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All tumors examined to date contain mutations in mitochondrial DNA (mtDNA). In addition, depletion of mtDNA is reported in a variety of tumors. Mitochondrial dysfunction resulting from changes in mtDNA invokes mitochondria-to-nucleus retrograde response in human cells. To identify proteins involved in retrograde response and their potential role in tumorigenesis, we carried out a comparative proteomic analysis using a cell line in which the mitochondrial genome was completely depleted (?0 cells lacking all mtDNA-encoded protein subunits), a cybrid cell line in which mtDNA was restored, and the parental cell line. Our comparative proteomic approach revealed marked changes in the cellular proteome and led us to identify quantitative changes in expression of several proteins. We found that subunits of complex I and complex III, molecular chaperones, and a protein involved in cell cycle control were down-regulated and Inosine 5’-monophosphate dehydrogenase type 2 (IMPDH2) involved in nucleotides biosynthesis ...

TACC1 is the founding member of the evolutionarily conserved transforming acidic coiled coil genes. These genes play a role in normal development and tumorigenesis through interactions with multiple complexes involved in transcription, translation, and centrosomal dynamics. Despite its importance, detailed examination of the expression of TACC1 and splice variants has not previously been performed. In this study, the spatiotemporal distribution of the Tacc1 protein was examined immunohistochemically in cross-sections of mouse embryonic tissues. We also report the distribution of currently known/predicted TACC1 splice variants in adult humans. These results indicate that Tacc1 is regulated in a dynamic manner during embryogenesis. In adult humans, ubiquitous expression of at least one TACC1 splice variant is noted, although specific combinations of variants are evident in individual differentiated tissues. An important observation is that in the in vivo three-dimensional tissue architecture of the growing organism, both the human and mouse TACC1 protein can be localized to different subcellular compartments in a cell- and tissue-specific manner. This indicates that exploration of TACC1 function must take into account the temporal expression of specific splice variants that may perform different cell-type and tissue-specific functions. Furthermore, this analysis will provide the groundwork from which future Tacc1 knockout strategies can be designed and properly interpreted.

Dysregulation of the human transforming acidic coiled coil (TACC) genes is thought to be important in the development of multiple myeloma, breast and gastric cancer. However, even though these proteins have been implicated in the control of cell growth and differentiation, the mechanism by which they function still remains to be clarified. Using the yeast two-hybrid assay, we have now identified the histone acetyltransferase (HAT) hGCN5L2 as a TACC2-binding protein. GST pull-down analysis subsequently confirmed that all human TACC family members can bind in vitro to hGCN5L2. The authenticity of these interactions was validated by coimmunoprecipitation assays within the human embryonic kidney cell line HEK293, which identified the TACC2s isoform as a component consistently bound to several different members of HAT family. This raises the possibility that aberrant expression of one or more TACC proteins may affect gene regulation through their interaction with components of chromatin remodeling complexes, thus contributing to tumorigenesis.

The increasing use of high-throughput and large-scale bioinformatics-based studies has generated a massive amount of data stored in a number of different databases. The major need now is to explore this disparate data to find biologically relevant interactions and pathways. Thus, in the post-genomic era, there is clearly a need for the development of algorithms that can accurately predict novel protein-protein interaction networks in silico. The evolutionarily conserved Aurora family kinases have been chosen as a model for the development of a method to identify novel biological networks by a comparison of human and various model organisms. Our search methodology was designed to predict and prioritize molecular targets for Aurora family kinases, so that only the most promising are subjected to empirical testing. Four potential Aurora substrates and/or interacting proteins, TACC3, survivin, Hec1, and hsNuf2, were identified and empirically validated. Together, these results justify the timely implementation of in silico biology in routine wet-lab studies and have also allowed the application of a new approach to the elucidation of protein function in the post-genomic era.

A highly specific t(8;13)(p11;q12) translocation has been consistently identified in bone marrow cells from patients with an atypical myeloproliferative disease that is associated with peripheral blood eosinophila and T- or B-cell leukemias. In all patients analysed to date, the translocation event results in a chimeric gene in which the atypical zinc-finger domain of ZNF198 is fused to the N-terminal end of the catalytic domain of the FGFR1 receptor tyrosine kinase. To understand more about the consequences of this rearrangement we have investigated the normal function of the ZNF198 gene. Using yeast two-hybrid analysis we identified HHR6 as a protein binding partner and confirmed this using immunoprecipitation studies. The ZNF198/FGFR1 fusion protein also binds to HHR6. We demonstrate here that the human RAD18 is also present in the ZNF198/HHR6 protein complex, although it does not coimmunoprecipitate with the fusion kinase. Cells expressing the fusion kinase gene show a marked increased sensitivity to UVB irradiation, suggesting that it acts in a dominant-negative way to affect DNA repair. These observations support the idea that ZNF198, through its interaction with HHR6 and RAD18, may be involved in the DNA repair process.

Review on TACC2, with data on DNA/RNA, on the protein encoded and where the gene is implicated.

We recently showed that the Drosophila transforming acidic coiled-coil (D-TACC) protein is located in the centrosome, interacts with microtubules, and is required for mitosis in the Drosophila embryo. There are three known human TACC proteins that share a conserved, C-terminal, coiled-coil region with D-TACC. These proteins have all been implicated in cancer, but their normal functions are unknown. We show that all three human TACC proteins are concentrated at centrosomes, but with very different characteristics: TACC1 is weakly concentrated at centrosomes during mitosis; TACC2 is strongly concentrated at centrosomes throughout the cell cycle; and TACC3 is strongly concentrated in a more diffuse region around centrosomes during mitosis. When the C-terminal TACC domain is overexpressed in HeLa cells, it forms large polymers in the cytoplasm that can interact with both microtubules and tubulin. The full-length TACC proteins form similar polymers when overexpressed, but their interaction with microtubules and tubulin is regulated during the cell cycle. At least one of the human TACC proteins appears to increase the number and/or stability of centrosomal microtubules when overexpressed during mitosis. Thus, the TACC domain identifies a family of centrosomal proteins that can interact with microtubules. This may explain the link between the TACC genes and cancer.

Amplification of several chromosomal regions have been observed in human breast carcinomas. One such region, 8p11, is amplified in 10 – 15% of tumor samples. Although the FGFR1 gene is located close to this region, and is often included within the amplicon, the observation that tumors exhibiting 8p11 amplification do not always overexpress FGFR1 suggests that another gene located close to FGFR1 is involved in the tumorigenic process. We now report the precise location of four expressed sequence tags (ESTs) within this region and the cloning of a novel gene, designated TACC1 (transforming acidic coiled coil gene 1), which encodes an 8 kb transcript and which is expressed at high levels during early embryogenesis. Constitutive expression of this gene under the control of the cytomegalovirus (CMV) promoter in mouse fibroblasts, results in cellular transformation and anchorage independent growth, suggesting that inappropriate expression can impart a proliferative advantage. This observation raises the possibility that amplification of TACC1 could promote malignant growth, thereby making TACC1 an attractive candidate for the gene promoting tumorigenicity as a result of the 8p11 amplification in human breast cancers.

We used fluorescence in situ hybridization to characterize the molecular position of the breakpoints in a t(8;13)(p11;q12) reciprocal translocation from a patient with an atypical myeloproliferative disorder. This structural chromosome abnormality is characteristic of this specific disease and occurs often as the only chromosome abnormality in the malignant cells. Yeast artificial chromosome (YAC) analysis has demonstrated that the 8p11 breakpoint lies within a region defined by YAC 959A4 and that the 13q12 breakpoint is spanned by YAC 769F9. Identifying the position of the breakpoints in this rearrangement provides the means to search for candidate genes rearranged by this highly specific structural chromosome abnormality.

A reciprocal chromosome translocation between 13q12 and 8p11 is the consistent cytogenetic abnormality seen in a nonspecific myeloproliferative disorder that is associated with T-cell leukemia/lymphoma and peripheral blood eosinophilia. Detailed molecular analyses of the translocation breakpoints associated with this rearrangement have not been reported to date. We have now generated somatic cell hybrids from a newly described patient with this specific structural rearrangement and analyzed the breakpoints on the derivative chromosomes. We have shown that the breakpoint on chromosome 13 lies within a 300- to 500-kb region defined by the KIAA177 gene and D13S1123 marker. In addition, we have identified a 1.2-Mb YAC, 959A4, that crosses the translocation breakpoint on the short arm of chromosome 8 in this patient. The location of this breakpoint in 8p11 is distinct from the t(8; 16) and t(8; 22) translocations associated with M4/M5 myeloid leukemias, and suggests that three distinct loci located within 8p11 are involved in the pathogenesis of myeloid neoplasias.

The identification of cDNA clones from genomic regions known to contain human genes is usually the rate-limiting factor in positional cloning strategies. We demonstrate here that human genes present on yeast artificial chromosomes (YACs) are transcribed in yeast host cells. We have used the arbitrarily primed RNA (RAP) fingerprinting method to identify human-specific, transcribed sequences from YACs located in the 13q12 chromosome region. By comparing the RAP fingerprints generated using defined, arbitrary primers from various fragmented YACs, megaYACs, and host yeast, we were able to identify and map 20 products transcribed from the human YAC inserts. This method, therefore, permits the simultaneous isolation and mapping of novel expressed sequences directly from whole YACs.

Through linkage analysis and the identification of structural chromosome rearrangements, a number of disease genes have been mapped to the pericentromeric region of the long arm of chromosome 13. Structural rearrangements, or deletions, of the 13q12 region have been implicated in a range of myeloproliferative neoplasms, and other haematopoietic malignancies. In particular, seven cases of a t(8;13) (p11; q12·1) rearrangement have been noted in patients with an atypical myeloproliferative disorder associated with T-cell leukemia and eosinophilia. We have previously identified a CEPH megaYAC, 943E4, which crosses the translocation breakpoint in archival tumour samples from two patients with this t(8;13) translocation. As an initial step in the characterisation of this translocation breakpoint, we have generated a fine structure physical map of this 1·9 Mb YAC. We have used the method of YAC fragmentation to generate a series of deletion constructs of known size, which provide discreet physical landmarks convenient for mapping genetic markers along the 943E4 YAC. Analysis of these deletion constructs defined the order of ESTs and microsatellite markers in 943E4 as: cen-NIB1257-(ATP1AL1/D13S283)- D13S179E-(D13S504E/D13S505E)-D13S824E-D13S182E-D13S221-tel. These markers have also been assigned to physically defined regions relative to the fragmented YAC endpoints and a derived NotI restriction map.

Review on TACC1 (transforming, acidic coiled-coil containing protein 1), with data on DNA, on the protein encoded, and where the gene is implicated.

Review on TACC3 (transforming, acidic coiled-coil containing protein 3), with data on DNA, on the protein encoded, and where the gene is implicated.

Human mitochondrial DNA (mtDNA) encodes 13 proteins involved in oxidative phosphorylation (OXPHOS). In order to investigate the role of mitochondrial OXPHOS genes in breast tumorigenesis, we have developed a breast epithelial cell line devoid of mtDNA (rho(0) cells). Our analysis revealed that depletion of mtDNA in breast epithelial cells results in in vitro tumorigenic phenotype as well as breast tumorigenesis in a xenograft model. We identified two major gene networks which were differentially regulated between parental and rho(0) epithelial cells. The focal proteins in these networks include (i) FN1 (fibronectin) and (ii) p53. Bioinformatic analyses of FN1 network identified laminin, integrin and 3 of 6 members of peroxiredoxin whose expression were altered in rho(0) epithelial cells. In the p53 network, we identified SMC4 and WRN whose changes in expression suggest that this network may affect chromosomal stability. Consistent with above finding our study revealed an increase in DNA double strand breaks and unique chromosomal rearrangements in rho(0) breast epithelial cells. Additionally, we identified tight junction proteins claudin-1 and claudin-7 in p53 network. To determine the functional relevance of altered gene expression, we focused on detailed analyses of claudin-1 and -7 proteins in breast tumorigenesis. Our study determined that (i) claudin-1 and 7 were indeed downregulated in rho(0) breast epithelial cells, (ii) downregulation of claudin-1 or -7 led to neoplastic transformation of breast epithelial cells, and (iii) claudin-1 and -7 were also downregulated in primary breast tumors. Together, our study suggest that mtDNA encoded OXPHOS genes play a key role in transformation of breast epithelial cells and that multiple pathway involved in mitochondria-to-nucleus retrograde regulation contribute to transformation of breast epithelial cells.

The Transforming acidic coiled coil (TACC) proteins play a conserved role in normal development and tumorigenesis through interactions with multiple complexes involved in transcription, translation, and centrosomal dynamics. However, despite significant work on the function of TACC3 in the control of centrosomal mechanics, relatively little functional data is known about the family’s founding member, TACC1. From a continued analysis of clones isolated by an unbiased yeast two-hybrid assay, we now show direct physical interactions between the TACC1 and the FHL (Four and a Half LIM-only) family of proteins. The authenticity of these interactions was validated both in vitro and in cellular systems. The FHLs exhibit diverse biological roles such as the regulation of the actin cytoskeleton and are promiscuous coregulators for several transcription factors. The interaction of the endogenous TACC-FHL proteins is primarily localized to the nucleus. However, similar to FHL2, overexpression of TACC1A in HEK293 is able to sequester serum activated ERK to the cytoplasm. This has the effect of reducing the serum induced transcriptional response of the c-fos and c-jun genes. The observation that TACCs can interact with the FHLs and alter their serum induced activities raises the possibility that the TACCs participate in crosstalk between cell signaling pathways important for cancer development and tumor progression. The transforming acidic coiled coil genes are known to be important prognostic indicators for breast, ovarian and lung cancer. In this manuscript, we identify a novel interaction between the TACCs and the FHL protein family. This interaction has an affect on ERK and may in part explain the variable associations and changes in subcellular locations of each family with specific subtypes of malignancy.

To The Editor: An atypical myeloproliferative disorder has been described that is associated with T-cell leukemia/lymphoma and peripheral blood eosinophilia.[1][1] All these cases are associated with a translocation between 8p11 and 13q11-12 or two rare variant translocations involving 8p11 and

BackgroundDysregulation of the human Transforming Acidic Coiled Coil (TACC) genes is thought to be important in the development and progression of multiple myeloma, breast and gastric cancer. Recent, large-scale genomic analysis and Serial Analysis of Gene Expression data suggest that TACC1 and TACC3 may also be involved in the etiology of ovarian tumors from both familial and sporadic cases. Therefore, the aim of this study was to determine the occurrence of alterations of these TACCs in ovarian cancer.MethodsDetection and scoring of TACC1 and TACC3 expression was performed by immunohistochemical analysis of the T-BO-1 tissue/tumor microarray slide from the Cooperative Human Tissue Network, Tissue Array Research Program (TARP) of the National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. Tumors were categorized as either positive (greater than 10% of cells staining) or negative. Statistical analysis was performed using Fisher's exact test and p < 0.05 (single comparisons), and p < 0.02 (multiple comparisons) were considered to be significant. Transgenomics WAVE high performance liquid chromatography (dHPLC) was used to pre-screen the TACC3 gene in constitutional DNA from ovarian cancer patients and their unaffected relatives from 76 families from the Gilda Radner Familial Ovarian Cancer Registry. All variant patterns were then sequenced.ResultsThis study demonstrated absence of at least one or both TACC proteins in 78.5% (51/65) of ovarian tumors tested, with TACC3 loss observed in 67.7% of tumors. The distribution pattern of expression of the two TACC proteins was different, with TACC3 loss being more common in serous papillary carcinoma compared with clear cell carcinomas, while TACC1 staining was less frequent in endometroid than in serous papillary tumor cores. In addition, we identified two constitutional mutations in the TACC3 gene in patients with ovarian cancer from the Gilda Radner Familial Ovarian Cancer Registry. These patients had previously tested negative for mutations in known ovarian cancer predisposing genes.ConclusionWhen combined, our data suggest that aberrations of TACC genes, and TACC3 in particular, underlie a significant proportion of ovarian cancers. Thus, TACC3 could be a hitherto unknown endogenous factor that contributes to ovarian tumorigenesis.

Mitochondrial dysfunction is a hallmark of cancer cells. Consistent with this phenotype mutations in mitochondrial genome have been reported in all cancers examined to date. However, it is not clear whether mitochondrial genomic status in human cells affects nuclear genome stability and whether proteins involved in inter-genomic cross talk are involved in tumorigenesis. Using cell culture model and cybrid cell technology, we provide evidence that mitochondrial genetic status impacts nuclear genome stability in human cells. In particular our studies demonstrate 1) that depletion of mitochondrial genome (rho0) leads to chromosomal instability (CIN) reported to be present in variety of human tumors and 2) rho0 cells show transformed phenotype. Our study also demonstrates that mitochondrial genetic status plays a key role in regulation of a multifunctional protein APE1 (also known as Ref1 or HAP1) involved in transcription and DNA repair in the nucleus and the mitochondria. Interestingly we found that altered expression of APE1 in rho0 cells and tumorigenic phenotype can be reversed by exogenous transfer of wild type mitochondria in rho0 cells. Furthermore, we demonstrate that APE1 expression is altered in variety of primary tumors. Taken together, these studies suggest that inter-genomic cross talk between mitochondria and the nucleus plays an important role in tumorigenesis and that APE1 mediates this process.

Dysregulation of the human Transforming acidic coiled coil (TACC) genes is thought to be important in the development of breast cancer. However, the mechanism by which they function still remains to be clarified. We have demonstrated that the lull length TACC2 protein can inhibit the tumorigenic phenotype of certain breast cancer cells. Similarly, the TACC2 interacting protein, hGCN5 can also reduces breast cancer cell survival. The catalytic domain of this histone acetyltransferase is critically important for this effect. Thus, modulation of hGCN5 and/or pCAF activity may represent a mechanism by which TACC2 may exert its tumor suppressive properties. We have furthered our analysis of TACC2 mediated transcriptional events by examining the effects of TACC2 on the BRCAl mediated regulation of the p21 gene. Our initial data suggests that basal levels of p21 and STAT1 are decreased in TACC2 transfected cell lines. Independently, we have found an interaction between the TACCs and the FHL family of transcriptional regulators. These latter proteins coactivate or corepress transcription, in a promoter specific context. We are now examining whether TACC2 possesses similar properties, or alternatively acts as a transcriptional switch, as previously indicated for the CBF- l/RBP-Jk splice variant of FHLl in the Notch signaling pathway.

The human transforming acidic coiled-coil 2 (TACC2) gene has been suggested recently to be a putative breast tumor suppressor. Now we can report the cloning of full length TACC2 cDNAs corresponding to the major isoforms expressed during development. The TACC2 gene is encoded by 23 exons, and spans 255 kb of chromosome 10q26. In breast cancer cell lines, TACC2 is expressed as a 120 kDa protein corresponding to the major transcript expressed in the mammary gland. Although only slight differences in the expression of TACC2 in normal versus breast tumors were observed, overexpression of TACC2 can alter the in vitro cellular dynamics of some breast cancer cell lines. Significantly, we demonstrate that TACC2 interacts with GAS41 and the SWI/SNF chromatin remodeling complex. This suggests that defects in TACC2 expression may affect gene regulation, thus contributing to the pathogenesis of some tumors.

Poly(ADP-ribosyl)ation of nuclear proteins plays a significant role in the maintenance of genomic DNA stability. To date, four poly(ADP-ribosyl)ating proteins have been identified in humans. We now report the full-length sequence, expression profile, and chromosomal localization of a novel gene, ADPRTL1, encoding an ADP-ribosyltransferase-like protein. The predicted open reading frame encodes a protein of 1724 amino acids with a molecular mass of 192.8 kDa. The protein contains a region showing homology to the catalytic domains of the nuclear-localized ADP-ribosyltransferase proteins (Adprt), two recently identified Adprt-like proteins (Adprtl2 and Adprtl3), and the telomere-associated protein tankyrase. Key amino acids known to be important for the activity of these enzymes are conserved within this region of the Adprtl1 protein, indicating that Adprtl1 is a functional poly(ADP-ribosyl)transferase. As has been noted for tankyrase, sequence analysis of the Adprtl1 protein suggests that it is not capable of binding DNA directly. Thus, the transferase activity of Adprtl1 may be activated by other factors such as protein–protein interaction mediated by the extensive carboxyl terminus. We have subsequently refined the location of the ADPRTL1 genomic locus to 13q11, close to the recently cloned ZNF198 gene.

We have recently identified a novel gene, TACC1 (transforming acidic coiled coil-containing gene 1), which is located close to FGFR1 within a region amplified in breast cancer on human chromosome 8p11. The coiled coil domain of this gene identified a series of cDNAs in the expressed sequence tag database, which suggested the existence of a family of TACC genes comprising at least three family members. We have now characterized the human and mouse TACC3 cDNAs, and demonstrate that this gene is upregulated in various cancer cell lines, and at Embryonic Day 15 in mice, suggesting that the TACC3 protein is involved in the control of cell growth and differentiation. The TACC3 gene maps telomeric to the FGFR3 gene in 4p16.3, close to a region disrupted by translocation breakpoints associated with multiple myeloma. Thus, TACC1, TACC2, and TACC3 map close to the corresponding FGFR1, FGFR2, and FGFR3 genes. The phylogenetic relationship among the three TACC genes is similar to that of the three FGFR family members. These relationships suggest that the FGFR and TACC genes arose from a physically linked ancestral gene pair. Subsequently, this gene pair has undergone two successive rounds of gene duplication to give rise to the three FGFR/TACC gene pairs on chromosomes 4, 8, and 10.

A panel of somatic cell hybrids carrying a defined set of rearrangements involving chromosome 13 has been used to assign 35 novel gene transcripts regionally. The positions of the chromosome 13 breakpoints in each somatic cell hybrid have previously been defined relative to the Genethon genetic linkage map. As a result, the position of each gene transcript has been determined relative to both the physical and the genetic linkage maps. Analysis of the distribution of these gene transcripts indicates a slight overrepresentation toward locations on the distal long arm of chromosome 13, with no localizations noted in the 13q22–q31 region. Seven of these novel gene transcripts and the gene encoding small ribonucleoprotein U6 have been mapped to YACs known to contain Genethon microsatellite markers, thereby providing further sublocalization relative to the genetic map. The positioning of these gene transcripts within the genetic and physical maps provides candidate genes for disease loci that are being mapped to the same intervals on the chromosome.

We have refined the location of the Brn-3a genomic locus (POU4F1), using somatic cell hybrid analysis, to 13q21.1→q22. Furthermore, we have shown that two YACs, known to contain the closest marker linked to neuronal ceroidlipofuscinosis (CLN5), also contains the Brn-3a gene. As one of these YACs is only 320 kb long, this localises POU4F1 within the critical region defining the CLN5 locus, and raises the possibility that POU4F1 is a candidate gene for CLN5.

Examination of ancient gene families can provide an insight into how the evolution of gene structure can relate to function. Functional homologs of the evolutionarily conserved transforming acidic coiled coil (TACC) gene family are present in organisms from yeast to man. However, correlations between functional interactions and the evolution of these proteins have yet to be determined. We have performed an extensive database analysis to determine the genomic and cDNA sequences of the TACCs from phylogenetically diverse organisms. This analysis has determined the phylogenetic relationship of the TACC proteins to other coiled coil proteins, the resolution of the placement of the rabbit TACC4 as the orthologue of human TACC3, and RHAMM as a distinct family of coiled coil proteins. We have also extended the analysis of the TACCs to the interaction databases of C. elegans and D. melanogaster to identify potentially novel TACC interactions. The validity of this modeling was confirmed independently by the demonstration of direct binding of human TACC2 to the nuclear hormone receptor RXRβ. The data so far suggest that the ancestral TACC protein played a role in centrosomal/mitotic spindle dynamics. TACC proteins were then recruited to complexes involved in protein translation, RNA processing and transcription by interactions with specific bridging proteins. However, during evolution, the TACC proteins have now acquired the ability to directly interact with components of these complexes (such as the LSm proteins, nuclear hormone receptors, GAS41, and transcription factors). This suggests that the function of the TACC proteins may have evolved from performing assembly or coordination functions in the centrosome to include a more intimate role in the functional evolution of chromatin remodeling, transcriptional and posttranscriptional complexes in the cell.