The KRAB repression domain is one of the most widely distributed transcriptional effector domains yet identified, but its mechanism of repression is unknown. We have cloned a corepressor, KAP-1, which associates with the KRAB domain but not with KRAB mutants that have lost repression activity. KAP-1 can enhance KRAB-mediated repression and is a repressor when directly tethered to DNA. KAP-1 contains a RING finger, B boxes, and a PHD finger; the RING.B1-B2 structure is required for KRAB binding and corepression. We propose that KAP-1 may be a universal eorepressor for the large family of KRAB domain-containing transcription factors.

We have identi®ed a novel protein, BAP1, which binds to the RING ®nger domain of the Breast/Ovarian Cancer Susceptibility Gene product, BRCA1. BAP1 is a nuclearlocalized, ubiquitin carboxy-terminal hydrolase, suggesting that deubiquitinating enzymes may play a role in BRCA1 function. BAP1 binds to the wild-type BRCA1-RING ®nger, but not to germline mutants of the BRCA1-RING ®nger found in breast cancer kindreds. BAP1 and BRCA1 are temporally and spatially coexpressed during murine breast development and remodeling, and show overlapping patterns of subnuclear distribution. BAP1 resides on human chromosome 3p21.3; intragenic homozgyous rearrangements and deletions of BAP1 have been found in lung carcinoma cell lines. BAP1 enhances BRCA1-mediated inhibition of breast cancer cell growth and is the ®rst nuclearlocalized ubiquitin carboxy-terminal hydrolase to be identi®ed. BAP1 may be a new tumor suppressor gene which functions in the BRCA1 growth control pathway.

An enzyme isolated from rat liver cytosol (native molecular mass 78. 3 kDa; polypeptide molecular mass 42.5 kDa) is capable of catalysing the NADH/NADPH-dependent degradation of S-nitrosoglutathione (GSNO). The activity utilizes 1 mol of coenzyme per mol of GSNO processed. The isolated enzyme has, as well, several characteristics that are unique to alcohol dehydrogenase (ADH) class III isoenzyme: it is capable of catalysing the NAD+-dependent oxidations of octanol (insensitive to inhibition by 4-methylpyrazole), methylcrotyl alcohol (stimulated by added pentanoate) and 12-hydroxydodecanoic acid, and also the NADH/NADPH-dependent reduction of octanal. Methanol and ethanol oxidation activity is minimal. The enzyme has formaldehyde dehydrogenase activity in that it is capable of catalysing the NAD+/NADP+-dependent oxidation of S-hydroxymethylglutathione. Treatment with the arginine-specific reagent phenylglyoxal prevents the pentanoate stimulation of methylcrotyl alcohol oxidation and markedly diminishes the enzymic activity towards octanol, 12-hydroxydodecanoic acid and S-hydroxymethylglutathione; the capacity to catalyse GSNO degradation is also checked. Additionally, limited peptide sequencing indicates 100% correspondence with known ADH class III isoenzyme sequences. Kinetic studies demonstrate that GSNO is an exceptionally active substrate for this enzyme. S-Nitroso-N-acetylpenicillamine and S-nitrosated human serum albumin are not substrates; the activity towards S-nitrosated glutathione mono- and di-ethyl esters is minimal. Product analysis suggests that glutathione sulphinamide is the major stable product of enzymic GSNO processing, with minor yields of GSSG and NH3; GSH, hydroxylamine, nitrite, nitrate and nitric oxide accumulations are minimal. Inclusion of GSH in the reaction mix decreases the yield of the supposed glutathione sulphinamide in favor of GSSG and hydroxylamine.

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. (1997) J. Cell. Biochem. 67, 24 -31). To investigate mechanisms underlying this induction, effects of serum stimulation on regulation of Sp1 were examined. In Balb/c 3T3 cells, serum stimulation did not affect Sp1 synthesis or the relative binding of Sp1 family members to DNA; however, it did result in a rapid, ϳ2-fold increase in Sp1 levels and an ϳ3-fold increase in specific Sp1 phosphorylation in mid-G 1 . In normal human diploid fibroblasts, serum stimulation also increased Sp1 phosphorylation in mid-G 1 but did not affect Sp1 levels. Therefore, Sp1 phosphorylation is regulated in a growth/cell cycle-dependent manner which correlates temporally with induction of dihydrofolate reductase transcription. Further studies revealed a kinase activity specifically associated with Sp1 in a growth-regulated manner. This activity is distinct from purified kinases previously shown to phosphorylate Sp1 in vitro and phosphorylates Sp1 between amino acids 612 and 678 in its C terminus, a region also phosphorylated in mid-G 1 in vivo. Therefore, this study indicates that phosphorylation of the C terminus of Sp1 may play a role in the cell cycle regulation of its transcriptional activity.Expression of a large number of genes associated with DNA synthesis, such as dihydrofolate reductase (DHFR), 1 is tightly regulated with cell growth and the cell cycle. Many of these genes have promoters which lack a TATAA element but contain binding sites for the transcription factors Sp1 and E2F (1). Although the role of E2F sites in growth/cell cycle regulation of transcription and the regulation of E2F by retinoblastoma protein (pRB) and related pocket proteins have been extensively characterized (see Refs. 2-4, for review), a role for Sp1 sites in growth/cell cycle regulation of transcription is only beginning to emerge (e.g. Refs. 5 and 6). We have determined that Sp1 and E2F sites have distinct roles in the growth/cell cycle regulation of the hamster DHFR promoter (6). Although complete repression of DHFR transcription in G 0 and early G 1 requires E2F sites, its induction in late G 1 is mediated by Sp1 sites. A direct role of Sp1-dependent transcription in growth regulation of transcription is supported by targeting of Sp1 by viral oncoproteins (e.g. Refs. 7-9), down-regulation of Sp1 expression, and/or DNA binding activity upon differentiation in some systems (10), and increased Sp1 expression during events associated with transformation (11).Sp1 is a ubiquitous, 778-amino acid transcription factor that recognizes GC-rich sequences present in many promoters (see Refs. 1 and 2, for review). Although Sp1 has been viewed as a constitutive transcriptional activator which acts as a basal factor for TATAA-less promoters, an increasing number of studies indicate that Sp1-dependent transcription is regulated in response to a variety of signals. For example, in addition to their role in growth/cell cycle regulation of transcription, Sp1 sites are involved in induction of DHFR transcription in response to methotrexate (12), induction of CYP1...