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The optical resolution of conventional far field fluorescence light microscopy is restricted to about 200 nm laterally and 600 nm axially and has been thought for many decades to be an insurmountable barrier for the quantitative spatial analysis of cellular and hence also nuclear constituents. Novel approaches in light microscopy have now overcome this barrier. Here, we report on a special method of localisation microscopy, spectral precision distance/position determination microscopy and its combination with fluorescence in situ hybridization to analyse the spatial distribution of specific DNA sequences in human cell nuclei at the macromolecular optical resolution level. As an example, repetitive DNA sequence DYZ2 located within the heterochromatin region on human chromosome Yq12 was labelled with clone pHY2.1. Between 300 and 700 single-probe molecules were resolved in individual chromatin domains, corresponding to a detected molecule density around 500/μm², i.e., many times higher than resolvable by conventional fluorescence microscopy. A mean localisation accuracy of about 20 nm indicated a mean optical resolution in the 50 nm range. Beyond new perspectives for light microscopic studies of specific chromatin nanostructures, this may open a new avenue towards the general analysis of copy number of specific DNA sequences in small regions of individual interphase nuclei.

The genome folds into a hierarchy of three-dimensional structures within the nucleus. At the sub-megabase scale, chromosomes form topologically associating domains (TADs) 1 – 4 . However, how TADs fold in single cells is elusive. Here, we reveal TAD features inaccessible to cell population analysis by using super-resolution microscopy. TAD structures and physical insulation associated with their borders are variable between individual cells, yet chromatin intermingling is enriched within TADs compared to adjacent TADs in most cells. The spatial segregation of TADs is further exacerbated during cell differentiation. Favored interactions within TADs are regulated by cohesin and CTCF through distinct mechanisms: cohesin generates chromatin contacts and intermingling while CTCF prevents inter-TAD contacts. Furthermore, TADs are subdivided into discrete nanodomains, which persist in cells depleted of CTCF or cohesin, whereas disruption of nucleosome contacts alters their structural organization. Altogether, these results provide a physical basis for the folding of individual chromosomes at the nanoscale. Super-resolution microscopy identifies sub-topologically associating domain (TAD) nanodomains and intercellular heterogeneity in TAD conformation and insulation. Cohesin or CTCF depletion regulates distinct types of chromatin contacts at the TAD but not nanodomain level.

Small molecules are useful tools for probing the biological function and therapeutic potential of individual proteins, but achieving selectivity is challenging when the target protein shares structural domains with other proteins. The Bromo and Extra-Terminal (BET) proteins have attracted interest because of their roles in transcriptional regulation, epigenetics, and cancer. The BET bromodomains (protein interaction modules that bind acetyl-lysine) have been targeted by potent small-molecule inhibitors, but these inhibitors lack selectivity for individual family members. We developed an ethyl derivative of an existing small-molecule inhibitor, I-BET/JQ1, and showed that it binds leucine/alanine mutant bromodomains with nanomolar affinity and achieves up to 540-fold selectivity relative to wild-type bromodomains. Cell culture studies showed that blockade of the first bromodomain alone is sufficient to displace a specific BETprotein, Brd4, from chromatin. Expansion of this approach could help identify the individual roles of single BETproteins in human physiology and disease.

Titanium is essentially absent from biological systems yet reliably integrates into bone. To achieve osseointegration, titanium must activate biological processes without entering cells, defining it as a bio-activating material. Nanostructuring bulk titanium reduces grain size, increases strength, and improves other quantifiable physical properties, including cytocompatibility. The biological processes activated by increasing grain boundary availability were detected with total RNA-sequencing in mouse pre-osteoblasts grown for 72 hours on nanometrically smooth substrates of either coarse grain or nanostructured ultrafine grain titanium. The average grain boundary length under cells on the conventional coarse grain substrates is 273.0 μm, compared to 70,881.5 μm for cells adhered to the nanostructured ultrafine grain substrates; a 260-fold difference. Cells on both substrates exhibit similar expression profiles for genes whose products are critical for mechanosensation and transduction of cues that trigger osteoconduction. Biological process Gene Ontology term enrichment analysis of differentially expressed genes reveals that cell cycle, chromatin modification, telomere maintenance, and RNA metabolism processes are upregulated on ultrafine grain titanium. Processes related to immune response, including apoptosis, are downregulated. Tumor-suppressor genes are upregulated while tumor-promoting genes are downregulated. Upregulation of genes involved in chromatin remodeling and downregulation of genes under the control of the peripheral circadian clock implicate both processes in the transduction of mechanosensory information. Non-coding RNAs may also play a role in the response. Merging transcriptomics with well-established mechanobiology principles generates a unified model to explain the bio-activating properties of titanium. The modulation of processes is accomplished through chromatin remodeling in which the nucleus responds like a rheostat to grain boundary concentration. This convergence of biological and materials science reveals a pathway toward understanding the biotic-abiotic interface and will inform the development of effective bio-activating and bio-inactivating materials.

Cancer has arisen from both genetic mutations and epigenetic changes, making epigenetics a crucial area of research for innovative cancer prevention and treatment strategies. This dual perspective has propelled epigenetics into the forefront of cancer research. This review highlights the important roles of DNA methylation, histone modifications and non-coding RNAs (ncRNAs), particularly microRNAs (miRNAs) and long non-coding RNAs, which are key regulators of cancer-related gene expression. It explores the potential of epigenetic-based therapies to revolutionize patient outcomes by selectively modulating specific epigenetic markers involved in tumorigenesis. The review examines promising epigenetic biomarkers for early cancer detection and prognosis. It also highlights recent progress in oligonucleotide-based therapies, including antisense oligonucleotides (ASOs) and antimiRs, to precisely modulate epigenetic processes. Furthermore, the concept of epigenetic editing is discussed, providing insight into the future role of precision medicine for cancer patients. The integration of nanomedicine into cancer therapy has been explored and offers innovative approaches to improve therapeutic efficacy. This comprehensive review of recent advances in epigenetic-based cancer therapy seeks to advance the field of precision oncology, ultimately culminating in improved patient outcomes in the fight against cancer.

Transcription factors OCT4 and NANOG are main constituents of a functional network that controls proliferation and pluripotency maintenance of stem cells as well as early lineage decisions. We investigated expression profiles of OCT4 and NANOG during the early phases of neural differentiation using NT2/D1 cells induced by retinoic acid as an in vitro model system of human neurogenesis. We demonstrated decrease in OCT4 and NANOG mRNA and protein levels following exposure to retinoic acid. Next, by employing chromatin immunoprecipitation, we investigated profiles of selected H3 and H2B histone marks deposited on the promoters of the OCT4 and NANOG genes. We found decline in H3K4me3, H2BK5ac, and H2BK120ac on both promoters, which paralleled the decrease in OCT4 and NANOG expression. Moreover, we found that the H2BK16ac mark is differentially enriched on these two promoters, pointing to differences in epigenetic regulation of OCT4 and NANOG gene expression. Finally, based on our data, we suggest that the early response of pluripotency genes OCT4 and NANOG to the differentiation-inducing stimuli is mediated by dynamic changes in chromatin marks, while DNA methylation is acquired in the later stages of neurogenesis.

Among the molecules to which the human skin is exposed, glyphosate is used as an herbicide. Glyphosate has been shown to induce in vitro cutaneous cytotoxic effects, concomitant with oxidative disorders. In this following study, we focused on dynamic events of the loss of HaCaT cell integrity appearing after a glyphosate treatment. In these conditions, we showed that glyphosate is able to disrupt HaCaT cells and to induce intracellular oxidative cascade. In this aim, we optimized the conditions of cell treatment playing on exposure time (from 24 h to 30 min), which directly modify the cell viability profile (glyphosate 50% inhibition concentration from 28 to 53 mM) and allow to track cells along the treatment as an “induction and visualization” process. The combination of atomic force and fluorescence microscopic approaches offered opportunities to lead in parallel an investigation of the membrane surface and of the intracellular disorders, through cytoskeleton, nuclear, and oxidative stress marker targeting. The originality of our approach relies on monitoring all events derived from oxidative stress in process and performed by simultaneous cytotoxic induction and nanoscale cell visualization. We revealed a transition from spread and globular to elongated cell morphology, with a drastic cell size reduction, after a dose- and time-dependent glyphosate treatment; a redistribution of cell surface protrusions was also pointed out. All these membrane damages, added to observations of disorganized cytoskeleton, condensed chromatin, and overproduction of oxidative reactive species, lead us to conclude that glyphosate acts in induction of apoptotic process.