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Traditional clinical research relies on conventional strategies to invite and enroll research participants. However, these strategies often fail to reach potential participants from marginalized communities or that reflect the diversity of the nation, such as race, ethnicity, or geography. As we discuss here, the digital clinical study model sets the stage for improved and equitable participation in biomedical research.

In Drosophila, dosage compensation is mediated by the MSL complex, which binds numerous sites on the X chromosome in males and enhances the transcriptional rate of a substantial number of X-linked genes. We have determined that topoisomerase II (Topo II) is enriched on dosage compensated genes, to which it is recruited by association with the MSL complex, in excess of the amount that is present on autosomal genes with similar transcription levels. Using a plasmid model, we show that Topo II is required for proper dosage compensation and that compensated chromatin is topologically different from non-compensated chromatin. This difference, which is not the result of the enhanced transcription level due of X-linked genes and which represents a structural modification intrinsic to the DNA of compensated chromatin, requires the function of Topo II. Our results suggest that Topo II is an integral part of the mechanistic basis of dosage compensation.

Brd4 is a double bromodomain protein that has been shown to interact with acetylated histones to regulate transcription by recruiting Positive Transcription Elongation Factor b to the promoter region. Brd4 is also involved in gene bookmarking during mitosis and is a therapeutic target for the treatment of acute myeloid leukemia. The Drosophila melanogaster Brd4 homologue is called Fs(1)h and, like its vertebrate counterpart, encodes different isoforms. We have used ChIP-seq to examine the genome-wide distribution of Fs(1)h isoforms. We are able to distinguish the Fs(1)h-L and Fs(1)h-S binding profiles and discriminate between the genomic locations of the two isoforms. Fs(1)h-S is present at enhancers and promoters and its amount parallels transcription levels. Correlations between the distribution of Fs(1)h-S and various forms of acetylated histones H3 and H4 suggest a preference for binding to H3K9acS10ph. Surprisingly, Fs(1)h-L is located at sites in the genome where multiple insulator proteins are also present. The results suggest that Fs(1)h-S may be responsible for the classical role assigned to this protein, whereas Fs(1)h-L may have a new and unexpected role in chromatin architecture by working in conjunction with insulator proteins to mediate intra- or inter-chromosome interactions.

Insulators are multi-protein-DNA complexes thought to affect gene expression by mediating inter- and intra-chromosomal interactions. Drosophila insulators contain specific DNA binding proteins plus common components, such as CP190, that facilitate these interactions. Here we examine changes in the distribution of Drosophila insulator proteins during the heat-shock and ecdysone responses. We find that CP190 recruitment to insulator sites is the main regulatable step in controlling insulator function during heat shock. In contrast, both CP190 and DNA binding protein recruitment are regulated during the ecdysone response. CP190 is necessary to stabilize specific chromatin loops and for proper activation of transcription of genes regulated by this hormone. These findings suggest that cells may regulate recruitment of insulator proteins to the DNA in order to activate insulator activity at specific sites and create distinct patterns of nuclear organization that are necessary to achieve proper gene expression in response to different stimuli.

Background Benign ethnic neutropenia (BEN) is a hematologic condition associated with people of African ancestry and specific Middle Eastern ethnic groups. Prior genetic association studies in large population showed that rs2814778 in Duffy Antigen Receptor for Chemokines (DARC) gene, specifically DARC null red cell phenotype, was associated with BEN. However, the mechanism of this red cell phenotype leading to low white cell count remained elusive. Methods We conducted an extreme phenotype design genome-wide association study (GWAS), analyzed ~16 million single nucleotide polymorphisms (SNP) in 1,178 African-Americans individuals from the Reasons for Geographic and Racial Differences in Stroke (REGARDS) study and replicated from 819 African-American participants in the Atherosclerosis Risk in Communities (ARIC) study. Conditional analyses on rs2814778 were performed to identify additional association signals on chromosome 1q22. In a separate cohort of healthy individuals with and without BEN, whole genome gene expression from peripheral blood neutrophils were analyzed for DARC. Results We confirmed that rs2814778 in DARC was associated with BEN (p = 4.09×10-53). Conditioning on rs2814778 abolished other significant chromosome 1 associations. Inflammatory cytokines (IL-2, 6, and 10) in participants in the Howard University Family Study (HUFS) and Multi-Ethnic Study in Atherosclerosis (MESA) showed similar levels in individuals homozygous for the rs2814778 allele compared to others, indicating cytokine sink hypothesis played a minor role in leukocyte homeostasis. Gene expression in neutrophils of individuals with and without BEN was also similar except for low DARC expression in BEN, suggesting normal function. BEN neutrophils had slightly activated profiles in leukocyte migration and hematopoietic stem cell mobilization pathways (expression fold change Conclusions These results in humans support the notion of DARC null erythroid progenitors preferentially differentiating to myeloid cells, leading to activated DARC null neutrophils egressing from circulation to the spleen, and causing relative neutropenia. Collectively, these human data sufficiently explained the mechanism DARC null red cell phenotype causing BEN and further provided a biologic basis that BEN is clinically benign.

Background Strains from a collection of Drosophila GFP protein trap lines express GFP in the normal tissues where the endogenous protein is present. This collection can be used to screen for proteins distributed in the nucleus in a non-uniform pattern. Methodology/Principal Findings We analyzed four lines that show peripheral or punctate nuclear staining. One of these lines affects an uncharacterized gene named CG11138. The CG11138 protein shows a punctate distribution in the nuclear periphery similar to that of Drosophila insulator proteins but does not co-localize with known insulators. Interestingly, mutations in Lamin proteins result in alterations in CG11138 localization, suggesting that this protein may be a novel component of the nuclear lamina. A second line affects the Decondensation factor 31 (Df31) gene, which encodes a protein with a unique nuclear distribution that appears to segment the nucleus into four different compartments. The X-chromosome of males is confined to one of these compartments. We also find that Drosophila Nucleoplasmin (dNlp) is present in regions of active transcription. Heat shock leads to loss of dNlp from previously transcribed regions of polytene chromosome without redistribution to the heat shock genes. Analysis of Stonewall (Stwl), a protein previously found to be necessary for the maintenance of germline stem cells, shows that Stwl is present in a punctate pattern in the nucleus that partially overlaps with that of known insulator proteins. Finally we show that Stwl, dNlp, and Df31 form part of a highly interactive network. The properties of other components of this network may help understand the role of these proteins in nuclear biology. Conclusions/Significance These results establish screening of GFP protein trap alleles as a strategy to identify factors with novel cellular functions. Information gained from the analysis of CG11138 Stwl, dNlp, and Df31 sets the stage for future studies of these proteins.

Insulin resistance (IR) is a key determinant of type 2 diabetes (T2D) and other metabolic disorders. This genome-wide association study (GWAS) was designed to shed light on the genetic basis of fasting insulin (FI) and IR in 927 non-diabetic African Americans. 5 396 838 single-nucleotide polymorphisms (SNPs) were tested for associations with FI or IR with adjustments for age, sex, body mass index, hypertension status and first two principal components. Genotyped SNPs (n = 12) with P5 × 10(-6) in African Americans were carried forward for de novo genotyping in 570 non-diabetic West Africans. We replicated SNPs in or near SC4MOL and TCERG1L in West Africans. The meta-analysis of 1497 African Americans and West Africans yielded genome-wide significant associations for SNPs in the SC4MOL gene: rs17046216 (P = 1.7 × 10(-8) and 2.9 × 10(-8) for FI and IR, respectively); and near the TCERG1L gene with rs7077836 as the top scoring (P = 7.5 × 10(-9) and 4.9 × 10(-10) for FI and IR, respectively). In silico replication in the MAGIC study (n = 37 037) showed weak but significant association (adjusted P-value of 0.0097) for rs34602777 in the MYO5A gene. In addition, we replicated previous GWAS findings for IR and FI in Europeans for GCKR, and for variants in four T2D loci (FTO, IRS1, KLF14 and PPARG) which exert their action via IR. In summary, variants in/near SC4MOL, and TCERG1L were associated with FI and IR in this cohort of African Americans and were replicated in West Africans. SC4MOL is under-expressed in an animal model of T2D and plays a key role in lipid biosynthesis, with implications for the regulation of energy metabolism, obesity and dyslipidemia. TCERG1L is associated with plasma adiponectin, a key modulator of obesity, inflammation, IR and diabetes.

Insulators are DNA sequences thought to be important for the establishment and maintenance of cell-type specific nuclear architecture. In Drosophila there are several classes of insulators that appear to have unique roles in gene expression. The mechanisms involved in determining and regulating the specific roles of these insulator classes are not understood. Here we report that DNA Topoisomerase II modulates the activity of the Su(Hw) insulator. Downregulation of Topo II by RNAi or mutations in the Top2 gene result in disruption of Su(Hw) insulator function. This effect is mediated by the Mod(mdg4)2.2 protein, which is a unique component of the Su(Hw) insulator complex. Co-immunoprecipitation and yeast two-hybrid experiments show that Topo II and Mod(mdg4)2.2 proteins directly interact. In addition, mutations in Top2 cause a slight decrease of Mod(mdg4)2.2 transcript but have a dramatic effect on Mod(mdg4)2.2 protein levels. In the presence of proteasome inhibitors, normal levels of Mod(mdg4)2.2 protein and its binding to polytene chromosomes are restored. Thus, Topo II is required to prevent Mod(mdg4)2.2 degradation and, consequently, to stabilize Su(Hw) insulator-mediated chromatin organization.

Background DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Most methods to scan the genome in different tissues for differentially methylated sites have focused on the methylation of CpGs in CpG islands, which are concentrations of CpGs often associated with gene promoters. Results Here, we use a methylation profiling strategy that is predominantly responsive to methylation differences outside of CpG islands. The method compares the yield from two samples of size-selected fragments generated by a methylation-sensitive restriction enzyme. We then profile nine different normal tissues from two human donors relative to spleen using a custom array of genomic clones covering the euchromatic portion of human chromosome 1 and representing 8% of the human genome. We observe gross regional differences in methylation states across chromosome 1 between tissues from the same individual, with the most striking differences detected in the comparison of cerebellum and spleen. Profiles of the same tissue from different donors are strikingly similar, as are the profiles of different lobes of the brain. Comparing our results with published gene expression levels, we find that clones exhibiting extreme ratios reflecting low relative methylation are statistically enriched for genes with high expression ratios, and vice versa, in most pairs of tissues examined. Conclusion The varied patterns of methylation differences detected between tissues by our methylation profiling method reinforce the potential functional significance of regional differences in methylation levels outside of CpG islands.

Boundary Element Associated Factor-32 (BEAF-32) is an insulator protein predominantly found near gene promoters and thought to play a role in gene expression. We find that mutations in BEAF-32 are lethal, show loss of epithelial morphology in imaginal discs and cause neoplastic growth defects. To investigate the molecular mechanisms underlying this phenotype, we carried out a genome-wide analysis of BEAF-32 localization in wing imaginal disc cells. Mutation of BEAF-32 results in miss-regulation of 3850 genes by at least 1.5-fold, 794 of which are bound by this protein in wing imaginal cells. Up-regulated genes encode proteins involved in cell polarity, cell proliferation and cell differentiation. Among the down-regulated genes are those encoding components of the wingless pathway, which is required for cell differentiation. Miss-regulation of these genes explains the unregulated cell growth and neoplastic phenotypes observed in imaginal tissues of BEAF-32 mutants.

SummaryInsulators mediate inter- and intrachromosomal contacts to regulate enhancer-promoter interactions and establish chromosome domains. The mechanisms by which insulator activity can be regulated to orchestrate changes in the function and three-dimensional arrangement of the genome remain elusive. Here, we demonstrate that Drosophila insulator proteins are poly(ADP-ribosyl)ated and that mutation of the poly(ADP-ribose) polymerase (Parp) gene impairs their function. This modification is not essential for DNA occupancy of insulator DNA-binding proteins dCTCF and Su(Hw). However, poly(ADP-ribosyl)ation of K566 in CP190 promotes protein-protein interactions with other insulator proteins, association with the nuclear lamina, and insulator activity in vivo. Consistent with these findings, the nuclear clustering of CP190 complexes is disrupted in Parp mutant cells. Importantly, poly(ADP-ribosyl)ation facilitates intrachromosomal interactions between insulator sites measured by 4C. These data suggest that the role of insulators in organizing the three-dimensional architecture of the genome may be modulated by poly(ADP-ribosyl)ation.