Protocol for siRNA-mediated TET1 knockdown during differentiation of human embryonic stem cells into definitive endoderm cells.

TET1-mediated active DNA demethylation is required for endogenous retrovirus (ERV) enhancer activation during human ES differentiation into definitive endoderm (DE) cells. Here we present a protocol for siRNA-mediated TET1 knockdown during this process to decipher TET1's role in ERV activation and DE differentiation. We describe steps for inducing ES into DE cells. We then detail steps for knocking down TET1 during differentiation and for examining the effects of TET1 knockdown on LTR6B methylation, cell morphology, and gene expression. For complete details on the use and execution of this protocol, please refer to Wu et al. (2022).1.

Canalization of Phenotypes-When the Transcriptome is Constantly but Weakly Perturbed.

Recent studies have increasingly pointed to microRNAs (miRNAs) as the agent of gene regulatory network (GRN) stabilization as well as developmental canalization against constant but small environmental perturbations. To analyze mild perturbations, we construct a Dicer-1 knockdown line (dcr-1 KD) in Drosophila that modestly reduces all miRNAs by, on average, ∼20%. The defining characteristic of stabilizers is that, when their capacity is compromised, GRNs do not change their short-term behaviors. Indeed, even with such broad reductions across all miRNAs, the changes in the transcriptome are very modest during development in stable environment. By comparison, broad knockdowns of other regulatory genes (esp. transcription factors) by the same method should lead to drastic changes in the GRNs. The consequence of destabilization may thus be in long-term development as postulated by the theory of canalization. Flies with modest miRNA reductions may gradually deviate from the developmental norm, resulting in late-stage failures such as shortened longevity. In the optimal culture condition, the survival to adulthood is indeed normal in the dcr-1 KD line but, importantly, adult longevity is reduced by ∼90%. When flies are stressed by high temperature, dcr-1 KD induces lethality earlier in late pupation and, as the perturbations are shifted earlier, the affected stages are shifted correspondingly. Hence, in late stages of development with deviations piling up, GRN would be increasingly in need of stabilization. In conclusion, miRNAs appear to be a solution to weak but constant environmental perturbations.

Species-specific rewiring of definitive endoderm developmental gene activation via endogenous retroviruses through TET1-mediated demethylation.

Transposable elements (TEs) are the major sources of lineage-specific genomic innovation and comprise nearly half of the human genome, but most of their functions remain unclear. Here, we identify that a series of endogenous retroviruses (ERVs), a TE subclass, regulate the transcriptome at the definitive endoderm stage with in vitro differentiation model from human embryonic stem cell. Notably, these ERVs perform as enhancers containing binding sites for critical transcription factors for endoderm lineage specification. Genome-wide methylation analysis shows most of these ERVs are derepressed by TET1-mediated DNA demethylation. LTR6B, a representative definitive endoderm activating ERV, contains binding sites for FOXA2 and GATA4 and governs the primate-specific expression of its neighboring developmental genes such as ERBB4 in definitive endoderm. Together, our study proposes evidence that recently evolved ERVs represent potent de novo developmental regulatory elements, which, in turn, fine-tune species-specific transcriptomes during endoderm and embryonic development.

A Rapid Evolving microRNA Cluster Rewires Its Target Regulatory Networks in Drosophila.

New miRNAs are evolutionarily important but their functional evolution remains unclear. Here we report that the evolution of a microRNA cluster, mir-972C rewires its downstream regulatory networks in Drosophila. Genomic analysis reveals that mir-972C originated in the common ancestor of Drosophila where it comprises six old miRNAs. It has subsequently recruited six new members in the melanogaster subgroup after evolving for at least 50 million years. Both the young and the old mir-972C members evolved rapidly in seed and non-seed regions. Combining target prediction and cell transfection experiments, we found that the seed and non-seed changes in individual mir-972C members cause extensive target divergence among D. melanogaster, D. simulans, and D. virilis, consistent with the functional evolution of mir-972C reported recently. Intriguingly, the target pool of the cluster as a whole remains relatively conserved. Our results suggest that clustering of young and old miRNAs broadens the target repertoires by acquiring new targets without losing many old ones. This may facilitate the establishment of new miRNAs in existing regulatory networks.

Rapid Evolution of Autosomal Binding Sites of the Dosage Compensation Complex in Drosophila melanogaster and Its Association With Transcription Divergence.

How pleiotropy influences evolution of protein sequence remains unclear. The male-specific lethal (MSL) complex in Drosophila mediates dosage compensation by 2-fold upregulation of the X chromosome in males. Nevertheless, several MSL proteins also bind autosomes and likely perform functions not related to dosage compensation. Here, we study the evolution of MOF, MSL1, and MSL2 biding sites in Drosophila melanogaster and its close relative Drosophila simulans. We found pervasive expansion of the MSL binding sites in D. melanogaster, particularly on autosomes. The majority of these newly-bound regions are unlikely to function in dosage compensation and associated with an increase in expression divergence between D. melanogaster and D. simulans. While dosage-compensation related sites show clear signatures of adaptive evolution, these signatures are even more marked among autosomal regions. Our study points to an intriguing avenue of investigation of pleiotropy as a mechanism promoting rapid protein sequence evolution.

Run or Die in the Evolution of New MicroRNAs-Testing the Red Queen Hypothesis on De Novo New Genes.

The Red Queen hypothesis depicts evolution as the continual struggle to adapt. According to this hypothesis, new genes, especially those originating from nongenic sequences (i.e., de novo genes), are eliminated unless they evolve continually in adaptation to a changing environment. Here, we analyze two Drosophila de novo miRNAs that are expressed in a testis-specific manner with very high rates of evolution in their DNA sequence. We knocked out these miRNAs in two sibling species and investigated their contributions to different fitness components. We observed that the fitness contributions of miR-975 in Drosophila simulans seem positive, in contrast to its neutral contributions in D. melanogaster, whereas miR-983 appears to have negative contributions in both species, as the fitness of the knockout mutant increases. As predicted by the Red Queen hypothesis, the fitness difference of these de novo miRNAs indicates their different fates.

Death of new microRNA genes in Drosophila via gradual loss of fitness advantages.

The prevalence of de novo coding genes is controversial due to length and coding constraints. Noncoding genes, especially small ones, are freer to evolve de novo by comparison. The best examples are microRNAs (miRNAs), a large class of regulatory molecules ∼22 nt in length. Here, we study six de novo miRNAs in Drosophila, which, like most new genes, are testis-specific. We ask how and why de novo genes die because gene death must be sufficiently frequent to balance the many new births. By knocking out each miRNA gene, we analyzed their contributions to the nine components of male fitness (sperm production, length, and competitiveness, among others). To our surprise, the knockout mutants often perform better than the wild type in some components, and slightly worse in others. When two of the younger miRNAs are assayed in long-term laboratory populations, their total fitness contributions are found to be essentially zero. These results collectively suggest that adaptive de novo genes die regularly, not due to the loss of functionality, but due to the canceling out of positive and negative fitness effects, which may be characterized as "quasi-neutrality." Since de novo genes often emerge adaptively and become lost later, they reveal ongoing period-specific adaptations, reminiscent of the "Red-Queen" metaphor for long-term evolution.

On the possibility of death of new genes - evidence from the deletion of de novo microRNAs.

BACKGROUND: New genes are constantly formed, sometimes from non-genic sequences, creating what is referred to as de novo genes. Since the total number of genes remains relatively steady, gene deaths likely balance out new births. In metazoan genomes, microRNAs (miRs) genes, small and non-coding, account for the bulk of functional de novo genes and are particularly suited to the investigation of gene death. RESULTS: In this study, we discover a Drosophila-specific de novo miRNA (mir-977) that may be facing impending death. Strikingly, after this testis-specific gene is deleted from D. melanogaster, most components of male fitness increase, rather than decrease as had been expected. These components include male viability, fertility and males' ability to repress female re-mating. Given that mir-977 has a negative fitness effect in D. melanogaster, this de novo gene with an adaptive history for over 60 Myrs may be facing elimination. In some other species where mir-977 is not found, gene death may have already happened. CONCLUSION: The surprising result suggests that de novo genes, constantly rising and falling during evolution, may often be transiently adaptive and then purged from the genome.

Regulation of Large Number of Weak Targets-New Insights from Twin-microRNAs.

Each animal microRNA (miRNA) targets many genes for repression. Down-regulation of most of these targets is weak and has no detectable individual phenotypic effect. Whether this extensive weak repression is biologically relevant is a central issue in the debate on miRNA functionality. In the "small (target) pool" view, weak repression is nonfunctional and should be gradually removed during evolution. However, since the selective advantage of removing individual targets is small, testing this hypothesis is a challenge. We propose a novel approach by using miRNAs we call twin-miRs, which produce two mature products from the hairpin of the same miRNA precursor. Loss of the minor miR partner would affect all its targets and thus could be visible to selection. Since the minor miRs repress all their targets weakly, the "small pool" hypothesis would predict the elimination of twin-miRs over time. Surveying and sequencing 45 small RNA libraries in Drosophila, we found that nearly 40% of miRNAs produce twin-miRs. The minor forms are expressed in nontrivial abundance and repress their targets weakly. Interestingly, twin-miRs are often evolutionarily old, highly conserved, and comparable to solo-miRs in expression. Since there is no measurable trend toward reduction in target pool size, we conclude that at least some of the weak repression interactions are functional. A companion study using the May-Wigner theory of network stability suggests that distributed weak repression cumulatively contributes to stability of gene regulatory networks.

Redundant and incoherent regulations of multiple phenotypes suggest microRNAs' role in stability control.

Each microRNA (miRNA) represses a web of target genes and, through them, controls multiple phenotypes. The difficulties inherent in such controls cast doubt on how effective miRNAs are in driving phenotypic changes. A "simple regulation" model posits "one target-one phenotype" control under which most targeting is nonfunctional. In an alternative "coordinate regulation" model, multiple targets are assumed to control the same phenotypes coherently, and most targeting is functional. Both models have some empirical support but pose different conceptual challenges. Here, we concurrently analyze multiple targets and phenotypes associated with the miRNA-310 family (miR310s) of Drosophila Phenotypic rescue in the mir310s knockout background is achieved by promoter-directed RNA interference that restores wild-type expression. For one phenotype (eggshell morphology), we observed redundant regulation, hence rejecting "simple regulation" in favor of the "coordinate regulation" model. For other phenotypes (egg-hatching and male fertility), however, one gene shows full rescue, but three other rescues aggravate the phenotype. Overall, phenotypic controls by miR310s do not support either model. Like a thermostat that controls both heating and cooling elements to regulate temperature, redundancy and incoherence in regulation generally suggest some capacity in stability control. Our results therefore support the published view that miRNAs play a role in the canalization of transcriptome and, hence, phenotypes.

Small RNA transcriptomes of mangroves evolve adaptively in extreme environments.

MicroRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) are key players in plant stress responses. Here, we present the sRNA transcriptomes of mangroves Bruguiera gymnorrhiza and Kandelia candel. Comparative computational analyses and target predictions revealed that mangroves exhibit distinct sRNA regulatory networks that differ from those of glycophytes. A total of 32 known and three novel miRNA families were identified. Conserved and mangrove-specific miRNA targets were predicted; the latter were widely involved in stress responses. The known miRNAs showed differential expression between the mangroves and glycophytes, reminiscent of the adaptive stress-responsive changes in Arabidopsis. B. gymnorrhiza possessed highly abundant but less conserved TAS3 trans-acting siRNAs (tasiRNAs) in addition to tasiR-ARFs, with expanded potential targets. Our results indicate that the evolutionary alteration of sRNA expression levels and the rewiring of sRNA-regulatory networks are important mechanisms underlying stress adaptation. We also identified sRNAs that are involved in salt and/or drought tolerance and nutrient homeostasis as possible contributors to mangrove success in stressful environments.