Charles G. Danko

Complete Khoisan and Bantu genomes from southern Africa

The genetic structure of the indigenous hunter-gatherer peoples of southern Africa, the oldest known lineage of modern human, is important for understanding human diversity. Studies based on mitochondrial and small sets of nuclear markers have shown that these hunter-gatherers, known as Khoisan, San, or Bushmen, are genetically divergent from other humans. However, until now, fully sequenced human genomes have been limited to recently diverged populations. Here we present the complete genome sequences of an indigenous hunter-gatherer from the Kalahari Desert and a Bantu from southern Africa, as well as protein-coding regions from an additional three hunter-gatherers from disparate regions of the Kalahari. We characterize the extent of whole-genome and exome diversity among the five men, reporting 1.3 million novel DNA differences genome-wide, including 13,146 novel amino acid variants. In terms of nucleotide substitutions, the Bushmen seem to be, on average, more different from each other than, for example, a European and an Asian. Observed genomic differences between the hunter-gatherers and others may help to pinpoint genetic adaptations to an agricultural lifestyle. Adding the described variants to current databases will facilitate inclusion of southern Africans in medical research efforts, particularly when family and medical histories can be correlated with genome-wide data.

A Rapid, Extensive, and Transient Transcriptional Response to Estrogen Signaling in Breast Cancer Cells

We report the immediate effects of estrogen signaling on the transcriptome of breast cancer cells using global run-on and sequencing (GRO-seq). The data were analyzed using a new bioinformatic approach that allowed us to identify transcripts directly from the GRO-seq data. We found that estrogen signaling directly regulates a strikingly large fraction of the transcriptome in a rapid, robust, and unexpectedly transient manner. In addition to protein-coding genes, estrogen regulates the distribution and activity of all three RNA polymerases and virtually every class of noncoding RNA that has been described to date. We also identified a large number of previously undetected estrogen-regulated intergenic transcripts, many of which are found proximal to estrogen receptor binding sites. Collectively, our results provide the most comprehensive measurement of the primary and immediate estrogen effects to date and a resource for understanding rapid signal-dependent transcription in other systems.

Bayesian inference of ancient human demography from individual genome sequences

Whole-genome sequences provide a rich source of information about human evolution. Here we describe an effort to estimate key evolutionary parameters based on the whole-genome sequences of six individuals from diverse human populations. We used a Bayesian, coalescent-based approach to obtain information about ancestral population sizes, divergence times and migration rates from inferred genealogies at many neutrally evolving loci across the genome. We introduce new methods for accommodating gene flow between populations and integrating over possible phasings of diploid genotypes. We also describe a custom pipeline for genotype inference to mitigate biases from heterogeneous sequencing technologies and coverage levels. Our analysis indicates that the San population of southern Africa diverged from other human populations approximately 108–157 thousand years ago, that Eurasians diverged from an ancestral African population 38–64 thousand years ago, and that the effective population size of the ancestors of all modern humans was ∼9,000.

Analysis of nascent RNA identifies a unified architecture of initiation regions at mammalian promoters and enhancers

Despite the conventional distinction between them, promoters and enhancers share many features in mammals, including divergent transcription and similar modes of transcription factor binding. Here we examine the architecture of transcription initiation through comprehensive mapping of transcription start sites (TSSs) in human lymphoblastoid B cell (GM12878) and chronic myelogenous leukemic (K562) ENCODE Tier 1 cell lines. Using a nuclear run-on protocol called GRO-cap, which captures TSSs for both stable and unstable transcripts, we conduct detailed comparisons of thousands of promoters and enhancers in human cells. These analyses identify a common architecture of initiation, including tightly spaced (110 bp apart) divergent initiation, similar frequencies of core promoter sequence elements, highly positioned flanking nucleosomes and two modes of transcription factor binding. Post-initiation transcript stability provides a more fundamental distinction between promoters and enhancers than patterns of histone modification and association of transcription factors or co-activators. These results support a unified model of transcription initiation at promoters and enhancers.

Signaling Pathways Differentially Affect RNA Polymerase II Initiation, Pausing, and Elongation Rate in Cells

RNA polymerase II (Pol II) transcribes hundreds of kilobases of DNA, limiting the production of mRNAs and lncRNAs. We used global run-on sequencing (GRO-seq) to measure the rates of transcription by Pol II following gene activation. Elongation rates vary as much as 4-fold at different genomic loci and in response to two distinct cellular signaling pathways (i.e., 17β-estradiol [E2] and TNF-α). The rates are slowest near the promoter and increase during the first ~15 kb transcribed. Gene body elongation rates correlate with Pol II density, resulting in systematically higher rates of transcript production at genes with higher Pol II density. Pol II dynamics following short inductions indicate that E2 stimulates gene expression by increasing Pol II initiation, whereas TNF-α reduces Pol II residence time at pause sites. Collectively, our results identify previously uncharacterized variation in the rate of transcription and highlight elongation as an important, variable, and regulated rate-limiting step during transcription.

A unified architecture of transcriptional regulatory elements

Gene expression is precisely controlled in time and space through the integration of signals that act at gene promoters and gene-distal enhancers. Classically, promoters and enhancers are considered separate classes of regulatory elements, often distinguished by histone modifications. However, recent studies have revealed broad similarities between enhancers and promoters, blurring the distinction: active enhancers often initiate transcription, and some gene promoters have the potential to enhance transcriptional output of other promoters. Here, we propose a model in which promoters and enhancers are considered a single class of functional element, with a unified architecture for transcription initiation. The context of interacting regulatory elements and the surrounding sequences determine local transcriptional output as well as the enhancer and promoter activities of individual elements.

Identification of active transcriptional regulatory elements from GRO-seq data

Modifications to the global run-on and sequencing (GRO-seq) protocol that enrich for 5′-capped RNAs can be used to reveal active transcriptional regulatory elements (TREs) with high accuracy. Here, we introduce discriminative regulatory-element detection from GRO-seq (dREG), a sensitive machine learning method that uses support vector regression to identify active TREs from GRO-seq data without requiring cap-based enrichment (https://github.com/Danko-Lab/dREG/). This approach allows TREs to be assayed together with gene expression levels and other transcriptional features in a single experiment. Predicted TREs are more enriched for several marks of transcriptional activation—including expression quantitative trait loci, disease-associated polymorphisms, acetylated histone 3 lysine 27 (H3K27ac) and transcription factor binding—than those identified by alternative functional assays. Using dREG, we surveyed TREs in eight human cell types and provide new insights into global patterns of TRE function.

Gene regulation by CcpA and catabolite repression explored by RNA-Seq in Streptococcus mutans

A bacterial transcriptome of the primary etiological agent of human dental caries, Streptococcus mutans, is described here using deep RNA sequencing. Differential expression profiles of the transcriptome in the context of carbohydrate source, and of the presence or absence of the catabolite control protein CcpA, revealed good agreement with previously-published DNA microarrays. In addition, RNA-seq considerably expanded the repertoire of DNA sequences that showed statistically-significant changes in expression as a function of the presence of CcpA and growth carbohydrate. Novel mRNAs and small RNAs were identified, some of which were differentially expressed in conditions tested in this study, suggesting that the function of the S. mutans CcpA protein and the influence of carbohydrate sources has a more substantial impact on gene regulation than previously appreciated. Likewise, the data reveal that the mechanisms underlying prioritization of carbohydrate utilization are more diverse than what is currently understood. Collectively, this study demonstrates the validity of RNA-seq as a potentially more-powerful alternative to DNA microarrays in studying gene regulation in S. mutans because of the capacity of this approach to yield a more precise landscape of transcriptomic changes in response to specific mutations and growth conditions.

Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq)

We provide a protocol for precision nuclear run-on sequencing (PRO-seq) and its variant, PRO-cap, which map the location of active RNA polymerases (PRO-seq) or transcription start sites (TSSs) (PRO-cap) genome-wide at high resolution. The density of RNA polymerases at a particular genomic locus directly reflects the level of nascent transcription at that region. Nuclei are isolated from cells and, under nuclear run-on conditions, transcriptionally engaged RNA polymerases incorporate one or, at most, a few biotin-labeled nucleotide triphosphates (biotin-NTPs) into the 3′ end of nascent RNA. The biotin-labeled nascent RNA is used to prepare sequencing libraries, which are sequenced from the 3′ end to provide high-resolution positional information for the RNA polymerases. PRO-seq provides much higher sensitivity than ChIP-seq, and it generates a much larger fraction of usable sequence reads than ChIP-seq or NET-seq (native elongating transcript sequencing). Similarly to NET-seq, PRO-seq maps the RNA polymerase at up to base-pair resolution with strand specificity, but unlike NET-seq it does not require immunoprecipitation. With the protocol provided here, PRO-seq (or PRO-cap) libraries for high-throughput sequencing can be generated in 4–5 working days. The method has been applied to human, mouse, Drosophila melanogaster and Caenorhabditis elegans cells and, with slight modifications, to yeast.

GAGA Factor Maintains Nucleosome-Free Regions and Has a Role in RNA Polymerase II Recruitment to Promoters

Previous studies have shown that GAGA Factor (GAF) is enriched on promoters with paused RNA Polymerase II (Pol II), but its genome-wide function and mechanism of action remain largely uncharacterized. We assayed the levels of transcriptionally-engaged polymerase using global run-on sequencing (GRO-seq) in control and GAF-RNAi Drosophila S2 cells and found promoter-proximal polymerase was significantly reduced on a large subset of paused promoters where GAF occupancy was reduced by knock down. These promoters show a dramatic increase in nucleosome occupancy upon GAF depletion. These results, in conjunction with previous studies showing that GAF directly interacts with nucleosome remodelers, strongly support a model where GAF directs nucleosome displacement at the promoter and thereby allows the entry Pol II to the promoter and pause sites. This action of GAF on nucleosomes is at least partially independent of paused Pol II because intergenic GAF binding sites with little or no Pol II also show GAF-dependent nucleosome displacement. In addition, the insulator factor BEAF, the BEAF-interacting protein Chriz, and the transcription factor M1BP are strikingly enriched on those GAF-associated genes where pausing is unaffected by knock down, suggesting insulators or the alternative promoter-associated factor M1BP protect a subset of GAF-bound paused genes from GAF knock-down effects. Thus, GAF binding at promoters can lead to the local displacement of nucleosomes, but this activity can be restricted or compensated for when insulator protein or M1BP complexes also reside at GAF bound promoters.