Analytical validation of a multi-cancer early detection test with cancer signal origin using a cell-free DNA-based targeted methylation assay.

The analytical validation is reported for a targeted methylation-based cell-free DNA multi-cancer early detection test designed to detect cancer and predict the cancer signal origin (tissue of origin). A machine-learning classifier was used to analyze the methylation patterns of >105 genomic targets covering >1 million methylation sites. Analytical sensitivity (limit of detection [95% probability]) was characterized with respect to tumor content by expected variant allele frequency and was determined to be 0.07%-0.17% across five tumor cases and 0.51% for the lymphoid neoplasm case. Test specificity was 99.3% (95% confidence interval, 98.6-99.7%). In the reproducibility and repeatability study, results were consistent in 31/34 (91.2%) pairs with cancer and 17/17 (100%) pairs without cancer; between runs, results were concordant for 129/133 (97.0%) cancer and 37/37 (100%) non-cancer sample pairs. Across 3- to 100-ng input levels of cell-free DNA, cancer was detected in 157/182 (86.3%) cancer samples but not in any of the 62 non-cancer samples. In input titration tests, cancer signal origin was correctly predicted in all tumor samples detected as cancer. No cross-contamination events were observed. No potential interferent (hemoglobin, bilirubin, triglycerides, genomic DNA) affected performance. The results of this analytical validation study support continued clinical development of a targeted methylation cell-free DNA multi-cancer early detection test.

ATL8, a RING E3 ligase, modulates root growth and phosphate homeostasis in Arabidopsis.

Ubiquitination-mediated post-translational modification of proteins is a pivotal regulatory mechanism involved in the growth and development of the plant. The Arabidopsis Tóxicos en Levadura (ATL) family is a group of RING-type ubiquitin ligases (E3) and ATL8 is a membrane-localized protein. Here, a reverse genetics approach was used to elucidate the role of ATL8 in phosphate (Pi) homeostasis. Deficiencies of Pi and sucrose (Suc) enhanced the relative expression level of ATL8 in different tissues of the wild-type (Wt). The relative expression level of ATL8 was attenuated and augmented in the mutant (atl8) and overexpression lines (Oe1 and Oe2), respectively. There were significant reductions in different root traits, root hairs, root to shoot ratio, and total Pi content in atl8 compared with the Wt under different Pi regimes. On the contrary, Oe1 and Oe2 lines exhibited enhancement in some of these traits. Noticeably, anthocyanin content was significantly reduced in Oe1 and Oe2 compared with the Wt and atl8 under P- condition. Abscisic acid (ABA) treatment led to an increase in the primary root length of atl8 compared with the Wt, suggesting a cross-talk between ABA and ATL8 on root growth. Furthermore, the relative expression levels of the genes involved in the maintenance of Pi homeostasis (WRKY75, RNS1, E3L, and ACP5) were differentially modulated in atl8, Oe1, and Oe2 compared with the Wt under different Pi regimes. The results revealed the pivotal role of ATL8 in mediating morphophysiological and molecular adaptive responses to Pi deficiency.

A microRNA cluster in the Fragile-X region expressed during spermatogenesis targets FMR1.

Testis-expressed X-linked genes typically evolve rapidly. Here, we report on a testis-expressed X-linked microRNA (miRNA) cluster that despite rapid alterations in sequence has retained its position in the Fragile-X region of the X chromosome in placental mammals. Surprisingly, the miRNAs encoded by this cluster (Fx-mir) have a predilection for targeting the immediately adjacent gene, Fmr1, an unexpected finding given that miRNAs usually act in trans, not in cis Robust repression of Fmr1 is conferred by combinations of Fx-mir miRNAs induced in Sertoli cells (SCs) during postnatal development when they terminate proliferation. Physiological significance is suggested by the finding that FMRP, the protein product of Fmr1, is downregulated when Fx-mir miRNAs are induced, and that FMRP loss causes SC hyperproliferation and spermatogenic defects. Fx-mir miRNAs not only regulate the expression of FMRP, but also regulate the expression of eIF4E and CYFIP1, which together with FMRP form a translational regulatory complex. Our results support a model in which Fx-mir family members act cooperatively to regulate the translation of batteries of mRNAs in a developmentally regulated manner in SCs.

Chromatin Modification and Global Transcriptional Silencing in the Oocyte Mediated by the mRNA Decay Activator ZFP36L2.

Global transcriptional silencing is a highly conserved mechanism central to the oocyte-to-embryo transition. We report the unexpected discovery that global transcriptional silencing in oocytes depends on an mRNA decay activator. Oocyte-specific loss of ZFP36L2 an RNA-binding protein that promotes AU-rich element-dependent mRNA decay prevents global transcriptional silencing and causes oocyte maturation and fertilization defects, as well as complete female infertility in the mouse. Single-cell RNA sequencing revealed that ZFP36L2 downregulates mRNAs encoding transcription and chromatin modification regulators, including a large group of mRNAs for histone demethylases targeting H3K4 and H3K9, which we show are bound and degraded by ZFP36L2. Oocytes lacking Zfp36l2 fail to accumulate histone methylation at H3K4 and H3K9, marks associated with the transcriptionally silent, developmentally competent oocyte state. Our results uncover a ZFP36L2-dependent mRNA decay mechanism that acts as a developmental switch during oocyte growth, triggering wide-spread shifts in chromatin modification and global transcription.

The Homeobox Transcription Factor RHOX10 Drives Mouse Spermatogonial Stem Cell Establishment.

The developmental origins of most adult stem cells are poorly understood. Here, we report the identification of a transcription factor-RHOX10-critical for the initial establishment of spermatogonial stem cells (SSCs). Conditional loss of the entire 33-gene X-linked homeobox gene cluster that includes Rhox10 causes progressive spermatogenic decline, a phenotype indistinguishable from that caused by loss of only Rhox10. We demonstrate that this phenotype results from dramatically reduced SSC generation. By using a battery of approaches, including single-cell-RNA sequencing (scRNA-seq) analysis, we show that Rhox10 drives SSC generation by promoting pro-spermatogonia differentiation. Rhox10 also regulates batteries of migration genes and promotes the migration of pro-spermatogonia into the SSC niche. The identification of an X-linked homeobox gene that drives the initial generation of SSCs has implications for the evolution of X-linked gene clusters and sheds light on regulatory mechanisms influencing adult stem cell generation in general.

Nonsense-Mediated RNA Decay Influences Human Embryonic Stem Cell Fate.

Nonsense-mediated RNA decay (NMD) is a highly conserved pathway that selectively degrades specific subsets of RNA transcripts. Here, we provide evidence that NMD regulates early human developmental cell fate. We found that NMD factors tend to be expressed at higher levels in human pluripotent cells than in differentiated cells, raising the possibility that NMD must be downregulated to permit differentiation. Loss- and gain-of-function experiments in human embryonic stem cells (hESCs) demonstrated that, indeed, NMD downregulation is essential for efficient generation of definitive endoderm. RNA-seq analysis identified NMD target transcripts induced when NMD is suppressed in hESCs, including many encoding signaling components. This led us to test the role of TGF-ß and BMP signaling, which we found NMD acts through to influence definitive endoderm versus mesoderm fate. Our results suggest that selective RNA decay is critical for specifying the developmental fate of specific human embryonic cell lineages.

The Antagonistic Gene Paralogs Upf3a and Upf3b Govern Nonsense-Mediated RNA Decay.

Gene duplication is a major evolutionary force driving adaptation and speciation, as it allows for the acquisition of new functions and can augment or diversify existing functions. Here, we report a gene duplication event that yielded another outcome--the generation of antagonistic functions. One product of this duplication event--UPF3B--is critical for the nonsense-mediated RNA decay (NMD) pathway, while its autosomal counterpart--UPF3A--encodes an enigmatic protein previously shown to have trace NMD activity. Using loss-of-function approaches in vitro and in vivo, we discovered that UPF3A acts primarily as a potent NMD inhibitor that stabilizes hundreds of transcripts. Evidence suggests that UPF3A acquired repressor activity through simple impairment of a critical domain, a rapid mechanism that may have been widely used in evolution. Mice conditionally lacking UPF3A exhibit "hyper" NMD and display defects in embryogenesis and gametogenesis. Our results support a model in which UPF3A serves as a molecular rheostat that directs developmental events.

Identification of novel post-transcriptional features in olfactory receptor family mRNAs.

Olfactory receptor (Olfr) genes comprise the largest gene family in mice. Despite their importance in olfaction, how most Olfr mRNAs are regulated remains unexplored. Using RNA-seq analysis coupled with analysis of pre-existing databases, we found that Olfr mRNAs have several atypical features suggesting that post-transcriptional regulation impacts their expression. First, Olfr mRNAs, as a group, have dramatically higher average AU-content and lower predicted secondary structure than do control mRNAs. Second, Olfr mRNAs have a higher density of AU-rich elements (AREs) in their 3'UTR and upstream open reading frames (uORFs) in their 5 UTR than do control mRNAs. Third, Olfr mRNAs have shorter 3' UTR regions and with fewer predicted miRNA-binding sites. All of these novel properties correlated with higher Olfr expression. We also identified striking differences in the post-transcriptional features of the mRNAs from the two major classes of Olfr genes, a finding consistent with their independent evolutionary origin. Together, our results suggest that the Olfr gene family has encountered unusual selective forces in neural cells that have driven them to acquire unique post-transcriptional regulatory features. In support of this possibility, we found that while Olfr mRNAs are degraded by a deadenylation-dependent mechanism, they are largely protected from this decay in neural lineage cells.

Ethylene Response Factor070 regulates root development and phosphate starvation-mediated responses.

Inorganic phosphate (Pi) availability is a major factor determining growth and consequently the productivity of crops. However, it is one of the least available macronutrients due to its high fixation in the rhizospheres. To overcome this constraint, plants have developed adaptive responses to better acquire, utilize, and recycle Pi. Molecular determinants of these adaptive mechanisms include transcription factors (TFs) that play a major role in transcriptional control, thereby regulating genome-scale networks. In this study, we have characterized the biological role of Arabidopsis thaliana Ethylene Response Factor070 (AtERF070), a Pi starvation-induced TF belonging to the Apetala2/Ethylene Response Factor family of TFs in Arabidopsis (Arabidopsis thaliana). It is localized to the nucleus and induced specifically in Pi-deprived roots and shoots. RNA interference-mediated suppression of AtERF070 led to augmented lateral root development resulting in higher Pi accumulation, whereas there were reductions in both primary root length and lateral root number in 12-d-old transgenic seedlings overexpressing AtERF070. When the overexpressing lines were grown to maturity under greenhouse conditions, they revealed a stunted bushy appearance that could be rescued by gibberellic acid application. Furthermore, a number of Pi starvation-responsive genes were modulated in AtERF070-overexpressing and RNA interference lines, thereby suggesting a potential role for this TF in maintaining Pi homeostasis.

How to activate a gene: decap its associated noncoding RNA.

Capped and polyadenylated long noncoding RNAs (lncRNAs) are shown to be degraded by a DCP2-mediated turnover mechanism by Geisler et al. (2012); this provides a new level of regulatory control for inducible genes by lncRNAs.

Characterization of the phosphate starvation-induced glycerol-3-phosphate permease gene family in Arabidopsis.

Phosphate (Pi) deficiency is one of the leading causes of loss in crop productivity. Plants respond to Pi deficiency by increasing Pi acquisition and remobilization involving organic and inorganic Pi transporters. Here, we report the functional characterization of a putative organic Pi transporter, Glycerol-3-phosphate permease (G3Pp) family, comprising five members (AtG3Pp1 to -5) in Arabidopsis (Arabidopsis thaliana). AtG3Pp1 and AtG3Pp2 showed 24-and 3-fold induction, respectively, in the roots of Pi-deprived seedlings, whereas Pi deficiency-mediated induction of AtG3Pp3 and -4 was evident in both roots and shoots. Furthermore, promoter-ß-glucuronidase (GUS) fusion transgenics were generated for AtG3Pp2 to -5 for elucidation of their in planta role in Pi homeostasis. During Pi starvation, there was a strong expression of the reporter gene driven by AtG3Pp4 promoter in the roots, shoots, anthers, and siliques, whereas GUS expression was specific either to the roots (AtG3Pp3) or to stamens and siliques (AtG3Pp5) in other promoter-GUS fusion transgenics. Quantification of reporter gene activities further substantiated differential responses of AtG3Pp family members to Pi deprivation. A distinct pattern of reporter gene expression exhibited by AtG3Pp3 and AtG3Pp5 during early stages of germination also substantiated their potential roles during seedling ontogeny. Furthermore, an AtG3Pp4 knockdown mutant exhibited accentuated total lateral root lengths under +phosphorus and -phosphorus conditions compared with the wild type. Several Pi starvation-induced genes involved in root development and/or Pi homeostasis were up-regulated in the mutant. A 9-fold induction of AtG3Pp3 in the mutant provided some evidence for a lack of functional redundancy in the gene family. These results thus reflect differential roles of members of the G3Pp family in the maintenance of Pi homeostasis.