Enhancing Biodiversity and Multifunctionality of an Organic Farmscape in California’s Central Valley

Organic farmers in the USA increasingly manage the margins of previously monocultured farmed landscapes to increase biodiversity, e.g. they restore and protect riparian corridors, plant hedgerows and construct vegetated tailwater ponds. This study attempts to link habitat enhancements, biodiversity and changes in ecosystem functions by: 1. inventorying the existing biodiversity and the associated belowground community structure and composition in the various habitats of an organic farm in California’s Central Valley; and 2. monitoring key ecosystem functions of these habitats. Two years of inventories show greater native plant diversity in non-cropped areas. While nematode diversity did not differ between habitats, functional groups were clearly associated with particular habitats as were soil microbial communities (phospholipid fatty acid analysis). Earthworm diversity did not differ between habitats, but biomass was higher in non-cropped areas. Habitats with woody vegetation stored 20% of the farmscape’s total carbon (C), despite their relatively small size (only 5% of the total farm). Two years of monitoring data of farmscape C and nitrogen (N) through emissions, run-off and leaching showed distinct tradeoffs in function associated with each habitat. Clearly habitat restoration in field margins will increase both landscape biodiversity and the multifunctionality of the farmscape as a whole

Protein Delivery of an Artificial Transcription Factor Restores Widespread Ube3a Expression in an Angelman Syndrome Mouse Brain.

Angelman syndrome (AS) is a neurological genetic disorder caused by loss of expression of the maternal copy of UBE3A in the brain. Due to brain-specific genetic imprinting at this locus, the paternal UBE3A is silenced by a long antisense transcript. Inhibition of the antisense transcript could lead to unsilencing of paternal UBE3A, thus providing a therapeutic approach for AS. However, widespread delivery of gene regulators to the brain remains challenging. Here, we report an engineered zinc finger-based artificial transcription factor (ATF) that, when injected i.p. or s.c., crossed the blood-brain barrier and increased Ube3a expression in the brain of an adult mouse model of AS. The factor displayed widespread distribution throughout the brain. Immunohistochemistry of both the hippocampus and cerebellum revealed an increase in Ube3a upon treatment. An ATF containing an alternative DNA-binding domain did not activate Ube3a. We believe this to be the first report of an injectable engineered zinc finger protein that can cause widespread activation of an endogenous gene in the brain. These observations have important implications for the study and treatment of AS and other neurological disorders

Translational outcomes in a full gene deletion of ubiquitin protein ligase E3A rat model of Angelman syndrome.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by developmental delay, impaired communication, motor deficits and ataxia, intellectual disabilities, microcephaly, and seizures. The genetic cause of AS is the loss of expression of UBE3A (ubiquitin protein ligase E6-AP) in the brain, typically due to a deletion of the maternal 15q11-q13 region. Previous studies have been performed using a mouse model with a deletion of a single exon of Ube3a. Since three splice variants of Ube3a exist, this has led to a lack of consistent reports and the theory that perhaps not all mouse studies were assessing the effects of an absence of all functional UBE3A. Herein, we report the generation and functional characterization of a novel model of Angelman syndrome by deleting the entire Ube3a gene in the rat. We validated that this resulted in the first comprehensive gene deletion rodent model. Ultrasonic vocalizations from newborn Ube3am-/p+ were reduced in the maternal inherited deletion group with no observable change in the Ube3am+/p- paternal transmission cohort. We also discovered Ube3am-/p+ exhibited delayed reflex development, motor deficits in rearing and fine motor skills, aberrant social communication, and impaired touchscreen learning and memory in young adults. These behavioral deficits were large in effect size and easily apparent in the larger rodent species. Low social communication was detected using a playback task that is unique to rats. Structural imaging illustrated decreased brain volume in Ube3am-/p+ and a variety of intriguing neuroanatomical phenotypes while Ube3am+/p- did not exhibit altered neuroanatomy. Our report identifies, for the first time, unique AS relevant functional phenotypes and anatomical markers as preclinical outcomes to test various strategies for gene and molecular therapies in AS

Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications.

Analysis of DNA methylation patterns relies increasingly on sequencing-based profiling methods. The four most frequently used sequencing-based technologies are the bisulfite-based methods MethylC-seq and reduced representation bisulfite sequencing (RRBS), and the enrichment-based techniques methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylated DNA binding domain sequencing (MBD-seq). We applied all four methods to biological replicates of human embryonic stem cells to assess their genome-wide CpG coverage, resolution, cost, concordance and the influence of CpG density and genomic context. The methylation levels assessed by the two bisulfite methods were concordant (their difference did not exceed a given threshold) for 82% for CpGs and 99% of the non-CpG cytosines. Using binary methylation calls, the two enrichment methods were 99% concordant and regions assessed by all four methods were 97% concordant. We combined MeDIP-seq with methylation-sensitive restriction enzyme (MRE-seq) sequencing for comprehensive methylome coverage at lower cost. This, along with RNA-seq and ChIP-seq of the ES cells enabled us to detect regions with allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression

ZNF274 Recruits the Histone Methyltransferase SETDB1 to the 3′ Ends of ZNF Genes

Only a small percentage of human transcription factors (e.g. those associated with a specific differentiation program) are expressed in a given cell type. Thus, cell fate is mainly determined by cell type-specific silencing of transcription factors that drive different cellular lineages. Several histone modifications have been associated with gene silencing, including H3K27me3 and H3K9me3. We have previously shown that genes for the two largest classes of mammalian transcription factors are marked by distinct histone modifications; homeobox genes are marked by H3K27me3 and zinc finger genes are marked by H3K9me3. Several histone methyltransferases (e.g. G9a and SETDB1) may be involved in mediating the H3K9me3 silencing mark. We have used ChIP-chip and ChIP-seq to demonstrate that SETDB1, but not G9a, is associated with regions of the genome enriched for H3K9me3. One current model is that SETDB1 is recruited to specific genomic locations via interaction with the corepressor TRIM28 (KAP1), which is in turn recruited to the genome via interaction with zinc finger transcription factors that contain a Kruppel-associated box (KRAB) domain. However, specific KRAB-ZNFs that recruit TRIM28 (KAP1) and SETDB1 to the genome have not been identified. We now show that ZNF274 (a KRAB-ZNF that contains 5 C2H2 zinc finger domains), can interact with KAP1 both in vivo and in vitro and, using ChIP-seq, we show that ZNF274 binding sites co-localize with SETDB1, KAP1, and H3K9me3 at the 3′ ends of zinc finger genes. Knockdown of ZNF274 with siRNAs reduced the levels of KAP1 and SETDB1 recruitment to the binding sites. These studies provide the first identification of a KRAB domain-containing ZNF that is involved in recruitment of the KAP1 and SETDB1 to specific regions of the human genome

Characterization of the Contradictory Chromatin Signatures at the 3′ Exons of Zinc Finger Genes

The H3K9me3 histone modification is often found at promoter regions, where it functions to repress transcription. However, we have previously shown that 3′ exons of zinc finger genes (ZNFs) are marked by high levels of H3K9me3. We have now further investigated this unusual location for H3K9me3 in ZNF genes. Neither bioinformatic nor experimental approaches support the hypothesis that the 3′ exons of ZNFs are promoters. We further characterized the histone modifications at the 3′ ZNF exons and found that these regions also contain H3K36me3, a mark of transcriptional elongation. A genome-wide analysis of ChIP-seq data revealed that ZNFs constitute the majority of genes that have high levels of both H3K9me3 and H3K36me3. These results suggested the possibility that the ZNF genes may be imprinted, with one allele transcribed and one allele repressed. To test the hypothesis that the contradictory modifications are due to imprinting, we used a SNP analysis of RNA-seq data to demonstrate that both alleles of certain ZNF genes having H3K9me3 and H3K36me3 are transcribed. We next analyzed isolated ZNF 3′ exons using stably integrated episomes. We found that although the H3K36me3 mark was lost when the 3′ ZNF exon was removed from its natural genomic location, the isolated ZNF 3′ exons retained the H3K9me3 mark. Thus, the H3K9me3 mark at ZNF 3′ exons does not impede transcription and it is regulated independently of the H3K36me3 mark. Finally, we demonstrate a strong relationship between the number of tandemly repeated domains in the 3′ exons and the H3K9me3 mark. We suggest that the H3K9me3 at ZNF 3′ exons may function to protect the genome from inappropriate recombination rather than to regulate transcription

MAGE I Transcription Factors Regulate KAP1 and KRAB Domain Zinc Finger Transcription Factor Mediated Gene Repression

Class I MAGE proteins (MAGE I) are normally expressed only in developing germ cells but are aberrantly expressed in many cancers. They have been shown to promote tumor survival, aggressive growth, and chemoresistance but the underlying mechanisms and MAGE I functions have not been fully elucidated. KRAB domain zinc finger transcription factors (KZNFs) are the largest group of vertebrate transcription factors and regulate neoplastic transformation, tumor suppression, cellular proliferation, and apoptosis. KZNFs bind the KAP1 protein and direct KAP1 to specific DNA sequences where it suppresses gene expression by inducing localized heterochromatin characterized by histone 3 lysine 9 trimethylation (H3me3K9). Discovery that MAGE I proteins also bind to KAP1 prompted us to investigate whether MAGE I can affect KZNF and KAP1 mediated gene regulation. We found that expression of MAGE I proteins, MAGE-A3 or MAGE-C2, relieved repression of a reporter gene by ZNF382, a KZNF with tumor suppressor activity. ChIP of MAGE I (-) HEK293T cells showed KAP1 and H3me3K9 are normally bound to the ID1 gene, a target of ZNF382, but that binding is greatly reduced in the presence of MAGE I proteins. MAGE I expression relieved KAP1 mediated ID1 repression, causing increased expression of ID1 mRNA and ID1 chromatin relaxation characterized by loss of H3me3K9. MAGE I binding to KAP1 also induced ZNF382 poly-ubiquitination and degradation, consistent with loss of ZNF382 leading to decreased KAP1 binding to ID1. In contrast, MAGE I expression caused increased KAP1 binding to Ki67, another KAP1 target gene, with increased H3me3K9 and decreased Ki67 mRNA expression. Since KZNFs are required to direct KAP1 to specific genes, these results show that MAGE I proteins can differentially regulate members of the KZNF family and KAP1 mediated gene repression

Genome-wide binding of the orphan nuclear receptor TR4 suggests its general role in fundamental biological processes

<p>Abstract</p> <p>Background</p> <p>The orphan nuclear receptor TR4 (human testicular receptor 4 or NR2C2) plays a pivotal role in a variety of biological and metabolic processes. With no known ligand and few known target genes, the mode of TR4 function was unclear.</p> <p>Results</p> <p>We report the first genome-wide identification and characterization of TR4 <it>in vivo </it>binding. Using chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq), we identified TR4 binding sites in 4 different human cell types and found that the majority of target genes were shared among different cells. TR4 target genes are involved in fundamental biological processes such as RNA metabolism and protein translation. In addition, we found that a subset of TR4 target genes exerts cell-type specific functions. Analysis of the TR4 binding sites revealed that less than 30% of the peaks from any of the cell types contained the DR1 motif previously derived from <it>in vitro </it>studies, suggesting that TR4 may be recruited to the genome via interaction with other proteins. A bioinformatics analysis of the TR4 binding sites predicted a <it>cis </it>regulatory module involving TR4 and ETS transcription factors. To test this prediction, we performed ChIP-seq for the ETS factor ELK4 and found that 30% of TR4 binding sites were also bound by ELK4. Motif analysis of the sites bound by both factors revealed a lack of the DR1 element, suggesting that TR4 binding at a subset of sites is facilitated through the ETS transcription factor ELK4. Further studies will be required to investigate the functional interdependence of these two factors.</p> <p>Conclusions</p> <p>Our data suggest that TR4 plays a pivotal role in fundamental biological processes across different cell types. In addition, the identification of cell type specific TR4 binding sites enables future studies of the pathways underlying TR4 action and its possible role in metabolic diseases.</p

Genome-Scale Validation of Deep-Sequencing Libraries

Chromatin immunoprecipitation followed by high-throughput (HTP) sequencing (ChIP-seq) is a powerful tool to establish protein-DNA interactions genome-wide. The primary limitation of its broad application at present is the often-limited access to sequencers. Here we report a protocol, Mab-seq, that generates genome-scale quality evaluations for nucleic acid libraries intended for deep-sequencing. We show how commercially available genomic microarrays can be used to maximize the efficiency of library creation and quickly generate reliable preliminary data on a chromosomal scale in advance of deep sequencing. We also exploit this technique to compare enriched regions identified using microarrays with those identified by sequencing, demonstrating that they agree on a core set of clearly identified enriched regions, while characterizing the additional enriched regions identifiable using HTP sequencing