56 research outputs found
A map of human PRDM9 binding provides evidence for novel behaviors of PRDM9 and other zinc-finger proteins in meiosis.
PRDM9 binding localizes almost all meiotic recombination sites in humans and mice. However, most PRDM9-bound loci do not become recombination hotspots. To explore factors that affect binding and subsequent recombination outcomes, we mapped human PRDM9 binding sites in a transfected human cell line and measured PRDM9-induced histone modifications. These data reveal varied DNA-binding modalities of PRDM9. We also find that human PRDM9 frequently binds promoters, despite their low recombination rates, and it can activate expression of a small number of genes including CTCFL and VCX. Furthermore, we identify specific sequence motifs that predict consistent, localized meiotic recombination suppression around a subset of PRDM9 binding sites. These motifs strongly associate with KRAB-ZNF protein binding, TRIM28 recruitment, and specific histone modifications. Finally, we demonstrate that, in addition to binding DNA, PRDM9's zinc fingers also mediate its multimerization, and we show that a pair of highly diverged alleles preferentially form homo-multimers
Using population admixture to help complete maps of the human genome
Tens of millions of base pairs of euchromatic human genome sequence, including many protein-coding genes, have no known location in the human genome. We describe an approach for localizing the human genome's missing pieces by utilizing the patterns of genome sequence variation created by population admixture. We mapped the locations of 70 scaffolds spanning four million base pairs of the human genome's unplaced euchromatic sequence, including more than a dozen protein-coding genes, and identified eight large novel inter-chromosomal segmental duplications. We find that most of these sequences are hidden in the genome's heterochromatin, particularly its pericentromeric regions. Many cryptic, pericentromeric genes are expressed in RNA and have been maintained intact for millions of years while their expression patterns diverged from those of paralogous genes elsewhere in the genome. We describe how knowledge of the locations of these sequences can inform disease association and genome biology studies
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Single-cell and single-molecule methods for mapping protein-DNA interactions
The same two meters of DNA is carefully packed into the nucleus of nearly every cell in a human’s body, where it encodes essentially all of the complex information required to build a complete human being. However, DNA by itself cannot give rise to life; it must be decoded and maintained by specialized macromolecules, including proteins that read, regulate, replicate, recombine, and repair DNA. Mapping where and how these life-giving proteins interact with DNA can provide key insights into how they function or malfunction in healthy and diseased cells. High-throughput DNA sequencing technologies form the basis of several powerful methods for mapping protein-DNA interactions across the genome, but they often require researchers to blend together many thousands or millions of cells to provide enough material to make an accurate measurement. Due to this blending, these bulk methods cannot capture the dynamic and heterogeneous nature of protein-DNA interactions as they regulate the genome in individual cells. While newer methods are beginning to enable protein-DNA mapping in single cells, they are incompatible with high-resolution microscopy, which can provide rich orthogonal information about nuclear organization and other complex phenotypes in single cells. Furthermore, existing protein-DNA mapping approaches fail almost completely within highly repetitive DNA sequences, which constitute roughly 5-10% of the human genome and play indispensable roles in maintaining genome stability.In this body of work, I have developed two new technologies to address each of these limitations in turn. Firstly, I designed an integrated microfluidic platform (μDamID) that combines high-resolution imaging and sequencing information in the same single cells, allowing for the joint analysis of the nuclear localization, sequence identity, and variability of protein-DNA interactions in single cells. Secondly, I worked collaboratively to develop DiMeLo-seq (Directed Methylation with Long-read sequencing), which uses cutting-edge DNA sequencing technologies to map protein-DNA interactions on long, single molecules of DNA that retain endogenous DNA methylation marks and can be mapped to highly repetitive regions of the genome. Together, these new methods expand the toolkit available to researchers to study the fundamental processes that regulate the genome, with the potential to enhance our understanding of embryo development, stem cell differentiation, and diseases resulting from genome misregulation
A classical revival: Human satellite DNAs enter the genomics era.
The classical human satellite DNAs, also referred to as human satellites 1, 2 and 3 (HSat1, HSat2, HSat3, or collectively HSat1-3), occur on most human chromosomes as large, pericentromeric tandem repeat arrays, which together constitute roughly 3% of the human genome (100 megabases, on average). Even though HSat1-3 were among the first human DNA sequences to be isolated and characterized at the dawn of molecular biology, they have remained almost entirely missing from the human genome reference assembly for 20 years, hindering studies of their sequence, regulation, and potential structural roles in the nucleus. Recently, the Telomere-to-Telomere Consortium produced the first truly complete assembly of a human genome, paving the way for new studies of HSat1-3 with modern genomic tools. This review provides an account of the history and current understanding of HSat1-3, with a view towards future studies of their evolution and roles in health and disease
Novel genetic and molecular properties of meiotic recombination protein PRDM9
Meiotic recombination is a fundamental biological process in sexually reproducing organisms, enabling offspring to inherit novel combinations of mutations, and ensuring even segregation of chromosomes into gametes. Recombination is initiated by programmed Double Strand Breaks (DSBs), the genomic locations of which are determined in most mammals by PRDM9, a rapidly evolving DNA-binding protein. In crosses between different mouse subspecies, certain Prdm9 alleles cause infertility in hybrid males, implying a critical role in fertility and speciation. Upon binding to DNA, PRDM9 deposits a histone modification (H3K4me3) typically found in the promoters of expressed genes, suggesting that binding might alter the expression of nearby genes. Many other questions have remained about how PRDM9 initiates recombination, how it causes speciation, and why it evolves so rapidly. This body of work investigates these questions using complementary experimental and analytical methodologies. By generating a map of human PRDM9 binding sites and applying novel sequence analysis methods, I uncovered new DNA-binding modalities of PRDM9 and identified sequence-independent factors that predict binding and recombination outcomes. I also confirmed that PRDM9 can affect gene expression by binding to promoters, identifying candidate regulatory targets in meiosis. Furthermore, I showed that PRDM9âÃôs DNA-binding domain also mediates strong protein-protein interactions that produce PRDM9 multimers, which may play an important functional role. Finally, by generating high-resolution maps of PRDM9 binding in hybrid mice, I provide evidence for a mechanism to explain PRDM9-mediated speciation as a consequence of the joint evolution of PRDM9 and its binding targets. This work reveals that PRDM9 binding on one chromosome strongly impacts DSB formation and/or repair on the homologue, suggesting a novel role for PRDM9 in promoting efficient homology search and DSB repair, both critical for meiotic progression and fertility. One consequence is that PRDM9 may play a wider role in mammalian speciation.</p
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