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Electron transfer dissociation (ETD) is an analytically useful tool for primary structure interrogation of intact proteins, but its utility is limited by higher-order reactions with the products. To inhibit these higher-order reactions, first-generation fragment ions are kinetically excited by applying an experimentally tailored parallel ion parking waveform during ETD (ETD-PIP). In combination with subsequent ion/ion proton transfer reactions, precursor-to-product conversion was maximized as evidenced by the consumption of more than 90% of the 21 kDa Protein G precursor to form ETD product ions. The employment of ETD-PIP increased sequence coverage to 90% from 80% with standard ETD. Additionally, the inhibition of sequential electron transfers was reflected in the high number of complementary ion pairs from ETD-PIP (90%) compared to standard ETD (39%).

Complete sequence coverage of monospecific antibodies was previously achieved using immobilized aspergillopepsin I in a single LC-MS/MS analysis. Bispecific antibodies are asymmetrical compared to their monospecific antibody counterparts, resulting in a decrease in the concentration of individual subunits. Four standard proteins were used to characterize the effect of a decrease in concentration when using this immobilized enzyme reactor. Low concentration samples resulted in the elimination of large peptide products due to a greater number of enzymatic cleavages. A competitive inhibitor rich in arginine residues reduced the number of enzymatic cleavages to the protein and retained large molecular weight products. The digestion of a bispecific antibody with competitive inhibition of aspergillopepsin I maintained large peptide products better suited for sequence reconstruction, resulting in complete sequence coverage from a single LC-MS/MS analysis.

Survival at high altitude requires adapting to extreme conditions such as environmental hypoxia. To understand high-altitude adaptations in a primate, we assembled the genome of the gelada (Theropithecus gelada), an endemic Ethiopian monkey, and complemented it with population resequencing, hematological, and morphometric data. Unexpectedly, we identified a novel karyotype that may contribute to reproductive isolation between gelada populations. We also identified genomic elements including protein-coding sequences and gene families that exhibit accelerated changes in geladas and may contribute to high-altitude adaptation. Our findings lend insight into mechanisms of speciation and adaptation while providing promising avenues for functional hypoxia research.

Protamines are small, highly-specialized, arginine-rich, and intrinsically-disordered chromosomal proteins that replace histones during spermiogenesis in many organisms. Previous evidence supports the notion that, in the animal kingdom, these proteins have evolved from a primitive replication-independent histone H1 involved in terminal cell differentiation. Nevertheless, a direct connection between the two families of chromatin proteins is missing. Here, we primarily used electron transfer dissociation MS-based analyses, revealing that the protamines in the sperm of the liverwort Marchantia polymorpha result from post-translational cleavage of three precursor H1 histones. Moreover, we show that the mature protamines are further post-translationally modified by di-aminopropanelation, and previous studies have reported that they condense spermatid chromatin through a process consisting of liquid-phase assembly likely involving spinodal decomposition. Taken together, our results reveal that the interesting evolutionary ancestry of protamines begins with histone H1 in both the animal and plant kingdoms.

Accurate sequence characterization is essential for the development of therapeutic antibodies by the pharmaceutical industry. Presented here is a methodology to obtain comprehensive sequence analysis of a monoclonal antibody. An enzyme reactor of immobilized Aspergillopepsin I, a highly stable nonspecific protease, was used to cleave reduced antibody subunits into a peptide profile ranging from 1 to 20 kDa. Utilizing the Thermo Orbitrap Fusion’s unique instrument architecture combined with state-of-the-art instrument control software allowed for dynamic instrument methods that optimally characterize eluting peptides based on their size and charge density. Using a data-dependent instrument method, both collisional dissociation and electron transfer dissociation were used to fragment the appropriate charge state of analyte peptides. The instrument layout also allowed for scans to be taken in parallel using both the ion trap and Orbitrap concurrently, thus allowing larger peptides to be analyzed in high resolution using the Orbitrap while simultaneously analyzing tryptic-like peptides using the ion trap. We harnessed these capabilities to develop a custom method to optimally fragment the eluting peptides based on their mass and charge density. Using this approach, we obtained 100% sequence coverage of the total antibody in a single chromatographic analysis, enabling unambiguous sequence assignment of all residues.

International audience; The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to 20 laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS.

Glucocorticoids are potent steroid hormones that regulate immunity and metabolism by activating the transcription factor (TF) activity of glucocorticoid receptor (GR). Previous models have proposed that DNA binding motifs and sites of chromatin accessibility predetermine GR binding and activity. However, there are vast excesses of both features relative to the number of GR binding sites. Thus, these features alone are unlikely to account for the specificity of GR binding and activity. To identify genomic and epigenetic contributions to GR binding specificity and the downstream changes resultant from GR binding, we performed hundreds of genome-wide measurements of TF binding, epigenetic state, and gene expression across a 12-h time course of glucocorticoid exposure. We found that glucocorticoid treatment induces GR to bind to nearly all pre-established enhancers within minutes. However, GR binds to only a small fraction of the set of accessible sites that lack enhancer marks. Once GR is bound to enhancers, a combination of enhancer motif composition and interactions between enhancers then determines the strength and persistence of GR binding, which consequently correlates with dramatic shifts in enhancer activation. Over the course of several hours, highly coordinated changes in TF binding and histone modification occupancy occur specifically within enhancers, and these changes correlate with changes in the expression of nearby genes. Following GR binding, changes in the binding of other TFs precede changes in chromatin accessibility, suggesting that other TFs are also sensitive to genomic features beyond that of accessibility.

Introduction: CDK7 is a key regulator of transcription and cell cycle progression and has been implicated in multiple tumor types driven by aberrant transcriptional (e.g., MYC-, ESR1-activation) and/or aberrant cell cycle control (e.g., loss of RB pathway checkpoint function) mechanisms. SY-5609 is a potent and selective CDK7 inhibitor currently in development in patients with solid tumors (NCT04247126). To assess the potential for SY-5609 development in heme malignancies, we evaluated SY-5609 activity in preclinical models of mantle cell lymphoma (MCL). MCL is an aggressive B cell lymphoma driven by genetic alterations in RB pathway genes (e.g. CCND1, RB1, CDKN2A) and by hyper-activation of B-cell receptor (BCR) signaling leading to activation of NF-kB-dependent transcriptional programs that drive cell proliferation and survival. Our results support evaluation of SY-5609 in MCL patients and highlight the potential for combining SY-5609 with Bruton's Tyrosine Kinase (BTK) inhibitors, inhibitors of BCR signal transduction approved for treatment of MCL. Methods: SY-5609 in vitro antiproliferative activity was assessed in a panel of 7 MCL cell lines by evaluating growth rate inhibition (GR) curve metrics GR 50 (drug concentration at which growth rate is inhibited by 50%) and GR max (maximum depth of response) after 5-days of treatment. SY-5609 pharmacodynamic (PD) responses and combination activity with the BTK inhibitor acalabrutinib were further assessed in MCL cell line Mino-1. PD activity was assessed by measuring transcriptional changes in POLR2A using a NanoString-based PD assay from the SY-5609 solid tumor trial. Combination activity in vitro was assessed by comparing GR curve metrics between acalabrutinib, SY-5609, and acalabrutinib in combination with SY-5609. Expression of CCND1 and E2F1, a key transcriptional regulator of DNA replication commitment and progression, were assayed via western blot. Combination activity in vivo was assessed in mice bearing Mino-1 xenograft tumors by comparing tumor growth rate inhibition (GRI) and body weight change in mice treated with acalabrutinib (15mg/kg, QD) and/or SY-5609 (0.5mg/kg, BID, days 1-7 and 15-21) over 25 days. Results: SY-5609, as a single agent, potently inhibited proliferation of all 7 MCL cell lines tested with a GR 50 geometric-mean of 7 nM (range: 2 to 20 nM) and a GR max geometric-mean of -0.2 (range: -0.6 to 0.2). In MCL cell line Mino-1, SY-5609 induced a dose-dependent increase in POLR2A expression to levels associated with SY-5609 antiproliferative activity in Mino-1 cells in vitro and to levels comparable with those observed in peripheral blood mononuclear cells (PBMCs) collected from SY-5609 solid tumor trial patients. In combination with acalabrutinib, SY-5609 demonstrated synergistic antiproliferative activity in Mino-1 cells in vitro, a dose-dependent decrease in acalabrutinib GR 50 (up to ~2-fold at the highest SY-5609 concentration tested, 5nM), and a dose-dependent increase in GR max (up to ~10-fold). In addition, the combination of SY-5609 and acalabrutinib caused dose-dependent decreases in CCND1 and E2F1 protein expression in vitro, which were not observed with either single agent alone. Finally, in mice bearing Mino-1 xenografts, the combination of SY-5609 and acalabrutinib significantly (p < 0.01) increased tumor GRI relative to each single agent at the doses and regimens tested and was well tolerated (no difference in body weight changes relative to vehicle treated mice). Conclusions: SY-5609 is a potent and selective CDK7 inhibitor that demonstrates antiproliferative activity in MCL cells in vitro, associated with PD changes comparable to those observed in patients enrolled in the SY-5609 solid tumor trial (ENA, 2020). The combination of SY-5609 and acalabrutinib is synergistic in MCL cells in vitro and inhibits expression of key cell cycle regulatory proteins CCND1 and E2F1 at concentrations that are preclinically subtherapeutic for either single agent alone. The combination of SY-5609 and acalabrutinib is also significantly more effective at inhibiting MCL xenograft growth in vivo than either single agent. These results support evaluation of SY-5609, including in combination with BTK inhibitors, in patients with mantle cell lymphoma. Disclosures Johannessen: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Henry: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Sawant: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. D'Ippolito: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Ke: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Lefkowitz: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Eaton: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Dworakowski: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Rosario: Syros Pharmaceuticals: Current Employment. Hodgson: Syros Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company.

Overexpression of exogenous fate-specifying transcription factors can directly reprogram differentiated somatic cells to target cell types. Here, we show that similar reprogramming can also be achieved through the direct activation of endogenous genes using engineered CRISPR/Cas9-based transcriptional activators. We use this approach to induce activation of the endogenous Brn2, Ascl1, and Myt1l genes (BAM factors) to convert mouse embryonic fibroblasts to induced neuronal cells. This direct activation of endogenous genes rapidly remodeled the epigenetic state of the target loci and induced sustained endogenous gene expression during reprogramming. Thus, transcriptional activation and epigenetic remodeling of endogenous master transcription factors are sufficient for conversion between cell types. The rapid and sustained activation of endogenous genes in their native chromatin context by this approach may facilitate reprogramming with transient methods that avoid genomic integration and provides a new strategy for overcoming epigenetic barriers to cell fate specification.

Environmental stimuli commonly act via changes in gene regulation. Human-genome-scale assays to measure such responses are indirect or require knowledge of the transcription factors (TFs) involved. Here, we present the use of human genome-wide high-throughput reporter assays to measure environmentally-responsive regulatory element activity. We focus on responses to glucocorticoids (GCs), an important class of pharmaceuticals and a paradigmatic genomic response model. We assay GC-responsive regulatory activity across >108 unique DNA fragments, covering the human genome at >50×. Those assays directly detected thousands of GC-responsive regulatory elements genome-wide. We then validate those findings with measurements of transcription factor occupancy, histone modifications, chromatin accessibility, and gene expression. We also detect allele-specific environmental responses. Notably, the assays did not require knowledge of GC response mechanisms. Thus, this technology can be used to agnostically quantify genomic responses for which the underlying mechanism remains unknown., Quantification of genomic responses to environmental stimuli by current genome-scale assays is limited to indirect measurements or requires knowledge of the transcription factors involved. Here, the authors use genome-wide high-throughput reporter assays to agnostically map enhancer activity in response to glucocorticoid treatment across the human genome.

Technologies that enable targeted manipulation of epigenetic marks could be used to precisely control cell phenotype or interrogate the relationship between the epigenome and transcriptional control. Here we describe a programmable, CRISPR-Cas9-based acetyltransferase consisting of the nuclease-null dCas9 protein fused to the catalytic core of the human acetyltransferase p300. The fusion protein catalyzes acetylation of histone H3 lysine 27 at its target sites, leading to robust transcriptional activation of target genes from promoters and both proximal and distal enhancers. Gene activation by the targeted acetyltransferase was highly specific across the genome. In contrast to previous dCas9-based activators, the acetyltransferase activates genes from enhancer regions and with an individual guide RNA. We also show that the core p300 domain can be fused to other programmable DNA-binding proteins. These results support targeted acetylation as a causal mechanism of transactivation and provide a robust tool for manipulating gene regulation.

Introduction: CDK7 is a key regulator of transcription and cell cycle progression and has been implicated in multiple tumor types driven by aberrant transcriptional control (e.g. MYC-, ESR1-activation) and/or aberrant cell cycle control (e.g. RB1, CCNE1, CDKN2A alterations). SY-5609 is an oral, potent, and highly selective CDK7 inhibitor that is advancing through IND-enabling studies to support initiation of a planned Phase 1 oncology trial in early 2020. Here we report on the relationship between pharmacokinetics (PK), pharmacodynamics (PD), and tumor growth inhibition (TGI) in xenograft models of tumor types with transcriptional and/or cell cycle aberrations including high grade serous ovarian cancer (HGSOC), small cell lung cancer (SCLC), triple negative breast cancer (TNBC), and estrogen receptor positive breast cancer (ER+BC). Methods: The relationship between SY-5609 PK, PD, and TGI was evaluated in xenograft models of HGSOC (OVCAR3) and TNBC (HCC70) using once daily (QD) or twice daily (BID) dosing via oral gavage. SY-5609 TGI was evaluated as a single agent (SA) QD in patient-derived xenograft (PDX) models of HGSOC (n=3), SCLC (n=4), TNBC (n=4), and in combination with once weekly fulvestrant in ER+BC PDX models selected in vivo for resistance to the CDK4/6 inhibitor palbociclib (n=1) or resistance to both palbociclib and fulvestrant (n=1). Results: SY-5609 plasma exposure was dose proportional and did not accumulate after repeated therapeutic doses. Dose-dependent transcriptional responses in xenograft tissue were observed within 4 hours of SY-5609 dosing and were sustained for 24 hours. TGI was dose-dependent, with tumor regressions observed at doses significantly below the maximum tolerated dose (MTD). Similar TGI was seen when the same daily dose was administered either QD or BID suggesting that the effect was driven by overall exposure or minimum concentration. In HGSOC, SCLC, and TNBC PDX models, SA SY-5609 induced >50% TGI in all models tested (11/11), with 7/11 (64%) demonstrating robust anti-tumor activity (≥90% TGI or regression): 3/3 HGSOC, 2/4 SCLC, and 2/4 TNBC. In a palbociclib-resistant ER+BC PDX model, the combination of SY-5609 and fulvestrant induced significant TGI (89%), with no evident tumor regrowth up to 21 days after dosing cessation, distinguishing the observed effects from SY-5609 SA or fulvestrant SA. In a palbociclib and fulvestrant double-resistant ER+ BC PDX model, SY-5609 SA resulted in 33% TGI and fulvestrant SA had no activity. In contrast, the combination of SY-5609 and fulvestrant demonstrated significant TGI (68%; p Citation Format: Liv H Johannessen, Shanhu Hu, Nan Ke, Anthony D'Ippolito, Nisha Rajagopal, Jason Marineau, Anneli Savinainen, William Zamboni, Graeme Hodgson. Preclinical evaluation of PK, PD, and antitumor activity of the oral, non-covalent, potent and highly selective CDK7 inhibitor, SY-5609, provides rationale for clinical development in multiple solid tumor indications [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C091. doi:10.1158/1535-7163.TARG-19-C091

Glycogen synthase kinase-3 (Gsk-3) isoforms, Gsk-3α and Gsk-3β, are constitutively active, largely inhibitory kinases involved in signal transduction. Underscoring their biological significance, altered Gsk-3 activity has been implicated in diabetes, Alzheimer disease, schizophrenia, and bipolar disorder. Here, we demonstrate that deletion of both Gsk-3α and Gsk-3β in mouse embryonic stem cells results in reduced expression of the de novo DNA methyltransferase Dnmt3a2, causing misexpression of the imprinted genes Igf2, H19, and Igf2r and hypomethylation of their corresponding imprinted control regions. Treatment of wild-type embryonic stem cells and neural stem cells with the Gsk-3 inhibitor, lithium, phenocopies the DNA hypomethylation at these imprinted loci. We show that inhibition of Gsk-3 by phosphatidylinositol 3-kinase (PI3K)-mediated activation of Akt also results in reduced DNA methylation at these imprinted loci. Finally, we find that N-Myc is a potent Gsk-3-dependent regulator of Dnmt3a2 expression. In summary, we have identified a signal transduction pathway that is capable of altering the DNA methylation of imprinted loci.

The glucocorticoid receptor (GR) is a hormone-inducible transcription factor involved in metabolic and anti-inflammatory gene expression responses. To investigate what controls interactions between GR binding sites and their target genes, we used in situ Hi-C to generate high-resolution, genome-wide maps of chromatin interactions before and after glucocorticoid treatment. We found that GR binding to the genome typically does not cause new chromatin interactions to target genes but instead acts through chromatin interactions that already exist prior to hormone treatment. Both glucocorticoid-induced and glucocorticoid-repressed genes increased interactions with distal GR binding sites. In addition, while glucocorticoid-induced genes increased interactions with transcriptionally active chromosome compartments, glucocorticoid-repressed genes increased interactions with transcriptionally silent compartments. Lastly, while the architectural DNA-binding proteins CTCF and RAD21 were bound to most chromatin interactions, we found that glucocorticoid-responsive chromatin interactions were depleted for CTCF binding but enriched for RAD21. Together, these findings offer new insights into the mechanisms underlying GC-mediated gene activation and repression.

Epigenome editing with the CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 platform is a promising technology for modulating gene expression to direct cell phenotype and to dissect the causal epigenetic mechanisms of gene regulation. Fusions of nuclease-inactive dCas9 to the Kruppel-associated box (KRAB) repressor (dCas9-KRAB) can silence target gene expression, but the genome-wide specificity and the extent of heterochromatin formation catalyzed by dCas9-KRAB are not known. We targeted dCas9-KRAB to the HS2 enhancer, a distal regulatory element that orchestrates the expression of multiple globin genes, and observed highly specific induction of H3K9 trimethylation (H3K9me3) at the enhancer and decreased chromatin accessibility of both the enhancer and its promoter targets. Targeted epigenetic modification of HS2 silenced the expression of multiple globin genes, with minimal off-target changes in global gene expression. These results demonstrate that repression mediated by dCas9-KRAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome.

A genome-wide analysis is given of DNA methylation in mouse embryonic stem cells in which both Gsk-3α and Gsk-3β have been genetically deleted. DNA methylation patterns are compared to those of wild-type cells. More than 75% of known imprinted loci have reduced DNA methylation in the Gsk-3–knockout cells., Glycogen synthase kinase-3 (Gsk-3) is a key regulator of multiple signal transduction pathways. Recently we described a novel role for Gsk-3 in the regulation of DNA methylation at imprinted loci in mouse embryonic stem cells (ESCs), suggesting that epigenetic changes regulated by Gsk-3 are likely an unrecognized facet of Gsk-3 signaling. Here we extend our initial observation to the entire mouse genome by enriching for methylated DNA with the MethylMiner kit and performing next-generation sequencing (MBD-Seq) in wild-type and Gsk-3α−/−;Gsk-3β−/− ESCs. Consistent with our previous data, we found that 77% of known imprinted loci have reduced DNA methylation in Gsk-3-deficient ESCs. More specifically, we unambiguously identified changes in DNA methylation within regions that have been confirmed to function as imprinting control regions. In many cases, the reduced DNA methylation at imprinted loci in Gsk-3α−/−;Gsk-3β−/− ESCs was accompanied by changes in gene expression as well. Furthermore, many of the Gsk-3–dependent, differentially methylated regions (DMRs) are identical to the DMRs recently identified in uniparental ESCs. Our data demonstrate the importance of Gsk-3 activity in the maintenance of DNA methylation at a majority of the imprinted loci in ESCs and emphasize the importance of Gsk-3–mediated signal transduction in the epigenome.

Glycogen synthase kinase-3 (Gsk-3) activity is an important regulator of numerous signal transduction pathways. Gsk-3 activity is the sum of two largely redundant proteins, Gsk-3α and Gsk-3β, and in general, Gsk-3 is a negative regulator of cellular signaling. Genetic deletion of both Gsk-3α and Gsk-3β in mouse embryonic stem cells (ESCs) has previously been shown to lead to the constitutive activation of the Wnt/β-catenin signaling pathway. However, in addition to Wnt signaling, all Gsk-3-regulated pathways, such as insulin signaling, are also affected simultaneously in Gsk-3α(-) (/) (-); Gsk-3β(-) (/) (-)ESCs. In an effort to better understand how specific signaling pathways contribute to the global pattern of gene expression in Gsk-3α(-) (/) (-); Gsk-3β(-) (/) (-)ESCs, we compared the gene expression profiles in Gsk-3α(-) (/) (-); Gsk-3β(-) (/) (-) ESCs to mouse ESCs in which either Wnt/β-catenin signaling or phosphatidylinositol 3-kinase (PI3K)-dependent insulin signaling are constitutively active. Our results show that Wnt signaling has a greater effect on up-regulated genes in the Gsk-3α(-) (/) (-); Gsk-3β(-) (/) (-)ESCs, whereas PI3K-dependent insulin signaling is more responsible for the down-regulation of genes in the same cells. These data show the importance of Gsk-3 activity on gene expression in mouse ESCs, and that these effects are due to the combined effects of multiple signaling pathways.

Personalizing treatments to account for genetically mediated differences in drug responses is an exciting opportunity to improve patient outcomes. In this issue, Soccio et al. reveal new mechanisms by which non-coding variants alter the activity of the anti-diabetic drug rosiglitazone.

The CRISPR/Cas9 genome engineering platform has emerged as a powerful tool for gene regulation. Catalytically inactivated, “dead” Cas9 (dCas9) can be fused to a variety of transcriptional modulators to activate or silence gene expression. The KRAB domain, a common heterochromatin-forming motif in naturally occurring zinc finger transcription factors, has been genetically linked to dCas9 to create an RNA-guided synthetic repressor. KRAB-based synthetic repressors can effectively silence the expression of single genes and have been used to repress oncogenes, inhibit viral replication, and treat dominant negative diseases. However, the specificity of silencing endogenous genes and the distance of heterochromatin formation catalyzed by dCas9-KRAB repressors have not been studied. Understanding the precision of dCas9-KRAB repressors is critical to their further therapeutic application. As a model system to investigate the epigenetic consequences of KRAB-based silencing, dCas9-KRAB was targeted to the HS2 enhancer of the globin locus in the K562 human erythroid leukemia cell line. Enhancers are regulatory elements characterized by open chromatin that can activate distal genes targets. Genetic variation in enhancer sequences has been linked to many disease states, including cancer and cardiovascular disease. The HS2 enhancer is a potent activator of the developmentally regulated globin genes, which are involved in sickle cell anemia and beta thalassemia. We demonstrated that dCas9-KRAB targeted to HS2 by a single guide RNA (sgRNA) can disrupt the expression of multiple downstream globin genes. These findings expand the capabilities of synthetic repressors by showing that dCas9-KRAB targeted to a distal regulatory element can silence expression of multiple genes located 10 to 50 kb away. Genome-wide analysis of DNA-binding and mRNA expression by ChIP-seq and RNA-seq, respectively, established that RNA-guided synthetic repressors were highly specific for targeting and silencing the globin genes. To further investigate the utility of dCas9-KRAB repressors as a targeted epigenetic modifier, we performed ChIP-seq for tri-methylation of H3K9, a mark of heterochromatin, and DNase I hypersensitivity-sequencing (DNase-seq), a genome-wide measure of chromatin accessibility. dCas9-KRAB repressors induced H3K9 methylation at the HS2 enhancer. Off-target H3K9 methylation events were minimal and generally depended on the sgRNA. Reduced DNase I hypersensitivity was also observed at the enhancer region and its promoter targets, indicating that dCas9-KRAB repressors can modulate the chromatin structure of active enhancers to affect the activity of downstream target genes. These studies establish that dCas9-KRAB repressors can interfere with distal enhancer activity by highly specific remodeling of the epigenetic state of targeted loci. Enhancers play critical roles in differentiation, development, and disease, and understanding the epigenetic implications of targeting engineered repressors to distal regulatory elements will inform further use of genome engineering technologies for therapeutic applications.

Although nuclear ribosomal DNA (rDNA) repeats evolve together through concerted evolution, some genomes contain a considerable diversity of paralogous rDNA. This diversity includes not only multiple functional loci but also putative pseudogenes and recombinants. We examined the occurrence of divergent paralogues and recombinants in Gossypium, Nicotiana, Tripsacum, Winteraceae, and Zea ribosomal internal transcribed spacer (ITS) sequences. Some of the divergent paralogues are probably rDNA pseudogenes, since they have low predicted secondary structure stability, high substitution rates, and many deamination-driven substitutions at methylation sites. Under standard PCR conditions, the low stability paralogues amplified well, while many high-stability paralogues amplified poorly. Under highly denaturing PCR conditions (i.e., with dimethylsulfoxide), both low- and high-stability paralogues amplified well. We also found recombination between divergent paralogues. For phylogenetics, divergent ribosomal paralogues can aid in reconstructing ancestral states and thus serveas good outgroups. Divergent paralogues can also provide companion rDNA phylogenies. However, phylogeneticists must discriminate among families of divergent paralogues and recombinants or suffer from muddled and inaccurate organismal phylogenies.

The glucocorticoid receptor (GR) binds the human genome at >10,000 sites, but only regulates the expression of hundreds of genes. To determine the functional effect of each site, we measured the glucocorticoid (GC) responsive activity of nearly all GR binding sites (GBSs) captured using chromatin immunoprecipitation (ChIP) in A549 cells. 13% of GBSs assayed had GC-induced activity. The responsive sites were defined by direct GR binding via a GC response element (GRE) and exclusively increased reporter-gene expression. Meanwhile, most GBSs lacked GC-induced reporter activity. The non-responsive sites had epigenetic features of steady state enhancers and clustered around direct GBSs. Together, our data support a model in which clusters of GBSs observed with ChIP-seq reflect interactions between direct and tethered GBSs over tens of kilobases. We further show that those interactions can synergistically modulate the activity of direct GBSs, and may therefore play a major role in driving gene activation in response to GCs.

The role of pollinators in plant speciation and maintenance of species boundaries is dubious because most plant species are visited by several types of pollinators, and most pollinator species visit several species of plants. We investigated pollinator preferences and their efficacy as ethological isolation mechanisms between two interfertile species, Nicotiana alata and N. forgetiana and their F1 hybrids. Hawkmoths pollinate N. alata, while primarily hummingbirds and occasionally small hawkmoths visit N. forgetiana. F1 hybrids are easily produced in the greenhouse and although the species grow in similar habitats, hybrids have not been found in nature. In Rio Grande do Sul, Brazil, near where both species are found, experimental plots were studied containing both species, and both species plus F1 hybrids. In the mixed-species plots, hawkmoths showed a strong preference for N. alata. Hummingbirds were less common and only visited N. forgetiana. Hybrid seed was produced but plants made significantly fewer hybrid offspring than predicted by the frequency of interspecific pollinator movements. Nicotiana forgetiana was the seed parent of 97% of the F1 offspring, suggesting an asymmetry in pollen delivery or postpollination processes. In plots containing F1 hybrids plus both parental species, hawkmoths preferred N. alata and undervisited the other two phenotypes, except that in the third plot they visited hybrids in proportion to the hybrid frequency. Hummingbirds strongly preferred N. forgetiana in all plots but also visited F1 hybrids in proportion to their frequency in the third plot. Overall, F1 hybrids were well pollinated and were frequently visited immediately before or after one of the parental species. Thus hybrids could facilitate gene flow between the parental species. We conclude that pollinator discrimination among species is strong but is an imperfect isolation mechanism, especially if hybrids are present.

Hornstedtia scottiana (Zingiberaceae) is a large, rhizomatous, rain forest herb with vegetative aerial shoots and short, lateral inflorescences. The floral characteristics, scarlet corolla, no odor, and copious nectar are typical of ornithophily, but the near ground position of the flowers seemed inappropriate for bird pollination. Also, the nectar of Hornstedtia scyphifera was reported to be largely sucrose, a sugar composition apparently atypical of plants pollinated by paleotropical passerines. The floral biology and pollination of Hornstedtia scottiana were studied in Queensland, Australia. Three species of honeyeaters (Melphagidae) were observed to be the only floral visitors capable of effecting pollination. Similar to Hornstedtia scyphifera, H. scottiana produced sucrose-rich nectar. Flowering was diurnal with anthesis beginning at dawn. Floral visits were most frequent during mid-mornings. By early afternoon, if flowers were not visitor-pollinated, elongation of the corolla tube effected self-pollination. Pollination manipulations determined that outcrossed flowers set significantly more fruit, with significantly heavier seeds, than self-pollinated flowers, suggesting an inbreeding depression. Mean seed mass data from open-pollinated controls suggested that the fruits produced were from a combination of self (65%) and outcrossed (35%) pollinations.