An optogenetics approach to decipher mammalian promoter decoding of transcription factor dynamics

Several transcription factors (TFs), such as the tumour suppressor p53, the immune response regulator NF-B, the yeast stress response regulator Msn2 and others, exhibit different nuclear accumulation patterns (dynamics) depending on the upstream activating stimulus. TF dynamics thus encode information about the type and intensity of the stimulus perceived by a eukaryotic cell. TF dynamics are believed to govern cell fate because they lead to the activation of distinct sets of target genes. Studies on how information about either internal or external stimuli is transmitted through signalling pathways into specific cell fates have shown that promoters of target genes play a critical role in decoding the information encoded in TF dynamics. Earlier studies suggested that the binding affinities of the TF for the promoters of the different target genes may orchestrate the observed differential gene expression under different TF dynamics. It was later shown that nucleosome positioning and, as a consequence, promoter accessibility determines how rapidly a promotergets activated, thus making it more or less sensitive to different TF dynamics. The distance between the core promoter and the TF binding sites as well as the core promoter itself have also been demonstrated to affect the expression of different target genes. Other studies on p53 and NF-Bmeasuring transcript levels of various target genes have revealed differences in the stability of transcripts belonging to early and late response genes. Some p53 target genes show oscillatory transcript levels in response to p53 pulses. In such studies, an external stimulus such as a cytokine, radiation or a chemical agent whose effects may not be fully understood were used to impose different TF dynamics. The lack of full clarity on the effects of the agents used to induce the TF dynamics may therefore undermine observations and the explanations given in such studies. Despite the progress made in understanding the role played by TF dynamics in gene expression regulation, it is still not clear which mammalian promoter elements contribute to decoding TF dynamics, and how they do so. In this study, I constructed a library of synthetic optogenetic circuits consisting of a library of synthetic light-responsive TFs and a library of promoters designed with well-studied elementsto investigate the relationship between TF dynamics and promoter activation in mammalian cells. Such a synthetic biology approach allows us to minimize the complexity, which is inevitable when studying endogenous pathways.I observed that there is a threshold for the time the TF must remain bound to the cognate responsive elements (REs) at the promoter (TF dwelling time) for transcription to be successfully initiated. The TF dwelling time is set by the affinity of the TATA binding protein (TBP) for the TATA-box (TB). A high-affinity TATA-box consents efficient assembly of the transcription pre-initiation complex (PIC), which reduces the TF dwelling time required for transcription initiation. The affinity of the TF for the REs defines the TF concentration (amplitude) threshold necessary to achieve the required TF dwelling time. Consequently, promoters with low-affinity REs and TATA-box filter out low-frequency pulsatile signals, but are activated by sustained TF signals. Additionally, reducing DNA looping efficiency by increasing the distance between the REs and the TATA-box, turns an otherwise TF dynamics-insensitive promoter into a promoter that can distinguish TF dynamics. I also show that the efficiency of translation initiation is critical for differential expression of target genes in response to different TF dynamics observed at the protein level. viiiFinally, I investigated a different type of synthetic TF bearing only the DNA binding domain (DBD)which interacts with a light-responsive co-regulator that bears thetransactivation domain(TAD). This scenario resembles several natural TFs, such as the TEAD/YAP pair. I found that this system is very sensitive to the interaction strength between the DBD-and TAD-bearing proteins. Furthermore, a high concentration of nuclear DBD-bearing TF impedes gene expression due to the competition for the REs between its free and TAD-bearing protein-bound fractions. These observations will help to further understand gene expression regulation by dynamics and how TEAD concentration in mammalian cells can be targeted in cancers where TEAD/YAP is dysregulate