Bidirectional regulation of postmitotic H3K27me3 distributions underlie cerebellar granule neuron maturation dynamics
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eLife assessment
This study presents a useful inventory of chromatin modifications and genes that are up- and down-regulated during cerebellar development in vivo and in primary culture. The main claims were incomplete and would benefit from further analysis and/or additional experiments. The work will be of interest to biologists working on brain development.
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Abstract
The functional maturation of neurons is a prolonged process that extends past the mitotic exit and is mediated by the chromatin-dependent orchestration of gene transcription programs. We find that expression of this maturation gene program in mouse cerebellar granule neurons (CGNs) requires dynamic changes in the genomic distribution of histone H3 lysine 27 trimethylation (H3K27me3), demonstrating a function for this chromatin modification beyond its role in cell fate specification. The developmental loss of H3K27me3 at promoters of genes activated as CGNs mature is facilitated by the lysine demethylase and ASD-risk gene, Kdm6b. Interestingly, inhibition of the H3K27 methyltransferase EZH2 in newborn CGNs not only blocks the repression of progenitor genes but also impairs the induction of mature CGN genes, showing the importance of bidirectional H3K27me3 regulation across the genome. These data demonstrate that H3K27me3 turnover in developing postmitotic neurons regulates the temporal coordination of gene expression programs that underlie functional neuronal maturation.
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eLife assessment
This study presents a useful inventory of chromatin modifications and genes that are up- and down-regulated during cerebellar development in vivo and in primary culture. The main claims were incomplete and would benefit from further analysis and/or additional experiments. The work will be of interest to biologists working on brain development.
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Reviewer #1 (Public Review):
In this work, authors seek to understand how the polycomb complex may coordinate gene expression changes that occur during sequential stages of neuronal maturation. The main strengths are 1) choice of cerebellar granule neurons which mature over a protracted period during normal cerebellar development and constitute a relatively homogeneous population of neurons, 2) use of a genetic in vivo mouse model where a histone demethylase is knocked out, combined with an in vitro culture model of maturing cerebellar granule neurons in which a histone methyltransferase is inhibited, 3) use of CUT & TAG in neuronal cultures to investigate how changes in the H3K27me3 repressor chromatin modification at promoters correlate with gene expression and chromatin accessibility changes. The authors propose a bidirectional …
Reviewer #1 (Public Review):
In this work, authors seek to understand how the polycomb complex may coordinate gene expression changes that occur during sequential stages of neuronal maturation. The main strengths are 1) choice of cerebellar granule neurons which mature over a protracted period during normal cerebellar development and constitute a relatively homogeneous population of neurons, 2) use of a genetic in vivo mouse model where a histone demethylase is knocked out, combined with an in vitro culture model of maturing cerebellar granule neurons in which a histone methyltransferase is inhibited, 3) use of CUT & TAG in neuronal cultures to investigate how changes in the H3K27me3 repressor chromatin modification at promoters correlate with gene expression and chromatin accessibility changes. The authors propose a bidirectional effect of the same chromatin repressor modification that is responsible, at least in part, for the timely loss of expression of early genes and the appearance of genes expressed later in maturation. This is the major impact of the work for those interested in cerebellar development. A weakness in the work lies in its narrow focus, which is on promoter regions almost exclusively.
The work is primarily bioinformatics driven and lacks physiological significance of the gene expression changes, or how the culture timing correlates with temporal regulation and chromatin changes in vivo. However, the results do support the proposal that polycomb-associated enzymatic activities play sequential roles during successive stages of cerebellar maturation.
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Reviewer #2 (Public Review):
Ramesh, Liu et al. investigated the dynamics of the histone H3 lysine 27 trimethyl mark (H3K27me3) in the cerebellum during postnatal development. They profile the mark and measure gene expression at three time points (P7, P14, P60) to show that there is a global increase in the amounts of H3K27me3 genome-wide, but a generalized loss of the mark at promoters. This loss is associated with neuronal genes that become expressed in the mature cerebellum. Through conditional knockout and transcription factor analysis, they implicate the autism-associated lysine demethylase gene KDM6B in the removal of H3K27me3 at genes that become active postnatally and show that the ZIC transcription factors are candidates to mediate some of these effects. They then use pharmacologic inhibition of KDM6B and the PRC2 component, …
Reviewer #2 (Public Review):
Ramesh, Liu et al. investigated the dynamics of the histone H3 lysine 27 trimethyl mark (H3K27me3) in the cerebellum during postnatal development. They profile the mark and measure gene expression at three time points (P7, P14, P60) to show that there is a global increase in the amounts of H3K27me3 genome-wide, but a generalized loss of the mark at promoters. This loss is associated with neuronal genes that become expressed in the mature cerebellum. Through conditional knockout and transcription factor analysis, they implicate the autism-associated lysine demethylase gene KDM6B in the removal of H3K27me3 at genes that become active postnatally and show that the ZIC transcription factors are candidates to mediate some of these effects. They then use pharmacologic inhibition of KDM6B and the PRC2 component, EZH2, in a granule neuron culture system to further dissect the function of these enzymes in H3K27me3 dynamics.
The authors employ multiple genomic methods to carry out rigorously controlled experiments and their conclusions are well supported by the data. The study provides fundamental insights into the dynamics of H3K27me3 during the postnatal development of circuits in the brain. In particular, the findings that substantial changes in the H3K27me3 mark continue through the later steps of cerebellar maturation (P14 to P60) and that the autism-associated gene KDM6B is involved in this process, will be of significant interest to the field.
The study has some limitations with regard to scope and mechanism. For example, given the importance of enhancers in the regulation of gene expression, the omission of any analysis of H3K27me3 at defined enhancer elements is a limitation of the study. In addition, while the observations supporting the role of ZIC proteins in the removal of H3K27me3 during gene activation are interesting, the lack of direct mechanistic analysis investigating this biology limits the strength of the conclusions that can be made about the direct function of these factors in H3K27me3 dynamics.
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