Mapping of the nuclear matrix-bound chromatin hubs by a new M3C experimental procedure

Abstract

We have developed an experimental procedure to analyze the spatial proximity of nuclear matrix-bound DNA fragments. This protocol, referred to as Matrix 3C (M3C), includes a high salt extraction of nuclei, the removal of distal parts of unfolded DNA loops using restriction enzyme treatment, ligation of the nuclear matrix-bound DNA fragments and a subsequent analysis of ligation frequencies. Using the M3C procedure, we have demonstrated that CpG islands of at least three housekeeping genes that surround the chicken α-globin gene domain are assembled into a complex (presumably, a transcription factory) that is stabilized by the nuclear matrix in both erythroid and non-erythroid cells. In erythroid cells, the regulatory elements of the α-globin genes are attracted to this complex to form a new assembly: an active chromatin hub that is linked to the pre-existing transcription factory. The erythroid-specific part of the assembly is removed by high salt extraction. Based on these observations, we propose that mixed transcription factories that mediate the transcription of both housekeeping and tissue-specific genes are composed of a permanent compartment containing integrated into the nuclear matrix promoters of housekeeping genes and a 'guest' compartment where promoters and regulatory elements of tissue-specific genes can be temporarily recruited.

Figure 1.

The principle behind the M3C protocol. (A) A schematic showing the main steps of the M3C procedure (see the text for explanations). (B) Microscope images of nuclei, nucleoids and nuclear matrices from proliferating HD3 cells. In each case, the same confocal section is shown with immunostained lamins A and C (upper row) and DNA stained by DAPI (lower row).

Figure 2.

Comparison of the results for the 3C and M3C experiments. (A) A schematic showing positions within the gene locus being studied of genes (open rectangles with arrows indicating direction of transcription), CpG islands (vertical open rectangles) and regulatory elements (closed ovals). Positions of Bam HI and BglII restriction sites are shown correspondingly by black and gray vertical lines above the scale. The horizontal tailless arrows show positions and directions of PCR primers. The primers on anchor fragments are encircled. The scale is in kilobase and ‘0’ point of the scale was arbitrarily placed at the 3′-end of the C16orf35 gene. (B) The results of the 3C experiments in which the anchor was placed on the C16orf35 CpG island. The graphs show the relative frequencies of interactions between the anchor fragment (dark gray shadowing) and upstream and downstream fragments (light shadowing). The borders between neighboring fragments are indicated by dark gray lines. The regions with a white background were not analyzed. The maximal interaction frequency (the amount of the most abundant ligation product that was normalized using 3C data for the ERCC3 gene domain) observed in experiments with the three cell lines studied was arbitrarily considered to be 100, and results were normalized to this value. Error bars represent the SEM for three independent experiments. (C) The results of the M3C experiments in which the anchor was placed on the C16orf35 CpG island. All designations are the same as in (B). The maximal interaction frequency (the amount of the most abundant ligation product) observed for each cell line in this experiment and in experiments with other anchors (Figure 4) was arbitrarily considered to be 100, and results were normalized to this value. Error bars represent the SEM for three independent experiments.

Figure 3.

A schematic illustrating the possibility of cross-ligation of nmDNA fragments from different nuclear matrix-bound chromatin hubs, and the experimental approach used to test this possibility. (A) A schematic showing the main steps of the control experiment with formaldehyde fixation of the nuclear matrix DNA complexes and subsequent analysis of ligation frequencies by normal 3C protocol. See the text for the details. (B) A schematic showing the main steps of the control experiment with lysis of non-fixed nuclear matrix with SDS. (C and D) Graphs showing the frequencies of interactions of the anchor placed on the C16orf35 CpG island with the downstream DNA fragments observed in experiments outlined in sections (A) and (B) correspondingly.

Figure 4.

The interaction frequencies observed in the M3C experiments in which the anchor was placed on different DNA fragments. (A) Anchor on the CpG island of the MPRL28 gene, (B) Anchor on the CpG island of the AXIN1 gene, (C) Anchor on the CpG island of the TMEM8 gene, and (D and E) Anchor on the promoter of the α^D gene. All designations are the same as in Figure 2. Error bars represent the SEM for at least two independent experiments.

Figure 5.

A schematic illustrating the model for a nuclear matrix-bound transcription factory in erythroid cells. CpG islands bearing promoters for the housekeeping genes C16orf35, MPRL28 and AXIN1 (indicated in the schematic Figures 1, 3 and 5) constitute a permanent compartment of a transcription factory. They are permanently bound to (partially integrated into) the nuclear matrix. The elements of a ‘guest compartment’ (the α^D gene promoter, the CpG island of the TMEM8 gene and the erythroid-specific major regulatory element) interact with the surface of the permanent compartment that is not immersed into the nuclear matrix. These interactions are not resistant to 2M NaCl extraction and become disassembled in a result of this extraction.