The ChAHP Complex Counteracts Chromatin Looping at CTCF Sites that Emerged from SINE Expansions in Mouse.

CCCTC-binding factor (CTCF) and cohesin are key players in three-dimensional chromatin organization. The topologically associating domains (TADs) demarcated by CTCF are remarkably well conserved between species, although genome-wide CTCF binding has diverged substantially following transposon-mediated motif expansions. Therefore, the CTCF consensus motif poorly predicts TADs, and additional factors must modulate CTCF binding and subsequent TAD formation. Here, we demonstrate that the ChAHP complex (CHD4, ADNP, HP1) competes with CTCF for a common set of binding motifs. In Adnp knockout cells, novel insulated regions are formed at sites normally bound by ChAHP, whereas proximal canonical boundaries are weakened. These data reveal that CTCF-mediated loop formation is modulated by a distinct zinc-finger protein complex. Strikingly, ChAHP-bound loci are mainly situated within less diverged SINE B2 transposable elements. This implicates ChAHP in maintenance of evolutionarily conserved spatial chromatin organization by buffering novel CTCF binding sites that emerged through SINE expansions.

Tdrd6a Regulates the Aggregation of Buc into Functional Subcellular Compartments that Drive Germ Cell Specification.

Phase separation represents an important form of subcellular compartmentalization. However, relatively little is known about how the formation or disassembly of such compartments is regulated. In zebrafish, the Balbiani body (Bb) and the germ plasm (Gp) are intimately linked phase-separated structures essential for germ cell specification and home to many germ cell-specific mRNAs and proteins. Throughout development, these structures occur as a single large aggregate (Bb), which disperses throughout oogenesis and upon fertilization accumulates again into relatively large assemblies (Gp). Formation of the Bb requires Bucky ball (Buc), a protein with prion-like properties. We found that the multi-tudor domain-containing protein Tdrd6a interacts with Buc, affecting its mobility and aggregation properties. Importantly, lack of this regulatory interaction leads to significant defects in germ cell development. Our work presents insights into how prion-like protein aggregations can be regulated and highlights the biological relevance of such regulatory events.

Systemic Loss and Gain of Chromatin Architecture throughout Zebrafish Development.

The spatial organization of chromosomes is critical in establishing gene expression programs. We generated in situ Hi-C maps throughout zebrafish development to gain insight into higher-order chromatin organization and dynamics. Zebrafish chromosomes segregate in active and inactive chromatin (A/B compartments), which are further organized into topologically associating domains (TADs). Zebrafish A/B compartments and TADs have genomic features similar to those of their mammalian counterparts, including evolutionary conservation and enrichment of CTCF binding sites at TAD borders. At the earliest time point, when there is no zygotic transcription, the genome is highly structured. After zygotic genome activation (ZGA), the genome loses structural features, which are re-established throughout early development. Despite the absence of structural features, we see clustering of super-enhancers in the 3D genome. Our results provide insight into vertebrate genome organization and demonstrate that the developing zebrafish embryo is a powerful model system to study the dynamics of nuclear organization.

Enhancers reside in a unique epigenetic environment during early zebrafish development.

BACKGROUND: Enhancers, not promoters, are the most dynamic in their DNA methylation status throughout development and differentiation. Generally speaking, enhancers that are primed to or actually drive gene expression are characterized by relatively low levels of DNA methylation (hypo-methylation), while inactive enhancers display hyper-methylation of the underlying DNA. The direct functional significance of the DNA methylation state of enhancers is, however, unclear for most loci. RESULTS: In contrast to conventional epigenetic interactions at enhancers, we find that DNA methylation status and enhancer activity during early zebrafish development display very unusual correlation characteristics: hypo-methylation is a unique feature of primed enhancers whereas active enhancers are generally hyper-methylated. The hypo-methylated enhancers that we identify (hypo-enhancers) are enriched close to important transcription factors that act later in development. Interestingly, hypo-enhancers are de-methylated shortly before the midblastula transition and reside in a unique epigenetic environment. Finally, we demonstrate that hypo-enhancers do become active at later developmental stages and that they are physically associated with the transcriptional start site of target genes, irrespective of target gene activity. CONCLUSIONS: We demonstrate that early development in zebrafish embodies a time window characterized by non-canonical DNA methylation-enhancer relationships, including global DNA hypo-methylation of inactive enhancers and DNA hyper-methylation of active enhancers.

PID-1 is a novel factor that operates during 21U-RNA biogenesis in Caenorhabditis elegans.

The Piwi-piRNA pathway represents a small RNA-based mechanism responsible for the recognition and silencing of invading DNA. Biogenesis of piRNAs (21U-RNAs) is poorly understood. In Caenorhabditis elegans, the piRNA-binding Argonaute protein PRG-1 is the only known player acting downstream from precursor transcription. From a screen aimed at the isolation of piRNA-induced silencing-defective (Pid) mutations, we identified, among known Piwi pathway components, PID-1 as a novel player. PID-1 is a mostly cytoplasmic, germline-specific factor essential for 21U-RNA biogenesis, affecting an early step in the processing or transport of 21U precursor transcripts. We also show that maternal 21U-RNAs are essential to initiate silencing.

piRNA dynamics in divergent zebrafish strains reveal long-lasting maternal influence on zygotic piRNA profiles.

Transposable elements (TEs) are mobile genetic elements that can have many deleterious effects on the fitness of their host. The germline-specific PIWI pathway guards the genome against TEs, deriving its specificity from sequence complementarity between PIWI-bound small RNAs (piRNAs) and the TEs. The piRNAs are derived from so-called piRNA clusters. Recent studies have demonstrated that the piRNA repertoire can be adjusted to accommodate recent TE invasions by capturing invading TEs in piRNA loci. Thus far, no information concerning piRNA divergence is available from vertebrates. We present piRNA analyses of two relatively divergent zebrafish strains. We find that significant differences in the piRNA populations have accumulated, most notably among active class I TEs. This divergence can be split into differences in piRNA abundance per element and differences in sense/antisense polarity ratios. In crosses between animals of the different strains, many of these differences are resolved in the progeny. However, some differences remain, often leaning to the maternally contributed piRNA population. These differences can be detected at least two generations later. Our data illustrate, for the first time, the fluidity of piRNA populations in vertebrates and how the established diversity is transmitted to future generations.

Extremely stable Piwi-induced gene silencing in Caenorhabditis elegans.

In recent years, the Piwi pathway has been shown to regulate the silencing of mobile genetic elements. However, we know little about how Piwi pathways impose silencing and even less about trans-generational stability of Piwi-induced silencing. We demonstrate that the Caenorhabditis elegans Piwi protein PRG-1 can initiate an extremely stable form of gene silencing on a transgenic, single-copy target. This type of silencing is faithfully maintained over tens of generations in the absence of a functional Piwi pathway. Interestingly, RNAi can also trigger permanent gene silencing of a single-copy transgene and the phenomenon will be collectively referred to as RNA-induced epigenetic silencing (RNAe). RNAe can act in trans and is dependent on endogenous RNAi factors. The involvement of factors known to act in nuclear RNAi and the fact that RNAe is accompanied by repressive chromatin marks indicate that RNAe includes a transcriptional silencing component. Our results demonstrate that, at least in C. elegans, the Piwi pathway can impose a state of gene silencing that borders on 'permanently silent'. Such a property may be more widely conserved among Piwi pathways in different animals.

Differential impact of the HEN1 homolog HENN-1 on 21U and 26G RNAs in the germline of Caenorhabditis elegans.

RNA interference (RNAi)-related pathways affect gene activity by sequence-specific recruitment of Ago proteins to mRNA target molecules. The sequence specificity of this process stems from small RNA (sRNA) co-factors bound by the Ago protein. Stability of sRNA molecules in some pathways is in part regulated by Hen1-mediated methylation of their 3' ends. Here we describe the effects of the Caenorhabditis elegans HEN1 RNA-methyl-transferase homolog, HENN-1, on the different RNAi pathways in this nematode. We reveal differential effects of HENN-1 on the two pathways that are known to employ methylated sRNA molecules: the 26G and 21U pathways. Surprisingly, in the germline, stability of 21U RNAs, the C. elegans piRNAs, is only mildly affected by loss of methylation; and introduction of artificial 21U target RNA does not further destabilize non-methylated 21U RNAs. In contrast, most 26G RNAs display reduced stability and respond to loss of HENN-1 by displaying increased 3'-uridylation frequencies. Within the 26G RNA class, we find that specifically ERGO-1-bound 26G RNAs are modified by HENN-1, while ALG-3/ALG-4-bound 26G RNAs are not. Global gene expression analysis of henn-1 mutants reveals mild effects, including down-regulation of many germline-expressed genes. Our data suggest that, apart from direct effects of reduced 26G RNA levels of henn-1 on gene expression, most effects on global gene expression are indirect. These studies further refine our understanding of endogenous RNAi in C. elegans and the roles for Hen1 like enzymes in these pathways.

Tdrd1 acts as a molecular scaffold for Piwi proteins and piRNA targets in zebrafish.

Piwi proteins function in an RNAi-like pathway that silences transposons. Piwi-associated RNAs, also known as piRNAs, act as a guide to identify Piwi targets. The tudor domain-containing protein Tdrd1 has been linked to this pathway but its function has thus far remained unclear. We show that zebrafish Tdrd1 is required for efficient Piwi-pathway activity and proper nuage formation. Furthermore, we find that Tdrd1 binds both zebrafish Piwi proteins, Ziwi and Zili, and reveals sequence specificity in the interaction between Tdrd1 tudor domains and symmetrically dimethylated arginines (sDMAs) in Zili. Finally, we show that Tdrd1 complexes contain piRNAs and RNA molecules that are longer than piRNAs. We name these longer transcripts Tdrd1-associated transcripts (TATs). TATs likely represent cleaved Piwi pathway targets and may serve as piRNA biogenesis intermediates. Altogether, our data suggest that Tdrd1 acts as a molecular scaffold for Piwi proteins, bound through specific tudor domain-sDMA interactions, piRNAs and piRNA targets.

The inactive X chromosome adopts a unique three-dimensional conformation that is dependent on Xist RNA.

Three-dimensional topology of DNA in the cell nucleus provides a level of transcription regulation beyond the sequence of the linear DNA. To study the relationship between the transcriptional activity and the spatial environment of a gene, we used allele-specific chromosome conformation capture-on-chip (4C) technology to produce high-resolution topology maps of the active and inactive X chromosomes in female cells. We found that loci on the active X form multiple long-range interactions, with spatial segregation of active and inactive chromatin. On the inactive X, silenced loci lack preferred interactions, suggesting a unique random organization inside the inactive territory. However, escapees, among which is Xist, are engaged in long-range contacts with each other, enabling identification of novel escapees. Deletion of Xist results in partial refolding of the inactive X into a conformation resembling the active X without affecting gene silencing or DNA methylation. Our data point to a role for Xist RNA in shaping the conformation of the inactive X chromosome at least partially independent of transcription.

Hen1 is required for oocyte development and piRNA stability in zebrafish.

Piwi-interacting RNAs (piRNAs) are germ line-specific small RNA molecules that have a function in genome defence and germ cell development. They associate with a specific class of Argonaute proteins, named Piwi, and function through an RNA interference-like mechanism. piRNAs carry a 2'-O-methyl modification at their 3' end, which is added by the Hen1 enzyme. We show that zebrafish hen1 is specifically expressed in germ cells and is essential for maintaining a female germ line, whereas it is dispensable in the testis. Hen1 protein localizes to nuage through its C-terminal domain, but is not required for nuage formation. In hen1 mutant testes, piRNAs become uridylated and adenylated. Uridylation frequency is highest on retro-transposon-derived piRNAs and is accompanied by decreased piRNA levels and mild derepression of transposon transcripts. Altogether, our data suggest the existence of a uridylation-mediated 3'-5' exonuclease activity acting on piRNAs in zebrafish germ cells, which is counteracted by nuage-bound Hen1 protein. This system discriminates between piRNA targets and is required for ovary development and fully efficient transposon silencing.

Dcr1 tracked down.

RNAi is essential for pericentromeric heterochromatic formation in S. pombe, and although Dcr1, the initiator protein of this process, has been biochemically well described, its subcellular localization has remained elusive. In this issue of Developmental Cell, Emmerth et al. now show that Dcr1 is dynamically shuttling between nucleus and cytoplasm, adding new insight into the subcellular mechanics of RNAi.