Abstract
(1) Little information exists on the role of clustered Hox genes in oligodendrocyte (OG) development. This study examines the expression profile of Hoxd1 and identifies a potential downstream target in the OG lineage. (2) Immunocytochemical analysis of primary mixed glial cultures demonstrated Hoxd1 was expressed throughout OG development. (3) A human myelin protein gene, myelin oligodendrocyte glycoprotein (MOG), was identified as a putative downstream target of Hoxdl through Genbank searches utilizing the Hoxdl homeodomain consensus binding sequence. (4) The dissociation coefficient constant (KD) and dissociation rate constant (kd) of the Hoxd1-MOG complex, determined using electrophoretic mobility shift assays (EMSAs), were estimated to be 1.9 x 10(-7) M and 1.3 x 10(-3) s(-1), respectively. The DNA-Hoxdl homeodomain complex had a half-life (t1/2) of 15 min. (5) Mutational analysis of Hoxd1-MOG complexes revealed the binding affinity of M1 (with mutation from (-1054)5'-TAAT-3'(-1051) to TACT within the consensus binding site) and M2 (with mutation from (-1054)5'-TAATTG-3'(-1049) to TAATCC within the consensus binding site) probes to the MOG promoter was severely affected. Thus the TAATTG core of the binding sequence appears important for Hoxd1 specificity. (6) Analysis of the involvement of TAAT sites adjacent to the consensus sequence in Hoxdl binding showed the binding affinity of the M3 probe was affected, but not as severely as the M1 and M2 probes. These in vitro results suggest the TTTAATTGTA sequence is involved in Hoxd1 binding to the MOG promoter but neighboring TAAT sites may also be needed. Thus, MOG may be a target of Hoxd1.