Effect of Expansion on Cystatin B Expression

The cystatin B gene is ubiquitously expressed with high levels of expression comparable to housekeeping genes in both human and mouse (Pennacchio et al. 1996; Lalioti et al. 1997b; Pennacchio and Myers 1997; Hsiao et al. 2001). Sensitive RNase protection experiments showed that the expression of cys-tatin B is greatly reduced in blood leukocytes from patients homozygous for the repeat expansion (Lalioti et al. 1997b) (Fig. 5). Antibody staining for cystatin B in the brain of EPM1 patients also showed reduced expression (Kinne et al. 2002). Some lymphoblastoid and fibroblast cell lines from EPM1 patients display reduced cystatin B expression, whereas in others normal cystatin B expression was restored following growth in culture (Pennacchio et al. 1996; Bespalova et al. 1997a; Lafreniere et al. 1997; Lalioti et al. 1997b). It is possible that the dodecamer repeat downregulates cystatin B expression in primary cells but occasionally fails to do so after the cells are transformed and/or cultured. Culture conditions and other unknown factors cause cell lines to acquire phenotypes different from those of the cells of origin. In these cell types, cystatin B expression, as a response to dodecamer expansion regulation, may vary. This is supported by further in vitro promoter assays showing that the dodecamer repeat shuts down expression of reporter genes in some cell types but not in others (Lalioti et al. 1999). Lymphoblastoid cell lines from the CEPH family, carrying the intermediate dodecamer expansions with 12 to 17 copies of the repeat also show reduced cystatin B expression (Alakurtti et al. 2000). The expression of cystatin B in primary cells of these unaffected individuals has not been examined; therefore, it is not known whether the expression in their cell lines is at all modified by growth in culture. As a result, it probe protected fragment

211 bp_f

102 bp

-161 bp

CSTB

-102 bp blood leukocyte RNA

cell line RNA

Fig. 5 Quantitation of cystatin B expression in EPM1 and normal samples. a Schematic representation of the plasmid used RNase protection experiment to produce the ribo-probe. The transcribed (probe) and protected fragments are shown with arrows under the plasmid. b Autoradiogram showing the cystatin B (CSTB) expression relative to that of TATA binding protein (TBP, control probe) in blood leukocyte RNA from patients and controls (N), or in lymphoblastoid cell lines. EPM1-11d, EPM1-11e, EPM1-11a, and EPM1-11c are siblings; EPM1-11d and EPM1-11e are homozygous for the expansion; EPM1-11a and EPM1-11c are unaffected. In blood leukocytes, there is a marked reduction of cystatin B RNA in patients, compared with that in controls. In cell lines the expression is either normal or slightly reduced. For patients 11d, and 12, there are both blood and lymphoblastoid cell lines data (asterisks). The bold arrow and bracket on the side highlight the aberrant protected products in patient EPM1-05, who is a compound heterozygote for a repeat expansion and a splicing mutation (IVS1-1G > C)

-161 bp

CSTB

-102 bp blood leukocyte RNA

cell line RNA

Fig. 5 Quantitation of cystatin B expression in EPM1 and normal samples. a Schematic representation of the plasmid used RNase protection experiment to produce the ribo-probe. The transcribed (probe) and protected fragments are shown with arrows under the plasmid. b Autoradiogram showing the cystatin B (CSTB) expression relative to that of TATA binding protein (TBP, control probe) in blood leukocyte RNA from patients and controls (N), or in lymphoblastoid cell lines. EPM1-11d, EPM1-11e, EPM1-11a, and EPM1-11c are siblings; EPM1-11d and EPM1-11e are homozygous for the expansion; EPM1-11a and EPM1-11c are unaffected. In blood leukocytes, there is a marked reduction of cystatin B RNA in patients, compared with that in controls. In cell lines the expression is either normal or slightly reduced. For patients 11d, and 12, there are both blood and lymphoblastoid cell lines data (asterisks). The bold arrow and bracket on the side highlight the aberrant protected products in patient EPM1-05, who is a compound heterozygote for a repeat expansion and a splicing mutation (IVS1-1G > C)

has not yet been determined beyond what threshold of cystatin B expression EPM1 symptoms are initiated.

Sensitive and quantitative in vitro reporter assays have shown that a repeat as short as 19 copies results in tenfold downregulation of the gene (Alakurtti et al. 2000).

Mechanism of Transcriptional Repression

The experiments already outlined provide evidence that the dodecamer repeat expansion has a direct effect on cystatin B transcription. Possible ex planations for the reduced transcription include altered spacing of promoter elements, hypermethylation, altered chromatin structure, and recruitment of transcription repressors to the repeat sequence.

The cystatin B minimal promoter was mapped using reporter gene assays and serial deletions of upstream sequences (Lalioti et al. 1999; Alakurtti et al. 2000). This approach demonstrated that there are important transcription factor binding sites or other regulatory sequences upstream of the dodecamer repeat. It was hypothesized that the insertion of a large DNA fragment could alter the spacing of transcription factor binding sites and/or the transcription initiation complex and result in gene suppression (Lalioti et al. 1999) (Fig. 6). This hypothesis was supported by the finding that a different sequence of the

3 copies

50 copies or foreign DNA

pE1(2)-luC

pN1-kanF(R)

pNI-PWPR

Fig. 6 Transcriptional repression of the expanded cystatin B promoter. a Schematic representation of the constructs. A 3.1-kb fragment of the cystatin B promoter containing three dodecamer repeats was cloned upstream of the luciferase reporter gene. A similar construct containing 50 copies of the repeat or heterologous DNA insertions instead of the repeat was also engineered. b Relative promoter activity in two different neuroblastoma cell lines. The activity of the wild-type promoter is set at 100%. Asterisks indicate loss of promoter activity in the SK-N-BE cell line due to expansion or introduction of heterol-ogous DNA. c Model of cystatin B transcriptional repression due to spacing of promoter elements or recruitment of repressors. For simplicity, all other regulators are referred as a basal transcription complex and are shown as vertically striped ovals. The dodecamers are shown as hatched boxes. An "activator" is shown as a dotted ball, and can normally interact with the complex. When the distance is increased the activator is no longer able to interact with the complex and activate transcription. Alternatively, the repeat may be able to bind transcriptional repressors gray octagons. The position of the critical AP1 binding site (Lalioti et al. 1999; Alakurtti et al. 2000) is shown

Fig. 6 Transcriptional repression of the expanded cystatin B promoter. a Schematic representation of the constructs. A 3.1-kb fragment of the cystatin B promoter containing three dodecamer repeats was cloned upstream of the luciferase reporter gene. A similar construct containing 50 copies of the repeat or heterologous DNA insertions instead of the repeat was also engineered. b Relative promoter activity in two different neuroblastoma cell lines. The activity of the wild-type promoter is set at 100%. Asterisks indicate loss of promoter activity in the SK-N-BE cell line due to expansion or introduction of heterol-ogous DNA. c Model of cystatin B transcriptional repression due to spacing of promoter elements or recruitment of repressors. For simplicity, all other regulators are referred as a basal transcription complex and are shown as vertically striped ovals. The dodecamers are shown as hatched boxes. An "activator" is shown as a dotted ball, and can normally interact with the complex. When the distance is increased the activator is no longer able to interact with the complex and activate transcription. Alternatively, the repeat may be able to bind transcriptional repressors gray octagons. The position of the critical AP1 binding site (Lalioti et al. 1999; Alakurtti et al. 2000) is shown same size inserted in the cystatin B promoter instead of the dodecamer repeat could suppress expression in a similar manner (Lalioti et al. 1999).

Methylation of CpG sites is a characteristic feature of the fragile X repeat (Oberle et al. 1991; Knight et al. 1993); however, both the HpalUMspl CpG islands throughout the cystatin B genomic area and the dodecamer repeat are unmethylated (Lalioti et al. 1997b; Weinhaeusel et al. 2003).

Secondary structures such as hairpins, tetraplexes, and l-motif structures of the expanded dodecamer repeat (Pataskar et al. 2001a,b; Saha and Usdin 2001) are likely to play a role in repeat instability. It is plausible that such structures also modify chromatin, making it inaccessible to transcription factors or other regulatory proteins, thus diminishing transcription (Li et al. 2004).

Recruitment of transcriptional repressors to the expanded repeat is another appealing, yet untested possibility. The repeat sequence contains several binding sites for the SP1 transcription factor (Lalioti et al. 1999; Alakurtti et al. 2000), which are multiplied upon expansion.

While spacing of promoter elements was shown to downregulate cystatin B expression, the reduction seen in EPM1 patients may be due to the synergistic effect of more than one mechanism.

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