Identification and characterization of hoxa2 target genes by ChIP
Akin, Zeynep Nesrin
Hox genes are evolutionarily conserved transcription factors which act to control important developmental pathways involved in morphogenesis of the embryo. Hoxa2 is expressed in the developing CNS in rhombomeres 2-7 in the presumptive hindbrain. During development Hoxa2 expression extends caudally throughout the spinal cord and persists into adulthood. Although previous analysis of Hoxa2 expression indicates its possible role in neuronal circuit specification and/or dorsal-ventral patterning within the spinal cord, the precise genetic pathways through which Hoxa2 affects spinal cord development have not been characterized. We have used immunoprecipitation of Hoxa2-target DNA complexes from chromatin preparations of E18 mouse spinal cord and hindbrain tissue to isolate in vivo downstream target genes of Hoxa2. Seven DNA fragments were isolated, sequenced and were shown to exhibit in vitro DNA binding by Hoxa2. A search of sequence databases for the target sequences revealed that of these, two displayed high identity with novel mouse genes: toll-associated serine protease (Tasp) and the murine homolog of the human dual specificity tyrosine phosphorylation regulated kinase 4 (Dyrk4). Also, two of the isolated clones are presumably bacterial sequences containing the canonical homeodomain binding site TAAT, and the remaining three clones have not yet been mapped in the mouse genome. A potential core Hoxa2 binding motif consisting of 5' CCATCA/T 3', which is based on a previously characterized Hoxa2-Pbx consensus sequence (Lampe et al., 2004), has been identified in both the Tasp and Dyrk4 intronic elements. Both Dyrk4 and Tasp mRNA have been detected within the developing mouse from E10-18 and in the adult CNS. Analysis by RT-PCR of Tasp expression in Hoxa2-/- newborn mice hindbrain and spinal cord tissues showed an upregulation of Tasp, and transient transfection experiments indicated that Hoxa2 may act as a transcriptional repressor of Tasp through an intronic regulatory element. Transfection studies using the intronic sequence of Dyrk4 indicated that it may function as an enhancer of transcription of Dyrk4 in the presence of Hoxa2. Both Dyrk4 and Tasp belong to large protein subfamilies whose members play a role in numerous developmental pathways in several organisms. Tasp, also known as HtrA3, interacts with TGFâ signaling molecules which are known to be key regulators of development, dorsoventral patterning and are involved in various neuronal pathways. Although the function of Dyrk4 is not known, many of its family members are involved in the regulation of transcription factors and signaling molecules via phosphorylation that are involved in neuronal pathways also. Hoxa2 may act in specifying neuronal subtypes and dorsoventral patterning in the CNS through down and upregulation of its downstream targets Dyrk4 and Tasp, respectively.
DegreeDoctor of Philosophy (Ph.D.)
CommitteeNazarali, Adil J.
Copyright DateSeptember 2004