Waves of retrotransposon expansion remodel genome organization and CTCF binding in multiple mammalian lineages.

CTCF-binding locations represent regulatory sequences that are highly constrained over the course of evolution. To gain insight into how these DNA elements are conserved and spread through the genome, we defined the full spectrum of CTCF-binding sites, including a 33/34-mer motif, and identified over five thousand highly conserved, robust, and tissue-independent CTCF-binding locations by comparing ChIP-seq data from six mammals. Our data indicate that activation of retroelements has produced species-specific expansions of CTCF binding in rodents, dogs, and opossum, which often functionally serve as chromatin and transcriptional insulators. We discovered fossilized repeat elements flanking deeply conserved CTCF-binding regions, indicating that similar retrotransposon expansions occurred hundreds of millions of years ago. Repeat-driven dispersal of CTCF binding is a fundamental, ancient, and still highly active mechanism of genome evolution in mammalian lineages.

Cell type-specific analysis by single-cell profiling identifies a stable mammalian tRNA-mRNA interface and increased translation efficiency in neurons.

The correlation between codon and anticodon pools influences the efficiency of translation, but whether differences exist in these pools across individual cells is unknown. We determined that codon usage and amino acid demand are highly stable across different cell types using available mouse and human single-cell RNA-sequencing atlases. After showing the robustness of ATAC-sequencing measurements for the analysis of tRNA gene usage, we quantified anticodon usage and amino acid supply in both mouse and human single-cell ATAC-seq atlases. We found that tRNA gene usage is overall coordinated across cell types, except in neurons, which clustered separately from other cell types. Integration of these data sets revealed a strong and statistically significant correlation between amino acid supply and demand across almost all cell types. Neurons have an enhanced translation efficiency over other cell types, driven by an increased supply of tRNAAla (AGC) anticodons. This results in faster decoding of the Ala-GCC codon, as determined by cell type-specific ribosome profiling, suggesting that the reduction of tRNAAla (AGC) anticodon pools may be implicated in neurological pathologies. This study, the first such examination of codon usage, anticodon usage, and translation efficiency resolved at the cell-type level with single-cell information, identifies a conserved landscape of translation elongation across mammalian cellular diversity and evolution.

Target-enriched nanopore sequencing and de novo assembly reveals co-occurrences of complex on-target genomic rearrangements induced by CRISPR-Cas9 in human cells.

The CRISPR-Cas9 system is widely used to permanently delete genomic regions via dual guide RNAs. Genomic rearrangements induced by CRISPR-Cas9 can occur, but continuous technical developments make it possible to characterize complex on-target effects. We combined an innovative droplet-based target enrichment approach with long-read sequencing and coupled it to a customized de novo sequence assembly. This approach enabled us to dissect the sequence content at kilobase scale within an on-target genomic locus. We here describe extensive genomic disruptions by Cas9, involving the allelic co-occurrence of a genomic duplication and inversion of the target region, as well as integrations of exogenous DNA and clustered interchromosomal DNA fragment rearrangements. Furthermore, we found that these genomic alterations led to functional aberrant DNA fragments and can alter cell proliferation. Our findings broaden the consequential spectrum of the Cas9 deletion system, reinforce the necessity of meticulous genomic validations, and introduce a data-driven workflow enabling detailed dissection of the on-target sequence content with superior resolution.

Estrogen receptor activation remodels TEAD1 gene expression to alleviate hepatic steatosis.

Sex-based differences in obesity-related hepatic malignancies suggest the protective roles of estrogen. Using a preclinical model, we dissected estrogen receptor (ER) isoform-driven molecular responses in high-fat diet (HFD)-induced liver diseases of male and female mice treated with or without an estrogen agonist by integrating liver multi-omics data. We found that selective ER activation recovers HFD-induced molecular and physiological liver phenotypes. HFD and systemic ER activation altered core liver pathways, beyond lipid metabolism, that are consistent between mice and primates. By including patient cohort data, we uncovered that ER-regulated enhancers govern central regulatory and metabolic genes with clinical significance in metabolic dysfunction-associated steatotic liver disease (MASLD) patients, including the transcription factor TEAD1. TEAD1 expression increased in MASLD patients, and its downregulation by short interfering RNA reduced intracellular lipid content. Subsequent TEAD small molecule inhibition improved steatosis in primary human hepatocyte spheroids by suppressing lipogenic pathways. Thus, TEAD1 emerged as a new therapeutic candidate whose inhibition ameliorates hepatic steatosis.

ZFP207 sustains pluripotency by coordinating OCT4 stability, alternative splicing and RNA export.

The pluripotent state is not solely governed by the action of the core transcription factors OCT4, SOX2, and NANOG, but also by a series of co-transcriptional and post-transcriptional events, including alternative splicing (AS) and the interaction of RNA-binding proteins (RBPs) with defined subpopulations of RNAs. Zinc Finger Protein 207 (ZFP207) is an essential transcription factor for mammalian embryonic development. Here, we employ multiple functional analyses to characterize its role in mouse embryonic stem cells (ESCs). We find that ZFP207 plays a pivotal role in ESC maintenance, and silencing of Zfp207 leads to severe neuroectodermal differentiation defects. In striking contrast to human ESCs, mouse ZFP207 does not transcriptionally regulate neuronal and stem cell-related genes but exerts its effects by controlling AS networks and by acting as an RBP. Our study expands the role of ZFP207 in maintaining ESC identity, and underscores the functional versatility of ZFP207 in regulating neural fate commitment.

Myeloid cell-specific topoisomerase 1 inhibition using DNA origami mitigates neuroinflammation.

Targeting myeloid cells, especially microglia, for the treatment of neuroinflammatory diseases such as multiple sclerosis (MS), is underappreciated. Our in silico drug screening reveals topoisomerase 1 (TOP1) inhibitors as promising drug candidates for microglial modulation. We show that TOP1 is highly expressed in neuroinflammatory conditions, and TOP1 inhibition using camptothecin (CPT) and its FDA-approved analog topotecan (TPT) reduces inflammatory responses in microglia/macrophages and ameliorates neuroinflammation in vivo. Transcriptomic analyses of sorted microglia from LPS-challenged mice reveal an altered transcriptional phenotype following TPT treatment. To target myeloid cells, we design a nanosystem using ß-glucan-coated DNA origami (MyloGami) loaded with TPT (TopoGami). MyloGami shows enhanced specificity to myeloid cells while preventing the degradation of the DNA origami scaffold. Myeloid-specific TOP1 inhibition using TopoGami significantly suppresses the inflammatory response in microglia and mitigates MS-like disease progression. Our findings suggest that TOP1 inhibition in myeloid cells represents a therapeutic strategy for neuroinflammatory diseases and that the myeloid-specific nanosystems we designed may also benefit the treatment of other diseases with dysfunctional myeloid cells.

CCT3-LINC00326 axis regulates hepatocarcinogenic lipid metabolism.

OBJECTIVE: To better comprehend transcriptional phenotypes of cancer cells, we globally characterised RNA-binding proteins (RBPs) to identify altered RNAs, including long non-coding RNAs (lncRNAs). DESIGN: To unravel RBP-lncRNA interactions in cancer, we curated a list of ~2300 highly expressed RBPs in human cells, tested effects of RBPs and lncRNAs on patient survival in multiple cohorts, altered expression levels, integrated various sequencing, molecular and cell-based data. RESULTS: High expression of RBPs negatively affected patient survival in 21 cancer types, especially hepatocellular carcinoma (HCC). After knockdown of the top 10 upregulated RBPs and subsequent transcriptome analysis, we identified 88 differentially expressed lncRNAs, including 34 novel transcripts. CRISPRa-mediated overexpression of four lncRNAs had major effects on the HCC cell phenotype and transcriptome. Further investigation of four RBP-lncRNA pairs revealed involvement in distinct regulatory processes. The most noticeable RBP-lncRNA connection affected lipid metabolism, whereby the non-canonical RBP CCT3 regulated LINC00326 in a chaperonin-independent manner. Perturbation of the CCT3-LINC00326 regulatory network led to decreased lipid accumulation and increased lipid degradation in cellulo as well as diminished tumour growth in vivo. CONCLUSIONS: We revealed that RBP gene expression is perturbed in HCC and identified that RBPs exerted additional functions beyond their tasks under normal physiological conditions, which can be stimulated or intensified via lncRNAs and affected tumour growth.

Noncoding RNAs and RNA-binding proteins: emerging governors of liver physiology and metabolic diseases.

The liver holds central roles in detoxification, energy metabolism, and whole body homeostasis but can develop malignant phenotypes when being chronically overwhelmed with fatty acids and glucose. The global rise of metabolic dysfunction-associated fatty liver disease (MAFLD) is already affecting a quarter of the global population. Pharmaceutical treatment options against different stages of MAFLD do not yet exist, and several clinical trials against hepatic transcription factors and other proteins have failed. However, emerging roles of noncoding RNAs, including long (lncRNA) and short noncoding RNAs (sRNA), in various cellular processes pose exciting new avenues for treatment interventions. Actions of noncoding RNAs mostly rely on interactions with proteins, whereby the noncoding RNA fine-tunes protein function in a process termed riboregulation. The developmental stage-, disease stage-, and cell type-specific nature of noncoding RNAs harbors enormous potential to precisely target certain cellular pathways in a spatiotemporally defined manner. Proteins interacting with RNAs can be categorized into canonical or noncanonical RNA-binding proteins (RBPs) depending on the existence of classical RNA-binding domains. Both, RNA- and RBP-centric methods have generated new knowledge of the RNA-RBP interface and added an additional regulatory layer. In this review, we summarize recent advances in how RBP-lncRNA interactions and various sRNAs shape cellular physiology and the development of liver diseases such as MAFLD and hepatocellular carcinoma.

Inhibition of Respiratory Syncytial Virus Infection by Small Non-Coding RNA Fragments.

Respiratory syncytial virus (RSV) causes acute lower respiratory tract infection in infants, immunocompromised individuals and the elderly. As the only current specific treatment options for RSV are monoclonal antibodies, there is a need for efficacious antiviral treatments against RSV to be developed. We have previously shown that a group of synthetic non-coding single-stranded DNA oligonucleotides with lengths of 25-40 nucleotides can inhibit RSV infection in vitro and in vivo. Based on this, herein, we investigate whether naturally occurring single-stranded small non-coding RNA (sncRNA) fragments present in the airways have antiviral effects against RSV infection. From publicly available sequencing data, we selected sncRNA fragments such as YRNAs, tRNAs and rRNAs present in human bronchoalveolar lavage fluid (BALF) from healthy individuals. We utilized a GFP-expressing RSV to show that pre-treatment with the selected sncRNA fragments inhibited RSV infection in A549 cells in vitro. Furthermore, by using a flow cytometry-based binding assay, we demonstrate that these naturally occurring sncRNAs fragments inhibit viral infection most likely by binding to the RSV entry receptor nucleolin and thereby preventing the virus from binding to host cells, either directly or via steric hindrance. This finding highlights a new function of sncRNAs and displays the possibility of using naturally occurring sncRNAs as treatments against RSV.

5´XP sRNA-seq: efficient identification of transcripts with and without 5´ phosphorylation reveals evolutionary conserved small RNA.

Small RNA (sRNA) sequencing has been critical for our understanding of many cellular processes, including gene regulation. Nonetheless, the varying biochemical properties of sRNA, such as 5´ nucleotide modifications, make many sRNA subspecies incompatible with common protocols for sRNA sequencing. Here we describe 5XP-seq that outlines a novel strategy that captures a more complete picture of sRNA. By tagging 5´P sRNA during library preparation, 5XP-seq combines an open approach that includes all types of 5'-terminal modifications (5´X), with a selective approach for 5-phosphorylated sRNA (5´P). We show that 5XP-seq not only enriches phosphorylated miRNA and piRNA but successfully discriminates these sRNA from all other sRNA species. We further demonstrate the importance of this strategy by successful inter-species validation of sRNAs that would have otherwise failed, including human to insect translation of several tRNA (tRFs) and rRNA (rRFs) fragments. By combining 5´ insensitive library strategies with 5´ sensitive tagging, we have successfully tackled an intrinsic bias in modern sRNA sequencing that will help us reveal the true complexity and the evolutionary significance of the sRNA world.

Latent regulatory potential of human-specific repetitive elements.

At least half of the human genome is derived from repetitive elements, which are often lineage specific and silenced by a variety of genetic and epigenetic mechanisms. Using a transchromosomic mouse strain that transmits an almost complete single copy of human chromosome 21 via the female germline, we show that a heterologous regulatory environment can transcriptionally activate transposon-derived human regulatory regions. In the mouse nucleus, hundreds of locations on human chromosome 21 newly associate with activating histone modifications in both somatic and germline tissues, and influence the gene expression of nearby transcripts. These regions are enriched with primate and human lineage-specific transposable elements, and their activation corresponds to changes in DNA methylation at CpG dinucleotides. This study reveals the latent regulatory potential of the repetitive human genome and illustrates the species specificity of mechanisms that control it.

Codon-Driven Translational Efficiency Is Stable across Diverse Mammalian Cell States.

Whether codon usage fine-tunes mRNA translation in mammals remains controversial, with recent papers suggesting that production of proteins in specific Gene Ontological (GO) pathways can be regulated by actively modifying the codon and anticodon pools in different cellular conditions. In this work, we compared the sequence content of genes in specific GO categories with the exonic genome background. Although a substantial fraction of variability in codon usage could be explained by random sampling, almost half of GO sets showed more variability in codon usage than expected by chance. Nevertheless, by quantifying translational efficiency in healthy and cancerous tissues in human and mouse, we demonstrated that a given tRNA pool can equally well translate many different sets of mRNAs, irrespective of their cell-type specificity. This disconnect between variations in codon usage and the stability of translational efficiency is best explained by differences in GC content between gene sets. GC variation across the mammalian genome is most likely a result of the interplay between genome repair and gene duplication mechanisms, rather than selective pressures caused by codon-driven translational rates. Consequently, codon usage differences in mammalian transcriptomes are most easily explained by well-understood mutational biases acting on the underlying genome.

Aberrant methylation of tRNAs links cellular stress to neuro-developmental disorders.

Mutations in the cytosine-5 RNA methyltransferase NSun2 cause microcephaly and other neurological abnormalities in mice and human. How post-transcriptional methylation contributes to the human disease is currently unknown. By comparing gene expression data with global cytosine-5 RNA methylomes in patient fibroblasts and NSun2-deficient mice, we find that loss of cytosine-5 RNA methylation increases the angiogenin-mediated endonucleolytic cleavage of transfer RNAs (tRNA) leading to an accumulation of 5' tRNA-derived small RNA fragments. Accumulation of 5' tRNA fragments in the absence of NSun2 reduces protein translation rates and activates stress pathways leading to reduced cell size and increased apoptosis of cortical, hippocampal and striatal neurons. Mechanistically, we demonstrate that angiogenin binds with higher affinity to tRNAs lacking site-specific NSun2-mediated methylation and that the presence of 5' tRNA fragments is sufficient and required to trigger cellular stress responses. Furthermore, the enhanced sensitivity of NSun2-deficient brains to oxidative stress can be rescued through inhibition of angiogenin during embryogenesis. In conclusion, failure in NSun2-mediated tRNA methylation contributes to human diseases via stress-induced RNA cleavage.

High-resolution mapping of transcriptional dynamics across tissue development reveals a stable mRNA-tRNA interface.

The genetic code is an abstraction of how mRNA codons and tRNA anticodons molecularly interact during protein synthesis; the stability and regulation of this interaction remains largely unexplored. Here, we characterized the expression of mRNA and tRNA genes quantitatively at multiple time points in two developing mouse tissues. We discovered that mRNA codon pools are highly stable over development and simply reflect the genomic background; in contrast, precise regulation of tRNA gene families is required to create the corresponding tRNA transcriptomes. The dynamic regulation of tRNA genes during development is controlled in order to generate an anticodon pool that closely corresponds to messenger RNAs. Thus, across development, the pools of mRNA codons and tRNA anticodons are invariant and highly correlated, revealing a stable molecular interaction interlocking transcription and translation.

Rapid turnover of long noncoding RNAs and the evolution of gene expression.

A large proportion of functional sequence within mammalian genomes falls outside protein-coding exons and can be transcribed into long RNAs. However, the roles in mammalian biology of long noncoding RNA (lncRNA) are not well understood. Few lncRNAs have experimentally determined roles, with some of these being lineage-specific. Determining the extent by which transcription of lncRNA loci is retained or lost across multiple evolutionary lineages is essential if we are to understand their contribution to mammalian biology and to lineage-specific traits. Here, we experimentally investigated the conservation of lncRNA expression among closely related rodent species, allowing the evolution of DNA sequence to be uncoupled from evolution of transcript expression. We generated total RNA (RNAseq) and H3K4me3-bound (ChIPseq) DNA data, and combined both to construct catalogues of transcripts expressed in the adult liver of Mus musculus domesticus (C57BL/6J), Mus musculus castaneus, and Rattus norvegicus. We estimated the rate of transcriptional turnover of lncRNAs and investigated the effects of their lineage-specific birth or death. LncRNA transcription showed considerably greater gain and loss during rodent evolution, compared with protein-coding genes. Nucleotide substitution rates were found to mirror the in vivo transcriptional conservation of intergenic lncRNAs between rodents: only the sequences of noncoding loci with conserved transcription were constrained. Finally, we found that lineage-specific intergenic lncRNAs appear to be associated with modestly elevated expression of genomically neighbouring protein-coding genes. Our findings show that nearly half of intergenic lncRNA loci have been gained or lost since the last common ancestor of mouse and rat, and they predict that such rapid transcriptional turnover contributes to the evolution of tissue- and lineage-specific gene expression.

Intrinsic deletion at 10q23.31, including the PTEN gene locus, is aggravated upon CRISPR-Cas9-mediated genome engineering in HAP1 cells mimicking cancer profiles.

The CRISPR-Cas9 system is a powerful tool for studying gene functions and holds potential for disease treatment. However, precise genome editing requires thorough assessments to minimize unintended on- and off-target effects. Here, we report an unexpected 283-kb deletion on Chromosome 10 (10q23.31) in chronic myelogenous leukemia-derived HAP1 cells, which are frequently used in CRISPR screens. The deleted region encodes regulatory genes, including PAPSS2, ATAD1, KLLN, and PTEN We found that this deletion was not a direct consequence of CRISPR-Cas9 off-targeting but rather occurred frequently during the generation of CRISPR-Cas9-modified cells. The deletion was associated with global changes in histone acetylation and gene expression, affecting fundamental cellular processes such as cell cycle and DNA replication. We detected this deletion in cancer patient genomes. As in HAP1 cells, the deletion contributed to similar gene expression patterns among cancer patients despite interindividual differences. Our findings suggest that the unintended deletion of 10q23.31 can confound CRISPR-Cas9 studies and underscore the importance to assess unintended genomic changes in CRISPR-Cas9-modified cells, which could impact cancer research.

Role of cis-12-oxo-phytodienoic acid in tomato embryo development.

Oxylipins including jasmonates are signaling compounds in plant growth, development, and responses to biotic and abiotic stresses. In Arabidopsis (Arabidopsis thaliana) most mutants affected in jasmonic acid (JA) biosynthesis and signaling are male sterile, whereas the JA-insensitive tomato (Solanum lycopersicum) mutant jai1 is female sterile. The diminished seed formation in jai1 together with the ovule-specific accumulation of the JA biosynthesis enzyme allene oxide cyclase (AOC), which correlates with elevated levels of JAs, suggest a role of oxylipins in tomato flower/seed development. Here, we show that 35S::SlAOC-RNAi lines with strongly reduced AOC in ovules exhibited reduced seed set similarly to the jai1 plants. Investigation of embryo development of wild-type tomato plants showed preferential occurrence of AOC promoter activity and AOC protein accumulation in the developing seed coat and the embryo, whereas 12-oxo-phytodienoic acid (OPDA) was the dominant oxylipin occurring nearly exclusively in the seed coat tissues. The OPDA- and JA-deficient mutant spr2 was delayed in embryo development and showed an increased programmed cell death in the developing seed coat and endosperm. In contrast, the mutant acx1a, which accumulates preferentially OPDA and residual amount of JA, developed embryos similar to the wild type, suggesting a role of OPDA in embryo development. Activity of the residual amount of JA in the acx1a mutant is highly improbable since the known reproductive phenotype of the JA-insensitive mutant jai1 could be rescued by wound-induced formation of OPDA. These data suggest a role of OPDA or an OPDA-related compound for proper embryo development possibly by regulating carbohydrate supply and detoxification.

Exposure of volunteers to microgravity by dry immersion bed over 21 days results in gene expression changes and adaptation of T cells.

The next steps of deep space exploration are manned missions to Moon and Mars. For safe space missions for crew members, it is important to understand the impact of space flight on the immune system. We studied the effects of 21 days dry immersion (DI) exposure on the transcriptomes of T cells isolated from blood samples of eight healthy volunteers. Samples were collected 7 days before DI, at day 7, 14, and 21 during DI, and 7 days after DI. RNA sequencing of CD3+ T cells revealed transcriptional alterations across all time points, with most changes occurring 14 days after DI exposure. At day 21, T cells showed evidence of adaptation with a transcriptional profile resembling that of 7 days before DI. At 7 days after DI, T cells again changed their transcriptional profile. These data suggest that T cells adapt by rewiring their transcriptomes in response to simulated weightlessness and that remodeling cues persist when reexposed to normal gravity.

Coinhibition of topoisomerase 1 and BRD4-mediated pause release selectively kills pancreatic cancer via readthrough transcription.

Pancreatic carcinoma lacks effective therapeutic strategies resulting in poor prognosis. Transcriptional dysregulation due to alterations in KRAS and MYC affects initiation, development, and survival of this tumor type. Using patient-derived xenografts of KRAS- and MYC-driven pancreatic carcinoma, we show that coinhibition of topoisomerase 1 (TOP1) and bromodomain-containing protein 4 (BRD4) synergistically induces tumor regression by targeting promoter pause release. Comparing the nascent transcriptome with the recruitment of elongation and termination factors, we found that coinhibition of TOP1 and BRD4 disrupts recruitment of transcription termination factors. Thus, RNA polymerases transcribe downstream of genes for hundreds of kilobases leading to readthrough transcription. This occurs during replication, perturbing replisome progression and inducing DNA damage. The synergistic effect of TOP1 + BRD4 inhibition is specific to cancer cells leaving normal cells unaffected, highlighting the tumor's vulnerability to transcriptional defects. This preclinical study provides a mechanistic understanding of the benefit of combining TOP1 and BRD4 inhibitors to treat pancreatic carcinomas addicted to oncogenic drivers of transcription and replication.

Systematic comparison of microarray profiling, real-time PCR, and next-generation sequencing technologies for measuring differential microRNA expression.

RNA abundance and DNA copy number are routinely measured in high-throughput using microarray and next-generation sequencing (NGS) technologies, and the attributes of different platforms have been extensively analyzed. Recently, the application of both microarrays and NGS has expanded to include microRNAs (miRNAs), but the relative performance of these methods has not been rigorously characterized. We analyzed three biological samples across six miRNA microarray platforms and compared their hybridization performance. We examined the utility of these platforms, as well as NGS, for the detection of differentially expressed miRNAs. We then validated the results for 89 miRNAs by real-time RT-PCR and challenged the use of this assay as a "gold standard." Finally, we implemented a novel method to evaluate false-positive and false-negative rates for all methods in the absence of a reference method.