David MacAlpine

Overview:

Our laboratory is interested in understanding the mechanisms by which the molecular architecture of the chromosome regulates fundamental biological processes such as replication and transcription. Specifically, how are replication, transcription and chromatin modification coordinated on a genomic scale to maintain genomic stability? We are addressing this question by using genomic, computational and biochemical approaches in the model organism Drosophila melanogaster.

DNA replication is an essential cell cycle event required for the timely and accurate duplication of chromosomes. Replication initiates at multiple sites (called origins of replication) distributed across each chromosome. The failure to properly regulate origin selection and activation may result in catastrophic genomic instability and potentially tumorigenesis. Recent metazoan genomic studies have demonstrated a correlation between time of DNA replication and transcriptional activity, with actively transcribed regions of the genome being replicated early. However, the underlying mechanism of this correlation remains unclear. By systematically characterizing the replication dynamics of multiple cell types, each with distinct transcriptional programs, we will be in a position to understand how these processes are coordinated.

Another goal of the laboratory is to identify the chromosomal features that direct and regulate metazoan DNA replication. Origins of DNA replication are marked by the formation of multi-protein complex, called the preRC. Despite conservation of the proteins that comprise the preRC in all eukaryotes, very little is known about the sequence elements required for the selection and regulation of metazoan origins. We are using genomic tiling microarrays to systematically map all the sites of preRC assembly in the Drosophila genome. The high resolution mapping of thousands of replication origins will provide an unprecedented opportunity to use both computational approaches and comparative genomics to identify cis-acting elements that may regulate replication.

Positions:

Professor of Pharmacology and Cancer Biology

Pharmacology & Cancer Biology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

Ph.D. 2001

University of Texas Southwestern Medical Center, Medical School

Grants:

Illumina Hi-Seq 2000 Sequencing System

Administered By
Institutes and Centers
Awarded By
National Institutes of Health
Role
Major User
Start Date
End Date

Defining RAS isoform- and mutation-specific roles in oncogenesis

Administered By
Pharmacology & Cancer Biology
Awarded By
University of North Carolina - Chapel Hill
Role
Collaborating Investigator
Start Date
End Date

Novel tissue injury regulation at an organ-organ junction

Administered By
Pharmacology & Cancer Biology
Awarded By
National Institutes of Health
Role
Collaborator
Start Date
End Date

Non-Canonical Responses to DNA damage in Drosophila Polyploid Cells

Administered By
Pharmacology & Cancer Biology
Awarded By
National Institutes of Health
Role
Collaborator
Start Date
End Date

High-performance Computing System for Bioinformatics

Administered By
Institutes and Centers
Awarded By
National Institutes of Health
Role
Major User
Start Date
End Date

Publications:

Disruption of origin chromatin structure by helicase activation in the absence of DNA replication.

Prior to initiation of DNA replication, the eukaryotic helicase, Mcm2-7, must be activated to unwind DNA at replication start sites in early S phase. To study helicase activation within origin chromatin, we constructed a conditional mutant of the polymerase α subunit Cdc17 (or Pol1) to prevent priming and block replication. Recovery of these cells at permissive conditions resulted in the generation of unreplicated gaps at origins, likely due to helicase activation prior to replication initiation. We used micrococcal nuclease (MNase)-based chromatin occupancy profiling under restrictive conditions to study chromatin dynamics associated with helicase activation. Helicase activation in the absence of DNA replication resulted in the disruption and disorganization of chromatin, which extends up to 1 kb from early, efficient replication origins. The CMG holohelicase complex also moves the same distance out from the origin, producing single-stranded DNA that activates the intra-S-phase checkpoint. Loss of the checkpoint did not regulate the progression and stalling of the CMG complex but rather resulted in the disruption of chromatin at both early and late origins. Finally, we found that the local sequence context regulates helicase progression in the absence of DNA replication, suggesting that the helicase is intrinsically less processive when uncoupled from replication.
Authors
MLA Citation
Hoffman, Rachel A., et al. “Disruption of origin chromatin structure by helicase activation in the absence of DNA replication.Genes Dev, vol. 35, no. 19–20, Oct. 2021, pp. 1339–55. Pubmed, doi:10.1101/gad.348517.121.
URI
https://scholars.duke.edu/individual/pub1497488
PMID
34556529
Source
pubmed
Published In
Genes Dev
Volume
35
Published Date
Start Page
1339
End Page
1355
DOI
10.1101/gad.348517.121

RoboCOP: jointly computing chromatin occupancy profiles for numerous factors from chromatin accessibility data.

Chromatin is a tightly packaged structure of DNA and protein within the nucleus of a cell. The arrangement of different protein complexes along the DNA modulates and is modulated by gene expression. Measuring the binding locations and occupancy levels of different transcription factors (TFs) and nucleosomes is therefore crucial to understanding gene regulation. Antibody-based methods for assaying chromatin occupancy are capable of identifying the binding sites of specific DNA binding factors, but only one factor at a time. In contrast, epigenomic accessibility data like MNase-seq, DNase-seq, and ATAC-seq provide insight into the chromatin landscape of all factors bound along the genome, but with little insight into the identities of those factors. Here, we present RoboCOP, a multivariate state space model that integrates chromatin accessibility data with nucleotide sequence to jointly compute genome-wide probabilistic scores of nucleosome and TF occupancy, for hundreds of different factors. We apply RoboCOP to MNase-seq and ATAC-seq data to elucidate the protein-binding landscape of nucleosomes and 150 TFs across the yeast genome, and show that our model makes better predictions than existing methods. We also compute a chromatin occupancy profile of the yeast genome under cadmium stress, revealing chromatin dynamics associated with transcriptional regulation.
Authors
Mitra, S; Zhong, J; Tran, TQ; MacAlpine, DM; Hartemink, AJ
MLA Citation
Mitra, Sneha, et al. “RoboCOP: jointly computing chromatin occupancy profiles for numerous factors from chromatin accessibility data.Nucleic Acids Research, vol. 49, no. 14, Aug. 2021, pp. 7925–38. Epmc, doi:10.1093/nar/gkab553.
URI
https://scholars.duke.edu/individual/pub1488871
PMID
34255854
Source
epmc
Published In
Nucleic Acids Research
Volume
49
Published Date
Start Page
7925
End Page
7938
DOI
10.1093/nar/gkab553

Stochastic initiation of DNA replication across the human genome.

Wang et al. (2021) comprehensively map DNA replication initiation events across the human genome using single-molecule optical resolution mapping and find that initiation events are randomly distributed across broad initiation zones that are only utilized in a stochastic fashion across a population of cells.
Authors
MLA Citation
MacAlpine, David M. “Stochastic initiation of DNA replication across the human genome.Mol Cell, vol. 81, no. 14, July 2021, pp. 2873–74. Pubmed, doi:10.1016/j.molcel.2021.06.022.
URI
https://scholars.duke.edu/individual/pub1488927
PMID
34270943
Source
pubmed
Published In
Mol Cell
Volume
81
Published Date
Start Page
2873
End Page
2874
DOI
10.1016/j.molcel.2021.06.022

Linking the dynamics of chromatin occupancy and transcription with predictive models.

Though the sequence of the genome within each eukaryotic cell is essentially fixed, it exists within a complex and changing chromatin state. This state is determined, in part, by the dynamic binding of proteins to the DNA. These proteins-including histones, transcription factors (TFs), and polymerases-interact with one another, the genome, and other molecules to allow the chromatin to adopt one of exceedingly many possible configurations. Understanding how changing chromatin configurations associate with transcription remains a fundamental research problem. We sought to characterize at high spatiotemporal resolution the dynamic interplay between transcription and chromatin in response to cadmium stress. Whereas gene regulatory responses to environmental stress in yeast have been studied, how the chromatin state changes and how those changes connect to gene regulation remain unexplored. By combining MNase-seq and RNA-seq data, we found chromatin signatures of transcriptional activation and repression involving both nucleosomal and TF-sized DNA-binding factors. Using these signatures, we identified associations between chromatin dynamics and transcriptional regulation, not only for known cadmium response genes, but across the entire genome, including antisense transcripts. Those associations allowed us to develop generalizable models that predict dynamic transcriptional responses on the basis of dynamic chromatin signatures.
Authors
Tran, TQ; MacAlpine, HK; Tripuraneni, V; Mitra, S; MacAlpine, DM; Hartemink, AJ
MLA Citation
Tran, Trung Q., et al. “Linking the dynamics of chromatin occupancy and transcription with predictive models.Genome Res, vol. 31, no. 6, June 2021, pp. 1035–46. Pubmed, doi:10.1101/gr.267237.120.
URI
https://scholars.duke.edu/individual/pub1480594
PMID
33893157
Source
pubmed
Published In
Genome Res
Volume
31
Published Date
Start Page
1035
End Page
1046
DOI
10.1101/gr.267237.120

Local nucleosome dynamics and eviction following a double-strand break are reversible by NHEJ-mediated repair in the absence of DNA replication.

We interrogated at nucleotide resolution the spatiotemporal order of chromatin changes that occur immediately following a site-specific double-strand break (DSB) upstream of the PHO5 locus and its subsequent repair by nonhomologous end joining (NHEJ). We observed the immediate eviction of a nucleosome flanking the break and the repositioning of adjacent nucleosomes away from the break. These early chromatin events were independent of the end-processing Mre11-Rad50-Xrs2 (MRX) complex and preceded the MRX-dependent broad eviction of histones and DNA end-resectioning that extends up to ∼8 kb away from the break. We also examined the temporal dynamics of NHEJ-mediated repair in a G1-arrested population. Concomitant with DSB repair by NHEJ, we observed the redeposition and precise repositioning of nucleosomes at their originally occupied positions. This re-establishment of the prelesion chromatin landscape suggests that a DNA replication-independent mechanism exists to preserve epigenome organization following DSB repair.
Authors
Tripuraneni, V; Memisoglu, G; MacAlpine, HK; Tran, TQ; Zhu, W; Hartemink, AJ; Haber, JE; MacAlpine, DM
MLA Citation
Tripuraneni, Vinay, et al. “Local nucleosome dynamics and eviction following a double-strand break are reversible by NHEJ-mediated repair in the absence of DNA replication.Genome Res, vol. 31, no. 5, May 2021, pp. 775–88. Pubmed, doi:10.1101/gr.271155.120.
URI
https://scholars.duke.edu/individual/pub1478350
PMID
33811083
Source
pubmed
Published In
Genome Res
Volume
31
Published Date
Start Page
775
End Page
788
DOI
10.1101/gr.271155.120