Biology subjects

Harden, T. T.

Publications and source records attributed to Harden, T. T..

3 recordsLinked to original sources

bioRxiv2021

Defining kinetic roles of transcriptional activators in the early Drosophila embryo

Most animal transcription factors are categorized as activators or repressors without specifying their mechanisms of action. Defining their specific roles is critical for deciphering the logic of transcriptional regulation and predicting the function of regulatory sequences. Here, we define the kinetic roles of three activating transcription factors in the Drosophila embryo--Zelda, Bicoid and Stat92E--by introducing their binding sites into the even skipped stripe 2 enhancer and measuring transcriptional output with live imaging. We find that these transcription factors act on different subsets of kinetic parameters, and these subsets can change over the course of nuclear cycle (NC) 14. These transcription factors all increase the fraction of active nuclei. Zelda dramatically shortens the time interval between the start of NC 14 and initial activation, and Stat92E increases the duration of active transcription intervals throughout NC 14. Zelda also decreases the time intervals between instances of active transcription early in NC 14, while Stat92E does so later. Different transcription factors therefore play distinct kinetic roles in activating transcription; this has consequences for understanding both regulatory DNA sequences as well as the biochemical function of transcription factors.

systems biology↗

bioRxiv2019

Alternative transcription cycle for bacterial RNA polymerase

RNA polymerases (RNAPs) transcribe genes through a cycle of recruitment to promoter DNA, initiation, elongation, and termination. After termination, RNAP is thought to initiate the next round of transcription by detaching from DNA and rebinding a new promoter. We used single-molecule fluorescence microscopy to observe individual RNAP molecules after transcript release at a terminator. Following termination, RNAP almost always remained bound to DNA and sometimes exhibited one-dimensional sliding over thousands of basepairs. Unexpectedly, the DNA-bound RNAP often restarted transcription, usually in reverse direction, thus producing an antisense transcript. Furthermore, we report evidence of this \"secondary initiation\" in live cells, using genome-wide RNA sequencing. These findings reveal an alternative transcription cycle that allows RNAP to reinitiate without dissociating from DNA, which is likely to have important implications for gene regulation.

biophysics↗

bioRxiv2018

Computations performed by shadow enhancers and enhancer duplications vary across the Drosophila embryo

Transcription of developmental genes is controlled by multiple enhancers. Frequently, more than one enhancer can activate transcription from the same promoter in the same cells. In these cases, how is regulatory information from multiple enhancers combined to determine the overall expression output of their shared promoter? To investigate this question, we quantified nascent transcription driven by a pair shadow enhancers, each individual of the pair, and their duplications in Drosophila embryos using live imaging. This set of constructs allows us to quantify the \"computation\" made by the pairs of enhancers: their combined output expression as a function of the expression that they drive separately. We show that the computation performed by these shadow enhancers and duplications varies across the expression pattern, implying that how their activities are combined depends on the transcriptional regulators bound to the enhancers in different parts of the embryo. Characterizing the computation made by multiple enhancers is a critical first step in developing conceptual and computational models of gene expression at the locus level, where multiple enhancers collaborate.

systems biology↗