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First published online March 2, 2004
doi: 10.1242/10.1242/jcs.00976

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Chromatin loops are selectively anchored using scaffold/matrix-attachment regions

¹ Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI 48202, USA
² Department of Pathology, Wayne State University School of Medicine, Detroit, MI 48202, USA
³ Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48202, USA
⁴ Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI 48202, USA
⁵ German Research Center for Biotechnology, RDIF/Epigenetic Regulation, Mascheroder Weg 1, 38124 Braunschweig, Germany
⁶ SeeDNA Biotech Inc, Windsor, ON N9A 4J2, Canada

View larger version (65K): [in a new window]   Fig. 1. The loop formation patterns of the lambda phage insert and the S/MAR-IFNB1 insert. Integrated lambda loops (A) and the integrated IFNBI construct loops (B) visualized with FISH probes to compare the loop size. (A) The lambda phage insertion (an example of non-S/MAR integrated construct) is shown as a green color that illustrates a large extended loop; the red color distinguishes the nuclear matrix. The integrated lambda loop detected as large released loops seems to lack endogenous MARs, and the fortuitous genomic MARs adjacent to the integrated sites are probably serving as anchoring sites. (B) The S/MAR-containing IFNB construct is shown as red and green signals (two-color FISH) in loop patterns closely resembling the endogenous loop pattern. The darker DAPI staining region in (B) represents the nuclear matrix and the lighter blue surrounding area illuminates the loop region. The IFNB1 construct displayed considerably smaller loop sizes than that of the lambda phage insert even though the total size of the insert was the same as the lambda insert. This is most probably due to the availability of S/MAR sequences within the IFNB1 construct.

View larger version (68K): [in a new window]   Fig. 2. FISH detection of inserted S/MARs (from the human IFN-ß locus) following DNase I digestion of the halo, demonstrating different nuclear matrix association patterns. The yellow FISH signals of the S/MAR anchor sequences show that they are tightly bound to the nuclear matrix and are therefore protected from nuclease digestion. (A) A 100-copy S/MAR-containing insert following DNase I digestion that displays multiple FISH signals in a pattern of many yellow dots. Because the halo or loop portion has been digested away, this pattern indicates that many but not all the S/MAR copies were tightly bound and were positioned to serve as loop anchors (indicated by arrows). (B) The FISH signal of a single-copy insert following DNase I digestion displays a single yellow spot localized to the nuclear matrix (indicated by the arrow). The great majority of single-copy S/MAR-containing inserts were detected on the nuclear matrix, whereas multiple-copy S/MAR-containing inserts all had a portion of the S/MARs associated with the nuclear matrix as well as detected on the loop. This illustrates that, regardless of the number of copies, those sequences that are anchored to the nuclear matrix are indeed strongly anchored and resistant to DNase I digestion.

View larger version (33K): [in a new window]   Fig. 3. The distribution of a multi-copy S/MAR-containing transgene on somatic loops as detected by FISH. (A) The majority of green FISH signals representing non-anchoring S/MAR sequences are located on the chromatin loop and not the nuclear matrix (NM) region. There is only one FISH signal that appears to be attached to the nuclear matrix. If all inserted S/MARs anchor on the nuclear matrix, all the FISH signals should be restricted to the nuclear matrix region. (B) Diagram of (A) illustrating the position of the nuclear matrix, the chromatin loops and the FISH signals. FISH analysis clearly demonstrated that the transgenic S/MARs were present in both the loop and nuclear matrix regions demonstrating that, when multiple S/MAR copies are available in tandem fashion, they do not all associate with the nuclear matrix.

View larger version (19K): [in a new window]   Fig. 4. (A) Expression results of the transgene by northern analysis: RNA was isolated from human, non-transgenic and transgenic mouse testes analyzed by northern analysis using PCR products specific to the human PRM1, PRM2 and conserved ß-actin transcripts as probes. Autoradiographic images were quantitated and the mean integrated intensities standardized to the 28S ribosomal RNA. The relative level of each of the PRM1, PRM2 transcripts in the transgenic mice were similar to that observed for human testes, indicating that only one copy of the transgene was being expressed. ß-actin, which hybridizes to both the mouse and human transcripts, served as the control. (B) Comparison of relative IFN-ß production and the copy number of transgenes. The IFN-ß protein level was determined by an IFN test. Four transfected cell lines with 2, 2-4, 20 and 100 copies of transgenes respectively, showing the non-linear relationship between the expression level and the number of insertions.

View larger version (28K): [in a new window]   Fig. 5. Distribution patterns of the S/MAR-IFNB1 insert detected by FISH. (A) Two independently integrated copies are clearly visible by FISH detection as two light spots within the nuclear matrix (indicated by the arrow). (B) Two independently integrated copies are clearly visible by FISH detection as two light spots on the loop portion (indicated by the arrow). (A,B) Even though the majority of single-copy integrations were detected on the nuclear matrix, some (a minority) were detected on the loop. (C) Approximately 20 integrated copies visualized by FISH detection as a large cluster of light blue spots located at the nuclear matrix and loop regions (indicated by the arrow). (D) Approximately 100 integrated copies visualized by FISH detection. The FISH signals of the S/MAR in the 100-copy construct were localized to both the nuclear matrix region (large light blue spot) and the loop region (multiple light spots). (C,D) Many multiple copy integrations serve as anchors (matrix location) whereas others do not (loop location). These data suggest that S/MARs are selectively used as anchors.

View larger version (43K): [in a new window]   Fig. 6. Two-color FISH showing the loop configuration changes of a 300 kb genomic region. (A) The V-shaped configuration of both red and green color probes were anchored on the nuclear matrix. (B,C) The linear-shaped configuration with only one probe (red or green) anchored on the nuclear matrix whereas the adjacent probe was not anchored on the matrix. The explanation for the configuration changes is that the anchor was not fixed.

View larger version (11K): [in a new window]   Fig. 7. A proposed model for the selective use of S/MAR for transcription/replication regulation. The left panel shows a gene located on the loop with a S/MAR in close proximity. When functional demands require the specific association of this gene with the transcriptional machinery located on the nuclear matrix, the S/MAR moves the gene to the nuclear matrix, thereby initiating gene expression (center panel). Following initiation, the gene is pulled in through the transcriptional machinery, thus completing the process (right panel). There are two types of S/MARs. Functional S/MARs serve as mediators to bring genes onto the nuclear matrix. Structural S/MARs serve as anchors, which are less dynamic compared with functional S/MARs.