Iestyn Whitehouse: Publications

Iestyn Whitehouse: Publications


Balancing act of a leading strand DNA polymerase-specific domain and its exonuclease domain promotes genome-wide sister replication fork symmetry. Meng X, Claussin C, Regan-Mochrie G, Whitehouse I, Zhao X.Genes Dev. 2023 Jan 26. doi: 10.1101/gad.350054.122.PMID: 36702483

Single-molecule mapping of replisome progression. Claussin C, Vazquez J, Whitehouse I.Mol Cell. 2022 Apr 7;82(7):1372-1382.e4. doi: 10.1016/j.molcel.2022.02.010. Epub 2022 Mar 2.PMID: 35240057

DNA replication through a chromatin environment. Bellush JM, Whitehouse I.Philos Trans R Soc Lond B Biol Sci. 2017 Oct 5;372(1731):20160287. doi: 10.1098/rstb.2016.0287.PMID: 28847824

DNA-mediated association of two histone-bound complexes of yeast Chromatin Assembly Factor-1 (CAF-1) drives tetrasome assembly in the wake of DNAreplication. Mattiroli F, Gu Y, Yadav T, Balsbaugh JL, Harris MR, Findlay ES, Liu Y, Radebaugh CA, Stargell LA, Ahn NG, Whitehouse I, Luger K.Elife. 2017 Mar 18;6:e22799. doi: 10.7554/eLife.22799.PMID: 28315523

Spatiotemporal coupling and decoupling of gene transcription with DNAreplication origins during embryogenesis in C. elegans. Pourkarimi E, Bellush JM, Whitehouse I.Elife. 2016 Dec 23;5:e21728. doi: 10.7554/eLife.21728.PMID: 28009254

Chromatin Constrains the Initiation and Elongation of DNA Replication. Devbhandari S, Jiang J, Kumar C, Whitehouse I, Remus D.Mol Cell. 2017 Jan 5;65(1):131-141. doi: 10.1016/j.molcel.2016.10.035. Epub 2016 Dec 15.PMID: 27989437

Replication-Coupled Nucleosome Assembly and Positioning by ATP-Dependent Chromatin-Remodeling Enzymes. Yadav T, Whitehouse I.Cell Rep. 2016 Apr 26;15(4):715-723. doi: 10.1016/j.celrep.2016.03.059. Epub 2016 Apr 14.PMID: 27149855 

Nucleosome repositioning underlies dynamic gene expression. Nocetti N, Whitehouse I.Genes Dev. 2016 Mar 15;30(6):660-72. doi: 10.1101/gad.274910.115. Epub 2016 Mar 10.PMID: 26966245

Post-licensing Specification of Eukaryotic Replication Origins by Facilitated Mcm2-7 Sliding along DNA. Gros J, Kumar C, Lynch G, Yadav T, Whitehouse I, Remus D.Mol Cell. 2015 Dec 3;60(5):797-807. doi: 10.1016/j.molcel.2015.10.022. Epub 2015 Nov 19.PMID: 26656162

Detection and Sequencing of Okazaki Fragments in S. cerevisiae. Smith DJ, Yadav T, Whitehouse I.Methods Mol Biol. 2015;1300:141-53. doi: 10.1007/978-1-4939-2596-4_10.PMID: 25916711

Quantitative, genome-wide analysis of eukaryotic replication initiation and termination. McGuffee SR, Smith DJ, Whitehouse I.Mol Cell. 2013 Apr 11;50(1):123-35. doi: 10.1016/j.molcel.2013.03.004. Epub 2013 Apr 4.PMID: 23562327

Chromatin dynamics at the replication fork: there’s more to life than histones. Whitehouse I, Smith DJ.Curr Opin Genet Dev. 2013 Apr;23(2):140-6. doi: 10.1016/j.gde.2012.12.007. Epub 2013 Jan 21.PMID: 23347596  Review.

An Eco1-independent sister chromatid cohesion establishment pathway in S. cerevisiae.Borges V, Smith DJ, Whitehouse I, Uhlmann F.Chromosoma. 2013 Mar;122(1-2):121-34. doi: 10.1007/s00412-013-0396-y. Epub 2013 Jan 20.PMID: 23334284

Intrinsic coupling of lagging-strand synthesis to chromatin assembly. Smith DJ, Whitehouse I.Nature. 2012 Mar 14;483(7390):434-8. doi: 10.1038/nature10895.PMID: 22419157

Overview of recent significant publications

Single-molecule mapping of replisome progression

Clémence Claussin, Jacob Vazquez, Iestyn Whitehouse

Mol Cell. (2022) Apr 7;82(7):1372-1382.e4

Cellular processes such as DNA replication are highly dynamic and display significant variation between cells. Such variation ensures that population-based approaches are incapable of detecting and quantifying rare or stochastic events that may occur. In the case of DNA replication, the replisome encounters numerous obstacles along the chromatin template but how these obstacles are overcome is poorly understood. In this paper we developed a novel approach to map DNA replication with single molecule precision. Our approach, called replicon-seq, utilizes Nanopore sequencing to capture and sequence nascent DNA molecules as they are being elongated. Because Nanopore is not limited in read-length, we can analyze nascent DNA molecules at various stages of synthesis. We show that replicon seq can provide unprecedented view of all stages of the replication reaction and so offers a unique methodology to address the precise mechanisms by which chromosomes are replicated.

Spatiotemporal coupling and decoupling of gene transcription with DNA replication origins during embryogenesis in C. elegans. Pourkarimi, E., J.M. Bellush, and I. Whitehouse (2016). Elife, 5.

DNA replication is a fundamental component of embryogenesis, yet how DNA replication is integrated into the transcription and developmental programs is not understood. To address these questions we have generated a genome-wide replication profile of C. elegans embryos; our results represent the first map of DNA replication from a living, multi-cellular organism. We show that histone modifications that define gene enhancers specify the location and efficiency of replication origins – a finding that resolves a longstanding issue regarding the determinants of replication origins within metazoan genomes. The association of gene activity with the replication program has sculpted the C. elegans genome and we show that genes involved in cell division, protein synthesis and growth are clustered near replication origins. Such association underlies a remarkable coordination of transcription and replication: when the first wave of zygotic transcription initiates, it does so in close proximity to pre-defined origins. Such “coupling” of transcription and replication persists through gastrulation but breaks down after the last wave of embryonic cell division when transcription activity gradually shifts from the now inactive origins. Ultimately, in differentiated cells, transcription occurs as far from origins as is possible. Our results are the first to show that the activation and inactivation of replication origins are integrated into the transcriptional program of developing embryos and address longstanding issues regarding the functional relationship between gene activity and DNA replication.

Nucleosome repositioning underlies dynamic gene expression. Nocetti, N., and Whitehouse, I. (2016). Genes Dev 30, 660-672.

Nucleosome repositioning at gene promoters is a fundamental aspect of gene expression. Yet the extent to which nucleosome repositioning occurs as genes are activated or repressed is poorly understood. Utilizing an ultradian cycle in which transcription of >50% of all budding yeast genes is highly synchronized, we found evidence of nucleosome repositioning at thousands of gene promoters as genes are activated and repressed. During activation, nucleosomes are relocated to allow sites of general transcription factor binding and transcription initiation to become accessible. The extent of nucleosome shifting is closely related to the dynamic range of gene transcription and generally related to DNA sequence properties and use of the coactivators TFIID or SAGA. However, dynamic gene expression is not limited to SAGA-regulated promoters and is an inherent feature of most genes. Contrary to widely held views, we present evidence that many so called “housekeeping” genes also utilize nucleosome remodeling during gene activation. While nucleosome repositioning occurs pervasively, we found that a class of genes required for cell growth experience acute nucleosome shifting as cells enter the cell cycle. Significantly, we found that the ATP-dependent chromatin-remodeling complex – SWI/SNF – plays a fundamental role in the expression of growth genes. Our use of a highly synchronized system has allowed us to uncover many events that are transient and therefore have been previously hidden by ensemble studies on mixed cell populations.

Replication-Coupled Nucleosome Assembly and Positioning by ATP-Dependent Chromatin-Remodeling Enzymes. Yadav, T., and Whitehouse, I. (2016). Cell Reports 15, 715-723.

There exists widespread interest in the inheritance of chromatin structures and transmission of chromatin states through generations. However, the re-establishment of mature chromatin after DNA replication remains poorly understood. A significant obstacle is the paucity of assays with sufficient spatiotemporal resolution to capture the transient events that occur at the replication fork in vivo. Thus, important questions remain: when and how are nucleosomes assembled? When are nucleosomes remodeled and by which factors? What are the mechanisms that govern the reestablishment of chromatin structures? We addressed each of these questions and our work provided evidence for the rapid assembly andpositioning of nucleosomes at the replication fork by chromatin remodeling enzymes. Our work offers new insight into the function of ATP dependent remodeling enzymes, whose biological roles are poorly understood. ATP dependent remodeling enzymes are best characterized for their roles in gene transcription; a subset of remodeling enzymes has long been suspected to load nucleosomes during DNA replication, but this has never been demonstrated. We showed that ATP dependent chromatin remodeling enzymes assemble and position nucleosomes on nascent DNA. Furthermore, we provided evidence that nucleosome assembly occurs in a progressive manner. Collectively our findings elucidate a general mechanism for faithful replication of nucleosome positioning, and hence chromatin structure.

Quantitative, genome-wide analysis of eukaryotic replication initiation and termination. McGuffee, S.R., Smith, D.J., and Whitehouse, I. (2013). Mol Cell 50, 123-135.

Sequencing Okazaki fragments provides a means with which to interrogate the dynamics of DNA replication. Analyzing Okazaki fragments from a large asynchronous culture allows us to capture a “snapshot” of DNA replication across the entire genome. Importantly, because the replisome is asymmetric and we sequence Okazaki fragments in a strand specific manner, patterns of DNA replication can be deduced from the asynchronous population. My lab developed computational tools with which to generate the first map of initiation sites at replication origins, we also provide the first quantitative measure of the efficiency of all replication origins and the direction of replication fork progression. Our analysis also provided the first genome-wide map of sites of DNA replication termination. Termination is very poorly understood in eukaryotes; previous reports have suggested that elements such as highly transcribed genes inhibit fork progression and may trigger termination. Contrary to this, our analysis showed that termination is largely a passive phenomenon that typically occurs midway between two adjacent origins – where converging forks meet. Indeed, by artificially altering replication origin firing time we could shift the positions at which termination occurred. Finally, we were able to accurately reconstruct the dynamics of DNA replication across the yeast genome by developing robust computational models.

Intrinsic coupling of lagging-strand synthesis to chromatin assembly.  Smith, D.J., and Whitehouse, I. (2012). Nature 483, 434-438.

How complex chromatin structures are established and maintained through generations is largely unknown. Because histones are rapidly deposited on nascent DNA, we considered whether the synthesis of the lagging strand might be coordinated with nucleosome assembly; if so, studying Okazaki fragments would potentially allow us to investigate nucleosome assembly pathways and also DNA replication in new ways. Surprisingly, eukaryotic Okazaki fragments had never been quantitatively purified from cells and there was little information regarding how they are processed in vivo. Thus, may lab established the first protocols to enrich purify and analyze Okazaki fragments from cells. Our study made several findings: 1, We developed deep sequencing and genomics approaches to precisely define the positions of Okazaki fragments in the yeast genome. 2, We determined that the ends of Okazaki fragments are strongly influenced by the positions of nucleosomes and certain DNA-bound transcription factors – indicating that both nucleosomes and key transcription factors are rapidly assembled onto the nascent DNA. 3, We presented and tested a new model to explain the relationship between Okazaki fragment processing and nucleosomes. The model describes a mechanism whereby nucleosome assembly behind the replication fork promotes Okazaki fragment processing and suggests why chromatin assembly is necessary to maintain genomic integrity.