A Drosophila Insulator Interacting Protein Suppresses Enhancer-Blocking Function and Modulates Replication Timing

Mapping Intimacies ◽

10.1101/661041 ◽

2019 ◽

Author(s):

Emily C. Stow ◽

Ran An ◽

Todd A. Schoborg ◽

Nastasya M. Davenport ◽

James R. Simmons ◽

...

Keyword(s):

Genome Structure ◽

Nuclear Lamina ◽

Replication Timing ◽

Interacting Protein ◽

Insulator Protein ◽

Protein Partners ◽

Insulator Proteins ◽

Enhancer Blocking ◽

And Function ◽

Partner Proteins

AbstractInsulators play important roles in genome structure and function in Drosophila and mammals. More than six different insulator proteins are required in Drosophila for normal genome function, whereas CTCF is the only identified protein contributing to insulator function in mammals. Interactions between a DNA binding insulator protein and its interacting partner proteins define the properties of each insulator site. The different roles of insulator protein partners in the Drosophila genome and how they confer functional specificity remain poorly understood. Functional analysis of insulator partner proteins in Drosophila is necessary to understand how genomes are compartmentalized and the roles that different insulators play in genome function. In Drosophila, the Suppressor of Hairy wing [Su(Hw)] insulator is targeted to the nuclear lamina, preferentially localizes at euchromatin/heterochromatin boundaries, and is associated with the Gypsy retrotransposon. The properties that the insulator confers to these sites rely on the ability of the Su(Hw) protein to bind the DNA at specific sites and interact with Mod(mdg4)-67.2 and CP190 partner proteins. HP1 and insulator partner protein 1 (HIPP1) is a recently identified partner of Su(Hw), but how HIPP1 contributes to the function of Su(Hw) insulators has not yet been elucidated. Here, we find that mutations in the HIPP1 crotonase-like domain have no impact on the function of Su(Hw) enhancer-blocking activity but do exhibit an impaired ability to repair double-strand breaks. Additionally, we find that the overexpression of each HIPP1 and Su(Hw) causes defects in cell proliferation by limiting the progression of DNA replication. We also find that HIPP1 overexpression suppresses the Su(Hw) insulator enhancer-blocking function.

Download Full-text

New Proteins Found Interacting with Brain Metallothionein-3 Are Linked to Secretion

International Journal of Alzheimer s Disease ◽

10.4061/2011/208634 ◽

2011 ◽

Vol 2011 ◽

pp. 1-9 ◽

Cited By ~ 3

Author(s):

Issam El Ghazi ◽

Bruce L. Martin ◽

Ian M. Armitage

Keyword(s):

Immunoaffinity Chromatography ◽

Glycolytic Metabolism ◽

Interacting Protein ◽

Aldolase C ◽

Growth Inhibitory Factor ◽

Protein Partners ◽

Growth Inhibitory ◽

Proposed Model ◽

The Brain ◽

Partner Proteins

Metallothionein 3 (MT-3), also known as growth inhibitory factor (GIF), exhibits a neuroinhibitory activity. Our lab and others have previously shown that this biological activity involves interacting protein partners in the brain. However, nothing specific is yet known about which of these interactions is responsible for the GIF activity. In this paper, we are reporting upon new proteins found interacting with MT-3 as determined through immunoaffinity chromatography and mass spectrometry. These new partner proteins—Exo84p, 14-3-3 Zeta,αandβEnolase, Aldolase C, Malate dehydrogenase, ATP synthase, and Pyruvate kinase—along with those previously identified have now been classified into three functional groups: transport and signaling, chaperoning and scaffolding, and glycolytic metabolism. When viewed together, these interactions support a proposed model for the regulation of the GIF activity of MT-3.

Download Full-text

Sites of chromosomal instability in the context of nuclear architecture and function

Cellular and Molecular Life Sciences ◽

10.1007/s00018-020-03698-2 ◽

2020 ◽

Author(s):

Constanze Pentzold ◽

Miriam Kokal ◽

Stefan Pentzold ◽

Anja Weise

Keyword(s):

Nuclear Architecture ◽

Nuclear Lamina ◽

Replication Timing ◽

Fragile Sites ◽

Chromosomal Breakage ◽

Metaphase Chromosomes ◽

Chromosomal Fragile Sites ◽

And Function ◽

Genomic Regions ◽

Mitotic Chromatin

AbstractChromosomal fragile sites are described as areas within the tightly packed mitotic chromatin that appear as breaks or gaps mostly tracing back to a loosened structure and not a real nicked break within the DNA molecule. Most facts about fragile sites result from studies in mitotic cells, mainly during metaphase and mainly in lymphocytes. Here, we synthesize facts about the genomic regions that are prone to form gaps and breaks on metaphase chromosomes in the context of interphase. We conclude that nuclear architecture shapes the activity profile of the cell, i.e. replication timing and transcriptional activity, thereby influencing genomic integrity during interphase with the potential to cause fragility in mitosis. We further propose fragile sites as examples of regions specifically positioned in the interphase nucleus with putative anchoring points at the nuclear lamina to enable a tightly regulated replication–transcription profile and diverse signalling functions in the cell. Consequently, fragility starts before the actual display as chromosomal breakage in metaphase to balance the initial contradiction of cellular overgrowth or malfunctioning and maintaining diversity in molecular evolution.

Download Full-text

Comparing 3D genome organization in multiple species using Phylo-HMRF

10.1101/552505 ◽

2019 ◽

Cited By ~ 1

Author(s):

Yang Yang ◽

Yang Zhang ◽

Bing Ren ◽

Jesse Dixon ◽

Jian Ma

Keyword(s):

Genome Organization ◽

Genome Mapping ◽

Mammalian Species ◽

Genome Structure ◽

Replication Timing ◽

Primate Species ◽

Spatial Constraints ◽

Evolutionary Patterns ◽

3D Genome ◽

And Function

AbstractRecent developments in whole-genome mapping approaches for the chromatin interactome (such as Hi-C) have offered new insights into 3D genome organization. However, our knowledge of the evolutionary patterns of 3D genome structures in mammalian species remains surprisingly limited. In particular, there are no existing phylogenetic-model based methods to analyze chromatin interactions as continuous features across different species. Here we develop a new probabilistic model, named phylogenetic hidden Markov random field (Phylo-HMRF), to identify evolutionary patterns of 3D genome structures based on multi-species Hi-C data by jointly utilizing spatial constraints among genomic loci and continuous-trait evolutionary models. The effectiveness of Phylo-HMRF is demonstrated in both simulation evaluation and application to real Hi-C data. We used Phylo-HMRF to uncover cross-species 3D genome patterns based on Hi-C data from the same cell type in four primate species (human, chimpanzee, bonobo, and gorilla). The identified evolutionary patterns of 3D genome organization correlate with features of genome structure and function, including long-range interactions, topologically-associating domains (TADs), and replication timing patterns. This work provides a new framework that utilizes general types of spatial constraints to identify evolutionary patterns of continuous genomic features and has the potential to reveal the evolutionary principles of 3D genome organization.

Download Full-text

Functional Phosphorylation Sites in the C-Terminal Region of the Multivalent Multifunctional Transcriptional Factor CTCF

Molecular and Cellular Biology ◽

10.1128/mcb.21.6.2221-2234.2001 ◽

2001 ◽

Vol 21 (6) ◽

pp. 2221-2234 ◽

Cited By ~ 67

Author(s):

Elena M. Klenova ◽

Igor V. Chernukhin ◽

Ayman El-Kady ◽

Robin E. Lee ◽

Elena M. Pugacheva ◽

...

Keyword(s):

Growth Regulation ◽

Wild Type ◽

Interacting Protein ◽

Binding Characteristics ◽

Protein Partners ◽

Enhancer Blocking ◽

Insulator Elements ◽

Necessary And Sufficient

ABSTRACT CTCF is a widely expressed and highly conserved multi-Zn-finger (ZF) nuclear factor. Binding to various CTCF target sites (CTSs) is mediated by combinatorial contributions of different ZFs. Different CTSs mediate distinct CTCF functions in transcriptional regulation, including promoter repression or activation and hormone-responsive gene silencing. In addition, the necessary and sufficient core sequences of diverse enhancer-blocking (insulator) elements, including CpG methylation-sensitive ones, have recently been pinpointed to CTSs. To determine whether a posttranslational modification may modulate CTCF functions, we studied CTCF phosphorylation. We demonstrated that most of the modifications that occur at the carboxy terminus in vivo can be reproduced in vitro with casein kinase II (CKII). Major modification sites map to four serines within the S604KKEDS609S610DS612E motif that is highly conserved in vertebrates. Specific mutations of these serines abrogate phosphorylation of CTCF in vivo and CKII-induced phosphorylation in vitro. In addition, we showed that completely preventing phosphorylation by substituting all serines within this site resulted in markedly enhanced repression of the CTS-bearing vertebrate c-myc promoters, but did not alter CTCF nuclear localization or in vitro DNA-binding characteristics assayed with c-myc CTSs. Moreover, these substitutions manifested a profound effect on negative cell growth regulation by wild-type CTCF. CKII may thus be responsible for attenuation of CTCF activity, either acting on its own or by providing the signal for phosphorylation by other kinases and for CTCF-interacting protein partners.

Download Full-text

Impact of DNA methylation on 3D genome structure

Nature Communications ◽

10.1038/s41467-021-23142-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Diana Buitrago ◽

Mireia Labrador ◽

Juan Pablo Arcon ◽

Rafael Lema ◽

Oscar Flores ◽

...

Keyword(s):

Dna Methylation ◽

Chromatin Structure ◽

Chromatin Condensation ◽

Genome Structure ◽

Dna Methyltransferases ◽

Model System ◽

Structure And Function ◽

3D Genome ◽

Cellular Machinery ◽

And Function

AbstractDetermining the effect of DNA methylation on chromatin structure and function in higher organisms is challenging due to the extreme complexity of epigenetic regulation. We studied a simpler model system, budding yeast, that lacks DNA methylation machinery making it a perfect model system to study the intrinsic role of DNA methylation in chromatin structure and function. We expressed the murine DNA methyltransferases in Saccharomyces cerevisiae and analyzed the correlation between DNA methylation, nucleosome positioning, gene expression and 3D genome organization. Despite lacking the machinery for positioning and reading methylation marks, induced DNA methylation follows a conserved pattern with low methylation levels at the 5’ end of the gene increasing gradually toward the 3’ end, with concentration of methylated DNA in linkers and nucleosome free regions, and with actively expressed genes showing low and high levels of methylation at transcription start and terminating sites respectively, mimicking the patterns seen in mammals. We also see that DNA methylation increases chromatin condensation in peri-centromeric regions, decreases overall DNA flexibility, and favors the heterochromatin state. Taken together, these results demonstrate that methylation intrinsically modulates chromatin structure and function even in the absence of cellular machinery evolved to recognize and process the methylation signal.

Download Full-text

Wheat genome structure and function: genome sequence data and the International Wheat Genome Sequencing Consortium

Australian Journal of Agricultural Research ◽

10.1071/ar06155 ◽

2007 ◽

Vol 58 (6) ◽

pp. 470 ◽

Cited By ~ 11

Author(s):

P. Moolhuijzen ◽

D. S. Dunn ◽

M. Bellgard ◽

M. Carter ◽

J. Jia ◽

...

Keyword(s):

Genome Sequencing ◽

Physical Map ◽

Genome Structure ◽

Genetic Research ◽

Structure And Function ◽

Wheat Genome ◽

D Genome ◽

Genome Sequencing Consortium ◽

And Function ◽

Bac Contigs

Genome sequencing and the associated bioinformatics is now a widely accepted research tool for accelerating genetic research and the analysis of genome structure and function of wheat because it leverages similar work from other crops and plants. The International Wheat Genome Sequencing Consortium addresses the challenge of wheat genome structure and function and builds on the research efforts of Professor Bob McIntosh in the genetics of wheat. Currently, expressed sequence tags (ESTs; ~500 000 to date) are the largest sequence resource for wheat genome analyses. It is estimated that the gene coverage of the wheat EST collection is ~60%, close to that of Arabidopsis, indicating that ~40% of wheat genes are not represented in EST collections. The physical map of the D-genome donor species Aegilops tauschii is under construction (http://wheat.pw.usda.gov/PhysicalMapping). The technologies developed in this analysis of the D genome provide a good model for the approach to the entire wheat genome, namely compiling BAC contigs, assigning these BAC contigs to addresses in a high resolution genetic map, filling in gaps to obtain the entire physical length of a chromosome, and then large-scale sequencing.

Download Full-text

Abstract 250: Haploinsufficiency of Muscle-Enriched A-Type Lamin Interacting Protein (MLIP) Manifests as Enlarged Dilated Hearts

Circulation Research ◽

10.1161/res.111.suppl_1.a250 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Patrick Burgon ◽

Julia Lockwood ◽

Glenn Wells ◽

Alexandre Blais

Keyword(s):

Heart Development ◽

Left Ventricular ◽

Lamin A ◽

Interacting Protein ◽

Cellular Hypertrophy ◽

Cellular Source ◽

Alternatively Spliced ◽

Normal Body Weight ◽

Heart Size ◽

And Function

Approximately 116 unique mutations in the lamin A/C gene have been described to date that are associated with dilated cardiomyopathy. We recently reported the discovery of MLIP through its interaction with lamin A/C. MLIP is expressed ubiquitously and most abundantly in heart, skeletal and smooth muscle of amniotes (mammals, reptiles and birds) and has no paralogous homologue suggesting no functional redundancy. The MLIP gene encodes at least seven, alternatively spliced, LMNA-interacting factors that possess several structural motifs not found in any other protein. The MLIP isoforms pattern of expression differs between each of the tissues with heart being the most heterogeneous. Down-regulation of lamin A/C expression by shRNA results in the up-regulation and mis-localization of MLIP. In addition to interacting and co-localizing with lamin A/C we also demonstrated that MLIP localizes to micro-domains in the nucleus with promyelocytic leukemia protein (PML) in close proximity to chromatin. MLIP's biological function still remains elusive. Eight week old hemizygous MLIP null mice develop enlarged hearts with a significant increase in heart to body weights (MLIP+/+ 5.62mg/g vs MLIP+/- 10.73mg/g, p<0.0001 n=7) with an overall 30% increase in the anterior-posterior ventricle length of MLIP hearts while maintaining a normal body weight (Figure). Echocardiographic analysis of MLIP+/- mice revealed that their hearts as having a significant (p3.93mm with a significant (p=0.011, n=12) reduction of left ventricular fractional shorting (LVFS) 31% when compare to littermate controls. Histological analysis of the hearts showed no overt phenotype other than an overall increase in the size of the MLIP+/- hearts. The cellular source for the increase in heart size and mass remains to be determined if it is the product of an increase in the number of cardiomyocytes due to aberrant hyperplasia or an increase in cardiomyocyte size through cellular hypertrophy. In conclusion, MLIP is a newly discovered lamin interacting protein that may serve as a transcriptional regulator that impact genes involved in heart development, growth and function and provides a new signaling paradigm.

Download Full-text

Abstract 37: Nrip Deficiency Leads to Dilated Cardiomyopathy

Circulation Research ◽

10.1161/res.117.suppl_1.37 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Show-Li Chen

Keyword(s):

Calcium Transient ◽

Receptor Interaction ◽

Cell Width ◽

Interacting Protein ◽

Cardiac Tissues ◽

Calmodulin Binding ◽

Calcineurin Phosphatase ◽

And Function

Previously, we demonstrate a gene, nuclear receptor interaction protein (NRIP, also named DCAF6 or IQWD1) as a Ca2+- dependent calmodulin binding protein that can activate calcineurin phosphatase activity. Here, we found that α-actinin-2 (ACTN2), is one of NRIP-interacting proteins from the yeast two-hybrid system using NRIP as a prey. We further confirmed the direct bound between NRIP and ACTN2 using in vitro protein-protein interaction and in vivo co-immunoprecipitation assays. To further map the binding domain of each protein, the results showed the IQ domain of NRIP responsible for ACTN2 binding, and EF hand motif of ACTN2 responsible for NRIP bound. Due to ACTN2 is a biomarker of muscular Z-disc complex; we found the co-localization of NRIP and ACTN2 in cardiac tissues by immunofluorescence assays. Taken together, NRIP is a novel ACTN2-interacting protein. To investigate insights into in vivo function of NRIP, we generated conventional NRIP-null mice. The H&E staining results are shown in the hearts of NRIP KO mice are enlarged and dilated and the cell width of NRIP KO cardiomyocyte is increased. The EM of NRIP KO heart muscles reveal the reduction of I-band width and extension length of Z-disc in sarcomere structure; and the echocardiography shows the diminished fractional shortening in heart functions. Additionally, the calcium transient and sarcomere contraction length in cardiomyocytes of NRIP KO is weaker and shorter than wt; respectively. In conclusion, NRIP is a novel Z-disc protein and has function for maintenance of sarcomere integrity structure and function for calcium transient and muscle contraction.

Download Full-text