Mono-methyl-H2AFZ (K7) Antibody

What is Mono-methyl-H2AFZ (K7) and why is it significant in epigenetic research?

Mono-methyl-H2AFZ (K7), also known as H2AZK7me1, is a specific post-translational modification where the histone variant H2AZ is monomethylated at lysine 7 in its N-terminal tail. This modification is catalyzed primarily by the lysine methyltransferase SETD6, which has been shown to effectively monomethylate the K7 position of H2AZ in vitro and contribute to H2AZ methylation in vivo . The significance of this modification lies in its role in chromatin regulation, particularly in embryonic stem cells where it appears to function as a marker of cellular differentiation. The H2AZK7me1 mark has been found to co-localize with the repressive H3K27me3 mark and the Polycomb Repressive Complex 2 (PRC2) complex subunits near the transcription start sites of developmental genes in mouse embryonic stem cells (mESCs) . These findings suggest that H2AZK7me1 plays a crucial role in maintaining the self-renewal capabilities of stem cells and regulates gene expression during cellular differentiation.

How does H2AZK7me1 differ from other H2AZ post-translational modifications?

H2AZ is subject to various post-translational modifications that regulate its function in chromatin. While H2AZ is known to be polyacetylated within the N-terminal tail (at residues K4, K7, K11, K13, and K15) as well as monoubiquitylated at the carboxy-terminus (at residues K120 and K121), H2AZ methylation was a novel discovery . The methylation of H2AZ at lysines 4 and 7 likely prevents subsequent acetylation at these sites, thereby potentially impairing the transcriptional functions associated with H2AZ acetylation . Interestingly, mass spectrometry analysis did not detect any acetylation on endogenous H2AZ in the contexts studied, suggesting potential competition between these modifications . Additionally, immunoblotting with a methyl-specific H2AZK7me1 antibody detected a slow-migrating band that, based on its molecular weight, was suggested to be an ubiquitylated form of H2AZ, indicating possible cross-talk between methylation and ubiquitylation of H2AZ .

What methods can be used to detect H2AZK7me1 in biological samples?

Detection of H2AZK7me1 in biological samples can be accomplished through several complementary techniques:

• Immunoblotting: Using a methyl-specific α-H2AZK7me1 antibody, researchers can detect the presence of this modification in protein lysates. This approach has been successfully employed to confirm the in vitro methylation of H2AZ by SETD6 and to detect changes in H2AZK7me1 levels during cellular differentiation .

• Mass Spectrometry (MS): This technique provides definitive identification of the specific methylation sites. MS analyses have successfully detected monomethylation of H2AZ at both K4 and K7 in vitro and in vivo. Detailed MS2 chromatograms can highlight the monomethylation of H2AZ at specific lysine residues with precise molecular masses (e.g., 766.49 for K7 methylation) .

• Chromatin Immunoprecipitation (ChIP): This method allows researchers to examine the genomic distribution of H2AZK7me1, particularly at promoters of interest such as developmental genes. ChIP analyses have revealed the co-localization of H2AZK7me1 with the repressive H3K27me3 mark at the promoters of differentiation marker genes .

• Immunohistochemistry (IHC): While not specifically mentioned for H2AZK7me1 in the provided search results, IHC is commonly used to detect protein expression levels in tissue samples, as demonstrated for H2AFZ in lung adenocarcinoma tissues .

Which enzymes are responsible for the methylation and potential demethylation of H2AZK7?

The primary enzyme responsible for H2AZK7 monomethylation is SETD6, a lysine methyltransferase that has been shown to effectively methylate H2AZ at lysine 7 in vitro and contribute to H2AZ methylation in vivo . In detailed in vitro experiments, SETD6 was demonstrated to preferentially monomethylate H2AZ at K7, although it can also methylate secondary sites such as K4 at lower levels .

Another enzyme, SET7, has also been identified as a potent lysine methyltransferase that can modify H2AZ, although it appears to indiscriminately methylate multiple sites on H2AZ rather than showing the specificity that SETD6 exhibits for K7 .

What is the relationship between H2AZK7me1 and embryonic stem cell differentiation?

H2AZK7me1 has a complex relationship with embryonic stem cell differentiation that appears to be context-dependent:

• Global Increase During Differentiation: Mass spectrometric analysis detected a significant (over 3-fold) increase in H2AZK7me1 and H2AZK4me1K7me1 following mouse embryonic stem cell (mESC) differentiation, whether induced by shRNA-mediated depletion of Nanog or by retinoic acid (RA) treatment .

• Promoter-Specific Dynamics: Intriguingly, while global levels of H2AZK7me1 increased upon differentiation, ChIP analyses revealed decreased levels of both H2AZK7me1 and the repressive H3K27me3 mark at the promoters of differentiation marker genes (Fgf5, FoxA2, and Hand2) upon retinoic acid-induced differentiation .

• Role in Self-Renewal: Depletion of Setd6 (the enzyme that catalyzes H2AZK7 methylation) in mESCs led to cellular differentiation, compromised self-renewal, and reduced clonogenicity, suggesting that Setd6-mediated H2AZ methylation is required for maintaining mESC self-renewal .

These findings suggest that H2AZK7me1 functions as part of an epigenetic program that regulates lineage commitment genes, potentially cooperating with H3K27me3 to maintain repression of differentiation markers in the self-renewal state .

How can researchers validate the specificity of a Mono-methyl-H2AFZ (K7) Antibody?

Validating the specificity of a Mono-methyl-H2AFZ (K7) Antibody requires a multi-faceted approach:

• Mutational Analysis: Generate H2AZ mutants where K7 is converted to an arginine (K7R) or other non-methylatable residue. The antibody should show significantly decreased or no signal with these mutants compared to wild-type H2AZ .

• Peptide Competition Assays: Test the antibody against a panel of synthetic peptides containing modified and unmodified versions of the target site (H2AZK7). The antibody should preferentially recognize the monomethylated K7 peptide over unmodified, di-, or tri-methylated versions .

• Knockdown/Knockout Validation: Deplete H2AZ using shRNA or CRISPR-Cas9 approaches and confirm reduced antibody signal. In the provided search results, the H2AZK7me1 antibody detected a decrease of H2AZ in knockdown cell extracts (shH2AZ) compared to control cells .

• Enzyme Depletion: Knockdown the methyltransferase responsible for the modification (SETD6) and demonstrate reduced antibody signal. Cells depleted of SETD6 by two independent shRNAs showed reduced levels of H2AZK7me1, confirming the antibody's specificity for the SETD6-mediated modification .

• Mass Spectrometry Correlation: Perform mass spectrometry analysis to confirm the presence of monomethylation at K7 in samples that show positive antibody reactivity, as was done in the reported research where MS confirmed the methylation detected by the antibody .

• In Vitro Methylation Assays: Conduct in vitro methylation reactions with recombinant H2AZ and SETD6, then analyze the products by immunoblotting with the methyl-specific antibody. A substantial increase in signal in the presence of both H2AZ and SETD6 would indicate specificity for the enzymatically generated modification .

What controls should be included when using H2AZK7me1 antibodies in ChIP experiments?

When conducting Chromatin Immunoprecipitation (ChIP) experiments with H2AZK7me1 antibodies, the following controls are essential:

How does H2AZK7me1 co-localize with other histone marks, and what does this tell us about its function?

H2AZK7me1 exhibits specific co-localization patterns with other histone modifications that provide insights into its functional role:

• Co-localization with H3K27me3: H2AZK7me1 co-localizes with the repressive H3K27me3 mark near the transcription start sites of developmental genes in mouse embryonic stem cells (mESCs) . This co-occurrence suggests a cooperative role in gene silencing.

• Association with PRC2 Complex: H2AZ, which can be methylated at K7, co-localizes with the PRC2 complex subunits SUZ12 and EED in mESCs near the transcription start sites of developmental genes . This association indicates that H2AZK7me1 may be part of the Polycomb-mediated gene repression machinery.

• Dynamic Changes During Differentiation: Upon retinoic acid-induced cellular differentiation, both H2AZK7me1 and H3K27me3 are concomitantly lost from the promoters of differentiation markers such as Fgf5, FoxA2, and Hand2 . This synchronized removal suggests a coordinated epigenetic program regulating developmental gene expression.

• Mutual Exclusivity with Acetylation: Methylation of H2AZ at K7 likely prevents acetylation at the same residue. Mass spectrometry analysis did not detect acetylation on endogenous H2AZ in the contexts studied, suggesting potential competition between these modifications .

These co-localization patterns suggest that H2AZK7me1 functions in an epigenetic program that, together with H3K27me3 and the PRC2 complex, maintains repression of differentiation-associated genes in the self-renewal state. The loss of both marks during differentiation allows for the activation of these genes and subsequent lineage commitment .

What is known about the biological significance of dual methylation at H2AZK4 and H2AZK7?

The dual methylation of H2AZ at both K4 and K7 (H2AZK4me1K7me1) appears to have specific biological significance:

• Co-occurrence In Vivo: Mass spectrometric analysis detected H2AZK7me1 in vivo exclusively in conjunction with H2AZK4me1, suggesting these modifications may function as a coordinated epigenetic mark .

• Differentiation-Associated Increase: The levels of H2AZK4me1K7me1 increased noticeably (over 3-fold) in response to cellular differentiation induced by either retinoic acid or the silencing of Nanog expression . This suggests a potential role in the differentiation process.

• Enzymatic Specificity: While SETD6 preferentially monomethylates H2AZ at K7, it can also methylate K4 at lower levels. Mass spectrometry detected low levels of simultaneous methylation on both K4 and K7 in in vitro reactions . This suggests that a single enzyme might coordinate the dual methylation pattern.

• Potential Functional Synergy: The consistent co-occurrence of these modifications suggests they may work synergistically rather than independently. Their coordinated increase during differentiation implies a functional role in this process .

• Distinction from Single Methylation: The dual methylation mark may have distinct functions from single methylation at either site. The research indicates that, while global levels of dual methylation increase during differentiation, locus-specific dynamics (such as at developmental gene promoters) may differ .

While the exact mechanism by which H2AZK4me1K7me1 influences gene expression remains to be fully elucidated, its differentiation-associated increase suggests it may be involved in the regulation of cell lineage commitment and developmental programs .

How do experimental approaches to studying H2AZK7me1 differ between embryonic stem cells and cancer contexts?

Experimental approaches to studying H2AZK7me1 differ significantly between embryonic stem cell and cancer research contexts:

In Embryonic Stem Cells:

• Differentiation Models: Research utilizes controlled differentiation systems such as retinoic acid treatment or shRNA-mediated depletion of pluripotency factors like Nanog to study dynamic changes in H2AZK7me1 during lineage commitment .

• Functional Assays: Alkaline phosphatase staining, clonogenic assays, and self-renewal competition assays are employed to assess the functional importance of H2AZK7me1 and its methyltransferase SETD6 in maintaining pluripotency .

• Developmental Gene Focus: ChIP analyses typically target the promoters of developmental and differentiation marker genes (e.g., Fgf5, FoxA2, and Hand2) to understand how H2AZK7me1 regulates lineage-specific expression .

In Cancer Research:

While some methodologies overlap (such as immunohistochemistry and expression analysis), the research questions and biological systems differ substantially. Stem cell research focuses on developmental transitions and differentiation processes, while cancer research emphasizes prognostic value, tumor biology, and potential therapeutic applications .

What technical challenges exist in distinguishing between H2AZK7me1 and other similar histone modifications in experimental systems?

Distinguishing H2AZK7me1 from other similar histone modifications presents several technical challenges:

• Antibody Cross-Reactivity: Generating antibodies that specifically recognize H2AZK7me1 without cross-reacting with unmethylated H2AZ, H2AZK4me1, or H2AZ acetylated at K7 requires extensive validation. The sequence surrounding K7 in H2AZ shares similarities with other histones, potentially leading to non-specific binding .

• Co-occurrence of Modifications: H2AZK7me1 has been found to co-occur with H2AZK4me1 in vivo, making it difficult to attribute biological effects specifically to K7 methylation versus K4 methylation or the dual modification . Mass spectrometry detected H2AZK7me1 exclusively in conjunction with H2AZK4me1, suggesting these modifications may function as a coordinated epigenetic mark .

• Low Abundance: Modified histones often exist at relatively low abundance compared to their unmodified counterparts, making detection challenging. This is especially true for site-specific methylation like H2AZK7me1, which may require highly sensitive detection methods .

• Similar Molecular Weights: Monomethylation adds only 14 Da to the molecular weight of a protein, making it difficult to distinguish methylated from unmethylated forms by gel electrophoresis alone. Additionally, different methylation sites (e.g., K4 vs. K7) are indistinguishable by simple molecular weight analysis .

• Mass Spectrometry Resolution: Even with high-resolution mass spectrometry, distinguishing between isomeric peptides with methylation at different lysine residues can be challenging and requires careful MS/MS fragmentation analysis . The research demonstrated this by using detailed MS2 chromatogram analysis to identify specific methylation sites .

• Mutational Approach Limitations: Site-directed mutagenesis (e.g., K7R) can help assign functional effects to specific lysine residues, but changing an amino acid can alter protein structure and function independently of the loss of methylation, complicating interpretation .

• Enzyme Specificity: While SETD6 preferentially methylates K7, it can also methylate K4 at lower levels. Similarly, SET7 can methylate multiple sites on H2AZ. This lack of absolute enzyme specificity makes it difficult to manipulate just one modification experimentally .

Addressing these challenges requires integrating multiple approaches, including site-specific antibodies, mass spectrometry, enzyme knockdown/knockout experiments, and careful controls to interpret the specific contribution of H2AZK7me1 to biological processes .

How can researchers optimize mass spectrometry protocols for detecting low abundance H2AZK7me1 in complex biological samples?

Optimizing mass spectrometry protocols for detecting low abundance H2AZK7me1 requires several specialized approaches:

• Enrichment of Histone Fractions: Begin with acid extraction of histones from nuclei to enrich for all histone proteins before further processing. This reduces sample complexity and increases the relative abundance of histone modifications .

• Targeted Immunoprecipitation: Use H2AZ-specific antibodies to enrich for the histone variant before mass spectrometry analysis. This can be followed by a second enrichment using methylation-specific antibodies if available .

• Chemical Derivatization: Modify histones with propionic anhydride before and after trypsin digestion to convert lysines to propionyl-lysines. This prevents trypsin from cleaving after every lysine and creates larger, more readily identifiable peptide fragments containing the modifications of interest .

• Sample Fractionation: Implement strong cation exchange (SCX) or hydrophilic interaction liquid chromatography (HILIC) fractionation prior to LC-MS/MS analysis to separate peptides based on charge or hydrophilicity, reducing sample complexity .

• Multiple Reaction Monitoring (MRM): Develop targeted MRM assays specific for H2AZK7me1-containing peptides. This approach increases sensitivity by monitoring specific precursor-to-fragment ion transitions characteristic of the modified peptide .

• Parallel Reaction Monitoring (PRM): Utilize high-resolution mass spectrometers with PRM capabilities to monitor all fragment ions from a selected precursor ion, increasing specificity and sensitivity for H2AZK7me1 detection .

• Internal Standards: Include isotopically labeled synthetic peptides corresponding to the H2AZK7me1 modification as internal standards for accurate quantification. The research used biotinylated peptides with specific modifications (mono-, di-, or tri-methylated H2AZK7) for in vitro methylation assays that could be adapted for MS quantification .

• Enhanced Chromatographic Separation: Optimize liquid chromatography parameters, including extended gradient times and specialized columns, to improve separation of isobaric peptides that differ only in the position of methylation (e.g., K4me1 vs. K7me1) .

• MS/MS Fragmentation Optimization: Fine-tune collision energies and fragmentation methods (CID, HCD, ETD) to maximize diagnostic fragment ions that distinguish between methylation at different sites. The research demonstrated this by using detailed MS2 chromatogram analysis to identify specific methylation sites with distinct molecular masses (e.g., 766.49 for K7 methylation) .

• Data Analysis Refinement: Employ specialized software and manual validation to accurately assign methylation sites, particularly when analyzing complex mixtures of modified peptides with similar masses and retention times .

By combining these approaches, researchers can significantly improve the detection sensitivity and quantification accuracy of low abundance H2AZK7me1 in complex biological samples.

What are the current hypotheses regarding the molecular mechanisms by which H2AZK7me1 influences gene expression?

Several hypotheses have been proposed regarding the molecular mechanisms by which H2AZK7me1 influences gene expression:

• Cooperative Repression with H3K27me3: H2AZK7me1 co-localizes with the repressive H3K27me3 mark near the transcription start sites of developmental genes in mESCs . This co-occurrence suggests these marks may cooperatively repress gene expression, potentially through recruitment of similar or coordinated repressive complexes.

• Inhibition of Activating Modifications: Methylation of H2AZ at K7 likely prevents acetylation at the same residue. Since H2AZ acetylation is associated with transcriptional activation, H2AZK7me1 may repress gene expression by blocking these activating marks . The research noted that "methylation of H2AZ at lysines 4 and 7 could prevent subsequent acetylation at these sites, thereby impairing the reported transcriptional functions associated with the acetylation of H2AZ" .

• Recruitment of Specific Reader Proteins: H2AZK7me1 may serve as a binding site for specific "reader" proteins that recognize this modification and subsequently recruit additional factors involved in transcriptional repression. The research suggests that "the eventual identification of H2AZ- or H2AZK7me1-specific readers will undoubtedly help concretize the molecular mechanism of chromatin signaling by H2AZK7me1" .

• Nucleosome Stability Regulation: The methylation of H2AZ may alter nucleosome stability or positioning, affecting the accessibility of DNA to transcription factors and the transcriptional machinery. This is supported by H2AZ's known role in PRC2 chromatin association .

• Crosstalk with Histone Ubiquitylation: The observation of a slow-migrating band detected by the H2AZK7me1 antibody suggests potential cross-talk between methylation and ubiquitylation of H2AZ . This interaction could create complex regulatory circuits controlling gene expression.

• Developmental Gene Regulation Program: The loss of both H2AZK7me1 and H3K27me3 from developmental gene promoters upon cellular differentiation suggests these marks function together in an epigenetic program that regulates expression of lineage commitment genes . The research states that "the loss of both H2AZK7me1 and H3K27me3 from Fgf5 upon cellular differentiation implies that H2AZK7me1 could be involved with H3K27me3 in an epigenetic program that regulates the expression of lineage commitment genes" .

• Context-Dependent Effects: While global levels of H2AZK7me1 increase during differentiation, the mark is specifically lost from certain developmental gene promoters, suggesting its effects on gene expression may be highly context-dependent and influenced by local chromatin environment .

These hypotheses are not mutually exclusive, and the actual mechanism likely involves multiple modes of action depending on the genomic context and cellular state. Further research, particularly the identification of specific H2AZK7me1 readers, is needed to fully elucidate these mechanisms .

What are the most promising approaches for identifying the readers and erasers of the H2AZK7me1 mark?

Identifying the readers and erasers of the H2AZK7me1 mark requires specialized techniques that can capture proteins specifically interacting with this modification. The most promising approaches include:

• Modified Peptide Pull-downs: Synthesize biotinylated peptides corresponding to the N-terminal tail of H2AZ with and without K7 methylation. Use these as baits to capture proteins from nuclear extracts that specifically bind to the methylated form. The research already utilized biotinylated and mono-, di-, or tri-methylated H2AZK7 peptides for in vitro studies , which could be adapted for pull-down experiments.

• CRISPR-based Proximity Labeling: Combine CRISPR-mediated targeting with proximity labeling enzymes (BioID, APEX2) fused to reader domains known to bind methylated lysines. Target these constructs to genomic regions enriched for H2AZK7me1 to identify proteins in proximity to this mark in living cells.

• Chemical Crosslinking Mass Spectrometry (XLMS): Use crosslinking agents to capture transient interactions between H2AZK7me1 and its binding partners, followed by mass spectrometry identification. This approach can preserve weak or dynamic interactions that might be lost in standard pull-down assays.

• Quantitative Interaction Proteomics: Compare the interactomes of wild-type H2AZ versus K7R mutant (cannot be methylated) or K7Q mutant (mimics acetylation) to identify differential binding partners dependent on the K7 methylation state.

• Domain-focused Protein Arrays: Screen libraries of known chromatin-associated domains (e.g., PHD fingers, chromodomains, Tudor domains) for specific binding to H2AZK7me1 peptides. These domains are common readers of histone methylation marks.

• Enzymatic Activity Assays for Erasers: Develop in vitro demethylation assays using purified H2AZK7me1 substrates and candidate lysine demethylases. The research noted that "whether H2AZK7me1 is demethylated by a demethylase or evicted by a histone exchange complex remains to be uncovered" , highlighting the need for such assays.

• Genomic Screens for Erasers: Perform CRISPR screens to identify enzymes whose loss leads to increased H2AZK7me1 levels, potentially indicating a role in removing this mark.

• Histone Dynamics Studies: Use techniques like SNAP-tag labeling or FRAP (Fluorescence Recovery After Photobleaching) to study the dynamics of H2AZK7me1-containing nucleosomes, potentially revealing mechanisms of histone exchange that might function alongside enzymatic demethylation.

• Candidate Approach Based on Known Methylation Readers/Erasers: Test known methyl-lysine readers (e.g., members of the MBT, PHD, Tudor protein families) and erasers (e.g., LSD1/KDM1A, JmjC domain proteins) for their ability to recognize or remove H2AZK7me1.

• Differential Gene Expression in SETD6-depleted Cells: Analyze gene expression changes in cells depleted of SETD6 to identify potential regulatory pathways affected by H2AZK7me1 loss, providing clues to proteins that might function downstream of this mark .

The research highlights that "the eventual identification of H2AZ- or H2AZK7me1-specific readers will undoubtedly help concretize the molecular mechanism of chromatin signaling by H2AZK7me1" , underscoring the importance of these approaches for understanding the complete regulatory circuit of this epigenetic modification.

How might researchers integrate H2AZK7me1 ChIP-seq data with other epigenomic datasets to gain comprehensive insights into its regulatory functions?

Integrating H2AZK7me1 ChIP-seq data with other epigenomic datasets requires sophisticated computational approaches to reveal comprehensive insights into its regulatory functions:

• Multi-mark Co-occupancy Analysis: Compare the genome-wide distribution of H2AZK7me1 with other histone modifications, particularly H3K27me3 which co-localizes with H2AZK7me1 at developmental gene promoters . Identify regions of overlap and exclusivity to map the combinatorial chromatin landscape.

• Chromatin State Segmentation: Apply algorithms like ChromHMM or Segway to integrate H2AZK7me1 data with other histone modifications, creating a comprehensive chromatin state map that defines functional genomic elements (e.g., poised promoters, repressed enhancers).

• Transcription Factor Binding Correlation: Integrate with transcription factor ChIP-seq datasets to identify factors that preferentially associate with or are excluded from H2AZK7me1-marked regions, potentially revealing functional interactions.

• RNA Expression Correlation: Combine with RNA-seq data to establish relationships between H2AZK7me1 occupancy and gene expression levels across different cellular states, particularly during differentiation when H2AZK7me1 levels change dynamically .

• Chromatin Accessibility Integration: Overlay with ATAC-seq or DNase-seq data to determine how H2AZK7me1 correlates with chromatin accessibility and nucleosome positioning, especially at developmental gene promoters.

• Three-dimensional Chromatin Organization: Integrate with Hi-C or other chromosome conformation capture datasets to examine how H2AZK7me1-marked regions participate in higher-order chromatin structures and long-range interactions.

• Methyltransferase Dependency Networks: Compare H2AZK7me1 ChIP-seq profiles between wild-type and SETD6-depleted cells to identify regions most dependent on SETD6 activity, potentially highlighting direct versus indirect regulation .

• Differentiation Dynamics Analysis: Perform time-course ChIP-seq during cellular differentiation to track the dynamic changes in H2AZK7me1 occupancy at specific genomic loci, correlating these changes with other epigenetic marks and gene expression changes .

• PRC2 Complex Co-occupancy: Integrate with ChIP-seq data for PRC2 complex components (e.g., SUZ12, EED) which co-localize with H2AZ at developmental gene promoters to examine their relationship with H2AZK7me1 specifically .

• Motif Enrichment Analysis: Identify DNA sequence motifs enriched at H2AZK7me1 sites to predict transcription factors that might influence H2AZK7me1 deposition or function.

• Cell Type-Specific Comparisons: Compare H2AZK7me1 profiles across different cell types, particularly contrasting pluripotent stem cells with differentiated lineages, to identify cell type-specific functions .

• Pathway and Ontology Enrichment: Analyze the biological pathways and gene ontology terms associated with H2AZK7me1-marked genes to understand the broader biological processes regulated by this modification.

The research suggests that "genome-wide studies, such as H2AZ and H2AZK7me1 ChIPseq, may reveal more precisely their biological functions" , highlighting the value of these integrative approaches. Such comprehensive analyses would provide a systems-level understanding of how H2AZK7me1 functions within the broader epigenetic landscape to regulate gene expression and cellular identity.