Tsinghua University's latest Nature article: NuA4 Mechanism of Selective Acetylated Histone H4

Organism genetic information DNA winding histone octamer 1.
7 circle forms the basic composition of chromosomes - nucleosomes
.
The N-terminal tail of histone H4 interacts with neighboring nucleosomes, promoting the formation of higher-level structures of chromosomes and heterochromatin silencing
.
Nucleosome assembly and heterochromatin formation hinder important biological processes
such as DNA replication, transcription, and damage repair.
Organisms have evolved a series of mechanisms to overcome the obstacles
of nucleosomes.
Among them, histone acetylation modification neutralizes the positive charge of lysine side chains and recruits other chromatin factors, thereby regulating the processes
of chromatin folding, gene transcription, and DNA damage repair.

On October 5, 2022, Professor Chen Zhucheng of the School of Life Sciences of Tsinghua University/Center for Advanced Innovation in Structural Biology/Tsinghua-Peking University Joint Center for Life Sciences cooperated with Associate Professor Li Xueming to publish online in the journal Nature entitled "Structure of the NuA4 acetyltransferase complex bound to the.
" nucleosome) research paper that reveals the mechanism of acetyltransferase NuA4 binding to nucleosomes and the spatial recognition of histone H4, and elucidating the structural basis for
the function of NuA4 as a transcriptional co-activator.

NuA4 and SAGA are two important acetotransferase complexes of Saccharomyces cerevisiae, selectively acetylated histone H4 and H3, respectively, that regulate gene transcription
.
NuA4 and SAGA are highly conserved in different species, the former being the only acetyltransferase
necessary for yeast survival.
As transcriptional co-activators, the two are recruited to the promoter region through the common subunit Tra1, in combination with transcription factors (Figure 1a
).
Because of its fundamental role, NuA4 has been continuously studied for more than 20 years since its discovery, but its structural history is quite tortuous, and it is not clear how fine molecular assembly patterns and the mechanism
of identifying substrate nucleosomes are unknown.

Figure 1 Diagram of the working mode of NuA4 and the structure of the combined nucleosome

(a) Diagram of the working mode of NuA4 and SAGA synergy to promote gene transcription; (b) Schematic diagram of NuA4 binding nucleosomes; (c) Electron microscopy density map
of NuA4-NCP complex.
(d) Schematic diagram of the composition of each subunit domain of NuA4, and the color is consistent
with that in the density map of electron microscopy.

Chen Zhucheng's research group reported the crystal structure of the NuA4 active center Piccolo subcomplex and its low-resolution cryo-electron microscopy structure
bound to nucleosomes.
On this basis, the research team continued to advance towards the structural resolution of NuA4 whole enzyme.

However, the researchers found that the NuA4 complex was structurally plastic, and the part of its active center that bound to the nucleosomes could not obtain a stable conformation
.
This creates key technical difficulties for the analysis of high-quality structures
.
To limit the flexibility of the complex structure, the research team added the transcription factor Gal4-VP16 to the sample and introduced the corresponding recognition DNA sequence at the nucleosome joint DNA as an upstream activation signal (UAS).

Moreover, the research team chemically modified the histone H4K16 site for CMC (carboxymethyl coenzyme A) and triphalated the
H3K36 site.
CMC binds NuA4's catalytic center Esa1 to stabilize the action of Esa1 with nucleosomes; H3K36 combines with the Eaf3 subunit
.
After breaking through various technical problems, the cryo-electron microscopy structure of NuA4 binding to nucleosomes (8.
8 Å) was finally resolved, and the local resolution was 2.
7-3.
4 Å
.

The NuA4 complex, consisting of 13 subunits, is divided into two large modules (Figure 1c): the histone acetyltransferase (HAT) and the transcription activator-binding (TRA
).
。 The HAT module is the piccolo subcomplex, which consists of Esa1, the N-terminal region of Epl1, Eaf6 and Yng2; The TRA module consists of
the C-end regions of Tra1, Eaf1, Eaf2, Act1, Arp4 and Epl1.
A disordered area of Epl1 connects the HAT module to
the TRA module.
In addition, there is a "bridge-shaped" structural flexibility zone
.
The researchers extrapolated from crosslinking mass spectrometry that this region was the Eaf3/5/7 subunit
.
The high degree of plasticity
of the NuA4 composite structure can be seen from here.

This structure shows that the linker DNA carrying the UAS extends toward the surface of the Tra1 subunit (Figure 2a
).
The researchers found that the surface was highly conserved in different species (Figure 2b), and that numerous transcription factors bound to Tra1 were located within
the surface.
Therefore, this conservative surface is named the transcription factor binding surface (ABS
).
The discovery of ABS provides the structural basis
for NuA4 to be recruited by transcription factors into the promoter region.

In the ABS periphery, the researchers found that a poly-basic amino acid interface (PBS) interacts with the linker DNA of the nucleosomes (Figure 2c
).
The researchers verified the acetyl-transfer activity of PBS and the importance of regulating yeast carbon source metabolism through biochemical and genetic experiments (Figure 2d
).

Figure 2 Structure of the TRA module and nucleosome recognition mechanism of NuA4

(a) Transcription factor binding surface (ABS); (b) Conservatory analysis of ABS (c) structure of polybasic amino acid surfaces (PBS); (d) Growth phenotypic analysis
of wild-type and PBS mutants.

The catalytic center HAT module combines two key elements on the surface
of the nucleosome disc structure.
Among them, Epl1's "arginine anchor" identifies the acidic region of the nucleosome, and "double function loop (DFL)" identifies the DNA at the superhelix 1.
5 position of the nucleosomes (Figures 3a, b, c).

This nucleosome recognition pattern places the active pocket of Esa1 just above
the N-terminal tail of H4.
This structure supports the H4 tail spatial position recognition mechanism at the whole enzyme level (Figure 3b
).
This mechanism differs from the common histone modifier enzyme working mechanism
based on amino acid sequence recognition.

Taken together, the study reveals the mechanism by which NuA4 coordinates the identification of nucleosomes through multiple structural elements (Figure 1b
).
ABS through transcription factors, PBS directly binds to the joint DNA, recruiting nucleosomes to the edges of the TRA module; This conformation allows the HAT module to identify the surface of the nucleosomes via DFL and arginine anchor, thus exerting the function
of selective acetylation of H4.
The plasticity between the TRA module and the HAT module allows NuA4 to adapt to complex chromosomal environments and be recruited
by different transcription factors.
In addition to the Eaf5 subunit, all other subunits are homologous proteins
present in the human TIP60 complex.
Therefore, this study provides a good model
for understanding the assembly and working mechanism of TIP60 complex.

It is worth mentioning that the structure of this working TRA module was validated by the structural research of three other separate NuA4 complexes (preprinted, see further reading
).

Figure 3 Mechanism of NuA4 HAT module identification of nucleosomes

(a) Structural model of the HAT module combined with nucleosomes; (b) pattern diagram of NuA4 identifying H4 by spatial position; (c) Arginine anchor local electron microscopy density map and structural details of its interaction with acidic regions; (d) Peptide competitive experiments show the importance of
Arginine anchor for NuA4 acetylation activity.

Professor Chen Zhucheng and Associate Professor Li Xueming of the School of Life Sciences of Tsinghua University are the co-corresponding authors of this paper, and Keke Qu (graduated), a 2014 doctoral student in the School of Life Sciences of Tsinghua University, Kangjing Chen (graduated), a 2017 doctoral student, and Wang Hao, a 2017 doctoral student, are the first authors
of the paper 。 This work has been strongly supported by the National Natural Science Foundation of China, the Ministry of Science and Technology Major Scientific Research Program Special Project, the Beijing Municipal High-Precision Innovation Center for Structural Biology, the Tsinghua-Peking University Life Science Joint Center, the Tsinghua Base of the National Protein Science Research (Beijing) Facility, and the cryo-electron microscopy platform and computing platform of the Tsinghua Base of the National Protein Science Research (Beijing) Facility provide support
for data collection and processing.

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