A combinatorial network of evolutionarily conserved myelin basic protein regulatory sequences confers distinct glial-specific phenotypes.
Myelin basic protein (MBP) is required for normal myelin compaction and is implicated in both experimental and human demyelinating diseases. In this study, as an initial step in defining the regulatory network controlling MBP transcription, we located and characterized the function of evolutionarily conserved regulatory sequences. Long-range human-mouse sequence comparison revealed over 1 kb of conserved noncoding MBP 5' flanking sequence distributed into four widely spaced modules ranging from 0.1 to 0.4 kb. We demonstrate first that a controlled strategy of transgenesis provides an effective means to assign and compare qualitative and quantitative in vivo regulatory programs. Using this strategy, single-copy reporter constructs, designed to evaluate the regulatory significance of modular and intermodular sequences, were introduced by homologous recombination into the mouse hprt (hypoxanthine-guanine phosphoribosyltransferase) locus. The proximal modules M1 and M2 confer comparatively low-level oligodendrocyte expression primarily limited to early postnatal development, whereas the upstream M3 confers high-level oligodendrocyte expression extending throughout maturity. Furthermore, constructs devoid of M3 fail to target expression to newly myelinating oligodendrocytes in the mature CNS. Mutation of putative Nkx6.2/Gtx sites within M3, although not eliminating oligodendrocyte targeting, significantly decreases transgene expression levels. High-level and continuous expression is conferred to myelinating or remyelinating Schwann cells by M4. In addition, when isolated from surrounding MBP sequences, M3 confers transient expression to Schwann cells elaborating myelin. These observations define the in vivo regulatory roles played by conserved noncoding MBP sequences and lead to a combinatorial model in which different regulatory modules are engaged during primary myelination, myelin maintenance, and remyelination.
Farhadi
Hooman F
HF
Royal Victoria Hospital, Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec H3A 1A1, Canada.
Lepage
Pierre
P
Forghani
Reza
R
Friedman
Hana C H
HC
Orfali
Wayel
W
Jasmin
Luc
L
Miller
Webb
W
Hudson
Thomas J
TJ
Peterson
Alan C
AC
D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D002772:Cholera Toxin; D017124:Conserved Sequence; D003711:Demyelinating Diseases / Q000139:chemically induced / Q000378:metabolism*; D005260:Female; D015870:Gene Expression / Q000502:physiology; D018390:Gene Targeting; D005809:Genes, Regulator / Q000502:physiology*; D017930:Genes, Reporter; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000235:genetics*; D009457:Neuroglia / Q000378:metabolism*; D009836:Oligodendroglia / Q000378:metabolism; D010641:Phenotype; D010940:Plant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D000076985:Saporins; D012689:Sequence Homology, Nucleic Acid
D004676:Myelin Basic Protein; D010940:Plant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; C420112:cholera toxin B-saporin conjugate; D002772:Cholera Toxin; D007041:Hypoxanthine Phosphoribosyltransferase; D000076985:Saporins