1 by the retinoblasma protein Rb is crucial for the proper control of cell proliferation. Rb has been proposed to function, at least in part, through the recruitment of histone deacetylases. However, recent results indicate that other chromatin-modifying enzymes are likely to be involved. Here, we show that Rb also interacts with a histone methyltransferase, which specifically methylates K9 of histone H3. The results of coimmunoprecipitation experiments of endogenous or transfected proteins indicate that this histone methyltransferase is the recently described heterochromatinassociated protein Suv39H1. Interestingly, phosphorylation of Rb in vitro as well as in vivo abolished the Rb-Suv39H1 interaction. We also found that Suv39H1 and Rb cooperate to repress E2F activity and that Suv39H1 could be recruited to E2F1 through its interaction with Rb. Taken together, these data indicate that Suv39H1 is involved in transcriptional repression by Rb and suggest an unexpected link between E2F regulation and heterochromatin.

The Cyclin E1 gene (CCNE1) is an ideal model to explore the mechanisms that control the transcription of cell cycle-regulated genes whose expression rises transiently before entry into S phase. E2F-dependent regulation of the CCNE1 promoter was shown to correlate with changes in the level of H3-K9 acetylation͞methyl-ation of nucleosomal histones positioned at the transcriptional start site region. Here we show that, upon growth stimulation, the same region is subject to variations of H3-R17 and H3-R26 methylation that correlate with the recruitment of coactivator-associated arginine methyltransferase 1 (CARM1) onto the CCNE1 and DHFR promoters. Accordingly, CARM1-deficient cells lack these modifications and present lowered levels and altered kinetics of CCNE1 and DHFR mRNA expression. Consistently, reporter gene assays demonstrate that CARM1 functions as a transcriptional coactivator for their E2F1͞DP1-stimulated expression. CARM1 recruitment at the CCNE1 gene requires activator E2Fs and ACTR, a member of the p160 coactivator family that is frequently overexpressed in human breast cancer. Finally, we show that grade-3 breast tumors present coelevated mRNA levels of ACTR and CARM1, along with their transcriptional target CCNE1. All together, our results indicate that CARM1 is an important regulator of the CCNE1 gene.ACTR ͉ CCNE1 ͉ histone ͉ arginine methylation ͉ breast tumor C yclin E1 (CCNE1) protein and mRNA levels are tightly regulated as an endpoint of several regulatory pathways that are critical for growth control and frequently altered in cancer cells (1, 2). CCNE1 gene transcription is undetectable in G 0 and G 1 phases of the cell cycle, whereas it rises sharply during a narrow window of time that precedes each entry into S phase. Several pieces of evidence suggest that the periodic association of activators E2Fs-and E2F-pocket protein complexes regulate CCNE1 gene expression (3-18). E2F complexes bound to this gene were found to recruit chromatin modifiers, including members of the SNF2-like helicase family, type I histone deacetylases, the acetyltransferase CBP͞p300, the lysine methyl transferase SUVAR39H1, and the protein arginine N-methyltransferase (PRMT) 5 (7, 9-14, 17, 18), suggesting that they foster periodic chromatin remodeling of the CCNE1 promoter region (11,12,14). Notably, repression of the CCNE1 gene in G 0 -G 1 correlates with the methylation of H3-K9 and H4-R3 on a single nucleosome positioned at the transcriptional start site (11)(12)(13)(14). Conversely, the late G 1 activation of the CCNE1 gene correlates with decreased H3-K9 methylation and with enhanced H3͞H4 acetylation of the same chromatin region (11)(12)(13)(14). Here, we reveal that this CCNE1 proximal promoter region is targeted by another histone arginine methyl-transferase, the type I enzyme PRMT4 [coactivator-associated arginine methyltransferase (CARM1)] (19-25). PRMT4͞CARM1 was initially described as a transcriptional coactivator of the p160 family of nuclear receptor-associated factors (Src-1͞NCoA1, GRIP1͞TIF2͞Src-2͞ NC...

The histone acetyltransferases CREB binding protein (CBP) and the related p300 protein function as key transcriptional co-activators in multiple pathways. In the case of transcriptional activation by nuclear receptors, ligand promotes the recruitment of co-activators of the p160 family, such as GRIP-1. Subsequently, the p160 co-activators recruit other co-activators via two activation domains, AD1 and AD2. AD1 binds CBP or p300, whereas AD2 has been shown to activate transcription through the recruitment of the arginine methyltransferase CARM1. Recently, the KIX domain of CBP has been shown to be methylated by CARM1 in vitro. Here, we report that another domain of CBP is specifically methylated by CARM1 on conserved arginine residues in vitro and in vivo. We also provide functional evidence that arginine residues methylated by CARM1 play a critical role in GRIP-1-dependent transcriptional activation and in hormone-induced gene activation. Altogether, our data provide strong evidence that arginine methylation represents an important mechanism for modulating co-activator transcriptional activity.

Serratia marcescens S6 produces a pl 9.7 carbapenem-hydrolyzing ,-lactamase that is probably encoded by the chromosome (Y. Yang, P. Wu, and D. M. Livermore, Antimicrob. Agents Chemother. 34:755-758, 1990). A total of 11.3 kb of genomic DNA from this strain was cloned into plasmid pACYC184 in Escherichia coli. After further subclonings, the carbapenem-hydrolyzing ,I-lactamase gene (blaSme1) was sequenced (EMBL accession number Z28968). The gene corresponded to an 882-bp open reading frame which encoded a 294-amino-acid polypeptide. This open reading frame was preceded by a -10 and a -35 region consistent with a putative promoter sequence of members of the family Enterobacteriaceae. This promoter was active in E. coli and S. marcescens, as demonstrated by primer extension analysis. N-terminal sequencing showed that the Sme-1 enzyme had a 27-amino-acid leader peptide and enabled calculation of the molecular mass of the mature protein (29.3 kDa). Sequence alignment revealed that Sme-i is a class A serine ,I-lactamase and not a class B metalloenzyme. The earlier view that the enzyme was zinc dependent was discounted. Among class A j8-lactamases, Sme-i had the greatest amino acid identity (70%) with the pl 6.9 carbapenem-hydrolyzing I-lactamase, NMC-A, from Enterobacter cloacae NOR-1. Comparison of these two protein sequences suggested a role for specific residues in carbapenem hydrolysis. The relatedness of Sme-i to other class A ,-lactamases such as the TEM and SHV types was remote. This work details the sequence of the second carbapenemhydrolyzing class A P-lactamase from an enterobacterial species and the first in the genus Serratia.Despite widespread therapeutic usage, resistance to imipenem remains very rare in clinical isolates of the family Enterobacteriaceae, but resistance can arise via two mechanisms. First, high-level production of chromosomal AmpC cephalosporinases combined with substantially decreased outer membrane permeability may result in carbapenem resistance in Enterobacter cloacae, Enterobacter aerogenes, and Proteus rettgeri (8,11,13,16,24,41). Second, resistance may result from the synthesis of ,-lactamases able to hydrolyze carbapenems (27). So far, only three enterobacterial strains have been well described as producing carbapenem-hydrolyzing ,-lactamases. Specifically, Serratia marcescens S6 and S8 produce similar pI 9.7 carbapenem-hydrolyzing P-lactamases and E. cloacae NOR-1 produces a pl 6.9 enzyme, NMC-A (34, 50). These strains also have AmpC cephalosporinases. The carbapenem-hydrolyzing 1-lactamases of S. marcescens S6 and E. cloacae NOR-1 have similar hydrolytic properties and are partially inhibited by similar concentrations of clavulanic acid. The NMC-A enzyme has been sequenced and has been shown to be a class A serine 1-lactamase (35), but the S. marcescens S6 enzyme remains unsequenced and was initially inferred to be a metalloenzyme on the basis of data suggesting inhibition by EDTA (50).In the present report, we describe the cloning and DNA and