An in vitro-identified high-affinity nucleosome-positioning signal is capable of transiently positioning a nucleosome in vivo

<p>Abstract</p> <p>Background</p> <p>The physiological function of eukaryotic DNA occurs in the context of nucleosomal arrays that can expose or obscure defined segments of the genome. Certain DNA sequences are capable of strongly positioning a nucleosome <it>in vitro</it>, suggesting the possibility that favorable intrinsic signals might reproducibly structure chromatin segments. As high-throughput sequencing analyses of nucleosome coverage <it>in vitro </it>and <it>in vivo </it>have become possible, a vigorous debate has arisen over the degree to which intrinsic DNA:nucleosome affinities orchestrate the <it>in vivo </it>positions of nucleosomes, thereby controlling physical accessibility of specific sequences in DNA.</p> <p>Results</p> <p>We describe here the <it>in vivo </it>consequences of placing a synthetic high-affinity nucleosome-positioning signal, the 601 sequence, into a DNA plasmid vector in mice. Strikingly, the 601 sequence was sufficient to position nucleosomes during an early phase after introduction of the DNA into the mice (when the plasmid vector transgene was active). This positioning capability was transient, with a loss of strong positioning at a later time point when the transgenes had become silent.</p> <p>Conclusions</p> <p>These results demonstrate an ability of DNA sequences selected solely for nucleosome affinity to organize chromatin <it>in vivo</it>, and the ability of other mechanisms to overcome these interactions in a dynamic nuclear environment.</p

Reassessment of Piwi Binding to the Genome and Piwi Impact on RNA Polymerase II Distribution

Drosophila Piwi was reported by Huang et al. (2013) to be guided by piRNAs to piRNA-complementary sites in the genome, which then recruits Heterochromatin Protein 1a and histone methyltransferase Su(Var)3-9 to the sites. Among additional findings, Huang et al. (2013) also reported Piwi binding sites in the genome and the reduction of RNA polymerase II in euchromatin but its increase in pericentric regions in piwi mutants. Marinov et al. (2015) disputed the validity of the Huang et al. bioinformatic pipeline that led to the last two claims. Here we report our independent reanalysis of the data using current bioinformatic methods. Our reanalysis agrees with Marinov et al. (2015) that Piwi’s genomic targets still remain to be identified, yet confirms the Huang et al. claim that Piwi influences RNA polymerase II distribution in the genome. This Response addresses the Marinov et al. (2015) Matters Arising, published concurrently in Developmental Cell

Transcriptional regulatory control of mammalian nephron progenitors revealed by multi-factor cistromic analysis and genetic studies

Nephron progenitor number determines nephron endowment; a reduced nephron count is linked to the onset of kidney disease. Several transcriptional regulators including Six2, Wt1, Osr1, Sall1, Eya1, Pax2, and Hox11 paralogues are required for specification and/or maintenance of nephron progenitors. However, little is known about the regulatory intersection of these players. Here, we have mapped nephron progenitor-specific transcriptional networks of Six2, Hoxd11, Osr1, and Wt1. We identified 373 multi-factor associated ‘regulatory hotspots’ around genes closely associated with progenitor programs. To examine their functional significance, we deleted ‘hotspot’ enhancer elements for Six2 and Wnt4. Removal of the distal enhancer for Six2 leads to a ~40% reduction in Six2 expression. When combined with a Six2 null allele, progeny display a premature depletion of nephron progenitors. Loss of the Wnt4 enhancer led to a significant reduction of Wnt4 expression in renal vesicles and a mildly hypoplastic kidney, a phenotype also enhanced in combination with a Wnt4 null mutation. To explore the regulatory landscape that supports proper target gene expression, we performed CTCF ChIP-seq to identify insulator-boundary regions. One such putative boundary lies between the Six2 and Six3 loci. Evidence for the functional significance of this boundary was obtained by deep sequencing of the radiation-induced Brachyrrhine (Br) mutant allele. We identified an inversion of the Six2/Six3 locus around the CTCF-bound boundary, removing Six2 from its distal enhancer regulation, but placed next to Six3 enhancer elements which support ectopic Six2 expression in the lens where Six3 is normally expressed. Six3 is now predicted to fall under control of the Six2 distal enhancer. Consistent with this view, we observed ectopic Six3 in nephron progenitors. 4C-seq supports the model for Six2 distal enhancer interactions in wild-type and Br/+ mouse kidneys. Together, these data expand our view of the regulatory genome and regulatory landscape underpinning mammalian nephrogenesis

Transcriptional regulatory control of mammalian nephron progenitors revealed by multi-factor cistromic analysis and genetic studies.

Nephron progenitor number determines nephron endowment; a reduced nephron count is linked to the onset of kidney disease. Several transcriptional regulators including Six2, Wt1, Osr1, Sall1, Eya1, Pax2, and Hox11 paralogues are required for specification and/or maintenance of nephron progenitors. However, little is known about the regulatory intersection of these players. Here, we have mapped nephron progenitor-specific transcriptional networks of Six2, Hoxd11, Osr1, and Wt1. We identified 373 multi-factor associated \u27regulatory hotspots\u27 around genes closely associated with progenitor programs. To examine their functional significance, we deleted \u27hotspot\u27 enhancer elements for Six2 and Wnt4. Removal of the distal enhancer for Six2 leads to a ~40% reduction in Six2 expression. When combined with a Six2 null allele, progeny display a premature depletion of nephron progenitors. Loss of the Wnt4 enhancer led to a significant reduction of Wnt4 expression in renal vesicles and a mildly hypoplastic kidney, a phenotype also enhanced in combination with a Wnt4 null mutation. To explore the regulatory landscape that supports proper target gene expression, we performed CTCF ChIP-seq to identify insulator-boundary regions. One such putative boundary lies between the Six2 and Six3 loci. Evidence for the functional significance of this boundary was obtained by deep sequencing of the radiation-induced Brachyrrhine (Br) mutant allele. We identified an inversion of the Six2/Six3 locus around the CTCF-bound boundary, removing Six2 from its distal enhancer regulation, but placed next to Six3 enhancer elements which support ectopic Six2 expression in the lens where Six3 is normally expressed. Six3 is now predicted to fall under control of the Six2 distal enhancer. Consistent with this view, we observed ectopic Six3 in nephron progenitors. 4C-seq supports the model for Six2 distal enhancer interactions in wild-type and Br/+ mouse kidneys. Together, these data expand our view of the regulatory genome and regulatory landscape underpinning mammalian nephrogenesis

MoCA: A Tool for Motif Conservation Analysis

<p>Poster presented at the USC MCB Retreat 2015.</p

Refinement of Optical Map Assemblies (original paper)

Motivation. Genomic mutations and variations provide insightful information about the functionality of sequence elements and their association with human diseases. Traditionally, variations are identified through analysis of short DNA sequences, usually shorter than 1000 nucleotides per fragment. Optical maps provide both faster and more cost-efficient means for detecting such differences, because a single map can span over 1 million bps. Optical maps are assembled to cover the whole genome, and the accuracy of assembly is critical. Results. We present a computationally efficient model-based method for improving quality of such assem-blies. Our method provides very high accuracy even with moderate coverage (&lt; 20x). We utilize a hidden Markov model to represent the consensus map and use the EM algorithm to drive the refinement process. We also provide quality scores to assess the quality of the finished map. Availability. Code is available from www.cmb.usc.edu/people/valouev. Contact

Copy Number Variation Is a Fundamental Aspect of the Placental Genome

<div><p>Discovery of lineage-specific somatic copy number variation (CNV) in mammals has led to debate over whether CNVs are mutations that propagate disease or whether they are a normal, and even essential, aspect of cell biology. We show that 1,000N polyploid trophoblast giant cells (TGCs) of the mouse placenta contain 47 regions, totaling 138 Megabases, where genomic copies are underrepresented (UR). UR domains originate from a subset of late-replicating heterochromatic regions containing gene deserts and genes involved in cell adhesion and neurogenesis. While lineage-specific CNVs have been identified in mammalian cells, classically in the immune system where V(D)J recombination occurs, we demonstrate that CNVs form during gestation in the placenta by an underreplication mechanism, not by recombination nor deletion. Our results reveal that large scale CNVs are a normal feature of the mammalian placental genome, which are regulated systematically during embryogenesis and are propagated by a mechanism of underreplication.</p></div