A wide variety of bioinformatic tools have been described to characterize potential transcriptional regulatory mechanisms based on genomic sequence analysis and microarray hybridization studies. However, these regulatory mechanisms are still experimentally verified using transient transfection methods. Current transfection methods are limited both by their large scale and by the low level of efficiency for certain cell types. Our goals were to develop a microarray-based transfection method that could be optimized for different cell types and that would be useful in reporter assays of transcriptional regulation. Here we describe a novel transfection method, termed STEP (surface transfection and expression protocol), which employs microarray-based DNA transfection of adherent cells in the functional analysis of transcriptional regulation. In STEP, recombinant proteins with biological activities designed to enhance transfection are complexed with expression vector DNAs prior to spotting on microscope slides. The recombinant proteins used in STEP complexes can be varied to increase the efficiency for different cell types. We demonstrate that STEP efficiently transfects both supercoiled plasmids and PCR-generated linear expression cassettes. A co-transfection assay using effector expression vectors encoding the cAMP-dependent protein kinase (PKA), as well as reporter vectors containing PKA-regulated promoters, showed that STEP transfection allows detection and quantitation of transcriptional regulation by this protein kinase. Because bioinformatic studies often result in the identification of many putative regulatory elements and signaling pathways, this approach should be of utility in high-throughput functional genomic studies of transcriptional regulation.

The basic helix-loop-helix transcription factor Ascl1 plays a critical role in the intrinsic genetic program responsible for neuronal differentiation. Here, we describe a novel model system of P19 embryonic carcinoma cells with doxycycline-inducible expression of Ascl1. Microarray hybridization and real-time PCR showed that these cells demonstrated increased expression of many neuronal proteins in a time-and concentration-dependent manner. Interestingly, the gene encoding the cell cycle regulator Gadd45γ was increased earliest and to the greatest extent following Ascl1 induction. Here, we provide the first evidence identifying Gadd45γ as a direct transcriptional target of Ascl1. Transactivation and chromatin immunoprecipitation assays identified two E-box consensus sites within the Gadd45γ promoter necessary for Ascl1 regulation, and demonstrated that Ascl1 is bound to this region within the Gadd45γ promoter. Furthermore, we found that overexpression of Gadd45γ itself is sufficient to initiate some aspects of neuronal differentiation independent of Ascl1.

As members of the proneural basic-helix-loop-helix (bHLH) family of transcription factors, Ascl1 and Neurog2 direct the differentiation of specific populations of neurons at various times and locations within the developing nervous system. In order to characterize the mechanisms employed by these two bHLH factors, we generated stable, doxycycline-inducible lines of P19 embryonic carcinoma cells that express comparable levels of Ascl1 and Neurog2. Upon induction, both Ascl1 and Neurog2 directed morphological and immunocytochemical changes consistent with initiation of neuronal differentiation. Comparison of Ascl1- and Neurog2-regulated genes by microarray analyses showed both shared and distinct transcriptional changes for each bHLH protein. In both Ascl1- and Neurog2-differentiating cells, repression of Oct4 mRNA levels was accompanied by increased Oct4 promoter methylation. However, DNA demethylation was not detected for genes induced by either bHLH protein. Neurog2-induced genes included glutamatergic marker genes while Ascl1-induced genes included GABAergic marker genes. The Neurog2-specific induction of a gene encoding a protein phosphatase inhibitor, Ppp1r14a, was dependent on distinct, canonical E-box sequences within the Ppp1r14a promoter and the nucleotide sequences within these E-boxes were partially responsible for Neurog2-specific regulation. Our results illustrate multiple novel mechanisms by which Ascl1 and Neurog2 regulate gene repression during neuronal differentiation in P19 cells.

Cell-based microarrays have been used for a wide variety of assays including gain-of-function, loss-of-function and compound screening. Many of these assays have employed fluorescent proteins as reporters. These fluorescent reporter proteins can be monitored in living cells but have low sensitivity of detection compared to enzymatic reporters. Here we have described a novel transcriptional reporter assay using the alkaline phosphatase reporter enzyme and a fluorescent substrate (ELF-97) to screen for gain-of-function mutations in the type-I cGMP-dependent protein kinase (PRKG1). We have identified a constitutively active mutant of this enzyme in which a conserved Glu at position 81 was mutated to Lys.

The proneural basic‐helix‐loop‐helix (bHLH) transcription factors Ascl1 and Neurog2 direct the differentiation of specific populations of neurons by regulating the transcription of specific sets of genes during embryonic development. Here we describe the generation of stable P19 embryonic carcinoma cells expressing comparable levels of Ascl1 and Neurog2 mRNA in a tetracycline inducible system and the resulting neuronal differentiation of these cell lines in response to doxycycline.Microarray analysis of these cell lines showed that Ascl1 and Neurog2 overlap in their gene regulation but each bHLH protein also regulated distinct genes. We demonstrated that DNA methylation accompanied repression of the Oct4 gene by both Ascl1 and Neurog2, although DNA demethylation was not observed for any genes induced by either Ascl1 or Neurog2. While the ID1 gene showed preferential induction by Ascl1 over Neurog2, the induction of ID1 reporter constructs was dependent on distinct sets of E‐boxes within the ID1 proximal promoter. Finally, the induction of the Ppp1r14a gene was specifically induced by Neurog2 and this induction was at least partially due to preferences of Neurog2 for distinct canonical E‐box sequences within the Ppp1r14a promoter.Together, these results suggest that further complexities in the mechanisms by which Ascl1 and Neurog2 regulate gene expression during neuronal differentiation.

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