Histone H3K79me1 Antibody

What is H3K79me1 and why is it significant in epigenetic research?

H3K79me1 refers to the monomethylation of lysine 79 on histone H3, one of the core components of the nucleosome. Unlike most histone modifications that occur on N-terminal tails, H3K79 methylation is located within the globular domain of histone H3. This positioning makes it particularly interesting as it affects nucleosome structure and DNA accessibility differently than tail modifications.

The nucleosome, as the fundamental unit of chromatin, consists of approximately 147 base pairs of DNA wrapped around an octamer of core histone proteins (two each of H2A, H2B, H3, and H4) . H3K79 methylation serves as an important regulatory mark affecting transcriptional regulation, with the monomethylated form (H3K79me1) representing a distinct functional state with potentially different protein interaction partners and genomic distribution patterns compared to H3K79me2/3 . Research has established that H3K79 methylation marks are critical for transcriptional regulation, DNA damage responses, and in specialized processes like immunoglobulin gene diversification .

How does H3K79me1 differ from H3K79me2/3 in terms of distribution and function?

H3K79me1, H3K79me2, and H3K79me3 represent sequential methylation states of the same lysine residue, differing in the number of methyl groups attached:

• H3K79me1: Single methyl group (monomethylation)

• H3K79me2: Two methyl groups (dimethylation)

• H3K79me3: Three methyl groups (trimethylation)

These methylation states exhibit distinct genomic distribution patterns and potentially different functions. Research has demonstrated that H3K79me2/3 tends to be more abundant in variable (V) regions than constant (C) regions of immunoglobulin genes . When Dot1L (the enzyme responsible for H3K79 methylation) is inhibited, H3K79me1 increases in V regions while H3K79me2/3 decreases throughout the immunoglobulin heavy chain (IgH) locus . This suggests that these methylation states have distinct regulatory roles and distribution patterns.

Functionally, H3K79me1 may represent an intermediate state in the progressive methylation of H3K79, with each methylation state potentially recruiting different protein complexes. During somatic hypermutation, the continued presence of H3K79me1 (while H3K79me2/3 decreases) after Dot1L inhibition suggests that H3K79me1 itself may play functional roles distinct from the di- and trimethylated forms .

What is the relationship between Dot1L and H3K79 methylation states?

Dot1L (Disruptor of Telomeric silencing 1-like) is the sole methyltransferase responsible for catalyzing H3K79 methylation. Unlike most histone methyltransferases, Dot1L does not contain a SET domain and targets a lysine within the globular domain rather than on histone tails.

Dot1L operates through a sequential methylation mechanism:

• First adding one methyl group to create H3K79me1

• Then potentially adding a second methyl group to generate H3K79me2

• Finally possibly adding a third methyl group to produce H3K79me3

This sequential activity is evidenced by studies using Dot1L inhibitors like EPZ004777, which cause dose-dependent decreases in H3K79me2/3 levels throughout the IgH locus while H3K79me1 increases in certain regions like the V region . The accumulation of H3K79me1 when further methylation is blocked confirms the sequential nature of Dot1L's activity.

Western blot analyses have shown that Dot1L inhibition results in global dose-dependent decreases in all three methylation states (H3K79me1, -me2, and -me3), but the effect on H3K79me1 can be region-specific, with accumulation occurring in certain genomic locations . Importantly, Dot1L inhibition does not affect methylation of other histone lysines such as H3K4me3 and H3K36me3, confirming Dot1L's specificity for H3K79 .

What are key criteria for selecting a high-quality H3K79me1 antibody?

When selecting an H3K79me1 antibody for research applications, several critical factors should be considered:

• Specificity: The antibody should specifically recognize H3K79me1 without cross-reactivity to unmethylated H3K79 or other methylation states (H3K79me2/3). Commercial antibodies are typically raised against synthetic peptides containing monomethylated lysine 79 of histone H3 . Validation data should demonstrate minimal cross-reactivity with other H3K79 methylation states.

• Validated applications: The antibody should be validated for your intended applications. Available H3K79me1 antibodies are validated for multiple techniques including Chromatin Immunoprecipitation (ChIP), Western blotting (WB), Immunofluorescence (IF), and Dot blot analysis .

• Species reactivity: Ensure the antibody recognizes H3K79me1 in your species of interest. Many H3K79me1 antibodies react with human, mouse, and monkey samples, with predicted reactivity across additional species due to the high conservation of histone H3 sequences .

• Format and concentration: Consider whether the antibody is available as purified IgG or unpurified serum, and at appropriate concentrations for your applications. Some suppliers offer both formats - for example, both purified IgG (Catalog No. 39921) and unpurified serum (Catalog No. 39145) versions .

• Storage conditions: Check recommended storage conditions - typically at -20°C with 30% glycerol and 0.035% sodium azide to maintain stability . Proper aliquoting to avoid freeze-thaw cycles is essential for maintaining antibody performance.

How can I validate the specificity of an H3K79me1 antibody?

Rigorous validation of H3K79me1 antibody specificity is crucial for generating reliable experimental results. Several approaches should be combined:

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide containing H3K79me1 modification. This should block specific binding and eliminate signal in subsequent applications. Include a control with an unrelated peptide that should not affect binding.

• Dot blot specificity testing: Apply a panel of synthetic peptides (unmodified H3, H3K79me1, H3K79me2, H3K79me3) to a membrane and probe with the H3K79me1 antibody. A specific antibody should show strong signal with the H3K79me1 peptide and minimal reactivity with other methylation states.

• Western blot with Dot1L inhibition: Treat cells with a Dot1L inhibitor like EPZ004777 at varying concentrations. This should produce a dose-dependent decrease in global H3K79 methylation levels . For H3K79me1, the pattern may be complex with region-specific increases and decreases.

• ChIP-qPCR on positive and negative control regions: Perform ChIP-qPCR at known positive regions (like the BTG1 gene body) and negative regions (like nontranscribed CD4, which shows no detectable H3K79 methylation) . This provides functional validation of specificity.

• siRNA/CRISPR knockout of Dot1L: Complete absence of Dot1L should eliminate all H3K79 methylation, providing a powerful negative control for antibody validation.

What are the optimized protocols for Chromatin Immunoprecipitation (ChIP) with H3K79me1 antibody?

For successful ChIP experiments using H3K79me1 antibody, follow these optimized procedures:

• Chromatin preparation:

  • Cross-link cells with 1% formaldehyde for 10 minutes at room temperature

  • Quench with 0.125M glycine for 5 minutes

  • Lyse cells and isolate nuclei

  • Sonicate chromatin to generate fragments of 200-500bp

  • Verify fragmentation by agarose gel electrophoresis

• Immunoprecipitation:

  • Use 10 μg of H3K79me1 antibody per ChIP reaction as recommended in manufacturer protocols

  • Include appropriate controls (input DNA, IgG control, positive/negative genomic regions)

  • Pre-clear chromatin with protein A/G beads

  • Incubate antibody with chromatin overnight at 4°C with rotation

• Washing and elution:

  • Perform stringent washing steps to remove non-specific binding

  • Elute protein-DNA complexes and reverse cross-links (typically 65°C for 4-6 hours)

  • Treat with RNase A and Proteinase K

  • Purify DNA for downstream analysis

• Analysis strategies:

  • For targeted analysis: design primers for regions of interest like gene bodies (where H3K79me1 is typically enriched) and perform qPCR

  • For genome-wide profiling: prepare libraries for ChIP-seq

  • Include known positive regions like BTG1 gene body and negative regions like nontranscribed CD4

• Data normalization:

  • Normalize to input DNA (typically 1-10% of starting chromatin)

  • Compare enrichment to IgG control

  • Consider percent input or fold enrichment over background for quantification

The ChIP protocol may require optimization based on cell type and experimental conditions. When designing primers for ChIP-qPCR validation, focus on regions where H3K79me1 is known to be present based on previous studies or pilot ChIP-seq experiments.

What are the recommended procedures for Western blotting with H3K79me1 antibody?

For optimal Western blot detection of H3K79me1, follow these specialized procedures:

• Sample preparation:

  • Extract histones using acid extraction method (preferred for histone analysis) or high salt/sonication protocol

  • Standard nuclear extraction protocols may be insufficient as "many chromatin-bound proteins are not soluble in a low salt nuclear extract and fractionate to the pellet. Therefore, a High Salt/Sonication Protocol is recommended when preparing nuclear extracts for Western Blot"

• Gel electrophoresis:

  • Use 15-18% SDS-PAGE gels for optimal separation of histone proteins

  • Load 10-20 μg of histone extract per lane

  • Include molecular weight markers that cover the ~15 kDa range (expected size of histone H3)

• Transfer conditions:

  • Transfer to PVDF membrane (preferred for histone proteins)

  • Use lower methanol concentration (10-15%) in transfer buffer to enhance transfer efficiency of small proteins

• Blocking and antibody incubation:

  • Block membrane with 5% non-fat dry milk or BSA in TBST

  • Critically, "the addition of 0.05% Tween 20 in the blocking buffer and primary antibody incubation buffer is recommended to aid in detection by Western blot"

  • Incubate with H3K79me1 antibody at 1-2 μg/ml dilution

  • Incubate overnight at 4°C

• Detection and controls:

  • Use appropriate HRP-conjugated secondary antibody (anti-rabbit for most H3K79me1 antibodies)

  • Include total H3 antibody as loading control on a separate blot or after stripping

  • Consider including samples treated with Dot1L inhibitors at various concentrations as verification controls

The Western blot should reveal a band at approximately 15 kDa corresponding to histone H3 . Multiple bands or unexpected molecular weights may indicate degradation or non-specific binding.

How can I effectively use H3K79me1 antibody for immunofluorescence studies?

For successful immunofluorescence (IF) detection of H3K79me1, implement the following specialized protocol:

• Cell preparation:

  • Culture cells on coverslips or use cytospin to deposit suspension cells on slides

  • Fix with 4% paraformaldehyde in PBS for 15 minutes at room temperature

  • Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes

• Antigen retrieval (essential for accessing nucleosomal epitopes):

  • Incubate in citrate buffer (pH 6.0) at 95°C for 20 minutes

  • Cool slowly to room temperature

  • This step is crucial for exposing the H3K79 epitope which resides within the nucleosome core

• Blocking and primary antibody:

  • Block with 5% normal goat serum in PBS containing 0.1% Triton X-100 for 1 hour

  • Dilute H3K79me1 antibody according to manufacturer recommendations (typically 1:200-1:500)

  • Incubate overnight at 4°C in a humidified chamber

• Secondary antibody and counterstaining:

  • Wash extensively with PBS (3-5 times, 5 minutes each)

  • Incubate with fluorophore-conjugated secondary antibody for 1 hour at room temperature

  • Counterstain nuclei with DAPI (1 μg/ml for 5 minutes)

  • Mount with anti-fade mounting medium

• Controls and pattern interpretation:

  • Include negative controls (primary antibody omitted, non-immune IgG)

  • Include positive controls (cell types known to express H3K79me1)

  • H3K79me1 should show nuclear localization with potential focal enrichment patterns

  • The signal may correlate with transcriptionally active regions

For co-localization studies, consider double immunostaining with antibodies against other active chromatin marks such as H3K4me3 or H3K27ac, which have been found to co-occur with H3K79 methylation in certain genomic regions .

How should I interpret ChIP-seq data generated with H3K79me1 antibody?

Interpreting ChIP-seq data for H3K79me1 requires understanding both the technical aspects of the experiment and the biological significance of this modification:

• Expected genomic distribution patterns:

  • H3K79me1 typically shows enrichment within gene bodies rather than sharp peaks at promoters

  • Distribution may vary between cell types and in response to stimuli

  • In B cells, H3K79me1 has been observed in active genes like BTG1 and in immunoglobulin variable regions

  • Region-specific patterns may emerge, as seen in the study where H3K79me1 increased in V regions but decreased in C regions after Dot1L inhibition

• Correlation with gene expression:

  • Assess correlation between H3K79me1 enrichment and gene expression data

  • Research indicates that a decrease in both H3K79me1 and H3K79me2/3 in the C region correlates with decreased RNA levels of that region

  • Compare with known active transcription marks (H3K4me3, H3K27ac) which have been shown to co-occur with H3K79 methylation in active regions

• Comparison with other histone modifications:

  • Examine the relationship between H3K79me1 and other histone marks

  • H3K79me1 may show distinct patterns compared to H3K36me3, which increases toward the 3' portion of gene bodies

  • Consider integrative analysis with datasets for other histone modifications

• Bioinformatic analysis approaches:

  • Use appropriate peak calling algorithms suitable for broad marks (e.g., SICER or MACS2 with broad peak settings)

  • Generate heatmaps centered on transcription start sites (TSS), gene bodies, and transcription end sites (TES)

  • Create aggregate plots to visualize average enrichment patterns across genomic features

  • Perform gene ontology analysis to identify biological processes associated with H3K79me1-enriched genes

• Validation of key findings:

  • Confirm significant peaks by ChIP-qPCR at selected loci

  • Correlate with functional assays to establish biological significance

  • Consider the effects of experimental manipulations (e.g., Dot1L inhibition) on H3K79me1 patterns

What are common technical challenges with H3K79me1 antibodies and their solutions?

Researchers working with H3K79me1 antibodies may encounter several technical challenges that require specific troubleshooting approaches:

• Low signal in ChIP experiments:

  • Challenge: Insufficient enrichment despite proper technique

  • Solutions:

    • Increase antibody amount (ensure using recommended 10 μg per ChIP)

    • Optimize chromatin fragmentation (aim for 200-500bp fragments)

    • Increase cell number to obtain more starting material

    • Extend antibody incubation time (overnight at 4°C)

    • Verify antibody activity with dot blot using modified peptides

• High background in Western blots:

  • Challenge: Non-specific bands or high background staining

  • Solutions:

    • Add 0.05% Tween 20 to blocking and antibody buffers as specifically recommended

    • Optimize antibody dilution (start with recommended 1-2 μg/ml)

    • Increase washing steps duration and number

    • Use freshly prepared buffers

    • Consider alternative blocking agents (BSA vs. milk)

• Cross-reactivity with other methylation states:

  • Challenge: Antibody detecting H3K79me2/3 in addition to H3K79me1

  • Solutions:

    • Validate antibody specificity using peptide arrays with all methylation states

    • Include specificity controls in each experiment

    • Compare results with a second H3K79me1 antibody from a different source

    • Use Dot1L inhibition as a biological tool to alter methylation patterns

• Epitope masking in fixed tissues/cells:

  • Challenge: Formaldehyde fixation can hide the H3K79 epitope

  • Solutions:

    • Implement antigen retrieval steps (citrate buffer, heat treatment)

    • Optimize fixation conditions (time, concentration)

    • Try alternative fixatives or dual fixation protocols

    • Increase permeabilization to improve antibody access to nucleosomes

• Inconsistent results between experiments:

  • Challenge: Variable enrichment patterns between replicates

  • Solutions:

    • Standardize protocols rigorously (timing, temperatures, reagent concentrations)

    • Use the same antibody lot for related experiments

    • Include internal control regions in ChIP-qPCR experiments

    • Normalize to consistent housekeeping genes or input fractions

How does H3K79me1 distribution relate to transcriptional regulation?

H3K79me1 exhibits specific distribution patterns related to transcriptional activity, providing insights into its regulatory functions:

• Gene body enrichment patterns:

  • H3K79me1 is typically found within gene bodies rather than concentrated at promoters

  • It accumulates in actively transcribed genes, as evidenced by its presence in the BTG1 gene body and absence in nontranscribed regions like CD4

  • The distribution may vary along the gene, with region-specific enrichment patterns

• Relationship with transcriptional activity:

  • Research indicates that all H3K79 methylation states are associated with transcription

  • Decreases in both H3K79me1 and H3K79me2/3 on the C region have been correlated with decreased RNA levels of that region

  • This suggests a functional role for H3K79me1 in transcriptional regulation

• Dynamics during gene activation:

  • Studies have shown changes in H3K79 methylation patterns after stimulation of B cells

  • After LPS+IL-4 stimulation, cells underwent class switch recombination (CSR) with concurrent changes in H3K79 methylation patterns at activated switch regions

  • This indicates that H3K79me1 distribution responds dynamically to cellular activation signals

• Relationship with RNA polymerase II:

  • H3K79 methylation may influence RNA polymerase II progression through gene bodies

  • Dot1L inhibition, which affects H3K79 methylation patterns, did not significantly affect RNA levels of some genes (like mCherry and VH4-34) but decreased RNA levels of the C region

  • This suggests gene- and region-specific roles in transcriptional regulation

• Connection with chromatin structure:

  • As a modification within the globular domain of histone H3, H3K79me1 may directly affect nucleosome structure and stability

  • This could influence chromatin accessibility to transcription factors and RNA polymerase

What is the relationship between H3K79me1 and somatic hypermutation?

Research has revealed intriguing connections between H3K79 methylation states and somatic hypermutation (SHM), a process critical for antibody diversification:

• Correlation with mutation-prone regions:

  • H3K79me2/3 and its methyltransferase Dot1L are more abundant on the variable (V) region of immunoglobulin genes than on the constant (C) region, which does not undergo mutation

  • This spatial correlation suggests a potential role for H3K79 methylation in the SHM process

• Effects of Dot1L inhibition on SHM:

  • Inhibition of Dot1L leads to reduction in SHM frequency in a dose-dependent manner

  • This indicates a functional role for H3K79 methylation in facilitating or regulating SHM

• Differential roles of methylation states:

  • After Dot1L inhibition, H3K79me1 increased in the V region while H3K79me2/3 decreased

  • The continued presence of H3K79me1 might explain why inhibition of Dot1L only partially blocked SHM

  • This suggests that H3K79me1 itself could play a role in SHM, possibly distinct from H3K79me2/3

• Mechanistic implications:

  • H3K79 methylation may create a chromatin environment permissive for AID (Activation-Induced Deaminase) access

  • Different methylation states might recruit distinct protein complexes involved in DNA repair and mutation

  • The spatial and temporal regulation of H3K79 methylation could help target SHM to appropriate genomic regions

• Correlation with other chromatin marks:

  • Active transcription marks H3K4me3 and H3K27ac are also enriched in regions undergoing SHM

  • This suggests a complex chromatin signature involving multiple histone modifications may define regions susceptible to SHM

How does Dot1L inhibition differentially affect H3K79 methylation states?

Dot1L inhibition reveals complex dynamics between different H3K79 methylation states, providing insights into the sequential methylation process:

How can I design experiments to study H3K79me1 dynamics during cellular differentiation?

To investigate H3K79me1 dynamics during cellular differentiation processes, consider these experimental approaches:

• Time-course ChIP-seq analysis:

  • Perform ChIP-seq for H3K79me1 at multiple timepoints during differentiation

  • Include parallel ChIP-seq for H3K79me2/3 to compare dynamics of different methylation states

  • Correlate with RNA-seq data to connect chromatin changes with transcriptional programs

  • Example timepoints: undifferentiated state, early commitment, mid-differentiation, terminal differentiation

• Cell type-specific analysis:

  • Compare H3K79me1 profiles between progenitor cells and differentiated cells

  • In models like B cell development, examine naive B cells, germinal center B cells, and plasma cells

  • This approach can reveal lineage-specific H3K79me1 patterns associated with cell identity

• Perturbation experiments:

  • Use Dot1L inhibitors like EPZ004777 at different stages of differentiation

  • Assess the impact on differentiation trajectory and cell fate decisions

  • Perform dose-response and time-course experiments to capture dynamic effects

  • Include ChIP-seq analysis to monitor changes in H3K79 methylation patterns

• Single-cell approaches:

  • Implement single-cell technologies (CUT&RUN, scATAC-seq with H3K79me1 antibodies)

  • This can capture heterogeneity in H3K79me1 distribution during differentiation

  • Connect with single-cell RNA-seq to correlate with gene expression changes at the single-cell level

• Multi-modal chromatin analysis:

  • Perform sequential ChIP or co-ChIP to examine co-occurrence of H3K79me1 with other modifications

  • Include active marks (H3K4me3, H3K27ac) and elongation-associated marks (H3K36me3) mentioned in the research

  • This integrated approach can reveal how H3K79me1 contributes to chromatin state transitions during differentiation

What is the relationship between H3K79me1 and other histone modifications?

Understanding the interplay between H3K79me1 and other histone modifications provides insights into the complex histone code governing gene regulation:

• Co-occurrence with active transcription marks:

  • H3K79me1 often co-occurs with active transcription marks like H3K4me3 and H3K27ac

  • Research shows that these active marks are enriched in regions that also contain H3K79 methylation, such as the V region exon and Sμ region in immunoglobulin genes

  • This suggests potential cooperative functions in regulating gene expression

• Relationship with transcription elongation marks:

  • H3K36me3, associated with transcription elongation and mismatch repair, shows a distribution pattern increasing toward the 3' portion of gene bodies

  • This distribution pattern may complement H3K79me1 enrichment patterns

  • The combinatorial presence of H3K79me1 and H3K36me3 may signal specific regulatory states during transcription elongation

• Independent regulation mechanisms:

  • Despite co-occurrence patterns, H3K79 methylation is regulated independently from other histone modifications

  • Inhibition of Dot1L does not affect genome-wide levels of H3K4me3 and H3K36me3, confirming the specificity of each modification pathway

  • This suggests that while these marks may function cooperatively, they are established through distinct enzymatic mechanisms

• Functional implications of modification patterns:

  • Different combinations of histone modifications may create specific "chromatin signatures"

  • For example, the combination of H3K79 methylation with H3K4me3 and H3K27ac may mark regions for specific regulatory events or protein complex recruitment

  • In the context of somatic hypermutation, these combinations may define regions susceptible to AID targeting

• Chromatin state transitions:

  • The dynamic interplay between H3K79me1 and other modifications may facilitate transitions between chromatin states

  • During cellular responses to stimuli, coordinated changes in multiple histone modifications including H3K79me1 may drive gene expression changes

How can advanced technologies enhance H3K79me1 research?

Emerging technologies offer powerful new approaches for studying H3K79me1 distribution, dynamics, and function:

• CUT&RUN and CUT&Tag technologies:

  • These techniques provide higher resolution and lower background than traditional ChIP

  • They require fewer cells, enabling analysis of rare cell populations

  • CUT&RUN and CUT&Tag are compatible with H3K79me1 antibodies

  • These methods can reveal fine-scale patterns of H3K79me1 distribution with unprecedented clarity

• Single-cell chromatin profiling:

  • Single-cell adaptations of ChIP-seq, CUT&RUN, or CUT&Tag

  • These approaches can reveal cell-to-cell variability in H3K79me1 patterns

  • Combining with single-cell RNA-seq enables direct correlation between H3K79me1 and gene expression at the single-cell level

  • This is particularly valuable for studying heterogeneous populations during differentiation or disease progression

• Live-cell imaging of H3K79me1:

  • Development of specific nanobodies or mintbodies against H3K79me1

  • These tools enable real-time visualization of H3K79me1 dynamics in living cells

  • Combined with other labeled histone modifications, this approach can reveal temporal relationships between different chromatin marks

• Mass spectrometry-based approaches:

  • Quantitative mass spectrometry to measure global levels of different H3K79 methylation states

  • Proteomics identification of proteins that specifically bind to H3K79me1

  • This can reveal novel readers of this modification and provide mechanistic insights

• CRISPR-based epigenome editing:

  • Targeted manipulation of H3K79me1 at specific genomic loci

  • Could involve tethering Dot1L to specific regions to increase H3K79me1, or targeted recruitment of potential demethylases

  • This approach allows direct testing of H3K79me1 function at specific genes or regulatory elements