Introduction
In eukaryotic cells, genomic DNA is tightly packaged into chromatin through the formation of nucleosomes—the fundamental structural units. Each nucleosome consists of approximately 147 base pairs of DNA wrapped around a histone octamer made of two copies each of histones H2A, H2B, H3, and H4. Protruding from this complex are the N-terminal tails (NTs) of the histones, which serve as critical platforms for post-translational modifications (PTMs). Among these, lysine acetylation (Kac) plays a pivotal role in regulating gene expression by neutralizing the positive charge on lysine residues, thereby loosening chromatin structure and promoting transcriptional activation.
Two major histone acetyltransferases, p300 and its homolog CBP (CREB-binding protein), are central to writing these acetylation marks. These enzymes not only transfer acetyl groups to lysine residues across all core histones but also contain bromodomains—specialized modules that "read" acetylated lysines. This dual function allows p300/CBP to act as both writers and readers of epigenetic signals, particularly recognizing acetylated lysines on histone H4 such as H4K12ac and H4K16ac.
Notably, acetylation of H2B’s N-terminal tail (H2BNTac) has emerged as a strong correlate of active enhancers and their target promoters—more so than even H3K27ac, traditionally considered a key enhancer marker. The acetyltransferase activity of p300 responsible for H2BNTac is essential for recruiting RNA polymerase II (RNAPII), enabling rapid enhancer activation. However, the precise mechanism by which p300/CBP propagates histone acetylation within a single nucleosome remained unclear—until recent structural insights revealed an elegant intranucleosomal read/write system.
How p300/CBP Reads and Writes Acetylation Marks
Dependence on Pre-existing H4 Acetylation
Studies using reconstituted nucleosomes containing pre-acetylated H4 (specifically H4K12ac/K16ac) demonstrated that p300's catalytic activity is significantly enhanced when it encounters these marks. Immunoblotting and mass spectrometry analyses revealed that prior acetylation of H4 stimulates p300-mediated acetylation of H2B and H3 N-terminal tails. This enhancement was particularly pronounced at multiple lysine sites on H2B (e.g., K11, K15, K16, K20), with increases ranging from 4.3- to 46-fold compared to unmodified nucleosomes.
This effect is bromodomain-dependent: inhibition of the bromodomain pocket with CBP30—a selective small-molecule inhibitor—reduced acetylation at key residues like H2BK16ac and H3K27ac. These findings support a model in which p300 first "reads" existing H4 acetylation via its bromodomain and then "writes" new acetylation marks on adjacent histones within the same nucleosome.
Structural Insights from Cryo-EM
Cryo-electron microscopy (cryo-EM) structures of p300 bound to acetylated nucleosomes provided direct visual evidence of this mechanism. When p300 engages a nucleosome bearing H4K12ac/K16ac, its bromodomain locks onto the acetylated tail while the histone acetyltransferase (HAT) domain positions itself near the N-terminal tail of H2B. The overall conformation resembles a bent Slinky, stabilized by interactions both inside the bromodomain pocket and with nucleosomal DNA.
Importantly, the distance between the HAT catalytic site and the beginning of the H2B tail (~10 Å) suggests that p300 can efficiently acetylate lysines up to K16 on H2B. Similar structural arrangements were observed for complexes targeting H3NT and H2ANT, indicating that p300 uses a rotational binding mechanism—anchored at the acetylated H4 tail—to access different histone substrates within the same nucleosome.
Multiple Binding Modes Enable Broad Substrate Targeting
p300 does not merely act on one histone at a time. Instead, cryo-EM data revealed several distinct binding conformations in which the HAT domain is oriented toward different N-terminal tails—H2B, H3, or H2A—while the bromodomain remains anchored to acetylated H4.
These alternative configurations depend on electrostatic interactions between basic patches on p300 and nucleosomal or linker DNA:
- The KJ basic patch consistently interacts with nucleosomal DNA.
- The KN basic patch engages linker DNA in certain orientations, facilitating access to H3NT.
- A third BC basic patch near the bromodomain loop mediates additional DNA contacts that stabilize positioning.
This multivalent DNA interaction allows p300 to pivot around its anchor point on H4K12ac/K16ac, enabling sequential acetylation of multiple non-H4 histones without dissociating from the nucleosome. This mechanism ensures efficient propagation of activating marks across the chromatin fiber.
The Critical Role of the BC Basic Patch
Sequence alignment across human bromodomain proteins shows that the RKxxRxxK motif forming the BC basic patch is uniquely conserved in p300 and CBP among all 61 human bromodomains. Mutational studies confirmed its functional importance: replacing these positively charged residues with alanine (4A) or glutamic acid (4E) dramatically reduced both nucleosome binding affinity and acetylation efficiency.
Even more strikingly, these mutations impaired not only H4-dependent acetylation but also baseline activity on unmodified nucleosomes, suggesting that the BC basic patch contributes broadly to p300’s chromatin engagement—both in a read-dependent and independent manner.
Directionality in Acetylation Propagation
Biochemical assays further revealed a directional flow in acetylation signaling:
- Bidirectional: Acetylation can propagate between H4NT and H3NT.
- Unidirectional: Acetylation flows from H4NT → H2BNT and from H3NT → H2BNT—but not vice versa.
This asymmetry arises because while p300’s bromodomain effectively binds H4K12ac/K16ac and H3K14ac/K18ac, it shows minimal affinity for di-acetylated H2B peptides. Consequently, once multisite H2BNTac is established, it does not recruit more p300 molecules or initiate further read/write cycles—it acts as an endpoint rather than a relay station.
Interestingly, when one H3 tail is pre-acetylated, p300 can propagate acetylation across to the second H3 tail within the same tetramer. This inter-tail communication enables coordinated modification of both copies of H3 in the nucleosome core.
Frequently Asked Questions (FAQ)
Q: What makes p300/CBP unique among histone acetyltransferases?
A: Unlike most acetyltransferases, p300/CBP possesses both catalytic (HAT) and reader (bromodomain) domains, allowing it to simultaneously recognize existing acetylation marks and deposit new ones—enabling self-propagation of epigenetic signals.
Q: Why is H2BNTac considered a better enhancer marker than H3K27ac?
A: While H3K27ac is widely used, studies show that 79% of regions marked by multisite H2BNTac are actively transcribed. Moreover, H2BNTac correlates more strongly with RNA polymerase II recruitment and enhancer activity than other known chromatin marks.
Q: Can p300 initiate acetylation without any pre-existing marks?
A: Yes—although less efficiently. p300 exhibits basal activity on unmodified nucleosomes, but its efficiency increases dramatically when it detects pre-acetylated H4 or H3 tails through its bromodomain.
Q: How does p300 avoid indiscriminate acetylation across chromatin?
A: Its activity is tightly regulated by multiple factors: recruitment via transcription factors, autoinhibitory loops, and dependency on pre-existing marks via its bromodomain. This ensures targeted modification only at specific genomic loci.
Q: Is this mechanism relevant beyond basic research?
A: Absolutely. Dysregulation of p300/CBP activity is linked to cancer, neurodevelopmental disorders, and inflammatory diseases. Understanding its read/write mechanism opens avenues for developing selective epigenetic therapies.
Functional Implications: From Epigenetic Memory to Transcriptional Output
The dual functionality of p300/CBP supports two key biological roles:
1. Epigenetic Inheritance via the H3-H4 Tetramer
The symmetric flow of acetylation between H3 and H4 enables self-perpetuation of epigenetic states during cell division. Because parental (H3-H4)₂ tetramers segregate conservatively during DNA replication, they carry legacy acetylation marks into daughter strands. p300/CBP can then restore full acetylation levels by reading residual marks and propagating them across both old and new histones—a process potentially aided by chaperones like ASF1.
This mechanism helps maintain cell identity across generations by preserving transcriptional programs through mitosis.
2. Transcriptional Activation via the H2A-H2B Dimer
In contrast to the stable memory function of the inner tetramer, the outer H2A-H2B dimers serve as transient effectors of gene expression. Multisite acetylation of H2B destabilizes the nucleosome by weakening dimer-histone interactions. Thermal stability assays confirmed that heavily acetylated H2B lowers the melting temperature of the nucleosome by nearly 2°C—promoting dimer eviction.
This structural loosening facilitates RNA polymerase II progression and enhances transcriptional output. Furthermore, since RNAPII-associated chaperones like FACT exchange H2A-H2B dimers during elongation, this system ensures that acetylation signals are reset after each round of transcription—maintaining signal fidelity.
A Unified Model: The Epi-Central Hypothesis
Based on these findings, researchers propose an epi-central model of chromatin regulation:
- The H3-H4 tetramer functions like DNA—it stores heritable epigenetic information.
- The H2A-H2B dimer acts like RNA—it expresses that information transiently.
- p300/CBP serves as the central processor: it replicates marks within the tetramer ("epigenetic storage") and transcribes them onto dimers ("epigenetic expression").
This model integrates sequence-specific targeting (via transcription factors) with local chromatin context (via pre-existing acetylation), ensuring precise spatiotemporal control over gene activation.
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Conclusion
The discovery that p300/CBP propagates histone acetylation through an intranucleosomal read/write mechanism represents a major advance in epigenetics. By anchoring to pre-acetylated H4 tails and rotating across the nucleosome surface, p300 efficiently modifies multiple histone substrates—linking epigenetic memory with dynamic gene regulation.
This mechanism explains how activating signals spread locally within chromatin without diffusing uncontrollably. It also highlights potential therapeutic targets: disrupting specific protein-DNA or protein-histone interactions could modulate enhancer activity in disease contexts without globally altering the epigenome.
As research continues to unravel the complexity of chromatin dynamics, tools inspired by nature’s precision—such as targeted epigenetic editors or synthetic reader-writer fusions—may soon transform medicine and biotechnology alike.
Core Keywords: histone acetylation, p300/CBP, epigenetic inheritance, bromodomain, nucleosome, HAT domain, chromatin regulation, transcriptional activation