(635e) Mapping the Relationship between Transcription Factor Concentration and Gene Expression Using Scanning Fluorescence Correlation Spectroscopy

Controlling gene expression – and hence, differentiation – through manipulating transcription factor concentrations is an important aspect in tissue engineering and regenerative medicine. However, the precise relationship between transcription factors and gene expression has not been quantified. Therefore, in this work, we measured the concentrations of multiple cooperative transcription factors over several cell cycles and correlated these concentrations to gene activation. As a model system, we used the earliest transcription factor events in the 1-3 h old Drosophila melanogaster (fruit fly) embryo. This model system has served as a paradigm for studying transcription factor/gene expression relationships.

During the earliest stages of embryogenesis, the zygotic genome in most metazoans, including the fruit fly, remains transcriptionally silent after fertilization, and early embryonic development is controlled by maternally contributed mRNAs and proteins. Transcriptional activation of the zygotic genome coordinated with the degradation of maternally provided mRNAs occurs hours after fertilization, at a period termed as maternal-to-zygotic transition. In Drosophila melanogaster, the pioneer factor Zelda (Zld) acts as a master regulator for the transition and transcriptional activation of the zygotic genome. It binds to the nucleosomal DNA and increases chromatin accessibility, thus facilitating the binding of other transcription factors (TFs) and early gene expression. The absence of Zld leads to embryonic lethality. Quantitative measurements of the biophysical parameters such as the nuclear concentration of Zld and the fraction bound to DNA as well as the change of these parameters over time are required for a better understanding of the system. Therefore, we performed Raster Image Correlation Spectroscopy (RICS), a type of scanning Fluorescence Correlation Spectroscopy, on images of live embryos between 1 and 3 h old carrying superfolder GFP-tagged Zld and a histone-RFP marker. We analyzed the statistics of the auto- and cross-correlation functions to quantify absolute concentrations and fraction of transcription factor bound to DNA as functions of time. Using the results from the quantitative measurements we were able to develop a model for the Zld-DNA interaction as well as a model for the interaction of Zld with other transcription factors, whose activities are known to be potentiated by Zld. The parameters such as dissociation constants as well as the cooperativity between Zld and the other transcription factors calculated from these models are expected to help us better elucidate the input/output map between transcription factors and gene expression.