Facioscapulohumeral muscular dystrophy (FSHD) is a degenerative disorder affecting skeletal muscles of the face, the shoulder blades, and the upper arms. It is caused by the misexpression of the double homeobox transcription factor DUX4 in skeletal muscle cells, which leads to muscle cell death. Although the mechanism behind toxicity of DUX4 protein has not been fully elucidated, in vitro experiments with cultured FSHD muscle cells demonstrated that a transient expression of DUX4 triggered a long-lasting pathological phenotype. These observations led the Tapscott lab (Human Biology Division) to hypothesize that this burst of DUX4 expression sets up a transcription factor cascade or a chromatin memory.
Histones are proteins around which DNA winds and gets packed. They play an essential role in the regulation of gene expression. Histone variants allow for different degree of compaction of the chromatin, which is further regulated by post-translational modifications such as methylation or acetylation. Domains of open chromatin make DNA more accessible to transcription factors and therefore tend to exhibit increased transcriptional activity in these domains. Recently, it has been demonstrated that histone variants H3.X and H3.Y were incorporated in actively transcribed genes and resulted in a more relaxed chromatin conformation. This suggests that these variants may help transcribed genes to remain active. In this context, the Tapscott lab investigated the effect of DUX4 expression on H3.X and H3.Y mediated chromatin modification. They recently published their results in Cell Reports.
Dr. Rebecca Resnick, first author of the study, and her colleagues used well-characterized human myoblasts engineered with a doxycycline inducible DUX4 transgene (MB135iDUX4 cells). They observed that H3.X and H3.Y expression was induced 24 hours after doxycycline treatment. In myoblast cell lines derived from healthy or FSHD individuals, they demonstrated that DUX4, H3.X and H3.Y were highly expressed in pathological cells and barely present in healthy cells. The researchers sought to identify the genes where these histone variants were incorporated after DUX4 induction.
In collaboration with the Henikoff Lab, they performed a “CUT&RUN” protocol on MB135iDUX4 cells, with or without doxycycline. In this procedure, antibodies against H3.X/Y recruit a nuclease that cuts DNA, which allows for isolation of small pieces of DNA where the histone variants are incorporated and subsequent sequencing of these regions. When comparing the frequency of H3.X/Y incorporation and the level of expression of the corresponding genes, the authors observed that these variants were mostly incorporated in transcribed regions of the genome, and particularly in DUX4-induced genes.
In order to understand the sequence of events and the dependence between DUX4 expression and H3.X/Y incorporation, Resnick and colleagues observed the effect of consecutive DUX4 pulses with or without siRNA targeting H3.X/Y. Whereas the second pulse of DUX4 triggers a higher expression of H3.X/Y and DUX4 target genes compared to the first pulse in the control condition, downregulation of H3.X/Y inhibited this enhanced induction, but did not affect the initial induction of DUX4 target genes. This demonstrates that induction of histone variants H3.X and H3.Y expression by an initial DUX4 pulse leads to the incorporation of these variants in DUX4 target genes, which potentiates the activation of these genes when DUX4 is reexpressed.
This is an important step toward understanding how a pulse of DUX4 expression can set up an open chromatin state or chromatin memory that favors persistence of the DUX4-induced transcriptional program and may explain the pathological toxicity resulting from DUX4 expression. Conceptually, this work reconciles two main mechanisms determining cell fate, as explains Dr. Tapscott: “I began my research career many years ago believing that chromatin memory and cell lineage conferred the differentiation potential of cells in the embryo. Then with the discovery of MyoD, NeuroD, and other master regulatory genes, there was more of an emphasis on stable transcriptional networks. I find this work on the H3.X/Y histone variants exciting because it bridges these two different frameworks: a master regulator that imprints a memory in the chromatin.”
This work was supported by the Friends of FSH research the National Institutes of Health.
Fred Hutch/UW Cancer Consortium members Drs. Henikoff and Tapscott contributed to this research.
Resnick R, Wong CJ, Hamm DC, et al. 2019. DUX4-Induced Histone Variants H3.X and H3.Y Mark DUX4 Target Genes for Expression. Cell Rep. 29(7):1812–1820.e5. doi:10.1016/j.celrep.2019.10.025