Biomedical subjects

Sven Heinz

Publications and source records attributed to Sven Heinz.

6 recordsLinked to original sources

PubMed2026

Improved spike-in normalization clarifies the relationship between active histone modifications and transcription.

Spike-in normalization enables quantitative analysis of chromatin immunoprecipitation sequencing (ChIP-seq) signal. Here we introduce a robust dual spike-in normalization approach for ChIP-seq (ChIP-wrangler), optimize parameters and verify its accuracy in quantifying changes in ChIP-seq signal and detecting technical artifacts. We use ChIP-wrangler to revisit recent claims that active histone marks depend on transcription. We show that acute depletion of RNA polymerase II (RNAPII) has a modest impact on H3K27ac levels, with only 6% of peaks significantly changing after RNAPII depletion, indicating that histone acetylation maintenance is not entirely dependent on ongoing transcription. Promoters and enhancers are differentially affected, with 82% of decreasing acetylation peaks located at promoter-distal elements with enhancer-related motifs. ChIP-wrangler provides increased rigor and 'guardrails' for successful spike-in normalization and, as applied here, refines the understanding of crosstalk between RNAPII activity and transcription-associated histone marks.

Histones↗

PubMed2025

Automated chromatin profiling with spa-ChIP-seq uncovers the impacts of condition variations.

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is widely used to study the genomic localization of DNA-associated proteins. However, conventional protocols include multiple manual steps that can introduce inconsistency and limit scalability, thereby restricting the inclusion of appropriate replicates and controls. Although the introduction of liquid handling platforms has improved reproducibility, most existing efforts have automated only a subset of the workflow, and extending automation to efficiently map non-histone proteins, such as chromatin regulators, remains challenging. Here, we present a fully automated implementation of our previously developed single-pot ChIP-seq protocol (Texari et al. 2021), named spa-ChIP-seq, which enables scalable processing of 8 to 96 ChIP-seq samples from crosslinked cells to sequencing-ready library in approximately three days with an estimated cost of $70 per sample. Benchmarking spa-ChIP-seq against manual ChIP-seq performed in parallel demonstrates comparable signal-to-noise ratio between the two workflows. Using spa-ChIP-seq, we systematically evaluate multiple parameters including shearing and crosslinking conditions, buffer compositions, and the ratio of antibody to cell-number. We find, for the first time to our knowledge, that weaker genomic localization signals are sensitive to changing the antibody to cell-number ratio, whereas the stronger signals remain unaffected. This finding underscores the importance of maintaining consistent antibody-to-cell-number ratio for comparative studies, such as treatment responses or chromatin-QTL mapping. The spa-ChIP-seq protocol is publicly available, including deck setups, operational parameters, and scripts. We envision that this robust, cost-efficient protocol will facilitate high-throughput, reproducible ChIP-seq analyses, supporting large-scale studies of antibody validation, compound screening, population genomics, and diagnostic frameworks.

Journal Article↗

PubMed2006

Interleukin-4 induction of the CC chemokine TARC (CCL17) in murine macrophages is mediated by multiple STAT6 sites in the TARC gene promoter.

BACKGROUND: Macrophages (Mtheta) play a central role in the innate immune response and in the pathology of chronic inflammatory diseases. Macrophages treated with Th2-type cytokines such as Interleukin-4 (IL-4) and Interleukin-13 (IL-13) exhibit an altered phenotype and such alternatively activated macrophages are important in the pathology of diseases characterised by allergic inflammation including asthma and atopic dermatitis. The CC chemokine Thymus and Activation-Regulated Chemokine (TARC/CCL17) and its murine homologue (mTARC/ABCD-2) bind to the chemokine receptor CCR4, and direct T-cell and macrophage recruitment into areas of allergic inflammation. Delineating the molecular mechanisms responsible for the IL-4 induction of TARC expression will be important for a better understanding of the role of Th2 cytokines in allergic disease. RESULTS: We demonstrate that mTARC mRNA and protein are potently induced by the Th2 cytokine, Interleukin-4 (IL-4), and inhibited by Interferon-gamma (IFN-gamma) in primary macrophages (Mtheta). IL-4 induction of mTARC occurs in the presence of PI3 kinase pathway and translation inhibitors, but not in the absence of STAT6 transcription factor, suggesting a direct-acting STAT6-mediated pathway of mTARC transcriptional activation. We have functionally characterised eleven putative STAT6 sites identified in the mTARC proximal promoter and determined that five of these contribute to the IL-4 induction of mTARC. By in vitro binding assays and transient transfection of isolated sites into the RAW 264.7 Mtheta cell-line, we demonstrate that these sites have widely different capacities for binding and activation by STAT6. Site-directed mutagenesis of these sites within the context of the mTARC proximal promoter revealed that the two most proximal sites, conserved between the human and mouse genes, are important mediators of the IL-4 response. CONCLUSION: The induction of mTARC by IL-4 results from cooperative interactions between STAT6 sites within the mTARC gene promoter. Significantly, we have shown that transfer of the nine most proximal mTARC STAT6 sites in their endogenous conformation confers potent (up to 130-fold) IL-4 inducibility on heterologous promoters. These promoter elements constitute important and sensitive IL-4-responsive transcriptional units that could be used to drive transgene expression in sites of Th2 inflammation in vivo.

Animals↗

PubMed2005

Transcription factor Tfec contributes to the IL-4-inducible expression of a small group of genes in mouse macrophages including the granulocyte colony-stimulating factor receptor.

Expression of the mouse transcription factor EC (Tfec) is restricted to the myeloid compartment, suggesting a function for Tfec in the development or function of these cells. However, mice lacking Tfec develop normally, indicating a redundant role for Tfec in myeloid cell development. We now report that Tfec is specifically induced in bone marrow-derived macrophages upon stimulation with the Th2 cytokines, IL-4 and IL-13, or LPS. LPS induced a rapid and transient up-regulation of Tfec mRNA expression and promoter activity, which was dependent on a functional NF-kappaB site. IL-4, however, induced a rapid, but long-lasting, increase in Tfec mRNA, which, in contrast to LPS stimulation, also resulted in detectable levels of Tfec protein. IL-4-induced transcription of Tfec was absent in macrophages lacking Stat6, and its promoter depended on two functional Stat6-binding sites. A global comparison of IL-4-induced genes in both wild-type and Tfec mutant macrophages revealed a surprisingly mild phenotype with only a few genes affected by Tfec deficiency. These included the G-CSFR (Csf3r) gene that was strongly up-regulated by IL-4 in wild-type macrophages and, to a lesser extent, in Tfec mutant macrophages. Our study also provides a general definition of the transcriptome in alternatively activated mouse macrophages and identifies a large number of novel genes characterizing this cell type.

Animals↗

PubMed2003

Species-specific regulation of Toll-like receptor 3 genes in men and mice.

Toll-like receptor 3 (TLR3) belongs to a family of evolutionary conserved innate immune recognition molecules and recognizes double-stranded RNA, a molecular pattern associated with viral infections. Earlier studies suggested a differential expression pattern in men and mice; the molecular basis for this observation, however, was unknown. Here we demonstrate that species-specific differences in tissue expression and responses to lipopolysaccaride (LPS) coincide with the presence of different, evolutionary non-conserved promoter sequences in both species. Despite the overall unrelatedness of TLR3 promoter sequences, mRNA expression of both TLR3 orthologues was induced by interferons, particularly by interferon (IFN)-beta. The basal and IFN-beta-induced activation of promoters from both species largely depended on similar interferon regulatory factor (IRF) elements, which constitutively bound IRF-2 and recruited IRF-1 after stimulation. In murine macrophages, IFN-beta-induced TLR3 up-regulation required IFNAR1, STAT1, and in part IRF-1, but not the Janus kinase (Jak) family member Tyk2. We also show that LPS specifically up-regulates TLR3 expression in murine cells through the induction of autocrine/paracrine IFN-beta. In humans, however, IFN-beta-induced up-regulation of TLR3 was blocked by pretreatment with LPS, despite the efficient induction of IRF-1. Our findings reveal a mechanistic basis for the observed differences as well as similarities in TLR3 expression in men and mice. The IFN-beta-TLR3 link further suggests a role of TLR3 in innate and adaptive immune responses to viral infections. It will be interesting and important to clarify whether the observed differences in the transcriptional regulation of TLR3 influence innate immune responses in a species-specific manner.

Animals↗

PubMed2002

Genomic organization of the human gene HEP27: alternative promoter usage in HepG2 cells and monocyte-derived dendritic cells.

We used representational difference analysis to discover new genes with specific expression in dendritic cells. Among other genes, we identified HEP27, encoding a member of the short chain alcohol dehydrogenase/reductase family to be upregulated during monocyte to dendritic cell differentiation. Originally cloned from hepatocellular carcinoma cells (HepG2), HEP27 was exclusively expressed in monocyte-derived dendritic cells within the hematopoietic system. The presence of different transcripts in monocyte-derived dendritic cells, HepG2 cells, and various tissues could be traced back to alternative splicing and alternative promoter usage. We describe here the complete genomic organization of HEP27, including two alternative promoter regions: a hepatocyte-specific promoter which was induced by the histone deacetylase inhibitor sodium butyrate in several other cell types, and a second upstream promoter which was specifically active in monocyte-derived dendritic cells. Its exclusive usage in monocyte-derived dendritic cells makes the alternative HEP27 promoter an interesting target to study dendritic-cell-specific gene regulation.

Alcohol Dehydrogenase↗