Hormonal regulation of metastasis-associated protein 3 transcription in breast cancer cells.

Metastasis-associated protein 3 (MTA3) is a cell type-specific subunit of the Mi-2/NuRD transcriptional corepressor complex. In breast cancer cells, MTA3 and the Mi-2/NuRD complex mediate repression of Snail, a transcription factor that promotes epithelial to mesenchymal transitions. Thus, MTA3 functions to maintain a differentiated, epithelial status in breast cancer. Interestingly, in mammary epithelial cells, MTA3 biosynthesis requires both functional estrogen receptor (ER) and estradiol. Here we have investigated the molecular basis for estrogen and ER-dependent expression of MTA3 in breast cancer cells. Molecular dissection of the MTA3 promoter using transient transfection assays identified a composite element required for high-level transcription consisting of an SP1 site in close proximity to a consensus estrogen response element half-site. Depletion of either SP1 or ER-alpha by RNA interference led to loss of MTA3 transcript in multiple breast cancer cell lines, indicating a requirement for both transcription factors in expression of endogenous MTA3. The MTA3 gene thus joins a growing list of loci regulated by both SP1 and ER.

Nuclear envelope irregularity is induced by RET/PTC during interphase.

Nuclear envelope (NE) irregularity is an important diagnostic feature of cancer, and its molecular basis is not understood. One possible cause is abnormal postmitotic NE re-assembly, such that a rounded contour is never achieved before the next mitosis. Alternatively, dynamic forces could deform the NE during interphase following an otherwise normal postmitotic NE re-assembly. To distinguish these possibilities, normal human thyroid epithelial cells were microinjected with the papillary thyroid carcinoma oncogene (RET/PTC1 short isoform, known to induce NE irregularity), an attenuated version of RET/PTC1 lacking the leucine zipper dimerization domain (RET/PTC1 Deltazip), H (V-12) RAS, and labeled dextran. Cells were fixed at 6 or 18 to 24 hours, stained for lamins and the products of microinjected plasmids, and scored blindly using previously defined criteria for NE irregularity. 6.5% of non-injected thyrocytes showed NE irregularity. Neither dextran nor RAS microinjections increased NE irregularity. In contrast, RET/PTC1 microinjection induced NE irregularity in 27% of cells at 6 hours and 37% of cells at 18 to 24 hours. RET/PTC1 Deltazip induced significantly less irregularity. Since irregularity develops quickly, and since no mitoses and only rare possible postmitotic cells were scored, postmitotic NE re-assembly does not appear necessary for RET/PTC signaling to induce an irregular NE contour.

The affinity of different MBD proteins for a specific methylated locus depends on their intrinsic binding properties.

The methyl-CpG binding domain (MBD) family of proteins was defined based on sequence similarity in their DNA binding domains. In light of their high degree of conservation, it is of inherent interest to determine the genomic distribution of these proteins, and their associated co-repressor complexes. One potential determinant of specificity resides in differences in the intrinsic DNA binding properties of the various MBD proteins. In this report, we use a capillary electrophoretic mobility shift assay (CEMSA) with laser-induced fluorescence (LIF) and neutral capillaries to calculate MBD-DNA binding affinities. MBD proteins were assayed on pairs of methylated and unmethylated duplex oligos corresponding to the promoter regions of the BRCA1, MLH1, GSTP1 and p16(INK4a) genes, and binding affinities for each case were calculated by Scatchard analyses. With the exception of mammalian MBD3 and Xenopus MBD3 LF, all the MBD proteins showed higher affinity for methylated DNA (in the nanomolar range) than for unmethylated DNA (in the micromolar range). Significant differences between MBD proteins in the affinity for methylated DNA were observed, ranging within two orders of magnitude. By mutational analysis of MBD3 and using CEMSA, we demonstrate the critical role of specific residues within the MBD in conferring selectivity for methylated DNA. Interestingly, the binding affinity of specific MBD proteins for methylated DNA fragments from naturally occurring sequences are affected by local methyl-CpG spacing.