Adaptive evolution targets a piRNA precursor transcription network [preprint]

In Drosophila, transposon-silencing piRNAs are derived from heterochromatic clusters and a subset of euchromatic transposon insertions, which are transcribed from internal non-canonical initiation sites and flanking canonical promoters. Rhino binds to Deadlock, which recruits TRF2 to promote non-canonical transcription of these loci. Cuff co-localizes with Rhino and Del. The role of Cuff is less well understood, but the cuff gene shows hallmarks of adaptive evolution, which frequently targets functional interactions within host defense systems. We show that Drosophila simulans cuff is a dominant negative allele when expressed in Drosophila melanogaster, where it traps Deadlock, TRF2 and the transcriptional co-repressor CtBP in stable nuclear complexes. Cuff promotes Rhino and Deadlock localization, driving non-canonical transcription. CtBP, by contrast, suppresses canonical cluster and transposon transcription, which interferes with downstream non-canonical transcription and piRNA production. Cuff, TRF2 and CtBP thus form a network that balances canonical and non-canonical piRNA precursor transcription

Adaptive Evolution Targets a piRNA Precursor Transcription Network

In Drosophila, transposon-silencing piRNAs are derived from heterochromatic clusters and a subset of euchromatic transposon insertions, which are bound by the Rhino-Deadlock-Cutoff complex. The HP1 homolog Rhino binds to Deadlock, which recruits TRF2 to promote non-canonical transcription from both genomic strands. Cuff function is less well understood, but this Rai1 homolog shows hallmarks of adaptive evolution, which can remodel functional interactions within host defense systems. Supporting this hypothesis, Drosophila simulans Cutoff is a dominant-negative allele when expressed in Drosophila melanogaster, in which it traps Deadlock, TRF2, and the conserved transcriptional co-repressor CtBP in stable complexes. Cutoff functions with Rhino and Deadlock to drive non-canonical transcription. In contrast, CtBP suppresses canonical transcription of transposons and promoters flanking the major germline clusters, and canonical transcription interferes with downstream non-canonical transcription and piRNA production. Adaptive evolution thus targets interactions among Cutoff, TRF2, and CtBP that balance canonical and non-canonical piRNA precursor transcription

Long first exons and epigenetic marks distinguish conserved pachytene piRNA clusters from other mammalian genes

In the male germ cells of placental mammals, 26-30-nt-long PIWI-interacting RNAs (piRNAs) emerge when spermatocytes enter the pachytene phase of meiosis. In mice, pachytene piRNAs derive from ~100 discrete autosomal loci that produce canonical RNA polymerase II transcripts. These piRNA clusters bear 5\u27 caps and 3\u27 poly(A) tails, and often contain introns that are removed before nuclear export and processing into piRNAs. What marks pachytene piRNA clusters to produce piRNAs, and what confines their expression to the germline? We report that an unusually long first exon ( \u3e /= 10 kb) or a long, unspliced transcript correlates with germline-specific transcription and piRNA production. Our integrative analysis of transcriptome, piRNA, and epigenome datasets across multiple species reveals that a long first exon is an evolutionarily conserved feature of pachytene piRNA clusters. Furthermore, a highly methylated promoter, often containing a low or intermediate level of CG dinucleotides, correlates with germline expression and somatic silencing of pachytene piRNA clusters. Pachytene piRNA precursor transcripts bind THOC1 and THOC2, THO complex subunits known to promote transcriptional elongation and mRNA nuclear export. Together, these features may explain why the major sources of pachytene piRNA clusters specifically generate these unique small RNAs in the male germline of placental mammals

A benchmark and an algorithm for detecting germline transposon insertions and measuring de novo transposon insertion frequencies

Transposons are genomic parasites, and their new insertions can cause instability and spur the evolution of their host genomes. Rapid accumulation of short-read whole-genome sequencing data provides a great opportunity for studying new transposon insertions and their impacts on the host genome. Although many algorithms are available for detecting transposon insertions, the task remains challenging and existing tools are not designed for identifying de novo insertions. Here, we present a new benchmark fly dataset based on PacBio long-read sequencing and a new method TEMP2 for detecting germline insertions and measuring de novo \u27singleton\u27 insertion frequencies in eukaryotic genomes. TEMP2 achieves high sensitivity and precision for detecting germline insertions when compared with existing tools using both simulated data in fly and experimental data in fly and human. Furthermore, TEMP2 can accurately assess the frequencies of de novo transposon insertions even with high levels of chimeric reads in simulated datasets; such chimeric reads often occur during the construction of short-read sequencing libraries. By applying TEMP2 to published data on hybrid dysgenic flies inflicted by de-repressed P-elements, we confirmed the continuous new insertions of P-elements in dysgenic offspring before they regain piRNAs for P-element repression. TEMP2 is freely available at Github: https://github.com/weng-lab/TEMP2

YAP1 withdrawal in hepatoblastoma drives therapeutic differentiation of tumor cells to functional hepatocyte-like cells

BACKGROUND and AIMS: Despite surgical and chemotherapeutic advances, the five-year survival rate for Stage IV Hepatoblastoma (HB), the predominant pediatric liver tumor, remains at 27%. YAP1 and beta-Catenin co-activation occurs in 80% of children\u27s HB; however, a lack of conditional genetic models precludes tumor maintenance exploration. Thus, the need for a targeted therapy remains unmet. Given the predominance of YAP1 and beta-Catenin activation in HB, we sought to evaluate YAP1 as a therapeutic target in HB. APPROACH and RESULTS: We engineered the first conditional HB murine model using hydrodynamic injection to deliver transposon plasmids encoding inducible YAP1(S127A) , constitutive beta-Catenin(DelN90) , and a luciferase reporter to murine liver. Tumor regression was evaluated using bioluminescent imaging, and tumor landscape characterized using RNA and ATAC sequencing, and DNA foot-printing. Here we show that YAP1(S127A) withdrawal mediates \u3e90% tumor regression with survival for 230+ days in mice. YAP1 (S127A) withdrawal promotes apoptosis in a subset of tumor cells and in remaining cells induces a cell fate switch driving therapeutic differentiation of HB tumors into Ki-67 negative hbHep cells with hepatocyte-like morphology and mature hepatocyte gene expression. YAP1 (S127A) withdrawal drives formation of hbHeps by modulating liver differentiation transcription factor (TF) occupancy. Indeed, tumor-derived hbHeps, consistent with their reprogrammed transcriptional landscape, regain partial hepatocyte function and rescue liver damage in mice. CONCLUSIONS: YAP1(S127A) withdrawal, without silencing oncogenic beta-Catenin, significantly regresses hepatoblastoma, providing the first in vivo data to support YAP1 as a therapeutic target for HB. YAP1(S127A) withdrawal alone sufficiently drives long-term regression in hepatoblastoma because it promotes cell death in a subset of tumor cells and modulates transcription factor occupancy to reverse the fate of residual tumor cells to mimic functional hepatocytes

A-MYB/TCFL5 regulatory architecture ensures the production of pachytene piRNAs in placental mammals

In male mice, the transcription factor A-MYB initiates the transcription of pachytene piRNA genes during meiosis. Here, we report that A-MYB activates the transcription factor Tcfl5 produced in pachytene spermatocytes. Subsequently, A-MYB and TCFL5 reciprocally reinforce their own transcription to establish a positive feedback circuit that triggers pachytene piRNA production. TCFL5 regulates the expression of genes required for piRNA maturation and promotes transcription of evolutionarily young pachytene piRNA genes, whereas A-MYB activates the transcription of older pachytene piRNA genes. Intriguingly, pachytene piRNAs from TCFL5-dependent young loci initiate the production of piRNAs from A-MYB-dependent older loci, ensuring the self-propagation of pachytene piRNAs. A-MYB and TCFL5 act via a set of incoherent feedforward loops that drive regulation of gene expression by pachytene piRNAs during spermatogenesis. This regulatory architecture is conserved in rhesus macaque, suggesting that it was present in the last common ancestor of placental mammals

A-MYB/TCFL5 regulatory architecture ensures the production of pachytene piRNAs in placental mammals

In male mice, the transcription factor A MYB initiates the transcription of pachytene piRNA genes during meiosis. Here, we report that A MYB activates the transcription factor Tcfl5 produced in pachytene spermatocytes. Subsequently, A MYB and TCFL5 reciprocally reinforce their own transcription to establish a positive feedback circuit that triggers pachytene piRNA production. TCFL5 regulates the expression of genes required for piRNA maturation and promotes transcription of evolutionarily young pachytene piRNA genes, whereas A-MYB activates the transcription of older pachytene piRNA genes. Intriguingly, pachytene piRNAs from TCFL5-dependent young loci initiates the production of piRNAs from A-MYB-dependent older loci ensuring the self-propagation of pachytene piRNAs. A MYB and TCFL5 act via a set of incoherent feedforward loops that drive regulation of gene expression by pachytene piRNAs during spermatogenesis. This regulatory architecture is conserved in rhesus macaque, suggesting that it was present in the last common ancestor of placental mammals

piRNA-independent transposon silencing by the Drosophila THO complex

piRNAs guide Piwi/Panoramix-dependent H3K9me3 chromatin modification and transposon silencing during Drosophila germline development. The THO RNA export complex is composed of Hpr1, Tho2, and Thoc5-7. Null thoc7 mutations, which displace Thoc5 and Thoc6 from a Tho2-Hpr1 subcomplex, reduce expression of a subset of germline piRNAs and increase transposon expression, suggesting that THO silences transposons by promoting piRNA biogenesis. Here, we show that the thoc7-null mutant combination increases transposon transcription but does not reduce anti-sense piRNAs targeting half of the transcriptionally activated transposon families. These mutations also fail to reduce piRNA-guided H3K9me3 chromatin modification or block Panoramix-dependent silencing of a reporter transgene, and unspliced transposon transcripts co-precipitate with THO through a Piwi- and Panoramix-independent mechanism. Mutations in piwi also dominantly enhance germline defects associated with thoc7-null alleles. THO thus functions in a piRNA-independent transposon-silencing pathway, which acts cooperatively with Piwi to support germline development

Co-dependent Assembly of Drosophila piRNA Precursor Complexes and piRNA Cluster Heterochromatin

Summary: In Drosophila, the piRNAs that guide germline transposon silencing are produced from heterochromatic clusters marked by the HP1 homolog Rhino. We show that Rhino promotes cluster transcript association with UAP56 and the THO complex, forming RNA-protein assemblies that are unique to piRNA precursors. UAP56 and THO are ubiquitous RNA-processing factors, and null alleles of uap56 and the THO subunit gene tho2 are lethal. However, uap56sz15 and mutations in the THO subunit genes thoc5 and thoc7 are viable but sterile and disrupt piRNA biogenesis. The uap56sz15 allele reduces UAP56 binding to THO, and the thoc5 and thoc7 mutations disrupt interactions among the remaining THO subunits and UAP56 binding to the core THO subunit Hpr1. These mutations also reduce Rhino binding to clusters and trigger Rhino binding to ectopic sites across the genome. Rhino thus promotes assembly of piRNA precursor complexes, and these complexes restrict Rhino at cluster heterochromatin. : Zhang et al. show that the piRNA-generating loci in Drosophila female germline are co-dependently assembled between unique chromatin factor Rhino (with Cutoff and Deadlock) and general RNA-processing factor TREX complex. Keywords: transposon silencing, heterochromatin, piRNA, Drosophili