Biology subjects

Shnaider, T.

Publications and source records attributed to Shnaider, T..

3 recordsLinked to original sources

bioRxiv2025

Condensins-mediated mitotic folding persists as a structural echo in interphase chromatin

The Structural Maintenance of Chromosomes (SMC) protein complexes, cohesin and condensins, orchestrate cell cycle-dependent transitions in chromosome folding: from the relatively decondensed interphase chromatin, organized into dynamic DNA loops by cohesin, to the highly condensed arrays of DNA loops in mitotic chromosomes formed by condensins. Here, using Hi-C data from mouse embryonic stem cell lines with auxin-inducible degron-tagged subunits of SMC complexes, we clarify the role of condensins in shaping chromatin contacts within the interphase nucleus. We found that depletion of condensin I leads to weakened segregation of chromatin into A and B compartments. We also show that the contact patterns established by condensins during mitosis are preserved in interphase, suggesting a structural memory of mitotic chromosome folding. Condensins influence chromocenter organization: in the absence of condensin II during mitosis, centromeres tend to cluster, forming hyperclusters of pericentric heterochromatin. In contrast, depletion of condensin I reduces the number of heterochromatin clusters, consistent with enhanced chromosome territoriality. By combining a cohesin-degron system with Mcph1 knockout, we established an artificial model in which condensin II becomes the major driver of chromatin folding in interphase nuclei following cohesin depletion. Unlike extrusive cohesin, condensin II does not stall at sequence-specific sites and forms large, randomly positioned loops on interphase chromatin. Moreover, condensin II activity during interphase does not interfere with cohesin-dependent structures such as TADs and chromatin loops. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/669285v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1926857org.highwire.dtl.DTLVardef@100640eorg.highwire.dtl.DTLVardef@a903dcorg.highwire.dtl.DTLVardef@3c6a16_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2022

TAD border deletion at the Kit locus causes tissue-specific ectopic activation of a neighboring gene

Topologically associated domains (TADs) restrict promoter-enhancer interactions, thereby maintaining the spatiotemporal pattern of gene activity. However, rearrangements of the TADs boundaries do not always lead to significant changes in the activity pattern. Here, we investigated the consequences of the TAD boundaries deletion on the expression of developmentally important genes encoding tyrosine kinase receptors: Kit, Kdr, Pdgfra. We used genome editing in mice to delete the TADs boundaries at the Kit locus and characterized chromatin folding and gene expression in pure cultures of fibroblasts, mast cells, and melanocytes. We found that although Kit is highly active in both mast cells and melanocytes, deletion of the TAD boundary between the Kit and Kdr genes results in ectopic activation only in melanocytes. Thus, the epigenetic landscape, namely the mutual arrangement of enhancers and actively transcribing genes, is important for predicting the consequences of the TAD boundaries removal. We also found that mice without a TAD border between the Kit and Kdr genes have a phenotypic manifestation of the mutation -- a lighter coloration. Thus, the data obtained shed light on the principles of interaction between the 3D chromatin organization and epigenetic marks in the regulation of gene activity.

genetics↗

bioRxiv2021

Evaluation of the OsTIR1 and AtAFB2 AID systems for chromatin protein degradation in mammalian cells

Auxin-inducible degron (AID) system is a promising tool for dynamic protein degradation. In mammalian cells, this approach has become indispensable to study fundamental molecular functions, such as replication, chromatin dynamics or transcription, that are otherwise difficult to dissect. We present evaluation of the two prominent AID systems based on OsTIR1 and AtAFB2 auxin receptor F-box proteins (AFBs). We analyzed degradation dynamics of cohesin/condensin complexes subunits in mouse embryonic stem cells (mRad21, mSMC2, mCapH, mCapH2) and human haploid HAP1 line (hRad21, hSMC2). Double antibiotic selection helped to achieve high homozygous AID targeting efficiency for all genes, ranging from 11 to 77%. We found that the main challenge for successful protein degradation is obtaining cell clones with high and stable AFB expression levels due to mosaic expression of AFBs, which also tends to decline with passages in the absence of constant puromycin selection, even at the AAVS1 safe-harbor locus. Comparing two AFBs, we found that OsTIR1 system showed weak dynamics of protein degradation. At the same time, AtAFB2 approach was very efficient even in random integration. Other factors such as degradation dynamics and low basal depletion were also in favor of AtAFB2 system. Our main conclusion is that repeated addition of puromycin to culture medium prevents AtAFB2 silencing and restores auxin sensitivity, facilitating robust protein degradation. We hope that our report will be useful for researchers that plan to establish AID method in their lab.

molecular biology↗