Weinmann, R. | Search bioRxiv

bioRxiv2024

HP1 binding creates a local barrier against transcription activation and persists during chromatin decondensation

Mouse pericentric repeats form transcriptionally silent and compacted domains known as chromocenters. These prototypic heterochromatin compartments are marked by heterochromatin protein 1 (HP1). However, its contributions to chromocenter structure and function remain debated. We investigated the role of HP1 by recruiting the activators VP16, p65, and VPR to mouse fibroblast chromocenters and analyzed its silencing activity with a transcription reporter. Upon chromocenter decondensation and transcription activation, interactions of HP1 with chromatin and H3K9 trimethylation remained stable, suggesting stoichiometric binding rather than higher-order assembly. HP1-mediated repression required promoter-proximal binding and effectively suppressed VP16-triggered activation but not the stronger activation by VPR. These observations are explained by a 1D lattice binding model, which conceptualizes chromocenters as arrays of repeat units that can independently switch between silenced and activated states. Our findings provide a quantitative framework that explains how chromocenter organization responds to transcriptional activation while maintaining local heterochromatin features. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=189 HEIGHT=200 SRC="FIGDIR/small/627308v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@83816corg.highwire.dtl.DTLVardef@1a603c6org.highwire.dtl.DTLVardef@41d8b4org.highwire.dtl.DTLVardef@fa4d0d_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIHP1 represses transcription at mouse chromocenters and an ectopic reporter when competing with transcriptional activators VP16, p65 and VPR C_LIO_LIHP1 repression requires promoter-proximal binding and effectively counteracts the weak activator VP16 C_LIO_LIThe strong activator VPR overcomes HP1-mediated repression while HP1 remains bound to chromatin C_LIO_LIHP1 binding and H3K9me3 persist during chromocenter decondensation and transcriptional activation C_LIO_LIA 1D lattice binding model explains how independent repeat units transition between silenced and activated states without requiring phase separation C_LI

cell biology↗

bioRxiv2021

PQN-59 antagonizes microRNA-mediated repression and functions in stress granule formation during C. elegans development

microRNAs (miRNAs) are potent regulators of gene expression that function in a variety of developmental and physiological processes by dampening the expression of their target genes at a post-transcriptional level. In many gene regulatory networks (GRNs), miRNAs function in a switch-like manner whereby their expression and activity elicit a transition from one stable pattern of gene expression to a distinct, equally stable pattern required to define a nascent cell fate. While the importance of miRNAs that function in this capacity are clear, we have less of an understanding of the cellular factors and mechanisms that ensure the robustness of this form of regulatory bistability. In a screen to identify suppressors of temporal patterning phenotypes that result from ineffective miRNA-mediated target repression during C. elegans development, we identified pqn-59, an ortholog of human UBAP2L, as a novel factor that antagonizes the activities of multiple heterochronic miRNAs. Specifically, we find that depletion of pqn-59 can restore normal development in animals with reduced miRNA activity. Importantly, inactivation of pqn-59 is not sufficient to bypass the requirement of these regulatory RNAs within the heterochronic GRN. The pqn-59 gene encodes an abundant, cytoplasmically localized and unstructured protein that harbors three essential "prion-like" domains. These domains exhibit LLPS properties in vitro and normally function to limit PQN-59 diffusion in the cytoplasm in vivo. Like human UBAP2L, PQN-59s localization becomes highly dynamic during stress conditions where it re-distributes to cytoplasmic stress granules and is important for their formation. Proteomic analysis of PQN-59 complexes from embryonic extracts indicates that PQN-59 and human UBAP2L interact with orthologous cellular components involved in RNA metabolism and promoting protein translation and that PQN-59 additionally interacts with proteins involved in transcription and intracellular transport. Finally, we demonstrate that pqn-59 depletion results in the stabilization of several mature miRNAs (including those involved in temporal patterning) without altering steady-state pre-miRNAs levels indicating that PQN-59 may ensure the bistability of some GRNs that require miRNA functions by promoting miRNA turnover and, like UBAP2L, enhancing protein translation. AUTHOR SUMMARYBistability plays a central role in many gene regulatory networks (GRNs) that control developmental processes where distinct and mutually exclusive cell fates are generated in a defined order. While genetic analysis has identified a number of gene types that promote these transitions, we know little regarding the mechanisms and players that ensure these decisions are robust. and in many cases, irreversible. We leveraged the robust genetics and phenotypes associated with temporal patterning mutants of C. elegans to identify genes whose depletion would restore normal regulation in animals that express miRNA alleles that do not sufficiently down-regulate their targets. These efforts identified pqn-59, the C. elegans ortholog of the human UBAP2L gene. Like UBAP2L, PQN-59 likely forms a hub for a number of RNA/RNA-binding protein mediated processes in cells including translational activation and in the formation of stress granules in adverse environmental conditions. Finally, we also demonstrate that pqn-59 depletion stabilizes mature miRNA levels further connecting this new family of RNA-binding proteins to translation and miRNA-mediated gene regulation.

developmental biology↗