Super resolution imaging of a distinct chromatin loop in human lymphoblastoid cells

Abstract

The three-dimensional genome structure plays a fundamental role in gene regulation 19 and cellular functions. Recent studies in genomics based on sequencing technologies inferred the 20 very basic functional chromatin folding structures of the genome known as chromatin loops, the 21 long-range chromatin interactions that are often mediated by protein factors. To visualize the 22 visualization of higher-order chromatin structures for the very short genomic segments can be 29 realized by microscopic imaging. 30 31 35 et al., 1997; Tsunaka et al., 2005) that are connected by dozens of bp of linker DNA, appearing as 36a "beads on a string" structure (Olins and Olins, 1974; Kornberg, 1974; Oudet et al., 1975; Finch 37 and Klug, 1976; Bustin et al., 1976; Leuba et al., 1994). The 10 nm "beads on a string" DNA fiber is 38 then folded into higher-order chromatin structures for further chromatin compaction. However, 39 1 of 13 Manuscript submitted to eLife the organization of higher-order chromatin structures was elusive for tens of years. Although the 40 30 nm chromatin fiber was observed and suggested to be the next organizational level of the 10 41 nm fiber, it is now debatable whether it exists in vivo (Felsenfeld and Groudine, 2003; van Holde 42 and Zlatanova, 2007; Nishino et al., 2012). Recently, technologies combining biochemistry and 43 high-throughput sequencing such as Hi-C (Lieberman-Aiden et al., 2009) and ChIA-PET (Fullwood 44 et al., 2009) have been developed to characterize genome-wide landscape of long-range chromatin 45 interactions (usually from several kilobases (kb) to hundreds of kilobases) that are considered as the 46 basis of higher-order chromatin organization. Chromatin interactions suggest the looping structure 47 119 the pre-processed images. We demonstrated a new image processing algorithm Figure 2-Figure 120 Supplement 2 that identifies the coordinates of the single molecules from pre-processed iPALM 121 images (Figure 1e) and returns points localizations in a PDB file format (Figure 2a), which can be 122 used in further modeling. Basically, we extracted significant signals from the images. We measured 123 the brightness of signals in relative luminosity units which range from 0 to 255. Brightness threshold 124 was chosen manually to cut off noise. In this way, we got different sets of dots for each image. 125 Borg I, Groenen PJ, Mair P. Applied multidimensional scaling and unfolding. Springer; 2017. 330 Bustin M, Goldblatt D, Sperling R. Chromatin structure visualization by immunoelectron microscopy. Cell. 1976; 331 7(2):297-304. 332 Dempsey GT, Vaughan JC, Chen KH, Bates M, Zhuang X. Evaluation of fluorophores for optimal performance in 333 localization-based super-resolution imaging. Nature methods. 2011; 8(12):domains in mammalian genomes 335 identified by analysis of chromatin interactions. Nature. 2012; 485(7398):376. 336 Felsenfeld G, Groudine M. Controlling the double helix. Nature. 2003; 421(6921):448. 337 Finch J, Klug A. Solenoidal model for superstructure in chromatin.

FISH Going Meso-Scale: A Microscopic Search for Chromatin Domains

“…Similarly, imaging-based models of a single 13 kb loop and its 10 kb flanking regions in T-cell receptor alpha locus, probed by FISH and iPALM, showed multiple loop conformations in single cells. Still, the pairwise distances for most of the conformations reproduced inverse correlation between frequency of interactions and distance when compared with Hi-C and ChIA-PET data ( Jufen Zhu et al, 2019 ).…”

“…Heterogeneity of chromatin-folding structures within distinct loop domains has also been demonstrated in human cell line by ISH combined with serial block-face scanning electron microscopy (3D-EMISH) ( Trzaskoma et al, 2020 ), FISH with interferometric PALM (iPALM; Jufen Zhu et al, 2019 ) and loop tracing with DNA-PAINT in non-denaturing conditions ( Beckwith et al, 2021 ). In the 3D-EMISH study, BAC-probes covering the 1.7-Mb region on chromosome 7 in lymphoblastoid GM12878 cells were detected with 1.4-nm-thick streptavidin-conjugated fluoronanogold, followed by analysis of density center distribution in reconstructed ultrastructural serial images of targeted chromatin region.…”