Biology subjects

Gordan, R.

Publications and source records attributed to Gordan, R..

3 recordsLinked to original sources

bioRxiv2018

DNA sequence context controls the binding and processivity of T7 DNA primase

Primases are key enzymes involved in DNA replication. They act on single-stranded DNA, and catalyze the synthesis of short RNA primers used by DNA polymerases. Here, we investigate the DNA-binding and activity of the bacteriophage T7 primase using a new workflow called High-Throughput Primase Profiling (HTPP). Using a unique combination of high-throughput binding assays and biochemical analyses, HTPP reveals a complex landscape of binding specificity and functional activity for the T7 primase, determined by sequences flanking the primase recognition site. We identified specific features, such as G/T-rich flanks, which increase primase-DNA binding up to 10-fold and, surprisingly, also increase the length of newly formed RNA (up to 3-fold). To our knowledge, variability in primer length has not been reported for this primase. We expect that applying HTPP to additional enzymes will reveal new insights into the effects of DNA sequence composition on the DNA recognition and functional activity of primases.

biophysics↗

bioRxiv2017

Intrinsic specificity differences between transcription factor paralogs partly explain their differential in vivo binding

Members of transcription factor (TF) families, i.e. paralogous TFs, are oftentimes reported to have identical DNA-binding motifs, despite the fact that they perform distinct regulatory functions in the cell. Differential genomic targeting by paralogous TFs is generally assumed to be due to interactions with protein cofactors or the chromatin environment. Contrary to previous assumptions, we find that paralogous TFs have different intrinsic preferences for DNA, not captured by current motif models, and these differences partly explain differential genomic binding and functional specificity. Our finding was possible due to a unique combination of carefully designed high-throughput assays and rigorous computation modeling, integrated into a unified framework called iMADS. We used iMADS to quantify, model, and analyze specificity differences between 11 paralogous TFs from 4 distinct human TF families. Our finding of differential specificity between closely related TFs has important implications for the interpretation of the regulatory effects of non-coding genetic variants.

genomics↗

bioRxiv2017

Incomplete MyoD-induced transdifferentiation is associated with chromatin remodeling deficiencies

Our current understanding of cellular transdifferentiation systems is limited. It is oftentimes unknown, at a genome-wide scale, how much transdifferentiated cells differ quantitatively from both the starting cells and the target cells. Focusing on transdifferentiation of primary human skin fibroblasts by forced expression of myogenic transcription factor MyoD, we performed quantitative analyses of gene expression and chromatin accessibility profiles of transdifferentiated cells compared to fibroblasts and myoblasts. In this system, we find that while many of the early muscle marker genes are reprogrammed, global gene expression and accessibility changes are still incomplete when compared to myoblasts. In addition, we find evidence of epigenetic memory in the transdifferentiated cells, with reminiscent features of fibroblasts being visible both in chromatin accessibility and gene expression. Quantitative analyses revealed a continuum of changes in chromatin accessibility induced by MyoD, and a strong correlation between chromatin-remodeling deficiencies and incomplete gene expression reprogramming. Classification analyses identified genetic and epigenetic features that distinguish reprogrammed from non-reprogrammed sites, and suggested ways to potentially improve transdifferentiation efficiency. Our approach for combining gene expression, DNA accessibility, and protein-DNA binding data to quantify and characterize the efficiency of cellular transdifferentiation on a genome-wide scale can be applied to any transdifferentiation system.

genomics↗