Histone Chaperones

Histone chaperones represent a structurally and functionally diverse family of histone-binding proteins that prevent promiscuous interactions of histones before their assembly into chromatin. Our understanding of the mechanisms of histone shuttling between different chaperone systems, and histone transfer onto and off DNA, has been hampered due to the availability of only a limited number of histone-chaperone complexes.

Identification of post-replicative chromatin by a histone H4K20me0 reader protein

Identification of post-replicative chromatin by a histone H4K20me0 reader protein

The eukaryotic genome is organized into chromatin and modifications on histones play a central role in regulating DNA-based processes. It is clear that genome maintenance depends on chromatin context, but mechanisms that integrate chromatin state with DNA repair remain largely unknown. In this structure-function study, we show that the homologous recombination (HR) complex TONSL–MMS22L recognizes post-replicative chromatin by binding to histone H4 unmodified at K20 (H4K20me0). Our structural analysis identifies the TONSL Ankyrin Repeat Domain (ARD) as a histone reader specific for H4K20me0, and pull-downs with modified peptides and recombinant nucleosomes confirm that H4K20 methylation abrogates TONSL–MMS22L binding. H4K20me0 is a signature of new histones incorporated during DNA replication, demarcating replicated chromatin until cells reach G2/M. Accordingly, TONSL binds new histones prior to and after their deposition onto DNA and accumulates in chromatin as cells progress through S phase. This recruitment of TONSL to post-replicative chromatin via ARD recognition of H4K20me0 is required for TONSL accumulation at DNA lesions. Together, these data uncover a mechanism for identification of post-replicative chromatin, which can allow cells to discriminate lesions with a sister chromatid available for HR repair.

Saredi, G., Huang, H., Hammond, C., Bekker-Jensen, S., Forne, I., Reveron-Gomez, N., Foster, B. M., Mlejnkova, L., Bartke, T., Cejka, P., Mailand, N., Imhof, A., Patel, D. J. and Groth, A. (2016). H4 K20me0 marks post-replicative chromatin and recruits the TONSL-MMS22L DNA repair complex. Nature 534, 714-718.

Structural Basis Underlying Viral Hijacking of a Histone Chaperone Complex

Structural Basis Underlying Viral Hijacking of a Histone Chaperone Complex

Histone H3.3 chaperone DAXX is implicated in formation of heterochromatin and transcription silencing, especially for newly infecting DNA virus genomes entering the nucleus. Epstein-Barr Virus (EBV) can efficiently establish stable latent infection as a chromatinized episome in the nucleus of infected cells. The EBV tegument BNRF1 is a DAXX-interacting protein required for the establishment of selective viral gene expression during latency. We report the structure of BNRF1 DAXX-interaction domain (DID) in complex with DAXX histone-binding domain (HBD) and histones H3.3-H4. BNRF1 DID contacts DAXX HBD and histones through nonconserved loops. The BNRF1-DAXX interface is responsible for BNRF1 localization to PML-nuclear bodies typically associated with host anti-viral resistance and transcriptional repression. Paradoxically, the interface is also required for selective transcription activation of viral latent cycle genes required for driving B-cell proliferation. These findings reveal molecular details of virus reprogramming of an anti-viral histone chaperone to promote viral latency and cellular immortalization.

Huang, H., Deng, Z., Vladimirova, O., Wiedmer, A., Lu, F., Lieberman, P. M. & Patel, D. J. (2016). Structural basis underlying viral hijacking of a histone chaperone complex. Nat. Commun. 7: 12707.

The CENP-T/-W Complex is a Binding Partner of the Histone Chaperone FACT

A project championed by the Genevieve Almouzni lab (Institut Pasteur, Paris) has demonstrated that the FACT chaperone stabilizes the soluble CENP-Y/-W complex in the cell and promotes dynamics of exchange, enabling CENP-T/-W deposition at centromeres.

Prendergast, L., Muller, S., Liu, Y., Huang, H., Dingli, F., Lowe, D., Vassias, I., Patel, D. J., Sullivan, K. F. and Almouzni, G. (2016). The CENP-T-CENP-W complex is a binding partner of the histone chaperone FACT. Genes Dev. 30, 1313-1326.

A unique binding mode enables MCM2 and ASF1to chaperone histones H3-H4 at the replication fork

A unique binding mode enables MCM2 and ASF1to chaperone histones H3-H4 at the replication fork

A unique binding mode enables MCM2 and ASF1to chaperone histones H3-H4 at the replication fork

During cell division, both DNA sequence and its organization into chromatin must be duplicated. At the replication fork, chromatin is re-assembled by the recycling of old histones combined with deposition of new ones. How histone dynamics are integrated with DNA replication to maintain genome and epigenome information is unclear. Here, we provide structure-function analyses showing how human MCM2, part of the replicative helicase, can chaperone histone H3-H4. Our first structure shows an H3-H4 tetramer bound by two MCM2 histone-binding domains (HBD), hijacking interaction sites used by nucleosomal DNA, while our second structure reveals how MCM2 and ASF1 co-chaperone an H3-H4 dimer. MCM2 histone chaperone activity relies on an intact HBD and is required for normal rates of cell proliferation. Further, we demonstrate that MCM2, as part of the MCM2-7 helicase, can chaperone both new and old canonical histones H3-H4, as well as H3.3 and CENPA variants. This unique mode of histone-binding thus provides MCM2 with ideal properties to handle histones evicted genome-wide during DNA replication, which bears on how dividing cells can maintain functional chromatin domains.

Huang, H., Stromme, C. B., Saredi, G., Hodl, M., Strandsby, A., Strandsby, A., Gonzalez-Aguilera, C., Chen, S., Groth, A. and Patel, D. J. (2015). A unique binding mode enables MCM2 to chaperone histones H3-H4 at replication forks. Nat. Struct. Mol. Biol. 22, 618-626.

Structure-function studies of histone H3/H4 tetramer maintenance during transcription by chaperone Spt2

Structure-function studies of histone H3/H4 tetramer maintenance during transcription by chaperone Spt2

Cells utilize specific mechanisms such as histone chaperones to abrogate the inherent barrier that the nucleosome poses to transcribing polymerases. The current model postulates that nucleosomes can be transiently disrupted to accommodate passage of RNA polymerases and that histones H3 and H4 possess their own chaperones dedicated to the recovery of nucleosomes. Here, we have determined the crystal structure of the conserved C-terminus of human Suppressors of Ty insertions 2 (hSpt2C) chaperone bound to an H3/H4 tetramer. The structural studies demonstrate that hSpt2C is bound to the periphery of the H3/H4 tetramer, mimicking the trajectory of nucleosomal bound DNA. These structural studies have been complemented with in vitro binding and in vivo functional studies on mutants that disrupt key intermolecular contacts involving two acidic patches and hydrophobic residues on Spt2C. We show that contacts between both human and yeast Spt2C with H3/H4 tetramer are required for the suppression of H3/H4 exchange as measured by H3K56ac and new H3 deposition. These interactions are also crucial for the inhibition of spurious transcription from within coding regions. Together our data indicate that Spt2 interacts with the periphery of the H3/H4 tetramer and promotes its recycling in the wake of RNA polymerase.

Chen, S., Ruflange, A., Huang, H., Nourani, A. and Patel, D. J. (2015). Structure-function studies of histone H3/H4 tetramer maintenance during transcription by chaperone Spt2. Genes Dev. 29, 1326-1340.

DAXX Envelops a Histone H3.3-H4 Dimer for H3.3-Specific Recognition

DAXX Envelops a Histone H3.3-H4 Dimer for H3.3-Specific Recognition

 

DAXX is a metazoan histone chaperone specific to the evolutionary conserved histone variant H3.3. Here in collaboration with the David Allis laboratory (Rockefeller University), we report the crystal structures of the DAXX histone-binding domain with a histone H3.3-H4 dimer, including mutants within DAXX and H3.3, together with in vitro and in vivo functional studies towards elucidation of the principles underlying H3.3 recognition specificity. Occupying 40% of the histone surface-accessible area, DAXX wraps around the H3.3-H4 dimer, with complex formation accompanied by structural transitions in the H3.3-H4 histone fold. DAXX employs an extended  α-helical conformation to compete with major inter-histone, DNA and ASF1 interaction sites. Our structural studies identify recognition elements that read out H3.3-specific residues, while functional studies address the contributions of Gly90 in H3.3 and Glu225 in DAXX to chaperone-mediated H3.3 variant recognition specificity.

Elsasser, S. J., Huang, H., Lewis, P. W., Allis, C. D. & Patel, D. J. (2012). DAXX histone chaperone envelops an H3.3/H4 dimer for H3.3-specific recognition. Nature 491, 560-565.