A functional role for 4qA/B in the structural rearrangement of the 4q35 region and in the regulation of FRG1 and ANT1 in facioscapulohumeral dystrophy

Abstract

The number of D4Z4 repeats in the subtelomeric region of chromosome 4q is strongly reduced in patients with Facio-Scapulo-Humeral Dystrophy (FSHD). We performed chromosome conformation capture (3C) analysis to document the interactions taking place among different 4q35 markers. We found that the reduced number of D4Z4 repeats in FSHD myoblasts was associated with a global alteration of the three-dimensional structure of the 4q35 region. Indeed, differently from normal myoblasts, the 4qA/B marker interacted directly with the promoters of the FRG1 and ANT1 genes in FSHD cells. Along with the presence of a newly identified transcriptional enhancer within the 4qA allele, our demonstration of an interaction occurring between chromosomal segments located megabases away on the same chromosome 4q allows to revisit the possible mechanisms leading to FSHD.

Figure 1. 3C analysis of nine landmarks in the 4q35 region.

A. Map and genomic coordinates (in bp) of primer pairs used for the 3C analysis. Genes are represented by unique arrows, promoters by ovals. The D4Z4 array is shown as green block arrows. B. Control digestion on crosslinked templates. Genomic DNA was digested with BglII and amplified using the primer pairs that allow only the amplification of non-digested DNA. No PCR products were observed in the absence of the ligation step. C. The PCR amplification linear range was obtained by titration of the template concentration and number of amplification cycles. Finally, 10 ng of crosslinked template and 100 ng of control template in 15 µl of reaction mixture were used in our experiments. The PCR cycling conditions were as follows: 94°C for 3 min; 94°C for 45 sec and 58°C for 30 sec, 72°C for 50 sec, followed by a final extension at 72°C for 10 min using Taq DNA Polymerase (Invitrogen). D. The DNA GM10115A human/rodent hybrid cell line containing a single chromosome 4 was digested with BglII, ligated and then amplified using specific primer pairs to verify the accuracy of the primer pairs for the chromosome 4 sequences. E. The D4Z4 repeat cloned into the pGEM42 plasmid was amplified using one primer pair specific for D4Z4 and two different primer pairs specific for DUX4c (DUX4c1 and DUX4c2). Two different template concentrations, 100 ng and 200 ng were used for amplification.

Figure 2. 3C Analysis of the 4q35 locus.

A–B. Representation of the spatial proximity in normal (A) and FSHD (B) myoblasts. The fragment tested for crosslinking is indicated in each panel. An arbitrary score of 10 corresponds to the PCR amplification obtained using primers located on either side of the restriction site separating two adjacent fragments within the corresponding genomic segment. The Y axis indicates relative levels of interaction with the other landmarks tested which are represented along the X axis according to their localization along chromosome 4q. The data represent the average results of three independent experiments. The panels below the charts show the 3C ligation products detected by PCR amplification using specific primers. One experiment out of three independent ones is represented in the Figure. C. The differences in 3C interactions between the normal (top) and FSHD myoblasts. Only interactions which are different between the normal and FSHD myoblasts are shown.

Figure 3

A. FISH analysis on primary human myoblasts. Nuclei from normal (left panels) and FSHD (right panel) primary myoblasts were hybridized to a FR-MAR probe (arrowed dots) and counterstained with DAPI. B. The 4qA allele contains a transcriptional enhancer. The transcriptional effect of the 4qA allele was tested 48 hrs after transfection in HeLa cells. The enhancer strength was quantified relative to the luciferase activity generated by the pGL3 plasmid with the SV40 enhancer (pGL3Con). Equal amounts of the plasmids were transfected. Luciferase signals were normalized to the total protein content in the extracts. pGL3Pro, enhancer-less, empty pGL3 plasmid; 4qA1 and 4qA2, 4qA allele cloned in the enhancer-less pGL3 plasmid.

Figure 4. A–B. Models schematizing the proximity between 4q subtelomeric fragments in control (A) and FSHD (B) nuclei.