Cohen syndrome is an autosomal recessive disorder caused by VPS13B (COH1) gene mutations. This syndrome is significantly underdiagnosed and is characterized by intellectual disability, microcephaly, autistic symptoms, hypotension, myopia, retinal dystrophy, neutropenia, and obesity. VPS13B regulates intracellular membrane transport and supports the Golgi apparatus structure, which is critical for neuron formation. We generated induced pluripotent stem cells from two patients with pronounced manifestations of Cohen syndrome and differentiated them into neural stem cells and neurons. Using transmission electron microscopy, we documented multiple new ultrastructural changes associated with Cohen syndrome in the neuronal cells. We discovered considerable disturbances in the structure of some organelles: Golgi apparatus fragmentation and swelling, endoplasmic reticulum structural reorganization, mitochondrial defects, and the accumulation of large autophagosomes with undigested contents. These abnormalities underline the ultrastructural similarity of Cohen syndrome to many neurodegenerative diseases. The cell models that we developed based on patient-specific induced pluripotent stem cells can serve to uncover not only neurodegenerative processes, but the causes of intellectual disability in general.

The phenomenal progress in biotechnology and genomics is both inspiring and overwhelming—a classic curse of choice, particularly when it comes to selecting methods for mapping transgene DNA integration sites. Transgene localization remains a crucial task for the validation of transgenic mouse or other animal models generated by pronuclear microinjection. Due to the inherently random nature of DNA integration, reliable characterization of the insertion site is essential. Over the years, a vast number of mapping methods have been developed, and new approaches continue to emerge, making the choice of the most suitable technique increasingly complex. Factors such as cost, required reagents, and the nature of the generated data require careful consideration. In this review, we provide a structured overview of current transgene mapping techniques, which we have broadly classified into three categories: classic PCR-based methods (such as inverse PCR and TAIL-PCR), next-generation sequencing with target enrichment, and long-read sequencing platforms (PacBio and Oxford Nanopore). To aid in decision-making, we include a comparative table summarizing approximate costs for the methods. While each approach has its own advantages and limitations, we highlight our top four recommended methods, which we believe offer the best balance of cost-effectiveness, reliability, and simplicity for identifying transgene integration sites.

The auxin-inducible degron (AID) system is widely used to study function of various proteins. The plant hormone auxin is used as an inducer in this system, which easily penetrates into the cells and causes proteasomal degradation of the protein of interest fused to a small degron tag. It is often assumed that as a plant hormone, auxin does not significantly affect physiology of animal cells. In order to test how auxin affects gene expression in human and mouse cells, we collected a set of published data on the levels of gene expression in various experiments with the auxin degradation system of various proteins. The analysis showed that in human HCT116, DLD1, and HAP1 cell lines, as well as in mouse embryonic stem cell lines, auxin treatment leads to activation of aryl hydrocarbon receptor (AHR)-related genes. However, activation of AHR pathway genes does not occur upon auxin treatment in the human IMR32 cells and mouse G1E-ER4 cells, which are characterized by low AHR gene expression. To verify this observation, we conducted an experiment treating human U87, A549, and HCT116 cells with auxin and demonstrated activation of one of the main AHR pathway responders, the CYP1B1 gene. We believe that activation of the AHR pathway should be taken into account by those using the auxin degradation system in their studies.

Many cellular processes, including gene expression regulation, DNA replication and repair, as well as proper condensation and segregation of chromosomes, require highly coordinated remodeling of chromatin. Cohesin and condensins, the structural maintenance of chromosomes (SMC) protein complexes that function as ATP-powered loop extrusion motors, are key determinants of chromatin structure. The genetic loss of their function is lethal, whereas inducible degradation approaches enable rapid, robust analysis of the depletion phenotype. In this review, we discuss new insights into chromatin folding through each cell cycle phase from the auxin-inducible degradation (AID) system. We review the mechanisms by which condensins and cohesins contribute to the helical organization of mitotic chromosomes and to the maintenance of chromosome territories in interphase. Additionally, we discuss studies examining the roles of TOP2A, KIF4A, and SRBD during mitosis using the AID system. We then outline emerging principles of the mitotic-to-interphase transition and how targeted degradation of chromatin proteins reshapes this process. Finally, we highlight and discuss new advances in understanding interphase chromatin organization revealed by AID-based studies.

The Notch signaling pathway is a critical regulator of embryonic brain development. Among its four mammalian receptors, Notch1 and Notch2 are particularly significant in the developing cortex, yet their roles in human neurodevelopment are not well understood. In murine cortex development, Notch1 primarily regulates early progenitor identity and neurogenesis, while Notch2 is required for maintaining radial glial cells at later stages. However, it is unclear whether these functions are conserved in the human developing brain. In this study, we used cerebral organoids as an in vitro model of early human corticogenesis and conducted lentiviral shRNA-mediated knockdowns of NOTCH1 and NOTCH2. Our findings indicate that NOTCH1 is essential for organoid growth, lumen morphogenesis, radial glial identity, and progenitor proliferation. In contrast, depleting NOTCH2 did not significantly affect these early developmental processes. These results demonstrate that NOTCH1 and NOTCH2 have potentially non-redundant and temporally distinct roles in early human corticogenesis, reflecting receptor-specific specialization within the Notch signaling pathway.

Chromatin is not randomly distributed within the nucleus, but organized in a three-dimensional structure that plays a critical role in genome functions. Сohesin and condensins are conserved multi-subunit protein complexes that participate in mammalian genome organization by extruding chromatin loops. The fine temporal regulation of these complexes is facilitated by a number of other proteins, one of which is microcephalin (Mcph1). Mcph1 prevents condensin II from associating with chromatin through interphase. Loss of Mcph1 induces chromosome hypercondensation; it is not clear to what extent this reorganization affects gene expression. In this study, we generated several mouse embryonic stem cell (mESC) lines with knockout of the Mcph1 gene and analyzed their gene expression profile. Gene Ontology analyses of differentially expressed genes (DEGs) after Mcph1 knockout revealed gene categories related to general metabolism and olfactory receptor function but not to cell cycle control previously described for Mcph1. We did not find a correlation between the DEGs and their frequency of lamina association. Thus, this evidence questions the hypothesis that Mcph1 knockout-mediated chromatin reorganization governs gene expression in mESCs. Among the negative effects of Mcph1 knockout, we observed numerous chromosomal aberrations, including micronucleus formation and chromosome fusion. This confirms the role of Mcph1 in maintaining genome integrity described previously. In our opinion, dysfunction of Mcph1 may be a kind of “Rosetta stone” for deciphering the function of condensin II in the interphase nucleus. Thus, the cell lines with knocked-out Mcph1 can be used to further study the influence of chromatin structural proteins on gene expression.