Membrane-less organelles (MLOs) are liquid-like subcellular compartments that form through phase separation of proteins and RNA. While their biophysical properties are increasingly understood, their regulation and the consequences of perturbed MLO states for cell physiology are less clear. To study the regulatory networks, we targeted 1,354 human genes and screened for morphological changes of nucleoli, Cajal bodies, splicing speckles, PML nuclear bodies (PML-NBs), cytoplasmic processing bodies, and stress granules. By multivariate analysis of MLO features we identified hundreds of genes that control MLO homeostasis. We discovered regulatory crosstalk between MLOs, and mapped hierarchical interactions between aberrant MLO states and cellular properties. We provide evidence that perturbation of pre-mRNA splicing results in stress granule formation and reveal that PML-NB abundance influences DNA replication rates and that PML-NBs are in turn controlled by HIP kinases. Together, our comprehensive dataset is an unprecedented resource for deciphering the regulation and biological functions of MLOs.