AHCY Mouse

What is AHCY and what is its function in mice?

Adenosylhomocysteinase (AHCY), also known as S-adenosyl-L-homocysteine hydrolase (SAHH), is a key enzyme in the methionine cycle that hydrolyzes S-adenosylhomocysteine (SAH). In mice, AHCY is encoded by the Ahcy gene located on chromosome 2, with several pseudogenes on chromosomes X, 1, and 16. AHCY is the only enzyme capable of hydrolyzing SAH in mammals, making it essential for metabolic homeostasis, epigenetic regulation, and transcriptional control. Functionally, it prevents the accumulation of SAH, which is a competitive inhibitor of S-adenosyl-L-methionine (SAM)-dependent methyltransferase reactions, thereby playing a crucial role in regulating methylation processes .

What are the molecular characteristics of mouse AHCY?

Mouse AHCY is a 47,688 Da protein (P50247) that shares 98.4% sequence identity with human AHCY. The protein functions as a homotetramer and contains binding sites for NAD+ and adenosine. Several aliases exist for this protein, including AdoHcyase, CUBP (Liver copper-binding protein), and SAHH. The mouse Ahcy gene (Gene ID: 11615) is highly conserved across species, reflecting its essential metabolic function. Structurally, AHCY contains domains responsible for substrate binding and catalytic activity, with key residues determining enzymatic efficiency .

How is AHCY expression regulated during mouse development?

AHCY expression increases gradually from the zygote to blastula stages during early mouse development, correlating with the acquisition of pluripotency. Single-cell RNA-seq data shows that Ahcy expression correlates with pluripotency markers such as Nanog during preimplantation development. Immunostaining and confocal microscopy analysis of mouse embryos at the blastula stage reveals that AHCY is primarily localized in the cell nucleus and is most highly expressed in NANOG-positive pluripotent cells, indicating a nuclear function during early mammalian development .

What happens when AHCY is knocked out in mice?

Homozygous deletion of Ahcy is embryonic lethal before embryonic day 9.5 (E9.5) in mice. Chromosomal microdeletions (approximately 100 kb) encompassing the Ahcy gene cause recessive lethality at the blastula stage. This demonstrates that AHCY is essential for early mouse embryo development, particularly at the pre-implantation stage. Pharmacological inhibition of AHCY with inhibitors severely compromises blastula progression, further confirming its critical role in early development .

How can researchers create and validate transgenic AHCY mouse models?

Researchers can generate transgenic AHCY mice by placing the AHCY cDNA (human or mouse) downstream of inducible promoters such as the zinc-inducible mouse MT-1 (metallothionein-1) promoter. For differential detection between endogenous and transgenic AHCY, strategies include:

• Attaching epitope tags (e.g., HA-tag) to the N-terminus of mouse AHCY

• Using human AHCY (which can be detected with human-specific antibodies)

• Including locus control regions to enhance expression

Validation should include:

• Western blot analysis to confirm protein expression

• Enzyme activity assays to measure functional AHCY levels

• Tissue-specific expression analysis (e.g., liver, kidney)

• Assessment of SAM/SAH ratio to verify metabolic impact

One important finding from existing transgenic models is that AHCY protein levels may be subject to post-translational regulation, as simple transgene expression doesn't always lead to increased total AHCY levels in all tissues .

What conditional and inducible AHCY knockout systems are available for mouse studies?

Several approaches are available for conditional AHCY manipulation:

• CRISPR/Cas9-based systems: Guide RNAs targeting exon 4 of Ahcy have been successfully used to generate knockouts in mouse embryonic fibroblasts (MEFs). The guide RNA sequence 5′-TGCGCACCTGACAGAAGCTG-3′ has proven effective .

• Inducible shRNA systems: Doxycycline-inducible shRNA vectors targeting mouse Ahcy allow temporal control of knockdown. These systems typically employ lentiviral delivery and can achieve significant reduction in AHCY levels within 24 hours of doxycycline administration .

• Tissue-specific Cre-loxP systems: Although not explicitly described in the search results, the lethality of constitutive knockout suggests that tissue-specific Cre-loxP systems would be valuable for studying AHCY function in specific tissues beyond early development .

For rescue experiments, stable expression of wild-type or mutant AHCY (e.g., K186N) can be achieved through transfection of linearized plasmids encoding AHCY variants followed by antibiotic selection .

How does AHCY affect mouse embryonic stem cell (mESC) maintenance?

AHCY plays multiple crucial roles in maintaining mESC pluripotency and self-renewal:

• Metabolic regulation: AHCY is required for metabolic homeostasis in mESCs, supporting the unique metabolic demands of pluripotent cells.

• Epigenetic control: AHCY maintains both H3K4me3 (activating) and H3K27me3 (repressive) histone modifications, thereby exerting dual roles in transcriptional activation and inhibition.

• Transcriptional regulation: AHCY is preferentially recruited to regions near transcription start sites (TSS) of highly expressed genes in ESCs, particularly those associated with the Myc network linked to proliferation and ribosomal activity.

• Pluripotency network: AHCY is a direct target of OCT4, a master regulator of pluripotency, placing it within the core pluripotency regulatory network.

Depletion of Ahcy drastically reduces mESC proliferation, and prolonged depletion promotes spontaneous differentiation, demonstrating its essential role in maintaining the pluripotent state .

What is the relationship between AHCY and circadian rhythms in mice?

AHCY links methionine metabolism to circadian rhythms in mice. Pharmacological inhibition of AHCY in the hypothalamus alters circadian locomotor activity and rhythmic transcription. Experimental approaches to study this connection include:

• Recording locomotor activity in light-dark cycles (LD 12:12) followed by constant darkness (DD) to calculate circadian period before and after AHCY inhibition.

• Using inhibitors like DZnep to target AHCY activity in specific brain regions.

• Analyzing the effects on circadian clock components using luciferase reporters for promoters of clock genes like Dbp and Per2.

• Examining the interaction between AHCY and circadian transcription factors like BMAL1.

These approaches reveal that AHCY functions as a metabolic link between the methionine cycle and circadian regulation, affecting both behavioral outputs and molecular clock mechanisms .

What are the recommended methods for measuring AHCY levels and activity in mouse samples?

For AHCY protein detection and quantification in mouse samples, sandwich ELISA kits are available with a detection range of 0.156-10 ng/mL. These kits can measure AHCY in serum, plasma, and lysate samples. For activity assays, enzyme function can be assessed by measuring the conversion of SAH to adenosine and homocysteine, though these typically require specialized techniques and equipment .

What approaches can be used to study AHCY's role in transcriptional regulation?

Several methodologies are effective for studying AHCY's transcriptional regulatory functions:

• Chromatin Immunoprecipitation (ChIP): AHCY ChIP followed by qPCR or sequencing (ChIP-seq) reveals that AHCY is preferentially recruited to regions near transcription start sites (TSS). AHCY target genes are among the most highly expressed genes in ESCs and are decorated with transcriptionally permissive histone modifications.

• Transcriptome analysis: RNA-seq following AHCY knockdown or inhibition can identify genes and pathways regulated by AHCY.

• Reporter assays: Luciferase reporters with promoters of interest (e.g., Dbp, Per2, PGC1α) can be used to assess AHCY's impact on transcriptional activity.

• Co-localization studies: Fluorescently tagged AHCY (e.g., GFP-AHCY) can be used to visualize its nuclear localization and potential co-localization with transcription factors.

• Epigenetic profiling: Assessment of histone modifications (H3K4me3, H3K27me3) following AHCY manipulation reveals its role in maintaining the epigenetic landscape.

These approaches have demonstrated that AHCY occupancy correlates with key factors in the cancer-related ESC-specific Myc network, including Myc, TRIM28, and DMAP1 .

How does AHCY interact with aging processes in mice?

While the direct interaction between AHCY and aging wasn't extensively detailed in the search results, metabolic pathways involving AHCY are known to change during aging. A recent metabolic atlas of mouse aging described changes in metabolism across 12 organs in male and female mice at 5 different ages. This atlas provides a resource for investigating age- and sex-specific processes and diseases, potentially including AHCY-related metabolic changes.

Researchers interested in AHCY's role in aging should consider:

• Age-related changes in AHCY expression and activity across different tissues

• The impact of age on methionine metabolism and SAM/SAH ratios

• Changes in methylation patterns associated with aging and their relationship to AHCY function

• Potential therapeutic interventions targeting AHCY to address age-related metabolic dysregulation

The identification of hydroxyproline as a new marker of aging may also have implications for understanding methionine pathway alterations, including AHCY function, during the aging process .

What are the critical considerations when designing AHCY inhibition studies in mice?

When designing studies using AHCY inhibitors in mice, researchers should consider:

• Inhibitor specificity: Select inhibitors with established specificity for AHCY to minimize off-target effects.

• Dosage determination: Conduct dose-response studies to identify effective concentrations that achieve the desired inhibition without toxicity.

• Delivery method:

  • For systemic effects: Intraperitoneal or intravenous administration

  • For tissue-specific effects: Stereotaxic injection (e.g., hypothalamus for circadian studies)

  • For in vitro studies: Direct media supplementation

• Timing considerations:

  • For developmental studies: Precise embryonic timing is critical

  • For circadian studies: Control for time-of-day effects and circadian phase

• Readouts and controls:

  • Metabolic parameters (SAM/SAH ratio, methionine, homocysteine levels)

  • Methylation status (global and gene-specific)

  • Transcriptional changes (RNA-seq, qPCR)

  • Phenotypic assessment (behavior, development, tissue-specific functions)

• Rescue experiments: Include rescue conditions with SAM supplementation or AHCY overexpression to confirm specificity of observed effects .

What are the implications of AHCY variants and mutations for mouse models of human disease?

Human AHCY deficiency is a rare autosomal recessive disorder characterized by elevated methionine, S-adenosylhomocysteine (SAH), and S-adenosylmethionine (SAM) levels in bodily fluids, with patients typically displaying severe hepatic, muscle, and cognitive dysfunction. Several pathogenic variants have been identified in humans (R49C, R49H, A50T, T57I, G71S, D86G, A89V, E108K, T112stop, Y143C, V217M, and Y328D).

For mouse models of AHCY deficiency:

These approaches could provide insights into the pathophysiology of AHCY deficiency and potential therapeutic strategies, with implications for understanding the underdiagnosed impact of AHCY variants in the general human population .

What are the optimal protocols for AHCY ChIP-seq in mouse embryonic stem cells?

For optimal AHCY ChIP-seq in mouse embryonic stem cells, the following protocol considerations are recommended:

• Cell preparation:

  • Culture mESCs in standard conditions (LIF + 2i or serum + LIF)

  • Use 10-20 million cells per ChIP experiment

  • Crosslink with 1% formaldehyde for 10 minutes at room temperature

• Chromatin preparation:

  • Sonicate to generate fragments of 200-500 bp

  • Verify fragment size by agarose gel electrophoresis

  • Pre-clear chromatin with protein A/G beads

• Immunoprecipitation:

  • Use validated anti-AHCY antibodies (commercial or custom)

  • Include appropriate controls (IgG control, input samples)

  • Verify antibody specificity using AHCY-knockdown cells

• Quality control:

  • Perform ChIP-qPCR on known AHCY targets before sequencing

  • Analyze enrichment at positive control regions (e.g., highly expressed genes)

  • Verify AHCY knockdown reduces ChIP signal at specific promoters

• Data analysis:

  • Focus analysis on regions near transcription start sites (TSS)

  • Examine correlation with transcriptionally permissive histone marks

  • Compare AHCY occupancy with factors in the Myc network

This approach has successfully revealed that AHCY preferentially binds to regions in close proximity to TSS, covering a narrow window around TSS of target genes that are among the most highly expressed in ESCs .

How can researchers effectively study the dual roles of AHCY in transcriptional activation and repression?

To investigate AHCY's dual roles in both transcriptional activation and repression:

• Integrated genomic approaches:

  • Combine ChIP-seq (for AHCY binding) with RNA-seq (for expression changes)

  • Perform histone modification ChIP-seq (H3K4me3 for activation, H3K27me3 for repression)

  • Conduct ATAC-seq to assess chromatin accessibility changes

• Epigenetic profiling:

  • Map genome-wide DNA methylation changes (WGBS or RRBS)

  • Correlate methylation patterns with gene expression and AHCY binding

  • Analyze changes in both activating and repressive histone marks

• Mechanistic studies:

  • Perform protein interaction studies (IP-MS) to identify AHCY-interacting partners

  • Use targeted ChIP to assess co-occupancy with transcriptional activators and repressors

  • Utilize reporter assays with promoters of both activated and repressed genes

• Temporal analysis:

  • Use inducible knockdown/knockout systems to track temporal changes

  • Perform time-course experiments to distinguish primary from secondary effects

  • Analyze immediate and delayed transcriptional responses

• Domain-specific mutants:

  • Generate AHCY mutants that selectively disrupt specific protein interactions

  • Test these mutants in rescue experiments to define domain-specific functions

  • Analyze their impact on both activating and repressive functions

This comprehensive approach will help elucidate how AHCY maintains H3K4me3 (activating) and H3K27me3 (repressive) marks, supporting its dual role in both transcriptional activation and inhibition as observed in mouse embryonic stem cells .

How does AHCY function differ across mouse tissues?

AHCY function and regulation vary considerably across different mouse tissues:

Transgenic studies have revealed tissue-specific regulation of AHCY. For example, the Tg-hAHCY line shows good transgene-driven protein expression in both kidney and liver, while the Tg-mAhcy line only has detectable transgene expression in the liver. Surprisingly, despite good protein expression, there was only a modest increase in total AHCY activity in the liver (16%, non-significant) compared to a 131% increase in the kidney, suggesting tissue-specific post-translational regulation .

What metabolic pathways interact with AHCY in mice, and how can these interactions be studied?

AHCY is a central component of several interconnected metabolic pathways in mice:

• Methionine cycle: AHCY hydrolyzes S-adenosylhomocysteine (SAH) to adenosine and homocysteine, maintaining the SAM/SAH ratio critical for methylation reactions.

• One-carbon metabolism: Connected to folate cycle and purine/pyrimidine synthesis.

• Transsulfuration pathway: Links to cysteine production and glutathione synthesis.

• Circadian metabolism: Influences and is influenced by circadian oscillators.

Methodological approaches to study these interactions include:

• Metabolomic profiling:

  • Targeted LC-MS/MS analysis of methionine cycle intermediates

  • Untargeted metabolomics to identify novel connections

  • Stable isotope tracing (13C-methionine, 13C-serine) to track flux

• Genetic interaction studies:

  • Cross AHCY transgenic mice with models of interacting pathways

  • Combined knockdown of AHCY with other metabolic enzymes

  • Epistasis analysis in cell culture systems

• Pharmacological approaches:

  • Use specific inhibitors like DZnep to target AHCY

  • Supplement with pathway metabolites (methionine, SAM)

  • Dietary interventions (methionine restriction)

• Multi-tissue analysis:

  • Comparative metabolic profiles across tissues

  • Analysis of metabolite transport between tissues

  • Organ-specific perturbations and systemic responses

One important example is the interaction between AHCY and CBS (cystathionine beta-synthase) pathways. Tg-hAHCY Tg-I278T Cbs-/- mice have been created to study the effects of AHCY overexpression in the context of severe hyperhomocysteinemia, demonstrating how genetic manipulation can illuminate metabolic pathway interactions .

What emerging technologies show promise for advancing AHCY research in mice?

Several cutting-edge technologies hold significant potential for AHCY research:

• Single-cell multi-omics:

  • scRNA-seq combined with scATAC-seq to correlate AHCY expression with chromatin accessibility

  • Single-cell proteomics to track AHCY protein levels and modifications

  • Spatial transcriptomics to map AHCY expression within tissue architecture

• Genome editing advances:

  • Base editing for precise introduction of AHCY variants

  • Prime editing for scarless modification of AHCY regulatory elements

  • Inducible, tissue-specific CRISPR systems for temporal control

• In vivo imaging:

  • AHCY activity biosensors based on fluorescence resonance energy transfer (FRET)

  • Intravital microscopy to track AHCY localization and dynamics

  • PET tracers for non-invasive monitoring of methionine metabolism

• Organoid systems:

  • AHCY manipulation in tissue-specific organoids

  • Brain organoids to study neurodevelopmental aspects

  • Liver organoids for metabolic studies

• AI-driven approaches:

  • Machine learning for prediction of AHCY-dependent pathways

  • Network analysis to identify novel AHCY interactors

  • In silico modeling of SAM/SAH dynamics

These technologies could help resolve current knowledge gaps, such as the precise mechanism by which AHCY controls developmental processes, tissue-specific functions, and the full spectrum of its non-enzymatic roles .

What are the most promising therapeutic applications emerging from AHCY mouse research?

Research on AHCY in mouse models points to several potential therapeutic applications:

• Rare disease treatments:

  • Development of enzyme replacement therapies for AHCY deficiency

  • Gene therapy approaches to restore AHCY function in affected tissues

  • Small molecule chaperones to stabilize mutant AHCY proteins

• Stem cell applications:

  • Modulation of AHCY to enhance pluripotent stem cell maintenance

  • Controlled inhibition to direct differentiation toward specific lineages

  • Metabolic optimization of stem cell therapies

• Metabolic disorders:

  • Targeting AHCY to normalize methionine metabolism in hyperhomocysteinemia

  • Dietary interventions informed by AHCY function in different tissues

  • Personalized approaches based on AHCY genetic variants

• Circadian rhythm disorders:

  • AHCY modulation to reset disrupted circadian clocks

  • Chronotherapy approaches targeting methionine metabolism

  • Nutritional interventions timed to optimize circadian function

• Aging and neurodegeneration:

  • Interventions targeting age-related changes in AHCY function

  • Methionine restriction or supplementation strategies

  • Neuroprotective approaches based on AHCY's role in brain methylation