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Supporting Information

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Supporting Table 1
Supporting Table 2
Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Figure 9
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Supporting Figure 6

Fig. 6. Derepression of Mmp12 in NCoR^-/- macrophages. Wild-type or NCoR^-/- macrophages were treated for 6 h with LPS or TPA. RNA was harvested and 5 mg was analyzed by Northern blotting using specific probes for the Mmp12.

Supporting Figure 7

Fig. 7. Knockdown of NCoR expression in RAW264.7 cells using an NCoR-specific siRNA increases basal and TPA-induced promoter activities of the Mmp12 promoter.

Supporting Figure 8

Fig. 8. Development of c-Jun^-/- macrophages. Breeding of c-Jun^f/f mice and MxCre transgenic mice was performed to obtain mice with c-Jun^f/f ´ MxCre^- or c-Jun^f/f ´ MxCre⁺ genotypes. Both genotypes were treated with polyinosinic-polycytidylic acid, with induction of Cre in MxCre⁺ mice, resulting in quantitative recombination of the c-Jun^f/f locus in bone marrow-derived macrophages, indicated by Southern blot analysis of macrophages from three independent mice.

Supporting Figure 9

Fig. 9. NCoR inhibits binding of c-Jun/c-Fos heterodimers. Baculovirus-expressed c-Jun and c-Fos were incubated with a [³²P] oligonucleotide containing a consensus AP-1-binding site in the presence of in vitro-translated NCoR or control reticulocyte lysate. Protein-DNA complexes were resolved by electrophoretic mobility shift assay.

Supporting Table 1

Table 1. A list of genes exhibiting significantly different levels of expression in wild-type and NCoR^-/- macrophages under basal conditions identified by the program SAM.

Supporting Table 2

Table 2. Interactive directed acyclic graph for the entire set of differentially expressed genes based on biological process annotations defined by Gene Ontology (GO) Consortium.

The graph at the bottom of the page provides a partial view of a directed acyclic graph of the GO Biological Process terms linked with genes over- or underexpressed in NCoR^-/- macrophages. The top of the page consists of a list of genes associated with a specific gene ontology term. Terms are listed in order of statistical significance, beginning with the most significant term at the top.

In the graph, each GO Biological Process is represented by an ellipse. The number in each ellipse is the GO annotation number for that term. Mousing over the annotation number results in display of the associated term, e.g., mousing over the ellipse numbered 9607 results in display of "response to biotic stimulus." The terms are organized according to the hierarchical structure of the GO database. In general, the most general terms are to the left, e.g., 9605, response to external stimulus, and the most specific terms to the right, e.g., 30595, immune cell chemotaxis. Terms are color-coded for the probability that the genes associated with that term occurred by chance. The most intensely colored ellipses are associated with the most significant P values. Paths in which functional annotations associated with genes differentially regulated in NCoR^-/- macrophages had P values >0.1 for all annotations in the path are not displayed. This results in a simplified graph that emphasizes the most significant classes of annotations. Double clicking an ellipse brings the list of genes associated with that annotation into view in a new window and displays the P value associated with the list. Additional annotations associated with this genes can then be accessed through the provided Locus Link accession number.

In the case of NCoR^-/- macrophages, the most highly significant categories related to "response to external stimulus" (including subannotations related to inflammatory responses, chemotaxis, and responses to pathogens), cell proliferation (including subannotations to M phase of the cell cycle), and collagen catabolism.

This program has been tested on several different computer platforms using the Microsoft Internet Explorer and Netscape browsers. The program is still in a relatively early phase of development and may not run on all systems.

Experimental Procedures. The following set of primers were used to amplify regions on the promoter of the genes
object of study:
Mmp12 proximal, 5'-ACTCTGCTAATGGAGTTCTG-3' and 5'-GCAGCTCATCAACCTGTTCC-3';
Mmp12 distal, 5'-ATTGTGTGAAAGAACAGTGA-3' and 5'-TATCAATGATATGCAATGCT-3';
Mmp9, 5'-AGAACCTACAGTGTGGGGAT-3' and 5'-AGTAAAACGGAATCAGTGAC-3';
Mmp13, 5'-AGAACTCATCAACCTATAAC-3' and 5'-GTCTCTCCTTCCCAGGGCAA-3';
Csf3, 5'-GTATAGGTGACTTGAGAATC-3' and 5'-TTATACATAAAGCCATCAAG-3';
Alcam, 5'-GTGGGAGGCGGAGTCGATGT-3' and 5'-ACCGTGCACGGATCACCGGG-3';
Usp18, 5'-TAAAAACATTTTATCCTATAG-3' and 5'-GCTTTCGTTTTCCCCTAGAT-3'; and
Cxcl1, 5'-TGCGTTAATCTGCCACACTC-3' and 5'-CTCGAAACTCCAGAGCTCCG-3'.