Therefore, this region is a target of upstream serotonergic transcriptional cascade. interact with GATA factors ETS transgene expression. Unexpectedly, function. Comparable numbers of in their midline organization. Our findings identify a direct transcriptional interaction between Gata-2 and and a unique marker for new insight into function PF 477736 in 5-HT neuron development. Keywords: (Hendricks et al., 2003). In expression is governed by a serotonergic transcriptional cascade that includes the proneural factor (Pattyn et al., 2004), the homeodomain factor (Pattyn et al., 2003), and the forkhead box factor (Jacob et al., 2007) in ventral hindbrain progenitors and the zinc finger factor in postmitotic precursors (Craven et al., 2004). We showed previously that a is sufficient to direct transgene reporter expression to developing and adult 5-HT neurons (Scott et al., 2005). Therefore, this region is a target of upstream serotonergic transcriptional cascade. However, the precise location of has not been determined, nor is it known whether any of the identified transcription factors in the cascade directly regulate encodes a protein that has 96% identity to and is expressed specifically in human raphe (Iyo et al., 2005). Recently, we showed that both serotonergic and nurturing deficits in (Lerch-Haner et al., 2008), hence demonstrating that is an ortholog of gene expression. These findings show subtle alterations in expression can influence serotonergic gene expression and the quality of nurturing behaviors. Thus, regulation and function may be relevant to disease pathogenesis (Rand et al., 2007). However, the mechanisms that control expression in 5-HT neurons have not been investigated. Here, we investigated the and report that sequences surrounding the transcriptional start site are sufficient to direct 5-HT neuron-specific transgene expression. Two conserved GATA sites in this region are required in a functionally redundant manner for serotonin neuron transgene expression. Finally, upstream fragment was subcloned into the modified BGZA vector. The vector sequences were removed before pronuclear injection with upstream sequences and transgene structure. Top, zPicture analysis of mouse and human conserved genomic sequences upstream of reveals blocks of human/mouse conservation. The LacZ transgenes tested in this study. The 5 ends of FEV2.2Z, FEV1.1Z, and FEV0.6Z are located at ?1924, ?787, and ?275 bp, respectively, relative to the transcriptional start site. The 3 end of all transgenes is a at E12.5, over the total number of lines evaluated for each construct. ?, Very weak expression detected in 11 of 27 lines. *Adult expression also examined: 11 of 12 FEV2.2Z and 1 of 4 FEV0.6Z lines showed adult serotonergic transgene expression. FEV2.2Zg. FEV2.2Z was digested with distal site (GATA1) 5-GGATGCGGGCAGAGATAAAGGGAGCAACGGCTGC-3 and complement; proximal site (GATA2) 5-GGAAATTTAAAAGTGAAGATGCAGATAACGCAGCCTGGAGACGGG-3 and complement. The inserts were fully sequenced, and fragment was obtained from RPCI-3304 and subcloned into pBACe3.6 using fragment to prepare FEV60Z. The vector backbone of FEV60Z transgene was removed PF 477736 with GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_017521″,”term_id”:”1707761915″,”term_text”:”NM_017521″NM_017521; GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_153111″,”term_id”:”237858809″,”term_text”:”NM_153111″NM_153111) and the ECR browser tools (Ovcharenko et al., 2004). Predicted transcription factor binding sites were obtained using rVista 2.0 (Loots and Ovcharenko, 2004) and MatInspector (Cartharius et al., 2005). were tested PF 477736 with the following biotinylated oligonucleotides (GATA motif PF 477736 underlined): GATA1 site, 5-CGGGCAGAGATAAAGGGAGC-3; GATA2 site, 5-AAGATGCAGATAACGCAGCC-3; and complementary oligonucleotides. Biotin-labeled oligonucleotides were annealed, and 60C80 fmol of double-stranded oligonucleotides were incubated with 1 g recombinant Gata-1 protein (Panomics) or 6.4C12.8 g Rabbit Polyclonal to MARK3 of HeLa nuclear extracts (Promega). Competition assays were performed using 100-fold excess of unlabeled wild-type or base-substituted oligonucleotides (in which the GATA motif was changed to AATT as in transgenic studies). For supershift experiments, 5 l of goat anti-Gata-2 (Santa Cruz Biotechnology) or rabbit anti-green fluorescent protein (GFP) (Invitrogen) were used. For both supershift and competition experiments, extracts were preincubated for 20 min in the absence of labeled DNA, followed by 20 min incubation with labeled oligonucleotide. Reactions were electrophoresed on 6% PAGE in 0.5 Tris-borate-EDTA and processed according the instructions of the manufacturer (Pierce). Chromatin immunoprecipitation assays Hindbrain tissues (from mesencephalic flexure to cervical flexure) were removed from 141 E11.5 embryos and quick frozen on dry ice. Gata-2 occupancy of genomic regions was tested by GenPathway, using rabbit anti-Gata-2 antibody (Santa Cruz Biotechnology) and quantitative PCR (QPCR) according to their protocols (Alexiadis et al., 2007). Supplemental Table 1 (available at www.jneurosci.org PF 477736 as supplemental material) gives the sequences of primers used for QPCR. Each primer pair gave a single product by melt-curve analysis and agarose gel electrophoresis. Binding was tested in triplicate for two negative control regions (untranscribed genomic regions Untr8, Untr17) and several upstream regions containing predicted GATA sites. Data are expressed as fold increase in binding for each sample relative to binding at Untr17. Differences in binding among regions were calculated using one-way ANOVA.
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