Recombinant Human Histone H2A type 2-A (H2AC18; H2AC19)

What is Recombinant Human Histone H2A type 2-A (H2AC18; H2AC19)?

Recombinant Human Histone H2A type 2-A (H2AC18; H2AC19), also known as HIST2H2AA3, is a member of the histone H2A family, which constitutes one of the core histone proteins involved in chromatin structure. This recombinant protein is typically produced in yeast expression systems and represents the full-length mature protein (amino acids 2-130). It has a UniProt accession number of Q6FI13 and is also referred to as Histone H2A.2 or Histone H2A/o in some literature . As a recombinant protein, it serves as a valuable research tool for in vitro nucleosome assembly, chromatin structure studies, and as standards in biochemical assays.

What are the key structural features of Histone H2A type 2-A?

Histone H2A type 2-A consists of 130 amino acids with the sequence "SGRGKQGGK ARAKAKSRSS RAGLQFPVGR VHRLLRKGNY AERVGAGAPV YMAAVLEYLT AEILELAGNA ARDNKKTRII PRHLQLAIRN DEELNKLLGK VTIAQGGVLP NIQAVLLPKK TESHHKAKGK" . Its structure includes several functional domains that contribute to nucleosome formation and chromatin dynamics:

• N-terminal tail: Rich in basic amino acids (lysine and arginine), serving as targets for various post-translational modifications

• Central globular domain: Participates in histone-histone interactions within the nucleosome

• C-terminal domain: Extends from the nucleosome core and can interact with linker DNA and regulatory proteins

The protein contains multiple lysine residues that can be acetylated, ubiquitylated, or otherwise modified to influence chromatin structure and function .

How does Histone H2A type 2-A differ from other H2A variants?

Histone H2A type 2-A belongs to the diverse H2A family, which is the most heterogeneous family of histones . The key differences include:

H2A variants are encoded by paralogous genes with sequences that are highly conserved with canonical H2A but differ particularly in their N- and C-termini . These differences contribute to specialized functions in processes such as transcription regulation, DNA repair, and chromosome structure.

What are the optimal conditions for handling and storing Recombinant Human Histone H2A type 2-A?

For optimal handling and storage of Recombinant Human Histone H2A type 2-A in research settings, observe the following guidelines:

Storage Conditions:

• Lyophilized form: Store at -20°C/-80°C for up to 12 months

• Liquid form: Store at -20°C/-80°C for up to 6 months

• Working aliquots: Store at 4°C for up to one week

Reconstitution Protocol:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is typically recommended)

• Prepare small aliquots for long-term storage to avoid repeated freeze-thaw cycles

Buffer Considerations:

• The protein is typically lyophilized from Tris/PBS-based buffer with 6% Trehalose at pH 8.0

• For functional studies, choose buffers appropriate for nucleosome reconstitution or specific enzymatic assays

Important Precautions:

• Avoid repeated freezing and thawing as this can lead to protein denaturation

• Verify protein integrity by SDS-PAGE before use in critical experiments

• Consider adding protease inhibitors when working with the protein in solution for extended periods

What methods can be used to detect Histone H2A type 2-A in cellular samples?

Several methods can be employed to detect Histone H2A type 2-A in cellular samples, each with specific advantages:

Western Blotting:

• Most common approach for protein detection

• Requires specific antibodies against H2A type 2-A

• Can distinguish between modified and unmodified forms using modification-specific antibodies

• Sample preparation typically involves acid extraction of histones from nuclei

Immunofluorescence:

• Enables visualization of H2A type 2-A distribution within the nucleus

• Can be combined with other markers to study colocalization

• Requires careful fixation to preserve nuclear architecture

Chromatin Immunoprecipitation (ChIP):

• Identifies genomic regions where H2A type 2-A is incorporated

• Can be coupled with sequencing (ChIP-seq) for genome-wide analysis

• Requires highly specific antibodies and careful optimization

Mass Spectrometry:

• Provides comprehensive analysis of histone variants and their modifications

• Sequential ion-ion reaction and top-down tandem MS-MS approaches have been used to distinguish between highly similar H2A variants

• Can detect and quantify post-translational modifications

Flow Cytometry:

• Allows quantification of H2A type 2-A levels in individual cells

• Can be combined with cell cycle analysis

• Requires permeabilization protocols optimized for nuclear proteins

How can researchers distinguish between H2A type 2-A and other H2A variants experimentally?

Distinguishing between highly similar H2A variants presents technical challenges requiring sophisticated approaches:

Antibody-Based Strategies:

• Use monoclonal antibodies targeting unique epitopes of H2A type 2-A

• Validate antibody specificity using recombinant proteins and knockout controls

• For western blotting, utilize high-percentage or specialized SDS-PAGE systems to resolve slight molecular weight differences

Mass Spectrometry Approaches:

• Top-down MS-MS approaches can differentiate proteins differing by only a few amino acids

• Sequential ion-ion reaction techniques have successfully identified distinct H2A.Z isoforms that differ by only 3-6 amino acids

• Sample preparation should include histone enrichment steps for optimal results

Genetic Approaches:

• Expression of tagged variants (FLAG, HA, or GFP)

• CRISPR/Cas9-mediated endogenous gene tagging

• Variant-specific knockdown/knockout followed by rescue experiments

Genomic Profiling:

• Variant-specific ChIP-seq using validated antibodies

• RNA-seq to distinguish expression of different variant-encoding genes

• CUT&RUN or CUT&Tag for higher resolution chromatin profiling

The most reliable results typically come from integrating multiple approaches to overcome the limitations of each individual method.

What experimental approaches are recommended for studying H2A type 2-A incorporation into nucleosomes?

Studying H2A type 2-A incorporation into nucleosomes requires approaches spanning from biochemical methods to genomic techniques:

In Vitro Nucleosome Reconstitution:

• Assemble nucleosomes using recombinant H2A type 2-A, other core histones, and DNA fragments

• Analyze by native gel electrophoresis, sucrose gradient ultracentrifugation, or electron microscopy

• Compare stability and structural properties with nucleosomes containing other H2A variants

Fluorescence-Based Approaches:

• FRAP (Fluorescence Recovery After Photobleaching) with tagged H2A type 2-A to study dynamic incorporation

• FRET (Förster Resonance Energy Transfer) to examine interactions with other nucleosomal proteins

• Single-molecule techniques to observe real-time incorporation events

Chromatin Profiling:

• ChIP-seq with H2A type 2-A specific antibodies to map genomic locations

• Sequential ChIP to identify co-occupancy with other variants or modifications

• CUT&RUN or CUT&Tag for higher resolution profiling with lower background

Biochemical Fractionation:

• Salt fractionation of chromatin to separate regions with different stability

• Sucrose gradient ultracentrifugation to isolate nucleosome populations

• Combine with proteomics to identify associated factors

Genetic Manipulation:

• CRISPR/Cas9-mediated tagging or mutation of endogenous H2A type 2-A

• Inducible expression systems to study dynamics of incorporation

• Functional complementation studies comparing different H2A variants

How does the expression of H2A type 2-A vary across different cell types and developmental stages?

The expression of H2A type 2-A varies across cellular contexts in important ways:

Developmental Regulation:

• As a canonical histone, H2A type 2-A expression is typically coupled to DNA replication and highly expressed in proliferating cells

• Expression patterns shift during differentiation

• The related H2A.Z variant has been shown to be required for early mammalian development

Tissue-Specific Patterns:

• Different tissues express distinct profiles of H2A variants

• Expression levels can be assessed through tissue-specific RNA-seq and proteomics

• In plants like Arabidopsis, certain H2A.Z-related genes show tissue-specific expression patterns

Cell Cycle Regulation:

• As a replication-dependent histone, H2A type 2-A expression typically peaks during S phase

• This contrasts with variants like H2A.Z, which are expressed throughout the cell cycle (replication-independent)

Methodological Approaches for Expression Analysis:

What is the role of Histone H2A type 2-A in DNA damage repair pathways?

While specific information about H2A type 2-A in DNA damage repair is limited in the available literature, its potential roles can be inferred from what is known about the H2A family:

Potential Functions in DNA Damage Response:

• Serves as the substrate that can be exchanged for specialized variants like H2A.X during DNA damage response

• May undergo similar post-translational modifications as other H2A variants in response to damage

• Influences chromatin accessibility at damage sites, affecting repair factor recruitment

Comparison with H2A.X Pathway:

• H2A.X becomes phosphorylated at serine 139 (forming γH2A.X) in response to double-strand breaks (DSBs)

• This phosphorylation is mediated by kinases ATM, ATR, and DNA-PKcs and serves as the first DSB-induced histone mark

• While H2A.X phosphorylation is not essential for initial recruitment of repair factors, its absence decreases recruitment of homologous recombination factors like BRCA1 and RAD51

Ubiquitylation Cascade:

• H2A family members, including canonical H2A, can be ubiquitylated at lysines 13 and 15 by RNF168 following DSB detection

• This modification is part of a signaling cascade initiated by ATM phosphorylation of H2A.X, followed by MDC1 binding, recruitment of RNF8, and finally RNF168-mediated ubiquitylation

• The ubiquitylation creates a binding platform for 53BP1, promoting non-homologous end joining (NHEJ) repair

How do post-translational modifications of H2A type 2-A influence its function?

Post-translational modifications (PTMs) of H2A family members significantly impact their functions in chromatin regulation:

Key Modifications and Their Functions:

Modification Crosstalk:

• The methylation state of histone H4K20 interacts with H2A modifications to influence repair pathway choice

• The pattern of modifications creates a "histone code" that directs recruitment of specific factors

• Modifications can be mutually exclusive (e.g., acetylation vs. ubiquitylation of the same lysine)

Methodological Approaches:

• Modification-specific antibodies for western blotting, ChIP, or immunofluorescence

• Mass spectrometry to identify and quantify modifications

• In vitro assays with modified histones to study effects on nucleosome structure

• CRISPR/Cas9-mediated mutation of modification sites to study functional impacts

What are the current challenges in studying specific functions of H2A type 2-A?

Studying H2A type 2-A presents several technical and conceptual challenges:

Sequence Similarity Issues:

• H2A variants share high sequence homology, making it difficult to generate truly specific antibodies

• Even minor differences in amino acid sequence can have significant functional consequences

• Top-down mass spectrometry approaches have been necessary to distinguish between similar variants like H2A.Z-1 and H2A.Z-2 that differ by only 3-6 amino acids

Redundancy and Compensation:

• Functional redundancy between H2A variants can mask phenotypes in single-variant manipulation studies

• In plants like Arabidopsis, single H2A.Z variant knockouts show limited phenotypes due to redundancy

• Compensatory upregulation of other variants may occur when one variant is depleted

Context-Dependent Functions:

• The same H2A variant can have different or even opposing functions depending on genomic context

• H2A.Z has been associated with both activation and repression of transcription in different contexts

• This context-dependency complicates interpretation of experimental results

Technical Limitations:

• Difficulty in specifically detecting or manipulating individual variants in vivo

• Challenges in distinguishing direct from indirect effects in functional studies

• Limited availability of variant-specific tools and reagents

How can researchers effectively study interactions between H2A type 2-A and chromatin remodeling factors?

Studying interactions between H2A type 2-A and chromatin remodeling factors requires a multi-faceted approach:

In Vitro Interaction Assays:

• Pull-down assays using recombinant H2A type 2-A as bait

• Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to measure binding kinetics

• In vitro reconstituted nucleosomes containing H2A type 2-A for remodeling assays

Cell-Based Interaction Studies:

• Co-immunoprecipitation (Co-IP) to identify endogenous interacting partners

• Proximity ligation assay (PLA) to visualize interactions in situ

• FRET/BRET approaches with fluorescently tagged proteins to study dynamics

• BioID or APEX2 proximity labeling to identify nearby proteins in living cells

Genomic Approaches:

• ChIP-seq for H2A type 2-A and chromatin remodelers to identify co-occupancy

• Sequential ChIP to directly demonstrate co-occupancy at specific loci

• ATAC-seq or DNase-seq to correlate H2A type 2-A presence with chromatin accessibility

Functional Interaction Assays:

• Genetic interaction screens (e.g., synthetic lethality, suppressor screens)

• Combine knockdown/knockout of H2A type 2-A with manipulation of remodeler activity

• In vitro nucleosome remodeling assays with reconstituted nucleosomes

Structural Approaches:

• Cryo-EM or X-ray crystallography of nucleosomes containing H2A type 2-A in complex with remodeling factors

• Molecular dynamics simulations to predict interaction interfaces

• Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

What are the implications of H2A type 2-A dysregulation in disease models?

Dysregulation of histone variants has been implicated in various pathological conditions:

Cancer Connections:

• Altered histone variant expression patterns are frequently observed in cancer

• H2A.X null mice show increased chromosomal aberrations and higher risk of tumor development

• Dysregulation of H2A variants affects genomic stability and DNA repair efficiency

Neurodevelopmental and Neurodegenerative Disorders:

• Proper chromatin regulation is essential for neural development and function

• Mutations in chromatin regulators that interact with H2A variants are associated with neurodevelopmental disorders

• Changes in histone variant composition affect neuronal gene expression patterns

Immunological Disorders:

• Defects in histone H2A-related pathways are associated with immunodeficiencies

• RIDDLE syndrome, linked to mutations in RNF168 (which ubiquitylates H2A), is characterized by immunodeficiency and increased cancer risk

Developmental Impacts:

• H2A.Z is required for early mammalian development

• Improper expression or regulation of H2A variants during development leads to developmental abnormalities

• In Drosophila, the H2Av variant (a hybrid of H2A.Z and H2A.X) is indispensable for survival

Research Approaches for Disease Studies:

• Analysis of H2A type 2-A expression, localization, and modifications in patient samples

• Development of disease models with altered H2A type 2-A expression

• Therapeutic approaches targeting enzymes that modify H2A type 2-A

• Correlation of genetic variants in H2A type 2-A or its regulators with disease phenotypes