Abstract A25: A novel spectroscopic technology to image the native chromatin nanostructure in live cells
Authors: Stypula Yolanda , Gladstein Scott , Almassalha Luay , Bauer Greta , Chandler John , Cherkezyan Lusik , Zhang Di , Subramanian Hariharan , Szleifer Igal , Backman Vadim 
Published: May 2016

Proper regulation of higher-order chromatin structure is essential for normal gene regulation and cellular function. We have previously found that the nanoscale chromatin structure is significantly altered in early and field carcinogenesis using novel spectroscopic methods in parallel with biological assays (Backman and Roy, J Cancer, 2013, 3:251-261; Subramanian et al, Cancer Res, 2009, 13:5357-63; Stypula-Cyrus et al, PLoS One, 2013, 5:e64600). This was done in fixed human and animal model samples, suggesting that genetic/epigenetic alterations can serve as the earliest marker for neoplastic transformation. While chromatin is well understood at the nucleosomal level (&lt;20nm) and chromosomal level (&gt;200nm), little is known about the higher-order chromatin structure between these length scales. Current techniques available to study cellular structures below the diffraction limit (&lt;200nm) require labeling that may alter the native cell structure, can only visualize a few molecules concurrently, and have poor temporal resolution. Here we present a new technique, BAck-Scattering Interference Spectroscopic (BaSIS) microscopy, with sensitivity to structures between 20-200nm that can quantify the dynamics of the nano-molecular organization in live cells without using exogenous labels.

The BaSIS instrument was built into a commercial inverted microscope (Leica DMIRB) equipped with a high NA oil immersion objective with broadband illumination provided by a Xenon lamp. Refractive index fluctuations are measured by sampling backscattered light at each wavelength 500-700nm using a combination of a liquid crystal tunable filter (LCTF) and a CMOS camera. HeLa and CHO cells were first imaged in petri dishes with coverslip bottoms, and then incubated with Hoechst 33342, a nuclear stain that binds to AT-rich regions of the genome and has been reported to cause double-stranded breaks (DSBs) in the DNA (Pfeiffer et al, Mutagenesis, 2000, 4: 289-302). Additionally, mock-staining experiments were performed to compare the changes in nuclear structure due to Hoechst 33342 excitation compared to UV light exposure alone. We utilized a γ-H2A.X-Alexa488 conjugated antibody after Hoechst- and mock-staining to compare observed changes in BaSIS signal with the formation of DSBs.

Using BaSIS, we show for the first time that the excitation of Hoechst 33342 immediately alters the native nuclear nanostructure and induces formation of DSBs, confirmed by the rapid phosphorylation of H2A.X. In our mock-stained control, we observed an average increase of 0.006% and 0.001% signal after UV exposure (p-value &gt; 0.5), whereas the stained cells display a 17.01% and 7.1% decrease in HeLa and CHO nuclei, respectively (p-value &lt; 0.001). Significantly, these changes in Hoechst-stained cells are detectable by BaSIS within seconds. This suggests that the mechanism responsible for the nuclear alteration detected by BaSIS is localized and immediate. We hypothesized that the observed transformation was due to the homogenization of chromatin through fragmentation and the concurrent chromatin decompaction, detected by refractive index fluctuations. Indeed, following Hoechst staining, we observed an accumulation of the γ-H2A.X antibody, whereas we observed little or no localization in the mock Hoechst-stained nuclei. Furthermore, traditional phase contrast microscopy, which is used for label-free imaging of living samples, did not show any changes in the higher-order chromatin structure following DSB formation in these experiments.

In conclusion, BaSIS is a powerful tool for studying the dynamics of chromatin nanostructure and can serve as a natural supplement to super-resolution fluorescence techniques, providing quantified information about native cellular organization. With this technique, we demonstrated that using the Hoechst DNA-binding dye causes irreversible alterations in chromatin structure at time-scales (seconds) not previously recognized. As a result, BASIS can be applied to a broad range of critical studies in chromatin research. Current and future research include: (i) mRNA transport and the accessibility of euchromatin and heterochromatin to transcription factors; (ii) why and how high-order chromatin structure changes in cancer progression; (iii) the role of nuclear architecture as an epigenetic regulator of gene expression; and (iv) the effect of metabolism on chromatin structure; (v) damage/repair mechanisms and potentially, chemotherapeutic efficacy.

Citation Format: Yolanda Stypula, Scott Gladstein, Luay Almassalha, Greta Bauer, John Chandler, Lusik Cherkezyan, Di Zhang, Hariharan Subramanian, Igal Szleifer, Vadim Backman. A novel spectroscopic technology to image the native chromatin nanostructure in live cells. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A25.