Biomedical subjects

Alon Goren

Publications and source records attributed to Alon Goren.

7 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↗

PubMed2025

Genome editing with programmable base editors in human cells.

Genome editing has garnered significant attention over the last decade, resulting in a massive expansion of the genome engineering toolbox. Base editors encompass a class of tools that enable installing single-nucleotide changes in genomic DNA without the use of double-strand breaks. With the ever-increasing development of new and/or improved base editor systems, it is easy to be overwhelmed by the abundance of options. Here, we provide clear guidance to facilitate the selection of a base editor and to design guide RNAs (gRNAs) to suit various needs. Additionally, we describe in detail how to generate gRNA plasmids, transfect various mammalian cell types, and evaluate editing efficiencies. Finally, we give alternative methods and troubleshooting tips for some common pitfalls encountered during base editing.

Humans↗

PubMed2006

Fine tuning of globin gene expression by DNA methylation.

Expression patterns in the globin gene cluster are subject to developmental regulation in vivo. While the gamma(A) and gamma(G) genes are expressed in fetal liver, both are silenced in adult erythrocytes. In order to decipher the role of DNA methylation in this process, we generated a YAC transgenic mouse system that allowed us to control gamma(A) methylation during development. DNA methylation causes a 20-fold repression of gamma(A) both in non-erythroid and adult erythroid cells. In erythroid cells this modification works as a dominant mechanism to repress gamma gene expression, probably through changes in histone acetylation that prevent the binding of erythroid transcription factors to the promoter. These studies demonstrate that DNA methylation serves as an elegant in vivo fine-tuning device for selecting appropriate genes in the globin locus. In addition, our findings provide a mechanism for understanding the high levels of gamma-globin transcription seen in patients with Hereditary Persistence of Fetal Hemoglobin, and help explain why 5azaC and butyrate compounds stimulate gamma-globin expression in patients with beta-hemoglobinopathies.

Acetylation↗

PubMed2004

Theoretical and experimental investigation of the thermal effects within body cavities during transendoscopical CO2 laser-based surgery.

BACKGROUND AND OBJECTIVES: The recent development of flexible hollow waveguides for MID-IR lasers may be utilized transendoscopically to ablate selectively neoplastic, superficial tissues within body cavities. Study goals are to investigate theoretically and experimentally heat distribution and thermal response of cavity lining, during CO2 laser minimally invasive surgery (MIS), and to thermally optimize the procedure under practical conditions. STUDY DESIGN/MATERIALS AND METHODS: Mathematical model was developed to predict temperature distribution along cavity lining. Experimental setup was built, including all the necessary components for a fully feedback-controlled MIS, i.e., laser generator, gas insufflating system, surgical suction, and infrared imaging feedback mechanism, all controlled by central PC-based program. Thermal images of cavity lining were recorded and analyzed throughout varying conditions. RESULTS: Thermal gradients along the cavity lining, during and after the laser irradiation, were obtained mathematically and experimentally. Diverse modes of heat dispersions were observed, as well as the relative contributions of user-controlled parameters to the maximal heat of cavity lining. The software-controlled setup has demonstrated the capacity to instantly manage varying conditions, by which it automatically protects cavity lining from getting overheated. CONCLUSIONS: Analytical predictions and experimental measurements were highly correlated. The software-controlled system may serve a powerful tool to control thermal side effects during MIS within body cavities.

Carbon Dioxide↗

PubMed2003

Natural thermoelectric heat pump in social wasps.

Photographs of wasps or hornets, taken with different temperature sensitive infrared cameras, reveal body temperatures that are sometimes significantly lower than the ambient temperature. This suggests that the hornets possess an intrinsic biological heat pump mechanism which can be used to achieve such cooling. Evidence is presented to substantiate this novel suggestion and to argue that the heat pump is most likely implemented by exploiting a thermoelectric effect in the hornet cuticle. Such a natural heat pump can conceivably also serve to cool the active hornet, engaged in daytime activities outside the nest at ambient temperatures exceeding 40 degrees C, to a body temperature that is low enough to allow its survival in extreme thermal conditions. It might also function as a means of raising the body temperature up to a level that enables the hornet to remain active even when the ambient temperature is as low as 10 degrees C.

Animals↗

PubMed2003

Replicating by the clock.

The eukaryotic genome is divided into well-defined DNA regions that are programmed to replicate at different times during S phase. Active genes are generally associated with early replication, whereas inactive genes replicate late. This expression pattern might be facilitated by the differential restructuring of chromatin at the time of replication in early or late S phase.

Animals↗