Histone H3


Table of contents

Assembly of histone H3 into nascent chromatin

During the organization and acetylation of nascent histones prior to their stable incorporation into chromatin two somatic non-nucleosomal histone complexes are detected: one containing nascent H3 and H4, and a second containing H2A (and probably H2B) in association with the nonhistone protein NAP-1. The H3/H4 complex has a sedimentation coefficient of 5-6S, consistent with the presence of one or more escort proteins. H4 in the cytosolic H3/H4 complex is diacetylated, fully in accord with the acetylation state of newly synthesized H4 in chromatin. The diacetylation of nascent human H4 is therefore completed prior to nucleosome assembly. As part of the study of the nascent H3/H4 complex, the cytoplasmic histone acetyltransferase most likely responsible for acetylating newly synthesized H4 was also investigated. HeLa histone acetyltransferase B (HAT B) acetylates H4 but not H3 in vitro, and maximally diacetylates H4 even in the presence of sodium butyrate. Human HAT B acetylates H4 exclusively on the lysine residues at positions 5 and 12, in complete agreement with the highly conserved acetylation pattern of nascent nucleosomal H4, and has a native molecular weight of approximately 100 kDa. Based on these findings a model has been presented for the involvement of histone acetylation and NAP-1 in H2A/H2B deposition and exchange, during nucleosome assembly and chromatin remodeling in vivo (Chang, 1997).

Structural basis for the histone chaperone activity of Asf1

Anti-silencing function 1 (see Drosophila Asf1) is a highly conserved chaperone of histones H3/H4 that assembles or disassembles chromatin during transcription, replication, and repair. The structure of the globular domain of Asf1 bound to H3/H4 determined by X-ray crystallography to a resolution of 1.7 Å shows how Asf1 binds the H3/H4 heterodimer, enveloping the C terminus of histone H3 and physically blocking formation of the H3/H4 heterotetramer. Unexpectedly, the C terminus of histone H4 that forms a mini-β sheet with histone H2A in the nucleosome undergoes a major conformational change upon binding to Asf1 and adds a β strand to the Asf1 β sheet sandwich. Interactions with both H3 and H4 are required for Asf1 histone chaperone function in vivo and in vitro. The Asf1-H3/H4 structure suggests a 'strand-capture' mechanism whereby the H4 tail acts as a lever to facilitate chromatin disassembly/assembly that may be used ubiquitously by histone chaperones (English, 2006).

The ubiquitous function of Asf1 in eukaryotes is highlighted by the sequence conservation of the residues involved in the interactions between Asf1 and histones H3/H4. Budding yeast Asf1 is 56% identical to Xenopus Asf1 in the conserved core, and the Xenopus histones are 88% and 92% identical to H3 and H4 from yeast, respectively. Only the following three residues of Xenopus H3 that contact Asf1 differ in other species: C110 (Ala in yH3), Q125 (Lys in yH3), and I130 (Leu in yH3); these substitutions would appear to cause only minor and possibly compensated differences in interprotein packing. Furthermore, none of these interspecies differences occur in residues of H4 that contact Asf1. Therefore, the interactions observed in this structure will likely be applicable to Asf1-histone H3/H4 complexes from different species (English, 2006).

The Asf1 histone chaperone forms extensive contacts with both histones H3 and H4. The Asf1-H3/H4 structure reveals the details of the interface between Asf1 and a3 of H3 and has identified a new interaction between Asf1 and a2 of H3. The implications of the mutagenesis study, with regard to Asf1 and H3, are that disruption of this intricate interface has severe consequences in the context of the cellular activity. For example, mutations in the regions of Asf1 that bind to only H3 (R145E/S48R, Y112A/R145E, V94R, and S48R) or the region of H3 that binds to Asf1 (K115 and K122) weakened the interaction between Asf1 and H3 and disrupted Asf1 function in vivo and in vitro. As such, the interaction between histone H3 and Asf1 is clearly critical for its cellular functions (English, 2006).

The Asf1-H3/H4 structure shows extensive contacts between Asf1 and histone H4. This interface has two parts: (1) the globular core of Asf1 interacts with the C-terminal tail of H4 to form a strand-swapped dimer and (2) the C-terminal tail of Asf1 binds to the histone-fold region (a3) of histone H4. These interactions are also important because mutations in residues of Asf1 that contact H4 (T147, L6, V109, and V146) weaken histone binding and alter the functions of Asf1 in yeast. Similarly, mutation of histone H4 residues R92, H75, Y72, Y88, and F100 that contact Asf1 in the Asf1-H3/H4 structure reduces the chromatin assembly and/or disassembly functions of Asf1 in vivo. Clearly, interactions of Asf1 with both histones H3 and H4 are required for Asf1 function, and neither interaction is sufficient (English, 2006).

The mutations that affect the interaction between Asf1 and H3/H4 fall into two distinct functional classes; (1) those that reduce the function of Asf1 and (2) those that cause a gain-of-function phenotype. The former was expected, but the latter uncovered specific mutations that overcome the requirement for CAF-1 in transcriptional silencing. These include Asf1 S48R, V109M, Y112E, and V146L that weaken the interaction with histones H3/H4 in vivo and in vitro. Interestingly, the histone H4 H75Y mutation that had reduced Asf1-mediated chromatin-disassembly activity and Zeocin sensitivity has also been shown to bypass the requirement for CAF-1 in silencing. The same ability to bypass the requirement for CAF-1 in silencing has been demonstrated by truncations or insertion mutations in the C terminus of Asf1. Specifically, inactivation of CAF-1 leads to reduced histone deposition onto DNA, while additional mutations in the C terminus of Asf1 restores the histone deposition onto DNA. Although the C terminus of Asf1 is not present in the determined structure, it may extend toward histone H4 from its current location in the structure and may contribute further to histone binding affinity. It is possible that the Asf1 L6M, S48R, V109M, Y112E, V146L, and T147E mutations enhance transcriptional silencing by the same mechanism as the C-terminal mutations in Asf1 (English, 2006).

Acetylated lysine 56 on Histone H3 drives chromatin assembly after repair

DNA damage causes checkpoint activation leading to cell cycle arrest and repair, during which the chromatin structure is disrupted. The mechanisms whereby chromatin structure and cell cycle progression are restored after DNA repair are largely unknown. Chromatin reassembly following double-strand break (DSB) repair requires the histone chaperone Asf1 and that absence of Asf1 causes cell death, as cells are unable to recover from the DNA damage checkpoint. Asf1 contributes toward chromatin assembly after DSB repair by promoting acetylation of free histone H3 on lysine 56 (K56) via the histone acetyl transferase Rtt109. Mimicking acetylation of K56 bypasses the requirement for Asf1 for chromatin reassembly and checkpoint recovery, whereas mutations that prevent K56 acetylation block chromatin reassembly after repair. These results indicate that restoration of the chromatin following DSB repair is driven by acetylated H3 K56 and that this is a signal for the completion of repair (C.-C. Chen, 2008).

Chromatin is taken apart and reassembled during DNA replication and transcription by chromatin assembly factors, including histone chaperones, and this is also likely to the be the case during double-strand DNA repair. The histone chaperone Anti-silencing Function 1 (Asf1) was identified biochemically by its ability to deposit histones H3 and H4 onto newly replicated DNA in vitro. Yeast deleted for ASF1 are highly sensitive to DNA damaging agents, which is likely to reflect a direct role for Asf1 in modulating chromatin structure during repair. Indeed, human Asf1 is required for the assembly of nucleosomes following nucleotide excision repair in vitro. Furthermore, yeast asf1 mutants have elevated rates of genomic instability. Furthermore, there exists a dynamic interaction between Asf1 and the Rad53 DNA damage checkpoint kinase, which suggests that activation of Asf1 may be an important cellular response to DNA damage. In addition to its role in chromatin assembly and disassembly, Asf1 is also essential for stimulating the acetylation of free histone H3 on lysine 56 (K56) by the histone acetyl transferase (HAT) Rtt109 (Recht, 2006; Tsubota, 2007). Despite its occurrence in eukaryotes from yeast to humans, the molecular function of acetylation of H3 K56 remains unknown (C.-C. Chen, 2008).

Although chromatin disassembly has been previously documented at a site of double-strand DNA damage, chromatin reassembly following double-strand DNA repair has not been reported. This work set out to discover why the Asf1 histone chaperone is required for rapid growth after DSB repair. In addition to finding a role for Asf1 in chromatin reassembly following DSB repair, a role was discovered for Asf1 in recovery and adaptation to the DNA damage checkpoint following repair, explaining why asf1 mutant yeast die after DNA repair. These roles for Asf1 can be bypassed by a mimic of permanent acetylation of histone H3 on lysine 56, whereas deletion of the gene encoding the K56 histone acetyl transferase, RTT109, also leads to persistent DNA damage checkpoint activation following DNA repair. As such, acetylated K56 on H3 is required to reinstate the chromatin structure over the repaired DNA, which, in turn, is a critical signal for turning off the DNA damage checkpoint, allowing cell cycle re-entry following DNA repair (C.-C. Chen, 2008).

Acetylation of Histone H3 lysine 56 regulates replication-coupled nucleosome assembly

Chromatin assembly factor 1 (CAF-1) and Rtt106 participate in the deposition of newly synthesized histones onto replicating DNA to form nucleosomes. This process is critical for the maintenance of genome stability and inheritance of functionally specialized chromatin structures in proliferating cells. However, the molecular functions of the acetylation of newly synthesized histones in this DNA replication-coupled nucleosome assembly pathway remain enigmatic. This study shows that histone H3 acetylated at lysine 56 (H3K56Ac) is incorporated onto replicating DNA and, by increasing the binding affinity of CAF-1 and Rtt106 for histone H3, H3K56Ac enhances the ability of these histone chaperones to assemble DNA into nucleosomes. Genetic analysis indicates that H3K56Ac acts in a nonredundant manner with the acetylation of the N-terminal residues of H3 and H4 in nucleosome assembly. These results reveal a mechanism by which H3K56Ac regulates replication-coupled nucleosome assembly mediated by CAF-1 and Rtt106 (Li, 2008).

In the yeast S. cerevisiae, three histone chaperones, CAF-1, Asf1, and Rtt106, have been implicated in the assembly of H3-H4 into nucleosomes. However, how the roles of these histone chaperones are coordinated to promote nucleosome assembly is largely unknown. The results suggest that these histone chaperones function in a hierarchical manner to promote nucleosome assembly. First, Asf1 binds to newly synthesized H3-H4 dimers and presents those dimers for acetylation of H3K56 by the Rtt109-Vps75 complex. H3K56Ac-H4 complexes are then transferred to Rtt106 and CAF-1 for deposition onto DNA and subsequent nucleosome formation (Li, 2008).

While the mechanism of parental histones segregation is still debated and it is not known whether this process requires CAF-1, it is clear that deposition of newly synthesized histones requires CAF-1 and Asf1 in yeast cells. In the crystal structures of Asf1-H3-H4 complexes, Asf1 binds to a surface of H3 that is critical for formation of (H3-H4)2 tetramers. In vitro, Asf1 disrupts (H3-H4)2 tetramers and forms Asf1-H3-H4 heterotrimeric complexes. Thus, it may not be energetically and/or kinetically possible for Asf1 alone to deposit histones onto replicating DNA for formation of (H3-H4)2 tetramers, the first building blocks needed for rapid de novo nucleosome assembly during S phase of the cell cycle. H3 lysine 56 is far away from the surface involved in the formation of (H3-H4)2 tetramers. Thus, the transfer of H3K56Ac-H4 dimers from Asf1 to CAF-1 and Rtt106, which bind preferentially to H3K56Ac, may ensure rapid formation of (H3-H4)2 tetramers and subsequent formation of nucleosomes (Li, 2008).

A histone deacetylase pathway regulates mitosis by modulating Aurora B kinase activity

Histone deacetylase (HDAC) inhibitors perturb the cell cycle and have great potential as anti-cancer agents, but their mechanism of action is not well established. HDACs classically function as repressors of gene expression, tethered to sequence-specific transcription factors. This study reports that HDAC3 is a critical, transcription-independent regulator of mitosis. HDAC3 forms a complex with A-Kinase-Anchoring Proteins AKAP95 and HA95, which are targeted to mitotic chromosomes. Deacetylation of H3 in mitosis requires AKAP95/HA95 and HDAC3 and provides a hypoacetylated H3 tail that is the preferred substrate for Aurora B kinase. Phosphorylation of H3S10 by Aurora B leads to dissociation of HP1 proteins from methylated H3K9 residues on mitotic heterochromatin. This transcription-independent pathway, involving interdependent changes in histone modification and protein association, is required for normal progression through mitosis and is an unexpected target of HDAC inhibitors, a class of drugs currently in clinical trials for treating cancer (Y. Li, 2006).

The classic role of HDAC3 has been that of a transcriptional repressor of gene expression, as part of a complex tethered to sequence-specific transcription factors. This study reports the unexpected finding that HDAC3 has a critical, transcription-independent function in mitosis. In interphase cells, AKAP95/HA95 binds to the nuclear matrix and is less associated with HDAC3. HP1 proteins are recruited to methylated H3K9 in heterochromatin. When cells enter into mitosis, AKAP95/HA95 may target the HDAC3 complex to deacetylate H3, in a reaction that is blocked by HDAC inhibitors, and thereby provides a hypoacetylated H3 tail as substrate for Aurora B to phosphorylate on S10. Phosphorylation of S10 by Aurora B then dissociates HP1 proteins from methylated H3K9 residues on mitotic heterochromatin, which has been referred to as the 'meth-phos switch'. These interdependent changes in histone modification and protein association are required for normal progression through mitosis, perhaps by facilitating chromosome condensation, or by serving as the indicator for the mitotic checkpoint to control proper cell division (Li, 2006).

While the transcriptional effect of HDAC inhibitors on specific genes, such as p21 and other cell cycle-regulated genes, has been reported to contribute to their anti-tumor actions, especially in G1-phase arrest, their direct effects on histone acetylation levels may be equally important for the anti-tumor activity because of the important functions of histones in different cellular processes, including mitosis. It is increasingly clear that HDAC inhibition induces G2/M arrest in many human cell lines and causes mitotic defects in different cancer cell lines. This effect of HDAC inhibition is independent of ongoing gene transcription, suggesting direct effects of histone hyperacetylation on mitosis. These results indicate that the hyperacetylation of histones induced by HDAC inhibitors directly interfere with mitotic progression (Li, 2006).

Global histone acetylation is reduced during mitosis. The current studies reveal that HDAC3 and its partner proteins AKAP95 and HA95 are required for global histone deacetylation during mitosis. Of note, the most dramatic change in acetylation that occurs during mitosis is hypoacetylation of Lys 5 of H4, which matches the substrate specificity of HDAC3. Moreover, the results clearly show that HDAC3 is required for normal mitotic progression. This is consistent with a recent study in which knockdown of HDAC3, but not HDAC1 or HDAC2, increased cells in G2/M phase in human colon cancer cells. Furthermore, knockdown of HDAC3 or AKAP95/HA95 also mimics the effects of nonselective HDAC inhibition on phosphorylation of H3S10 and retention of HP1β proteins on mitotic chromosomes. Inhibition of HDAC3 is therefore likely to be the mechanism by which HDAC inhibitors induce the G2/M block in the cell cycle. The transcription independence of this effect, while unexpected, is completely consistent with a direct mitotic function of HDAC3 in the context of the novel pathway that that is reported here (Li, 2006).

Specific patterns of histone modification at gene promoters regulate transcription via a 'histone code'. Notably, the transient phosphorylation of H3S10 has been reported in the promoter region of many mammalian immediate-early genes, which are rapidly induced in response to extracellular stimuli including UV radiation, growth factors, and cytokines. On these promoters, the phosphorylation of H3S10 precedes the H3K14 acetylation, resulting in multiple modifications of H3 that facilitate gene activation. On the contrary, this study found that the phosphorylation of H3S10 by Aurora B during mitosis requires the previous deacetylation of histones by HDAC3. Thus, in contrast to the phosphorylation of H3S10 by other kinases that prefer preacetylated histone tails, the mitotic phosphorylation of H3S10 by Aurora B kinase is linked to the deacetylation of H3, specifically by HDAC3. This characteristic of Aurora B may be specific to metazoans because IPL1, the yeast homolog of Aurora kinase, phosphorylated both monoacetylated and unacetylated H3. In addition to H3S10, Aurora B also phosphorylates H3S28 and other proteins including his- tone H3 variant centromere protein A (CENP-A). In human cell systems, Aurora B also seems to prefer hypoacetylated H3 and CENP-A H3 as substrate for phosphorylation of H3S28 and CENP-A Ser7, respectively. The global hypoacetylation of H3 tail lysines in mitotic cells and their proximity to the major sites of phosphorylation by Aurora B kinase suggest that deacetylation of histone substrates may be a general preference for Aurora B function. The relative importance of specific hypoacetylated lysines for phosphorylation of specific serine residues remains to be elucidated (Li, 2006).

The specificity of Aurora B toward hypoacetylated histone substrate suggests a mechanistic link between HDAC3-dependent histone deacetylation and a transcription-independent mechanism of mitotic arrest. H3S10 phosphorylation during mitosis is characteristic of many organisms, and is dependent on Aurora B kinase, which plays a central role throughout different stage of mitosis, including chromosome condensation, alignment, and segregation, spindle assembly, and cytokinesis. The recent finding that Aurora-dependent phosphorylation of H3S10 dissociates HP1 from mitotic heterochromatin provides molecular insight into the function of Aurora B. The current findings implicate AKAP95/HA95 and HDAC3 as upstream regulators of this "meth-phos switch", and provide a molecular mechanism to explain the anti-cancer effects of HDAC inhibitors. Aurora B kinase itself is overexpressed in a large number of cancers. The finding that Aurora B is present in HDAC3 complexes and that its kinase activity is dramatically greater when the H3 tail is hypoacetylated suggests that the interdependence of Aurora B and HDAC3 may be a novel and specific target for cancer therapies that would overcome the toxicity of nonspecific HDAC inhibitors (Li, 2006).

Histone deacetylase 3 is required for centromeric H3K4 deacetylation and sister chromatid cohesion

This study describes the role of histone deacetylase 3 (HDAC3) in sister chromatid cohesion and the deacetylation of histone H3 Lys 4 (H3K4) at the centromere. HDAC3 knockdown induces spindle assembly checkpoint activation and sister chromatid dissociation. The depletion of Polo-like kinase 1 (Plk1) or Aurora B restores cohesion in HDAC3-depleted cells. HDAC3 is also required for Shugoshin localization at centromeres. Finally, HDAC3 depletion is shown to result in the acetylation of centromeric H3K4, correlated with a loss of dimethylation at the same position (Eot-Houllier, 2008). These findings provide a functional link between sister chromatid cohesion and the mitotic 'histone code' (Eot-Houllier, 2008).

A chromatin-wide transition to H4K20 monomethylation impairs genome integrity and programmed DNA rearrangements in the mouse

H4K20 methylation is a broad chromatin modification that has been linked with diverse epigenetic functions. Several enzymes target H4K20 methylation, consistent with distinct mono-, di-, and trimethylation states controlling different biological outputs. To analyze the roles of H4K20 methylation states, conditional null alleles were generated for the two Suv4-20h histone methyltransferase (HMTase) genes in the mouse. Suv4-20h-double-null (dn) mice are perinatally lethal and have lost nearly all H4K20me3 and H4K20me2 states. The genome-wide transition to an H4K20me1 state results in increased sensitivity to damaging stress, since Suv4-20h-dn chromatin is less efficient for DNA double-strand break (DSB) repair and prone to chromosomal aberrations. Notably, Suv4-20h-dn B cells are defective in immunoglobulin class-switch recombination, and Suv4-20h-dn deficiency impairs the stem cell pool of lymphoid progenitors. Thus, conversion to an H4K20me1 state results in compromised chromatin that is insufficient to protect genome integrity and to process a DNA-rearranging differentiation program in the mouse (Schotta, 2008).

Functional dynamics of histones H3 and H4 during gametogenesis

Profound epigenetic differences exist between genomes derived from male and female gametes; however, the nature of these changes remains largely unknown. A systematic investigation was undertaken of chromatin reorganization during gametogenesis, using the model eukaryote Saccharomyces cerevisiae to examine sporulation, which has strong similarities with higher eukaryotic spermatogenesis. A mutational screen of histones H3 and H4 was undertaken to uncover substitutions that reduce sporulation efficiency. Two patches of residues -- one on H3 and a second on H4 -- were discovered that are crucial for sporulation but not critical for mitotic growth, and likely comprise interactive nucleosomal surfaces. Furthermore, novel histone post-translational modifications were discoved that mark the chromatin reorganization process during sporulation. First, phosphorylation of H3T11 appears to be a key modification during meiosis, and requires the meiotic-specific kinase Mek1. Second, H4 undergoes amino tail acetylation at Lys 5, Lys 8, and Lys 12, and these are synergistically important for post-meiotic chromatin compaction, occurring subsequent to the post-meiotic transient peak in phosphorylation at H4S1, and crucial for recruitment of Bdf1, a bromodomain protein, to chromatin in mature spores. Strikingly, the presence and temporal succession of the new H3 and H4 modifications are detected during mouse spermatogenesis, indicating that they are conserved through evolution. Thus, the results show that investigation of gametogenesis in yeast provides novel insights into chromatin dynamics, which are potentially relevant to epigenetic modulation of the mammalian process (Govin, 2010).

JMJD-5/KDM8 regulates H3K36me2 and is required for late steps of homologous recombination and genome integrity

Methylation of lysine 36 of histone 3 (H3K36) is a post-translational modification functionally relevant during early steps of DNA damage repair (see Drosophila DNA damage repair). This study shows that the JMJD-5 (see Drosophila CG13902) regulates H3K36 di-methylation and is required at late stages of double strand break repair mediated by homologous recombination. Loss of jmjd-5 results in hypersensitivity to ionizing radiation and meiotic defects, and it is associated with aberrant retention of RAD-51 (see Drosophila spn-A) at sites of double strand breaks. Analyses of jmjd-5 genetic interactions with genes required for resolving recombination intermediates (rtel-1 (see Drosophila CG4078) or promoting the resolution of RAD-51 double stranded DNA filaments (rfs-1 and helq-1 (see Drosophila mus301)) suggest that jmjd-5 prevents the formation of stalled postsynaptic recombination intermediates and favors RAD-51 removal. As these phenotypes are all recapitulated by a catalytically inactive jmjd-5 mutant, the study proposes a novel role for H3K36me2 regulation during late steps of homologous recombination critical to preserve genome integrity (Amendola, 2017).

Evolutionary homologs: Table of contents

Histone H3: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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