Tm7sf2 Disruption Alters Radial Gene Positioning in Mouse Liver Leading to Metabolic Defects and Diabetes Characteristics

Tissue-specific patterns of radial genome organization contribute to genome regulation and can be established by nuclear envelope proteins. Studies in this area often use cancer cell lines, and it is unclear how well such systems recapitulate genome organization of primary cells or animal tissues; so, we sought to investigate radial genome organization in primary liver tissue hepatocytes. Here, we have used a NET47/Tm7sf2–/– liver model to show that manipulating one of these nuclear membrane proteins is sufficient to alter tissue-specific gene positioning and expression. Dam-LaminB1 global profiling in primary liver cells shows that nearly all the genes under such positional regulation are related to/important for liver function. Interestingly, Tm7sf2 is a paralog of the HP1-binding nuclear membrane protein LBR that, like Tm7sf2, also has an enzymatic function in sterol reduction. Fmo3 gene/locus radial mislocalization could be rescued with human wild-type, but not TM7SF2 mutants lacking the sterol reductase function. One central pathway affected is the cholesterol synthesis pathway. Within this pathway, both Cyp51 and Msmo1 are under Tm7sf2 positional and expression regulation. Other consequences of the loss of Tm7sf2 included weight gain, insulin sensitivity, and reduced levels of active Akt kinase indicating additional pathways under its regulation, several of which are highlighted by mispositioning genes. This study emphasizes the importance for tissue-specific radial genome organization in tissue function and the value of studying genome organization in animal tissues and primary cells over cell lines.

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Methylation of the Cyclin A1 Promoter Correlates with Gene Silencing in Somatic Cell Lines, while Tissue-Specific Expression of Cyclin A1 Is Methylation Independent

Molecular and Cellular Biology ◽

10.1128/mcb.20.9.3316-3329.2000 ◽

2000 ◽

Vol 20 (9) ◽

pp. 3316-3329 ◽

Cited By ~ 55

Author(s):

Carsten Müller ◽

Carol Readhead ◽

Sven Diederichs ◽

Gregory Idos ◽

Rong Yang ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Promoter Methylation ◽

Cpg Island ◽

Trichostatin A ◽

Specific Gene ◽

Cyclin A1 ◽

Specific Expression ◽

Tissue Specific ◽

Tissue Specific Expression

ABSTRACT Gene expression in mammalian organisms is regulated at multiple levels, including DNA accessibility for transcription factors and chromatin structure. Methylation of CpG dinucleotides is thought to be involved in imprinting and in the pathogenesis of cancer. However, the relevance of methylation for directing tissue-specific gene expression is highly controversial. The cyclin A1 gene is expressed in very few tissues, with high levels restricted to spermatogenesis and leukemic blasts. Here, we show that methylation of the CpG island of the human cyclin A1 promoter was correlated with nonexpression in cell lines, and the methyl-CpG binding protein MeCP2 suppressed transcription from the methylated cyclin A1 promoter. Repression could be relieved by trichostatin A. Silencing of a cyclin A1 promoter-enhanced green fluorescent protein (EGFP) transgene in stable transfected MG63 osteosarcoma cells was also closely associated with de novo promoter methylation. Cyclin A1 could be strongly induced in nonexpressing cell lines by trichostatin A but not by 5-aza-cytidine. The cyclin A1 promoter-EGFP construct directed tissue-specific expression in male germ cells of transgenic mice. Expression in the testes of these mice was independent of promoter methylation, and even strong promoter methylation did not suppress promoter activity. MeCP2 expression was notably absent in EGFP-expressing cells. Transcription from the transgenic cyclin A1 promoter was repressed in most organs outside the testis, even when the promoter was not methylated. These data show the association of methylation with silencing of the cyclin A1 gene in cancer cell lines. However, appropriate tissue-specific repression of the cyclin A1 promoter occurs independently of CpG methylation.

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238 PRODUCTION OF CLEAVAGE-RESISTANT PHYTASE TRANSGENIC PIGS BY HANDMADE CLONING

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab238 ◽

2016 ◽

Vol 28 (2) ◽

pp. 251

Author(s):

M. Zhang ◽

S. Chen ◽

X. Chen ◽

Y. Huang ◽

L. Wei ◽

...

Keyword(s):

Cell Lines ◽

Animal Husbandry ◽

Phytase Activity ◽

Specific Gene ◽

Transgenic Pigs ◽

Specific Expression ◽

Phytase Gene ◽

Tissue Specific ◽

Fetal Fibroblasts ◽

Handmade Cloning

Rapidly developing and intensive animal husbandry of livestock is a major contributor to global environmental pollutions. Large quantities and high concentrations of manure waste that contains phytate phosphorus are generated. The use of phytase can effectively solve the problem of high phosphorus pollution in the fecal material of monogastric animals. Enviropigs, producing phytase in the salivary glands and secreting the enzyme in the saliva, were first generated at the University of Guelph (Guelph, ON, Canada) in 1999. However, phytase is easily inactivated in digestive processing. To address this problem, we improved the transgene construct and successfully generated phytase transgenic pigs by handmade cloning. The Escherichia coli periplasmic phosphoanhydride phosphohydrolase (appA) gene was subcloned. Using fragment substitution method, we designed a phytase gene that was insensitive to cleavage by pepsin and trypsin and had a higher affinity for the substrate. After codon optimization, the designed phytase gene was named Cafp and subcloned downstream of the pig parotid secretory protein (PSP) gene promoter. The tissue-specific vector p-PSP-Intron-Cafp was constructed and transferred into Landrace fetal fibroblasts by electroporation. The cell lines carrying Cafp were used as nuclear donors in handmade cloning. Cloned embryos were cultured in vitro to blastocysts and transferred to recipient sows. The presence of Cafp was tested by PCR and sequencing of cloned pigs. Phytase activity in saliva, feed, and feces was detected by the ammonium molybdate method with a slight modification. Immunohistochemistry (IHC) was used to determine tissue-specific expression. Three cell lines carrying Cafp were obtained. We generated 1027 blastocysts; 712 were of good quality and transferred to 6 recipients. Fourteen piglets were born, of which 6 survived. The PCR and sequencing results showed that 7 (3 live and 4 dead) of the 14 piglets carried Cafp. Phytase activity in the saliva of the 6 live cloned pigs was tested at 4 months of age and only 1 pig had 0.155 FTU mL–1 enzyme activity. The enzyme in the other 2 pigs may be inactivated in the transgenic parotid gland. Among all the transgenic pigs, the highest phosphorus digestion rate was 59.2% of intake, which represents a 25.4% decrease in fecal emissions compared with the average of controls. The IHC results on the 3 later dead, Cafp-positive pigs showed that the transgene was expressed only in parotids, confirming tissue-specific gene expression. In summary, cleavage-resistant phytase transgenic pigs were successfully produced through handmade cloning. The cloned pigs offer a unique biological approach to manage phosphorus nutrition and environmental pollution in animal husbandry.

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Assessment of the Utility of Gene Positioning Biomarkers in the Stratification of Prostate Cancers

10.1101/728972 ◽

2019 ◽

Author(s):

Karen J. Meaburn ◽

Tom Misteli

Keyword(s):

Genome Organization ◽

Metastatic Cancer ◽

Cell Types ◽

Specific Gene ◽

Gene Positioning ◽

Spatial Reorganization ◽

Prostate Cancers ◽

Spatial Positioning ◽

Context Specific ◽

Spatial Genome Organization

AbstractIn eukaryotic cells, the genome is spatially organized in a non-random fashion within the confines of the interphase nucleus, and most genes occupy preferred nuclear positions. For some genomic loci these positioning patterns are context specific, reflected in the distinct location of certain genes and chromosomes in different cell types and in disease. Disease-related differential spatial positioning of genes has led to the hypothesis that the spatial reorganization of the genome may be utilized as a diagnostic biomarker. In keeping with this possibility, the positioning patterns of specific genes can be used to reproducibly discriminate benign tissues from cancerous ones. In addition to the use of spatial genome organization for diagnostic purposes, we explore here the potential use of spatial genome organization as a prognostic tool. This is a pressing need since in many cancer types there is a lack of accurate markers to predict the aggressiveness of individual tumors. We find that directional repositioning of SP100 and TGFB3 gene loci stratifies prostate cancers of differing Gleason scores. A more peripheral position of SP100 and TGFB3 in the nucleus, compared to benign tissues, is associated with low Gleason score cancers, whereas more internal positioning correlates with higher Gleason scores. Conversely, LMNA is more internally positioned in many non-metastatic prostate cancers, while its position is indistinguishable from benign tissue in metastatic cancer. Our findings of subtype-specific gene positioning patterns in prostate cancer provides a proof-of-concept for the potential usefulness of spatial gene positioning as a prognostic biomarker.

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The 3D enhancer network of the developing T cell genome is controlled by SATB1

10.1101/2021.07.09.451769 ◽

2021 ◽

Author(s):

Tomas Zelenka ◽

Antonios Klonizakis ◽

Despina Tsoukatou ◽

Sören Franzenburg ◽

Petros Tzerpos ◽

...

Keyword(s):

Immune System ◽

T Cell ◽

Genome Organization ◽

Molecular Mechanisms ◽

Regulatory Elements ◽

Conditional Knockout ◽

Specific Factor ◽

Specific Gene ◽

Gene Promoters ◽

Tissue Specific

Mechanisms of tissue-specific gene expression regulation via spatial coordination of gene promoters and distal regulatory elements are still poorly understood. We investigated the 3D genome organization of developing murine T cells and identified SATB1, a tissue-specific genome organizer, enriched at the anchors of promoter-enhancer chromatin loops. We assessed the function of SATB1 in T cell chromatin organization and compared it to the conventional genome organizer CTCF. SATB1 builds a more refined layer of genome organization upon a CTCF scaffold. To understand the regulatory implications of SATB1 loopscape structure, we generated Satb1fl/flCd4-Cre+ (Satb1 cKO) conditional knockout animals which suffered from autoimmunity. We aimed to identify molecular mechanisms responsible for the deregulation of the immune system in Satb1 cKO animals. H3K27ac HiChIP and Hi-C experiments indicated that SATB1 primarily mediates promoter-enhancer loops affecting master regulator genes (such as Bcl6), the T cell receptor locus and adhesion molecule genes, collectively being critical for cell lineage specification and immune system homeostasis. Our findings unravel the function of a tissue-specific factor that controls transcription programs, via spatial chromatin arrangements complementary to the chromatin structure imposed by ubiquitously expressed genome organizers.

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Faculty Opinions recommendation of Tissue-Specific Gene Repositioning by Muscle Nuclear Membrane Proteins Enhances Repression of Critical Developmental Genes during Myogenesis.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726403555.793523818 ◽

2016 ◽

Author(s):

Lucio Comai

Keyword(s):

Membrane Proteins ◽

Nuclear Membrane ◽

Specific Gene ◽

Developmental Genes ◽

Tissue Specific ◽

Tissue Specific Gene

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Tissue-Specific Gene Repositioning by Muscle Nuclear Membrane Proteins Enhances Repression of Critical Developmental Genes during Myogenesis

Molecular Cell ◽

10.1016/j.molcel.2016.04.035 ◽

2016 ◽

Vol 62 (6) ◽

pp. 834-847 ◽

Cited By ~ 84

Author(s):

Michael I. Robson ◽

Jose I. de las Heras ◽

Rafal Czapiewski ◽

Phú Lê Thành ◽

Daniel G. Booth ◽

...

Keyword(s):

Membrane Proteins ◽

Nuclear Membrane ◽

Specific Gene ◽

Developmental Genes ◽

Tissue Specific ◽

Tissue Specific Gene

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T cell receptor beta gene has two downstream DNase I hypersensitive regions. Possible mechanisms of tissue- and stage-specific gene regulation.

Journal of Experimental Medicine ◽

10.1084/jem.169.6.2097 ◽

1989 ◽

Vol 169 (6) ◽

pp. 2097-2107 ◽

Cited By ~ 7

Author(s):

Y Hashimoto

Keyword(s):

Gene Expression ◽

T Cell ◽

Cell Lines ◽

Regulatory Elements ◽

Dnase I ◽

The Other ◽

Specific Gene ◽

Enhancer Element ◽

Tissue Specific ◽

T Cell Lines

Two DNase I-hypersensitive regions were identified downstream of the TCR gene constant region. One of these regions is located at the site of a putative enhancer element and was observed only in T cell lines and not in cell lines derived from other tissues. The other DNase-hypersensitive region was also detected only in T cell lines but only in those expressing TCR-beta RNA. Thus, the first region is probably tissue specific, while the second region is probably tissue and stage specific. The DNA sequence of the second DNase I-hypersensitive region revealed several stretches of nucleotides that are characteristic of consensus sequences for regulatory elements. These results, together with the observations in transgenic mice that indicate a requirement for two distinct regions for optimal TCR gene expression, suggest the presence of at least two regulatory regions downstream of the C-beta-2 region; one is an enhancer region and the other is a transcriptionally related regulatory region. The tissue/stage specificity of these DNase I-hypersensitive regions supports the notion that changes in chromatin structure control tissue-specific gene expression.

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Toward an understanding of the relation between gene regulation and 3D genome organization

10.1101/2020.01.13.903872 ◽

2020 ◽

Author(s):

Hao Tian ◽

Ying Yang ◽

Sirui Liu ◽

Hui Quan ◽

Yi Qin Gao

Keyword(s):

Gene Regulation ◽

Chromatin Structure ◽

Genome Organization ◽

Network Formation ◽

Expression Profiles ◽

Chromosome Conformation ◽

Tissue Specific ◽

3D Genome ◽

Gene Positioning ◽

3D Chromatin Structure

AbstractThe development and usage of chromosome conformation capture technologies have provided great details on 3D genome organization and provide great opportunities to understand how gene regulation is affected by the 3D chromatin structure. Previously, we identified two types of sequence domains, CGI forest and CGI prairie, which tend to segregate spatially, but to different extent in different tissues/cell states. To further quantify the association of domain segregation with gene regulation and differentiation, we analyzed in this study the distribution of genes of different tissue specificities along the linear genome, and found that the distribution patterns are distinctly different in forests and prairies. The tissue-specific genes (TSGs) are significantly enriched in the latter but not in the former and genes of similar expression profiles among different cell types (co-activation/repression) also tend to cluster in specific prairies. We then analyzed the correlation between gene expression and the spatial contact revealed in Hi-C measurement. Tissue-specific forest-prairie contact formation was found to correlate with the regulation of the TSGs, in particular those in the prairie domains, pointing to the important role gene positioning, in the linear DNA sequence as well as in 3D chromatin structure, plays in gene regulatory network formation.

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