Pentapeptide insertion mutagenesis of the Hoxa1 protein: Mapping of transcription activation and DNA‐binding regulatory domains

Lambert, Barbara; Vandeputte, Julie; Desmet, Pierre-Marie; Hallet, Bernard; Remacle, Sophie; Rezsöhazy, René

doi:10.1002/jcb.22563

Cited by 6 publications

(6 citation statements)

References 63 publications

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“…These two HOXA1 variants are impaired in their DNA-binding capacity. mHOXA1 Δ71-199 (hereafter referred to as mHOXA1 ΔCenter ) lacks a central region of the protein, extending from amino-acid 71 to 199. mHOXA1 WM-AA has a mutant hexapeptide and has lost its capacity to interact with PBX ( 32 , 46 , 52 , 53 ). MCF10A cells were transfected with HOXA1 expression plasmids to assess their effect on ERα activity, as determined by ERE::luc activity.…”

Section: Resultsmentioning

confidence: 99%

HOXA1 Is an Antagonist of ERα in Breast Cancer

et al. 2021

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Breast cancer is a heterogeneous disease and the leading cause of female cancer mortality worldwide. About 70% of breast cancers express ERα. HOX proteins are master regulators of embryo development which have emerged as being important players in oncogenesis. HOXA1 is one of them. Here, we present bioinformatic analyses of genome-wide mRNA expression profiles available in large public datasets of human breast cancer samples. We reveal an extremely strong opposite correlation between HOXA1 versus ER expression and that of 2,486 genes, thereby supporting a functional antagonism between HOXA1 and ERα. We also demonstrate in vitro that HOXA1 can inhibit ERα activity. This inhibition is at least bimodal, requiring an intact HOXA1 DNA-binding homeodomain and involving the DNA-binding independent capacity of HOXA1 to activate NF-κB. We provide evidence that the HOXA1-PBX interaction known to be critical for the transcriptional activity of HOXA1 is not involved in the ERα inhibition. Finally, we reveal that HOXA1 and ERα can physically interact but that this interaction is not essential for the HOXA1-mediated inhibition of ERα. Like other HOX oncoproteins interacting with ERα, HOXA1 could be involved in endocrine therapy resistance.

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Section: Resultsmentioning

confidence: 99%

HOXA1 Is an Antagonist of ERα in Breast Cancer

et al. 2021

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“…To identify the structural determinants involved in the HOXA1-mediated activation of NF-κB, we used HOXA1 mutants that were previously generated by random pentapeptide insertion mutagenesis (45). All the insertion mutants activated the NF-κB reporter to similar levels as wild-type HOXA1 (Supplementary Figure S3).…”

Section: Resultsmentioning

confidence: 99%

“…Consistent with the results obtained for the transcriptionally inactive HOXA1 QN-AA protein mutated in the homeodomain, HOXA1 insertion mutants P14, P15 and P16, showing a disrupted homeodomain, were not affected in their ability to activate the NF-κB reporter. Interestingly, mutants displaying a loss-of-activity on a HOXA1 reporter (P1, P5, P13, P15, P16, P17) (45) could still activate the NF-κB reporter as efficiently as wild-type HOXA1. This again confirms the transcription-independent activity of HOXA1 in the NF-κB signaling pathway.…”

Section: Resultsmentioning

confidence: 99%

“…Reporter plasmids pML-EPHA2-r4-Luc (40) and pCMV-LacZ (41), expression plasmids for HOXA1 WT (pGIH-309), HOXA1 QN-AA (pGIH-512), HOXA1 WM-AA (pGIH-328) Flag-HOXA2 (42) and PBX1 (pCMV-PBX1) (43), the expression plasmid for PREP1 (44), the pEXP-FLAG(Nter)-HOXA1, pEXP-VC155(Nter)-HOXA1, pEXP-VN173(Nter)-HOXA1, pEXP-GST(Nter)-RBCK1, pEXP-VN173(Nter)-RBCK1, pEXP-GST(Nter)-TRAF2 and pEXP-VN173(Nter)-TRAF2 vectors (22) and HOXA1 mutants generated by pentapeptide insertion mutagenesis (45) were all described previously. The HOXA1 deletion derivatives HOXA1 ΔNter , HOXA1 Δ11His , HOXA1 Δ77-205 , HOXA1 ΔHD and HOXA1 ΔCter were generated by overlapping polymerase chain reaction using the primers listed in Supplementary Table S2 and swapping in pEXP expression vectors for HOXA1 using the Gateway® system (Life Technologies, Carlsbad, California, USA) following manufacturers's instructions.…”

Section: Methodsmentioning

confidence: 99%

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HOXA1 binds RBCK1/HOIL-1 and TRAF2 and modulates the TNF/NF-κB pathway in a transcription-independent manner

et al. 2016

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HOX proteins define a family of key transcription factors regulating animal embryogenesis. HOX genes have also been linked to oncogenesis and HOXA1 has been described to be active in several cancers, including breast cancer. Through a proteome-wide interaction screening, we previously identified the TNFR-associated proteins RBCK1/HOIL-1 and TRAF2 as HOXA1 interactors suggesting that HOXA1 is functionally linked to the TNF/NF-κB signaling pathway. Here, we reveal a strong positive correlation between expression of HOXA1 and of members of the TNF/NF-κB pathway in breast tumor datasets. Functionally, we demonstrate that HOXA1 can activate NF-κB and operates upstream of the NF-κB inhibitor IκB. Consistently, we next demonstrate that the HOXA1-mediated activation of NF-κB is non-transcriptional and that RBCK1 and TRAF2 influences on NF-κB are epistatic to HOXA1. We also identify an 11 Histidine repeat and the homeodomain of HOXA1 to be required both for RBCK1 and TRAF2 interaction and NF-κB stimulation. Finally, we highlight that activation of NF-κB is crucial for HOXA1 oncogenic activity.

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“…Based on this we assessed the activity of wild-type and mutated HOXA1 proteins using a luciferase reporter gene controlled by the "r4" enhancer of the EphA2 gene (EphA2-r4). PBX1a and PREP proteins are known to be critical cofactors of HOXA1 and to play an important role in HOXA1-mediated transcriptional activation 23,24 . HEK293T cells were co-transfected with different vectors for each of the HOXA1 proteins in conjunction with PBX1a and PREP and the EphA2-r4 luciferase reporter plasmid (Fig.…”

Section: Resultsmentioning

confidence: 99%

Variations in the poly-histidine repeat motif of HOXA1 predispose individuals to bicuspid aortic valve

Odelin

Faucherre

Marchese

et al. 2022

Preprint

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Bicuspid aortic valve (BAV) is the most common cardiovascular malformation (0.5–1.2% of the population) and is often associated with premature aortic valve stenosis or insufficiency, aortic aneurysm and other congenital cardiac heart defects. Although highly heritable, few causal mutations have been identified in BAV patients. Here, we report the association of novel variants in the transcription factor HOXA1 with BAV in humans. Targeted sequencing of HOXA1 in a cohort of 333 BAV patients identified rare indel variants in the homopolymeric histidine tract of HOXA1. In vitro analysis revealed that disruption of the histidine repeat motif causes a significant reduction in the half-life of the protein, and that these variants are associated with a defective transcriptional activity of the HOXA1 protein. Targeting the zebrafish hoxa1a ortholog in vivo resulted in aortic valve defects that could be rescued by expressing the wild-type human HOXA1. Furthermore, expression of HOXA1 Histidine variants in zebrafish also impacted aortic valve development indicating a dominant negative effect of these mutated proteins. Lastly, homozygous knockout mice for Hoxa1 develop a BAV phenotype, which is associated with a very small, rudimentary non-coronary leaflet. Genetic lineage analysis indicates that this defect is caused by a strong reduction of mesenchymal cells in the intercalated cushion due to a failure of neural crest cell migration toward the heart which is consistent with our transcriptomic analysis showing a down-regulation of premigratory and migratory neural crest markers in Hoxa1−/− compared with control embryos. Together, these findings indicate that variants causing HOXA1 dysfunction play a significant role in the genetic cause of BAV in humans.

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Pentapeptide insertion mutagenesis of the Hoxa1 protein: Mapping of transcription activation and DNA‐binding regulatory domains

Cited by 6 publications

References 63 publications

HOXA1 Is an Antagonist of ERα in Breast Cancer

HOXA1 Is an Antagonist of ERα in Breast Cancer

HOXA1 binds RBCK1/HOIL-1 and TRAF2 and modulates the TNF/NF-κB pathway in a transcription-independent manner

Variations in the poly-histidine repeat motif of HOXA1 predispose individuals to bicuspid aortic valve

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