Biology subjects

Hirsekorn, A.

Publications and source records attributed to Hirsekorn, A..

3 recordsLinked to original sources

bioRxiv2018

Global identification of functional microRNA::mRNA interactions in Drosophila

MicroRNAs (miRNAs) are key mediators of post-transcriptional gene expression silencing. Although Drosophila has been of critical importance for miRNA discovery, biogenesis and function, there has been no comprehensive experimental annotation of functional miRNA target sites. To close this gap, we generated the first in vivo map of miRNA::mRNA interactions in Drosophila melanogaster, making use of crosslinked nucleotides in Argonaute (AGO) crosslinking and immunoprecipitation (CLIP) experiments that enable an unambiguous assignment of miRNAs to AGO binding sites at much higher signal-to-noise ratio than computational predictions alone.\n\nAbsolute quantification of cellular miRNA levels showed the miRNA pool in Drosophila cell lines to be more diverse than previously reported. Benchmarking two different CLIP approaches, we identified a similar predictive potential to unambiguously assign thousands of miRNA::mRNA pairs from AGO1 interaction data at unprecedented depth. Quantitative RNA-Seq and subcodon-resolution ribosomal footprinting data upon AGO1 depletion enabled the determination of miRNA-mediated effects on target expression and translation. We thus provide the first comprehensive resource of miRNA target sites as well as their quantitative functional impact in Drosophila.

systems biology↗

bioRxiv2018

Reproducible inference of transcription factor footprints in ATAC-seq and DNase-seq datasets via protocol-specific bias modeling

DNase-seq and ATAC-seq are broadly used methods to assay open chromatin regions genome-wide. The single nucleotide resolution of DNase-seq has been further exploited to infer transcription factor binding sites (TFBS) in regulatory regions via footprinting. Recent studies have demonstrated the sequence bias of DNase I and its adverse effects on footprinting efficiency. However, footprinting and the impact of sequence bias have not been extensively studied for ATAC-seq. Here, we undertake a systematic comparison of the two methods and show that a modification to the ATAC-seq protocol increases its yield and its agreement with DNase-seq data from the same cell line. We demonstrate that the two methods have distinct sequence biases and correct for these protocol-specific biases when performing footprinting. Despite differences in footprint shapes, the locations of the inferred footprints in ATAC-seq and DNase-seq are largely concordant. However, the protocol-specific sequence biases in conjunction with the sequence content of TFBSs impacts the discrimination of footprint from background, which leads to one method outperforming the other for some TFs. Finally, we address the depth required for reproducible identification of open chromatin regions and TF footprints.

genomics↗

bioRxiv2017

Determinants of Transcription Initiation Directionality in Metazoans

Divergent transcription from promoters and enhancers is pervasive in many species, but it remains unclear if it is a general and passive feature of all eukaryotic cis regulatory elements. To address this, we define promoters and enhancers in C. elegans, D. melanogaster and H. sapiens using ATAC-Seq and investigate the determinants of their transcription initiation directionalities by analyzing genome-wide nascent, cap-selected, polymerase run-on assays. All three species initiate divergent transcription from separate core promoter sequences. Sequence asymmetry downstream of forward and reverse initiation sites, known to be important for termination and stability in H. sapiens, is unique in each species. Chromatin states of divergent promoters are not entirely conserved, but in all three species, the levels of histone modifications on the +1 nucleosome are independent from those on the -1 nucleosome, arguing for independent initiation events. This is supported by an integrative model of H3K4me3 levels and core promoter sequence that is highly predictive of promoter directionality and of two types of promoters: those with balanced initiation directionality and those with skewed directionality. Lastly, D. melanogaster enhancers display variation in chromatin architecture depending on enhancer location, and D. melanogaster promoter regions with dual enhancer/promoter potential are enriched for divergent transcription. Our results point to a high degree of variation in regulatory element transcription initiation directionality within and between metazoans, and to non-passive regulatory mechanisms of transcription initiation directionality in those species.

molecular biology↗