AGG/CCT interruptions affect nucleosome formation and positioning of healthy-length CGG/CCG triplet repeats

Abstract

Background: Fragile X Syndrome (FXS), the most common inherited form of mental retardation, is caused by expansion of a CGG/CCG repeat tract in the 5'-untranslated region of the fragile X mental retardation (FMR1) gene, which changes the functional organization of the gene from euchromatin to heterochromatin. Interestingly, healthy-length repeat tracts possess AGG/CCT interruptions every 9-10 repeats, and clinical data shows that loss of these interruptions is linked to expansion of the repeat tract to disease-length. Thus, it is important to understand how these interruptions alter the behavior of the repeat tract in the packaged gene.

Results: To investigate how uninterrupted and interrupted CGG/CCG repeat tracts interact with the histone core, we designed experiments using the nucleosome core particle, the most basic unit of chromatin packaging. Using DNA containing 19 CGG/CCG repeats, flanked by either a nucleosome positioning sequence or the FMR1 gene sequence, we determined that the addition of a single AGG/CCT interruption modulates both the ability of the CGG/CCG repeat DNA to incorporate into a nucleosome and the rotational and translational position of the repeat DNA around the histone core when flanked by the nucleosome positioning sequence. The presence of these interruptions also alters the periodicity of the DNA in the nucleosome; interrupted repeat tracts have a greater periodicity than uninterrupted repeats.

Conclusions: This work defines the ability of AGG/CCT interruptions to modulate the behavior of the repeat tract in the packaged gene and contributes to our understanding of the role that AGG/CCT interruptions play in suppressing expansion and maintaining the correct functional organization of the FMR1 gene, highlighting a protective role played by the interruptions in genomic packaging.

Figure 1

Schematic representations of the DNA used in this study. S1 serves as a control and contains no triplet repeat tracts. S1-CGG19 contains 19 CGG/CCG repeats (black) within the S1 flanking sequence (gray). S1-CAG19 contains 19 CAG/CTG repeats (blue) within the S1 flanking sequence (gray). S1-1AGGa, S1-1AGGb, and S1-2AGG contain AGG/CCT interruptions (orange) within the repeat tract. FMR1-CCG19, FMR1-1AGGa, FMR1-1AGGb, and FMR1-2AGG also contain CGG/CCG repeats (black) and AGG/CCT interruptions (orange), however they contain the FMR1 flanking sequence (NG_007529.1, dashed). Please refer to the supporting information for the DNA sequences.

Figure 2

Representative competitive nucleosome incorporation reactions. To analyze the ability of each substrate to incorporate into a nucleosome, competitive nucleosome incorporation assays were performed under equilibrium conditions and the products of the reaction were resolved by native PAGE. An incorporation performed in the absence of chicken nucleosomes (NCP) (−NCP) and an incorporation performed in the presence of NCP (+NCP) are shown. The incorporation assays with the S1 series of substrates are shown in panel A and the incorporation assays with FMR1 series of substrates are shown in panel B. By quantifying the amount of radioactivity in both the incorporated DNA and free DNA bands, the ratio of incorporated DNA to free DNA was calculated for each substrate and is shown in panel C. The error bars represent the standard error for each substrate. Data represent a total of three biological replicates per substrate, each consisting of three technical replicates, for nine total values. The standard used for statistical comparison of the S1 series was the S1 substrate while the FMR1-CGG19 was used as the standard for statistical comparison of the FMR1 series. *p ≤ 0.05, **p ≤ 0.005 by Student’s T-Test.

Figure 3

Exonuclease III digestion reveals weaker interactions between the ends of the DNA and the histone core. Exo III digestion of the CGG-containing strands of the S1 substrates are shown in panel A, while digestion of the CGG-containing strands of the FMR1 substrates are shown in panel B. Reactions were performed on both free duplex (labeled in blue) and nucleosome (NCP, labeled in red) substrates. In panel A, lanes 1 contain the S1 control, lanes 2 contain S1-CGG19, lanes 3 contain S1-1AGGa, lanes 4 contain S1-1AGGb, and lanes 5 contain S1-2AGG. In panel B, lanes 1 contain FMR1-CGG19, lanes 2 contain FMR1-1AGGa, lanes 3 contain FMR1-1AGGb, and lanes 4 contain FMR1-2AGG. The marker lane consists of the Maxam-Gilbert A/G sequencing reaction performed on S1a. The location of the repeat tract and the A of the interruptions (arrows) are indicated on the right-hand side of the gel.

Figure 4

Hydroxyl radical footprinting reveals the periodicity of the DNA around the histone core. Radiolabeled samples, both free duplex (labeled in blue) and nucleosomes (NCP, labeled in red) substrates, were exposed to hydroxyl radicals, revealing a characteristic pattern of oscillating high and low reactivity as the DNA wraps around the histone core. Reactivity toward hydroxyl radical for the CGG-containing strands of the S1 substrates are shown in panel A. Reactions were performed on both free duplex (labeled in blue) and nucleosome (NCP, labeled in red) substrates. In panel A, lanes 1 contain the S1 control, lanes 2 contain S1-CGG19, lanes 3 contain S1-1AGGa, lanes 4 contain S1-1AGGb, and lanes 5 contain S1-2AGG. The marker lane consists of the Maxam-Gilbert A/G sequencing reaction performed on S1a. The location of the repeat tract and interruptions are indicated on the right-hand side of the gel. Panel B shows the reactivity at each nucleotide, generated from the gel presented in A. The dashed lines indicate the maxima of the S1 substrate and the arrows indicate the position of the A of the AGG interruptions.

Figure 5

Translational positioning curves reveal a change in the position of the dyad axis for the S1 series. Translational positioning curves were created by determining the location of and distance between each consecutive maxima and minima in the hydroxyl radical footprinting data. The locations of the maxima and minima are then graphed with respect to their periodicity. The curve reveals the variation in periodicity around the histone core, and the highest peak indicates the location of the dyad axis. In the S1 series, the translational positioning curves reveal 2–5 base pair offsets in the location of the dyad axis.

Figure 6

Hydroxyl radical footprinting reveals the periodicity of the DNA around the histone core. Radiolabeled samples, both free duplex (labeled in blue) and nucleosomes (NCP, labeled in red) substrates, were exposed to hydroxyl radicals. Reactivity toward hydroxyl radical for the CGG-containing strands of the FMR1 substrates are shown in panel A. Reactions were performed on both free duplex (labeled in blue) and nucleosome (NCP, labeled in red) substrates. In panel A, lanes 1 contain FMR1-CGG19, lanes 2 contain FMR1-1AGGa, lanes 3 contain FMR1-1AGGb, and lanes 4 contain FMR1-2AGG. The marker lane consists of the Maxam-Gilbert A/G sequencing reaction performed on S1a. The location of the repeat tract and interruptions are indicated on the right-hand side of the gel. Panel B shows the reactivity at each nucleotide, generated from the gel presented in A. The arrows indicate the position of the A in the AGG interruptions.