The microsatellite sequence (CT)n x (GA)n promotes stable chromosomal integration of large tandem arrays of functional human U2 small nuclear RNA genes

Abstract

The multigene family encoding human U2 small nuclear RNA (snRNA) is organized as a single large tandem array containing 5 to 25 copies of a 6.1-kb repeat unit (the RNU2 locus). Remarkably, each of the repeat units within an individual U2 tandem array appears to be identical except for an irregular dinucleotide tract, known as the CT microsatellite, which exhibits minor length and sequence polymorphism. Using a somatic cell genetic assay, we previously noticed that the CT microsatellite appeared to stabilize artificial tandem arrays of U2 snRNA genes. We now demonstrate that the CT microsatellite is required to establish large tandem arrays of transcriptionally active U2 genes, increasing both the average and maximum size of the resulting arrays. In contrast, the CT microsatellite has no effect on the average or maximal size of artificial arrays containing transcriptionally inactive U2 genes that lack key promoter elements. Our data reinforce the connection between recombination and transcription. Active U2 transcription interferes with establishment or maintenance of the U2 tandem array, and the CT microsatellite opposes these effects, perhaps by binding GAGA or GAGA-related factors which alter local chromatin structure. We speculate that the mechanisms responsible for maintenance of tandem arrays containing active promoters may differ from those that maintain tandem arrays of transcriptionally inactive sequences.

FIG. 1

Constructs and ligation conditions. (A) An intact U2 repeat unit and the five fragments used to construct artificial tandem arrays. Key restriction sites are shown (Bg, BglII; K, KpnI; Ea, EagI; A, AseI; N, NdeI; Hc, HincII; Sm, SmaI; D, DraI; RI, EcoRI; Bm, BamHI; Sf, SfuI; F, FokI). The 5′ and 3′ ends of each construct are BglII and BamHI, respectively. Sites in parentheses were destroyed by ligation. Open arrow, U2 snRNA gene; asterisk, U87C mutation; open circle and square, DSE and PSE, respectively; hatched rectangle, d(CT)_n · d(GA)_n or CT microsatellite, where n ≈ 75; shaded rectangle, truncated L1 repeat. LTR, long terminal repeat. (B) Representative ligation reactions. The mU2 and mU2+CT constructs were subjected to the two-step ligation as described in Materials and Methods, and the products were separated by agarose FIGE under conditions that resolve both large and small fragments. FIGE gels were then blotted and probed with the labeled mU2+CT construct. Either 1.0 (left) or 0.02 (right) μg of each ligation reaction was loaded per lane. As an internal control, 100 ng of the mU2 fragment was loaded to the right of the mU2 lane in each of these panels. The residual monomer and dimer are indicated; these species account for <30% of the input fragments compared to DNA mass markers (data not shown). Size markers are shown in kilobases (center).

FIG. 2

Characterization of artificial arrays by genomic blotting. Only representative blots are shown. The probe was the labeled mU2 repeat unit in every case; signal strength was corrected for the absence of PSE and DSE sequences in mU2ΔDSEΔPSE lines. Exogenous and resident U2 gene fragments are indicated (in kilobases). (A) mU2 cell lines, SfuI digest; (B) mU2+CT cell lines, SfuI digest; (C) mU2ΔDSEΔPSE cell lines, SmaI digest; (D) mU2ΔDSEΔPSE+CT cell lines, DraI digest; (E) mixed mU2 and mU2+CT cell lines, SfuI digest; (F) mixed mU2 and CT cell lines, SfuI digest.

FIG. 3

Distribution of cell lines by number of exogenous marked U2 genes. For each cell line, the ratio of marked U2 genes to resident U2 genes was determined by phosphorimager analysis of genomic blots, and the number of marked U2 genes was calculated by assuming 22 resident U2 genes in pseudodiploid HT1080 cells (49). In representative cases, pseudodiploidy for chromosome 17 was confirmed by fluorescent in situ hybridization. For each transfection, a tally was made of the number of cell lines having n marked U2 genes where n ranged from 0 to 105. The tally data were plotted as the number of cell lines with n marked U2 genes versus n. Insets in each panel indicate the average n and the percentages of empty sites containing the neomycin resistance marker but no detectable U2 genes. (A) mU2 transfection; (B) mU2+CT transfection; (C) mU2ΔDSEΔPSE transfection; (D) mU2ΔDSEΔPSE+CT transfection; (E) mixed mU2 and mU2+CT transfection; (F) mixed mU2 and CT transfection.

FIG. 4

Artificial tandem arrays are integrated at a single site. Genomic DNA from representative mU2+CT cell lines was digested with BamHI, and the fragments were resolved by agarose FIGE. All tandem arrays are excised intact; BamHI does not cut the resident U2 repeat unit, nor the artificial arrays which are entirely free of tail-to-tail junctions (see Fig. 1 and text). The two resident U2 arrays are indicated; the artificial arrays (marked by asterisks) vary in size, but only one is seen in each cell line. Note that the average size of a genomic BamHI fragment is roughly 8 to 10 kb; thus, the intensity of an artificial array correlates only with the number of exogenous U2 repeats (as determined in Fig. 3) and not with the size of the artificial array excised by BamHI. Fragment sizes (kilobases) were estimated relative to Midrange Markers I (Gibco/BRL). The intense signal below 30 kb reflects a weak cross-reaction of the probe with bulk genomic DNA, possibly with abundant U2 retropseudogenes (58).

FIG. 5

The CT microsatellite was unaltered during generation of cell lines containing artificial tandem arrays of mU2+CT and mU2ΔDSEΔPSE+CT. Marked and resident arrays were isolated from the indicated cell lines by preparative FIGE. CT microsatellites were amplified by PCR in the presence of [α-³²P]dCTP, digested with MspI and MnlI, and separated by electrophoresis on a 6% denaturing acrylamide gel as described previously (33) and in Materials and Methods. Three cell lines were examined from each transfection. Control reactions with mU2ΔDSEΔPSE+CT, iU2, CT, and mU2+CT plasmid DNA or with undigested HT1080 genomic DNA are shown in the rightmost panel. An M13 sequencing ladder provided size markers (base pairs).

FIG. 6

The marked U2 genes are efficiently expressed in stable mU2 and mU2+CT lines. RNA from representative mU2, mU2+CT, and mU2ΔDSEΔPSE lines and from the parental HT1080 line was analyzed by primer extension with ddATP and a ³²P-labeled oligonucleotide complementary to positions 89 to 109 of U2 snRNA as described in Materials and Methods. Extension products corresponding to resident U2 snRNA and marked U2 snRNA are indicated on the left.