RNA inhibits dMi-2/CHD4 chromatin binding and nucleosome remodeling.

The ATP-dependent nucleosome remodeler Mi-2/CHD4 broadly modulates chromatin landscapes to repress transcription and to maintain genome integrity. Here we use individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) to show that Drosophila Mi-2 associates with thousands of mRNA molecules in vivo. Biochemical data reveal that recombinant dMi-2 preferentially binds to G-rich RNA molecules using two intrinsically disordered regions of unclear function. Pharmacological inhibition of transcription and RNase digestion approaches establish that RNA inhibits the association of dMi-2 with chromatin. We also show that RNA inhibits dMi-2-mediated nucleosome mobilization by competing with the nucleosome substrate. Importantly, this activity is shared by CHD4, the human homolog of dMi-2, strongly suggesting that RNA-mediated regulation of remodeler activity is an evolutionary conserved mechanism. Our data support a model in which RNA serves to protect actively transcribed regions of the genome from dMi-2/CHD4-mediated establishment of repressive chromatin structures.

Bistable Photoswitch Allows in Vivo Control of Hematopoiesis.

Optical control has enabled functional modulation in cell culture with unparalleled spatiotemporal resolution. However, current tools for in vivo manipulation are scarce. Here, we design and implement a genuine on-off optochemical probe capable of achieving hematopoietic control in zebrafish. Our photopharmacological approach first developed conformationally strained visible light photoswitches (CS-VIPs) as inhibitors of the histone methyltransferase MLL1 (KMT2A). In blood homeostasis MLL1 plays a crucial yet controversial role. CS-VIP 8 optimally fulfils the requirements of a true bistable functional system in vivo under visible-light irradiation, and with unprecedented stability. These properties are exemplified via hematopoiesis photoinhibition with a single isomer in zebrafish. The present interdisciplinary study uncovers the mechanism of action of CS-VIPs. Upon WDR5 binding, CS-VIP 8 causes MLL1 release with concomitant allosteric rearrangements in the WDR5/RbBP5 interface. Since our tool provides on-demand reversible control without genetic intervention or continuous irradiation, it will foster hematopathology and epigenetic investigations. Furthermore, our workflow will enable exquisite photocontrol over other targets inhibited by macrocycles.

Ush regulates hemocyte-specific gene expression, fatty acid metabolism and cell cycle progression and cooperates with dNuRD to orchestrate hematopoiesis.

The generation of lineage-specific gene expression programmes that alter proliferation capacity, metabolic profile and cell type-specific functions during differentiation from multipotent stem cells to specialised cell types is crucial for development. During differentiation gene expression programmes are dynamically modulated by a complex interplay between sequence-specific transcription factors, associated cofactors and epigenetic regulators. Here, we study U-shaped (Ush), a multi-zinc finger protein that maintains the multipotency of stem cell-like hemocyte progenitors during Drosophila hematopoiesis. Using genomewide approaches we reveal that Ush binds to promoters and enhancers and that it controls the expression of three gene classes that encode proteins relevant to stem cell-like functions and differentiation: cell cycle regulators, key metabolic enzymes and proteins conferring specific functions of differentiated hemocytes. We employ complementary biochemical approaches to characterise the molecular mechanisms of Ush-mediated gene regulation. We uncover distinct Ush isoforms one of which binds the Nucleosome Remodeling and Deacetylation (NuRD) complex using an evolutionary conserved peptide motif. Remarkably, the Ush/NuRD complex specifically contributes to the repression of lineage-specific genes but does not impact the expression of cell cycle regulators or metabolic genes. This reveals a mechanism that enables specific and concerted modulation of functionally related portions of a wider gene expression programme. Finally, we use genetic assays to demonstrate that Ush and NuRD regulate enhancer activity during hemocyte differentiation in vivo and that both cooperate to suppress the differentiation of lamellocytes, a highly specialised blood cell type. Our findings reveal that Ush coordinates proliferation, metabolism and cell type-specific activities by isoform-specific cooperation with an epigenetic regulator.

Photoswitchable peptides for spatiotemporal control of biological functions.

Light is unsurpassed in its ability to modulate biological interactions. Since their discovery, chemists have been fascinated by photosensitive molecules capable of switching between isomeric forms, known as photoswitches. Photoswitchable peptides have been recognized for many years; however, their functional implementation in biological systems has only recently been achieved. Peptides are now acknowledged as excellent protein-protein interaction modulators and have been important in the emergence of photopharmacology. In this review, we briefly explain the different classes of photoswitches and summarize structural studies when they are incorporated into peptides. Importantly, we provide a detailed overview of the rapidly increasing number of examples, where biological modulation is driven by the structural changes. Furthermore, we discuss some of the remaining challenges faced in this field. These exciting proof-of-principle studies highlight the tremendous potential of photocontrollable peptides as optochemical tools for chemical biology and biomedicine.

Modulating Protein-Protein Interactions with Visible-Light-Responsive Peptide Backbone Photoswitches.

Life relies on a myriad of carefully orchestrated processes, in which proteins and their direct interplay ultimately determine cellular function and disease. Modulation of this complex crosstalk has recently attracted attention, even as a novel therapeutic strategy. Herein, we describe the synthesis and characterization of two visible-light-responsive peptide backbone photoswitches based on azobenzene derivatives, to exert optical control over protein-protein interactions (PPI). The novel peptidomimetics undergo fast and reversible isomerization with low photochemical fatigue under alternatively blue-/green-light irradiation cycles. Both bind in the nanomolar range to the protein of interest. Importantly, the best peptidomimetic displays a clear difference between isomers in its protein-binding capacity and, in turn, in its potential to inhibit enzymatic activity through PPI disruption. In addition, crystal structure determination, docking and molecular dynamics calculations allow a molecular interpretation and open up new avenues in the design and synthesis of future photoswitchable PPI modulators.

Controlled inhibition of methyltransferases using photoswitchable peptidomimetics: towards an epigenetic regulation of leukemia.

We describe a cell-permeable photoswitchable probe capable of modulating epigenetic cellular states by disruption of an essential protein-protein interaction within the MLL1 methyltransferase core complex. Our azobenzene-containing peptides selectively block the WDR5-MLL1 interaction by binding to WDR5 with high affinity (Ki = 1.25 nM). We determined the co-crystal structure of this photoswitchable peptiomimetic with WDR5 to understand the interaction at the atomic level. Importantly, the photoswitchable trans and cis conformers of the probe display a clear difference in their inhibition of MLL1. We further demonstrate that the designed photo-controllable azo-peptidomimetics affect the transcription of the MLL1-target gene Deptor, which regulates hematopoiesis and leukemogenesis, and inhibit the growth of leukemia cells. This strategy demonstrates the potential of photopharmacological inhibition of methyltransferase protein-protein interactions as a novel method for external epigenetic control, providing a new toolbox for controlling epigenetic states.