific enhancers fostering the transcription, amongst other targets, of KRAB-zinc finger proteins (KZFPs) of comparable evolutionary age, which in turn corral the activity of TE-embedded regulatory sequences in a similarly lineage-restricted fashion. Thus, TEs and their KZFP controllers play broad roles in shaping transcriptional networks during early human development.

Transposon-activated POU5F1B promotes colorectal cancer growth and metastasis.

The treatment of colorectal cancer (CRC) is an unmet medical need in absence of early diagnosis. Here, upon characterizing cancer-specific transposable element-driven transpochimeric gene transcripts (TcGTs) produced by this tumor in the SYSCOL cohort, we find that expression of the hominid-restricted retrogene POU5F1B through aberrant activation of a primate-specific endogenous retroviral promoter is a strong negative prognostic biomarker. Correlating this observation, we demonstrate that POU5F1B fosters the proliferation and metastatic potential of CRC cells. We further determine that POU5F1B, in spite of its phylogenetic relationship with the POU5F1/OCT4 transcription factor, is a membrane-enriched protein that associates with protein kinases and known targets or interactors as well as with cytoskeleton-related molecules, and induces intracellular signaling events and the release of trans-acting factors involved in cell growth and cell adhesion. As POU5F1B is an apparently non-essential gene only lowly expressed in normal tissues, and as POU5F1B-containing TcGTs are detected in other tumors besides CRC, our data provide interesting leads for the development of cancer therapies.

Transposable elements contribute to the spatiotemporal microRNA landscape in human brain development.

Transposable elements (TEs) contribute to the evolution of gene regulatory networks and are dynamically expressed throughout human brain development and disease. One gene regulatory mechanism influenced by TEs is the miRNA system of post-transcriptional control. miRNA sequences frequently overlap TE loci and this miRNA expression landscape is crucial for control of gene expression in adult brain and different cellular contexts. Despite this, a thorough investigation of the spatiotemporal expression of TE-embedded miRNAs in human brain development is lacking. Here, we identify a spatiotemporally dynamic TE-embedded miRNA expression landscape between childhood and adolescent stages of human brain development. These miRNAs sometimes arise from two apposed TEs of the same subfamily, such as for L2 or MIR elements, but in the majority of cases stem from solo TEs. They give rise to in silico predicted high-confidence pre-miRNA hairpin structures, likely represent functional miRNAs, and have predicted genic targets associated with neurogenesis. TE-embedded miRNA expression is distinct in the cerebellum when compared to other brain regions, as has previously been described for gene and TE expression. Furthermore, we detect expression of previously nonannotated TE-embedded miRNAs throughout human brain development, suggestive of a previously undetected miRNA control network. Together, as with non-TE-embedded miRNAs, TE-embedded sequences give rise to spatiotemporally dynamic miRNA expression networks, the implications of which for human brain development constitute extensive avenues of future experimental research. To facilitate interactive exploration of these spatiotemporal miRNA expression dynamics, we provide the "Brain miRTExplorer" web application freely accessible for the community.

Transposable elements and their KZFP controllers are drivers of transcriptional innovation in the developing human brain.

Transposable elements (TEs) account for more than 50% of the human genome and many have been co-opted throughout evolution to provide regulatory functions for gene expression networks. Several lines of evidence suggest that these networks are fine-tuned by the largest family of TE controllers, the KRAB-containing zinc finger proteins (KZFPs). One tissue permissive for TE transcriptional activation (termed "transposcription") is the adult human brain, however comprehensive studies on the extent of this process and its potential contribution to human brain development are lacking. To elucidate the spatiotemporal transposcriptome of the developing human brain, we have analyzed two independent RNA-seq data sets encompassing 16 brain regions from eight weeks postconception into adulthood. We reveal a distinct KZFP:TE transcriptional profile defining the late prenatal to early postnatal transition, and the spatiotemporal and cell type-specific activation of TE-derived alternative promoters driving the expression of neurogenesis-associated genes. Long-read sequencing confirmed these TE-driven isoforms as significant contributors to neurogenic transcripts. We also show experimentally that a co-opted antisense L2 element drives temporal protein relocalization away from the endoplasmic reticulum, suggestive of novel TE dependent protein function in primate evolution. This work highlights the widespread dynamic nature of the spatiotemporal KZFP:TE transcriptome and its importance throughout TE mediated genome innovation and neurotypical human brain development. To facilitate interactive exploration of these spatiotemporal gene and TE expression dynamics, we provide the "Brain TExplorer" web application freely accessible for the community.

Integrated proteogenomic deep sequencing and analytics accurately identify non-canonical peptides in tumor immunopeptidomes.

Efforts to precisely identify tumor human leukocyte antigen (HLA) bound peptides capable of mediating T cell-based tumor rejection still face important challenges. Recent studies suggest that non-canonical tumor-specific HLA peptides derived from annotated non-coding regions could elicit anti-tumor immune responses. However, sensitive and accurate mass spectrometry (MS)-based proteogenomics approaches are required to robustly identify these non-canonical peptides. We present an MS-based analytical approach that characterizes the non-canonical tumor HLA peptide repertoire, by incorporating whole exome sequencing, bulk and single-cell transcriptomics, ribosome profiling, and two MS/MS search tools in combination. This approach results in the accurate identification of hundreds of shared and tumor-specific non-canonical HLA peptides, including an immunogenic peptide derived from an open reading frame downstream of the melanoma stem cell marker gene ABCB5. These findings hold great promise for the discovery of previously unknown tumor antigens for cancer immunotherapy.

KAP1 targets actively transcribed genomic loci to exert pleomorphic effects on RNA polymerase II activity.

KAP1 (KRAB-associated protein 1) is best known as a co-repressor responsible for inducing heterochromatin formation, notably at transposable elements. However, it has also been observed to bind the transcription start site of actively expressed genes. To address this paradox, we characterized the protein interactome of KAP1 in the human K562 erythro-leukaemia cell line. We found that the regulator can associate with a wide range of nucleic acid binding proteins, nucleosome remodellers, chromatin modifiers and other transcription modulators. We further determined that KAP1 is recruited at actively transcribed polymerase II promoters, where its depletion resulted in pleomorphic effects, whether expression of these genes was normally constitutive or inducible, consistent with the breadth of possible KAP1 interactors. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.

Hominoid-Specific Transposable Elements and KZFPs Facilitate Human Embryonic Genome Activation and Control Transcription in Naive Human ESCs.

Expansion of transposable elements (TEs) coincides with evolutionary shifts in gene expression. TEs frequently harbor binding sites for transcriptional regulators, thus enabling coordinated genome-wide activation of species- and context-specific gene expression programs, but such regulation must be balanced against their genotoxic potential. Here, we show that Krüppel-associated box (KRAB)-containing zinc finger proteins (KZFPs) control the timely and pleiotropic activation of TE-derived transcriptional cis regulators during early embryogenesis. Evolutionarily recent SVA, HERVK, and HERVH TE subgroups contribute significantly to chromatin opening during human embryonic genome activation and are KLF-stimulated enhancers in naive human embryonic stem cells (hESCs). KZFPs of corresponding evolutionary ages are simultaneously induced and repress the transcriptional activity of these TEs. Finally, the same KZFP-controlled TE-based enhancers later serve as developmental and tissue-specific enhancers. Thus, by controlling the transcriptional impact of TEs during embryogenesis, KZFPs facilitate their genome-wide incorporation into transcriptional networks, thereby contributing to human genome regulation.

ZNF445 is a primary regulator of genomic imprinting.

Genomic imprinting is an epigenetic process regulated by germline-derived DNA methylation, causing parental origin-specific monoallelic gene expression. Zinc finger protein 57 (ZFP57) is critical for maintenance of this epigenetic memory during post-fertilization reprogramming, yet incomplete penetrance of ZFP57 mutations in humans and mice suggests additional effectors. We reveal that ZNF445/ZFP445, which we trace to the origins of imprinting, binds imprinting control regions (ICRs) in mice and humans. In mice, ZFP445 and ZFP57 act together, maintaining all but one ICR in vivo, whereas earlier embryonic expression of ZNF445 and its intolerance to loss-of-function mutations indicate greater importance in the maintenance of human imprints.

Pharmacological Induction of a Progenitor State for the Efficient Expansion of Primary Human Hepatocytes.

The liver is an organ with strong regenerative capacity, yet primary hepatocytes have a low amplification potential in vitro, a major limitation for the cell-based therapy of liver disorders and for ex vivo biological screens. Induced pluripotent stem cells (iPSCs) may help to circumvent this obstacle but often harbor genetic and epigenetic abnormalities, limiting their potential. Here, we describe the pharmacological induction of proliferative human hepatic progenitor cells (HPCs) through a cocktail of growth factors and small molecules mimicking the signaling events involved in liver regeneration. Human HPCs from healthy donors and pediatric patients proliferated vigorously while maintaining their genomic stability and could be redifferentiated in vitro into metabolically competent cells that supported the replication of hepatitis B and delta viruses. Redifferentiation efficiency was boosted by three-dimensional culture. Finally, transcriptome analysis showed that HPCs were more closely related to mature hepatocytes than iPSC-derived hepatocyte-like cells were. Conclusion: HPC induction holds promise for a variety of applications such as ex vivo disease modeling, personalized drug testing or metabolic studies, and development of a bioartificial liver.

Properties of LINE-1 proteins and repeat element expression in the context of amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving loss of motor neurons and having no known cure and uncertain etiology. Several studies have drawn connections between altered retrotransposon expression and ALS. Certain features of the LINE-1 (L1) retrotransposon-encoded ORF1 protein (ORF1p) are analogous to those of neurodegeneration-associated RNA-binding proteins, including formation of cytoplasmic aggregates. In this study we explore these features and consider possible links between L1 expression and ALS. RESULTS: We first considered factors that modulate aggregation and subcellular distribution of LINE-1 ORF1p, including nuclear localization. Changes to some ORF1p amino acid residues alter both retrotransposition efficiency and protein aggregation dynamics, and we found that one such polymorphism is present in endogenous L1s abundant in the human genome. We failed, however, to identify CRM1-mediated nuclear export signals in ORF1p nor strict involvement of cell cycle in endogenous ORF1p nuclear localization in human 2102Ep germline teratocarcinoma cells. Some proteins linked with ALS bind and colocalize with L1 ORF1p ribonucleoprotein particles in cytoplasmic RNA granules. Increased expression of several ALS-associated proteins, including TAR DNA Binding Protein (TDP-43), strongly limits cell culture retrotransposition, while some disease-related mutations modify these effects. Using quantitative reverse transcription PCR (RT-qPCR) of ALS tissues and reanalysis of publicly available RNA-Seq datasets, we asked if changes in expression of retrotransposons are associated with ALS. We found minimal altered expression in sporadic ALS tissues but confirmed a previous report of differential expression of many repeat subfamilies in C9orf72 gene-mutated ALS patients. CONCLUSIONS: Here we extended understanding of the subcellular localization dynamics of the aggregation-prone LINE-1 ORF1p RNA-binding protein. However, we failed to find compelling evidence for misregulation of LINE-1 retrotransposons in sporadic ALS nor a clear effect of ALS-associated TDP-43 protein on L1 expression. In sum, our study reveals that the interplay of active retrotransposons and the molecular features of ALS are more complex than anticipated. Thus, the potential consequences of altered retrotransposon activity for ALS and other neurodegenerative disorders are worthy of continued investigation.

Mitochondrial DNA and TLR9 drive muscle inflammation upon Opa1 deficiency.

Opa1 participates in inner mitochondrial membrane fusion and cristae morphogenesis. Here, we show that muscle-specific Opa1 ablation causes reduced muscle fiber size, dysfunctional mitochondria, enhanced Fgf21, and muscle inflammation characterized by NF-κB activation, and enhanced expression of pro-inflammatory genes. Chronic sodium salicylate treatment ameliorated muscle alterations and reduced the muscle expression of Fgf21. Muscle inflammation was an early event during the progression of the disease and occurred before macrophage infiltration, indicating that it is a primary response to Opa1 deficiency. Moreover, Opa1 repression in muscle cells also resulted in NF-κB activation and inflammation in the absence of necrosis and/or apoptosis, thereby revealing that the activation is a cell-autonomous process and independent of cell death. The effects of Opa1 deficiency on the expression NF-κB target genes and inflammation were absent upon mitochondrial DNA depletion. Under Opa1 deficiency, blockage or repression of TLR9 prevented NF-κB activation and inflammation. Taken together, our results reveal that Opa1 deficiency in muscle causes initial mitochondrial alterations that lead to TLR9 activation, and inflammation, which contributes to enhanced Fgf21 expression and to growth impairment.

Early-onset and classical forms of type 2 diabetes show impaired expression of genes involved in muscle branched-chain amino acids metabolism.

The molecular mechanisms responsible for the pathophysiological traits of type 2 diabetes are incompletely understood. Here we have performed transcriptomic analysis in skeletal muscle, and plasma metabolomics from subjects with classical and early-onset forms of type 2 diabetes (T2D). Focused studies were also performed in tissues from ob/ob and db/db mice. We document that T2D, both early and late onset, are characterized by reduced muscle expression of genes involved in branched-chain amino acids (BCAA) metabolism. Weighted Co-expression Networks Analysis provided support to idea that the BCAA genes are relevant in the pathophysiology of type 2 diabetes, and that mitochondrial BCAA management is impaired in skeletal muscle from T2D patients. In diabetic mice model we detected alterations in skeletal muscle proteins involved in BCAA metabolism but not in obese mice. Metabolomic analysis revealed increased levels of branched-chain keto acids (BCKA), and BCAA in plasma of T2D patients, which may result from the disruption of muscle BCAA management. Our data support the view that inhibition of genes involved in BCAA handling in skeletal muscle takes place as part of the pathophysiology of type 2 diabetes, and this occurs both in early-onset and in classical type 2 diabetes.

DUX-family transcription factors regulate zygotic genome activation in placental mammals.

In animal embryos, transcription is mostly silent for several cell divisions, until the release of the first major wave of embryonic transcripts through so-called zygotic genome activation (ZGA). Maternally provided ZGA-triggering factors have been identified in Drosophila melanogaster and Danio rerio, but their mammalian homologs are still undefined. Here, we provide evidence that the DUX family of transcription factors is essential to this process in mice and potentially in humans. First, human DUX4 and mouse Dux are both expressed before ZGA in their respective species. Second, both orthologous proteins bind the promoters of ZGA-associated genes and activate their transcription. Third, Dux knockout in mouse embryonic stem cells (mESCs) prevents the cells from cycling through a 2-cell-like state. Finally, zygotic depletion of Dux leads to impaired early embryonic development and defective ZGA. We conclude that DUX-family proteins are key inducers of zygotic genome activation in placental mammals.