Exchange of associated factors directs a switch in HBO1 acetyltransferase histone tail specificity

Marie-Eve Lalonde1, Nikita Avvakumov1, Karen C. Glass2*, France-Hélène Joncas1, Nehmé Saksouk1, Michael Holliday3, Eric Paquet1, Kezhi Yan5, Qiong Tong2, Brianna J.

Klein2, Song Tan4, Xiang Jiao Yang‑ 5,6, Tatiana G. Kutateladze2,3 and Jacques Côté1#

1Laval University Cancer Research Center, Hôtel-Dieu de Québec (CHUQ), Quebec City, Québec G1R 2J6, Canada. 2Department of Pharmacology, and 3Molecular Biology Program, University of Colorado School of Medicine, Aurora, CO 80045, USA. 4Center for Gene Regulation, Department of Biochemistry & Molecular Biology, 108 Althouse Laboratory, The Pennsylvania University, University Park, PA 16802-1014, USA. 5The Rosalind & Morris Goodman Cancer Research Center and Department of Biochemistry, McGill University, Montreal, Québec H3A 1A1, Canada. 6Department of Medicine, McGill University Health Center, Montreal, Québec H3A 1A1, Canada. *Present address: Department of Pharmaceutical Sciences, Albany College of Pharmacy and Health Sciences, Colchester VT # Corresponding author: (418) 525-4444 ext. 15545; Jacques.Cote@crhdq.ulaval.ca

Genes and Development (2013), Sep 15;27(18):2009-2024PMID: 24065767

From Musselman et al., NSMB 2012

Each modification is recognized by different domains:

Chromatin Binding Domains:

MYST Acetyltransferases-Conservation from Yeast to Human

From Avvakumov*, Lalonde* et al., MCB 2012

MYST Acetyltransferases-Protein Domains:

From Avvakumov*, Lalonde* et al., MCB 2012

PHD domains (plant homeodomain) can recognize different marks of the histone H3 tail.

PHD domains

PHD domains

Binding specificity Examples

H3K4me3 INGs, BPTF, PHF2/8

H3 unmodified AIRE, DNMT3

H3K9me3 UHRF1, KDM5C

2 different marksRag2 (H3R2me2K4me3)

DPF3 (H3K4unK14ac)

BRPF1-MOZ/MORF complex

MOZ and MORF are implicated in transription of HOX genes as well as in embryonic development in mouse (hematopoïetic cells, neurogenesis, etc.)

MOZ -/- mice show a decrease in expression level of Hoxa et Hoxb clusters.

BRPF1 was also shown to play a role in the transcription of HOX genes and in developmental processes in zebrafish.

Hypothesis

The chromatin binding modules found within the MOZ/MORF-BRPF1 complex regulate its genome-wide localization and its acetylation specificity.

Specific goals:1. Characterize the different chromatin binding domains in BRPF1 protein.2. Map the genome-wide localization of the MYST complex on chromatin.3. Study how these different chromatin binding domains are implicated in

histone acetylation specificity.

PHD1 of BRPF1

GST-PHD1 purification, peptide pull-downs with biotinylated histone peptides.

NMR:

1 2

PHD2 of BRPF11 2

PZP Domain (PHD1+PHD2) of BRPF1

PHD1 is a dominant recognition module over PHD2 within the PZP domain.

1 2

Both PHD Domains are Essential for Chromatin Binding and Acetylation

Anti-HA IP on transfected 293T with HA-BRPF1 (wt or mutant) , Flag-ING5, Flag-Eaf6 and Flag-MOZ:

In vitro acetylation assays on chromatin or free histones with overexpressed complexes:

Binding of the Complex to Chromatin: The ING5 PHD Domain

ING5

The ING5 PHD targets the entire complex to H3K4me3 on chromatin.PZP domain of BRPF1 inhibits the recognition of H3K4me3.

In vitro acetylation assays on histone peptides, using purified complexes from SF9 cells:

Genome-Wide Localization:

ChIP-seq anti-Brpf2, anti-ING5 and anti-H3K4me3 in RKO cells:

Colocalization of Brpf2, ING5 and H3K4me3 genome-wide, on Transcription Start Sites (TSS).

Brpf2

Brpf2

Genome-Wide Localization:

Colocalization of Brpf1/2, HBO1, H3K4me3 shown by heatmaps on TSS of genes sorted by their expression levels

Brpf1 Complex Purification

Anti-Flag IP in 3xFlag Brpf1 stable HeLa cells :

All these proteins have also been confirmed by mass spectrometry.

TAP purification with HBO1-TAP stable HeLa S3 cells:

HBO1 catalytic subunit can also co-purify with the BRPF1 scaffold subunit.Two different HBO1 complexes : HBO1-JADE1 and HBO1-BRPF1

Acetylation assays with purified native BRPF1 complexes (Free histones vs Chromatin):

Brpf1 Complex Purification

Native BRPF1 complexes (HBO1-BRPF1 + MOZ/MORF + BRPF1) can acetylate both H3 and H4 on free histones, but only target H3 acetylation on chromatin.In vivo increase of H3 acetylation but not H4 on p21 TSS by ChIP in 3xFlag BRPF1 HeLa cells

ChIP of H3K14/23ac, H4ac histone marks on stable 3xFlag BRPF1 HeLa cells :

Complex Acetylation Specificity :

?

?

Acetylation Specificity HBO1-BRPF1 vs HBO1-JADE1

In vitro acetylation assays on free histones or chromatin with overexpressed complexes purified from 293T cells with Flag-BRPF1 ou Flag-JADE1:

The HBO1-BRPF1 complex acetylates H3K14/K23 on chromatin while the HBO1-JADE1 complex acetylates H4 (K5,8,12).

Fluorogram(3H acetyl coA)

WB detection of different H3 marks on acetylated chromatin with mock vs HBO1-BRPF1 complexes:

?

PWWP and Bromodomains of BRPF1

Additional Bromo and PWWP domains in BRPFs scaffold proteins compared to JADEs.

BRPF1 PWWP and Bromodomains:

The bromodomain and PWWP domain of BRPF1 do not influence histone acetylation specificity of the complex.

Anti-Flag IP on transfected 293T with Flag-BRPF1 (wt or mutant) , HA-ING5, HA-Eaf6 and HA-HBO1:

In vitro acetylation assays on free histones or chromatin with purified complexes:

Fluorogram(3H acetyl coA)

N-term Region of EPC Domain Containing Proteins:

N-term region of MYST acetyltransferase scaffold subunits:

N-term Deletions of BRPF1 and JADE1

• N-term domain deletions of JADE1 lose acetylation of H4 and gain acetylation of H3. • N-term domain deletions of BRPF1 decrease the activity of complexes on H3.

• Deletion of the first 52 amino acids of JADE1

• Deletion of the first 124 amino acids of BRPF1

In vitro acetylation assays on chromatin with overexpressed complexes purified from 293T by anti-Flag IP of BRPF1 or JADE1:

N-term Region of EPC1

N-term domains of EPC1 and Epl1 or also essential for H4 acetylation on chromatin.

N-term domains of MYST acetyltransferase scaffold subunits are targeting the histone acetylation specificity.

The MYST TIP60 (human) or NuA4 (Yeast) complexes also have a scaffold EPC domain containing protein, EPC1 /2 or Epl1.Deletion of the first 13 amino acids of EPC1.Deletion of the first 71 amino acids of Epl1.

Conclusions

• The PZP domain of BRPF1 is controlled by PHD1 and blocks binding to H3K4me3.

• Both PHD domains are essential for binding to chromatin.

• The MOZ/MORF-BRPF1 complex colocalizes with H3K4me3 on TSS of genes through the ING5 PHD.

• The scaffold subunits (NOT the catalytic subunits) of MYST acetyltransferase complexes are responsable for histone acetylation specificity.

• The bromo and PWWP domains of Brpf1 are not involved in histone tail specificity.

• Deletion of the N-term domains of JADE1 and EPC1 lose H4 acetylation.

• N-term domains of scaffold subunits are regulating the histone acetylation specificity.

Conclusion-Final Model (1)

Conclusion-Final Model (2)

Ackowledgments

• Jacques Côté• Nikita Avvakumov• France-Hélène Joncas• Céline Roques• Nehmé Saksouk• Eric Paquet

• Tatiana G. Kutateladze• Karen C. Glass• Michael Holliday• Brianna J. Klein• Qiong Tong• Xiang-Jiao Yang• Kezhi Yan• Song Tan