Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome.

Isogenic settings are routine in model organisms, yet remain elusive for genetic experiments on human cells. We describe the use of designed zinc finger nucleases (ZFNs) for efficient transgenesis without drug selection into the PPP1R12C gene, a "safe harbor" locus known as AAVS1. ZFNs enable targeted transgenesis at a frequency of up to 15% following transient transfection of both transformed and primary human cells, including fibroblasts and hES cells. When added to this locus, transgenes such as expression cassettes for shRNAs, small-molecule-responsive cDNA expression cassettes, and reporter constructs, exhibit consistent expression and sustained function over 50 cell generations. By avoiding random integration and drug selection, this method allows bona fide isogenic settings for high-throughput functional genomics, proteomics, and regulatory DNA analysis in essentially any transformed human cell type and in primary cells.

Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology.

We previously demonstrated high-frequency, targeted DNA addition mediated by the homology-directed DNA repair pathway. This method uses a zinc-finger nuclease (ZFN) to create a site-specific double-strand break (DSB) that facilitates copying of genetic information into the chromosome from an exogenous donor molecule. Such donors typically contain two approximately 750 bp regions of chromosomal sequence required for homology-directed DNA repair. Here, we demonstrate that easily-generated linear donors with extremely short (50 bp) homology regions drive transgene integration into 5-10% of chromosomes. Moreover, we measure the overhangs produced by ZFN cleavage and find that oligonucleotide donors with single-stranded 5' overhangs complementary to those made by ZFNs are efficiently ligated in vivo to the DSB. Greater than 10% of all chromosomes directly incorporate this exogenous DNA via a process that is dependent upon and guided by complementary 5' overhangs on the donor DNA. Finally, we extend this non-homologous end-joining (NHEJ)-based technique by directly inserting donor DNA comprising recombinase sites into large deletions created by the simultaneous action of two separate ZFN pairs. Up to 50% of deletions contained a donor insertion. Targeted DNA addition via NHEJ complements our homology-directed targeted integration approaches, adding versatility to the manipulation of mammalian genomes.

EGF regulates plasminogen activator inhibitor-1 (PAI-1) by a pathway involving c-Src, PKCdelta, and sphingosine kinase 1 in glioblastoma cells.

Patients with gliomas expressing high levels of epidermal growth factor receptor (EGFR) and plasminogen activator inhibitor-1 (PAI-1) have a shorter overall survival prognosis. Moreover, EGF enhances PAI-1 expression in glioma cells. Although multiple known signaling cascades are activated by EGF in glioma cells, we show for the first time that EGF enhances expression of PAI-1 via sequential activation of c-Src, protein kinase C delta (PKCdelta), and sphingosine kinase 1 (SphK1), the enzyme that produces sphingosine-1-phosphate. EGF induced rapid phosphorylation of c-Src and PKCdelta and concomitant translocation of PKCdelta as well as SphK1 to the plasma membrane. Down-regulation of PKCdelta abolished EGF-induced SphK1 translocation and up-regulation of PAI-1 by EGF; whereas, down-regulation of PKCalpha had no effect on the EGF-induced PAI-1 activation but enhanced its basal expression. Similarly, inhibition of c-Src activity by PP2 blocked both EGF-induced translocation of SphK1 and PKCdelta to the plasma membrane and up-regulation of PAI-1 expression. Furthermore, SphK1 was indispensable for both EGF-induced c-Jun phosphorylation and PAI-1 expression. Collectively, our results provide a functional link between three critical downstream targets of EGF, c-Src, PKCdelta, and SphK1 that have all been implicated in regulating motility and invasion of glioma cells.

Nuclear factor-1-X regulates astrocyte-specific expression of the alpha1-antichymotrypsin and glial fibrillary acidic protein genes.

Discrete tissue-specific changes in chromatin structure of the distal serpin subcluster on human chromosome 14q32.1 allow a single gene encoding alpha1-antichymotrypsin (ACT) to be expressed in astrocytes and glioma cells. This astrocyte-specific regulation involves activatory protein-1 (AP-1) because overexpression of dominant-negative c-jun(TAM67) abolishes ACT expression in glioma cells. Here we identify a new regulatory element, located within the -13-kb enhancer of the ACT gene, that binds nuclear factor-1 (NFI) and is indispensable for the full basal transcriptional activity of the ACT gene. Furthermore, down-regulation of NFI expression by siRNA abolishes basal ACT expression in glioma cells. However, NFI does not mediate astrocyte-specific expression by itself, but likely cooperates with AP-1. A detailed analysis of the 14-kb long 5'-flanking region of the ACT gene indicated the presence of adjacent NFI and AP-1 elements that colocalized with DNase I-hypersensitive sites found in astrocytes and glioma cells. Interestingly, knock-down of NFI expression also specifically abrogates the expression of glial acidic fibrillary protein (GFAP), which is an astrocyte-specific marker protein. Mutations introduced into putative NFI and AP-1 elements within the 5'-flanking region of the GFAP gene also diminished basal expression of the reporter. In addition, we found, using isoform-specific siRNAs, that NFI-X regulates the astrocyte-specific expression of ACT and GFAP. We propose that NFI-X cooperates with AP-1 by an unknown mechanism in astrocytes, which results in the expression of a subset of astrocyte-specific genes.

Astrocyte-specific expression of the alpha1-antichymotrypsin and glial fibrillary acidic protein genes requires activator protein-1.

An amyloid-associated serine proteinase inhibitor (serpin), alpha(1)-antichymotrypsin (ACT), is encoded by a gene located within the distal serpin subcluster on human chromosome 14q32.1. The expression of these distal serpin genes is determined by tissue-specific chromatin structures that allow their ubiquitous expression in hepatocytes; however, their expression is limited to a single ACT gene in astrocytes. In astrocytes and glioma cells, six specific DNase I-hypersensitive sites (DHSs) were found located exclusively in the 5'-flanking region of the ACT gene. We identified two enhancers that mapped to the two DHSs at -13 kb and -11.5 kb which contain activator protein-1 (AP-1) binding sites, both of which are critical for basal astrocyte-specific expression of ACT reporters. In vivo, these elements are occupied by c-jun homodimers in unstimulated cells and c-jun/c-fos heterodimers in interleukin-1-treated cells. Moreover, functional c-jun is required for the expression of ACT in glioma cells because both transient and stable inducible overexpression of dominant-negative c-jun(TAM67) specifically abrogates basal and reduces cytokine-induced expression of ACT. Expression-associated methylation of lysine 4 of histone H3 was also lost in these cells, but the DHS distribution pattern and global histone acetylation were not changed upstream of the ACT locus. Interestingly, functional AP-1 is also indispensable for the expression of glial fibrillary acidic protein (GFAP), which is an astrocyte-specific marker. We propose that AP-1 is a key transcription factor that, in part, controls astrocyte-specific expression of genes including the ACT and GFAP genes.

A novel mechanism of tissue inhibitor of metalloproteinases-1 activation by interleukin-1 in primary human astrocytes.

Reactive astrogliosis is the gliotic response to brain injury with activated astrocytes and microglia being the major effector cells. These cells secrete inflammatory cytokines, proteinases, and proteinase inhibitors that influence extracellular matrix (ECM) remodeling. In astrocytes, the expression of tissue inhibitor of metalloproteinases-1 (TIMP-1) is up-regulated by interleukin-1 (IL-1), which is a major neuroinflammatory cytokine. We report that IL-1 activates TIMP-1 expression via both the IKK/NF-kappaB and MEK3/6/p38/ATF-2 pathways in astrocytes. The activation of the TIMP-1 gene can be blocked by using pharmacological inhibitors, including BAY11-7082 and SB202190, overexpression of the dominant-negative inhibitor of NF-kappaB (IkappaBalphaSR), or by the knock-down of p65 subunit of NF-kappaB. Binding of activated NF-kappaB (p50/p65 heterodimer) and ATF-2 (homodimer) to two novel regulatory elements located -2.7 and -2.2 kb upstream of the TIMP-1 transcription start site, respectively, is required for full IL-1-responsiveness. Mutational analysis of these regulatory elements and their weak activity when linked to the minimal tk promoter suggest that cooperative binding is required to activate transcription. In contrast to astrocytes, we observed that TIMP-1 is expressed at lower levels in gliomas and is not regulated by IL-1. We provide evidence that the lack of TIMP-1 activation in gliomas results from either dysfunctional IKK/NF-kappaB or MEK3/6/p38/ATF-2 activation by IL-1. In summary, we propose a novel mechanism of TIMP-1 regulation, which ensures an increased supply of the inhibitor after brain injury, and limits ECM degradation. This mechanism does not function in gliomas, and may in part explain the increased invasiveness of glioma cells.