The high mobility group AT-hook 2 (HMGA2), a DNA architectural protein, is highly regulated during development and plays an important role in tumorigenesis. Indeed, HMGA2 was overexpressed in many different kinds of tumors. However, the mechanisms regulating HMGA2 expression remain elusive. Using microarray analysis, we found that HMGA2, along with a dozen of other genes, was co-repressed by ZBRK1, BRCA1, and CtIP. BRCA1 exerts its transcriptional repression activity through interaction with the transcriptional repressor ZBRK1 in the central domain, and with CtIP in the C-terminal BRCT domain. Here, we show that ZBRK1, BRCA1, and CtIP form a repression complex that coordinately regulates HMGA2 expression via a ZBRK1 recognition site in the HMGA2 promoter. Depletion of any of the proteins in this complex via adenoviral RNA interference in MCF10A mammary epithelial cells activates HMGA2 expression, resulting in increased colony formation in soft agar. Similarly, depletion of ZBRK1, or ectopic overexpression of HMGA2, in MCF10A cells induces abnormal acinar size with increased cell number and inhibits normal acinar formation. Consistently, many BRCA1-deficient mouse breast tumors express higher levels of HMGA2 than BRCA1-proficient tumors. These results suggest that activation of HMGA2 gene expression through derepression of the ZBRK1/ BRCA1/CtIP complex is a significant step in accelerating breast tumorigenesis.The HMGA family consists of four proteins: HMGA1a, HMGA1b, HMGA1c, and HMGA2.2 HMGA1a, -b, and -c are all encoded by the same gene but vary in length due to alternative splicing (1-3), whereas HMGA2 is encoded by a distinctive gene (4). The common structural motifs in this group include an acidic C terminus and three DNA binding domains called A-T hooks, because they bind short (4 Ϯ 6 bp) AT-rich sequences in the minor groove (5-8). HMGA proteins regulate the expression of many genes through architectural remodeling of the chromatin structure and the formation of multiprotein complexes on promoter/enhancer regions. In accordance with their many roles in transcriptional regulation, aberrant expression of HMGA proteins has been observed in a large number of human cancers (reviewed by Farnet et al. (9)). The HMGA2 architectural protein is critical for a variety of cellular processes, including gene transcription, induction of neoplastic transformation, and promotion of metastatic progression (10, 11). Importantly, HMGA2 overexpression in tumors is associated with poor prognosis and metastasis in breast cancer patients (12). Although it is known that transcriptional repression of HMGA2 may prevent mammary tumorigenesis, the mechanisms governing repression remain elusive.The potential role of BRCA1 in transcriptional regulation has been revealed by discovering its ability to bind many important transcription factors, including p53, c-Myc, and STAT1 (13-15). Expression of several target proteins, including p21 WAF1 , cyclin B1, and EGR1, is activated or repressed by the presence of BRCA1. BRCA1 lacks DNA sequence...

Proper assembly of mitotic spindles requires Hice1, a spindleassociated protein. Hice1 possesses direct microtubule binding activity at its N-terminal region and contributes to intraspindle microtubule nucleation as a subunit of the Augmin complex. However, whether microtubule binding activity of Hice1 is modulated by mitotic regulators remains unexplored. Here, we found that Aurora-A kinase, a major mitotic kinase, specifically binds to and phosphorylates Hice1. We identified four serine/ threonine clusters on Hice1 that can be phosphorylated by Aurora-A in vitro. Of the four clusters, the Ser/Thr-17-21 cluster was the most critical for bipolar spindle assembly, whereas other phospho-deficient point mutants had a minimal effect on spindle assembly. Immunostaining with a phospho-Ser-19/20 phospho-specific antibody revealed that phosphorylated Hice1 primarily localizes to spindle poles during prophase to metaphase but gradually diminishes after anaphase. Consistently, the phospho-mimic 17-21E mutant reduced microtubule binding activity in vitro and diminished localization to spindles in vivo. Furthermore, expression of the 17-21E mutant led to decreased association of Fam29a, an Augmin component, with spindles. On the other hand, expression of the phospho-deficient 17-21A mutant permitted intraspindle nucleation but delayed the separation of early mitotic spindle poles and the timely mitotic progression. Taken together, these results suggest that Aurora-A modulates the microtubule binding activity of Hice1 in a spatiotemporal manner for proper bipolar spindle assembly.

Class switch DNA recombination (CSR) and somatic hypermutation (SHM) are central to the maturation of the Ab response. Both processes involve DNA mismatch repair (MMR). MMR proteins are recruited to dU:dG mispairs generated by activation-induced cytidine deaminase-mediated deamination of dC residues, thereby promoting S-S region synapses and introduction of mismatches (mutations). The MutL homolog Mlh3 is the last complement of the mammalian set of MMR proteins. It is highly conserved in evolution and is essential to meiosis and microsatellite stability. We used the recently generated knockout mlh3−/− mice to address the role of Mlh3 in CSR and SHM. We found that Mlh3 deficiency alters both CSR and SHM. mlh3−/− B cells switched in vitro to IgG and IgA but displayed preferential targeting of the RGYW/WRCY (R = A or G, Y = C or T, W = A or T) motif by Sγ1 and Sγ3 breakpoints and introduced more insertions and fewer donor/acceptor microhomologies in Sμ-Sγ1 and Sμ-Sγ3 DNA junctions, as compared with mlh3+/+ B cells. mlh3−/− mice showed only a slight decrease in the frequency of mutations in the intronic DNA downstream of the rearranged JH4 gene. However, the residual mutations were altered in spectrum. They comprised a decreased proportion of mutations at dA/dT and showed preferential RGYW/WRCY targeting by mutations at dC/dG. Thus, the MMR Mlh3 protein plays a role in both CSR and SHM.

RB plays an essential role in DNA damage-induced growth arrest and regulates the expression of several factors essential for DNA repair machinery. However, how RB coordinates DNA damage response through transcriptional regulation of genes involved in growth arrest remains largely unexplored. We examined whether RB can mediate the response to DNA damage through modulation of ZBRK1, a zinc finger-containing transcriptional repressor that can modulate the expression of GADD45A, a DNA damage response gene, to induce cell cycle arrest in response to DNA damage. We found that the ZBRK1 promoter contains an authentic E2F-recognition sequence that specifically binds E2F1, but not E2F4 or E2F6, together with chromatin remodeling proteins CtIP and CtBP to form a repression complex that suppresses ZBRK1 transcription. Furthermore, loss of RB-mediated transcriptional repression led to an increase in ZBRK1 transcript levels, correlating with increased sensitivity to ultraviolet (UV) and methyl methanesulfonate-induced DNA damage. Taken together, these results suggest that the RB⅐CtIP (CtBP interacting protein)/CtBP (C terminus-binding protein) /E2F1 complex plays a critical role in ZBRK1 transcriptional repression, and loss of this repression may contribute to cellular sensitivity of DNA damage, ultimately leading to carcinogenesis.The DNA damage response that responds to genotoxic stress induced by radiation, chemicals, and endogenous reactive oxygen species is highly conserved in higher eukaryotes (1, 2). The cellular responses to DNA damage include activation of cell-cycle arrest, apoptosis, and DNA damage repair (3). In mammals, multiple partially overlapping DNA repair mechanisms, including base excision, recombination, and mismatch repair, are required for proper DNA damage repair. Defects in these repair pathways frequently lead to irreparable DNA damage, aging, and cancer.RB, a prototypic tumor suppressor (4), is essential for regulating cell cycle progression through interaction with binding partners such as E2F (5). In quiescent and early G 1 cells, RB associates with the E2F family of transcription factors to repress the expression of E2F-responsive genes involved in cell cycle progression (6). As cells progress toward S-phase, RB is phosphorylated by cyclin-dependent kinases, releasing E2F, which opens the DNA replication origin, and induces transcription of S-phase genes (7). When RB is lost or inactivated, DNA replication origins become readily accessible, resulting in uncontrolled transcription of S-phase genes and ultimately leading to premature S-phase progression (8). RB also plays an essential role in DNA damage-induced growth arrest (9) and transcriptional regulation of several DNA damage-repair factors involved in the ultraviolet (UV) damage repair process, including FEN1, XPC, RPA2-3, RFC4, and proliferating cell nuclear antigen (10). Although previous studies have established that phosphorylation of RB inactivates its binding activity with its interacting partners and promotes cell cycle progression (1...

Organization and dynamics of focal adhesion proteins have been well characterized in cells grown on two-dimensional (2D) cell culture surfaces. However, much less is known about the dynamic association of these proteins in the 3D microenvironment. Limited imaging technologies capable of measuring protein interactions in real time and space for cells grown in 3D is a major impediment in understanding how proteins function under different environmental cues. In this study, we applied the nano-scale precise imaging by rapid beam oscillation (nSPIRO) technique and combined the scaning-fluorescence correlation spectroscopy (sFCS) and the number and molecular brightness (N&B) methods to investigate paxillin and actin dynamics at focal adhesions in 3D. Both MDA-MB-231 cells and U2OS cells produce elongated protrusions with high intensity regions of paxillin in cell grown in 3D collagen matrices. Using sFCS we found higher percentage of slow diffusing proteins at these focal spots, suggesting assembling/disassembling processes. In addition, the N&B analysis shows paxillin aggregated predominantly at these focal contacts which are next to collagen fibers. At those sites, actin showed slower apparent diffusion rate, which indicated that actin is either polymerizing or binding to the scaffolds in these locals. Our findings demonstrate that by multiplexing these techniques we have the ability to spatially and temporally quantify focal adhesion assembly and disassembly in 3D space and allow the understanding tumor cell invasion in a more complex relevant environment.