Diane I. Schroeder

Green Fluorescent Protein Functions as a Reporter for Protein Localization in Escherichia coli

The use of green fluorescent protein (GFP) as a reporter for protein localization in Escherichia coli was explored by creating gene fusions between malE (encoding maltose-binding protein [MBP]) and a variant of gfp optimized for fluorescence in bacteria (GFPuv). These constructs encode hybrid proteins composed of GFP fused to the carboxy-terminal end of MBP. Fluorescence was not detected when the hybrid protein was synthesized with the MBP signal sequence. In contrast, when the MBP signal sequence was deleted, fluorescence was observed. Cell fractionation studies showed that the fluorescent MBP-GFP hybrid protein was localized in the cytoplasm, whereas the nonfluorescent version was localized to the periplasmic space. Smaller MBP-GFP hybrid proteins, however, exhibited abnormal fractionation. Expression of the gene fusions in different sec mutants, as well as signal sequence processing assays, confirmed that the periplasmically localized hybrid proteins were exported by the sec-dependent pathway. The distinction between fluorescent and nonfluorescent colonies was exploited as a scorable phenotype to isolate malE signal sequence mutations. While expression of hybrid proteins comprised of full-length MBP did not result in overproduction lethality characteristic of some exported beta-galactosidase hybrid proteins, synthesis of shorter, exported hybrid proteins was toxic to the cells. Purification of MBP-GFP hybrid protein from the different cellular compartments indicated that GFP is improperly folded when localized outside of the cytoplasm. These results suggest that GFP could serve as a useful reporter for genetic analysis of bacterial protein export and of protein folding.

The human placenta methylome

Tissue-specific DNA methylation is found at promoters, enhancers, and CpG islands but also over larger genomic regions. In most human tissues, the vast majority of the genome is highly methylated (>70%). Recently, sequencing of bisulfite-treated DNA (MethylC-seq) has revealed large partially methylated domains (PMDs) in some human cell lines. PMDs cover up to 40% of the genome and are associated with gene repression and inactive chromatin marks. However, to date, only cultured cells and cancers have shown evidence for PMDs. Here, we performed MethylC-seq in full-term human placenta and demonstrate it is the first known normal tissue showing clear evidence of PMDs. We found that PMDs cover 37% of the placental genome, are stable throughout gestation and between individuals, and can be observed with lower sensitivity in Illumina 450K Infinium data. RNA-seq analysis confirmed that genes in PMDs are repressed in placenta. Using a hidden Markov model to map placental PMDs genome-wide and compare them to PMDs in other cell lines, we found that genes within placental PMDs have tissue-specific functions. For regulatory regions, methylation levels in promoter CpG islands are actually higher for genes within placental PMDs, despite the lower overall methylation of surrounding regions. Similar to PMDs, polycomb-regulated regions are hypomethylated but smaller and distinct from PMDs, with some being hypermethylated in placenta compared with other tissues. These results suggest that PMDs are a developmentally dynamic feature of the methylome that are relevant for understanding both normal development and cancer and may be of use as epigenetic biomarkers.

15q11.2–13.3 chromatin analysis reveals epigenetic regulation of CHRNA7 with deficiencies in Rett and autism brain

Copy number variations (CNVs) within human 15q11.2-13.3 show reduced penetrance and variable expressivity in a range of neurologic disorders. Therefore, characterizing 15q11.2-13.3 chromatin structure is important for understanding the regulation of this locus during normal neuronal development. Deletion of the Prader-Willi imprinting center (PWS-IC) within 15q11.2-13.3 disrupts long-range imprinted gene expression resulting in Prader-Willi syndrome. Previous results establish that MeCP2 binds to the PWS-IC and is required for optimal expression of distal GABRB3 and UBE3A. To examine the hypothesis that MeCP2 facilitates 15q11.2-13.3 transcription by linking the PWS-IC with distant elements, chromosome capture conformation on chip (4C) analysis was performed in human SH-SY5Y neuroblastoma cells. SH-SY5Y neurons had 2.84-fold fewer 15q11.2-13.3 PWS-IC chromatin interactions than undifferentiated SH-SY5Y neuroblasts, revealing developmental chromatin de-condensation of the locus. Out of 68 PWS-IC interactions with15q11.2-13.3 identified by 4C analysis and 62 15q11.2-13.3 MeCP2-binding sites identified by previous ChIP-chip studies, only five sites showed overlap. Remarkably, two of these overlapping PWS-IC- and MeCP2-bound sites mapped to sites flanking CHRNA7 (cholinergic receptor nicotinic alpha 7) encoding the cholinergic receptor, nicotinic, alpha 7. PWS-IC interaction with CHRNA7 in neurons was independently confirmed by fluorescent in situ hybridization analysis. Subsequent quantitative transcriptional analyses of frontal cortex from Rett syndrome and autism patients revealed significantly reduced CHRNA7 expression compared with controls. Together, these results suggest that transcription of CHRNA7 is modulated by chromatin interactions with the PWS-IC. Thus, loss of long-range chromatin interactions within 15q11.2-13.3 may contribute to multiple human neurodevelopmental disorders.

Large-scale methylation domains mark a functional subset of neuronally expressed genes

DNA methylation is essential for embryonic and neuronal differentiation, but the function of most genomic DNA methylation marks is poorly understood. Generally the human genome is highly methylated (>70%) except for CpG islands and gene promoters. However, it was recently shown that the IMR90 human fetal lung fibroblast cells have large regions of the genome with partially methylated domains (PMDs, <70% average methylation), in contrast to the rest of the genome which is in highly methylated domains (HMDs, >70% average methylation). Using bisulfite conversion followed by high-throughput sequencing (MethylC-seq), we discovered that human SH-SY5Y neuronal cells also contain PMDs. We developed a novel hidden Markov model (HMM) to computationally map the genomic locations of PMDs in both cell types and found that autosomal PMDs can be >9 Mb in length and cover 41% of the IMR90 genome and 19% of the SH-SY5Y genome. Genomic regions marked by cell line specific PMDs contain genes that are expressed in a tissue-specific manner, with PMDs being a mark of repressed transcription. Genes contained within N-HMDs (neuronal HMDs, defined as a PMD in IMR90 but HMD in SH-SY5Y) were significantly enriched for calcium signaling, synaptic transmission, and neuron differentiation functions. Autism candidate genes were enriched within PMDs and the largest PMD observed in SH-SY5Y cells marked a 10 Mb cluster of cadherin genes with strong genetic association to autism. Our results suggest that these large-scale methylation domain maps could be relevant to interpreting and directing future investigations into the elusive etiology of autism.

Role of DNMT3B in the regulation of early neural and neural crest specifiers.

The de novo DNA methyltransferase DNMT3B functions in establishing DNA methylation patterns during development. DNMT3B missense mutations cause immunodeficiency, centromere instability and facial anomalies (ICF) syndrome. The restriction of Dnmt3b expression to neural progenitor cells, as well as the mild cognitive defects observed in ICF patients, suggests that DNMT3B may play an important role in early neurogenesis. We performed RNAi knockdown of DNMT3B in human embryonic stem cells (hESCs) in order to investigate the mechanistic contribution of DNMT3B to DNA methylation and early neuronal differentiation. While DNMT3B was not required for early neuroepithelium specification, DNMT3B deficient neuroepithelium exhibited accelerated maturation with earlier expression, relative to normal hESCs, of mature neuronal markers (such as NEUROD1) and of early neuronal regional specifiers (such as those for the neural crest). Genome-wide analyses of DNA methylation by MethylC-seq identified novel regions of hypomethylation in the DNMT3B knockdowns along the X chromosome as well as pericentromeric regions, rather than changes to promoters of specific dysregulated genes. We observed a loss of H3K27me3 and the polycomb complex protein EZH2 at the promoters of early neural and neural crest specifier genes during differentiation of DNMT3B knockdown but not normal hESCs. Our results indicate that DNMT3B mediates large-scale methylation patterns in hESCs and that DNMT3B deficiency in the cells alters the timing of their neuronal differentiation and maturation.

Early Developmental and Evolutionary Origins of Gene Body DNA Methylation Patterns in Mammalian Placentas.

Over the last 20-80 million years the mammalian placenta has taken on a variety of morphologies through both divergent and convergent evolution. Recently we have shown that the human placenta genome has a unique epigenetic pattern of large partially methylated domains (PMDs) and highly methylated domains (HMDs) with gene body DNA methylation positively correlating with level of gene expression. In order to determine the evolutionary conservation of DNA methylation patterns and transcriptional regulatory programs in the placenta, we performed a genome-wide methylome (MethylC-seq) analysis of human, rhesus macaque, squirrel monkey, mouse, dog, horse, and cow placentas as well as opossum extraembryonic membrane. We found that, similar to human placenta, mammalian placentas and opossum extraembryonic membrane have globally lower levels of methylation compared to somatic tissues. Higher relative gene body methylation was the conserved feature across all mammalian placentas, despite differences in PMD/HMDs and absolute methylation levels. Specifically, higher methylation over the bodies of genes involved in mitosis, vesicle-mediated transport, protein phosphorylation, and chromatin modification was observed compared with the rest of the genome. As in human placenta, higher methylation is associated with higher gene expression and is predictive of genic location across species. Analysis of DNA methylation in oocytes and preimplantation embryos shows a conserved pattern of gene body methylation similar to the placenta. Intriguingly, mouse and cow oocytes and mouse early embryos have PMD/HMDs but their placentas do not, suggesting that PMD/HMDs are a feature of early preimplantation methylation patterns that become lost during placental development in some species and following implantation of the embryo.

MeCP2 is required for global heterochromatic and nucleolar changes during activity-dependent neuronal maturation

Mutations in MECP2, encoding methyl CpG binding protein 2, cause the neurodevelopmental disorder Rett syndrome. MeCP2 is an abundant nuclear protein that binds to chromatin and modulates transcription in response to neuronal activity. Prior studies of MeCP2 function have focused on specific gene targets of MeCP2, but a more global role for MeCP2 in neuronal nuclear maturation has remained unexplored. MeCP2 levels increase during postnatal brain development, coinciding with dynamic changes in neuronal chromatin architecture, particularly detectable as changes in size, number, and location of nucleoli and perinucleolar heterochromatic chromocenters. To determine a potential role for MeCP2 in neuronal chromatin maturational changes, we measured nucleoli and chromocenters in developing wild-type and Mecp2-deficient mouse cortical sections, as well as mouse primary cortical neurons and a human neuronal cell line following induced maturation. Mecp2-deficient mouse neurons exhibited significant differences in nucleolar and chromocenter number and size, as more abundant, smaller nucleoli in brain and primary neurons compared to wild-type, consistent with delayed neuronal nuclear maturation in the absence of MeCP2. Primary neurons increased chromocenter size following depolarization in wild-type, but not Mecp2-deficient cultures. Wild-type MECP2e1 over-expression in human SH-SY5Y cells was sufficient to induce significantly larger nucleoli, but not a T158M mutation of the methyl-binding domain. These results suggest that, in addition to the established role of MeCP2 in transcriptional regulation of specific target genes, the global chromatin-binding function of MeCP2 is essential for activity-dependent global chromatin dynamics during postnatal neuronal maturation.

Automated data-driven discovery of motif-based protein function classifiers

This paper describes an approach to data-driven discovery of decision trees or rules for assigning protein sequences to functional families using sequence motifs. This method is able to capture regularities that can be described in terms of presence or absence of arbitrary combinations of motifs, A training set of peptidase sequences labeled with the corresponding MEROPS functional families or clans is used to automatically construct decision trees that capture regularities sufficient to assign the sequences to their respective functional families. The performance of the resulting decision tree classifiers is then evaluated on an independent test set. We compared the rules constructed using motifs generated by a multiple sequence alignment based motif discovery tool (MEME) with rules constructed using expert annotated PROSITE motifs (patterns and profiles). Our results indicate that the former provide a potentially powerful high throughput technique for constructing protein function classifiers when adequate training data are available. Examination of the generated rules in relation to known three-dimensional structures of members in the case of two families (MEROPS families C14 and M12) suggests that the proposed technique may be able to identify combinations of sequence motifs that characterize functionally significant three-dimensional structural features of proteins.

Methylation and gene expression responses to ethanol feeding and betaine supplementation in the cystathionine beta synthase-deficient mouse

BACKGROUND Alcoholic steatohepatitis (ASH) is caused in part by the effects of ethanol (EtOH) on hepatic methionine metabolism. METHODS To investigate the phenotypic and epigenetic consequences of altered methionine metabolism in this disease, we studied the effects of 4-week intragastric EtOH feeding with and without the methyl donor betaine in cystathionine beta synthase (CβS) heterozygous C57BL/6J mice. RESULTS The histopathology of early ASH was induced by EtOH feeding and prevented by betaine supplementation, while EtOH feeding reduced and betaine supplementation maintained the hepatic methylation ratio of the universal methyl donor S-adenosylmethionine (SAM) to the methyltransferase inhibitor S-adenosylhomocysteine (SAH). MethylC-seq genomic sequencing of heterozygous liver samples from each diet group found 2 to 4% reduced methylation in gene bodies, but not promoter regions of all autosomes of EtOH-fed mice, each of which were normalized in samples from mice fed the betaine-supplemented diet. The transcript levels of nitric oxide synthase (Nos2) and DNA methyltransferase 1 (Dnmt1) were increased, while those of peroxisome proliferator receptor-α (Pparα) were reduced in EtOH-fed mice, and each was normalized in mice fed the betaine-supplemented diet. DNA pyrosequencing of CβS heterozygous samples found reduced methylation in a gene body of Nos2 by EtOH feeding that was restored by betaine supplementation and was correlated inversely with its expression and positively with SAM/SAH ratios. CONCLUSIONS The present study has demonstrated relationships among EtOH induction of ASH with aberrant methionine metabolism that was associated with gene body DNA hypomethylation in all autosomes and was prevented by betaine supplementation. The data imply that EtOH-induced changes in selected gene transcript levels and hypomethylation in gene bodies during the induction of ASH are a result of altered methionine metabolism that can be reversed through dietary supplementation of methyl donors.

How has the study of the human placenta aided our understanding of partially methylated genes

While the human genome sequence is relatively uniform between the cells of an individual, the DNA methylation of the genome (methylome) has unique features in different cells, tissues and stages of development. Recent genome-wide sequencing of the methylome has revealed large partially methylated domains (PMDs) in the human placenta. Unlike CpG islands and Polycomb-regulated regions, which can also have low levels of methylation, placental PMDs cover approximately 37% of the human genome and are associated with inaccessible chromatin and the repression of tissue-specific genes. Here, we summarize the interesting biological questions that have arisen as a result of finding PMDs in the human placenta, including how PMDs form, what they do, how they evolved and how they might be relevant to human disease.