Project description:Many genes harbour multiple transcriptional enhancers that act concomitantly to achieve robust and precise spatial-temporal expression. In vertebrates, however, the mechanisms underlying cooperation between cis-acting elements are poorly documented. The mouse gene Krox20 encodes a transcription factor required for the specification of two segments (rhombomeres) of the developing hindbrain. In rhombomere 3, Krox20 is subject to positive feedback, governed by enhancer A, which is directly bound by the KROX20 protein, whereas another element, C, distant from 70 kb, was supposed to be only required for initiation of expression. Here, using both enhancer knock-outs and investigations of chromatin organisation, we show that element C possesses a dual activity: besides its classical enhancer function, it is also permanently required in cis to potentiate element A autoregulatory activity, by increasing its chromatin accessibility. This work uncovers a novel, asymmetrical, long-range mode of cooperation between cis-acting elements that might be essential to avoid promiscuous activation of positive autoregulatory elements.
Project description:Many genes harbour multiple transcriptional enhancers that act concomitantly to achieve robust and precise spatial-temporal expression. In vertebrates, however, the mechanisms underlying cooperation between cis-acting elements are poorly documented. The mouse gene Krox20 encodes a transcription factor required for the specification of two segments (rhombomeres) of the developing hindbrain. In rhombomere 3, Krox20 is subject to positive feedback, governed by enhancer A, which is directly bound by the KROX20 protein, whereas another element, C, distant from 70 kb, was supposed to be only required for initiation of expression. Here, using both enhancer knock-outs and investigations of chromatin organisation, we show that element C possesses a dual activity: besides its classical enhancer function, it is also permanently required in cis to potentiate element A autoregulatory activity, by increasing its chromatin accessibility. This work uncovers a novel, asymmetrical, long-range mode of cooperation between cis-acting elements that might be essential to avoid promiscuous activation of positive autoregulatory elements.
Project description:Many genes harbour multiple transcriptional enhancers that act concomitantly to achieve robust and precise spatial-temporal expression. In vertebrates, however, the mechanisms underlying cooperation between cis-acting elements are poorly documented. The mouse gene Krox20 encodes a transcription factor required for the specification of two segments (rhombomeres) of the developing hindbrain. In rhombomere 3, Krox20 is subject to positive feedback, governed by enhancer A, which is directly bound by the KROX20 protein, whereas another element, C, distant from 70 kb, was supposed to be only required for initiation of expression. Here, using both enhancer knock-outs and investigations of chromatin organisation, we show that element C possesses a dual activity: besides its classical enhancer function, it is also permanently required in cis to potentiate element A autoregulatory activity, by increasing its chromatin accessibility. This work uncovers a novel, asymmetrical, long-range mode of cooperation between cis-acting elements that might be essential to avoid promiscuous activation of positive autoregulatory elements.
Project description:Gene regulation can evolve either by cis-acting local changes to regulatory element DNA sequences or by global changes to the trans-acting regulatory environment; however, the modes favored during recent human evolution are unknown. To date, studies investigating gene regulatory divergence between closely-related species have produced limited estimates on the relative contributions of cis and trans effects on DNA regulatory element activities at a global-scale. By leveraging a comparative ATAC-STARR-seq framework, we identified 10,779 regulatory regions with divergent activity in cis and 10,608 regulatory regions with divergent activity in trans between human and rhesus macaque lymphoblastoid cell lines (LCLs). This revealed substantially more trans effects than predicted and indicates trans-regulatory mechanisms play a larger role in human evolution than previously expected. We also discover that most species-specific regulatory elements (67%) diverge in both cis and trans, suggesting these two mechanisms jointly drive divergent regulatory activity in a single sequence.
Project description:In the present study, we have identified six enhancers located within the zebrafish krox20 locus, using accessibility data generated by ATAC-seq and characterized their activity profiles as well as their functions and interactions in the zebrafish hindbrain . We also investigated the orthologues in six evolutionary distant vertebrates of one of these element, showing a great variety of activity profiles demonstrating the striking evolutionary flexibility of this element .
Project description:Primary piRNAs in Drosophila ovarian somatic cells arise from piRNA cluster transcripts and the 3′ UTRs of a subset of mRNAs, including Traffic jam (Tj) mRNA. However, it is unclear how these RNAs are determined as primary piRNA sources. Here, we identify a cis-acting 100-nt fragment in the Tj 3′ UTR that is sufficient for producing artificial piRNAs from unintegrated DNA. These artificial piRNAs were effective in endogenous gene transcriptional silencing. Yb, a core component of primary piRNA biogenesis center Yb bodies, directly bound the Tj-cis-element. Disruption of this interaction markedly reduced piRNA production. Thus, Yb is the trans-acting partner of the Tj-cis-element. Yb-CLIP revealed that Yb-binding correlated with somatic piRNA production but Tj-cis-element downstream sequences produced few artificial piRNAs. Thus, Yb determines primary piRNA sources by two modes of action; primary binding to cis-elements to specify substrates, and secondary binding to downstream regions to increase diversity in piRNA populations. HITS-CLIP of Yb in OSCs (Ovarian Somatic Cells) depleted for tj cis-element, and small RNA sequencing of Piwi-piRNAs in OSCs depleted for tj cis-element.