MODULATING TRANSCRIPTIONAL NOISE THROUGH GENETIC MODIFICATION OF EUKARYOTIC PROMOTERS

IUNGMAN MARTÍN; SCHOR IGNACIO ESTEBAN

Salta

Congreso; Reunión Conjunta por el XIV Congreso de la Pan-American Association for Biochemistry and Molecular Biology y LV Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular; 2019

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

The promoter regions of eukaryotic genes control both the average expression levels and their cell-to-cell variability, a quality known as intrinsictranscriptional noise. We are interested in how the transcriptional noise affects other cellular processes, such as the coupling between transcription and RNA processing or the robustness of regulatory networks directing cellular processes like differentiation. Here we present the development of tools attempting to modulate transcriptional noise independently from transcript levels, the first step for our long-term aim. To measure intrinsic transcriptional noise, we use a dual fluorescent reporter containing the genes sfGFP under the control of the test promoter and mCherry under the regulation of a constitutive mammalian promoter. This plasmid was obtained from TIPR-cherry, a similar vector developed forusage in Drosophila cells, by replacing the fly constitutive actin promoter with the hPGK human promoter from the Tet-ONE vector (Clontech).For this purpose, we amplified by PCR a fragment containing the hPGK promoter from Tet-ONE and cloned it using Gibson cloning into a HindIIIKpnI fragment from TIPR-cherry. This vector, which we termed hTIPR-cherry, has an inducible promoter controlling sfGFP expression, with the MMTV minimal promoter downstream seven Tet-responsive elements (TRE). At this time, we are testing the transcriptional properties of this initial reporter through analytic flow cytometry, using the single-cell sfGFP/mCherry fluorescence ratio as readout. We have also engineered different variants of this promoter region in order to uncouple variations in transcriptional noise from those in average transcriptional strength.The hypothesis is that these two parameters are controlled by the number and affinity of transcription factor binding sites, which are expected to increase both, and the presence of nucleosome positioning elements, which have been reported to increase strength but not noise. In the first place, we are obtaining promoters with a different number of TREs, which we will subsequently combine with single nucleotide substitutions on these elements to modify their affinity. In parallel, we have obtained two different nucleosome positioning elements: a 20bp-long dA:dT stretch, which has been used as a nucleosome exclusion sequence in yeast, and a nucleosome positioning region already tested in human IFN-beta gene. We are currently performing overlap extension PCR to amplify these two parts of the test regulatory region and combine them, and Gibson cloning to introduce these new variants onto hTIPR-cherry. We expect that these synthetic promoters allow us to scan different combinations of transcriptional noise vs. strength, becoming useful tools for testing the separate effects of these two parameters of transcription.