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ARMC5 controls the degradation of most Pol II subunits, and ARMC5 mutation increases neural tube defect risks in mice and humans

ABSTRACT: ARMC5 is an armadillo domain (ARM)-containing protein. We found that Armc5 KO mice had an increased incidence of neural tube defects (NTDs). ARMC5 is the substrate recognition component of a ubiquitin ligase that targets POLR2A, the largest subunit of RNA polymerase II (Pol II). Surprisingly, the absence of ARMC5 caused the accumulation of not only POLR2A, but most of the other 11 Pol II subunits associated with POLR2A, indicating that the degradation of the whole Pol II complex is compromised. This did not lead to generalized Pol II stalling or a generalized decrease in mRNA transcription. In neural progenitor cells, ARMC5 KO only dysregulated 106 genes, some of which are known to be involved in neural tube development. FOLH1, critical for folate metabolism and vital in neural tube development, was downregulated in the KO intestine, suggesting that it is a downstream effector gene for NTD. To assess whether ARMC5 gene mutation was associated with human NTD, we conducted whole-exome sequencing of 511 patients with myelomeningocele, a severe form of NTD. Nine deleterious single nucleotide variants were discovered in the ARMC5 coding sequence. Four of them were validated in that they weakened the interaction between ARMC5 and POLR2A; consequently, POLR2A ubiquitination was decreased. This proves that our findings in mice are relevant to human NTD; it also supports the role of Pol II in mediating NTD pathogenesis. Our findings indicate that mutations in ARMC5 increase the risk of NTD and support the role of Pol II in NTD pathogenesis. Further investigation is needed to determine the cause-and-effect relationship between an enlarged Pol II pool and NTD and to validate the potential role of downregulated FOLH1 expression in NTD pathogenesis.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hek-293 Cell

SUBMITTER: Christian Poitras

LAB HEAD: Benoit Coulombe

PROVIDER: PXD047533 | Pride | 2023-12-06

REPOSITORIES: Pride

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Project description:BAF155, as a component of a chromatin remodeling complex, is thought to be involved in the regulation of gene expression. Baf155 knockdown in ES cells results in misregulation of hundreds of genes (Ho et al., 2009). The Baf155msp3 allele presents a unique case for gene expression analysis wherein the BAF complex appears to be intact, in contrast to disruption of the BAF complex when BAF155 function is lost (Chen and Archer, 2005; Sohn et al., 2007). To examine gene expression in the Baf155msp3 mutant at the time of neural tube closure, we performed RNA-Seq and compared gene expression levels between homozygous wildtype and homozygous mutant embryos. After extensive analysis of the RNA-Seq data comparing all 3 mutants to all 3 WT replicates, 78 genes were identified as being upregulated on average in the mutant samples while 22 genes were downregulated. To facilitate an understanding of global gene expression differences, Ingenuity Pathway Analysis (IPA) was used to assign functional categories to the up- and down-regulated genes in the mutants. The most significant molecular and cellular functions of these genes were: 1. cellular movement (1.46E-04>p>4.12E-02), 2. cell death and survival (2.69E-04>p>4.12E-02), and 3. cell growth and proliferation (2.69E-04>p>3.41E-02). Many neuronal genes were significantly misregulated as well. Overall, the RNA-Seq analysis revealed that surprisingly few genes showed altered expression in Baf155 mutant neural tissue, given the broad epigenetic role of the BAF complex, but included genes involved in neural development and cell survival. Moreover, gene expression changes between individual mutants were variable even though the NTD was consistently observed. This suggests that inconsistent gene regulation contributes to failed neural tube closure. RNA was isolated from somite matched (21-23 somites) and phenotype matched cranial tissue of homozygous wildtype and homozygous mutant embryos (3 of each genotype) at E9.5 just after the time of neural tube closure. At E9.5, the vast majority of the embryonic neural tube is comprised of proliferating neural progenitor cells, constituting a relatively homogeneous cell population.

			Action	DRS
	OF_20180723_COU_02.mgf	Mgf
	OF_20180723_COU_02.mzid.gz	Mzid
	OF_20180723_COU_02.raw	Raw
	OF_20180723_COU_03.mgf	Mgf
	OF_20180723_COU_03.mzid.gz	Mzid

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ARMC5 controls the degradation of most Pol II subunits, and ARMC5 mutation increases neural tube defect risks in mice and humans

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