Activation of the Notch Signaling Pathway In Vivo Elicits Changes in CSL Nuclear Dynamics.

A key feature of Notch signaling is that it directs immediate changes in transcription via the DNA-binding factor CSL, switching it from repression to activation. How Notch generates both a sensitive and accurate response-in the absence of any amplification step-remains to be elucidated. To address this question, we developed real-time analysis of CSL dynamics including single-molecule tracking in vivo. In Notch-OFF nuclei, a small proportion of CSL molecules transiently binds DNA, while in Notch-ON conditions CSL recruitment increases dramatically at target loci, where complexes have longer dwell times conferred by the Notch co-activator Mastermind. Surprisingly, recruitment of CSL-related corepressors also increases in Notch-ON conditions, revealing that Notch induces cooperative or "assisted" loading by promoting local increase in chromatin accessibility. Thus, in vivo Notch activity triggers changes in CSL dwell times and chromatin accessibility, which we propose confer sensitivity to small input changes and facilitate timely shut-down

The mlpt/Ubr3/Svb module comprises an ancient developmental switch for embryonic patterning

Small open reading frames (smORFs) encoding 'micropeptides' exhibit remarkable evolutionary complexity. Conserved peptides encoded by mille-pattes (mlpt)/polished rice (pri)/tarsal less (tal) are essential for embryo segmentation in Tribolium but, in Drosophila, function in terminal epidermal differentiation and patterning of adult legs. Here, we show that a molecular complex identified in Drosophila epidermal differentiation, comprising Mlpt peptides, ubiquitinligase Ubr3 and transcription factor Shavenbaby (Svb), represents an ancient developmental module required for early insect embryo patterning. We find that loss of segmentation function for this module in flies evolved concomitantly with restriction of Svb expression in early Drosophila embryos. Consistent with this observation, artificially restoring early Svb expression in flies causes segmentation defects that depend on mlpt function, demonstrating enduring potency of an ancestral developmental switch despite evolving embryonic patterning modes. These results highlight the evolutionary plasticity of conserved molecular complexes under the constraints of essential genetic networks. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter)

Genome-wide analyses of Shavenbaby target genes reveals distinct features of enhancer organization

International audienceBackground: Developmental programs are implemented by regulatory interactions between Transcription Factors (TFs) and their target genes, which remain poorly understood. While recent studies have focused on regulatory cascades of TFs that govern early development, little is known about how the ultimate effectors of cell differentiation are selected and controlled. We addressed this question during late Drosophila embryogenesis, when the finely tuned expression of the TF Ovo/Shavenbaby (Svb) triggers the morphological differentiation of epidermal trichomes.Results: We defined a sizeable set of genes downstream of Svb and used in vivo assays to delineate 14 enhancers driving their specific expression in trichome cells. Coupling computational modeling to functional dissection, we investigated the regulatory logic of these enhancers. Extending the repertoire of epidermal effectors using genome-wide approaches showed that the regulatory models learned from this first sample are representative of the whole set of trichome enhancers. These enhancers harbor remarkable features with respect to their functional architectures, including a weak or non-existent clustering of Svb binding sites. The in vivo function of each site relies on its intimate context, notably the flanking nucleotides. Two additional cis-regulatory motifs, present in a broad diversity of composition and positioning among trichome enhancers, critically contribute to enhancer activity.Conclusions: Our results show that Svb directly regulates a large set of terminal effectors of the remodeling of epidermal cells. Further, these data reveal that trichome formation is underpinned by unexpectedly diverse modes of regulation, providing fresh insights into the functional architecture of enhancers governing a terminal differentiation program

Deciphering the molecular mechanisms underpinning the transcriptional control of gene expression by L-AFL proteins in Arabidopsis seed.

International audienceIn Arabidopsis (Arabidopsis thaliana), transcriptional control of seed maturation involves three related regulators with a B3domain, namely LEAFY COTYLEDON2 (LEC2), ABSCISIC ACID INSENSITIVE3 (ABI3), and FUSCA3 (ABI3/FUS3/LEC2[AFLs]). Although genetic analyses have demonstrated partially overlapping functions of these regulators, the underlyingmolecular mechanisms remained elusive. The results presented here confirmed that the three proteins bind RY DNA elements(with a 59-CATG-39 core sequence) but with different specificities for flanking nucleotides. In planta as in the moss Physcomitrellapatens protoplasts, the presence of RY-like (RYL) elements is necessary but not sufficient for the regulation of the OLEOSIN1 (OLE1)promoter by the B3AFLs. G box-like domains, located in the vicinity of the RYL elements, also are required for proper activation ofthe promoter, suggesting that several proteins are involved. Consistentwith this idea, LEC2 andABI3 showed synergistic effects onthe activation of the OLE1 promoter. What is more, LEC1 (a homolog of the NF-YB subunit of the CCAAT-binding complex)further enhanced the activation of this target promoter in the presence of LEC2 and ABI3. Finally, recombinant LEC1 and LEC2proteins produced in Arabidopsis protoplasts could form a ternary complex with NF-YC2 in vitro, providing a molecular explanationfor their functional interactions. Taken together, these results allow us to propose a molecularmodel for the transcriptional regulationof seed genes by the L-AFL proteins, based on the formation of regulatory multiprotein complexes between NF-YBs, which carry aspecific aspartate-55 residue, and B3 transcription factors