1. A Functional Enhancer Suppresses Silencing of a Transgene and Prevents Its Localization Close to Centromeric Heterochromatin 
Claire Francastel, Mark C Walters, Mark Groudine, David I.K Martin 
Cell 
Volume 99, Issue 3, Pages (October 1999)
DOI: /S (00)

2. Figure 1
DNA Constructs Used to Evaluate the Role of Transcription Factors in Maintaining Expression
5′HS2 consists of a 1 kb SmaI/BglII fragment (S/Bg), and the core enhancer is a 380 bp XbaI/HindIII fragment (X/H). The major binding sites in the core enhancer are boxed. Part of the core enhancer sequence is shown, and the bases modified by oligonucleotide-directed mutagenesis are indicated. The two single base mutations in the tandem NF-E2-binding site (mNF-E2) have been shown to abolish NF-E2 binding to this site. The GATA and the CACC sites have been scrambled. m3sites is a combination of mutations in the GATA, CACC, and GT boxes, leaving the NF-E2 site intact. m4sites disrupts all major binding sites in the core enhancer. The (HS2core)γ construct links the HindIII/XbaI fragment to the γ-globin promoter, preserving the spacing between the Xba site and the 3′end of the promoter found in the constructs with the full-length enhancer. 5′HS2 or the different mutated versions of it were linked to the γ-globin promoter and inserted in the single LoxP or pL1L2 vectors, for targeted reintegration by Cre.
Cell  , DOI: ( /S (00) )

3. Figure 2
Site-Specific Integration of β-Geo Constructs by Expression Trap Strategy or RMCE
(A) Expression trap strategy. A parental β-geo construct, in which promoter and enhancer sequences were flanked by LoxP elements, was integrated into the genome of K562 erythroleukemia cells. Transient expression of Cre recombinase deletes promoter and enhancer, leaving a single LoxP element upstream of β-geo. This LoxP site can be then used as a trap for integration of the enhancer/promoter cassettes described in Figure 1. Reintegration of a promoter will result in expression of β-geo and so can be selected for with G418.
(B) RMCE. A CMV/HyTK construct flanked by two incompatible LoxP elements is integrated into the K562 genome. A second plasmid, in which β-geo and the control elements to be tested are also flanked by the incompatible LoxP elements, is then cotransfected with a Cre expression plasmid. Recombination between compatible LoxP elements exchanges the β-geo cassettes and CMV/HyTK cassette; this event can be selected for with G418 and Gancyclovir and confirmed by Southern blot.
Cell  , DOI: ( /S (00) )

4. Figure 3
Contribution of Core Enhancer Motifs to Suppression of Transgene Silencing
FACS-Gal assays of K562 cells carrying wild-type and mutant 5′HS2 enhancer constructs at three integration sites. At sites 6.2 and C30, the intact 5′HS2 enhancer suppresses silencing of the β-geo reporter; mutations in the core enhancer abolish this effect. At site 1, little silencing occurs even in the absence of the enhancer. Cells were removed from G418 selection at time point zero, at which all cells are expressing. Proportion of expressing and silent viable cells was measured with FACS-Gal. The silencing of the different constructs over time is represented as the percentage of expressing cells at a given time (y axis) versus the time in weeks during which cells were expanded in the absence of G418 (x axis). Each point represents the mean of at least two clones per construct and two or three independent experiments. Error bars represent SD.
Cell  , DOI: ( /S (00) )

5. Figure 4 CpG Methylation Analysis of the Transgene
MspI/HpaII digests of genomic DNA from expressing and silent cells (separated by FACS-Gal), Southern blotted and probed with the transgene. Both enzymes recognize the sequence CCGG; when the internal C is methylated, MspI but not HpaII cleaves the sequence (methylation of the external C inhibits both enzymes). An identical digestion pattern with the two enzymes indicates a lack of CpG methylation, whereas the presence of large HpaII fragments indicates methylation. This analysis shows that the transgene is heavily methylated in silent cells (−) and has little CpG methylation in expressing cells (+). There is a small amount of CpG methylation in the expressing transgene at site C30. Mutation of the NF-E2 site in the core 5′HS2 does not affect the methylation pattern of the active transgene.
Cell  , DOI: ( /S (00) )

6. Figure 5 DNase I and RE Accessibility Analysis of the Transgene
(A) Map of the transgene showing DNase I hypersensitive and restriction enzymes sites. The large and small arrows indicate the hypersensitive sites at the enhancer and the promoter, respectively. BspHI cuts in the NF-E2 site of 5′HS2, and HindIII cuts at the 3′ end of the γ-globin promoter. EcoRV or PstI, which cuts only once in the transgene, was used to digest genomic DNA prepared from nuclei. The 1 kb fragment for LacZ, used as a probe, is indicated.
(B) Silencing is associated with loss of DNase I hypersensitivity at the promoter and the enhancer; mutation of core enhancer motifs abolishes the 5′HS2 hypersensitive site. Nuclei from expressing (+) or nonexpressing (−) sorted populations of cells carrying the transgene at site 6.2 were digested with increasing amounts of DNase I; genomic DNA was then isolated, cut with EcoRV, Southern blotted, and probed with the transgene. The presence of a hypersensitive site at the promoter generates a 1.2 kb fragment (arrow), and at the enhancer a 1.8 kb fragment (arrowhead). The silent transgene (−) is relatively insensitive to DNase I treatment compared to active transgenes (+) in which an HS is detected at the promoter. The enhancer HS is present in a construct containing a wild-type 5′HS2 and disappears with mutation of the NF-E2 site, whereas disruption of the GATA sites does not affect its formation.
(C) Restriction enzyme accessibility is lost with silencing but does not require an intact enhancer. Nuclei were digested with BspHI or HindIII; genomic DNA was extracted and digested with PstI. Cleavage at the BspHI site generates a 3.8 kb fragment, and at the HindIII site a 3.5 kb fragment. The transgene is accessible to restriction enzyme digestion only in active transgenes. Mutation of the NF-E2 site does not affect this accessibility.
Cell  , DOI: ( /S (00) )

7. Figure 6 Localization of the Transgene Relative to Centromeres
Fluorescence in situ hybridization (FISH) of K562 cells with probes hybridizing to the transgene (green) and the centromeres (red). DAPI staining is shown as blue.
(A) Metaphase spreads from cells carrying the HS2γ construct integrated at sites 6.2, C30, and 1. The transgene integration sites are on the long arms of three different chromosomes; none of the sites are proximal to a centromere or a telomere.
(B) Images of interphase nuclei from cells carrying a construct containing an intact 5′HS2 (HS2γ) or 5′HS2 in which the NF-E2 site was disrupted (mNF-E2), integrated at sites 6.2, C30, and 1. The pictures shown represent the projection of 20 Z sections taken through the nucleus. In sites 6.2 and C30, the green signal for the transgene is found at a distance from a red signal for centromeres in expressing cells (+), whereas it is in close proximity in silent cells (−) or when the NF-E2 site is disrupted (mNF-E2). In site 1, the spatial position of the transgene relative to centromeres is similar for all three examples.
Cell  , DOI: ( /S (00) )

8. Figure 7 Distance between the Transgene and the Closest Centromere
Distances between the transgene and the nearest centromeric signal were measured for each construct from about 50 interphase nuclei like those shown in Figure 5. Horizontal bars represent the 10th, 25th, 50th (median), 75th, and 90th percentiles, and p represents the p value for a pair of samples (see Experimental Procedures). This type of representation indicates that 50% of the population is found between the 25th and 75th percentiles, and represented as a box. Silent cells are represented by white boxes. Expressing cells are divided into two categories. Gray boxes denote expressing cells in which the transgene contains either wild-type 5′HS2 or 5′HS2 with a mutation that does not affect its ability to retard silencing (mGATA). Expressing cells carrying a transgene with a mutation in 5′HS2 that impairs its ability to retard silencing (mNF-E2 and m4Sites) are shown as gray boxes with hatched bars. The box plots show that silencing is associated with significantly closer proximity of the transgene and centromeric signals, at sites 6.2 and C30 but not site 1. Disruption of the enhancer, as reflected in loss of its ability to suppress silencing, is associated with significantly closer proximity of the two signals in active cells when compared with active cells carrying a functional enhancer. This occurs at sites 6.2 and C30, but not at site 1.
Cell  , DOI: ( /S (00) )