Biology subjects

Hirsekorn, A.

Publications and source records attributed to Hirsekorn, A..

9 recordsLinked to original sources

bioRxiv2024

Cell-type specific prediction of RNA stability from RNA-protein interactions

RNA-binding proteins (RBPs) are important contributors to post-transcriptional regulatory processes. The combinatorial action of expressed RBPs and non-coding factors bound to the same transcript determines post-transcriptional properties of the mRNA in a context-dependent manner. To gain a better understanding of RNA stability and translational activity across different conditions, we have compiled and analyzed a set of ribosome profiling datasets for four human cell lines and used existing and newly generated metabolic labeling data to determine matching RNA degradation rates. We then used machine learning methods to predict RNA degradation rate and translation level from RBP binding information, which comprised existing in vivo binding datasets and computationally predicted binding sites. Utilizing this new RNA stability resource, we predicted RNA degradation rate and translation level from RBP binding alone. In vivo binding sites had higher importance for prediction compared to computationally predicted binding sites, likely due to confounding effects. We further explored the feature importance of different RBPs for stability prediction in the context of differential stability conferred by 3UTR isoforms. Taken together, an RNA stability machine learning model trained on one context successfully generalizes but is impacted by the availability and reliability of current data.

bioinformatics↗

bioRxiv2024

Sensitive dissection of a genomic regulatory landscape using bulk and targeted single-cell activation

Transcriptional enhancers are non-coding DNA elements that regulate gene transcription in a temporal and tissue-specific manner. Despite advances in computational and experimental methods, identifying enhancers and their target genes essential for specific biological processes remains challenging. Determining target genes for enhancers is also complex and often relies on indirect, low-resolution, and/or assumptive methodologies. To identify and functionally perturb enhancers at their endogenous sites without altering their sequence, we performed a pooled tiling CRISPR activation (CRISPRa) screen surrounding PHOX2B, a master regulator of neuronal cell fate and a key player in neuroblastoma development. This screen allowed the de novo identification of CRISPRa responsive elements (CaREs) that alter cellular growth within the 2 Mb genomic region. To determine CaRE target genes, we developed TESLA-seq (TargEted SingLe cell Activation), which combines CRISPRa screening with targeted single-cell RNA-sequencing and enables the parallel readout of the effect of hundreds of enhancers on all genes in the locus. While most TESLA-revealed CaRE-gene relationships involved neuroblastoma-related regulatory elements already active in the system, we found many CaREs and target connections normally active only in other tissue types or with no previous evidence and induced out of context by CRISPRa. This highlights the power of TESLA-seq to reveal gene regulatory networks, including edges active outside of a given experimental system. HighlightsO_LISystematically perturbed regulatory landscape in a 2 Mb genomic region surrounding PHOX2B to identify hundreds of CRISPRa-responsive elements that affect cellular growth C_LIO_LIDeveloped TESLA-seq as a principled molecular approach to find gene targets of dozens of candidate regulatory elements C_LIO_LIValidated interactions between identified regulatory elements and target genes and characterized their genomic features C_LIO_LIIntegrated a compendium of epigenomic datasets to identify regulatory relationships induced out of context or with no previous evidence C_LI

genomics↗

bioRxiv2021

RNA-binding proteins regulate aldosterone homeostasis in human steroidogenic cells

Angiotensin II (AngII) binds to the type I angiotensin receptor in the adrenal cortex to initiate a cascade of events leading to the production of aldosterone, a master regulator of blood pressure. Despite extensive characterization of the transcriptional and enzymatic control of adrenocortical steroidogenesis, there are still major gaps in our knowledge related to precise regulation of AII-induced gene expression kinetics. Specifically, we do not know the regulatory contribution of RNA-binding proteins (RBPs) and RNA decay, which can control the timing of stimulus-induced gene expression. To investigate this question, we performed a high-resolution RNA-seq time course of the AngII stimulation response and 4-thiouridine pulse labeling in a steroidogenic human cell line (H295R). We identified twelve temporally distinct gene expression responses that contained mRNA encoding proteins known to be important for various steps of aldosterone production, such as cAMP signaling components and steroidogenic enzymes. AngII response kinetics for many of these mRNAs revealed a coordinated increase in both synthesis and decay. These findings were validated in primary human adrenocortical cells stimulated ex vivo with AngII. Using a candidate siRNA screen, we identified a subset of RNA-binding protein and RNA decay factors that activate or repress AngII-stimulated aldosterone production. Among the repressors of aldosterone were BTG2, which promotes deadenylation and global RNA decay. BTG2 was induced in response to AngII stimulation and promoted the repression of mRNAs encoding pro-steroidogenic factors indicating the existence of an incoherent feedforward loop controlling aldosterone homeostasis. Together, these data support a model in which coordinated increases in transcription and regulated RNA decay facilitates the major transcriptomic changes required to implement a pro-steroidogenic gene expression program that is temporally restricted to prevent aldosterone overproduction.

genomics↗

bioRxiv2020

Single-cell-resolved dynamics of chromatin architecture delineate cell and regulatory states in wildtype and cloche/npas4l mutant zebrafish embryos

DNA accessibility of cis regulatory elements (CREs) dictates transcriptional activity and drives cell differentiation during development. While many of the genes that regulate embryonic development have been described, the underlying CRE dynamics controlling their expression remain largely unknown. To address this, we applied single-cell combinatorial indexing ATAC-seq (sci-ATAC-seq) to whole 24 hours post fertilization (hpf) stage zebrafish embryos and developed a new computational tool, ScregSeg, that selects informative genome segments and classifies complex accessibility dynamics. We integrated the ScregSeg output with bulk measurements for histone post-translational modifications and 3D genome organization, expanding knowledge of regulatory principles between chromatin modalities. Sci-ATAC-seq profiling of npas4l/cloche mutant embryos revealed novel cellular roles for this hemato-vascular transcriptional master regulator and suggests an intricate mechanism regulating its expression. Our work constitutes a valuable resource for future studies in developmental, molecular, and computational biology.

genomics↗

bioRxiv2019

Integrated Proteogenomic Deep Sequencing and Analytics Accurately Identify Non-Canonical Peptides in Tumor Immunopeptidomes

Efforts to precisely identify tumor human leukocyte antigen (HLA) bound peptides capable of mediating T cell-based tumor rejection still face important challenges. Recent studies suggest that non-canonical tumor-specific HLA peptides that derive from annotated non-coding regions could elicit anti-tumor immune responses. However, sensitive and accurate mass-spectrometry (MS)-based proteogenomics approaches are required to robustly identify these non-canonical peptides. We present an MS-based analytical approach that characterizes the non-canonical tumor HLA peptide repertoire, by incorporating whole exome sequencing, bulk and single cell transcriptomics, ribosome profiling, and a combination of two MS/MS search tools. This approach results in the accurate identification of hundreds of shared and tumor-specific non-canonical HLA peptides and of an immunogenic peptide from a downstream reading frame in the melanoma stem cell marker gene ABCB5. It holds great promise for the discovery of novel cancer antigens for cancer immunotherapy.

cancer biology↗

bioRxiv2019

SaTAnn quantifies translation on the functionally heterogeneous transcriptome

At the center of the gene expression cascade, translation is fundamental in defining the fate of much of the transcribed genome. RNA sequencing enables the quantification of complex transcript mixtures, often detecting several splice isoforms of unknown functions for one gene. We have developed ORFquant, a new approach to annotate and quantify translation at the single open reading frame (ORF) level, using information from Ribo-seq data. Relying on a novel approach for transcript filtering, we quantify translation on thousands of ORFs, showing the power of Ribo-seq in revealing alternative ORFs on multiple isoforms per gene. While we find that one ORF represents the dominant translation product for most genes, we also detect genes with translated ORFs on multiple transcript isoforms, including targets of RNA surveillance mechanisms. Assessing the translation output across human cell lines reveals the extent of gene-specific differences in protein production, which are supported by steady-state protein abundance estimates. Computational analysis of Ribo-seq data with ORFquant (available at https://github.com/lcalviell/ORFquant) provides a window into the heterogeneous functions of complex transcriptomes.

genomics↗

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↗