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.

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.

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While feedback loops are essential for robustness in signaling systems, they make 9 discerning the role of individual components challenging. Here we introduce temperature as a 10 powerful perturbation method for uncoupling enzymatic processes, by exposing the differential 11 sensitivity of limiting reactions to temperature due to their activation energies. Using this method, 12 we study the sensitivity to temperature of different cell cycle events of early fly embryos. While the 13 subdivision of cell cycle steps is conserved across a wide range of temperatures (5-35 • C), the 14 transition into prometaphase exhibits the most sensitivity, arguing that it has a different 15 mechanism of regulation. Using a biosensor, we quantify the activity of Cdk1 and show that the 16 activation of Cdk1 drives entry into prometaphase but is not required for earlier events. In fact, 17 chromosome condensation can still occur when Cdk1 is inhibited pharmacologically. These results 18 demonstrate that different kinases are rate-limiting for different steps of mitosis.