30 research outputs found
Nuclear envelope transmembrane proteins in differentiation systems
Historically, our perception of the nuclear envelope has evolved from a
simple barrier isolating the genome from the rest of a cell to a complex system that
regulates functions including transcription, splicing, DNA replication and repair and
development. Several recent proteomic studies uncovered a great variety of nuclear
envelope transmembrane proteins (NETs). Diseases associated with several nuclear
envelope proteins, mostly NETs, affect many tissues e.g. muscle, adipose tissue, skin,
bones. Many NETs of the inner nuclear membrane have been shown to interact with
chromatin, suggesting that their influencing gene expression might explain NET
roles in disease.
This work is focused on finding novel interactions of NETs with chromatin.
First, SUN2 post-translational modifications were analysed and the effect of
phosphomimetic and phospho-null mutants on heterochromatin and the cytoskeleton
was tested by overexpression. However, no obvious changes were found. Second,
several tissue-preferential NETs were tested in an adipocyte differentiation system.
NET29 changed chromosome 6 position in pre-adipocytes. This matched changes in
chromosome positioning that occur during adipocyte differentiation when NET29 is
normally induced. Post-translational modifications of NET29 are likely to play a
vital role in this process because a phospho-null mutant dominantly blocked
chromosome repositioning. The effect of over-expression and down-regulation of
NET29 on transcription was tested and results suggest that NET29 negatively
regulates expression of myogenic genes during adipogenesis.
This thesis is split into six chapters. Chapter I is an overview of the nuclear
envelope, adipogenesis and chromatin remodelling, Chapter II is a detailed
description of methods used in this study. Chapter III focuses on post-translational
modifications of SUN2, as well as trials to identify novel partners of SUN2. Chapter
IV and V deal with a novel nuclear envelope transmembrane protein and its role in
adipogenesis. Finally, the last chapter includes a discussion and recommended future
directions
Tissue-specific NETs alter genome organization and regulation even in a heterologous system
Different cell types exhibit distinct patterns of 3D genome organization that correlate with changes in gene expression in tissue and differentiation systems. Several tissue-specific nuclear envelope transmembrane proteins (NETs) have been found to influence the spatial positioning of genes and chromosomes that normally occurs during tissue differentiation. Here we study 3 such NETs: NET29, NET39, and NET47, which are expressed preferentially in fat, muscle and liver, respectively. We found that even when exogenously expressed in a heterologous system they can specify particular genome organization patterns and alter gene expression. Each NET affected largely different subsets of genes. Notably, the liver-specific NET47 upregulated many genes in HT1080 fibroblast cells that are normally upregulated in hepatogenesis, showing that tissue-specific NETs can favor expression patterns associated with the tissue where the NET is normally expressed. Similarly, global profiling of peripheral chromatin after exogenous expression of these NETs using lamin B1 DamID revealed that each NET affected the nuclear positioning of distinct sets of genomic regions with a significant tissue-specific component. Thus NET influences on genome organization can contribute to gene expression changes associated with differentiation even in the absence of other factors and overt cellular differentiation changes
Genomic loci mispositioning in Tmem120a knockout mice yields latent lipodystrophy
Little is known about how the observed fat-specific pattern of 3D-spatial genome organisation is established. Here we report that adipocyte-specific knockout of the gene encoding nuclear envelope transmembrane protein Tmem120a disrupts fat genome organisation, thus causing a lipodystrophy syndrome. Tmem120a deficiency broadly suppresses lipid metabolism pathway gene expression and induces myogenic gene expression by repositioning genes, enhancers and miRNA-encoding loci between the nuclear periphery and interior. Tmem120a(−/−) mice, particularly females, exhibit a lipodystrophy syndrome similar to human familial partial lipodystrophy FPLD2, with profound insulin resistance and metabolic defects that manifest upon exposure to an obesogenic diet. Interestingly, similar genome organisation defects occurred in cells from FPLD2 patients that harbour nuclear envelope protein encoding LMNA mutations. Our data indicate TMEM120A genome organisation functions affect many adipose functions and its loss may yield adiposity spectrum disorders, including a miRNA-based mechanism that could explain muscle hypertrophy in human lipodystrophy
Specific nuclear envelope transmembrane proteins can promote the location of chromosomes to and from the nuclear periphery
BACKGROUND: Different cell types have distinctive patterns of chromosome positioning in the nucleus. Although ectopic affinity-tethering of specific loci can be used to relocate chromosomes to the nuclear periphery, endogenous nuclear envelope proteins that control such a mechanism in mammalian cells have yet to be widely identified. RESULTS: To search for such proteins twenty three nuclear envelope transmembrane proteins were screened for their ability to promote peripheral localization of human chromosomes in HT1080 fibroblasts. Five of these proteins had strong effects on chromosome 5, but individual proteins affected different subsets of chromosomes. The repositioning effects were reversible and the proteins with effects all exhibited highly tissue-restricted patterns of expression. Depletion of two nuclear envelope transmembrane proteins that were preferentially expressed in liver each reduced the normal peripheral positioning of chromosome 5 in liver cells. CONCLUSIONS: The discovery of nuclear envelope transmembrane proteins that can modulate chromosome position and have restricted patterns of expression may enable dissection of the functional relevance of tissue-specific patterns of radial chromosome positioning.Publisher PDFPeer reviewe
TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation
<div><p>Recent work indicates that the nuclear envelope is a major signaling node for the cell that can influence tissue differentiation processes. Here we present two nuclear envelope trans-membrane proteins TMEM120A and TMEM120B that are paralogs encoded by the <i>Tmem120A</i> and <i>Tmem120B</i> genes. The TMEM120 proteins are expressed preferentially in fat and both are induced during 3T3-L1 adipocyte differentiation. Knockdown of one or the other protein altered expression of several genes required for adipocyte differentiation, <i>Gata3</i>, <i>Fasn</i>, <i>Glut4</i>, while knockdown of both together additionally affected <i>Pparg</i> and <i>Adipoq</i>. The double knockdown also increased the strength of effects, reducing for example <i>Glut4</i> levels by 95% compared to control 3T3-L1 cells upon pharmacologically induced differentiation. Accordingly, TMEM120A and B knockdown individually and together impacted on adipocyte differentiation/metabolism as measured by lipid accumulation through binding of Oil Red O and coherent anti-Stokes Raman scattering microscopy (CARS). The nuclear envelope is linked to several lipodystrophies through mutations in lamin A; however, lamin A is widely expressed. Thus it is possible that the TMEM120A and B fat-specific nuclear envelope transmembrane proteins may play a contributory role in the tissue-specific pathology of this disorder or in the wider problem of obesity.</p></div
Comparative proteomic analyses of the nuclear envelope and pore complex suggests a wide range of heretofore unexpected functions
DNA copy-number measurement of genome replication dynamics by high-throughput sequencing: the sort-seq, sync-seq and MFA-seq family
Genome replication follows a defined temporal programme that can change during cellular differentiation and disease onset. DNA replication results in an increase in DNA copy number that can be measured by high-throughput sequencing. Here we present a protocol to determine genome replication dynamics using DNA copy-number measurements. Cell populations can be obtained in three variants of the method. First, sort-seq reveals the average replication dynamics across S phase in an unperturbed cell population; FACS is used to isolate replicating and non-replicating subpopulations from asynchronous cells. Second, sync-seq measures absolute replication time at specific points during S phase using a synchronized cell population. Third, marker frequency analysis can be used to reveal the average replication dynamics using copy-number analysis in any proliferating asynchronous cell culture. These approaches have been used to reveal genome replication dynamics in prokaryotes, archaea and a wide range of eukaryotes, including yeasts and mammalian cells. We have found this approach straightforward to apply to other organisms and highlight example studies from across the three domains of life. Here we present a Saccharomyces cerevisiae version of the protocol that can be performed in 7–10 d. It requires basic molecular and cellular biology skills, as well as a basic understanding of Unix and R
A Flow Cytometry-Based Screen of Nuclear Envelope Transmembrane Proteins Identifies NET4/Tmem53 as Involved in Stress-Dependent Cell Cycle Withdrawal
Disruption of cell cycle regulation is one mechanism proposed for how nuclear envelope protein mutation can cause disease. Thus far only a few nuclear envelope proteins have been tested/found to affect cell cycle progression: to identify others, 39 novel nuclear envelope transmembrane proteins were screened for their ability to alter flow cytometry cell cycle/DNA content profiles when exogenously expressed. Eight had notable effects with seven increasing and one decreasing the 4N∶2N ratio. We subsequently focused on NET4/Tmem53 that lost its effects in p53−/− cells and retinoblastoma protein-deficient cells. NET4/TMEM53 knockdown by siRNA altered flow cytometry cell cycle/DNA content profiles in a similar way as overexpression. NET4/TMEM53 knockdown did not affect total retinoblastoma protein levels, unlike nuclear envelope-associated proteins Lamin A and LAP2α. However, a decrease in phosphorylated retinoblastoma protein was observed along with a doubling of p53 levels and a 7-fold increase in p21. Consequently cells withdrew from the cell cycle, which was confirmed in MRC5 cells by a drop in the percentage of cells expressing Ki-67 antigen and an increase in the number of cells stained for ß-galactosidase. The ß-galactosidase upregulation suggests that cells become prematurely senescent. Finally, the changes in retinoblastoma protein, p53, and p21 resulting from loss of NET4/Tmem53 were dependent upon active p38 MAP kinase. The finding that roughly a fifth of nuclear envelope transmembrane proteins screened yielded alterations in flow cytometry cell cycle/DNA content profiles suggests a much greater influence of the nuclear envelope on the cell cycle than is widely held
Induction of TMEM120A, TMEM120B and adipogenic markers during adipogenesis.
<p>(A) The 3T3-L1 <i>in vitro</i> differentiation system. Left panel, 3T3-L1 pre-adipocytes have a normal fibroblast morphology and do not stain with LipidTOX which stains lipid droplets. Right panel, after induction by treatment with iso-butyl methyl xanthine, dexamethasone and insulin the cells begin to accumulate lipid droplets by day 4 and this increases so that by day 8 (shown) the vast majority of cells have generated many large lipid droplets that stain with LipidTOX. Scale bar, 100 μm. (B) Lysates were generated from cells over a timecourse of 3T3-L1 induction and reacted with Tmem120A antibodies or with histone H3 antibodies as a loading control. The protein is induced over time, with the major accumulation between days 3 and 8. (C) RNA was extracted from a timecourse of 3T3-L1 adipogenesis and the relative mRNA levels for <i>Tmem120A</i>, <i>Tmem120B</i>, <i>Gata3</i>, <i>Pparg</i>, <i>AdipoQ</i>, <i>Glut4</i> and controls <i>Sun1</i> and <i>Tpm1</i> measured by qRT-PCR. Asterisks indicate statistical significance (* p<0.05, ** p<0.01, *** p<0.001). (D) A subset of the same data overlaid as line plots for direct comparison clearly shows the earlier activated <i>Gata3</i> dropping prior to activation of the <i>Tmem120A</i> and <i>B</i> genes which become activated in relative levels at a faster rate than the master regulator <i>Pparg</i>.</p