Measurement of histone replacement dynamics with genetically encoded exchange timers in yeast

Yaakov, Gilad; Jonas, Felix; Barkai, Naama

doi:10.1038/s41587-021-00959-8

Cited by 16 publications

(55 citation statements)

References 66 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…All strains were generated from YGY663 (Yaakov et al 2021) using standard yeast transformation (Gietz and Schiestl 2007). To prevent competition between tagged and non-tagged histone variants, we fused the HA-TEVsite-MYC sensor to Hht2 in YGY663 (in which only Hht1 is tagged), resulting in the H3 double-tagged replicate strains YGY672 and YGY673.…”

Section: Methodsmentioning

confidence: 99%

“…The 400 ul supernatant was divided into 4 separate wells of a 96-well LoBind Eppendorf plate as follows: 110 ul lysate for anti-HA IP (added 10 ul of 12CA5 hybridoma supernatant), 110 ul lysate for anti-myc (added 10 ul of 9E10 hybridoma supernatant), 70 ul lysate for anti-H3K9ac (added 5ug of ab4441) and 70 ul for anti H3K56ac (added 10 ul of Rabbit polyclonal anti-H3K56ac kindly provided by Alain Verreault (Masumoto et al 2005)). Sequencing libraries were prepared as in (Yaakov et al 2021). Full time courses were repeated for all strains: wildtype cells n=4 for myc & HA, n=2 for H3K9ac and H3K56ac.…”

Section: Methodsmentioning

confidence: 99%

“…(added 5ug of ab4441) and 70 ul for anti H3K56ac (added 10 ul of Rabbit polyclonal anti-H3K56ac kindly provided by Alain Verreault (Masumoto et al 2005)). Sequencing libraries were prepared as in (Yaakov et al 2021). Full time courses were repeated for all strains: wildtype cells n=4 for myc & HA, n=2 for H3K9ac and H3K56ac.…”

Section: Chromatin Immunoprecipitation (Chip-seq)mentioning

confidence: 99%

“…These therefore involve temporal perturbations and are subject to significant time delays from histone labeling to exchange measurements, limiting their use during rapid dynamic processes such as DNA replication (Ahmad and Henikoff 2002; Keppler et al 2004; Schermer et al 2005; Dion et al 2007; Rufiange et al 2007; Kaplan et al 2008; Verzijlbergen et al 2010; Radman-Livaja et al 2011; Verzijlbergen et al 2011; Smolle et al 2012; Venkatesh et al 2012; Das and Tyler 2013; Huang et al 2013; Kraushaar et al 2013; Ha et al 2014; Yildirim et al 2014; Sadeghi et al 2015; Siwek et al 2018). Recently, we developed a new histone exchange sensor that alleviates the need for pulse-chase by using genetically encoded exchange sensors, and allows for exchange measurements during a dynamic process using a single sample (Yaakov et al 2021). Here, we use wild type and mutant strains carrying an H3 exchange sensor to define the time-resolved, genome-wide H3 exchange pattern during slowed replication.…”

Section: Introductionmentioning

confidence: 99%

“…Figure1) Chromatin replication dynamics during slowed S phase A, B) Profiling histone H3 modifications and exchange dynamics during slowed replication: Time-resolved Chromatin ImmunoPrecipitation (ChIP-seq) experiments were performed to follow replication in cells synchronously released into hydroxyurea (HU) to slow replication. H3 abundance (HA), H3 incorporation (myc, see(Yaakov et al, 2021)) and H3K56 & H3K9 acetylations were measured (A). Replication dynamics are captured by the change in read coverage, shown here on a segment of Chromosome X (B).…”

mentioning

confidence: 99%

See 4 more Smart Citations

Rtt109 promotes nucleosome replacement ahead of the replicating fork

Jonas

Yaakov

Barkai

2022

Preprint

Self Cite

View full text Add to dashboard Cite

DNA replication perturbs chromatin by triggering the eviction, replacement and incorporation of nucleosomes. How this dynamic is orchestrated in time and space is poorly understood. Here, we apply a recently established sensor for histone exchange to follow the time-resolved histone H3 exchange profile in budding yeast cells undergoing synchronous replication. We find that new histones are incorporated not only behind, but also ahead of the replication fork. We provide evidence that Rtt109, the S phase-specific acetyltransferase, stabilizes nucleosomes behind the fork, but promotes H3 replacement ahead of the fork. Unexpectedly, increased replacement ahead of the fork is independent of the primary Rtt109 acetylation target H3K56, and rather results from Rtt109 activity towards the H3 N-terminus. Our results suggest that selective incorporation of differentially modified H3s behind and ahead of the replication fork results in opposing effects on histone exchange, which may contribute to genome stability by overcoming replication-associated challenges.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Chromatin Immunoprecipitation (Chip-seq)mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Rtt109 promotes nucleosome replacement ahead of the replicating fork

Jonas

Yaakov

Barkai

2022

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

SETD2: from chromatin modifier to multipronged regulator of the genome and beyond

Molenaar

Leeuwen

2022

Cell. Mol. Life Sci.

View full text Add to dashboard Cite

Histone modifying enzymes play critical roles in many key cellular processes and are appealing proteins for targeting by small molecules in disease. However, while the functions of histone modifying enzymes are often linked to epigenetic regulation of the genome, an emerging theme is that these enzymes often also act by non-catalytic and/or non-epigenetic mechanisms. SETD2 (Set2 in yeast) is best known for associating with the transcription machinery and methylating histone H3 on lysine 36 (H3K36) during transcription. This well-characterized molecular function of SETD2 plays a role in fine-tuning transcription, maintaining chromatin integrity, and mRNA processing. Here we give an overview of the various molecular functions and mechanisms of regulation of H3K36 methylation by Set2/SETD2. These fundamental insights are important to understand SETD2’s role in disease, most notably in cancer in which SETD2 is frequently inactivated. SETD2 also methylates non-histone substrates such as α-tubulin which may promote genome stability and contribute to the tumor-suppressor function of SETD2. Thus, to understand its role in disease, it is important to understand and dissect the multiple roles of SETD2 within the cell. In this review we discuss how histone methylation by Set2/SETD2 has led the way in connecting histone modifications in active regions of the genome to chromatin functions and how SETD2 is leading the way to showing that we also have to look beyond histones to truly understand the physiological role of an ‘epigenetic’ writer enzyme in normal cells and in disease.

show abstract