Transcriptional activation of eukaryotic genes depends on the precise and ordered recruitment of activators, chromatin modifiers/remodelers, coactivators, and general transcription factors to the promoters of target genes. Using the human matrix metalloproteinase 9 (MMP-9) gene as a model system, we investigated the sequential assembly and dynamic formation of transcription complexes on a human promoter under the influence of mitogen signaling. We find that, coincident with activation of the MMP-9 gene, activators, chromatin remodeling complexes, and coactivators are recruited to the preassembled MMP-9 promoter in a stepwise and coordinated order, which is dependent on activation of MEK-1/extracellular signal-regulated kinase and NF-B signaling pathways. Conversely, corepressor complexes are released from the MMP-9 promoter after transcriptional activation. Histone modifications shift from repressive to permissive modifications concurrent with activation of the MMP-9 gene. Chromatin remodeling induced by Brg-1 is required for MMP-9 gene transcription, which is concomitant with initiation of transcription. Therefore, coordination of cell signaling, chromatin remodeling, histone modifications, and stepwise recruitment of transcription regulators is critical to precisely regulate MMP-9 gene transcription in a temporally and spatially dependent manner. Given the important role of MMP-9 in both normal development and pathological conditions, understanding MMP-9 gene regulation is of great relevance.Gene transcription in eukaryotic cells is controlled by protein complexes, including general and tissue-specific transcription factors, coregulators, chromatin-remodeling complexes, and complexes responsible for signal-specific histone modifications (26). As eukaryotic DNA is packaged into chromatin, generally a repressive structure for transcriptional activation, transcription in the context of chromatin requires remodeling processes to reconfigure the chromatin, so that activators, coactivators, and general transcription factors (GTFs) have access to promoters of target genes (12). Chromatin remodeling is dependent on either ATP-dependent chromatin-remodelingcomplex-induced structural modifications of nucleosomes or histone acetyltransferase-(HAT) and histone methyltransferase-mediated covalent modifications of the N-terminal tails of core histones (12). The SWI/SNF chromatin-remodeling complex can alter chromatin structure by either shifting nucleosomes along the DNA or twisting DNA to modulate the nucleosome structure (42). Brg-1 and Brm are two ATPase subunits of the SWI/SNF complex. Recruitment of the SWI/ SNF complex to target promoters requires protein-protein interactions through Brg-1 and other transcription regulators, as Brg-1 does not recognize sequence-specific DNA (21).

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Interferon-gamma (IFN-gamma) is a pleiotropic cytokine involved in aspects of immune regulation, cell proliferation, and host defense mechanisms directed toward various cancers. Some of the biological functions of IFN-gamma are achieved through inhibition of gene expression, although the mechanisms by which IFN-gamma suppresses gene transcription are poorly understood. Herein, we demonstrate the molecular basis by which IFN-gamma mediates suppression of the matrix metalloproteinase-9 (MMP-9) gene. IFN-gamma-activated signal transducer and activator of transcription-1alpha (STAT-1alpha) suppresses MMP-9 gene transcription, which is dependent on phosphorylation of tyrosine 701 but not phosphorylation of serine 727. The coactivator cyclic AMP response element-binding protein-binding protein (CBP) is an important component of induction of MMP-9 gene transcription. IFN-gamma induces the in vivo association of STAT-1alpha and CBP and decreases the association of CBP to the MMP-9 promoter. IFN-gamma does not influence the stability of CBP nor does IFN-gamma affect chromatin-remodeling events on the MMP-9 promoter. IFN-gamma inhibits the assembly of the MMP-9 transcription complex by suppressing H3/H4 acetylation and inhibiting recruitment of Pol II to the MMP-9 promoter. These findings indicate that IFN-gamma/STAT-1alpha exert their inhibitory effects by affecting multiple aspects of MMP-9 gene transcription.

Matrix metalloproteinases (MMPs) are a family of structurally related proteins with the collective capability to degrade all components of the extracellular matrix. Although MMP-mediated degradation of the extracellular matrix occurs physiologically, numerous pathological conditions exhibit increased MMP levels and excessive matrix degradation. Previous work from our laboratory has shown that interferon-␥ inhibits MMP-9 expression in a manner dependent upon STAT-1␣. Here we extend our previous observations and show that the class II major histocompatibility complex transactivator (CIITA), a transcriptional target of STAT-1␣, is also capable of inhibiting MMP-9 expression. By using stable cell lines that inducibly express CIITA or various mutant forms of CIITA, we show that CIITA requires the ability to bind the CREB-binding protein (CBP) to effectively inhibit MMP-9 expression. Furthermore, we show that CIITA-mediated inhibition of the MMP-9 gene does not rely on the transcriptional capability of CIITA. These findings support a model wherein CIITA inhibits MMP-9 expression by binding to and sequestering CBP, which reduces the levels of CBP at the MMP-9 promoter, inhibits levels of acetylated histone 3 at the MMP-9 promoter, and subsequently inhibits MMP-9 expression.The matrix metalloproteinases (MMPs) 1 are a family of Zn 2ϩ -dependent endopeptidases (1). To date, there are more than 20 members of the MMP family, and they are classified into four groups based on structural similarities, substrate preferences, and sequence homology. The four groups include the collagenases, gelatinases, stromelysins, and the membrane-type

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases whose aberrant expression are correlated with tumor invasion and angiogenesis. The transcription factors Sp1, Sp3, and AP-2 are required for constitutive expression of MMP-2 in tumor cells; however, the regulatory mechanisms of MMP-2 expression are not well understood. We investigated the involvement of Brg-1, the ATPase subunit of the SWI/SNF complex, in human MMP-2 gene transcription. Reconstitution of Brg-1 enhances MMP-2 transcription in Brg-1-deficient SW-13 cells. Chromatin immunoprecipitation assay demonstrates that Brg-1 is required for recruitment of Sp1, AP-2, and polymerase II to the MMP-2 promoter, whereas the binding of Sp3 to the MMP-2 promoter is decreased upon Brg-1 reconstitution. Furthermore, Sp1 interacts with Brg-1 in vivo. Restriction enzyme accessibility assays indicate that accessibility of the MMP-2 promoter region is not changed in the absence or presence of Brg-1. These results illustrate the connection between the SWI/SNF complex and optimal expression of MMP-2 and highlight the critical function of Brg-1 in regulating the recruitment of Sp1, Sp3, AP-2, and polymerase II to the MMP-2 promoter.Matrix metalloproteinase-2 (MMP-2), 1 also called 72-kDa type IV collagenase, belongs to the MMP family, which is composed of structurally conserved zinc-dependent endopeptidases that are involved in proteolytic modeling of the extracellular matrix. MMP-2 has been shown to play a critical role in invasion/metastasis and angiogenesis of malignant tumors (1, 2). MMP-2 is constitutively expressed by normal and transformed cells, and enhanced expression of MMP-2 is found in a variety of tumor cells that correlates with the malignancy grade of the original tumor (1, 2). We have previously shown that the transcription factors Sp1, Sp3, and AP-2 are functionally important in regulating constitutive expression of the MMP-2 gene (3). However, how the MMP-2 gene is constitutively transcribed in the generally repressive chromatin context under the regulation of Sp1, Sp3, and AP-2 is unknown.The Sp family of transcription factors, including Sp1, Sp2, Sp3, and Sp4, play a major role in regulating a large number of eukaryotic genes, which in turn, have indispensable effects in controlling development, differentiation, tumorigenesis, and viral infection (4, 5). Sp1 and Sp3 are ubiquitously expressed in eukaryotic cells, and both can recognize and bind GC boxes present in the promoters of a variety of constitutive or inducible genes (4, 5). Although Sp1 and Sp3 share a high degree of homology in structure, they have distinct functions in transcriptional regulation. Sp1 is generally a transcriptional activator, whereas Sp3 is considered as a relatively weak activator or a transcriptional repressor depending on the context of the gene and the assay system (5-8). In addition, the ratio of Sp1/Sp3 that binds on the GC box of a specific gene is also dynamically changed in response to regulatory signals (9 -11). Therefore, differential binding between S...