X-chromosome-linked miR548am-5p is a key regulator of sex disparity in the susceptibility to mitochondria-mediated apoptosis

Abstract Sex dimorphism in cell response to stress has previously been investigated by different research groups. This dimorphism could be at least in part accounted for by sex-biased expression of regulatory elements such as microRNAs (miRs). In order to spot previously unknown miR expression differences we took advantage of prior knowledge on specialized databases to identify X chromosome-encoded miRs potentially escaping X chromosome inactivation (XCI). MiR-548am-5p emerged as potentially XCI escaper and was experimentally verified to be significantly up-regulated in human XX primary dermal fibroblasts (DFs) compared to XY ones. Accordingly, miR-548am-5p target mRNAs, e.g. the transcript for Bax, was differently modulated in XX and XY DFs. Functional analyses indicated that XY DFs were more prone to mitochondria-mediated apoptosis than XX ones. Experimentally induced overexpression of miR548am-5p in XY cells by lentivirus vector transduction decreased apoptosis susceptibility, whereas its down-regulation in XX cells enhanced apoptosis susceptibility. These data indicate that this approach could be used to identify previously unreported sex-biased differences in miR expression and that a miR identified with this approach, miR548am-5p, can account for sex-dependent differences observed in the susceptibility to mitochondrial apoptosis of human DFs.

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Molecular biology approaches in bioadhesion research

Beilstein Journal of Nanotechnology ◽

10.3762/bjnano.5.112 ◽

2014 ◽

Vol 5 ◽

pp. 983-993 ◽

Cited By ~ 12

Author(s):

Marcelo Rodrigues ◽

Birgit Lengerer ◽

Thomas Ostermann ◽

Peter Ladurner

Keyword(s):

Molecular Biology ◽

Biological Function ◽

Molecular Approach ◽

Research Groups ◽

Blast Search ◽

Search Facility ◽

New Research ◽

And Function ◽

Functional Analyses

The use of molecular biology tools in the field of bioadhesion is still in its infancy. For new research groups who are considering taking a molecular approach, the techniques presented here are essential to unravelling the sequence of a gene, its expression and its biological function. Here we provide an outline for addressing adhesion-related genes in diverse organisms. We show how to gradually narrow down the number of candidate transcripts that are involved in adhesion by (1) generating a transcriptome and a differentially expressed cDNA list enriched for adhesion-related transcripts, (2) setting up a BLAST search facility, (3) perform an in situ hybridization screen, and (4) functional analyses of selected genes by using RNA interference knock-down. Furthermore, latest developments in genome-editing are presented as new tools to study gene function. By using this iterative multi-technologies approach, the identification, isolation, expression and function of adhesion-related genes can be studied in most organisms. These tools will improve our understanding of the diversity of molecules used for adhesion in different organisms and these findings will help to develop innovative bio-inspired adhesives.

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Notch-dependent DNA cis-regulatory elements and their dose-dependent control of C. elegans stem cell self-renewal

10.1101/2021.11.09.467950 ◽

2021 ◽

Author(s):

Tina R. Lynch ◽

Mingyu Xue ◽

Cazza W. Czerniak ◽

ChangHwan Lee ◽

Judith Kimble

Keyword(s):

Stem Cell ◽

Regulatory Elements ◽

Single Element ◽

Developmental Context ◽

C Elegans ◽

Stem Cell Maintenance ◽

Nascent Transcripts ◽

Functional Analyses ◽

Dose Dependent

A long-standing biological question is how DNA cis-regulatory elements shape transcriptional patterns during metazoan development. The use of reporter constructs, cell culture and computational modeling has made enormous contributions to understanding this fundamental question, but analysis of regulatory elements in their natural developmental context is an essential but rarely used complement. Here, we edited Notch-dependent cis-regulatory elements in the endogenous C. elegans sygl-1 gene, which encodes a key stem cell regulator. We then analyzed the in vivo consequences of those mutations – on both gene expression (nascent transcripts, mRNA, protein) and stem cell maintenance. Mutation of a single element in a three-element homotypic cluster reduced expression as well as stem cell pool size by about half, while mutation of two elements essentially abolished them. We find that LBS number and LBS neighborhood are both important to activity: elements on separate chromosomes function additively, while elements in the same cluster act synergistically. Our approach of precise CRISPR/Cas9 gene editing coupled with quantitation of both molecular and biological readouts establishes a powerful model for in vivo functional analyses of DNA cis-regulatory elements.

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Studies of esterase 6 in Drosophila melanogaster: XIV. Variation of esterase 6 levels controlled by unlinked genes in natural populations

Genetics Research ◽

10.1017/s0016672300025891 ◽

1984 ◽

Vol 43 (2) ◽

pp. 181-190 ◽

Cited By ~ 7

Author(s):

Craig S. Tepper ◽

Anne L. Terry ◽

James E. Holmes ◽

Rollin C. Richmond

Keyword(s):

Drosophila Melanogaster ◽

Structural Gene ◽

X Chromosome ◽

Genetic Background ◽

Natural Populations ◽

Regulatory Elements ◽

Regulatory Locus ◽

The Third ◽

Esterase 6 ◽

Activity Modifiers

SUMMARYThe esterase 6 (Est-6) locus in Drosophila melanogaster is located on the third chromosome and is the structural gene for a carboxylesterase (E.C.3.1.1.1) and is polymorphic for two major electromorphs (slow and fast). Isogenic lines containing X chromosomes extracted from natural populations and substituted into a common genetic background were used to detect unlinked factors that affect the activity of the Est-6 locus. Twofold activity differences of esterase 6 (EST 6) were found among males from these derived lines, which differ only in their X chromosome. These unlinked activity modifiers identify possible regulatory elements. Immunoelectrophoresis was used to estimate quantitatively the levels of specific cross-reacting material in the derived lines. The results show that the variation in activity is due to differences in the amount of EST 6 present. The data are consistent with the hypothesis that there is at least one locus on the X chromosome that regulates the synthesis of EST 6 and that this regulatory locus may be polymorphic in natural populations.

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A Functional Chromatin Domain Does Not Resist X Chromosome Inactivation: Silencing of cLys Correlates with Methylation of a Dual Promoter-Replication Origin

Molecular and Cellular Biology ◽

10.1128/mcb.22.13.4667-4676.2002 ◽

2002 ◽

Vol 22 (13) ◽

pp. 4667-4676 ◽

Cited By ~ 11

Author(s):

Suyinn Chong ◽

Joanna Kontaraki ◽

Constanze Bonifer ◽

Arthur D. Riggs

Keyword(s):

X Chromosome ◽

Copy Number ◽

Cpg Island ◽

Regulatory Elements ◽

X Chromosome Inactivation ◽

Chromosome Inactivation ◽

Chromatin Domain ◽

Specific Expression ◽

Position Effects ◽

Chicken Lysozyme

ABSTRACT To investigate the molecular mechanism(s) involved in the propagation and maintenance of X chromosome inactivation (XCI), the 21.4-kb chicken lysozyme (cLys) chromatin domain was inserted into the Hprt locus on the mouse X chromosome. The inserted fragment includes flanking matrix attachment regions (MARs), an origin of bidirectional replication (OBR), and all the cis-regulatory elements required for correct tissue-specific expression of cLys. It also contains a recently identified and widely expressed second gene, cGas41. The cLys domain is known to function as an autonomous unit resistant to chromosomal position effects, as evidenced by numerous transgenic mouse lines showing copy-number-dependent and development-specific expression of cLys in the myeloid lineage. We asked the questions whether this functional chromatin domain was resistant to XCI and whether the X inactivation signal could spread across an extended region of avian DNA. A generally useful method was devised to generate pure populations of macrophages with the transgene either on the active (Xa) or the inactive (Xi) chromosome. We found that (i) cLys and cGas41 are expressed normally from the Xa; (ii) the cLys chromatin domain, even when bracketed by MARs, is not resistant to XCI; (iii) transcription factors are excluded from lysozyme enhancers on the Xi; and (iv) inactivation correlates with methylation of a CpG island that is both an OBR and a promoter of the cGas41 gene.

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Getting to the center of X-chromosome inactivation: the role of transgenesThis paper is one of a selection of papers published in this Special Issue, entitled 30th Annual International Asilomar Chromatin and Chromosomes Conference, and has undergone the Journal’s usual peer review process.

Biochemistry and Cell Biology ◽

10.1139/o09-040 ◽

2009 ◽

Vol 87 (5) ◽

pp. 759-766 ◽

Cited By ~ 3

Author(s):

Jakub Minks ◽

Carolyn J. Brown

Keyword(s):

X Chromosome ◽

Peer Review Process ◽

Regulatory Elements ◽

X Chromosome Inactivation ◽

X Inactivation ◽

Chromosome Inactivation ◽

Xist Expression ◽

Inactivation Center ◽

Xist Rna ◽

Epigenetic Phenomenon

X-chromosome inactivation is a fascinating epigenetic phenomenon that is initiated by expression of a noncoding (nc)RNA, XIST, and results in transcriptional silencing of 1 female X. The process requires a series of events that begins even before XIST expression, and culminates in an active and a silent X within the same nucleus. We will focus on the role that transgenic systems have served in the current understanding of the process of X-chromosome inactivation, both in the initial delineation of an active and inactive X, and in the function of the XIST RNA. X inactivation is strictly cis-limited; recent studies have revealed elements within the X-inactivation center, the region required for inactivation, that are critical for the initial regulation of Xist expression and chromosome pairing. It has been revealed that the X-inactivation center contains a remarkable compendium of cis-regulatory elements, ncRNAs, and trans-acting pairing regions. We review the functional componentry of the X-inactivation center and discuss experiments that helped to dissect the XIST/Xist RNA and its involvement in the establishment of facultative heterochromatin.

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Functional analyses of PAU genes in Saccharomyces cerevisiae

Microbiology ◽

10.1099/mic.0.030726-0 ◽

2009 ◽

Vol 155 (12) ◽

pp. 4036-4049 ◽

Cited By ~ 33

Author(s):

Zongli Luo ◽

Hennie J. J. van Vuuren

Keyword(s):

Saccharomyces Cerevisiae ◽

Pairwise Comparison ◽

Expression Data ◽

Coding Regions ◽

Response To Stress ◽

Flanking Sequences ◽

The Stability ◽

The Individual ◽

Functional Analyses ◽

Subtelomeric Regions

PAU genes constitute the largest gene family in Saccharomyces cerevisiae, with 24 members mostly located in the subtelomeric regions of chromosomes. Little information is available about PAU genes, other than expression data for some members. In this study, we systematically compared the sequences of all 24 members, examined the expression of PAU3, PAU5, DAN2, PAU17 and PAU20 in response to stresses, and investigated the stability of all Pau proteins. The chromosomal localization, synteny and sequence analyses revealed that PAU genes could have been amplified by segmental and retroposition duplication through mechanisms of chromosomal end translocation and Ty-associated recombination. The coding sequences diverged through nucleotide substitution and insertion/deletion of one to four codons, thus causing changes in amino acids, truncation or extension of Pau proteins. Pairwise comparison of non-coding regions revealed little homology in flanking sequences of some members. All 24 PAU promoters contain a TATA box, and 22 PAU promoters contain at least one copy of the anaerobic response element and the aerobic repression motif. Differential expression was observed among PAU3, PAU5, PAU17, PAU20 and DAN2 in response to stress, with PAU5 having the highest capacity to be induced by anaerobic conditions, low temperature and wine fermentations. Furthermore, Pau proteins with 124 aa were less stable than those with 120 or 122 aa. Our results indicate that duplicated PAU genes have been evolving, and the individual Pau proteins might possess specific roles for the adaptation of S. cerevisiae to certain environmental stresses.

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Functional Analysis of the DXPas34Locus, a 3′ Regulator of Xist Expression

Molecular and Cellular Biology ◽

10.1128/mcb.19.12.8513 ◽

1999 ◽

Vol 19 (12) ◽

pp. 8513-8525 ◽

Cited By ~ 72

Author(s):

E. Debrand ◽

C. Chureau ◽

D. Arnaud ◽

P. Avner ◽

E. Heard

Keyword(s):

X Chromosome ◽

Yeast Artificial Chromosome ◽

Es Cells ◽

Artificial Chromosome ◽

Antisense Transcription ◽

Regulatory Elements ◽

X Inactivation ◽

X Chromosomes ◽

Inactivation Center ◽

Controlling Element

ABSTRACT X inactivation in female mammals is controlled by a key locus on the X chromosome, the X-inactivation center (Xic). The Xic controls the initiation and propagation of inactivation in cis. It also ensures that the correct number of X chromosomes undergo inactivation (counting) and determines which X chromosome becomes inactivated (choice). The Xist gene maps to the Xic region and is essential for the initiation of X inactivation in cis. Regulatory elements of X inactivation have been proposed to lie 3′ toXist. One such element, lying 15 kb downstream ofXist, is the DXPas34 locus, which was first identified as a result of its hypermethylation on the active X chromosome and the correlation of its methylation level with allelism at the X-controlling element (Xce), a locus known to affect choice. In this study, we have tested the potential function of theDXPas34 locus in Xist regulation and X-inactivation initiation by deleting it in the context of largeXist-containing yeast artificial chromosome transgenes. Deletion of DXPas34 eliminates both Xistexpression and antisense transcription present in this region in undifferentiated ES cells. It also leads to nonrandom inactivation of the deleted transgene upon differentiation. DXPas34 thus appears to be a critical regulator of Xist activity and X inactivation. The expression pattern of DXPas34 during early embryonic development, which we report here, further suggests that it could be implicated in the regulation of imprintedXist expression.

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Correction: X-chromosome-linked miR548am-5p is a key regulator of sex disparity in the susceptibility to mitochondria-mediated apoptosis

Cell Death and Disease ◽

10.1038/s41419-019-2069-0 ◽

2019 ◽

Vol 10 (11) ◽

Cited By ~ 1

Author(s):

Paola Matarrese ◽

Paolo Tieri ◽

Simona Anticoli ◽

Barbara Ascione ◽

Maria Conte ◽

...

Keyword(s):

X Chromosome ◽

Sex Disparity

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Identification of Developmentally Specific Enhancers for Tsix in the Regulation of X Chromosome Inactivation

Molecular and Cellular Biology ◽

10.1128/mcb.25.7.2757-2769.2005 ◽

2005 ◽

Vol 25 (7) ◽

pp. 2757-2769 ◽

Cited By ~ 55

Author(s):

Nicholas Stavropoulos ◽

Rebecca K. Rowntree ◽

Jeannie T. Lee

Keyword(s):

X Chromosome ◽

Regulatory Elements ◽

X Chromosome Inactivation ◽

Chromosome Inactivation ◽

X Chromosomes ◽

Binary Switch ◽

Inactive X Chromosome ◽

The Future ◽

Hypersensitive Sites ◽

Mammalian Female

ABSTRACT X chromosome inactivation silences one of two X chromosomes in the mammalian female cell and is controlled by a binary switch that involves interactions between Xist and Tsix, a sense-antisense pair of noncoding genes. On the future active X chromosome, Tsix expression suppresses Xist upregulation, while on the future inactive X chromosome, Tsix repression is required for Xist-mediated chromosome silencing. Thus, understanding the binary switch mechanism depends on ascertaining how Tsix expression is regulated. Here we have taken an unbiased approach toward identifying Tsix regulatory elements within the X chromosome inactivation center. First, we defined the major Tsix promoter and found that it cannot fully recapitulate the developmental dynamics of Tsix expression, indicating a requirement for additional regulatory elements. We then delineated two enhancers, one classical enhancer mapping upstream of Tsix and a bipartite enhancer that flanks the major Tsix promoter. These experiments revealed the intergenic transcription element Xite as an enhancer of Tsix and the repeat element DXPas34 as a component of the bipartite enhancer. Each enhancer contains DNase I-hypersensitive sites and appears to confer developmental specificity to Tsix expression. Characterization of these enhancers will facilitate the identification of trans-acting regulatory factors for X chromosome counting and choice.

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