Analyses of SUM1-1-mediated long-range repression

In Saccharomyces cerevisiae, local repression is promoter specific and localized to a small region on the DNA, while silencing is promoter nonspecific, encompasses large domains of chromatin, and is stably inherited for multiple generations. Sum1p is a local repressor protein that mediates repression of meiosis-specific genes in mitotic cells while the Sir proteins are long-range repressors that stably silence genes at HML, HMR, and telomeres. The SUM1-1 mutation is a dominant neomorphic mutation that enables the mutant protein to be recruited to the HMR locus and repress genes, even in the absence of the Sir proteins. In this study we show that the mutation in Sum1-1p enabled it to spread, and the native HMR barrier blocked it from spreading. Thus, like the Sir proteins, Sum1-1p was a long-range repressor, but unlike the Sir proteins, Sum1-1p-mediated repression was more promoter specific, repressing certain genes better than others. Furthermore, repression mediated by Sum1-1p was not stably maintained or inherited and we therefore propose that Sum1-1p-mediated long-range repression is related but distinct from silencing.     

Plant Mobile Small RNAs

In plants, RNA silencing is a fundamental regulator of gene expression, heterochromatin formation, suppression of transposable elements, and defense against viruses. The sequence specificity of these processes relies on small noncoding RNA (sRNA) molecules. Although the spreading of RNA silencing across the plant has been recognized for nearly two decades, only recently have sRNAs been formally demonstrated as the mobile silencing signals. Here, we discuss the various types of mobile sRNA molecules, their short- and long-range movement, and their function in recipient cells.RNA silencing is a regulatory mechanism that controls the expression of endogenous genes and exogenous molecular parasites such as viruses, transgenes, and transposable elements. One of the most fascinating aspects of RNA silencing found in plants and invertebrates is its mobile nature—in other words, its ability to spread from the cell where it has been initiated to neighboring cells. This phenomenon relies on the movement of small noncoding RNA molecules (sRNA, 21–24 nucleotides [nt] in length) that provide the sequence specificity of the silencing effects. In plants, there are two major classes of sRNAs: short interfering RNAs (siRNAs) and micro RNAs (miRNAs). These sRNAs are generated by diverse and sometimes interacting biochemical pathways, which may influence their mobility. Movement of plant sRNAs falls into two main categories: cell-to-cell (short-range) and systemic (long-range) movement (Melnyk et al. 2011).     

Spreading of Heterochromatin Is Limited to Specific Families of Maize Retrotransposons

Transposable elements (TEs) have the potential to act as controlling elements to influence the expression of genes and are often subject to heterochromatic silencing. The current paradigm suggests that heterochromatic silencing can spread beyond the borders of TEs and influence the chromatin state of neighboring low-copy sequences. This would allow TEs to condition obligatory or facilitated epialleles and act as controlling elements. The maize genome contains numerous families of class I TEs (retrotransposons) that are present in moderate to high copy numbers, and many are found in regions near genes, which provides an opportunity to test whether the spreading of heterochromatin from retrotransposons is prevalent. We have investigated the extent of heterochromatin spreading into DNA flanking each family of retrotransposons by profiling DNA methylation and di-methylation of lysine 9 of histone 3 (H3K9me2) in low-copy regions of the maize genome. The effects of different retrotransposon families on local chromatin are highly variable. Some retrotransposon families exhibit enrichment of heterochromatic marks within 800–1,200 base pairs of insertion sites, while other families exhibit very little evidence for the spreading of heterochromatic marks. The analysis of chromatin state in genotypes that lack specific insertions suggests that the heterochromatin in low-copy DNA flanking retrotransposons often results from the spreading of silencing marks rather than insertion-site preferences. Genes located near TEs that exhibit spreading of heterochromatin tend to be expressed at lower levels than other genes. Our findings suggest that a subset of retrotransposon families may act as controlling elements influencing neighboring sequences, while the majority of retrotransposons have little effect on flanking sequences.     

Phloem flow strongly influences the systemic spread of silencing in GFP Nicotiana benthamiana plants

The term 'RNA silencing' describes a process that results in the specific degradation of an RNA target. In plants, silenced tissues can initiate the spreading of the process into non-silenced regions by a mobile signal that can be transmitted over long distances. In the present work, we made use of a modified grafting approach to elucidate the driving force behind long-distance transport of the silencing signal. We made reciprocal grafts of two GFP-transgenic Nicotiana benthamiana lines, the non-silenced line 16c (sensor) and the silenced line 6.4 (inducer). We show that the direction of systemic spread of silencing from inducer to sensor can be manipulated by altering sink/source relations in the plant. Using radioactive phosphate as a phloem tracer, we demonstrated that plants that transmitted silencing from silenced scion to non-silenced rootstock had developed a persisting phloem flow from scion to rootstock. These data provide experimental proof of what has been hypothesized so far, that the silencing signal travels via phloem from source to sink. We present here evidence that the appearance of systemic silencing is not an accidental stochastic process, but can be predicted on the basis of the direction of phloem flow.     

Clustering heterochromatin: Sir3 promotes telomere clustering independently of silencing in yeast

A general feature of the nucleus is the organization of repetitive deoxyribonucleic acid sequences in clusters concentrating silencing factors. In budding yeast, we investigated how telomeres cluster in perinuclear foci associated with the silencing complex Sir2-Sir3-Sir4 and found that Sir3 is limiting for telomere clustering. Sir3 overexpression triggers the grouping of telomeric foci into larger foci that relocalize to the nuclear interior and correlate with more stable silencing in subtelomeric regions. Furthermore, we show that Sir3's ability to mediate telomere clustering can be separated from its role in silencing. Indeed, nonacetylable Sir3, which is unable to spread into subtelomeric regions, can mediate telomere clustering independently of Sir2-Sir4 as long as it is targeted to telomeres by the Rap1 protein. Thus, arrays of Sir3 binding sites at telomeres appeared as the sole requirement to promote trans-interactions between telomeres. We propose that similar mechanisms involving proteins able to oligomerize account for long-range interactions that impact genomic functions in many organisms.     

Region-specific requirement for cholesterol modification of sonic hedgehog in patterning the telencephalon and spinal cord

Sonic hedgehog (Shh) secreted from the axial signaling centers of the notochord and prechordal plate functions as a morphogen in dorsoventral patterning of the neural tube. Active Shh is uniquely cholesterol-modified and the hydrophobic nature of cholesterol suggests that it might regulate Shh spreading in the neural tube. Here, we examined the capacity of Shh lacking the cholesterol moiety (ShhN) to pattern different cell types in the telencephalon and spinal cord. In mice expressing ShhN, we detected low-level ShhN in the prechordal plate and notochord, consistent with the notion that ShhN can rapidly spread from its site of synthesis. Surprisingly, we found that low-level ShhN can elicit the generation of a full spectrum of ventral cell types in the spinal cord, whereas ventral neuronal specification and ganglionic eminence development in the Shh(N/-) telencephalon were severely impaired, suggesting that telencephalic patterning is more sensitive to alterations in local Shh concentration and spreading. In agreement, we observed induction of Shh pathway activity and expression of ventral markers at ectopic sites in the dorsal telencephalon indicative of long-range ShhN activity. Our findings indicate an essential role for the cholesterol moiety in restricting Shh dilution and deregulated spread for patterning the telencephalon. We propose that the differential effect of ShhN in patterning the spinal cord versus telencephalon may be attributed to regional differences in the maintenance of Shh expression in the ventral neuroepithelium and differences in dorsal tissue responsiveness to deregulated Shh spreading behavior.     

Introns reduce transitivity proportionally to their length, suggesting that silencing spreads along the pre-mRNA

Endogenes rarely support transitive silencing, whereas most transgenes generally allow the spread of silencing to occur along the primary target. To determine whether the presence of introns might explain the difference, we investigated the influence of introns in the primary target on 3'–5' silencing transitivity. When present in a transgene, an intron-containing endogene fragment does not prohibit the spread of silencing across this fragment, indicating that introns do not preclude silencing transitivity along endogenes. Also, a multiple intron-containing genomic gene fragment that had previously been shown not to support transitivity in an endogenous context could support transitivity when present in a transgene. Nevertheless, genomic intron-containing fragments delayed the onset and diminished the efficiency of transitive silencing of a secondary target compared with the corresponding cDNA fragments. Remarkably, transitivity was impaired proportionally with the length of the pre-mRNA, and not of the mRNA. The latter result suggests that the RNA dependent RNA polymerase-based spreading of silencing progresses along the non-spliced rather than the fully processed mature mRNA.     

HIV-1 Suppressive Sequences Are Modulated by Rev Transport of Unspliced RNA and Are Required for Efficient HIV-1 Production

The unspliced human immunodeficiency virus type 1 (HIV-1) RNAs are translated as Gag and Gag-Pol polyproteins or packaged as genomes into viral particles. Efficient translation is necessary before the transition to produce infective virions. The viral protein Rev exports all intron-containing viral RNAs; however, it also appears to enhance translation. Cellular microRNAs target cellular and viral mRNAs to silence their translation and enrich them at discrete cytoplasmic loci that overlap with the putative interim site of Gag and the genome. Here, we analyzed how Rev-mediated transport and the splicing status of the mRNA influenced the silencing status imposed by microRNA. Through identification and mutational analysis of the silencing sites in the HIV-1 genome, we elucidated the effect of silencing on virus production. Renilla luciferase mRNA, which contains a let-7 targeting site in its 3′ untranslated region, was mediated when it was transported by Rev and not spliced, but it was either not mediated when it was spliced even in a partial way or it was Rev-independent. The silencing sites in the pol and env-nef regions of the HIV-1 genome, which were repressed in T cells and other cell lines, were Drosha-dependent and could also be modulated by Rev in an unspliced state. Mutant viruses that contained genomic mutations that reflect alterations to show more derepressive effects in the 3′ untranslated region of the Renilla luciferase gene replicated more slowly than wild-type virus. These findings yield insights into the HIV-1 silencing sites that might allow the genome to avoid translational machinery and that might be utilized in coordinating virus production during initial virus replication. However, the function of Rev to modulate the silencing sites of unspliced RNAs would be advantageous for the efficient translation that is required to support protein production prior to viral packaging and particle production.     

Barriers and silencers: a theoretical toolkit for control and containment of nucleosome-based epigenetic states

Positive feedback in nucleosome modification has been proposed to allow large chromatin regions to exist stably and heritably in distinct expression states. However, modeling has shown that such epigenetic bistability requires that modifying enzymes recruited by nucleosomes are active on distant nucleosomes, potentially allowing uncontrollable spreading of modification. By modeling the silencing of mating-type loci in Saccharomyces cerevisiae, we show that a modification reaction that combines a long-range component and a locally acting component can provide bistability and can be blocked by simple barriers that interrupt the nucleosome chain. We find that robust containment of the silenced region could be achieved by the presence of a number of weak simple barriers in the surrounding chromatin and a limited capacity of the positive feedback reaction. In addition, we show that the state of the silenced region can be regulated by silencer elements acting only on neighboring nucleosomes. Thus, a relatively simple set of nucleosome-modifying enzymes and recognition domains is all that is needed to make chromatin-based epigenetics useful and safe.     

siRNA Has Greatly Elevated Mismatch Tolerance at 3′-UTR Sites

It has been noted that target sites located in the coding region or the 3′-untranslated region (3′-UTR) can be silenced to significantly different levels by the same siRNA, but little is known about at what specificity the silencing was achieved. In an exploration of positional effects on siRNA specificity by luciferase reporter system, we surprisingly discovered that siRNA had greatly elevated tolerance towards mismatches in target sites in the 3′-UTR of the mRNA compared with the same target sites cloned in the coding region. Assessment of changes in protein and mRNA levels suggested that the differential mismatch tolerance might have resulted from location-specific translational repression in the 3′-UTR. Ablation of argonaute proteins by AGO-specific siRNAs revealed that the AGO2 had major impact on siRNA silencing activity against sites in both coding region and 3′-UTR, while the silencing of nonnucleolytic AGO proteins (AGO1, AGO3 and AGO4) did not significantly affect silencing of sites in either region. This paper revealed the discovery that the specificity of an siRNA can be affected by the location of its target site.