The histone demethylase Kdm6b regulates a mature gene expression program in differentiating cerebellar granule neurons

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Highlights

  • Characterizes expression of Kdm6a and Kdm6b across multiple stages of cerebellar granule neuron differentiation

  • Demonstrates a requirement for Kdm6b in the maturation but not the formation of cerebellar granule neurons in vivo

  • Identifies a requirement for Kdm6b expression of genes that play important roles in development and function of synapses

Abstract

The histone H3 lysine 27 (H3K27) demethylase Kdm6b (Jmjd3) can promote cellular differentiation, however its physiological functions in neurons remain to be fully determined. We studied the expression and function of Kdm6b in differentiating granule neurons of the developing postnatal mouse cerebellum. At postnatal day 7, Kdm6b is expressed throughout the layers of the developing cerebellar cortex, but its expression is upregulated in newborn cerebellar granule neurons (CGNs). Atoh1-Cre mediated conditional knockout of Kdm6b in CGN precursors either alone or in combination with Kdm6a did not disturb the gross morphological development of the cerebellum. Furthermore, RNAi-mediated knockdown of Kdm6b in cultured CGN precursors did not alter the induced expression of early neuronal marker genes upon cell cycle exit. By contrast, knockdown of Kdm6b significantly impaired the induction of a mature neuronal gene expression program, which includes gene products required for functional synapse maturation. Loss of Kdm6b also impaired the ability of Brain-Derived Neurotrophic Factor (BDNF) to induce expression of Grin2c and Tiam1 in maturing CGNs. Taken together, these data reveal a previously unknown role for Kdm6b in the postmitotic stages of CGN maturation and suggest that Kdm6b may work, at least in part, by a transcriptional mechanism that promotes gene sensitivity to regulation by BDNF.

Introduction

Neuronal differentiation is comprised of sequential steps that include progenitor proliferation, exit from the cell cycle, migration, synapse formation, and functional maturation. Chromatin regulators play a key role in these processes by dynamically remodeling the chromatin landscape to orchestrate the temporal regulation of gene expression programs. The developing mouse cerebellum serves as an ideal model to identify and examine genetic programs that mediate neuronal differentiation (Wang and Zoghbi, 2001). The cerebellum is particularly useful in chromatin studies because of its utility for studying fate determination and postmitotic maturation of a single predominant neuronal cell-type over the full time course of differentiation (Frank et al., 2015).

Cerebellar granule neurons (CGNs) comprise > 99% of cerebellar neurons and > 85% of all cerebellar cells (Altman and Bayer, 1997). These cells are derived during the first two postnatal weeks from committed granule neuron precursors (GNPs) that proliferate in the outer portion of the external granular layer (EGL) of the developing cerebellar cortex. Following exit from the cell cycle, GNPs first differentiate into immature CGNs in the inner layer of the EGL then migrate past the Purkinje cells to the inner granular layer (IGL), where they mature and form synaptic connections. We have demonstrated that changes in chromatin accessibility, and the consequent regulation of transcription factor binding at distal enhancer elements, controls the developmentally-regulated programs of gene expression that accompany these stages of CGN differentiation (Frank et al., 2015). The key chromatin regulatory factors that coordinate these steps of CGN differentiation remain to be identified.

In addition to changes in chromatin accessibility, the functional state of enhancer and promoter elements is regulated by the deposition of specific combinations of histone marks (Ernst et al., 2011). Histone methylation is a particularly complex modification that can be associated with either the activation or the repression of gene transcription depending on the specific histone residue that is modified (Black et al., 2012). Methylation of histone H3 at lysine 4 is generally associated with transcriptional activity, with trimethylation (H3K4me3) marking active promoters and monomethylation (H3K4me1) marking transcriptional enhancers. Conversely, trimethylation of histone H3 at lysine 27 (H3K27me3) is associated with gene repression. During cell fate determination, a subset of regulatory elements that control cell-type specific genes is marked by both activating and repressive sites of histone methylation (Bernstein et al., 2006, Mohn et al., 2008). These elements are thought to be “poised” such that the corresponding genes can be rapidly turned on or off during subsequent stages of fate determination by the selective loss of either the activating or the repressive mark. Whether such processes play a role in the postmitotic timing of gene expression in a fate-committed neuron remains unknown.

Steady-state levels of H3K27me3 are established by the functional balance between the enzymes that add this modification (the histone methyltransferases Ezh1 and Ezh2) and the enzymes that remove it (Margueron and Reinberg, 2011). Within the Jumonji C (JmjC) domain-containing histone demethylase family, there are two selective H3K27me3 demethylases named Kdm6a (UTX) and Kdm6b (Jmjd3) (Agger et al., 2007, Hong et al., 2007, Hubner and Spector, 2010, Lan et al., 2007). Kdm6a is on the X chromosome and escapes X inactivation (Greenfield et al., 1998); its Y chromosome homolog, called Uty, lacks H3K27 demethylase activity (Hong et al., 2007, Lan et al., 2007, Shpargel et al., 2012).

Several studies have implicated Kdm6b in multiple aspects of neuronal differentiation and function. Consistent with the evidence from other cell linages that Kdm6b promotes cellular differentiation (Manna et al., 2015, Pan et al., 2015), loss of Kdm6b has been observed to impair neurogenesis in embryonic stem cells (Burgold et al., 2008) as well as neural stem cells in culture (Jepsen et al., 2007) and in vivo (Park et al., 2014). Furthermore, constitutive Kdm6b knockout mice die at birth of respiratory failure that appears to be due to impaired maturation and function of respiratory circuits in the brain (Burgold et al., 2012), suggesting that Kdm6b may function in the maturation of postmitotic neurons as well. Indeed, we have previously found that Kdm6b is a neural activity-regulated gene product that functions in differentiated hippocampal neurons to promote cell survival (Wijayatunge et al., 2014). However, the functions of Kdm6b in postmitotic stages of neuronal differentiation have not yet been established.

To fill this gap in knowledge, we generated GNP-conditional Kdm6b knockout mice and examined the expression and function of Kdm6b in developing CGNs of the mouse cerebellum. We find that Kdm6b expression is induced when CGN precursors exit the cell cycle and that Kdm6b is required in CGNs for developmental induction of a gene expression program that mediates mature CGN functions. These data implicate Kdm6b as a regulator of neuronal maturation in addition to its functions at early stages of neuronal differentiation.

Section snippets

Mice

We performed all procedures under an approved protocol from the Duke University Institutional Animal Care and Use Committee. LoxP-conditional Kdm6a and Kdm6b mice were described in (Shpargel et al., 2012) and (Shpargel et al., 2014) respectively. Briefly, the conditional Kdm6a strain has loxP sites flanking exon 3 of the 29-exon Kdm6a gene on the X chromosome, and the conditional Kdm6b strain has loxP sites flanking exons 14–20 (which encode the enzymatic JmjC domain) of the 23-exon Kdm6b gene

Kdm6b expression is induced in newborn CGNs of the postnatal mouse cerebellum

We performed in situ hybridization to determine the expression pattern of Kdm6b mRNA in the developing mouse cerebellum. At postnatal day 7 (P7), all of the stages of CGN differentiation can be observed within a single parasagittal section (Fig. 1A). Hybridization with an antisense probe against Kdm6b at P7 showed that Kdm6b mRNA is found in both the EGL and IGL of the developing cerebellum (Fig. 1B–C). This in situ signal is specific, because it was not observed upon hybridization with the

Discussion

In this study, we report the characterization of CGN differentiation in the cerebellum of Kdm6b conditional knockout mice and following Kdm6b knockdown in GNPs in culture. Our data show that the expression of Kdm6b is not required in GNPs in vivo for the generation of CGNs, however Kdm6b does contribute to the induction of a late program of gene expression, which includes gene products that regulate synaptic functions in the mature CGN state. These data implicate Kdm6b in the late postmitotic

Acknowledgements

This work was supported by NIH grants R21NS084336 and R01NS098804 (A.E.W.), and R01GM101974 (T.M.). We thank David Gallegos for assistance with the confocal microscopy, Caitlin Paisley for assistance with the westerns, Katherine Misuraca for help with the neuronal cultures, and Josh Starmer for advice on the statistics.

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