Comprehensive In Vivo Interrogation Reveals Phenotypic Impact of Human Enhancer Variants.

Establishing causal links between non-coding variants and human phenotypes is an increasing challenge. Here, we introduce a high-throughput mouse reporter assay for assessing the pathogenic potential of human enhancer variants in vivo and examine nearly a thousand variants in an enhancer repeatedly linked to polydactyly. We show that 71% of all rare non-coding variants previously proposed as causal lead to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knockin mice. We also used combinatorial and single-nucleotide mutagenesis to evaluate the in vivo impact of mutations affecting all positions of the enhancer and identified additional functional substitutions, including potentially pathogenic variants hitherto not observed in humans. Our results uncover the functional consequences of hundreds of mutations in a phenotype-associated enhancer and establish a widely applicable strategy for systematic in vivo evaluation of human enhancer variants.

Bacterial Retrons Function In Anti-Phage Defense.

Retrons are bacterial genetic elements comprised of a reverse transcriptase (RT) and a non-coding RNA (ncRNA). The RT uses the ncRNA as template, generating a chimeric RNA/DNA molecule in which the RNA and DNA components are covalently linked. Although retrons were discovered three decades ago, their function remained unknown. We report that retrons function as anti-phage defense systems. The defensive unit is composed of three components: the RT, the ncRNA, and an effector protein. We examined multiple retron systems and show that they confer defense against a broad range of phages via abortive infection. Focusing on retron Ec48, we show evidence that it "guards" RecBCD, a complex with central anti-phage functions in bacteria. Inhibition of RecBCD by phage proteins activates the retron, leading to abortive infection and cell death. Thus, the Ec48 retron forms a second line of defense that is triggered if the first lines of defense have collapsed.

Transcriptional and epigenetic control of T helper cell specification: molecular mechanisms underlying commitment and plasticity.

T helper cell differentiation occurs in the context of the extracellular cytokine milieu evoked by diverse microbes and other pathogenic stimuli along with T cell receptor stimulation. The culmination of these signals results in specification of T helper lineages, which occurs through the combinatorial action of multiple transcription factors that establish distinctive transcriptomes. In this manner, inducible, but constitutively active, master regulators work in conjunction with factors such as the signal transducer and activator of transcriptions (STATs) that sense the extracellular environment. The acquisition of a distinctive transcriptome also depends on chromatin modifications that impact key cis elements as well as the changes in global genomic organization. Thus, signal transduction and epigenetics are linked in these processes of differentiation. In this review, recent advances in understanding T helper lineage specification and deciphering the action of transcription factors are summarized with emphasis on comprehensive views of the dynamic T cell epigenome.

The Role of Non-coding RNAs in Oncology.

For decades, research into cancer biology focused on the involvement of protein-coding genes. Only recently was it discovered that an entire class of molecules, termed non-coding RNA (ncRNA), plays key regulatory roles in shaping cellular activity. An explosion of studies into ncRNA biology has since shown that they represent a diverse and prevalent group of RNAs, including both oncogenic molecules and those that work in a tumor suppressive manner. As a result, hundreds of cancer-focused clinical trials involving ncRNAs as novel biomarkers or therapies have begun and these are likely just the beginning.

Widespread and Functional RNA Circularization in Localized Prostate Cancer.

The cancer transcriptome is remarkably complex, including low-abundance transcripts, many not polyadenylated. To fully characterize the transcriptome of localized prostate cancer, we performed ultra-deep total RNA-seq on 144 tumors with rich clinical annotation. This revealed a linear transcriptomic subtype associated with the aggressive intraductal carcinoma sub-histology and a fusion profile that differentiates localized from metastatic disease. Analysis of back-splicing events showed widespread RNA circularization, with the average tumor expressing 7,232 circular RNAs (circRNAs). The degree of circRNA production was correlated to disease progression in multiple patient cohorts. Loss-of-function screening identified 11.3% of highly abundant circRNAs as essential for cell proliferation; for ∼90% of these, their parental linear transcripts were not essential. Individual circRNAs can have distinct functions, with circCSNK1G3 promoting cell growth by interacting with miR-181. These data advocate for adoption of ultra-deep RNA-seq without poly-A selection to interrogate both linear and circular transcriptomes.

Biology and Clinical Implications of the 19q13 Aggressive Prostate Cancer Susceptibility Locus.

Genome-wide association studies (GWAS) have identified rs11672691 at 19q13 associated with aggressive prostate cancer (PCa). Here, we independently confirmed the finding in a cohort of 2,738 PCa patients and discovered the biological mechanism underlying this association. We found an association of the aggressive PCa-associated allele G of rs11672691 with elevated transcript levels of two biologically plausible candidate genes, PCAT19 and CEACAM21, implicated in PCa cell growth and tumor progression. Mechanistically, rs11672691 resides in an enhancer element and alters the binding site of HOXA2, a novel oncogenic transcription factor with prognostic potential in PCa. Remarkably, CRISPR/Cas9-mediated single-nucleotide editing showed the direct effect of rs11672691 on PCAT19 and CEACAM21 expression and PCa cellular aggressive phenotype. Clinical data demonstrated synergistic effects of rs11672691 genotype and PCAT19/CEACAM21 gene expression on PCa prognosis. These results provide a plausible mechanism for rs11672691 associated with aggressive PCa and thus lay the ground work for translating this finding to the clinic.

A Suite of Transgenic Driver and Reporter Mouse Lines with Enhanced Brain-Cell-Type Targeting and Functionality.

Modern genetic approaches are powerful in providing access to diverse cell types in the brain and facilitating the study of their function. Here, we report a large set of driver and reporter transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.

Linking Protein and RNA Function within the Same Gene.

Exposure to ultraviolet light leads to a cell-wide DNA damage response that includes a global reduction in transcription. Williamson et al., identify a protein involved in this process as well as a noncoding RNA produced by alternative processing of RNA transcribed from the same gene that promotes recovery from the repressed state.

Approaching TERRA Firma: Genomic Functions of Telomeric Noncoding RNA.

Functions of the telomeric repeat-containing RNA (TERRA), the long noncoding RNA (lncRNA) transcribed from telomeres, have eluded researchers. In this issue of Cell, Graf el al. and Chu et al. uncover new regulatory roles for TERRA at the telomere and at distant genomic sites.

UV Irradiation Induces a Non-coding RNA that Functionally Opposes the Protein Encoded by the Same Gene.

The transcription-related DNA damage response was analyzed on a genome-wide scale with great spatial and temporal resolution. Upon UV irradiation, a slowdown of transcript elongation and restriction of gene activity to the promoter-proximal ∼25 kb is observed. This is associated with a shift from expression of long mRNAs to shorter isoforms, incorporating alternative last exons (ALEs) that are more proximal to the transcription start site. Notably, this includes a shift from a protein-coding ASCC3 mRNA to a shorter ALE isoform of which the RNA, rather than an encoded protein, is critical for the eventual recovery of transcription. The non-coding ASCC3 isoform counteracts the function of the protein-coding isoform, indicating crosstalk between them. Thus, the ASCC3 gene expresses both coding and non-coding transcript isoforms with opposite effects on transcription recovery after UV-induced DNA damage.

Non-coding Transcription Instructs Chromatin Folding and Compartmentalization to Dictate Enhancer-Promoter Communication and T Cell Fate.

It is now established that Bcl11b specifies T cell fate. Here, we show that in developing T cells, the Bcl11b enhancer repositioned from the lamina to the nuclear interior. Our search for factors that relocalized the Bcl11b enhancer identified a non-coding RNA named ThymoD (thymocyte differentiation factor). ThymoD-deficient mice displayed a block at the onset of T cell development and developed lymphoid malignancies. We found that ThymoD transcription promoted demethylation at CTCF bound sites and activated cohesin-dependent looping to reposition the Bcl11b enhancer from the lamina to the nuclear interior and to juxtapose the Bcl11b enhancer and promoter into a single-loop domain. These large-scale changes in nuclear architecture were associated with the deposition of activating epigenetic marks across the loop domain, plausibly facilitating phase separation. These data indicate how, during developmental progression and tumor suppression, non-coding transcription orchestrates chromatin folding and compartmentalization to direct with high precision enhancer-promoter communication.

HSP70 binds to specific non-coding RNA and regulates human RNA polymerase III.

Molecular chaperones are critical for protein homeostasis and are implicated in several human pathologies such as neurodegeneration and cancer. While the binding of chaperones to nascent and misfolded proteins has been studied in great detail, the direct interaction between chaperones and RNA has not been systematically investigated. Here, we provide the evidence for widespread interaction between chaperones and RNA in human cells. We show that the major chaperone heat shock protein 70 (HSP70) binds to non-coding RNA transcribed by RNA polymerase III (RNA Pol III) such as tRNA and 5S rRNA. Global chromatin profiling revealed that HSP70 binds genomic sites of transcription by RNA Pol III. Detailed biochemical analyses showed that HSP70 alleviates the inhibitory effect of cognate tRNA transcript on tRNA gene transcription. Thus, our study uncovers an unexpected role of HSP70-RNA interaction in the biogenesis of a specific class of non-coding RNA with wider implications in cancer therapeutics.

The challenges of investigating RNA function.

High-throughput sequencing methods have led to the discovery of many non-coding RNAs, RNA modifications, and protein-RNA interactions. While the list keeps growing, the challenge of determining their functions remains. For our focus issue on RNA biology, we spoke with several researchers about their perspective on investigating the functions of RNA.

3D RNA and Functional Interactions from Evolutionary Couplings.

Non-coding RNAs are ubiquitous, but the discovery of new RNA gene sequences far outpaces the research on the structure and functional interactions of these RNA gene sequences. We mine the evolutionary sequence record to derive precise information about the function and structure of RNAs and RNA-protein complexes. As in protein structure prediction, we use maximum entropy global probability models of sequence co-variation to infer evolutionarily constrained nucleotide-nucleotide interactions within RNA molecules and nucleotide-amino acid interactions in RNA-protein complexes. The predicted contacts allow all-atom blinded 3D structure prediction at good accuracy for several known RNA structures and RNA-protein complexes. For unknown structures, we predict contacts in 160 non-coding RNA families. Beyond 3D structure prediction, evolutionary couplings help identify important functional interactions-e.g., at switch points in riboswitches and at a complex nucleation site in HIV. Aided by increasing sequence accumulation, evolutionary coupling analysis can accelerate the discovery of functional interactions and 3D structures involving RNA.

Enhancer Variants Synergistically Drive Dysfunction of a Gene Regulatory Network In Hirschsprung Disease.

Common sequence variants in cis-regulatory elements (CREs) are suspected etiological causes of complex disorders. We previously identified an intronic enhancer variant in the RET gene disrupting SOX10 binding and increasing Hirschsprung disease (HSCR) risk 4-fold. We now show that two other functionally independent CRE variants, one binding Gata2 and the other binding Rarb, also reduce Ret expression and increase risk 2- and 1.7-fold. By studying human and mouse fetal gut tissues and cell lines, we demonstrate that reduced RET expression propagates throughout its gene regulatory network, exerting effects on both its positive and negative feedback components. We also provide evidence that the presence of a combination of CRE variants synergistically reduces RET expression and its effects throughout the GRN. These studies show how the effects of functionally independent non-coding variants in a coordinated gene regulatory network amplify their individually small effects, providing a model for complex disorders.

In Search of Lost Small Peptides.

A large body of evidence indicates that genome annotation pipelines have biased our view of coding sequences because they generally undersample small proteins and peptides. The recent development of genome-wide translation profiling reveals the prevalence of small/short open reading frames (smORFs or sORFs), which are scattered over all classes of transcripts, including both mRNAs and presumptive long noncoding RNAs. Proteomic approaches further confirm an unexpected variety of smORF-encoded peptides (SEPs), representing an overlooked reservoir of bioactive molecules. Indeed, functional studies in a broad range of species from yeast to humans demonstrate that SEPs can harbor key activities for the control of development, differentiation, and physiology. Here we summarize recent advances in the discovery and functional characterization of smORF/SEPs and discuss why these small players can no longer be ignored with regard to genome function.

A restrictor complex of ZC3H4, WDR82, and ARS2 integrates with PNUTS to control unproductive transcription.

The transcriptional termination of unstable non-coding RNAs (ncRNAs) is poorly understood compared to coding transcripts. We recently identified ZC3H4-WDR82 ("restrictor") as restricting human ncRNA transcription, but how it does this is unknown. Here, we show that ZC3H4 additionally associates with ARS2 and the nuclear exosome targeting complex. The domains of ZC3H4 that contact ARS2 and WDR82 are required for ncRNA restriction, suggesting their presence in a functional complex. Consistently, ZC3H4, WDR82, and ARS2 co-transcriptionally control an overlapping population of ncRNAs. ZC3H4 is proximal to the negative elongation factor, PNUTS, which we show enables restrictor function and is required to terminate the transcription of all major RNA polymerase II transcript classes. In contrast to short ncRNAs, longer protein-coding transcription is supported by U1 snRNA, which shields transcripts from restrictor and PNUTS at hundreds of genes. These data provide important insights into the mechanism and control of transcription by restrictor and PNUTS.

Non-coding RNAs in disease: from mechanisms to therapeutics.

Non-coding RNAs (ncRNAs) are a heterogeneous group of transcripts that, by definition, are not translated into proteins. Since their discovery, ncRNAs have emerged as important regulators of multiple biological functions across a range of cell types and tissues, and their dysregulation has been implicated in disease. Notably, much research has focused on the link between microRNAs (miRNAs) and human cancers, although other ncRNAs, such as long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), are also emerging as relevant contributors to human disease. In this Review, we summarize our current understanding of the roles of miRNAs, lncRNAs and circRNAs in cancer and other major human diseases, notably cardiovascular, neurological and infectious diseases. Further, we discuss the potential use of ncRNAs as biomarkers of disease and as therapeutic targets.

Ten principles of heterochromatin formation and function.

Heterochromatin is a key architectural feature of eukaryotic chromosomes, which endows particular genomic domains with specific functional properties. The capacity of heterochromatin to restrain the activity of mobile elements, isolate DNA repair in repetitive regions and ensure accurate chromosome segregation is crucial for maintaining genomic stability. Nucleosomes at heterochromatin regions display histone post-translational modifications that contribute to developmental regulation by restricting lineage-specific gene expression. The mechanisms of heterochromatin establishment and of heterochromatin maintenance are separable and involve the ability of sequence-specific factors bound to nascent transcripts to recruit chromatin-modifying enzymes. Heterochromatin can spread along the chromatin from nucleation sites. The propensity of heterochromatin to promote its own spreading and inheritance is counteracted by inhibitory factors. Because of its importance for chromosome function, heterochromatin has key roles in the pathogenesis of various human diseases. In this Review, we discuss conserved principles of heterochromatin formation and function using selected examples from studies of a range of eukaryotes, from yeast to human, with an emphasis on insights obtained from unicellular model organisms.

Disruptions of topological chromatin domains cause pathogenic rewiring of gene-enhancer interactions.

Mammalian genomes are organized into megabase-scale topologically associated domains (TADs). We demonstrate that disruption of TADs can rewire long-range regulatory architecture and result in pathogenic phenotypes. We show that distinct human limb malformations are caused by deletions, inversions, or duplications altering the structure of the TAD-spanning WNT6/IHH/EPHA4/PAX3 locus. Using CRISPR/Cas genome editing, we generated mice with corresponding rearrangements. Both in mouse limb tissue and patient-derived fibroblasts, disease-relevant structural changes cause ectopic interactions between promoters and non-coding DNA, and a cluster of limb enhancers normally associated with Epha4 is misplaced relative to TAD boundaries and drives ectopic limb expression of another gene in the locus. This rewiring occurred only if the variant disrupted a CTCF-associated boundary domain. Our results demonstrate the functional importance of TADs for orchestrating gene expression via genome architecture and indicate criteria for predicting the pathogenicity of human structural variants, particularly in non-coding regions of the human genome.