Yeast surface display libraries of human IgG1 Fc regions were prepared in which loop sequences at the C-terminal tip of the CH3 domain were randomized. A high percentage of these library members bound to soluble CD64 and Protein A indicating that the randomization step did not grossly interfere with the overall structure of the displayed Fc. Sorting these libraries by FACS for binders against HER2/neu yielded antigen-specific Fc binders (Fcab; Fc antigen binding) of which one was affinity matured, resulting in Fcab clone H10-03-6 which showed >10-fold improvement in antigen-binding activity versus the parental clone. Pre-equilibrium surface plasmon resonance experiments revealed a K(D) value of 69 nM. H10-03-6 did not react with other members of the HER family and specifically interacted with HER2-positive but not with HER2-negative cells. Importantly, Fcab H10-03-6 elicited potent antibody-dependent cellular cytotoxicity in vitro. Finally, the in vivo half-life in mice was similar to wild-type Fc indicating that the amino acid changes in the CH3 domain did not affect the pharmacokinetic behavior of the recombinant Fc. Our data demonstrate that the Fcab scaffold combines all features of normal antibodies in a small 50 kD homodimeric protein: antigen binding, effector functions and long half-life in vivo.
Reversible acetylation of core histones plays an important role in transcriptional regulation, cell cycle progression, and developmental events. The acetylation state of histones is controlled by the activities of acetylating and deacetylating enzymes. By using differential mRNA display, we have identified a mouse histone deacetylase gene, HD1, as an interleukin-2-inducible gene in murine T cells. Sequence alignments revealed that murine HD1 is highly homologous to the yeast RPD3 pleiotropic transcriptional regulator. Indirect immunofluorescence microscopy proved that mouse HD1 is a nuclear protein. When expressed in yeast, murine HD1 was also detected in the nucleus, although it failed to complement the rpd3⌬ deletion phenotype. HD1 mRNA expression was low in G 0 mouse cells but increased when the cells crossed the G 1 /S boundary after growth stimulation. Immunoprecipitation experiments and functional in vitro assays showed that HD1 protein is associated with histone deacetylase activity. Both HD1 protein levels and total histone deacetylase activity increased upon interleukin-2 stimulation of resting B6.1 cells. When coexpressed with a luciferase reporter construct, HD1 acted as a negative regulator of the Rous sarcoma virus enhancer/promoter. HD1 overexpression in stably transfected Swiss 3T3 cells caused a severe delay during the G 2 /M phases of the cell cycle. Our results indicate that balanced histone acetylation/deacetylation is crucial for normal cell cycle progression of mammalian cells.
The amino-terminal tails of core histones are targets for multiple modifications such as acetylation, phosphorylation, and methylation. Generation of modification-specific antibodies and the identification of some of the modifying enzymes allow us to begin to understand the impact of these modifications on several cellular processes, including DNA replication and transcription. Reversible histone acetylation emerged during recent years as an important mechanism for the chromatindependent regulation of gene expression. Acetylation of ε-amino groups of lysine residues results in reduced interaction between positively charged histone tails and negatively charged DNA. Local or wide-range histone deacetylation leads to chromatin condensation, while acetylation is believed to increase the accessibility of particular genomic regions for high-molecular-weight protein complexes, thereby setting the stage for transcription.In addition to acetylation, histone phosphorylation has been recently shown to play an important role for chromatin-associated processes. Distinct sets of kinases have been implicated in these events (references 4 and 41 and references cited therein). On one hand, H3 phosphorylation correlates with entry into mitosis, suggesting a link between chromatin condensation and histone modification by kinases. On the other hand, histone H3 phosphorylation at serine 10 was found to be an important step of the so-called nucleosomal response (26; reviewed in reference 41). This term describes the phosphorylation of histone H3, which leads to the concomitant activation of the immediate-early genes c-fos, c-jun, and c-myc (3, 26). The nucleosomal response can be induced through stimulation of the mitogen-activated protein (MAP) kinase cascade by growth factors, pharmacological agents, or stress. Induction of the MAP kinase pathway leads to the activation of effector kinases (MSK1/Rsk-2) which can phosphorylate histone H3 (33,40).Only a small fraction of histone H3 is transiently phosphorylated at the G 0 /G 1 transition in growth factor-stimulated cells (1). This subset of phosphorylated histone H3 proteins is highly susceptible to hyperacetylation induced by histone deacetylase (HDAC) inhibitors. One possible explanation for this finding is the strong preference in in vitro experiments of several acetylating enzymes for histone H3 phosphorylated at serine 10 (5, 24). Indeed, a number of recent observations strongly suggest the presence of cross talk between the different histone modifying mechanisms (reviewed in references 19, 35, and 43). A link between histone acetylation and phosphorylation is provided by studies reporting the association of simultaneously acetylated and phosphorylated (in this report referred to as "phosphoacetylated") histone H3 with nucleosomes of activated immediate-early genes (5, 6, 23). A concerted action of acetyltransferases and kinases was also demonstrated in yeast (25) and in mammalian cells (28).An alternative model predicts the independent targeting of histone H3 by kinases and ac...
To dissect the p53-dependent apoptotic pathway, events following induction of temperature sensitive (ts) p53val138 were studied in a Ewing tumor cell line. Transcriptional deregulation of p53 targets ®rst observable after 1 h at 328C preceded activation of caspases and the break-down of mitochondrial respiratory activity. Activation of caspases was ®rst observed 4 h after p53 induction. Using peptide inhibitors we identi®ed activation of caspase 8 upstream of caspases-9 and -3. Although the caspase 8 speci®c inhibitor z-IETD.fmk did not aect translocation of BAX to the mitochondrial membrane and cytochrome C release it almost completely blocked cleavage of the prototype caspase substrate PARP and DNA fragmentation while enforcing mitochondrial depolarization and production of reactive oxygene species (ROS). Activation of caspase 8 did not involve death-domain receptor signaling. Expression of BCL2 only partially suppressed caspase activation but blocked apoptosis. Replacement of the Nterminus of p53val138 by the related VP16 transactivation domain created a ts p53 with a tanscriptional activity indistinguishable from p53val138 until the time of caspase activation. However, the VP16 ± p53 fusion failed to trigger caspases and subsequent induction of the ROS producing gene pig3 paralleled by complete loss of apoptotic activity. These results indicate that p53-dependent transcriptional deregulation, triggering of the caspase cascade and the mitochondrial break-down occur in a timely ordered sequence coordinated by the genuine p53 amino terminus and suggest caspase 8 and PIG3 as key regulatory elements in this process. Oncogene (2000) 19, 4096 ± 4107.
The expression of the salvage pathway enzyme thymidine kinase (TK) is very low in resting mammalian cells, but increases dramatically when growth-stimulated cells enter S phase. The 30-fold rise in TK mRNA levels in response to growth factors is due to a well-characterized transcriptional activation and less defined post-transcriptional mechanisms. A minigene containing the murine TK promoter and the TK cDNA showed a 3-fold increase in TK mRNA levels after growth induction in stably transfected mouse TK-deficient L fibroblasts. Introduction of the first three TK introns resulted in a 10-fold regulation of TK expression which was predominantly due to repressed TK mRNA levels in serum-deprived cells. Removal of intron 3 from this construct or replacement of the TK promoter by a constitutive SV40 promoter led to a reduced, but still significant increase in TK mRNA levels during the onset of proliferation. These results indicate that both the TK promoter and specific TK introns contribute independently to the growth-dependent regulation of TK mRNA expression. To examine the regulatory mechanisms in more detail we analyzed TK transcription rates and steady-state levels of nuclear transcripts from an SV40 promoter-driven minigene that contains introns 2 and 3 of the TK gene. Using a set of single-stranded probes we detected TK-specific antisense transcription that was up-regulated in resting cells. Similarly, antisense transcription of the endogenous TK gene in Swiss 3T3 cells rose during serum deprivation while sense transcription was regulated in the opposite way. Luciferase reporter assays revealed the presence of a putative antisense promoter in intron 3 of the murine TK gene. These results suggest a negative role for intron-dependent antisense transcription in the regulation of TK mRNA expression in mouse fibroblasts.
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