The jump from unicellular to multicellular organisms is one of the major evolutionary events. Emerging evidence suggests this transition is mainly mediated by gene expression regulation rather than gene innovation. However, despite the importance of such an event, the regulatory elements driving this jump are surprisingly scarcely described. Without understanding the molecular mechanisms driving the regulation of developmental factors in our unicellular relatives, we cannot aspire to enlighten the regulatory processes behind the evolution of complex life. With this in mind, the main aim of the PROSPER project is to identify and describe regulatory elements driving life cycle transitions in unicellular relatives of Metazoans. To achieve this goal, I will increase my newly formed Croatian Evolutionary Regulatory Group (croERG), which specialises in regulatory and comparative genomics, producing and analysing experimental datasets by employing and developing state-of-the-art computational algorithms, aiming to understand the dynamic gene expression of proliferative stages and tissues in organisms across the tree of life. With the proposed research, we will expand the current knowledge with the following analyses: 1. Characterising cis-regulatory elements in selected protists. 2. Linking non-coding conservation to gene expression regulation. 3. Distinguishing evolutionary shared features from lineage-specific innovations of regulatory landscape using deep learning approach. The PROSPER project's result will not only identify the regulatory landscape and link it to evolutionary innovation but also set a scaffold for future advancement in this fundamental yet unexplored field.