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

Abstract

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.

Genomics analysis ofDrosophila sechelliaresponse toMorinda citrifoliafruit diet

“…All 5 members of the GATA family of transcription factors are predicted to regulate the expression of DEGs in noni and L-DOPA treatment, along with srp being predicted to also regulate DEG expression in HA treatment, but only GATAd is significantly upregulated in L-DOPA treatment. U-shaped, the Friend of GATA protein that binds GATA family of transcription factors that has been previously shown to be involved in fatty acid metabolism (Lenz et al 2021) is significantly upregulated in L-DOPA treatment. Expression of GATA factors may be responding to noni and L-DOPA treatment in order to help metabolize volatile fatty acids found in noni fruit that are concurrently experienced by D. sechellia in their natural environment when feeding on noni fruit.…”

RNA inhibits dMi-2/CHD4 chromatin binding and nucleosome remodelling

“…iCLIP2: Individual nucleotide-resolution UV-crosslinking and immunoprecipitation 2 was performed as described in (Buchbender et al, 2020). Briefly, S2 cells expressing GFP-tagged dMi-2 (Lenz et al, 2021) and untagged S2 cells were washed with cold 1× PBS. Protein-RNA cross-linking was performed at 254 nm UV light (300 mJ/cm 2 ) using a Stratagene Stratalinker.…”

“…We asked if the propensity of dMi-2 to crosslink to a given mRNA was a function of the expression level of that mRNA. We plotted the number of iCLIP2 crosslinks to a given mRNA against the expression level of that mRNA as determined by RNA-seq (Lenz et al, 2021) (Figure 1C, see Methods for details). This demonstrated that there was no strict linear correlation between RNA expression level and the number of dMi-2 iCLIP2 crosslinks.…”

“…However, a metagene analysis of dMi-2 chromatin binding sites at these genes as determined by ChIP-seq (Lenz et al, 2021) highlights a clear preference for promoter sequences and the 5' region of genes (Figure 2A). In fact, dMi-2 chromatin occupancy is lowest towards the 3' end of genes.…”

“…DE-Seq analysis-We compared iCLIP crosslinking results and mRNA expression level, as determined by RNA-Seq (Lenz et al, 2021) to identify mRNAs that preferentially associate with dMi-2. RNA-seq reads were mapped in the same manner as iCLIP reads but without prior trimming.…”