Stephen M. Hart

T:G Mismatch-specific Thymine-DNA Glycosylase Potentiates Transcription of Estrogen-regulated Genes through Direct Interaction with Estrogen Receptor α

Nuclear receptors (NR) classically regulate gene expression by stimulating transcription upon binding to their cognate ligands. It is now well established that NR-mediated transcriptional activation requires the recruitment of coregulator complexes, which facilitate recruitment of the basal transcription machinery through direct interactions with the basal transcription machinery and/or through chromatin remodeling. However, a number of recently described NR coactivators have been implicated in cross-talk with other nuclear processes including RNA splicing and DNA repair. T:G mismatch-specific thymine DNA glycosylase (TDG) is required for base excision repair of deaminated methylcytosine. Here we show that TDG is a coactivator for estrogen receptor α (ERα). We demonstrate that TDG interacts with ERα in vitro and in vivo and suggest a separate role for TDG to its established role in DNA repair. We show that this involves helix 12 of ERα. The region of interaction in TDG is mapped to a putative α-helical motif containing a motif distinct from but similar to the LXXLL motif that mediates interaction with NR. Together with recent reports linking TFIIH in regulating NR function, our findings provide new data to further support an important link between DNA repair proteins and nuclear receptor function.

T:G mismatch-specific thymine-DNA glycosylase (TDG) as a coregulator of transcription interacts with SRC1 family members through a novel tyrosine repeat motif

Gene activation involves protein complexes with diverse enzymatic activities, some of which are involved in chromatin modification. We have shown previously that the base excision repair enzyme thymine DNA glycosylase (TDG) acts as a potent coactivator for estrogen receptor-α. To further understand how TDG acts in this context, we studied its interaction with known coactivators of nuclear receptors. We find that TDG interacts in vitro and in vivo with the p160 coactivator SRC1, with the interaction being mediated by a previously undescribed motif encoding four equally spaced tyrosine residues in TDG, each tyrosine being separated by three amino acids. This is found to interact with two motifs in SRC1 also containing tyrosine residues separated by three amino acids. Site-directed mutagenesis shows that the tyrosines encoded in these motifs are critical for the interaction. The related p160 protein TIF2 does not interact with TDG and has the altered sequence, F-X-X-X-Y, at the equivalent positions relative to SRC1. Substitution of the phenylalanines to tyrosines is sufficient to bring about interaction of TIF2 with TDG. These findings highlight a new protein–protein interaction motif based on Y-X-X-X-Y and provide new insight into the interaction of diverse proteins in coactivator complexes.

Inhibiting estrogen responses in breast cancer cells using a fusion protein encoding estrogen receptor-α and the transcriptional repressor PLZF

Estrogen receptor α (ERα) is a ligand-inducible transcription factor that acts to regulate gene expression by binding to palindromic DNA sequence, known as the estrogen response element, in promoters of estrogen-regulated genes. In breast cancer ERα plays a central role, where estrogen-regulated gene expression leads to tumor initiation, growth and survival. As an approach to silencing estrogen-regulated genes, we have studied the activities of a fusion protein between ERα and the promyelocytic leukemia zinc-finger (PLZF) protein, a transcriptional repressor that acts through chromatin remodeling. To do this, we have developed lines from the estrogen-responsive MCF-7 breast cancer cell line in which the expression of the fusion protein PLZF-ERα is conditionally regulated by tetracycline and shows that these feature long-term silencing of the expression of several well-characterized estrogen-regulated genes, namely pS2, cathepsin-D and the progesterone receptor. However, the estrogen-regulated growth of these cells is not inhibited unless PLZF-ERα expression is induced, an observation that we have confirmed both in vitro and in vivo. Taken together, these results show that PLZF-ERα is a potent repressor of estrogen-regulated gene expression and could be useful in distinguishing estrogen-regulated genes required for the growth of breast cancer cells.

Pinhole hologram and its applications

We propose the use of a pinhole-camera approach to make holograms. On recording the object beam passes through a pinhole, forming an image plane hologram, but on reconstruction the pinhole can be taken away. The reconstructed image can be programmed and processed by using optical information-processing elements placed in the position of the reconstructed pinhole or pinholes. Pinhole holography can also suppress intermodulation effects. We present brief theoretical considerations and experimental results for pinhole holograms produced for programmable holographic optical interconnects.

Deep-red holography using a junction laser and silver-halide holographic emulsion

We have investigated exposure parameters for deep-red holography using a junction laser and silver-halide holographic emulsion to record single-beam reflection phase-volume holograms recorded and reconstructed at a wavelength of 750 nm. We present measurements of the variation of diffraction efficiency and its derivative efficiency with exposure energy. These measurements are compared with the corresponding values obtained when using a He-Ne laser for recording; similar diffraction efficiencies have been obtained with both kinds of laser.

Pinhole Imaging Holograms For Optical Interconnects

We suggest the use of a pinhole imaging hologram as a new approach for optical interconnects. It can avoid the formation of intermodulation gratings and has some other advantages. On recording, the object beam passes through a pinhole thus forming an image plane hologram, but when reconstructing the pinhole can be removed. The reconstructed image can be programmed and processed by using optical information processing elements placed in the position of the reconstructed pinhole or pinholes. We present theoretical considerations and experimental results for pinhole holograms produced for holographic optical interconnects.

Erratum: Inhibiting estrogen responses in breast cancer cells using a fusion protein encoding estrogen receptor-α and the transcriptional repressor PLZF (Gene Therapy (2005) vol. 12 (452-460) 10.1038/sj.gt.3302421)

Correction to: Gene Therapy (2005) 12, 452–460. doi:10.1038/sj.gt.3302421 The above author name was published incorrectly, the correct name is given below: N Sarwar The publishers apologise for this error.