Regulatory modules function in a non-autonomous manner to control transcription of the mbp gene

Abstract

Multiple regulatory modules contribute to the complex expression programs realized by many loci. Although long thought of as isolated components, recent studies demonstrate that such regulatory sequences can physically associate with promoters and with each other and may localize to specific sub-nuclear transcription factories. These associations provide a substrate for putative interactions and have led to the suggested existence of a transcriptional interactome. Here, using a controlled strategy of transgenesis, we analyzed the functional consequences of regulatory sequence interaction within the myelin basic protein (mbp) locus. Interactions were revealed through comparisons of the qualitative and quantitative expression programs conferred by an allelic series of 11 different enhancer/inter-enhancer combinations ligated to a common promoter/reporter gene. In a developmentally contextual manner, the regulatory output of all modules changed markedly in the presence of other sequences. Predicted by transgene expression programs, deletion of one such module from the endogenous locus reduced oligodendrocyte expression levels but unexpectedly, also attenuated expression of the overlapping golli transcriptional unit. These observations support a regulatory architecture that extends beyond a combinatorial model to include frequent interactions capable of significantly modulating the functions conferred through regulatory modules in isolation.

Figure 1.

Constructs docked as single copy at the hprt locus in transgenic mice. The constructs generated in this study contain different deletions and combinations of mbp regulatory sequences, ligated to a lacZ reporter gene. Mbp regulatory modules (M1–M4) are represented as colored boxes whereas non-conserved inter-modular sequences are shown in white. The first construct contains 9.5 kb of mbp 5′-flanking sequence while the others bear deletions of modules and/or intermodular sequences.

Figure 2.

Expression phenotypes of mbp reporter constructs in oligodendrocytes. The function of mbp regulatory sequences in oligodendrocytes was analyzed by measuring the β-galactosidase activity at post-natal Days 14, 21 and 90 in cervical spinal cords. Significant differences (when P < 0.01) are represented by asterisk or hash compared to 9.5 kb or M1M2M3M4, respectively. Comparisons between all constructs are shown in Supplementary Table S1.

Figure 3.

M1 restricts expression of M3 to oligodendrocytes. Whole mount histochemical preparations of mouse spinal cords at post-natal Day 9 show that M3 ligated to the hsp minimal promoter drives lacZ expression in both spinal cord (oligodendrocytes) and spinal roots (Schwann cells). In contrast, expression is limited to oligodendrocytes when M3 is ligated to the mbp M1 promoter.

Figure 4.

Expression phenotypes of mbp reporter constructs in Schwann cells. Graphs show levels of β-galactosidase activity realized from mbp regulated reporter constructs in the sciatic nerves of transgenic mice at P14, P21 and P90. Significant differences (when P < 0.01) are represented by asterisk or hash compared to 9.5 kb or M1M2M3M4, respectively. Comparisons between all constructs are shown in Supplementary Table S2.

Figure 5.

M3 knockout from the golli-mbp locus. (A) Gene targeting strategy for knocking out M3 in the endogenous mbp gene. Diagrams of the targeting vector, endogenous mbp gene, recombined allele bearing 3 lox P sites, and the excised allele or M3KO are shown. Homology arms are shown as gray rectangles and LoxP sites as gray triangles. M3, the neomycin resistance gene and the thymidine kinase gene are shown as white, labeled boxes. Relevant restriction sites for Southern blots are indicated in the diagrams. Probe sequences are shown as black rectangles underneath each diagram. (B) Identification of ES clones with the recombined +/3Lox allele using external and internal probes by Southern blot analysis. The correct targeting event was confirmed by the appearance of an 8.7-kb band after digestion with AseI and hybridization with the 5′-probe and a 9.5-kb band after digestion with EcoRI and hybridization with the 3′-probe. With the same probes, the wild-type allele revealed 6.8- and 7.6-kb bands, respectively. After SacI digestion, an internal probe (not shown) detected 6.3- and 2.3-kb bands with the wild-type allele and 8.2- and 2.3-kb bands with the +/3lox allele. (2.3-kb bands are not shown). (C) Strategy to discriminate between wt and M3KO alleles by PCR.

Figure 6.

Expression of mbp is reduced in oligodendrocytes of M3KO mice. At P12, 21 and 60, real-time RT–PCR evaluation of mbp mRNA from the cervical spinal cords of M3KO homozygotes revealed accumulation to 60% of the wild-type level (*P < 0.01). Number of mice analyzed: P12 (2+/+ and 5−/−); P21 (6+/+ and 5−/−); P60 (5+/+ and 5−/−). In P14 sciatic nerves, no difference between WT and M3KO mbp expression was observed.

Figure 7.

Expression of golli is reduced in M3KO mice. Real-time PCR analysis of golli mRNA from the cervical spinal cord showed reduced accumulation in M3KO homozygous mice (−/−) compared to wild-type (+/+). Similar relative reductions were observed at all ages investigated. The same dramatic reduction in golli mRNA accumulation also was observed in the optic nerves of M3KO (−/−) mice where no neuronal cell bodies are located.