A number of signalling pathways stimulate transcription of target genes through nuclear factors whose activities are primarily regulated by phosphorylation. Cyclic AMP regulates the expression of numerous genes, for example, through the protein kinase-A (PKA)-mediated phosphorylation of transcription factor CREB at Ser 133. Although phosphorylation may stimulate transcriptional activators by modulating their nuclear transport or DNA-binding affinity, CREB belongs to a class of proteins whose phosphorylation appears specifically to enhance their trans-activation potential. Recent work describing a phospho-CREB binding protein (CBP) which interacts specifically with the CREB trans-activation domain prompted us to examine whether CBP is necessary for cAMP regulated transcription. We report here that microinjection of an anti-CBP antiserum into fibroblasts can inhibit transcription from a cAMP responsive promoter. Surprisingly, CBP also cooperates with upstream activators such as c-Jun, which are involved in mitogen responsive transcription. We propose that CBP is recruited to the promoter through interaction with certain phosphorylated factors, and that CBP may thus play a critical role in the transmission of inductive signals from cell surface receptor to the transcriptional apparatus.

Efforts to elucidate the contributions by transcription factors to plant gene expression will require increasing knowledge of their specific in vivo regulatory associations. We are systematically investigating the role of individual TGA factors in the transcriptional control of pathogenesis-related (PR) defense genes, whose expression is stimulated in leaves by salicylic acid (SA) through a stimulus pathway involving NPR1. We focused on PR-1 because its SA-induced expression in Arabidopsis is mediated by an as-1 -type promoter cis element ( LS7 ) that is recognized in vitro by TGA factors. We found that two NPR1-interacting TGA factors, TGA2 and TGA3, are the principal contributors to an LS7 binding activity of leaves that is enhanced by SA through NPR1. The relevance of these findings to PR-1 expression was investigated by the use of chromatin immunoprecipitation, which demonstrated that in vivo these TGA factors are strongly recruited in an SA-and NPR1-dependent manner to the LS7 -containing PR-1 promoter. Significantly, the timing of promoter occupancy by these factors is linked to the SA-induced onset and sustained expression of PR-1 . Because leaf transfection assays indicate that TGA3 activates transcription, as noted previously for TGA2, these two TGA factors are predicted to make positive contributions to the expression of this target gene. Thus, the findings presented here distinguish among different modes of regulation by these transcription factors and provide strong support for their direct role in the stimulus-activated expression of an endogenous defense gene.

The mechanism by which CBP in turn mediates induction of cAMP-responsive genes is unknown but is thought to involve recruitment of basal transcription factors to the promoter. Here we demonstrate that CBP associates specifically with RNA polymerase II in HeLa nuclear extracts. This association in turn permits RNA polymerase II to be recruited to CREB in a phospho-(Ser-133)-dependent manner. As anti-CBP antiserum, which inhibits recruitment of CBP and RNA polymerase II to phospho-(Ser-133) CREB, attenuates transcriptional induction by protein kinase A in vitro, our results demonstrate that the CBP⅐RNA polymerase II complex is critical for expression of cAMP-responsive genes.A number of hormones and growth factors stimulate the expression of target genes by inducing the reversible phosphorylation of specific transcription factors (4). Although phosphorylation has been shown to regulate a number of nuclear factors by inducing their nuclear targeting or DNA binding activities, the cAMP-responsive transcription factor CREB belongs to a group whose transactivation potential is affected (1,5). In this regard, Chrivia et al. (3) have characterized a nuclear CREB binding protein, termed CBP, which binds to CREB in a phospho-(Ser-133)-dependent manner. The requirement for CBP in mediating cAMP-dependent transcription has been demonstrated by cellular microinjection experiments in which CBP antisera blocked transcriptional induction by cAMP (2) and by transient transfection experiments in which overexpression of CBP could potentiate CREB activity in response to agonist (2, 6). Here we examine the mechanism by which CBP interacts with the transcriptional apparatus to induce target gene expression in response to hormonal stimulation. Our results suggest that CBP is constitutively associated with specific components of the transcriptional apparatus and that this association in turn permits recruitment of certain basal factors to promoters of cAMP-responsive genes. EXPERIMENTAL PROCEDURES Preparation of Nuclear Extracts and in Vitro Transcription Assays-Nuclear extract preparations and in vitro transcription assays were carried out as described previously (5). To evaluate the effect of PKA 1 on in vitro transcription reactions, purified recombinant PKA catalytic subunit (1 g) (kindly provided by S. Taylor) was added to HeLa nuclear extracts during transcription reactions. CREB activity was monitored with an adenovirus major late promoter template containing three cAMP-responsive elements (CREs) from the rat somatostatin promoter (Ϫ56 to Ϫ32). Affinity-purified antisera were added to in vitro transcription assays as reported (5).Immunoprecipitation Assays-For immunoprecipitation assays, HeLa nuclear extract (100 g) was precleared with protein A-Sepharose for 30 min at 4°C. Precleared extract was incubated with primary antibody for 1 h at 4°C. To detect ␣ RNA polymerase II, a monoclonal antiserum, raised against a C-terminal domain polypeptide of the large subunit (Promega), was used. Antibody complexes were recovered by incuba...

SummaryGenome editing and human induced pluripotent stem cells hold great promise for the development of isogenic disease models and the correction of disease-associated mutations for isogenic tissue therapy. CRISPR-Cas9 has emerged as a versatile and simple tool for engineering human cells for such purposes. However, the current protocols to derive genome-edited lines require the screening of a great number of clones to obtain one free of random integration or on-locus non-homologous end joining (NHEJ)-containing alleles. Here, we describe an efficient method to derive biallelic genome-edited populations by the use of fluorescent markers. We call this technique FACS-assisted CRISPR-Cas9 editing (FACE). FACE allows the derivation of correctly edited polyclones carrying a positive selection fluorescent module and the exclusion of non-edited, random integrations and on-target allele NHEJ-containing cells. We derived a set of isogenic lines containing Parkinson's-disease-associated mutations in α-synuclein and present their comparative phenotypes.