Irina Solovei

Chromatin has a complex 3D structure and diverse binding proteins that coordinate the genome’s most essential functions. Many microscopy and genomics technologies that map chromatin proteins and modifications rely on the diffusion of antibodies (Abs) to target epitopes within whole nuclei. Here, we reveal a critical flaw in such methods that arises when Abs become trapped at the edge of nuclear structures and fail to reach internally positioned epitopes. This “Ab-trapping” results in artifactual peripheral signal that fundamentally distorts the apparent positions of chromatin features across the genome and nucleus. Using computational modeling and experimental validation, we demonstrate that Ab-trapping is caused by a combination of three compounding factors—high epitope abundance, high Ab affinity, and low Ab diffusion rates. Ab-trapping can thus systematically misrepresent the localization of many prevalent chromatin features like histone modifications, transcription factors, nucleolar proteins, and protein tags. We also show that this artifact manifests in multiple technologies, including immunofluorescence microscopy, more recent CUT&Tag-seq, and likely any method relying on Ab diffusion. Finally, we outline readily implementable strategies to identify and mitigate Ab-trapping. Combined, our work presents a previously unrecognized yet prevalent artifact in Ab-based chromatin mapping methods and the means to resolve it.

Diverse forms of heterochromatin block inappropriate transcription and safeguard differentiation and cell identity. Yet, how and when heterochromatin is reconfigured to facilitate changes in cell- fate remains a key open question. Here, we address this by mapping a prevalent heterochromatic feature - genome-lamina interactions - relative to transcription in single-cells during mouse embryogenesis. We find that genome-lamina interactions remain relatively uniform between germ layers following gastrulation but are extensively reconfigured in diverse tissues during later organogenesis. Focusing on limb development, we demonstrate that genome-lamina interactions are selectively released in early multipotent progenitors at key developmental genes and their surrounding regulatory domains. This “lamina-release” often precedes gene expression at later developmental stages, suggesting it primes regulatory domains for future potential activation. Conversely, lamina-release coincides with chromatin opening at sites of crucial limb transcription factor binding, and so is closely intertwined with the regulatory machinery driving limb formation. Finally, we show that the boundaries of topologically-associated domains (TADs) constrain the spread of lamina-release at a limb gene locus. This ensures independent lamina dynamics between neighbouring domains. Together, our data suggest a previously unrecognised process where genome-lamina interactions are selectively released at regulatory domains to transition loci toward more permissive chromatin states, thereby potentiating cell type specific activation. Our work thus reveals how systematic heterochromatin reorganization links to developmental multipotency, providing mechanistic insights into cell-fate decisions in vivo .

Antibodies (Ab) are essential for detecting specific epitopes in microscopy and genomics, but can produce artifacts leading to erroneous interpretations. Here, we characterize a novel artifact, Ab-trapping, in which antibodies bind at the periphery of a cellular structure and do not diffuse further into its interior. This causes anomalous peripheral staining for multiple critical targets, including endogenous or ectopically expressed nuclear proteins like nucleolar proteins, histone variants and their modifications like H3K9me2. Ab-trapping can affect any assay relying on Ab diffusion, including immunofluorescence microscopy and recent genomics approaches like CUT&Tag. Critically, computational modeling and experimental validation reveal that Ab-trapping is caused by high epitope abundance, high Ab affinity, and low diffusion rates. Consequently, its effects can be mitigated by using alternative Abs and optimizing incubation conditions. Ab-trapping is therefore a considerable artifact that should be considered when designing experiments and interpreting results.

Transcription of the majority of eukaryotic genes is accompanied by splicing. The timing of splicing varies significantly between introns, transcripts, genes and species. Although quick co-transcriptional intron removal has been demonstrated for many mammalian genes, most splicing events do not occur immediately after intron synthesis. In this study, we utilized the highly expressed Tg gene, which forms exceptionally long transcription loops, providing a convenient model for studying splicing dynamics using advanced light microscopy. Using single-cell oligopainting, we observed a splicing delay occurring several tens of kilobases downstream of a transcribed intron, a finding supported by standard cell population analyses. We speculate that this phenomenon is due to the abnormally high transcriptional rate of the Tg gene, which might lead to a localized deficiency in splicing factors and, consequently, delayed spliceosome assembly on thousands of nascent transcripts decorating the gene. Additionally, we found that, in contrast to what is seen for short introns (50 kb) is spliced promptly, providing further support for the idea that intron length might modulate splicing speed.

Corrigendum: THE HIGHLY AND PERPETUALLY UPREGULATED THYROGLOBULIN GENE IS A HALLMARK OF FUNCTIONAL THYROCYTESSimon Ullrich 1, Susanne Leidescher 1, Yana Feodorova 1,2, Katharina Thanisch 1, Jean-Baptiste Fini 3, Bernd Kaspers 4, Frank Weber 5, Boyka Markova 6, Dagmar Führer 6, Mirian Romitti 7, Stefan Krebs8, Helmut Blum 8, Heinrich Leonhardt 1, Sabine Costagliola 7, Heike Heuer 6, Irina Solovei 1*1Biocenter, Ludwig Maximilians University Munich, Munich, Germany2Department of Medical Biology, Medical University of Plovdiv, Division of Molecular and Regenerative Medicine, Research Institute at Medical University of Plovdiv, Plovdiv, Bulgaria3Département Adaptations du Vivant (AVIV), Physiologie Moléculaire et Adaptation (PhyMA UMR 7221 CNRS), Muséum National d’Histoire Naturelle, CNRS, CP 32, Paris, France4Department for Veterinary Sciences, Ludwig Maximilians University Munich, Planegg, Germany5Department of General, Visceral and Transplantation Surgery, Section of Endocrine Surgery, University Duisburg-Essen, University Hospital Essen, Essen, Germany6Department of Endocrinology, Diabetes and Metabolism, University Duisburg-Essen, University Hospital Essen, Essen, Germany7IRIBHM ULB, Brussels, Belgium8Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, Ludwig Maximilians University Munich, Munich, Germany* Correspondence: irina.solovei@lrz.uni-muenchen.deKeywords: gene upregulation; thyroglobulin gene; thyroid hormones; transcription; transcription loop.Corrigendum on Ullrich S, Leidescher S, Feodorova Y, Thanisch K, Fini JB, Kaspers B, Weber F, Markova B, Führer D, Romitti M, Krebs S, Blum H, Leonhardt H, Costagliola S, Heuer H, Solovei I. The highly and perpetually upregulated thyroglobulin gene is a hallmark of functional thyrocytes. Front Cell Dev Biol. 2023 Oct 4;11:1265407. doi: 10.3389/fcell.2023.1265407. PMID: 37860816; PMCID: PMC10582334.Additional Affiliation(s)In the published article, there was an error regarding the affiliation(s) for Yana Feodorova. As well as having affiliation 1, they should also have Department of Medical Biology, Medical University of Plovdiv; Division of Molecular and Regenerative Medicine, Research Institute at Medical University of Plovdiv, Plovdiv, Bulgaria. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.

The nocturnal aardvark Orycteropus afer is the only extant species in the mammalian order Tubulidentata. Previous studies have claimed that it has an all-rod retina. In the retina of one aardvark, we found rod densities ranging from 124,000/mm² in peripheral retina to 214,000/mm² in central retina; the retina of another aardvark had 182,000 – 245,000 rods/mm². This is moderate in comparison to other nocturnal mammals. With opsin immunolabelling we found that the aardvark also has a small population of cone photoreceptors. Cone densities ranged from 300 to 1,300/mm² in one animal, and from 1,100 to 1,600/mm² in the other animal, with large local variations and no large central-peripheral density gradient. Overall, cones comprised 0.25-0.9% of the photoreceptors. Both typical mammalian cone opsins, longwave-sensitive (L) and shortwave-sensitive (S), were present. However, there was colocalization of the two opsins in many cones across the retina (35 – 96% dual pigment cones). Pure L cones and S cones formed smaller populations. This probably results in poor colour discrimination. Thyroid hormones, important regulators of cone opsin expression, showed normal blood serum levels. The relatively low rod density and hence a relatively thin retina may be related to the fact that the aardvark retina is avascular and its oxygen and nutrient supply have to come from the choriocapillaris by diffusion. In contrast to some previous studies, we found that the aardvark eye has a reflective tapetum lucidum with features of a choroidal tapetum fibrosum, in front of which the retinal pigment epithelium is unpigmented. The discussion considers these findings from a comparative perspective.

Transcription of the majority of eukaryotic genes is accompanied by splicing, a process that depends on the assembly of the spliceosome on introns. The timing of spliceosome assembly varies significantly between introns, transcripts, genes and species. While quick co-transcriptional intron removal has been demonstrated for many mammalian genes, most splicing events do not occur immediately after intron synthesis. In this study, we utilized the highly expressed Tg gene, which forms exceptionally long transcription loops (Leidescher et al., 2022), providing a convenient model for studying splicing dynamics using advanced light microscopy. Our single-cell oligopainting-based analysis revealed a delay in splicing several tens of kilobases downstream of a transcribed intron, a finding further supported by standard cell population analyses. We speculate that this phenomenon is due to the abnormally high transcription rate of the Tg gene, which may lead to a localized deficiency in splicing factors and, consequently, delayed spliceosome assembly on thousands of nascent transcripts decorating the gene. Additionally, we found that, in contrast to short introns (50 kb) is spliced promptly, providing further support for the idea that intron length may modulate splicing speed.

Abnormalities are indispensable for studying normal biological processes and mechanisms. In the present work, we draw attention to the remarkable phenomenon of a perpetually and robustly upregulated gene, the thyroglobulin gene (Tg). The gene is expressed in the thyroid gland and, as it has been recently demonstrated, forms so-called transcription loops, easily observable by light microscopy. Using this feature, we show that Tg is expressed at a high level from the moment a thyroid cell acquires its identity and both alleles remain highly active over the entire life of the cell, i.e., for months or years depending on the species. We demonstrate that this high upregulation is characteristic of thyroglobulin genes in all major vertebrate groups. We provide evidence that Tg is not influenced by the thyroid hormone status, does not oscillate round the clock and is expressed during both the exocrine and endocrine phases of thyrocyte activity. We conclude that the thyroglobulin gene represents a unique and valuable model to study the maintenance of a high transcriptional upregulation.

Abnormalities are indispensable for studying normal biological processes and mechanisms. In the present work, we draw attention to the remarkable phenomenon of a perpetually and robustly upregulated gene, the thyroglobulin gene ( Tg ). The gene is expressed in the thyroid gland and, as it has been recently demonstrated, forms so-called transcription loops, easily observable by light microscopy. Using this feature, we show that Tg is expressed at a high level from the moment a thyroid cell acquires its identity and both alleles remain highly active over the entire life of the cell, i.e. for months or years depending on the species. We demonstrate that this high upregulation is characteristic of thyroglobulin genes in all major vertebrate groups. We provide evidence that Tg is not influenced by the thyroid hormone status, does not oscillate round the clock and is expressed during both the exocrine and endocrine phases of thyrocyte activity. We conclude that the thyroglobulin gene represents a valuable model to study the maintenance of a high transcriptional upregulation. SUMMARY STATEMENT: The thyroglobulin gene is highly and permanently expressed in thyrocytes of all vertebrates, at any condition and round the clock, offering a unique model to study mechanisms of high upregulation maintenance and chromatin dynamics