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The ATP-dependent nucleosome remodeller Mi-2/CHD4 broadly modulates epigenetic 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 previously undefined 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 remodeller 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/CHD4mediated establishment of repressive chromatin structures.

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., Author summary In multicellular organisms common progenitors differentiate into various kinds of specialised cells. During differentiation metabolic profiles and proliferation potentials are progressively adjusted and cell type-specific traits are established by the coordinated activation and inactivation of genes. Here we study U-shaped (Ush), a conserved gene regulator that acts during macrophage differentiation in Drosophila melanogaster. We uncover that Ush coordinates the activation and inactivation of three differentiation-related gene groups, thereby modulating lipid metabolism, promoting cell division and maintaining a progenitor state. These functions are conferred by different Ush protein isoforms and their associated co-factors. One such co-factor, the nucleosome remodeling and deacetylation complex dNuRD, contributes to progenitor state maintenance but is not required for other Ush-regulated processes. This exemplifies how a single gene regulator can simultaneously influence different aspects of cellular differentiation by employing protein isoforms and isoform-specific co-regulator interactions.

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.

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.

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.