CDK-regulated phase separation seeded by histone genes ensures precise growth and function of Histone Locus Bodies

Abstract

Many membrane-less organelles form through liquid-liquid phase separation, but how their size is controlled and whether size is linked to function remain poorly understood. The Histone Locus Body (HLB) is an evolutionarily conserved nuclear body that regulates the transcription and processing of histone mRNAs. Here, we show that Drosophila HLBs form through phase separation of the scaffold protein multi-sex combs (Mxc). The size of HLBs is controlled in a precise and dynamic manner that is dependent on the cell cycle and zygotic gene activation. Control of HLB growth is achieved by a mechanism integrating nascent mRNAs at the histone locus, which catalyzes phase separation, and the nuclear concentration of Mxc, which is controlled by the activity of cyclin-dependent kinases. Reduced Cdk2 activity results in smaller HLBs and the appearance of nascent, misprocessed histone mRNAs. Our experiments thus identify a mechanism linking nuclear body growth and size with gene expression.

DrosophilaHistone Locus Body assembly and function involves multiple interactions

“…Indeed, the dynamics of HLB assembly in single early Drosophila embryos display properties consistent with liquid-liquid phase transition seeded by HisC (Hur, 2019). Blocking transcription in the early embryo prevents normal HLB growth (Hur, 2019), and a defective H3-H4 promoter (with mutated TATA boxes) does not support HLB formation in the context of a single copy histone gene repeat in salivary glands (Salzler et al ., 2013). These data suggest that active transcription is essential for forming a complete HLB.…”

“…Drosophila HLB components also stochastically assemble smaller and more unstable foci in embryos lacking the RD histone locus (White et al ., 2007; Salzler et al ., 2013; Hur, 2019), suggesting that HLBs and the nucleolus form similar seeding events. Indeed, the dynamics of HLB assembly in single early Drosophila embryos display properties consistent with liquid-liquid phase transition seeded by HisC (Hur, 2019).…”

“…Drosophila HLB components also stochastically assemble smaller and more unstable foci in embryos lacking the RD histone locus (White et al ., 2007; Salzler et al ., 2013; Hur, 2019), suggesting that HLBs and the nucleolus form similar seeding events. Indeed, the dynamics of HLB assembly in single early Drosophila embryos display properties consistent with liquid-liquid phase transition seeded by HisC (Hur, 2019). Blocking transcription in the early embryo prevents normal HLB growth (Hur, 2019), and a defective H3-H4 promoter (with mutated TATA boxes) does not support HLB formation in the context of a single copy histone gene repeat in salivary glands (Salzler et al ., 2013).…”

“…In some instances, RNA is thought to help seed NB assembly, and NBs such as the nucleolus and ectopic paraspeckles can form at sites of specific transcription (Matera et al ., 2009; Mao et al ., 2011; Falahati et al ., 2016). Blocking transcription prevents complete HLB assembly in both zebrafish and flies (Salzler et al ., 2013; Heyn et al ., 2017; Hur, 2019). However, the HLB is present at RD histone genes even in G1 when the genes are not active, raising the possibility that histone genes themselves participate in seeding HLB assembly (Zhao et al ., 1998; Liu et al ., 2006).…”

A region of Drosophila SLBP distinct from the histone pre-mRNA binding and processing domains is essential for deposition of histone mRNA in the oocyte

“…The samples were then gradually re-hydrated into PBST. The samples were then washed for 10 minutes in Wash Buffer The probes used were obtained from Stellaris and have been described previously (Hur, 2019).…”