Formamide denaturation of double-stranded DNA for fluorescence in situ hybridization (FISH) distorts nanoscale chromatin structure.

As imaging techniques rapidly evolve to probe nanoscale genome organization at higher resolution, it is critical to consider how the reagents and procedures involved in sample preparation affect chromatin at the relevant length scales. Here, we investigate the effects of fluorescent labeling of DNA sequences within chromatin using the gold standard technique of three-dimensional fluorescence in situ hybridization (3D FISH). The chemical reagents involved in the 3D FISH protocol, specifically formamide, cause significant alterations to the sub-200 nm (sub-Mbp) chromatin structure.

DNA density is a better indicator of a nuclear bleb than lamin B loss.

Nuclear blebs are herniations of the nucleus that occur in diseased nuclei that cause nuclear rupture leading to cellular dysfunction. Chromatin and lamins are two of the major structural components of the nucleus that maintain its shape and function, but their relative roles in nuclear blebbing remain elusive. Lamin B is reported to be lost in blebs by qualitative data while quantitative studies reveal a spectrum of lamin B levels in nuclear blebs dependent on perturbation and cell type.

Local Volume Concentration, Packing Domains and Scaling Properties of Chromatin.

We propose the Self Returning Excluded Volume (SR-EV) model for the structure of chromatin based on stochastic rules and physical interactions that is able to capture the observed behavior across imaging and sequencing based measures of chromatin organization. The SR-EV model takes the return rules of the Self Returning Random Walk, incorporates excluded volume interactions, chain connectivity and expands the length scales range from 10 nm to over 1 micron. The model is computationally fast and we created thousands of configurations that we grouped in twelve different ensembles according to the two main parameters of the model.

Depletion of lamins B1 and B2 alters chromatin mobility and induces differential gene expression by a mesoscale-motion dependent mechanism.

Background: B-type lamins are critical nuclear envelope proteins that interact with the 3D genomic architecture. However, identifying the direct roles of B-lamins on dynamic genome organization has been challenging as their joint depletion severely impacts cell viability. To overcome this, we engineered mammalian cells to rapidly and completely degrade endogenous B-type lamins using Auxin-inducible degron (AID) technology.

Chromatin reprogramming and bone regeneration in vitro and in vivo via the microtopography-induced constriction of cell nuclei.

Topographical cues on cells can, through contact guidance, alter cellular plasticity and accelerate the regeneration of cultured tissue. Here we show how changes in the nuclear and cellular morphologies of human mesenchymal stromal cells induced by micropillar patterns via contact guidance influence the conformation of the cells' chromatin and their osteogenic differentiation in vitro and in vivo. The micropillars impacted nuclear architecture, lamin A/C multimerization and 3D chromatin conformation, and the ensuing transcriptional reprogramming enhanced the cells' responsiveness to osteogenic differentiation factors and decreased their plasticity and off-target differentiation.

Nanoscale chromatin imaging and analysis platform bridges 4D chromatin organization with molecular function.

Extending across multiple length scales, dynamic chromatin structure is linked to transcription through the regulation of genome organization. However, no individual technique can fully elucidate this structure and its relation to molecular function at all length and time scales at both a single-cell level and a population level. Here, we present a multitechnique nanoscale chromatin imaging and analysis (nano-ChIA) platform that consolidates electron tomography of the primary chromatin fiber, optical super-resolution imaging of transcription processes, and label-free nano-sensing of chromatin packing and its dynamics in live cells.