Transcription regulation by methylated DNA-binding proteins (MDBP) from the 5'-untranslated region of the human alpha-Galactosidase A gene
Description
Methylated DNA binding proteins (MDBP) are a unique class of sequence-specific DNA-binding proteins present ubiquitously in mammalian cells. A subset of the MDBP-binding sites are methylation-dependent sites, and MDBP binds better to these sites when they contain 5-methylcytosine. Because cytosine methylation has been implicated in gene inactivation, we proposed MDBP might negatively regulate transcription from its methylation-dependent sites. To test our hypothesis, we studied one such site that occurs at position +48 relative to the major transcription start site in the $5\sp\prime$-untranslated region $(5\sp\prime$-UTR) of the human $\alpha$-Galactosidase A gene, an X-linked, housekeeping gene. The bifilarly methylated site binds MDBP nearly five-fold better than the low-affinity unmethylated site. Site-specific substitutions were introduced to mutate the unmethylated wild-type MDBP site to either high-affinity methylation-independent MDBP binding sites (Up-MDBP), or to non-binding sequences (Down-MDBP) in $\alpha$-GAL A promoter-$5\sp\prime$-UTR/reporter constructs. Data from transient transfection assays of the reporter plasmids in a mammalian cell-line suggest that MDBP represses gene expression upon binding to its site in the $\alpha$-GAL A $5\sp\prime$-UTR. The NF-$\kappa$B-like binding site identified 9-bp upstream from the MDBP site might also be involved in down-regulating $\alpha$-GAL A expression, as individuals with a mutation in this site that decreased specific protein binding had higher-than-normal $\alpha$-GAL A activity in their plasma We wanted to determine whether activation domains of other transcription factors would also repress reporter gene expression from this site in the $\alpha$-GAL A promoter/reporter construct. Therefore, the +48 MDBP binding site was replaced by a GAL4 binding site and this reporter construct was cotransfected with several GAL4-based activation domain plasmids, including GAL4(1-147), GAL4(1-93), GAL4-Sp1B, GAL4-Oct-1, GAL4-VP16, GAL4-NF-kB p65, GAL4-AAD, GAL 4-AP2, GAL 4-CTF, GAL4-E1A and GAL4-RFX1. Reporter gene activity decreased many-fold in the presence of glutamine-rich activation domains (Sp1 and Oct-1) but showed a large increase in the presence of acidic activators (VP16, NF-kB p65 and GAL4-AAD). Activation domains of several transcription factors interact with different components of the transcription initiation complex. Interaction between the N-terminal glutamine-rich activation domain of MDBP/RFX1 and $\rm dTAF\sb{II}110$ was shown by gel-shift and yeast two-hybrid assays