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Nanobody-mediated control of gene expression and epigenetic memory.

ABSTRACT: Targeting chromatin regulators to specific genomic locations for gene control is emerging as a powerful method in basic research and synthetic biology. However, many chromatin regulators are large, making them difficult to deliver and combine in mammalian cells. Here, we develop a strategy for gene control using small nanobodies that bind and recruit endogenous chromatin regulators to a gene. We show that an antiGFP nanobody can be used to simultaneously visualize GFP-tagged chromatin regulators and control gene expression, and that nanobodies against HP1 and DNMT1 can silence a reporter gene. Moreover, combining nanobodies together or with other regulators, such as DNMT3A or KRAB, can enhance silencing speed and epigenetic memory. Finally, we use the slow silencing speed and high memory of antiDNMT1 to build a signal duration timer and recorder. These results set the basis for using nanobodies against chromatin regulators for controlling gene expression and epigenetic memory.

SUBMITTER: Van MV

PROVIDER: S-EPMC7822885 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Nanobody-mediated control of gene expression and epigenetic memory.

Van Mike V MV Fujimori Taihei T Bintu Lacramioara L

Nature communications 20210122 1

Targeting chromatin regulators to specific genomic locations for gene control is emerging as a powerful method in basic research and synthetic biology. However, many chromatin regulators are large, making them difficult to deliver and combine in mammalian cells. Here, we develop a strategy for gene control using small nanobodies that bind and recruit endogenous chromatin regulators to a gene. We show that an antiGFP nanobody can be used to simultaneously visualize GFP-tagged chromatin regulators ...[more]

PMID: 33483487

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Project description:RATIONALE:The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. OBJECTIVE:We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. METHODS AND RESULTS:We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5, vascular endothelial-protein tyrosine phosphatase (VE-PTP), and von Willebrand factor (vWf). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and ?-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5, VE-PTP, and vWf. VEC/?-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5, VE-PTP, and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP. CONCLUSIONS:These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system.

Project description:A fundamental question in neuroscience is how memories are stored and retrieved in the brain. Long-term memory formation requires transcription, translation and epigenetic processes that control gene expression. Thus, characterizing genome-wide the transcriptional changes that occur after memory acquisition and retrieval is of broad interest and importance. Genome-wide technologies are commonly used to interrogate transcriptional changes in discovery-based approaches. Their ability to increase scientific insight beyond traditional candidate gene approaches, however, is usually hindered by batch effects and other sources of unwanted variation, which are particularly hard to control in the study of brain and behavior.We examined genome-wide gene expression after contextual conditioning in the mouse hippocampus, a brain region essential for learning and memory, at all the time-points in which inhibiting transcription has been shown to impair memory formation. We show that most of the variance in gene expression is not due to conditioning and that by removing unwanted variance through additional normalization we are able provide novel biological insights. In particular, we show that genes downregulated by memory acquisition and retrieval impact different functions: chromatin assembly and RNA processing, respectively. Levels of histone 2A variant H2AB are reduced only following acquisition, a finding we confirmed using quantitative proteomics. On the other hand, splicing factor Rbfox1 and NMDA receptor-dependent microRNA miR-219 are only downregulated after retrieval, accompanied by an increase in protein levels of miR-219 target CAMKIIγ.We provide a thorough characterization of coding and non-coding gene expression during long-term memory formation. We demonstrate that unwanted variance dominates the signal in transcriptional studies of learning and memory and introduce the removal of unwanted variance through normalization as a necessary step for the analysis of genome-wide transcriptional studies in the context of brain and behavior. We show for the first time that histone variants are downregulated after memory acquisition, and splicing factors and microRNAs after memory retrieval. Our results provide mechanistic insights into the molecular basis of cognition by highlighting the differential involvement of epigenetic mechanisms, such as histone variants and post-transcriptional RNA regulation, after acquisition and retrieval of memory.

Dataset Information

Nanobody-mediated control of gene expression and epigenetic memory.

Publications

Nanobody-mediated control of gene expression and epigenetic memory.

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