Super-resolution imaging reveals three-dimensional folding dynamics of the -globin locus upon gene activation
Journal of Cell Science
Running head CLSM and 3D-SIM show chromatin dynamics Keywords Chromatin dynamics, super-resolution, 3D structured illumination microscopy, long-range chromatin interaction Journal of Cell Science Accepted manuscript Summary The chromatin architecture is constantly changing due to cellular processes such as cell proliferation, differentiation and changes in the expression profile such as gene activation or silencing. Unraveling the changes that occur in the chromatin structure during these
... during these processes has been a topic of interest for many years. It is known that gene activation of large gene loci is thought to occur by means of active looping mechanism. It was also shown for the β-globin locus that the gene's promotor interacts with an active chromatin hub by means of an active looping mechanism. This predicts that the locus changes in 3D nuclear volume occupation and chromatin shape. In search to visualize and measure these dynamic changes in chromatin structure of the β-globin locus, we used a 3D DNA-FISH method in combination with 3D image acquisition to volume render fluorescent signals into 3D objects. These 3D chromatin structures were geometrically analyzed and results prior to and after gene activation were quantitatively compared. Confocal and super-resolution imaging reveal that the inactive locus occurs in several different conformations. These conformations change in shape and surface structure upon cell differentiation into a more folded and rounded structure that has a substantially smaller size and volume. These physical measurements represent the first nonbiochemical evidence that upon gene activation an actively transcribing chromatin hub is formed by means of additional chromatin looping. Journal of Cell Science Accepted manuscript Chromatin is organized into regions of uncondensed euchromatin and more condensed heterochromatin in the cell nucleus (Fedorova and Zink, 2008; Schneider and Grosschedl, 2007). It has also been shown that heterochromatin appears to be more prevalent near the nuclear envelope but not at the nuclear pores (Schermelleh et al., 2008). Most of the active genes and gene rich regions are situated in the euchromatin. Heterochromatin contains only a few active genes (gene deserts) but has many repetitive sequences such as centromeres. In spite of this clear morphologically visible distinction in chromatin compaction, all chromosomes are contained in their own nuclear space, the chromosome territories, of which each territory contains a preferred arrangement of gene rich and gene poor regions (Cremer and Cremer, 2006a; Cremer and Cremer, 2006b). This grouping of transcriptionally active versus inactive domains can also be found in smaller regions on single chromosomes where gene rich and gene poor (deserts) domains of ~5Mb can be found (Ferreira et al., 1997; Zink et al., 1999). In spite of the more or less fixed nuclear position of chromosomes, a single chromatin fiber is very flexible and can be present in multiple 3D conformations. It is known that neighboring DNA sequences can activate gene transcription in cis by means of looping of the DNA, bringing promoter regions in close vicinity to the transcription start site of the genes. Examples for sub-mega base interactions by means of a looping mechanism are the V(D)J recombination in T cells and B cells (Jhunjhunwala et al., 2008), the Th2 LCR (Spilianakis et al., 2005), the H19 imprinting control region (Alzhanov et al., 2010) and the β-haemoglobin gene cluster (de Laat et al., 2008; Tolhuis et al., 2002). The mouse β-globin locus (Fig. 1A) contains four β-globin like genes that are arranged on the DNA in the order of their developmental expression and is embedded within a cluster of olfactory receptor (mOR) genes which are silenced in erythroid cells. The Locus Control Region (LCR) contains a cluster of erythroid specific cis-regulatory elements which are essential for high globin expression and are located upstream of the globin genes (Fiering et al., 1995; Grosveld et al., 1987). Next to the DNase I hypersensitive sites (HS) in the LCR the β-globin locus contains three additional HS clusters flanking the locus, two are positioned at 85 and 84Kb (5'HS-85/-84) and 62 and 60Kb (5'HS-62/-60) upstream of the εy gene and one approx 20kb downstream side of the most 3' β-gene (3'HS1) (Bulger et al., 1999). The first changes before the activation of globin transcription is the binding of transcription factors and their cognate chromatin modification proteins to the different HS sites. Chromatin conformation capture (3C) experiments showed that interacting loops are formed (a chromatin Journal of Cell Science Accepted manuscript another. As this is very time consuming we used an earlier described 3D DNA-FISH technique (Rauch et al., 2008; Solovei et al., 2002) and developed this further to perform a quantitative 3D size and shape analysis on the chromatin region containing the β-globin locus that changes conformation upon gene activation. To do this we used a 128 or a 175 Kb fluorescent labeled probe that contains the LCR and β-globin genes and included up-and downstream regions that were shown by 3C to be involved in the formation of a chromatin hub. The fluorescent signals were volume rendered into a 3D object from which the geometric size and shape can be quantitatively determined and compared. Journal of Cell Science Accepted manuscript Alzhanov, D. T., McInerney, S. F. and Rotwein, P. (2010). Long range interactions regulate Igf2 gene transcription during skeletal muscle differentiation. J Biol Chem 285, 38969-77. Renom, M. A. (2011). The three-dimensional folding of the alphaglobin gene domain reveals formation of chromatin globules. Nat Struct Mol Biol 18, 107-14. Birk, U. J., Baddeley, D. and Cremer, C. (2009). Nanosizing by spatially modulated illumination (SMI) microscopy and applications to the nucleus. Methods Mol Biol 464, 389-401. A., Felsenfeld, G., Axel, R. and Groudine, M. (1999). 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