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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-alpha. 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.

Estrogen receptor alpha (ERalpha) 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 ERalpha 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 ERalpha 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-ERalpha 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-ERalpha expression is induced, an observation that we have confirmed both in vitro and in vivo. Taken together, these results show that PLZF-ERalpha 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.

Background: The core binding factor (CBF) transcription complex, consisting of the interacting proteins RUNX1 and CBFb, is essential for normal hematopoiesis. Recent studies have shown that mutations and gene rearrangements involving this complex are frequently implicated in leukemogenesis. Understanding the molecular events leading to the disruption of CBF has provided important insights into our understanding of the normal regulatory pathways that control hematopoiesis and has begun to reveal how alterations in these pathways induce leukemia., Information Sources: Both authors are involved in the identification and characterization of chromosomal abnormalities associated with hematologic malignancy. This has led to contributions to multicenter clinical and laboratory investigations as well as publications in peer-reviewed journals. All of the references cited in this review are published in journals covered by Medline. State of the Art. The core binding factor (CBF) is a heterodimeric transcription factor composed of the RUNX1 and CBFb subunits. RUNX1 is the DNA binding element of the complex and its affinity is greatly increased in the presence of CBFb. Knock-out studies in mice have demonstrated that both RUNX1 and CBFb are necessary for definitive hematopoiesis. Furthermore, reciprocal chromosomal translocations involving both partners have been directly implicated in leukemogenesis. Evidence is now emerging that at least some of the resulting fusion proteins, namely ETV6-RUNX1, RUNX1-MTG8 and CBFb-MYH11 dominantly inhibit the function of native CBF by recruiting transcriptional co-repressor complexes. However, knock-in studies have shown that whilst expression of these fusion genes may disrupt normal hematopoiesis, this, by itself, is not sufficient for the subsequent development of leukemia. Mutations of RUNX1 have been identified in familial platelet disorder (FDP), in which there is a congenital predisposition to the development of AML and heterozygous point mutations have been identified in the RUNX1 gene in some leukemias. Moreover, a small number of cases have been reported in which amplification of RUNX1 has been detected in childhood ALL suggesting mechanisms other than loss of function, such as gene dosage may also play a role., Conclusions: Understanding the role CBF plays in normal hematopoiesis and hematologic malignancies has provided critical reagents for the accurate identification of the broad group of leukemias harboring alterations of CBF. The application of these molecular approaches has already shown an impact on the clinical management of these patients and as more information becomes available, the ability to tailor therapy to improve each patient's chance of a cure becomes feasible.

Nuclear receptors comprise a family of transcription factors that regulate gene expression in a ligand dependent manner. They can activate or repress target genes by binding directly to DNA response elements as homo- or hetero-dimers or by binding to other classes of DNA-bound transcription factors. These activities have been linked to the formation of complexes with molecules that appear to serve as coactivators or corepressors, causing local modification of chromatin structure in order to regulate expression of their target genes. Several members of nuclear receptor family are directly associated with human malignancies including breast cancer, prostate cancer and leukaemia. The pathogenesis of each of these diseases is underpinned by the activities of a member of the superfamily; estrogen receptor-alpha (ER alpha) in breast cancer, androgen receptor (AR) in prostate cancer, and retinoic acid receptor alpha (RAR alpha) in acute promyelocytic leukaemia.

The t(12;21)(p13;q22) translocation, resulting in the fusion of the ETV6 and AML1 genes, occurs in 20-25% of paediatric B-lineage acute lymphoblastic leukaemias (ALL). The identification of the fusion product has important prognostic and therapeutic implications as the translocation has been associated with a favourable clinical outcome. The aim of this study was threefold: (i) to assess the frequency and clinical association of the fusion gene in patients with and without a cytogenetically detectable chromosome 12 and/or 21 abnormality or failed cytogenetic results, (ii) to characterize alternative forms of ETV6/AML1 transcripts, and (iii) to use ETV6/AML1 as a molecular marker for the investigation of minimal residual disease (MRD). ETV6/AML1 fusion was detected in 22 (39%) of 56 cases studied by reverse transcriptase polymerase chain reaction (RT-PCR). ETV6/AML1 fusion was found in nine out of 16 (56%) cases with a cytogenetically visible chromosome 12 abnormality, but also in nine out of 29 patients (31%) without a chromosome 12 abnormality or patients with failed cytogenetics (four out of 11 patients, 36%), making this the most common cytogenetic abnormality in childhood ALL. Alternatively spliced ETV6/AML1 forms were investigated in 14 of the positive patients. Exon 5 of ETV6 was fused in frame to all AML1 exons, except exon 4. Fusion to exon 6 of AML1 resulted in one amino acid change. The presence of ETV6/AML1 was associated with a lower white blood cell count (Student's t-test; P = 0.009) and common (c)ALL phenotype (chi(2) test; P > 0.001), but no better disease-free survival. Our study shows that (i) RT-PCR is the most effective approach for the detection of t(12;21) in childhood ALL, (ii) the association of ETV6/AML1 and chromosome 12 and/or 21, seen in 56% of our cases, further confirms existing data, (iii) overall survival of patients with t(12;21) was not better than other cytogenetics groups, and (d) MRD analysis using ETV6/AML1 fusion is specific, but not sensitive enough to avoid false negative results.