MBD2 and MeCP2 regulate distinct transitions in the stage-specific differentiation of olfactory receptor neurons

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Abstract

DNA methylation-dependent gene silencing is initiated by DNA methyltransferases (DNMTs) and mediated by methyl-binding domain proteins (MBDs), which recruit histone deacetylases (HDACs) to silence DNA, a process that is essential for normal development. Here, we show that the MBD proteins MBD2 and MeCP2 regulate distinct transitional stages of olfactory receptor neuron (ORN) differentiation in vivo. Mbd2 null progenitors display enhanced proliferation, recapitulated by HDAC inhibition, and Mbd2 null ORNs have a decreased lifespan. Mecp2 null ORNs, on the other hand, temporarily stall at the stage of terminal differentiation, retaining expression of the immature neuronal protein GAP43 after initiating expression of mature neuronal genes. The Gap43 promoter is highly methylated in the mature, but not embryonic olfactory epithelium (OE), suggesting that Gap43 may be regulated by DNA methylation during ORN differentiation. Thus, MBD2 and MeCP2 may mediate distinct, sequential transitions of ORN differentiation—an epigenetic mechanism that may be relevant to developmental regulation throughout the nervous system.

Introduction

Modification of mammalian DNA by cytosine (CpG) methylation is a heritable component of epigenetic gene regulation (Jaenisch and Bird, 2003) that is essential for normal mammalian development (Li et al., 1992, Okano et al., 1999). DNA methylation silences gene expression by sterically hindering transcription factors from binding to their promoter, or by providing binding sites for methyl-binding domain (MBD) proteins which recruit histone deacetylase (HDAC)-containing repressor complexes to modify chromatin structure (reviewed in Bird, 2002). Abnormalities in DNA methylation can contribute to aberrant mitosis (Jones and Baylin, 2002), developmental disorders (Hansen et al., 1999, Xu et al., 1999), and are implicated in polygenic neuropsychiatric disorders such as autism and schizophrenia (Abdolmaleky et al., 2004, Jiang et al., 2004).

The nervous system appears to be particularly vulnerable to changes in the activity of MBD proteins, evidenced by the neurodevelopmental defects of Rett syndrome, a leading cause of mental retardation in females, which results from a mutation in the MBD protein, Mecp2 (Amir et al., 1999). In addition, Mbd1 null mice display impaired adult hippocampal neurogenesis and increased genomic instability in neural stem cells (Zhao et al., 2003), while Mbd2 null mice display maternal behaviour defects (Hendrich et al., 2001). Furthermore, DNMT1 and MBDs have been implicated in the differential vulnerability of neurons following ischemia or kindling (Endres et al., 2000, Jung et al., 2002). The nervous system may display heightened sensitivity to epigenetic chromatin modifications because of a requirement for balanced positive and negative gene regulation mechanisms to define shifts in state as neurons and glia become progressively more specialized as development proceeds.

Olfactory receptor neurons (ORNs) in the olfactory epithelium (OE) of the nasal cavity undergo continuous neuronal replacement throughout adulthood. The postnatal OE thus contains cells at all stages of neuronal development—proliferating progenitors, immature neurons, functionally mature neurons, and dying ORNs—organized in a stratified, developmentally hierarchical manner (Graziadei and Graziadei, 1979, Huard et al., 1998). As such, the OE is a valuable model for identifying and testing mechanisms that regulate distinct stages in neuroglial development (Murdoch and Roskams, 2007). The OE also displays tightly regulated expression of the de novo DNA methyltransferases, which catalyze new methylation patterns (Li et al., 1992, Okano et al., 1999), at successive stages of ORN differentiation (MacDonald et al., 2005). DNMT3b is induced in cycling progenitors and is downregulated postmitotically, whereas DNMT3a is first induced in postmitotic immature ORNs and downregulated as they functionally mature. HDAC1 is similarly found in cycling progenitors and is downregulated following mitosis, whereas HDAC2 is induced in postmitotic immature ORNs and downregulated as they functionally mature, at which time HDAC1 is reinduced in a subset of terminally differentiating ORNs (MacDonald et al., 2005), a pattern of expression that is also repeated in the developing CNS (MacDonald and Roskams, 2008).

The segregation of genes associated with gene silencing and chromatin remodelling to distinct stages in ORN development (MacDonald et al., 2005, Shetty et al., 2005) suggests that a progressive silencing of genes may be important in restricting an ORN's gene expression repertoire during neural development. Here we demonstrate that the MBD proteins MBD2 and MeCP2 are expressed at defined, sequential stages of olfactory neurogenesis, and that they centrally contribute to the development of ORNs as they traverse sequential transitional checkpoints in differentiation that require the silencing of genes no longer necessary in future development to traverse or maintain the next stage of differentiation.

Section snippets

Results

The tightly regulated and sequential expression of DNMT3a and DNMTb suggests at least two transitional checkpoints in ORN development that may be regulated by DNA methylation: (1) as dividing progenitors exit mitosis and commit to the neuronal lineage, initiated by DNMT3b, and (2) as immature receptor neurons lose developmental plasticity and transition into a mature ORN, initiated by DNMT3a (MacDonald et al., 2005). MeCP2 expression in neurons increases progressively with maturation, both in

Discussion

As a neuron differentiates through successive stages of development, there is a progressive restriction in the expression of developmentally regulated genes. Activation of transcriptional networks direct subsequent stages to drive differentiation forward, but how gene silencing via methylation-dependent chromatin remodelling contributes to increasing specialization in the developing CNS is just beginning to be appreciated (MacDonald and Roskams, 2009). Having already established potential time

Mice

Mbd2 mice were a gift from Dr. Adrian Bird (University of Edinburgh) (Hendrich et al., 2001). Mecp2 mice were obtained from Jackson Laboratories (Strain Name B6.129P2(C)-Mecp2tm1.1Bird, Stock Number 003890). Hemizygous null male mice (Mecp2/y) were used in all analyses.

Olfactory bulbectomies

Unilateral bulbectomies were performed as described previously (Carter et al., 2004, Cowan et al., 2001) on adult male Mbd2 null mice and WT littermates. Half of the WT mice were administered 250 mg/kg sodium valproate (Sigma) by

Acknowledgments

We would like to thank Drs. Adrian Bird and Brian Hendrich (University of Edinburgh) for the Mbd2 null mice and Dr. Frank Margolis (University of Maryland) for the OMP antibody. We would also like to thank Drs. Hugh Brock and Douglas Allan for critical reading of the manuscript, Erin Currie for excellent technical assistance, Dr. Matt Lorincz for considerable help in bisulphite sequencing analysis, and Dr. Jacob Hodgson and members of the Roskams Lab for experimental insights. This work was

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