Tao Lu

Cell-Type-Dependent Activity of the Ubiquitous Transcription Factor USF in Cellular Proliferation and Transcriptional Activation

ABSTRACT USF1 and USF2 are basic helix-loop-helix transcription factors implicated in the control of cellular proliferation. In HeLa cells, the USF proteins are transcriptionally active and their overexpression causes marked growth inhibition. In contrast, USF overexpression had essentially no effect on the proliferation of the Saos-2 osteosarcoma cell line. USF1 and USF2 also lacked transcriptional activity in Saos-2 cells when assayed by transient cotransfection with USF-dependent reporter genes. Yet, there was no difference in the expression, subcellular localization, or DNA-binding activity of the USF proteins in HeLa and Saos-2 cells. Furthermore, Gal4-USF1 and Gal4-USF2 fusion proteins activated transcription similarly in both cell lines. Mutational analysis and domain swapping experiments revealed that the small, highly conserved USF-specific region (USR) was responsible for the inactivity of USF in Saos-2 cells. In HeLa, the USR serves a dual function. It acts as an autonomous transcriptional activation domain at promoters containing an initiator element and also induces a conformational change that is required for USF activity at promoters lacking an initiator. Taken together, these results suggest a model in which the transcriptional activity of the USF proteins, and consequently their antiproliferative activity, is tightly controlled by interaction with a specialized coactivator that recognizes the conserved USR domain and, in contrast to USF, is not ubiquitous. The activity of USF is therefore context dependent, and evidence for USF DNA-binding activity in particular cells is insufficient to indicate USF function in transcriptional activation and growth control.

NGAL decreases E-cadherin-mediated cell–cell adhesion and increases cell motility and invasion through Rac1 in colon carcinoma cells

Expression of neutrophil gelatinase-associated lipocalin (NGAL)/lipocalin2, a recently recognized iron regulatory protein that binds to matrix metalloproteinase-9 (MMP9), is increased in a spectrum of cancers, including those of the colorectum. Using colon carcinoma cell lines stably transfected with NGAL or antisense NGAL, we showed that NGAL overexpression altered subcellular localization of E-cadherin and catenins, decreased E-cadherin-mediated cell–cell adhesion, enhanced cell–matrix attachment, and increased cell motility and in vitro invasion. Conversely, a decrease in NGAL enhanced more aggregated growth pattern and decreased in vitro invasion. We further showed that NGAL exerted these effects through the alteration of the subcellular localization of Rac1 in an extracellular matrix-dependent, but MMP9-independent, manner. Furthermore, we observed that the NGAL-overexpressing cells tolerated increased iron levels in the culture environment, whereas the NGAL-underexpressing cells showed significant cell death after prolonged incubation in high-iron condition. Thus, overexpressing NGAL in colon carcinomas is an important regulatory molecule that integrates extracellular environment cues, iron metabolism, and intracellular small GTPase signaling in cancer migration and invasion. NGAL may therefore be a new target for therapeutic intervention in colorectal carcinoma.

Loss of USF transcriptional activity in breast cancer cell lines.

USF is a family of transcription factors that are structurally related to the Myc oncoproteins and also share with Myc a common DNA-binding specificity. USF overexpression can prevent c-Myc-dependent cellular transformation and also inhibit the proliferation of certain transformed cells. These antiproliferative activities suggest that USF inactivation could be implicated in carcinogenesis. To explore this possibility, we compared the activities of the ubiquitous USF1 and USF2 proteins in several cell lines derived from either normal breast epithelium or breast tumors. The DNA-binding activities of USF1 and USF2 were present at similar levels in all cell lines. In the non-tumorigenic MCF-10A cells, USF in general, and USF2 in particular, exhibited strong transcriptional activities. In contrast, USF1 and USF2 were completely inactive in three out of six transformed breast cell lines investigated, while the other three transformed cell lines exhibited loss of USF2 activity. Analyses in cells cultured from healthy tissue confirmed the transcriptional activity of USF in normal human mammary epithelial cells. These results demonstrate that a partial or complete loss of USF function is a common event in breast cancer cell lines, perhaps because, like Myc overexpression, it favors rapid proliferation.

Disruption of the Rad9/Rad1/Hus1 (9–1–1) complex leads to checkpoint signaling and replication defects

The checkpoint sliding-clamp complex, Rad9/Rad1/Hus1, plays a critical role during initiation of checkpoint signals in response to DNA damage and replication disruption. We investigated the impact of loss of Rad1 on checkpoint function and on DNA replication in mammalian cells. We show that RAD1 is an essential gene for sustained cell proliferation and that loss of Rad1 causes destabilization of Rad9 and Hus1 and consequently disintegration of the sliding-clamp complex. In Rad1-depleted cells, Atr-dependent Chk1 activation was impaired whereas Atm-mediated Chk2 activation was unaffected, suggesting that the sliding clamp is required primarily in Atr-dependent signal activation. Disruption of sliding-clamp function also caused a major defect in S-phase control. Rad1-depleted cells exhibited an RDS phenotype, indicating that damage-induced S-phase arrest was compromised by Rad1 loss. Furthermore, lack of Rad1 also affected the efficiency of replication recovery from DNA synthesis blockage, resulting in a prolonged Su2009phase. These deficiencies may perpetually generate DNA strand breakage as we have found chromosomal abnormalities in Rad1-depleted cells. We conclude that the Rad9/Rad1/Hus1 complex is essential for Atr-dependent checkpoint signaling, which may play critical roles in the facilitation of DNA replication and in the maintenance of genomic integrity.

hSnm1 Colocalizes and Physically Associates with 53BP1 before and after DNA Damage

ABSTRACT snm1 mutants of Saccharomyces cerevisiae have been shown to be specifically sensitive to DNA interstrand crosslinking agents but not sensitive to monofunctional alkylating agents, UV, or ionizing radiation. Five homologs of SNM1 have been identified in the mammalian genome and are termed SNM1, SNM1B, Artemis, ELAC2, and CPSF73. To explore the functional role of human Snm1 in response to DNA damage, we characterized the cellular distribution and dynamics of human Snm1 before and after exposure to DNA-damaging agents. Human Snm1 was found to localize to the cell nucleus in three distinct patterns. A particular cell showed diffuse nuclear staining, multiple nuclear foci, or one or two larger bodies confined to the nucleus. Upon exposure to ionizing radiation or an interstrand crosslinking agent, the number of cells exhibiting Snm1 bodies was reduced, while the population of cells with foci increased dramatically. Indirect immunofluorescence studies also indicated that the human Snm1 protein colocalized with 53BP1 before and after exposure to ionizing radiation, and a physical interaction was confirmed by coimmunoprecipitation assays. Furthermore, human Snm1 foci formed after ionizing radiation were largely coincident with foci formed by human Mre11 and to a lesser extent with those formed by BRCA1, but not with those formed by human Rad51. Finally, we mapped a region of human Snm1 of approximately 220 amino acids that was sufficient for focus formation when attached to a nuclear localization signal. Our results indicate a novel function for human Snm1 in the cellular response to double-strand breaks formed by ionizing radiation.