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RNA-Seq of neural stem cells from Wild Type (WT) and Armc5 knockout mice

ABSTRACT: ARMC5 is a protein containing an armadillo domain (ARM) and a BTB domain. Its gene knockout caused many phenotypes, including dwarfism, compromise T-cell immunity, and adrenal gland hypertrophy. ARMC5 mutation in humans is associated with bilateral macronodular adrenal gland hypertrophy. We found that AMC5 KO mice suffered from an increased incidence of neural tube defects (NTDs). We revealed that ARMC5 complexed with CUL3 and POLR2A and was part of a novel POLR2A-specific ubiquitin ligase (E3). This E3 was the dominant DNA damage-independent POLR2A-specific E3 in developing neural tubes and neural precursor cells under a physiological condition. ARMC5 gene knockout (KO) caused diminished POLR2A ubiquitination and compromised POLR2A degradation via proteasomes. Surprisingly, the absence of this E3 did not lead to generalized Pol II stalling and the subsequent generalized decrease of mRNA transcription but caused an enlarged Pol II pool size, which dysregulated 108 genes in NPCs, including some known to neural development. ARMC5 KO in the intestine downregulated FOHL1 expression, which was essential in folate absorption. Whole-exome sequencing of 511 myelomeningocele (MM) patients revealed nine highly deleterious mutations in the ARMC5 coding sequence. A significant deleterious mutation Arg429Cys found in MM patients drastically weakened the interaction between ARMC5 and POLR2A, supporting our hypothesis that such mutations in ARMC5 increased the NTD risks by compromising the POLR2A-specific E3 activity. Our results indicated that this novel ARMC5-CUL3-RBX1 E3 played a critical role in Pol II pool homeostasis, and ARMC5 mutation was a modifier of NTD risks in mice and humans.

ORGANISM(S): Mus musculus

PROVIDER: GSE169350 | GEO | 2023/12/01

REPOSITORIES: GEO

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RPB1 ChIP-Seq of NSC from Wild Type (WT) and Armc5 knockout mice

Project description:ARMC5 is a protein containing an armadillo domain (ARM) and a BTB domain. Its gene knockout caused many phenotypes, including dwarfism, compromise T-cell immunity, and adrenal gland hypertrophy. ARMC5 mutation in humans is associated with bilateral macronodular adrenal gland hypertrophy. We found that AMC5 KO mice suffered from an increased incidence of neural tube defects (NTDs). We revealed that ARMC5 complexed with CUL3 and POLR2A and was part of a novel POLR2A-specific ubiquitin ligase (E3). This E3 was the dominant DNA damage-independent POLR2A-specific E3 in developing neural tubes and neural precursor cells under a physiological condition. ARMC5 gene knockout (KO) caused diminished POLR2A ubiquitination and compromised POLR2A degradation via proteasomes. Surprisingly, the absence of this E3 did not lead to generalized Pol II stalling and the subsequent generalized decrease of mRNA transcription but caused an enlarged Pol II pool size, which dysregulated 108 genes in NPCs, including some known to neural development. ARMC5 KO in the intestine downregulated FOHL1 expression, which was essential in folate absorption. Whole-exome sequencing of 511 myelomeningocele (MM) patients revealed nine highly deleterious mutations in the ARMC5 coding sequence. A significant deleterious mutation Arg429Cys found in MM patients drastically weakened the interaction between ARMC5 and POLR2A, supporting our hypothesis that such mutations in ARMC5 increased the NTD risks by compromising the POLR2A-specific E3 activity. Our results indicated that this novel ARMC5-CUL3-RBX1 E3 played a critical role in Pol II pool homeostasis, and ARMC5 mutation was a modifier of NTD risks in mice and humans.

2023-12-01 | GSE169582 | GEO

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

Project description: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.

2023-12-06 | PXD047572 | Pride

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

2023-12-06 | PXD047533 | Pride

RPB1 ChIP-Seq of adrenal glands tissue from Wild Type (WT) and Armc5 knockout mice

Project description:RPB1-specific ubiquitin ligase (E3) controls the degradation of this largest subunit of Pol II and consequently the Pol II pool size. Although several RPB1-specific E3s have been documented, their function is only revealed in cells with massive DNA damage. We demonstrated that ARMC5 physically interacted with cullin3 (CUL3) and RPB1. ARMC5 is an armadillo domain-containing protein with unknown mechanisms of action. ARMC5 deletion caused significant RPB1 accumulation in all the major organs under a physiological condition in the absence of artificially induced DNA damage. This is accompanied by reduced RPB1 ubiquitination. In vitro ubiquitination assay proved that ARMC5, CUL3, and RBX1 formed an effective E3 for RPB1. RPB1 was confirmed as being highly accumulated in the adrenal gland nodules from PBMAH patients with mutations in the ARMC5 coding sequence. Surprisingly, the compromised RPB1 degradation did not lead to increased Pol II stalling according to ChIP-Seq, or a general decrease of gene transcription according to RNA-seq. On the contrary, among 1486 differentially expressed genes in the KO adrenal glands, 93.5% of them were upregulated, probably due to the enlarged Pol II pool size. Our results showed that the ARMC3-CUL3 E3 was the dominant E3 in normal cells and organs and controlled Pol II pool size. The abnormally large Pol II pool size due to ARMC5 mutation dysregulated a large number of genes that in turn led to many phenotypes, including PBMAH in humans.

2022-05-20 | GSE169578 | GEO

RNA-Seq of adrenal glands tissue from Wild Type (WT) and Armc5 knockout mice

2022-05-20 | GSE169263 | GEO

ARMC5 selectively degrades SCAP-free SREBF1 and is essential for fatty acid desaturation in adipocytes

Project description:Background: Armadillo repeat containing 5 (ARMC5)-CUL3 complex was recently identified as an E3 ubiquitin ligase of full-length SREBF. Although ARMC5 was involved in SREBF2 pathway in the adrenocortical cells and might participate in the development of primary bilateral macronodular adrenal hyperplasia (PBMAH), the role of ARMC5 in adipocytes has not been investigated. Methods and Results: Adipocyte-specific Armc5 knockout mice were generated. In the white adipose tissue (WAT) of these mice, all the stearoyl-CoA desaturase (Scd) were drastically downregulated under refed condition and high fat-high sucrose diet. Consistently, unsaturated fatty acids were decreased, while saturated fatty acids were increased in the WAT of these mice. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) peaks at the SREBF1-binding sites were markedly diminished around the Scd1 locus in the WAT of adipocyte-specific Armc5 knockout mice. Armc5-deficient 3T3-L1 adipocytes also exhibited downregulation of Scd. Mechanistically, disruption of Armc5 restored decreased full-length SREBF1 in the CHO cells deficient for Scap, an escort protein required for the transport of SREBF from the ER to the Golgi. In HEK293T cells, overexpression of Scap inhibited ARMC5-mediated degradation of full-length SREBF1, and overexpression of Armc5 increased nuclear SREBF1/full-length SREBF1 ratio in the presence of exogenous Scap in HEK293T cells. Conclusions: ARMC5 exerts selective removal of SCAP-free SREBF1 and stimulates SCAP-mediated SREBF1 processing, hence is essential for fatty acid desaturation in vivo.

2024-12-19 | GSE262941 | GEO

Distinct pathways for removal of defective RNA polymerase II transcription complexes at a promoter-proximal pause checkpointm [RN21236 dTAG TTchem-seq]

Project description:The purpose and function of Integrator and RNA polymerase II (RNAPII) promoter-proximal pausing remain uncertain. Here, we show that when Integrator function is compromised by loss of INTS6, RNAPII interacts increasingly with proteins from alternative pathways for its DNA dissociation, including CUL3-ARMC5 (CRL3ARMC5), which ubiquitylates Ser5-phosphorylated Rpb1 and targets it for degradation. ARMC5-dependent RNAPII ubiquitylation is activated by defects in factors required for correctly regulated promoter-proximal pausing, including Integrator, DSIF and mRNA capping enzyme. This ARMC5 checkpoint curtails an appreciable fraction of RNAPII transcription, with ARMC5 knockout cells producing uncapped, nascent RNA transcripts that fail to mature into stable mRNA. Concomitant loss of INTS6 and ARMC5 greatly stabilizes RNAPII at the pause and has severe consequences for cell growth and viability. Our data support a model in which CRL3ARMC5 functions alongside Integrator in a checkpoint mechanism that removes faulty RNAPII complexes at promoter-proximal pause sites to safeguard transcription integrity.

2024-11-07 | GSE253354 | GEO

Distinct pathways for removal of defective RNA polymerase II transcription complexes at a promoter-proximal pause checkpoint

Project description:The Integrator complex interacts with RNA polymerase II (RNAPII) at promoter-proximal pause sites but its function there remains uncertain. Here, we show that when Integrator function is compromised by loss of INTS6, RNAPII interacts increasingly with proteins that mediate its dissociation from DNA, including CUL3-ARMC5, which ubiquitylates Ser5-phosphorylated Rpb1 and targets it for degradation. ARMC5-dependent RNAPII ubiquitylation is activated by defects in factors required for correctly regulated promoter-proximal pausing, including Integrator, DSIF and mRNA capping enzyme. This ARMC5 checkpoint curtails an appreciable fraction of RNAPII transcription, with ARMC5 knockout cells producing uncapped, nascent RNA transcripts that fail to mature into stable mRNA. Concomitant loss of INTS6 and ARMC5 greatly stabilizes RNAPII at the pause and has severe consequences for cell growth and viability. Our data support a model in which CRL3ARMC5 functions alongside Integrator in a checkpoint mechanism that removes faulty RNAPII complexes at promoter-proximal pause sites to safeguard transcription integrity.

2024-11-05 | GSE251941 | GEO

Distinct pathways for removal of defective RNA polymerase II transcription complexes at a promoter-proximal pause checkpoint [RN21114 Ttchemn-Seq]

2024-11-05 | GSE253353 | GEO

Ubiquitylation of Pol II controls early stages of the transcription cycle [dxChIP-seq]

Project description:Control of RNA Polymerase II (Pol II) through ubiquitylation is essential for the DNA-damage response. Here we reveal a distinct ubiquitylation pathway in human cells that targets excessive and defective Pol II molecules at the initial stages of the transcription cycle. This pathway, mediated by ARMC5CUL3 ubiquitin ligase, drives homeostatic Pol II turnover, and is further enhanced when early stages of transcription - initiation and pausing - are perturbed. Upon ARMC5 loss, Pol II accumulates in the free pool and in the promoter-proximal zone, but is not permitted into elongation. We identify Integrator subunit 8 (INTS8) as a gatekeeper preventing the release of excess Pol II molecules into gene bodies. Combined loss of ARMC5 and INTS8 has strong detrimental effects on cell growth, with ARMC5 loss exacerbating transcriptional defects seen in INTS8-depleted cells. These findings uncover that ARMC5CUL3 and INTS8 form a collaborative checkpoint, monitoring the quantity and quality of transcription complexes before they are licensed into elongation.

2024-09-07 | GSE276375 | GEO

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