Biology subjects

Hoepfner, D.

Publications and source records attributed to Hoepfner, D..

6 recordsLinked to original sources

bioRxiv2024

The transcription factor ZNF469 regulates collagen production in liver fibrosis

Non-alcoholic fatty liver disease (NAFLD) - characterized by excess accumulation of fat in the liver - now affects one third of the worlds population. As NAFLD progresses, extracellular matrix components including collagen accumulate in the liver causing tissue fibrosis, a major determinant of disease severity and mortality. To identify transcriptional regulators of fibrosis, we computationally inferred the activity of transcription factors (TFs) relevant to fibrosis by profiling the matched transcriptomes and epigenomes of 108 human liver biopsies from a deeply-characterized cohort of patients spanning the full histopathologic spectrum of NAFLD. CRISPR-based genetic knockout of the top 100 TFs identified ZNF469 as a regulator of collagen expression in primary human hepatic stellate cells (HSCs). Gain- and loss-of-function studies established that ZNF469 regulates collagen genes and genes involved in matrix homeostasis through direct binding to gene bodies and regulatory elements. By integrating multiomic large-scale profiling of human biopsies with extensive experimental validation we demonstrate that ZNF469 is a transcriptional regulator of collagen in HSCs. Overall, these data nominate ZNF469 as a previously unrecognized determinant of NAFLD-associated liver fibrosis.

molecular biology↗

bioRxiv2023

Convergence of multiple RNA-silencing pathways on GW182/TNRC6

The RNA-binding protein TRIM71/LIN-41 is a phylogenetically conserved developmental regulator that functions in mammalian stem cell reprogramming, brain development and cancer. TRIM71 recognizes target mRNAs through hairpin motifs and silences them through molecular mechanisms that await identification. Here, we uncover that TRIM71 silences its targets by recruiting TNRC6/GW182, a core component of the miRNA-induced silencing complex (miRISC). Co-immunoprecipitation reveals interaction of TNRC6A with additional RNA-binding proteins and we demonstrate that AGO2, TRIM71, and UPF1 each recruit TNRC6 to specific, largely distinct sets of transcripts to silence them. As cellular TNRC6 levels are limiting, competition occurs among the silencing pathways, such that loss of AGO2 protein, or of AGO2 binding to TNRC6, enhances the activities of the other pathways. We conclude that a miRNA-like silencing activity is shared among different mRNA silencing pathways and that use of TNRC6 as a central hub provides a means to integrate their activities.

molecular biology↗

bioRxiv2022

A common mechanism of Sec61 translocon inhibition by small molecules

The Sec61 complex forms a protein-conducting channel in the endoplasmic reticulum (ER) membrane that is required for secretion of soluble proteins and production of many membrane proteins. Several natural and synthetic small molecules specifically inhibit the Sec61 channel, generating cellular effects that are potentially useful for therapeutic purposes, but their inhibitory mechanisms remain unclear. Here we present near-atomic-resolution structures of the human Sec61 channel inhibited by a comprehensive panel of structurally distinct small molecules-- cotransin, decatransin, apratoxin F, ipomoeassin F, mycolactone, cyclotriazadisulfonamide (CADA) and eeyarestatin I (ESI). Remarkably, all inhibitors bind to a common lipid-exposed pocket formed by the partially open lateral gate and plug domain of the channel. Mutations conferring resistance to the inhibitors are clustered at this binding pocket. The structures indicate that Sec61 inhibitors stabilize the plug domain of Sec61 in a closed state, thereby preventing the protein-translocation pore from opening. Our study reveals molecular interactions between Sec61 and its inhibitors in atomic detail and offers the structural framework for further pharmacological studies and drug design.

biochemistry↗

bioRxiv2022

Robust and Versatile Arrayed Libraries for Human Genome-Wide CRISPR Activation, Deletion and Silencing

Arrayed CRISPR libraries extend the scope of gene-perturbation screens but require large numbers of efficacious sgRNA-expressing vectors. Using a newly invented liquid-phase plasmid cloning methodology, we constructed genome-wide arrayed libraries for human gene ablation (19,936 plasmids), activation, and epigenetic silencing (22,442 plasmids). At least 76% of each plasmid preparation encoded an intact array of 4 non-overlapping sgRNAs designed to tolerate most human DNA polymorphisms. We achieved perturbation efficacies of 75-99%, 76-92% and up to 10,000x in deletion, silencing and activation experiments, respectively. Upon conversion into massively parallel lentiviral vectors, an arrayed activation screen of 1,634 human transcription factors yielded 11 novel regulators of the cellular prion protein PrPC. Furthermore, a screen using a pooled version of the ablation library identified 5 novel modifiers of autophagy that went undetected with either of two 1sgRNA libraries. The CRISPR libraries described here represent a powerful resource for the targeted perturbation of human protein-coding genes.

genomics↗

bioRxiv2021

Genome-wide screening in human kidney organoids identifies novel aspects of nephrogenesis

Human organoids allow studying proliferation, lineage specification, and three-dimensional tissue development. Due to the inherent multicellular complexity, interrogation by systematic genetic methodologies is challenging. Here, we present the first genome-wide CRISPR screen in iPSC-derived kidney organoids. The combination of genome editing, longitudinal sampling and sorting of specific cell populations enabled us to uncover novel biology from development to tubular morphogenesis. We validated the high quality of our screen by individual hit follow up and comparisons to kidney disease datasets. The discovery of a novel regulatory mechanism which controls epithelial proliferation via a trans-activating but cis-inhibitory effect of the Notch ligand Jagged1 proves how mosaic knockouts generated by pooled CRISPR screening in organoids can identify novel ways of communication between heterogeneous cell populations in complex tissues. Collectively, these data demonstrate the feasibility of using complex iPSC- derived organoids for genome-scale screening and serves as a benchmark for future organoid CRISPR screens.

cell biology↗

bioRxiv2020

Monoclonal anti-AMP-antibodies reveal broad and diverse AMPylation patterns in cancer cells

AMPylation is a post-translational modification that modifies amino acid side chains with adenosine monophosphate (AMP). Recent progress in the field reveals an emerging role of AMPylation as a universal regulatory mechanism in infection and cellular homeostasis, however, generic tools to study AMPylation are required. Here, we describe three monoclonal anti-AMP antibodies (mAbs) from mouse which are capable of protein backbone independent recognition of AMPylation, in denatured (Western Blot) as well as native (ELISA, IP) applications, thereby outperforming previously reported tools. These antibodies are highly sensitive and specific for AMP modifications, highlighting their potential as tools for new target identification, as well as for validation of known targets. Interestingly, applying the anti-AMP mAbs to various cancer cell lines reveals a previously undescribed broad and diverse AMPylation pattern. In conclusion, the anti-AMP mABs will aid the advancement of understanding AMPylation and the spectrum of modified targets.

biochemistry↗