Krüppel (Kr), one of the zygotically active Drosophila segmentation genes, is expressed in a restricted domain during the blastoderm stage of embryogenesis and is involved in the control of development of the thoracic and abdominal segments of the fly. Kr encodes a polypeptide containing DNA-binding zinc-finger motifs, disruptions of which yield Kr mutants. We have assayed the transcriptional activities of wild-type Kr protein as well as Lac repressor/Kr fusion proteins in HeLa and CV-1 cells. Wild-type Kr and a Lac-Kr chimaeric protein repressed transcription from reporter promoters in which a consensus Kr binding site derived from sequences within the even-skipped promoter had been inserted in an upstream position. We mapped the repression function of Kr to an alanine-rich amino-terminal region of the protein, as a Lac/Kr fusion protein containing only amino acids 26-110 of Kr repressed transcription from a reporter promoter containing upstream lac operators. This demonstrates that the DNA-binding and repression activities of the Kr protein are distinct. These data are consistent with genetic evidence that Kr represses even-skipped and hunchback expression, and suggest that Kr is a negative regulator of transcription in Drosophila.
The transactivation domain (AD) of bovine papillomavirus type 1 E2 stimulates gene expression and DNA replication. To identify cellular proteins that interact with this 215-amino-acid domain, we used a transactivation-defective mutant as bait in the yeast two-hybrid screen. In vitro and in vivo results demonstrate that the cDNA of one plasmid isolated in this screen encodes a 37-kDa nuclear protein that specifically binds to an 82-amino-acid segment within the E2 AD. Mutants with point mutations within this E2 domain were isolated based on their inability to interact with AMF-1 and were found to be unable to stimulate transcription. These mutants also exhibited defects in viral DNA replication yet retained binding to the viral E1 replication initiator protein. Overexpression of AMF-1 stimulated transactivation by both wild-type E2 and a LexA fusion to the E2 AD, indicating that AMF-1 is a positive effector of the AD of E2. We conclude that interaction with AMF-1 is necessary for the transcriptional activation function of the E2 AD in mammalian cells.Components of the transcriptional apparatus that mediate the function of the transactivation domain (AD) of bovine papillomavirus type 1 (BPV1) E2 have not been characterized. The E2 AD encompasses its N-terminal 215 amino acids, and the C-terminal 125 amino acids bind the DNA sequence ACCGN 4 CGGT. Separating the AD and the DNA binding domain (DBD) is a region of approximately 100 amino acids termed the hinge. In addition to the full-length 410-amino-acid E2 protein (E2TA), the C-terminal 248 amino acids encoded by the E2 open reading frame (ORF) constitute a species (E2TR) lacking most of the AD, which retains DNA binding and represses transcription by E2TA.Within the E2 AD are two regions of net negative charge (14), a motif common in viral and cellular transcriptional activators. Specific amino acids in these regions are essential for transcriptional activation, although the distal portion of the E2 AD (amino acids 100 to 215) is also required for activity in yeast and mammalian cells (14,20,23,28). Whereas other ADs have been shown to interact with the basal factors TATA binding protein (TBP) and TFIIB, these associations have been reported not for the E2 AD but rather for its C-terminal DNA binding and dimerization domain (34, 39). These Cterminal interactions are dispensable for the function of E2, since it was observed that chimeric E2 proteins in which the E2 DBD was replaced with the GAL4 or LexA DBD activate transcription in vivo (7, 46). E2 cooperatively stimulates transcription with several cellular transcription factors, including Sp1 (27) and USF and CTF (8,19,42). Of these factors, only Sp1 has been reported to physically interact with E2 (27).E2TA also cooperates with the papillomavirus E1 protein to initiate papillomavirus DNA replication. E1 is a site-specific DNA binding protein that can unwind DNA and associate with DNA polymerase (5,36,41,48). The viral origin of replication contains binding sites for both E1 and E2 proteins (43). Although BPV1 ...
Over 10 years ago, cdk6 was identified as a new member in a family of vertebrate cdc-2 related kinases. This novel kinase was found to partner with the D-type cyclins and to possess pRb kinase activity in vitro and has since been understood to function solely as a pRb kinase in the regulation of the G 1 phase of the cell cycle. In the past 2 years, several independent studies in multiple cell types have indicated a novel role for cdk6 in differentiation. For example, cdk6 expression must be reduced to allow proper osteoblast and osteoclast differentiation, forced cdk6 expression blocked differentiation of mouse erythroid leukemia cells and cdk6 expression in primary astrocytes favors the expression of progenitor cell markers. Since exit from the cell cycle is a necessary step in terminal differentiation, down-regulation of a mitogenic factor may be expected in this process, however it is surprising that this association has not been previously uncovered and that it is apparently not shared with cdk4, long understood to be a functional homolog of cdk6. The mechanism of cdk6 function in differentiation is not understood, but it may extend beyond the established role of cdk6 as a pRb kinase. As this story unfolds it will be important to discover if the function of cdk6 in differentiation is pRb-dependent or pRb-independent, since pRb has long been established as a key factor in initiating and maintaining cell cycle exit during differentiation. J. Cell. Key words: cdk6; differentiation; G 1 phase; pRb; cdk4; cyclin DThe cell cycle is divided into four main phases: G 1 , S, G 2 , and M phase. G 1 and G 2 phase were originally named gap phases since the early microscopists saw these as breaks between the visible activities of S-phase-where DNA replication occurred and M-phase-where cytokinesis and mitosis occurred. We now understand that these gaps contain crucial regulatory events that prepare the cell for the events of DNA replication and mitosis.In G 1 phase and early S-phase, positive and negative regulatory proteins control the onset of DNA replication. The tightly regulated entry into S-phase ensures that damaged DNA is not copied and passed on to new cells and that the DNA replication necessary for cell division only occurs under the appropriate conditions. To regulate the onset of DNA replication, cyclin dependent kinases (cdk) partner with cyclins to phosphorylate the retinoblastoma (pRb) protein. When sufficiently phosphorylated, pRb releases its binding partner E2F. E2F is a transcriptional activator that, once freed from pRb, is able to activate the transcription of genes necessary to replicate DNA. Working against the mitogenic activity of the cdks are cell cycle inhibitor proteins. Two families of proteins-the INK family (containing p15 INK4b , p16 INK4a , p18 INK4c , and p19 INK4d ) and the CIP/ KIP family (containing p21 Cip1/Waf1 , p27 Kip1 , and p57 Kip2 ) have been shown to regulate the capacity of the cyclin/cdk complex for pRb phosphorylation. The relative abundance of inhibitors may dete...
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