Identification of a direct Dlx homeodomain target in the developing mouse forebrain and retina by optimization of chromatin immunoprecipitation

Abstract

Understanding homeobox gene specificity and function has been hampered by the lack of proven direct transcriptional targets during development. Dlx genes are expressed in the developing forebrain, retina, craniofacial structures and limbs. Dlx1/Dlx2 double knockout mice die at birth with multiple defects including abnormal forebrain development and decreased Dlx5 and Dlx6 expression. We have successfully applied chromatin immunoprecipitation (ChIP) to identify a direct transcriptional target of DLX homeoproteins from embryonic tissues in vivo. We optimized cross-linking conditions to enrich for protein-DNA complexes, then using specific high affinity DLX antibodies captured immunoenriched DLX genomic DNA transcriptional targets. DLX homeobox proteins bind differentially to the Dlx5/Dlx6 intergenic enhancer in newborn retina (DLX2) and embryonic striatum (DLX1, DLX2) in situ. Reporter gene assays demonstrated the functional significance of the binding of DLX proteins to this regulatory element, confirmed in vitro by electrophoretic mobility shift assays, using tissue extracts or recombinant DLX proteins. ChIP provides the best approach to identify direct Dlx homeoprotein targets from developing tissues in situ. The use of this technology will advance our understanding of Dlx gene function in the vertebrate in vivo and can be applied to examine targets of other homeobox genes and other classes of transcription factors.

Identification and characterization of a novel transcript down‐regulated in Dlx1/Dlx2 and up‐regulated in Pax6 mutant telencephalon

“…For example, the intergenic regions of the Drosophila bithorax complex (BX-C) genes contain cis-regulatory elements that are transcribed, their transcription preceding and regulating activation of abdominal genes Drewell et al, 2002). The evidence that the novel noncoding RNA identified in this study may be implicated in a similar transcriptional regulation of Dlx5 and Dlx6 comes from the finding that the first exon of EST AK032537 overlaps in part with the I56i enhancer element (Zerucha et al, 2000) to which DLX1 and DLX2 proteins bind (Zhou et al, 2004) to regulate Dlx5 and Dlx6 transcription. Moreover, this hypothesis is strengthened by the overlap between mEvf-1 and Dlx5-Dlx6 expression (Anderson et al, 1997b;Eisenstat et al, 1999).…”

“…AK032537 partially overlaps with enhancer I56i (nucleotides 1-114 of AK032537). Of interest, it has been shown that both DLX1 and DLX2 proteins bind to I56i (Zhou et al, 2004).…”

Dlx Homeobox Genes Promote Cortical Interneuron Migration from the Basal Forebrain by Direct Repression of the Semaphorin Receptor Neuropilin-2

“…Chromatin Immunoprecipitation Assays-ChIP was performed under the following conditions as described (17). 1-2 ϫ 10 7 E13.5 ganglionic eminence cells were fixed with 1% paraformaldehyde for 90 min at room temperature.…”

“…The binding reactions were incubated for 30 min at room temperature. We added unlabeled probes for "cold competition" assays and specific polyclonal DLX1 or DLX2 antibodies for "supershift" assays and polyclonal Secretory Component antibody (Dako, Mississauga, Ontario) to control for nonspecific binding (17).…”

“…We have commenced isolating and characterizing candidate Dlx gene targets in the developing mouse, particularly in the forebrain, using chromatin immunoaffinity purification (ChIP) of embryonic tissues that regionally express Dlx genes. We have optimized the ChIP protocol using embryonic ganglionic eminence tissues and specific affinity-purified rabbit polyclonal antibodies to the proteins encoded by the Dlx1 and Dlx2 genes (1) to isolate the Dlx5/Dlx6 intergenic enhancer, a DLX target gene promoter sequence (17). In this study, we exploited this approach to identify a novel DLX target in the embryonic forebrain.…”