Kathy Howe

The solution structure of the RING finger domain from the acute promyelocytic leukaemia proto-oncoprotein PML.

Acute promyelocytic leukaemia (APL) has been ascribed to a chromosomal translocation event which results in a fusion protein comprising the PML protein and the retinoic acid receptor alpha. PML is normally a component of a nuclear multiprotein complex (termed ND10, Kr bodies, nuclear bodies, PML oncogenic domains or PODs) which is disrupted in the APL disease state. PML contains a number of characterized motifs including a Zn2+ binding domain called the RING or C3HC4 finger. Here we describe the solution structure of the PML RING finger as solved by 1H NMR methods at physiological pH with r.m.s. deviations for backbone atoms of 0.88 and 1.39 A for all atoms. Additional biophysical studies including CD and optical spectroscopy, show that the PML RING finger requires Zn2+ for autonomous folding and that cysteines are used in metal ligation. A comparison of the structure with the previously solved equine herpes virus IE110 RING finger, shows significant differences suggesting that the RING motif is structurally diverse. The role of the RING domain in PML nuclear body formation was tested in vivo, by using site‐directed mutagenesis and immunofluorescence on transiently transfected NIH 3T3 cells. Independently mutating two pairs of cysteines in each of the Zn2+ binding sites prevents PML nuclear body formation, suggesting that a fully folded RING domain is necessary for this process. These results suggest that the PML RING domain is probably involved in protein‐protein interactions, a feature which may be common to other RING finger domains.

Structure, organization, and dynamics of promyelocytic leukemia protein nuclear bodies.

The interphase nucleus compartmentalizes its components to give rise to a highly organized and tightly controlled environment. Individual chromosomes occupy discrete areas, termed “chromosome territories,” that are separated from each other by a channel called the “interchromosomal domain” (reviewed in Lamond and Earnshaw 1998). Actively transcribed genes tend to be at the periphery of chromosomal territories, whereas newly made RNA transcripts localize into the interchromosomal domain, where they can undergo further processing and transport. Movement within the nucleus (Ferreira et al. 1997) may permit chromosomes to enter “factories” that contain all the necessary enzymatic machinery for replication (reviewed in Jackson 1995). Of the many discrete domains identified throughout the nucleus, the largest are nucleoli, sites of ribosomal RNA synthesis and processing, and sites of preribosomal particle assembly (reviewed in Scheer and Weisenberger 1994). Other subnuclear bodies that appear as punctate structures under immunofluorescence (IF) microscopy include various dynamic structures involved in the maintenance and replication of DNA and RNA synthesis, processing, and transport (reviewed in Nickerson et al. 1995): replication foci, transcript foci, speckled domains, coiled bodies, gems, and promyelocytic leukemia protein (PML) nuclear bodies. Spliceosomal small nuclear (sn) ribonucleoprotein (RNP) components and a subset of non-snRNP splicing factors can be found concentrated in discrete subnuclear domains called “coiled bodies” (Matera and Frey 1998 [in this issue]). It is becoming increasingly apparent that the nucleus has an organization and contains a number of discrete macromolecular domains that coordinate a variety of nuclear processes.

Expression of a truncated Brca1 protein delays lactational mammary development in transgenic mice

To address the hypothesis that certain disease-associated mutants of the breast-ovarian cancer susceptibility gene BRCA1 have biological activity in vivo, we have expressed a truncated Brca1 protein (trBrca1) in cell-lines and in the mammary gland of transgenic mice. Immunofluorescent analysis of transfected cell-lines indicates that trBRCA1 is a stable protein and that it is localized in the cell cytoplasm. Functional analysis of these cell-lines indicates that expression of trBRCA1 confers an increased radiosensitivity phenotype on mammary epithelial cells, consistent with abrogation of the BRCA1 pathway. MMTV-trBrca1 transgenic mice from two independent lines displayed a delay in lactational mammary gland development, as demonstrated by altered histological profiles of lobuloalveolar structures. Cellular and molecular analyses indicate that this phenotype results from a defect in differentiation, rather than altered rates of proliferation or apoptosis. The results presented in this paper are consistent with trBrca1 possessing dominant-negative activity and playing an important role in regulating normal mammary development. They may also have implications for germline carriers of BRCA1 mutations.

HLS5, a Novel RBCC (Ring Finger, B Box, Coiled-coil) Family Member Isolated from a Hemopoietic Lineage Switch, Is a Candidate Tumor Suppressor

Hemopoietic cells, apparently committed to one lineage, can be reprogrammed to display the phenotype of another lineage. The J2E erythroleukemic cell line has on rare occasions developed the features of monocytic cells. Subtractive hybridization was used in an attempt to identify genes that were up-regulated during this erythroid to myeloid transition. We report here on the isolation of hemopoietic lineage switch 5 (Hls5), a gene expressed by the monocytoid variant cells, but not the parental J2E cells. Hls5 is a novel member of the RBCC (Ring finger, B box, coiled-coil) family of genes, which includes Pml, Herf1, Tif-1α, and Rfp. Hls5 was expressed in a wide range of adult tissues; however, at different stages during embryogenesis, Hls5 was detected in the branchial arches, spinal cord, dorsal root ganglia, limb buds, and brain. The protein was present in cytoplasmic granules and punctate nuclear bodies. Isolation of the human cDNA and genomic DNA revealed that the gene was located on chromosome 8p21, a region implicated in numerous leukemias and solid tumors. Enforced expression of Hls5 in HeLa cells inhibited cell growth, clonogenicity, and tumorigenicity. It is conceivable that HLS5 is one of the tumor suppressor genes thought to reside at the 8p21 locus.