Biology subjects

Krebs, A.

Publications and source records attributed to Krebs, A..

8 recordsLinked to original sources

bioRxiv2026

ChromSMF: integrated profiling of histone modifications, protein-DNA interactions and DNA methylation on multi-kilobase DNA molecules

Chromatin accessibility and histone post-translational modifications are widely used to identify and characterize cis-regulatory elements. Yet, these are typically measured separately, precluding direct linkage between them. Here, we present Chromatin-informed Single Molecule Footprinting (ChromSMF), a method that simultaneously quantifies histone modifications, transcription factor binding, and nucleosome occupancy, while measuring DNA methylation and sequence variations on the same multi-kilobase DNA molecules. ChromSMF combines antibody-guided tethering of the adenine methyltransferase Hia5 to modified histones, protein-DNA footprinting using the cytosine methyltransferase M.CviPI, and direct methylation detection by Nanopore sequencing. We benchmark ChromSMF across five histone modifications, uncovering relationships between epigenetic states and chromatin opening across entire cis-regulatory landscapes. We further present a computational framework for integrated analysis of multiple layers of epigenetic regulation on individual haplotypes. Together, ChromSMF provides an integrated genome-wide genomic method to investigate the combinatorial function of multiple genetic and epigenetic factors on gene regulation across diverse cellular contexts.

genomics↗

bioRxiv2025

Single-Molecule DNA Footprinting and Transcription Imaging Reveal the Molecular Mechanisms of Promoter Dynamics

Live cell RNA imaging revealed that transcription levels are encoded by the intrinsic dynamics of promoters. However, capturing both kinetic and molecular aspects of promoter fluctuations has been challenging. Here, we resolve this key issue by combining Single Molecule DNA footprinting (SMF) with live transcription imaging. Using HIV-1 as a model, SMF reveals that the promoter functions in two modes depending on the viral transactivator Tat. Without Tat, a nucleosome occupies the core promoter and prevents assembly of the pre-initiation complex. With Tat, this nucleosome is absent while TBP and initiating polymerases are frequently detected. Combining live imaging with SMF provides a mechanistic model of promoter dynamics, which estimates the rates of deposition and removal of promoter nucleosomes (0.7 h-1), TBP binding (0.04 min-1) and polymerase loading (seconds). The data further reveal a kinetic proofreading mechanism of initiating polymerases, which enables Tat to indirectly control promoter nucleosomes by promoting elongation.

molecular biology↗

bioRxiv2025

Enhancer-promoter compatibility is mediated by the promoter-proximal region

Gene promoters induce transcription in response to distal enhancers. How enhancers specifically activate their target promoter while bypassing other promoters remains unclear. Here, we find that the promoter-proximal region is critical for cell-type specific enhancer-promoter compatibility. Using high-throughput genome-engineering in mouse embryonic stem cells (mESC), we systematically replace the endogenous Sox9 promoter with different libraries of core and extended (i.e. full) promoters and assess their response to long-range regulatory elements in mESCs and neural progenitor cells. We find that only a subset of full promoters is activated by distal neuronal enhancers and that the promoter-proximal region is necessary for this enhancer-promoter compatibility. Core promoters alone are insufficient to respond to distal enhancers but modulate the transcriptional output of responsive promoters. Our results suggest that within multipartite regulatory domains, the promoter-proximal region fulfills a facilitator-like function that filters and transmits signal from distal enhancers, ultimaltely conferring enhancer-promoter compatibility.

genetics↗

bioRxiv2025

Human iPSC derived alveolar macrophages reveal macrophage subtype specific functions of itaconate in M. tuberculosis host defense

Mycobacterium tuberculosis (Mtb) must survive within multiple macrophage populations during infection, including alveolar macrophages (AM) and recruited inflammatory macrophages. In mice, itaconate, produced in macrophages by ACOD1 mediated decarboxylation of aconitate, has direct antimicrobial activity, modulates inflammatory cytokines, and is required for resistance to M. tuberculosis (Mtb) infection. The role of itaconate in human macrophages is less clear and whether itaconate mediates distinct effects in macrophage subtypes is unknown. Here, we investigated the role of itaconate in human iPSC-derived macrophages, either induced by GM-CSF to resemble alveolar macrophages (AM-Like cells), or treated with M-CSF to generate control macrophages (MCDM cells). Both types of human macrophages produce substantially less itaconate than mouse macrophages and AM-Ls produced 4-fold less itaconate than MCDMs. Surprisingly, ACOD1 deficient AM-L macrophages, but not MCDM macrophages, were permissive for Mtb growth. Moreover, itaconate functioned to dampen the Mtb induced inflammatory response in MCDMs, but not AM-L macrophages, affecting both the Type I IFN and TNF pathways. These results indicate that itaconate is involved in human macrophage responses to TB, with distinct roles in different macrophage subsets. These results also show that genetically tractable hiPSC-derived macrophages are a robust and versatile model to dissect cellular host pathogen interactions.

immunology↗

bioRxiv2024

Mammalian promoters are characterised by low occupancy and high turnover of RNA polymerase II

The general transcription machinery and its occupancy at promoters are highly conserved across metazoans. This contrasts with the kinetics of mRNA production that considerably differ between model species such as Drosophila and mouse. The molecular basis for these kinetic differences is currently unknown. Here, we used Single Molecule Footprinting to measure RNA Polymerase II (Pol II) occupancy, the fraction of DNA molecules bound, at promoters in mouse and Drosophila cell lines. Single molecule data reveals that Pol II occupancy is on average 3-5 times more frequent at transcriptionally active Drosophila promoters than active mouse promoters. Kinetic modelling of the occupancy states suggests that these differences in Pol II occupancy are determined by the ratio between the transcription initiation and Pol II turnover rates. We used chemical perturbation of transcription initiation to determine Pol II turnover rate in both species. Integration of these data into the model shows that infrequent Pol II occupancy in mammals is explained by the combination of high Pol II turnover and low transcription initiation rates.

genomics↗

bioRxiv2023

JAK/STAT mediated insulin resistance in muscles is essential for effective immune response

The metabolically demanding nature of immune response requires nutrients to be preferentially directed towards the immune system at the expense of peripheral tissues. We study the mechanisms by which this metabolic reprograming occurs using the parasitoid infection of Drosophila larvae. To overcome such an immune challenge hemocytes differentiate into lamellocytes, which encapsulate and melanize the parasitoid egg. Hemocytes acquire the energy for this process by expressing JAK/STAT ligands upd2 and upd3, which activates JAK/STAT signaling in muscles and redirects carbohydrates away from muscles in favor of immune cells. Absence of upd/JAK/STAT signaling leads to an impaired immune response and increased mortality. We demonstrate how JAK/STAT signaling in muscles leads to suppression of insulin signaling through activation of ImpL2, the inhibitor of Drosophila insulin like peptides. We reveal the crucial function of muscles during immune response and show the benefits of insulin resistance as an adaptive mechanism that is necessary for survival.

immunology↗

bioRxiv2020

Paternal exposure to DDT programs breast cancer development in daughters via sperm miRNA in a mouse model

DNA sequence accounts for the majority of disease heritability, including cancer. Yet, not all familial cancer cases can be explained by genetic factors. It is becoming clear that environmentally induced epigenetic inheritance occurs and that the progenys traits can be shaped by parental environmental experiences. In humans, epidemiological studies have implicated environmental toxicants, such as the pesticide DDT, in intergenerational cancer development, including breast and childhood tumors. Here, we show that the female progeny of males exposed to DDT in the pre-conception period have higher susceptibility to developing aggressive tumors in mouse models of breast cancer. Sperm of DDT-exposed males exhibited distinct patterns of small non-coding RNAs, with an increase in miRNAs and a specific surge in miRNA-10b levels. Remarkably, embryonic injection of the entire sperm RNA load of DDT-exposed males, or synthetic miRNA-10b, recapitulated the tumor phenotypes observed in DDT offspring. Mechanistically, miR-10b injection altered the transcriptional profile in early embryos with enrichment of genes associated with cell differentiation, tissue and immune system development. In adult DDT-derived progeny, transcriptional and protein analysis of mammary tumors revealed alterations in stromal and in immune system compartments. Our findings reveal a causal role for sperm RNAs in environmentally induced inheritance of cancer predisposition and, if confirmed in humans, this could help partially explain some of the "missing heritability" of breast, and other, malignancies.

cancer biology↗

bioRxiv2018

Targeting neuronal and glial cell types with synthetic promoter AAVs in mice, non-human primates, and humans

Targeting genes to specific neuronal or glial cell types is valuable both for understanding and for repairing brain circuits. Adeno-associated viral vectors (AAVs) are frequently used for gene delivery, but targeting expression to specific cell types is a challenge. We created a library of 230 AAVs, each with a different synthetic promoter designed using four independent strategies. We show that ~11% of these AAVs specifically target expression to neuronal and glial cell types in the mouse retina, mouse brain, non-human primate retina in vivo, and in the human retina in vitro. We demonstrate applications for recording, stimulation, and molecular characterization, as well as the intersectional and combinatorial labeling of cell types. These resources and approaches allow economic, fast, and efficient cell-type targeting in a variety of species, both for fundamental science and for gene therapy.

neuroscience↗