A comprehensive guide of Histone H2A relating antibodies

1. A brief of Histone H2A

Histone H2A is one of the five main histone proteins (including Histone H1/H5, Histone H2A, Histone H2B, Histone H3 and Histone H4) involved in the structure of chromatin in eukaryotic cells. Histone H2A consists of approximately 130 amino acid residues, which is a 14kDa protein. So far, there are 265 different members of Histone H2A family. Histone H2A forms a dimer with Histone H2B, and then two H2A/H2B dimer binds to a Histone H3/H4 tetramer to form a histone octamer – this is the core of nucleosomes.

Histone H2A

(Image from Fastbleep.com)

2. Histone H2A structure

H2A consists of a main globular domain and a long N-terminal tail or C-terminal on one end of the molecule. The N-terminal tail or C-terminal tail is the location of post-translational modification. Thus far, researchers have not identified any secondary structures that arise in the tail. H2A utilizes a protein fold known as the ‘histone fold.’ The histone fold is a three-helix core domain that is connected by two loops. This connection forms a ‘handshake arrangement.’ Most notably, this is termed the helix-turn-helix motif, which allows for dimerization with H2B. The ‘histone fold’ is conserved among H2A at the structural level; however the genetic sequence that encodes for this structure differs between variants[1].

3. Histone HA2 Variants

Because core histone proteins have specific protein-protein interactions and protein-DNA interactions, they are subject to different degrees of structural constraint probably resulting in different potentials to evolve variants . [2] Interestingly, Histone H2A has the maximum number of variants among all other core histones (totally 19 variants). These are coded by 26 genes with majority of them present in cluster 1 and 2 and a single gene in cluster 3, notably Histone H2A.1, Histone H2A.2, Histone H2A.X and Histone H2A.Z etc. Out of these, 9 genes are not part of any cluster and code for atypical histone H2A such as macro-H2A, H2A-Bbd etc.

A number of Histone H2A variants were listed below:

http://www.actrec.gov.in/histome/histones.php?histone=H2A

In general, H2A variants function by conferring characteristic properties to chromatin on the nucleosomal and higher-order structural level. The following table is an overview of the influences of H2A variants on nucleosome stability and chromatin structure:

Histone VariantNucleosome stability Chromatin structure
 
OutputRelevant domainsOutputRelevant domains
H2A.XDestabilization-ND-
H2A.ZControversial-CompactionAcidic patch (extended)
Stabilization---
Destabilization---
No or subtle effects---
H2A.Z.2.2DestabilizationDocking domainND-
H2A.BbdDestabilizationDocking domainDecompactionAcidic patch (decreased)
MacroH2AStabilizationL1 loopCompactionH1-like linker (without macro domain)

Variants abundance and chromatin composition during development:

histone h2a variants

In differentiated oocytes, chromatin contains the canonical histone H2A and its variants H2A.X, H2A.Z and macroH2A (mH2A). However, after fertilization, the quantity of H2A in the nucleus decreases, and H2A.Z and macroH2A almost disappear, whereas the amount of H2A.X increases. Thus, chromatin mostly contains H2A.X during the early pre-implantation stage; the embryos are totipotent and genome reprogramming occurs. At the late pre-implantation stage, when differentiation begins to occur, H2A and variant histones reappear in the nucleus, and the chromatin again contains all the histone H2A species[3].

4. Histone H2A modification

Since H2A packages DNA molecules into chromatin, the packaging process will effect gene expression and Histone H2A is correlated with DNA modification and epigenetics. H2A plays a major role in determining the overall structure of chromatin. Inadvertently, H2A has been found to regulate gene expression[4]. Modifications of Histone H2A change the “histone code” of Histone H2A and thus alters the function of Histone H2A. These modifications include serine phosphorylation/acetylation, arginine methylation, lysine methylation/acetylation/ubiquitination. threonine methylation.

HistoneSiteMod.Catalog NumberAntibody name
H2ASTJ96865Rabbit Polyclonal Histone H2A antibody
H2AK15Acetyl STJ97173Rabbit Polyclonal Histone H2A (Acetyl Lys15) antibody
H2AK9AcetylSTJ97172Rabbit Polyclonal Histone H2A (Acetyl Lys9) antibody
H2AK5AcetylSTJ97171Rabbit Polyclonal Histone H2A (Acetyl Lys5) antibody
H2A S129PhosphoSTJ97152Rabbit Polyclonal Histone H2A (Phospho-Ser129) antibody
H2A T121PhosphoSTJ91231Rabbit Polyclonal Phospho-Histone H2A (T121) antibody
(For research use only)

5. Main Histone H2A variants

5.1 Histone H2A.X

H2AX a histone that replaces canonical H2A in a subset of nucleosomes. It is required for checkpoint-mediated arrest of cell cycle progression in response to low doses of ionizing radiation and for efficient repair of DNA double strand breaks (DSBs) specifically when modified by C-terminal phosphorylation.

5.1.1 Phosphorylation

Phosphorylation on Ser-140 (to form gamma-H2AX or H2AX139ph) in response to DNA double strand breaks (DSBs) generated by exogenous genotoxic agents and by stalled replication forks, and may also occur during meiotic recombination events and immunoglobulin class switching in lymphocytes.Phosphorylation can extend up to several thousand nucleosomes from the actual site of the DSB and may mark the surrounding chromatin for recruitment of proteins required for DNA damage signaling and repair[5].

Phosphorylation at Tyr-143 (H2AXY142ph) by BAZ1B/WSTF determines the relative recruitment of either DNA repair or pro-apoptotic factors. It favors the recruitment of APBB1/FE65 and pro-apoptosis factors such as MAPK8/JNK1, triggering apoptosis[6].

5.1.2 Ubiquitination

Monoubiquitination of Lys-120 (H2AXK119ub) by RING1 and RNF2/RING2 complex gives a specific tag for epigenetic transcriptional repression. Following DNA double-strand breaks (DSBs), it is ubiquitinated through ‘Lys-63’ linkage of ubiquitin moieties by the E2 ligase UBE2N and the E3 ligases RNF8 and RNF168, leading to the recruitment of repair proteins to sites of DNA damage[7].

Ubiquitination at Lys-14 and Lys-16 (H2AK13Ub and H2AK15Ub, respectively) in response to DNA damage is initiated by RNF168 that mediates monoubiquitination at these 2 sites[8].

5.1.3 Acetylation

Acetylation at Lys-37 increases in S and G2 phases. This modification has been proposed to play a role in DNA double-strand break repair.

5.2 Histone H2A.Z

Histone H2AZ is a variant of histone H2A, and is used to mediate the thermosensory response, and is essential to perceive the ambient temperature. Nucleosome occupancy of H2A.Z decreases with temperature, and in vitro assays show that H2A.Z-containing nucleosomes wrap DNA more tightly than canonical H2A nucleosomes in Arabidopsis.Recent research suggests that H2AZ is incorporated into the nucleosome using a Swr1, a Swi2/Snf2- related adenosine triphosphatase[9]

Recent evidence also points to a role for H2A.Z in repressing a subset of ncRNAs, derepressing CUTs, as well as mediation higher order chromatin structure formation[10].

HistoneSiteMod.Catalog NumberAntibody name
H2A.XS139phosphoAP0245Mouse Monoclonal Phospho-H2AFX-S139 antibody
H2A.XS139phosphoAP0099Rabbit Polyclonal Phospho-H2AFX-S139 antibody
H2A.XA2082Rabbit Polyclonal H2AFX antibody
H2A.XK5AcetylSTJ97189Rabbit Polyclonal Histone H2A.X (Acetyl Lys5) antibody
H2A.XY142PhosphoSTJ97187Rabbit Polyclonal Histone H2A.X (Phospho-Tyr142) antibody
H2A.X T120PhosphoSTJ97185Rabbit Polyclonal Histone H2A.X (Phospho-Thr120) antibody
H2A.ZA6614Rabbit polyclonal H2AFZ antibody
H2A.ZK4AcetylSTJ97190Rabbit Polyclonal Histone H2A.Z (Acetyl Lys4) antibody
H2A.ZK7AcetylSTJ97174Rabbit Polyclonal Histone H2A.Z (Acetyl Lys7) antibody
H2A-Bbd type 1CSB-PA010091LA01HUAnti-H2AFB1 Antibody
macro-H2A.1A7045Rabbit polyclonal H2AFY antibody
macro-H2A.2CSB-PA010098DSR2HUAnti-H2AFY Antibody

5.2.1 Ubiquitination

Monoubiquitination of Lys-122 gives a specific tag for epigenetic transcriptional repression[11].

5.2.2 Acetylation

Acetylated on Lys-5, Lys-8 and Lys-12 during interphase. Acetylation disappears at mitosis.

5.3.3 Methtylation

Monomethylated on Lys-5 and Lys-8 by SETD6. SETD6 predominantly methylates Lys-8, lys-5 being a possible secondary site[12].

5.3 Histone H2A.Bbd

Histone H2A.Bbd has only been found in mammals and testis-specific, which is encoded by a polyadenylated mRNA. Histone H2A.Bbd is involved in creating a specific chromatin landscape at the promoters of active genes, during spermatogenesis, in a temporally specific manner, where H2A.Z occupies the 2 nucleosome and H2A. However, in contrast with mouse endogenous H2A.Bbd, tagged human H2A.Bbd is depleted at the TSS but enriched over gene bodies(in HeLa cells), suggestion the function of Histone H2A.Bbd in mRNA processing[13].

5.4 Histone macro H2A

MacroH2A is the most divergent from its canonical histone and is the least understood in its function. MacroH2A is expressed in the inactive X chromosome in females. MacroH2A variants are generally considered transcriptionally repressive in nature due to their association with forms of condensed chromatin such as the inactive X chromosome and inactive genes. Studies suggested an involvement of MacroH2A in gene repression and heterochromatinization.

5.4.1 Unbiquitination

Monoubiquitinated at either Lys-116 or Lys-117. May also be polyubiquitinated. Ubiquitination is mediated by the CUL3/SPOP E3 complex and does not promote proteasomal degradation. Instead, it is required for enrichment in inactive X chromosome chromatin[14].

[1] Mariño-Ramírez, L; Jordan, I. K.; Landsman, D (2006). Multiple independent evolutionary solutions to core histone gene regulation. Genome Biology. 7 (12): R122

[2] Malik HS,  Henikoff S. Phylogenomics of the nucleosome, Nat. Struct. Biol. , 2003, vol. 10 (pg. 882-891)

[3] Changes in the nuclear deposition of histone H2A variants during pre-implantation development in mice

[4] Marino-Ramirez L, Jordan IK, Landsman D. Multiple independent evolutionary solutions to core histone gene regulation. Genome Biol. 2006;7:R12

[5] DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139.

[6] WSTF regulates the H2A.X DNA damage response via a novel tyrosine kinase activity.

[7] RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins.

[8] RNF168 ubiquitinates K13-15 on H2A/H2AX to drive DNA Damage signaling.”

[9] Mizuguchi, G; Shen, X; Landry, J; Wu, W. H.; Sen, S; Wu, C (2004). “ATP-driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin remodeling complex”. Science. 303 (5656): 343–8.

[10]  Mayuri Rege, Vidya Subramanian, Chenchen Zhu, Tsung-Han S. Hsieh, Assaf Weiner,Nir Friedman,Sandra Clauder-Mu€nster, Lars M. Steinmetz, Oliver J. Rando,Laurie A. Boyer, and Craig L. Peterson. (2015) Chromatin Dynamics and the RNA Exosome Function in Concert to Regulate Transcriptional Homeostasis

[11]  “Histone ubiquitination: a tagging tail unfolds?” Jason L.J.M., Moore S.C., Lewis J.D., Lindsey G., Ausio J. Bioessays 24:166-174(2002)

[12]  “SETD6 monomethylates H2AZ on lysine 7 and is required for the maintenance of embryonic stem cell self-renewal.” Binda O., Sevilla A., LeRoy G., Lemischka I.R., Garcia B.A., Richard S. Epigenetics 8:177-183(2013)

[13]  Tolstorukov MY,  Goldman JA,  Gilbert C,  Ogryzko V,  Kingston RE,  Park PJ. Histone variant H2A.Bbd is associated with active transcription and mRNA processing in human cells, Mol. Cell. , 2012, vol. 47 (pg. 596-607)

[14]  “Histone variant macroH2A1.2 is mono-ubiquitinated at its histone domain.” Ogawa Y., Ono T., Wakata Y., Okawa K., Tagami H., Shibahara K. Biochem. Biophys. Res. Commun. 336:204-209(2005)