Transcriptional regulators of the Golli/myelin basic protein locus integrate additive and stealth activities

Abstract

Myelin is composed of plasma membrane spirally wrapped around axons and compacted into dense sheaths by myelin-associated proteins. Myelin is elaborated by neuroepithelial derived oligodendrocytes in the central nervous system (CNS) and by neural crest derived Schwann cells in the peripheral nervous system (PNS). While some myelin proteins accumulate in only one lineage, myelin basic protein (Mbp) is expressed in both. Overlapping the Mbp gene is Golli, a transcriptional unit that is expressed widely both within and beyond the nervous system. A super-enhancer domain within the Golli/Mbp locus contains multiple enhancers shown previously to drive reporter construct expression specifically in oligodendrocytes or Schwann cells. In order to determine the contribution of each enhancer to the Golli/Mbp expression program, and to reveal if functional interactions occur among them, we derived mouse lines in which they were deleted, either singly or in different combinations, and relative mRNA accumulation was measured at key stages of early development and at maturity. Although super-enhancers have been shown previously to facilitate interaction among their component enhancers, the enhancers investigated here demonstrated largely additive relationships. However, enhancers demonstrating autonomous activity strictly in one lineage, when missing, were found to significantly reduce output in the other, thus revealing cryptic "stealth" activity. Further, in the absence of a key oligodendrocyte enhancer, Golli accumulation was markedly and uniformly attenuated in all cell types investigated. Our observations suggest a model in which enhancer-mediated DNA-looping and potential super-enhancer properties underlie Golli/Mbp regulatory organization.

Fig 1. Organization of the Golli/Mbp locus.

(A) Golli/Mbp locus with identified transcripts, CTCF binding signals and super-enhancer domain. (B) Mbp 5’ flanking sequence indicating five modules of high interspecies sequence conservation in selected vertebrates with demonstrated autonomous enhancer activity (adapted from UCSC browser; see full species comparisons in mouse genome assembly NCBI37/mm9). Only representative Golli/Mbp transcripts are shown. (C) The position and length of individual and combined enhancer deletions derived into individual mouse lines.

Fig 2. Relative Mbp mRNA accumulation in cervical spinal cord.

M1E, M3 and M5 are major enhancers of Mbp in oligodendrocytes. X-axis = post-natal age in days and Y-axis = Mbp/Gapdh mRNA ratio. Dots represent values at P7, 14, 21, 30 and 90 and connecting lines are the predicted developmental programs calculated as trendlines (Excel). Error bars = SEM.

Fig 3. Relative Mbp mRNA accumulation in sciatic nerves.

M4 is the major Mbp enhancer in Schwann cells. Loss of M3 and/or M5 alone resulted in lower accumulation at several ages while loss of M1E has no additional affect. X-axis = age in days and Y-axis = Mbp/Gapdh mRNA ratio. Dots represent post-natal days P4, 7, 14, 21, 30 and 90 and connecting lines are the predicted developmental programs calculated as trendlines (Excel). Error bars = SEM.

Fig 4. M3 is a major Golli enhancer in spinal cord.

All alleles deleted of M3 demonstrate an approximately 90% reduction in Golli mRNA accumulation in spinal cord. In contrast, the partially deleted M3(225)KO allele supported accumulation ranging from a mild decrease to a modest increase at different ages. X-axis = age in days and Y-axis = Golli/Gapdh mRNA ratio. Dots represent post-natal days P7, 14, 21, 30 and 90 and connecting lines (both solid and dashed) are the predicted developmental programs calculated as trendlines (Excel). Error bars = SEM.

Fig 5. M3 is also the major Golli enhancer in thymus and retina.

All alleles deleted of M3, but not the partially deleted M3(225)KO allele, reduced Golli accumulation to a similar extent. X-axis = age in days and Y-axis = %Golli/Gapdh mRNA. Error bars = SEM.

Fig 6. A tentative DNA-looping model for enhancer engagement.

(A) The predicted direct enhancer-promoter interaction for Mbp expression in oligodendrocytes (top with blue arrows) and Schwann cells (bellow with the red arrow). (B) The predicted relationship between M3 and the Mbp and Golli promoters. White boxes = enhancers and proximal promoters. Deleted sequences are highlighted in grey. Arrows = potential interactions. Red triangles = CTCF binding sites.