Multigenerational Regulation of the Caenorhabditis elegans Chromatin Landscape by Germline Small RNAs.

In animals, small noncoding RNAs that are expressed in the germline and transmitted to progeny control gene expression to promote fertility. Germline-expressed small RNAs, including endogenous small interfering RNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs), drive the repression of deleterious transcripts such as transposons, repetitive elements, and pseudogenes. Recent studies have highlighted an important role for small RNAs in transgenerational epigenetic inheritance via regulation of heritable chromatin marks; therefore, small RNAs are thought to convey an epigenetic memory of genomic self and nonself elements. Small RNA pathways are highly conserved in metazoans and have been best described for the model organism Caenorhabditis elegans. In this review, we describe the biogenesis, regulation, and function of C. elegans endo-siRNAs and piRNAs, along with recent insights into how these distinct pathways are integrated to collectively regulate germline gene expression, transgenerational epigenetic inheritance, and ultimately, animal fertility.

ecDNA hubs drive cooperative intermolecular oncogene expression.

Extrachromosomal DNA (ecDNA) is prevalent in human cancers and mediates high expression of oncogenes through gene amplification and altered gene regulation1. Gene induction typically involves cis-regulatory elements that contact and activate genes on the same chromosome2,3. Here we show that ecDNA hubs-clusters of around 10-100 ecDNAs within the nucleus-enable intermolecular enhancer-gene interactions to promote oncogene overexpression. ecDNAs that encode multiple distinct oncogenes form hubs in diverse cancer cell types and primary tumours. Each ecDNA is more likely to transcribe the oncogene when spatially clustered with additional ecDNAs. ecDNA hubs are tethered by the bromodomain and extraterminal domain (BET) protein BRD4 in a MYC-amplified colorectal cancer cell line. The BET inhibitor JQ1 disperses ecDNA hubs and preferentially inhibits ecDNA-derived-oncogene transcription. The BRD4-bound PVT1 promoter is ectopically fused to MYC and duplicated in ecDNA, receiving promiscuous enhancer input to drive potent expression of MYC. Furthermore, the PVT1 promoter on an exogenous episome suffices to mediate gene activation in trans by ecDNA hubs in a JQ1-sensitive manner. Systematic silencing of ecDNA enhancers by CRISPR interference reveals intermolecular enhancer-gene activation among multiple oncogene loci that are amplified on distinct ecDNAs. Thus, protein-tethered ecDNA hubs enable intermolecular transcriptional regulation and may serve as units of oncogene function and cooperative evolution and as potential targets for cancer therapy.

Acute exposure to carbon monoxide inhalation and/or hot water immersion transiently increases erythropoietin in females but not in males.

The use of acute carbon monoxide inhalation (COi) and hot water immersion (HWI) are of growing interest as interventions to stimulate erythropoietin (EPO) production. However, whether EPO production is further augmented when combining these stressors and whether there are sex differences in this response are poorly understood. Therefore, we measured circulating EPO concentration in response to acute COi and HWI independently and in combination and determined whether the responses were altered by sex. Participants completed three study visits-COi, HWI, and combined COi and HWI-separated by 1 week in a randomized, balanced, crossover design. Renal blood velocity was measured during all interventions, and carboxyhaemoglobin was measured during and after COi. Serum samples were analysed every hour for 6 h post-intervention for EPO concentration. HWI decreased renal blood velocity (46.2 cm/s to 36.2 cm/s) (P < 0.0001), and COi increased carboxyhaemoglobin (1.5%-12.8%) (P < 0.0001) without changing renal blood velocity (46.4-45.2 cm/s) (P = 0.4456). All three interventions increased peak EPO concentration from baseline (COi: 6.02-9.74 mIU/mL; HWI: 6.80-11.10 mIU/mL; COi + HWI: 6.71-10.91 mIU/mL) (P = 0.0048) and to the same extent (P = 0.3505). On average, females increased EPO while males did not in response to COi (females: 6.17 mIU/mL; males: 1.27 mIU/mL) (P = 0.0010), HWI (females: 6.47 mIU/mL; males: 2.14 mIU/mL) (P = 0.0104), and COi and HWI (females: 6.65 mIU/mL; males: 1.76 mIU/mL) (P = 0.0256). These data emphasize that combining these interventions does not augment EPO secretion and that these interventions may work better in females.

Enhancer activation from transposable elements in extrachromosomal DNA.

Extrachromosomal DNA (ecDNA) is a hallmark of aggressive cancer, contributing to both oncogene amplification and tumor heterogeneity. Here, we used Hi-C, super-resolution imaging, and long-read sequencing to explore the nuclear architecture of MYC-amplified ecDNA in colorectal cancer cells. Intriguingly, we observed frequent spatial proximity between ecDNA and 68 repetitive elements which we called ecDNA-interacting elements or EIEs. To characterize a potential regulatory role of EIEs, we focused on a fragment of the L1M4a1#LINE/L1 which we found to be co-amplified with MYC on ecDNA, gaining enhancer-associated chromatin marks in contrast to its normally silenced state. This EIE, in particular, existed as a naturally occurring structural variant upstream of MYC, gaining oncogenic potential in the transcriptionally permissive ecDNA environment. This EIE sequence is sufficient to enhance MYC expression and is required for cancer cell fitness. These findings suggest that silent repetitive genomic elements can be reactivated on ecDNA, leading to functional cooption and amplification. Repeat element activation on ecDNA represents a mechanism of accelerated evolution and tumor heterogeneity and may have diagnostic and therapeutic potential.

Oncogene Convergence in Extrachromosomal DNA Hubs.

Extrachromosomal DNA circles (ecDNA) are a common mechanism for oncogene amplification and are associated with worse clinical outcomes compared with other types of oncogene amplification. Several recent discoveries of ecDNA hubs-local congregations of ecDNAs in the nucleus-highlight unique features of ecDNA biology that may contribute to higher oncogene expression and rapid tumor evolution.

Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH.

Extrachromosomal DNA (ecDNA) is a common mode of oncogene amplification but is challenging to analyze. Here, we adapt CRISPR-CATCH, in vitro CRISPR-Cas9 treatment and pulsed field gel electrophoresis of agarose-entrapped genomic DNA, previously developed for bacterial chromosome segments, to isolate megabase-sized human ecDNAs. We demonstrate strong enrichment of ecDNA molecules containing EGFR, FGFR2 and MYC from human cancer cells and NRAS ecDNA from human metastatic melanoma with acquired therapeutic resistance. Targeted enrichment of ecDNA versus chromosomal DNA enabled phasing of genetic variants, identified the presence of an EGFRvIII mutation exclusively on ecDNAs and supported an excision model of ecDNA genesis in a glioblastoma model. CRISPR-CATCH followed by nanopore sequencing enabled single-molecule ecDNA methylation profiling and revealed hypomethylation of the EGFR promoter on ecDNAs. We distinguished heterogeneous ecDNA species within the same sample by size and sequence with base-pair resolution and discovered functionally specialized ecDNAs that amplify select enhancers or oncogene-coding sequences.

MORC-1 Integrates Nuclear RNAi and Transgenerational Chromatin Architecture to Promote Germline Immortality.

Germline-expressed endogenous small interfering RNAs (endo-siRNAs) transmit multigenerational epigenetic information to ensure fertility in subsequent generations. In Caenorhabditis elegans, nuclear RNAi ensures robust inheritance of endo-siRNAs and deposition of repressive H3K9me3 marks at target loci. How target silencing is maintained in subsequent generations is poorly understood. We discovered that morc-1 is essential for transgenerational fertility and acts as an effector of endo-siRNAs. Unexpectedly, morc-1 is dispensable for siRNA inheritance but is required for target silencing and maintenance of siRNA-dependent chromatin organization. A forward genetic screen identified mutations in met-1, which encodes an H3K36 methyltransferase, as potent suppressors of morc-1(-) and nuclear RNAi mutant phenotypes. Further analysis of nuclear RNAi and morc-1(-) mutants revealed a progressive, met-1-dependent enrichment of H3K36me3, suggesting that robust fertility requires repression of MET-1 activity at nuclear RNAi targets. Without MORC-1 and nuclear RNAi, MET-1-mediated encroachment of euchromatin leads to detrimental decondensation of germline chromatin and germline mortality.