JMJD6 Human

What is JMJD6 and what are its primary biochemical functions?

JMJD6 is a member of the superfamily of non-haem iron(II) and 2-oxoglutarate (2OG)-dependent oxygenases with a nuclear localization and a characteristic JmjC domain comprising a distorted double-stranded β-helical structure. It possesses dual enzymatic activities as both a lysyl hydroxylase and an arginine demethylase, though the latter function remains controversial in the literature. The protein has been conclusively demonstrated to catalyze 2OG-dependent C-5 hydroxylation of lysine residues in mRNA splicing-regulatory proteins and histones, thereby affecting multiple cellular processes . Recent mass spectrometry analyses have identified more than 150 sites of JMJD6-catalyzed lysine hydroxylation on 48 protein substrates, particularly in unstructured lysine-rich regions of the proteome .

How is the JMJD6 gene regulated at the transcriptional and post-transcriptional levels?

JMJD6 regulation involves complex mechanisms operating at multiple levels. At the transcriptional level, JMJD6 is a direct binding target of master pluripotency factors Oct4/Sox2 in embryonic stem cells, suggesting its expression is tied to pluripotency networks . The gene has been associated with transcriptional super-enhancers, indicating sophisticated regulatory control mechanisms . Post-transcriptionally, JMJD6 itself affects alternative splicing by catalyzing posttranslational lysyl-5-hydroxylation of RNA splicing factors such as U2AF65 . The regulatory mechanisms vary across different cell types and developmental contexts, with evidence suggesting auto-regulatory loops where JMJD6 can influence its own expression through interaction with RNA processing machinery.

What are the most effective approaches for detecting JMJD6-mediated hydroxylation of lysine residues?

Detecting JMJD6-mediated lysine hydroxylation requires specialized mass spectrometry methods due to the challenging nature of analyzing lysine-rich regions. Researchers have developed robust approaches including:

• Lysine derivatization with propionic anhydride to improve the analysis of tryptic peptides

• Non-tryptic proteolysis to better capture lysine-rich regions that are typically refractory to conventional trypsin-based analyses

• Targeted mass spectrometry methods that can detect the mass shift associated with hydroxylation (+16 Da)

• Combined immunoprecipitation and mass spectrometry workflows to enrich for JMJD6-interacting proteins

These approaches have been instrumental in identifying the broad substrate range of JMJD6, particularly in unstructured protein regions associated with biomolecular condensates.

How can researchers effectively modulate JMJD6 activity in experimental systems?

Researchers can modulate JMJD6 activity through several complementary approaches:

• Genetic manipulation: RNAi-mediated knockdown and CRISPR/Cas9-mediated knockout are commonly used to reduce JMJD6 expression. Studies have demonstrated that shRNA or siRNA against JMJD6 can effectively reduce its expression in various cell types including embryonic stem cells .

• Overexpression systems: Lentiviral or retroviral vectors containing JMJD6 cDNA can be used for gain-of-function studies. This approach has been employed to enhance reprogramming efficiency of mouse embryonic fibroblasts (MEFs) .

• Small molecule inhibitors: Structure-based design has produced potential JMJD6 inhibitors such as SKLB325, which was designed based on the crystal structure of the jmjC domain of JMJD6 and has shown promising results in cancer models .

• Domain-specific mutations: Introducing point mutations in catalytic residues can distinguish between the different enzymatic activities of JMJD6.

For monitoring efficacy, researchers typically combine these approaches with functional readouts such as gene expression analysis, protein hydroxylation assays, or cellular phenotyping.

What role does JMJD6 play in embryonic stem cell biology and somatic cell reprogramming?

JMJD6 serves as a critical regulator of embryonic stem (ES) cell homeostasis and enhances somatic cell reprogramming through multiple mechanisms:

• Maintenance of pluripotency genes: JMJD6 depletion in ES cells results in downregulation of key pluripotency genes, suggesting its role in maintaining the pluripotent state .

• Cell proliferation and survival: JMJD6 affects ES cell proliferation as evidenced by reduced BrdU incorporation in JMJD6-depleted cells. Microarray analysis revealed that JMJD6 knockdown upregulates genes related to apoptosis and negative regulation of the cell cycle .

• Metabolic regulation: JMJD6 influences glycolysis-related gene expression, which is particularly significant given that ES cells and iPSCs predominantly rely on glycolysis for ATP production .

• Enhancement of reprogramming efficiency: Overexpression of JMJD6 during OKSM (Oct4, Klf4, Sox2, c-Myc)-mediated reprogramming of MEFs significantly increases the efficiency of iPSC generation (approximately 1.6-fold), while its depletion reduces reprogramming efficiency .

• Transcriptional pause release: JMJD6 cooperates with BRD4 at enhancers to release RNA polymerase II promoter-proximal pause in many transcription units, thereby facilitating productive transcriptional elongation of pluripotency genes .

These findings establish JMJD6 as an important factor in both maintaining ES cell identity and promoting the conversion of somatic cells to a pluripotent state.

How does JMJD6 contribute to embryonic development and what are the consequences of its depletion?

JMJD6 plays essential roles in embryonic development, with its knockout causing severe developmental abnormalities:

• Prenatal lethality: JMJD6 knockout in mice leads to prenatal death, highlighting its crucial developmental functions .

• Tissue-specific defects: JMJD6 deficiency causes severe developmental defects in multiple tissues including heart, kidney, eyes, and brain .

• Angiogenesis regulation: Gene ontology analysis of JMJD6-regulated genes shows enrichment of angiogenesis-related functions, corresponding with observations of severe cardiopulmonary malformations in JMJD6 knockout mice .

• Differentiation processes: JMJD6 is essential for proper cell differentiation during development, likely through its effects on gene expression regulation and RNA splicing .

The developmental consequences of JMJD6 depletion likely stem from its roles in regulating transcription, alternative splicing, and posttranslational modifications of proteins involved in key developmental pathways.

What evidence supports JMJD6 as a biomarker or therapeutic target in cancer?

Multiple lines of evidence establish JMJD6 as both a potential biomarker and therapeutic target in various cancers:

• Prognostic biomarker: High JMJD6 expression independently predicts poor patient prognosis in neuroblastoma and serves as a marker of poor prognosis in ovarian cancer . Similar associations have been reported in breast, lung, and colon cancers .

• Chromosome location: The JMJD6 gene is located at chromosome 17qter, a region commonly gained in neuroblastoma, suggesting its potential role in tumorigenesis .

• Oncogenic mechanisms:

  • JMJD6 forms protein complexes with N-Myc and BRD4 in neuroblastoma, promoting the expression of oncogenic drivers including E2F2, N-Myc, and c-Myc .

  • In colon cancer, JMJD6 negatively regulates p53 through hydroxylation .

  • JMJD6 can affect angiogenesis, as demonstrated by its inhibition reducing capillary tube organization and migration in HUVECs .

• Therapeutic targeting: Structure-based designed inhibitors like SKLB325 have shown promise in ovarian cancer models, significantly suppressing proliferation, inducing apoptosis, reducing intraperitoneal tumor weight, and prolonging survival of tumor-bearing mice .

These findings collectively position JMJD6 as a valuable cancer biomarker and potential therapeutic target, particularly in tumors with high JMJD6 expression.

What are the known molecular mechanisms through which JMJD6 contributes to cancer progression?

JMJD6 contributes to cancer progression through several molecular mechanisms:

• Oncogenic transcription factor interaction: JMJD6 forms protein complexes with critical oncogenic factors such as N-Myc and BRD4 in neuroblastoma, thereby activating transcription of E2F2, N-Myc and c-Myc, which drives tumor progression .

• Regulation of p53 signaling: JMJD6 can hydroxylate and negatively regulate p53, a key tumor suppressor. In ovarian cancer cells treated with the JMJD6 inhibitor SKLB325, both mRNA and protein expression of p53 and its downstream effectors significantly increased, suggesting that JMJD6 normally suppresses this tumor-suppressive pathway .

• Angiogenesis promotion: JMJD6 appears to support tumor angiogenesis, as inhibition of JMJD6 with SKLB325 inhibits capillary tube organization and migration in endothelial cells .

• Super-enhancer association: JMJD6 is associated with transcriptional super-enhancers, suggesting it may participate in driving the expression of key oncogenes controlled by these regulatory elements .

• Cell cycle and survival regulation: JMJD6 promotes cancer cell proliferation and survival, as its knockdown reduces proliferation in multiple cancer cell models .

These diverse mechanisms highlight the multifaceted role of JMJD6 in promoting cancer development and progression across different tumor types.

How can researchers reconcile conflicting reports on JMJD6's arginine demethylase activity?

The conflicting reports on JMJD6's arginine demethylase activity present a significant challenge in the field. Researchers can address this controversy through several methodological approaches:

• Improved biochemical assays: Developing more sensitive and specific assays for detecting arginine demethylation, such as combining mass spectrometry with stable isotope labeling, could provide clearer evidence of JMJD6's demethylase activity.

• Structural studies: Crystal structures of JMJD6 in complex with arginine-methylated substrates could clarify whether the protein has the structural features necessary for demethylation.

• Domain-specific mutations: Creating JMJD6 variants with targeted mutations in domains putatively involved in either hydroxylase or demethylase activity could help separate these functions experimentally.

• Substrate specificity profiling: Comprehensive screening of potential substrates under varying conditions might reveal context-dependent demethylase activity.

• Physiological relevance assessment: Even if arginine demethylase activity is detected in vitro, evaluating its importance in vivo through quantitative proteomics approaches would help determine its biological significance .

Chang et al. initially reported JMJD6's histone arginine demethylase activity, but this finding has been challenged by multiple research groups who could not detect such activity . The field now generally acknowledges JMJD6's lysyl-hydroxylase activity while maintaining that its arginine demethylase function remains controversial and requires further investigation.

What are the most promising therapeutic strategies targeting JMJD6 in cancer and other diseases?

Several therapeutic strategies targeting JMJD6 show promise for cancer treatment and potentially other diseases:

• Direct JMJD6 inhibition: Structure-based designed inhibitors such as SKLB325 have demonstrated efficacy in ovarian cancer models, suggesting that direct inhibition of JMJD6's enzymatic activities is a viable approach .

• Combination therapies: Particularly promising is the combination of CDK7/super-enhancer inhibitors (such as THZ1) with histone deacetylase inhibitors (such as panobinostat), which synergistically reduces JMJD6, E2F2, N-Myc, and c-Myc expression, induces apoptosis in vitro, and leads to neuroblastoma tumor regression in mice .

• Disruption of protein-protein interactions: Given JMJD6's interactions with proteins like BRD4 and N-Myc, targeting these specific protein-protein interactions could offer a more selective approach than inhibiting enzymatic activity.

• Substrate-specific targeting: Identifying and targeting specific JMJD6-substrate interactions that are particularly important in disease contexts could provide more selective therapeutic strategies.

• Transcriptional modulation: Since JMJD6 is associated with super-enhancers, approaches that specifically target super-enhancer-driven transcription might indirectly modulate JMJD6's oncogenic functions .

The effectiveness of these approaches will likely depend on the specific disease context and the particular molecular mechanisms through which JMJD6 contributes to pathogenesis in each case.

What are the current technical challenges in identifying the complete set of JMJD6 substrates?

Researchers face several technical challenges in comprehensively identifying JMJD6 substrates:

• Analysis of lysine-rich regions: JMJD6 frequently targets unstructured lysine-rich regions that are refractory to conventional trypsin-based proteomic analyses. This requires specialized approaches such as non-tryptic proteolysis and lysine derivatization with propionic anhydride to improve peptide detection .

• Transient interactions: Many JMJD6-substrate interactions may be transient and context-dependent, making them difficult to capture using standard immunoprecipitation approaches.

• Low abundance modifications: Hydroxylation events often occur substoichiometrically, making their detection challenging without enrichment strategies.

• Distinguishing direct from indirect effects: Determining which proteins are directly modified by JMJD6 versus those affected indirectly through JMJD6's effects on other processes (like splicing) requires careful experimental design.

• Cellular heterogeneity: JMJD6 substrates may vary across different cell types, developmental stages, and disease states, necessitating analysis across multiple cellular contexts.

Recent advances using mass spectrometry methods have identified more than 150 sites of JMJD6-catalyzed lysine hydroxylation on 48 protein substrates, particularly in regions of the proteome that are involved in multiple levels of gene expression control . These findings represent significant progress, but the complete substrate repertoire and the functional consequences of each modification remain to be fully elucidated.

How does JMJD6 contribute to RNA splicing and mRNA processing?

JMJD6 plays a significant role in RNA splicing and mRNA processing through several mechanisms:

• Lysyl hydroxylation of splicing factors: JMJD6 catalyzes posttranslational lysyl-5-hydroxylation of the RNA splicing factor U2 small nuclear ribonucleoprotein auxiliary factor 65-kilodalton subunit (U2AF65), affecting its function in the splicing machinery .

• Interaction with splicing regulators: JMJD6 has been found to interact with multiple components of the splicing machinery, suggesting a direct role in regulating this process.

• Oxygen-dependent regulation: There is evidence that JMJD6's role in splicing is potentially iron- and oxygen-dependent, providing a potential link between cellular oxygen sensing and RNA processing .

• Influence on alternative splicing patterns: Through its interactions with splicing regulators, JMJD6 can influence alternative splicing decisions, potentially affecting the isoform distribution of numerous genes.

• Cotranscriptional processing: Cellular studies have implicated JMJD6 in cotranscriptional processing, suggesting it may serve as a bridge between transcription and RNA processing events .

These mechanisms collectively establish JMJD6 as an important regulator of RNA processing, though the full spectrum of its targets and the functional consequences of its activity in this context remain areas of active investigation.

What are the most informative animal models for studying JMJD6 function in vivo?

Several animal models have proven valuable for investigating JMJD6 function in vivo:

• Conventional knockout mice: Complete JMJD6 knockout in mice leads to prenatal death with severe developmental defects in multiple tissues including heart, kidney, eyes, and brain. While lethal, these models have provided insights into JMJD6's essential developmental roles .

• Conditional knockout models: Tissue-specific and inducible JMJD6 knockout models allow for more focused analysis of JMJD6 function in specific cell types and developmental stages, circumventing the embryonic lethality of conventional knockouts.

• Xenograft models: Human cancer cells with manipulated JMJD6 expression transplanted into immunodeficient mice have been valuable for assessing JMJD6's role in tumor progression. For example, neuroblastoma cells with JMJD6 knockdown showed reduced tumor progression in mice .

• Therapeutic testing models: Intraperitoneal ovarian cancer models have been used to demonstrate that JMJD6 inhibitors like SKLB325 can significantly reduce tumor weight and prolong survival of tumor-bearing mice .

• Gain-of-function models: Transgenic mice overexpressing JMJD6 in specific tissues can provide insights into the consequences of elevated JMJD6 activity, particularly relevant for modeling cancer contexts where JMJD6 is often overexpressed.

The choice of model system should be guided by the specific aspect of JMJD6 biology under investigation, with consideration of the developmental stage, tissue context, and particular function being studied.

What bioinformatic approaches are most effective for analyzing JMJD6-regulated gene expression networks?

Effective bioinformatic approaches for analyzing JMJD6-regulated gene expression networks include:

• Integrated multi-omics analysis: Combining transcriptomic, proteomic, and epigenomic data can provide a comprehensive view of JMJD6's impact on gene regulation. This approach has been used to identify genes affected by JMJD6 depletion in embryonic stem cells .

• Gene ontology (GO) analysis: Applied to datasets from JMJD6 manipulation experiments, GO analysis has revealed enrichment of genes related to angiogenesis, apoptosis, cell cycle regulation, and glycolysis, providing insights into JMJD6's functional impact .

• Network analysis: Protein-protein interaction networks and gene regulatory networks can help identify hub genes and pathways particularly sensitive to JMJD6 regulation.

• Super-enhancer association analysis: Given JMJD6's association with super-enhancers, computational approaches that map these regulatory elements and their target genes can reveal important JMJD6-dependent transcriptional programs .

• RNA splicing analysis: Specialized bioinformatic pipelines for detecting alternative splicing events are crucial for understanding JMJD6's impact on RNA processing.

• Motif analysis: Examining sequence motifs around JMJD6 binding sites or hydroxylation sites can provide insights into substrate recognition patterns.

The application of these approaches has revealed that JMJD6 influences diverse biological processes including pluripotency maintenance, cell cycle regulation, metabolism, and apoptosis, highlighting its multifaceted role in cellular homeostasis.