The E3 Ligases Spsb1 and Spsb4 Regulate RevErbα Degradation and Circadian Period

Mekbib, Tsedey; Suen, Ting-Chung; Rollins-Hairston, Aisha; DeBruyne, Jason P.

doi:10.1177/0748730419878036

“…These results are somewhat different to our previous results in U2OS cells (which are female [21]) where SIAH2 knockdown blunted expression of REVERBα targets coincident with altered REVERBα protein turnover [10]. However, in the liver, we were surprised to find that SIAH2 loss had little effect on rhythms of REVERBα protein abundance rhythms of either sex (S1 Fig), possibly owing to tissue-specific compensation by other ubiquitin ligases [22][23][24]. Thus, it is not clear if these effects of SIAH2 loss are through alterations in REVERBα function, though female-specific regulation of REVERBα is not expected given its role in circadian regulation.…”

Section: Plos Geneticscontrasting

confidence: 99%

“The ubiquitin ligase SIAH2 is a female-specific regulator of circadian rhythms and metabolism”

Mekbib

¹

,

Suen

²

,

Rollins-Hairston

³

et al. 2022

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Circadian clocks enable organisms to predict and align their behaviors and physiologies to constant daily day-night environmental cycle. Because the ubiquitin ligase Siah2 has been identified as a potential regulator of circadian clock function in cultured cells, we have used SIAH2-deficient mice to examine its function in vivo. Our experiments demonstrate a striking and unexpected sexually dimorphic effect of SIAH2-deficiency on the regulation of rhythmically expressed genes in the liver. The absence of SIAH2 in females, but not in males, altered the expression of core circadian clock genes and drastically remodeled the rhythmic transcriptome in the liver by increasing the number of day-time expressed genes, and flipping the rhythmic expression from nighttime expressed genes to the daytime. These effects are not readily explained by effects on known sexually dimorphic pathways in females. Moreover, loss of SIAH2 in females, not males, preferentially altered the expression of transcription factors and genes involved in regulating lipid and lipoprotein metabolism. Consequently, SIAH2-deficient females, but not males, displayed disrupted daily lipid and lipoprotein patterns, increased adiposity and impaired metabolic homeostasis. Overall, these data suggest that SIAH2 may be a key component of a female-specific circadian transcriptional output circuit that directs the circadian timing of gene expression to regulate physiological rhythms, at least in the liver. In turn, our findings imply that sex-specific transcriptional mechanisms may closely interact with the circadian clock to tailor overt rhythms for sex-specific needs.

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“…However, in the liver, we were surprised to find that SIAH2 loss had little effect on rhythms of REVERBα protein abundance rhythms of either sex (Suppl. Fig S1 ), possibly owing to tissue-specific compensation by other ubiquitin ligases (21)(22)(23). Thus, it is not clear if these effects of SIAH2 loss are through alterations in REVERBα function, though female-specific regulation of REVERBα is not expected given its role in circadian regulation.…”

Section: Resultsmentioning

confidence: 99%

“A novel female-specific circadian clock mechanism regulating metabolism”

Mekbib

¹

,

Suen

²

,

Rollins-Hairston

³

et al. 2020

Preprint

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Circadian clocks enable organisms to predict and align their behaviors and physiologies to constant daily day-night environmental cycle. Because the ubiquitin ligase Siah2 has been identified as a potential regulator of circadian clock function in cultured cells, we have used Siah2-deficient mice to examine its function in vivo. Our experiments demonstrate a striking and unexpected sexually dimorphic effect of Siah2 deficiency on the regulation of rhythmically expressed genes. The absence of Siah2 in females, but not in males, altered the expression of core circadian clock genes and drastically remodeled the rhythmic hepatic transcriptome. Siah2 loss, only in females, increased the expression of 100’s of genes selectively at mid-day, resulting in a ∼70% increase in the number of rhythmically expressed genes, and shifted the expression of 100’s of other genes from a mid-night peak, to a mid-day peak. The combined result is a near inversion of overall rhythmicity in gene expression selectively in Siah2-deficient females. This dramatic reorganization created a substantial misalignment between rhythmic liver functions and feeding/behavioral rhythms, and consequently impaired daily patterns of lipid/lipoprotein metabolism and metabolic responses to high-fat diet. Collectively, our results suggest that Siah2 is part of a female-specific circadian mechanism important for maintaining metabolic homeostasis and may play a key role in the establishing sexual dimorphisms in metabolism.Signficance statementCircadian clocks drive daily rhythms in many aspects of our physiology, optimally aligning functions across the day-night cycle. How circadian clocks drives these rhythms is thought to be due to largely similar transcriptional pathways and mechanisms in males and females, although some rhythms are modulated by sex and growth hormones. In this study, we present data that uncover the surprising existence of a female-specific transcriptional mechanism that is essential for the proper rhythmic control of gene expression in the liver. Disrupting this mechanism substantially impairs the circadian regulation of lipid and cholesterol metabolism selectively in females, impairing their resistance to diet-induced obesity. These results reveal that circadian clocks may be broadly coupled to physiological rhythms using unexpected sex-specific mechanisms.

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“…These two apparently contradictory functions likely depend on how subsegments within these first 25AAs interact with each other. The mapping of the SPSB4 binding site to AA4-9 of REV-ERB is exciting (Figures 5 and 6), since our previous experiments showed that SPSB4knockdown will result in the stabilization of REV-ERB, as well as a longer circadian period (13). The shortfall of these ubiquitin ligase knockdown experiments is that one can always argue that the observed effects may be due to other possible targets of the ubiquitin ligase.…”

Section: Discussionmentioning

confidence: 99%

“…We and others have investigated how various ubiquitin ligases contribute to the degradation of REV-ERB and their effects on the circadian clock (12)(13)(14)(15)(16). In this study, we focused on trying to understand the central events that need to happen to REV-ERB protein itself for degradation to take place.…”

Section: Discussionmentioning

confidence: 99%

“…Lanes "M" were lysates from cells treated with MG132 for 3 hours (lanes 6, 12, 18, and 24). C. WT and the rearranged mutant AA2-9insAA25 were co-transfected with Sport6 (lanes 1-12) or Sport6-SPSB4 (lanes [13][14][15][16][17][18][19][20][21][22][23][24], into HEK293 cells. 36-48 hours after transfection, cells were treated with cycloheximide for the indicated number of hours, and the Western blot was probed with anti-REV-ERB antibody Lanes "M" were lysates from cells treated with MG132 for 4 hours (lanes 6, 12, 18, and 24).…”

Section: Discussionmentioning

confidence: 99%

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Lysine-independent ubiquitination and degradation of REV-ERBα involves a bi-functional degradation control sequence at its N-terminus

Suen

¹

,

DeBruyne

²

2023

Preprint

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REV-ERB α and REV-ERBβ are important components of the mammalian circadian clock and play a crucial role in linking the circadian system to overt daily rhythms in physiology and behavior. Expression of these paralogs is driven by the circadian clockwork, and in most tissues, the abundance of REV-ERBα proteins robustly cycles such that they are detected only within a tight interval of 4-6 hours each day, suggesting control of both their synthesis and degradation are tightly controlled. Indeed, several different ubiquitin ligases have been shown to mediate REV-ERBα degradation, but how they interact with REV-ERBα and which lysine residues they ubiquitinate to drive its degradation are unknown. Here, we used a mutagenesis approach to functionally identify both binding and ubiquitination sites within REV-ERBα required for its regulation by the ubiquitin ligases Spsb4 and Siah2. Surprisingly, we found that REV-ERB α mutants with all 20 lysines changed to arginine (K20R) can be efficiently ubiquitinated and degraded in the absence or presence of these E3 ligases, consistent with N-terminal ubiquitination. To explore this, we examined if small deletions at the N-terminus of REV-ERBα will alter its degradation. Interestingly, deletion of amino acid (AA) residues 2 to 9 (delAA2-9) clearly resulted in a less stable REV-ERBα. We found that it was the length (i.e. 8 AA), and not the specific sequence, that confers stability in this region. Simultaneously, we also mapped the interaction site of the E3 ligase Spsb4 to this same region, specifically requiring AA4-9 of REV-ERBα. Thus, the first 9 AA of REV-ERBα has two opposing roles in regulating REV-ERBα turnover. Additionally, deleting eight addition additional AAs (delAA2-17) from REV-ERBα almost prevents its degradation. Combined, these results suggest that complex interactions within the first 25AAs that potentially act as a REV-ERBα switch that allows a protected/stabilized conformation to accumulate at one time of day, but then rapidly shifts to a destabilized form, to enhance its removal at the end of the daily cycle.

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The E3 Ligases Spsb1 and Spsb4 Regulate RevErbα Degradation and Circadian Period

Cited by 7 publications

References 49 publications

“The ubiquitin ligase SIAH2 is a female-specific regulator of circadian rhythms and metabolism”

“The ubiquitin ligase SIAH2 is a female-specific regulator of circadian rhythms and metabolism”

“A novel female-specific circadian clock mechanism regulating metabolism”

Lysine-independent ubiquitination and degradation of REV-ERBα involves a bi-functional degradation control sequence at its N-terminus

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