Epigenetic inheritance based evolution of antibiotic resistance in bacteria

Authors- Mike Adam, Bhuvana Murali, Nicole O Glenn and S Steven Potter

The evolution of antibiotic resistance in bacteria is a topic of paramount medical Importance. Evolution is the result of natural selection acting on variant phenotypes. Here is an isogenic population showing phenotypic variation, that is mediated by epigenetic inheritance.
When working with E.coli that was exposed to low concentrations of antibiotics, it became evident that within that isogenic group of microorganisms, there were heritable variations in the pattern of gene expression.
The antibiotics ampicillin, tetracycline and nalidixic acid act by inhibiting cell wall synthesis, protein synthesis and DNA synthesis, respectively. When these microorganisms were grown on antibiotic containing media successively, their survival rates were too high that it couldn’t be due to spontaneous DNA mutation. In addition, resistance levels could be ramped higher by successive exposures to increasing antibiotic concentrations. Genes, whose altered expression might increase survival, were tested by driving constitutive over-expression and determining antibiotic resistance. Three types of resistance genes were identified. The endogenous β-lactamase gene represented a cryptic gene- normally inactive, but when expressed becomes ampicillin resistance. The glutamate decarboxylase gene, in contrast, is normally expressed, but when over expressed has the capacity to increase ampicillin resistance. Finally, the DAM methylase gene regulates the expression of other genes, including multidrug efflux pumps. The semi-stable epigenetic inheritance is mediated by DNA methylation.


Pridhuvi Thavaraj
I.D number 41475937

dedifferentiation in plants

Hi Paul

I'm Shivani Naghnoor.My student ID NO is 41336337. The title of my blog is Large-scale dissociation and sequential reassembly of pericentric heterochromatin in dedifferentiated Arabidopsis cells.

Global DNA Methylation influenced by smoking

One of the very important factor in pathogenesis of human diseases is the level of epigenetic methylation. The association of the offspring’s DNA methylation is the strongest with the paternal DNA methylation if both of them are non smokers but on the other hand, if the offspring smoked, it completely vanished.
The epigenetic promoter methylation of the p16gene has been one of the major pathways responsible for the pathogenesis of the lung cancer. The major aim of the paper has been to find out a relation between epigenetics and the DNA methylation. The influence of environmental factors on the epigenetic mechanisms helps us understand the pathological mechanisms of a lot of diseases. This paper highlights on the possibility of the long term effects of smoking on global DNA methylation which was done by using the paternal heredity because the epigenetic global DNA methylation is paternal.
Global DNA methylation is not a marker for gene specific epigenetic changes, as global DNA hypomethylation can also be seen in gene specific hypomethylation or vice versa. Thus the effect of smoking on promoter specific DNA methylation of selected protooncogenes and tumor suppressor genes should be investigated on which would help in assessing clinical importance of possible epigenetic changes caused due to smoking habits.


Suneha Mohanty

SET8-Mediated Methylations of Histone H4 Lysine 20 Mark Silent Heterochromatic Domains in Apicomplexan Genomes

Enzymes of the SET family, namely histone lysine methyltransferases (HKMT) mediate the promotion or repression of genes, depending on their methylation activity at certain lysine residues on H3. Sautel et al shows that the SET8 enzyme of medically important parasites, Plasmodium sp and Toxoplasma gondii, members of the Apicomplexan genus, methylates lysine residue 20 on H4 (H4K20) in vivo.

Historically, it is thought that methylation of H4K20 played a pivotal role in DNA integrity and repair. Recently, methylation activity at H4K20 has been linked to gene silencing and cell cycle signalling. Moreover, this study finds that parasitic SET8 has mono-, di-, and trimethylase activity, unlike the SET8 enzymes of higher eukaryotes which display only monomethylase activity.

This study provides some insight into the substrates required and biochemical pathways used by metozoan parasites during growth, differentiation and parasitic life cycle. Although a vaccine or anti-protozaon targeted at an enzyme possessed by both host and parasite could lead to cytotoxicity in the host, further characterization of the enzymes involved in the complicated life cycle of the parasites may lead to the development of an effective agent to break the parasitic life cycle.

Katrina McMahon 40094797

Dual siRNA expression system targeting HIV and HBV supression

Human immunodeficiency virus (HIV) and hepatitis B virus (HBV) co-infection is a real and emerging condition, with an approximated 10% of the HIV infected population also carrying HBV. This co-infection causes very complex interactions, amplifying the effects of both HIV and HBV and greatly increasing the mortality rate compared to either individual viral infection. Although treatments do exist, there is no cure at this time.

Wu et al have observed the limited treatments towards HIV-HBV co-infection and have used RNA interference (RNAi) in attempts to produce novel therapies. Wu et al have developed a dual RNAi expression system to target two key features of HIV and HBV propagation: the gp120 gene and HBs gene respectively. Using this dual expression system, Wu et al observed simultaneous inhibition of gp120 and HBs expression, as well as decreased HIV and HBV replication. This study has provided novel insights into the simultaneous study of multiple genes, as well as providing a potential treatment for HIV-HBV co-infection.

Ref:
WU, K., MU, Y., HU, J., LU, L., ZHANG, X., YANG, Y., LI, Y., LIU, F., SONG, D., ZHU, Y. & WU, J. (2007) Simultaneously inhibition of HIV and HBV replication through a dual small interfering RNA expression system. Antiviral Research, 74, 142-149.

-Dominic Bruzzone-

Manipulating Epigenetic Processes to Fight Developing Cancers

Article: The commonality of plasticity underlying multipotent tumor cells and embryonic stem cells
Author(s): Postovit LM, Costa FF, Bischof JM, et al.
Source: JOURNAL OF CELLULAR BIOCHEMISTRY Volume: 101 Issue: 4 Pages: 908-917 Published: JUL 1 2007

Over the recent few years, there has been an exploding amount of interest and information gathered about epigenetic processes. These mechanisms play a large role in governing the expression of our inherited genes, dictating the road our development takes. How can we use the information on these processes though, to avoid the development of unwanted conditions such as cancer?

Postovit et al. (2007) has managed to achieve the reprogramming of an aggressive tumor cell to a more benign state by exposing it to embryonic microenvironments. The authors have established that at least a part of this reversion was achieved via the epigenetic processes of the healthy embryonic stem cells, which share similar processes in development to the aggressive cancer cells. The embryonic microenvironment was shown to facilitate inhibition of the production of the Nodal protein, which promotes melanoma cell plasticity and tumor progression. The protein's dna sequence was observed to undergo methylation at specific sites responsible for binding of transcription factors, restricting further Nodal protein production. The findings of this study provide great hope towards reversing cancerous activities in our body.

by Marina Donskoi

EPIGENETIC BASIS OF HUMAN DISEASES

Original article: Gene-Environmant Interactions and Epigenetic Basis of Human Diseases.
Published by: Liang Lui, Yuanyuan Li and Trygve O. Tollefsbol
Published in: Current issues in molecular biology.


Loss or gain in gene function is a major phenomenon which leads to human diseases. Epigenetic mechanisms effect gene deregulation resulting in human diseases.

Histone modification is a process that is involved in Chromatin remodeling, which is a phenomenon that helps facilitate transcription. Aberrancies in chromatin remodeling are found to be associated with both genetically and environmentally related diseases.

Complex human diseases like Cancer and Type 2 Diabetes have been found to have environmental component, affecting DNA methylation patters, in addition to genetic causes.

Many imprinted genes are found to be involved in regulation of growth and metabolism. Disruption in function of these imprinted genes in developmental syndromes, have been found to display features of Diabetes and Obesity. The expression of these genes is controlled by epigenetic mechanisms.

Another instance where environment influences epigenetic factors, is in the mechanism of Adipogenesis, which refers to the formation of adipocytes. The activity of adipogenic genes like transcript1 (apM 1), glucose transporter (Glut4) and glycerol phosphate dehydrogenase (Gpd1) are found to be regulated by epigenetics.

Dietary components such as Folic acid and Choline can interact with biological DNA methylation process. Nutritional imbalance can lead to DNA methylation aberrancies which affect the activities of adipogenic genes facilitating the development of Obesity and other Metabolic syndromes.

Thus, epigenetic mechanisms that modulate gene activity during development is found to be influenced by the environment.



Posted by: Mariska Miranda

Epigenetic regulation of telomerase in retinoid-induced differentiation of human leukemia cells

Author(s): Love WK (Love, William K.), Berletch JB (Berletch, Joel B.), Andrews LG (Andrews, Lucy G.), Tollefsbol TO (Tollefsbol, Trygve O.)
Source: INTERNATIONAL JOURNAL OF ONCOLOGY
Volume: 32 Issue: 3 Pages: 625-631 Published: MAR 2008

Telomerase activity is repressed in differentiated cells thus allowing senescence and apoptosis to occur in due time because of telomere shortening with repeated cell division. Cancer cells exhibit a high level of telomerase activity leading to their 'immortal' state.

All-trans retinoic acid (ATRA) is capable of inducing differentiation of human leukemia cells and has been proven to be significantly successful in treating promyelocytic leukemia. This treatment modality is termed as 'differentiation therapy'.

Previous studies have shown that ATRA down-regulates telomerase but its exact mechanism of action has not been clearly elucidated. This study explores the ATRA-induced epigenetic changes of telomerase activity. These changes include hypermethylation and hypoacetylation of the hTERT (human telomerase gene) promoter in HL60 human leukemia cells. Differential expression of the three DNA methyltransferases has also been observed.

These results suggest that altered DNA methylation may lead to activation of telomerase in cancer cells and epigenetic changes can be exploited for 'differentiation therapy' mechanisms in other tumors.

Good luck everyone... 8)
Sohinee Sarkar.
41513428

mircoRNA cluster controlling DNA methylation and Telomere recombination

DNA methylation is one of the major type of epigenetic control. Here we discuss the role of the microRNA clusters in the controlling the DNA methylation indirectly controlling the telomere recombination.
This entire mechanism is Dicer dependent, a RNase III family nuclease that helps in formation of the microRNAs. Dicer helps in maintaining the level of DNA methyltransferases (Dnmts) in the cell which are responsible for the DNA CpG methylation, H3K9 and H4K20 methylation and also telomere methylation. The study shows that with the decrease in the expression or deletion of Dicer, DNA methylation defects are seen due to repression of Dnmts coding gene.
The research paper suggests a mechanism which makes use of Rbl2 protein to control the expression of the Dnmts genes, in a way controlling the Dnmts proteins. Rbl2 uses the enzyme histone deacetylases, causing deacetylation of Dnmts gene promoter (acetylation helps in activating of particular nucleosome) thereby repressing the formation of the Dnmts. The expression of these Rbl2 proteins is in-turn controlled (post transcriptional control) by Dicer dependent microRNA cluster (miR290).
Thus here we can see a cycle of events, where, if Dicer is present, we get expression of miR 290 cluster, which down-regulates the expression of Rbl2. Down regulation of Rbl2 leads to expression of Dnmts, thereby resulting in proper methylation pattern of the DNA. On contrary, Dicer abrogation, causes repression of miR 290 cluster, thus leading to formation of Rbl2 protein. This Rbl2 further on down-regulates the expression of Dnmts, thereby affecting the normal methylation pattern.
Here we can see that Dnmts repression causes defects in the DNA methylation. These defects are found especially in heterochromatic region of the chromosome (telomeric and subtelomeric regions). Methylation in case of the telomere is the major controlling factor of their length. Methylation of the telomere prevents the telomeric recombination and also telomeric elongation. Thus any defect in the methylation pattern of telomere can cause telomeric recombination/elongation which might become cancerous.
Finally we can say that the presence or absence of Dicer controls the entire mechanism of Telomeric recombination/ elongation, in a way controlling the abnormal cell growth.


Original Article, 'A mammalian microRNA cluster controls DNA methylation and telomere recombination via Rbl2-dependent regulation of DNA methyltransferses'

Authors: Roberta Benetti et. al.

Journal: Nature Structural and Molecular Biology, Volume 15 (3), March 2008. 268-279

Akshay Bhumkar.

Role of Epigenetics in Specification of Neural Stem Cells

Original: Epigenetic mechanisms regulating fate specification of neural stem cells
Authors: Masakazu Namihira, Jun Kohyama, Masahiko Abematsu and Kinichi Nakashima
Published in:Philosophical transactions of the Royal Society of London. Series B, Biological sciences

Over the last four decades, it has been observed that the adult brain like other tissues of the body, also retains stem cells which are known as Neural Stem Cells (NSCs). These neural stem cells are self- renewable and can differentiate into the three major cell types of the CNS: oligodendrocytes, neurons and astrocytes. The NSCs divide symmetrically, to increase their numbers, this occurs during brain development in the early gestation period. The cells obtained from the symmetric division further undergo asymmetric division to produce two daughter cells i.e. a NSC which is similar to the parent cell and a neuron.

Although not proven, it is becoming obvious that epigenetics and its processes such as DNA methylation, Histone modification and non–coding RNA expression, apart from other regulatory factors, may play a major role in ensuring the proper regulation of neural stem cells differentiation into its different CNS cell types.

This article by Masakazu N. et al solely focuses on bringing to light, the regulatory role of epigenetics and the mechanisms by which the epigenetic factors regulate neural stem cell differentiation, and how a disturbance or an imbalance of/in these factors can lead to a complication in the process of differentiation.

Posted by: Priyadarshini Faleiro

Lifespan Regulation By Evolutionarily Conserved Genes Essential For Viability in C. elegans - Curran SP, Ruvkun G

The nematode Caenorhabditis elegans is a good model for studying longevity and DNA repair because of key advances in understanding the genetics of aging in this organism. Evolutionarily conserved mechanisms that control aging are predicted to have prereproductive functions in order to be subject to natural selection. Genes that are essential for growth and development are highly conserved in evolution, but their role in longevity has not previously been assessed.
2,700 genes essential for Caenorhabditis elegans development were screened and 64 genes that extend lifespan when inactivated postdevelopmentally, were identified. Many of these essential gene inactivations extend lifespan as much as the strongest known regulators of aging. Early gene inactivations of these essential genes caused growth arrest at larval stages, and some of these arrested animals live much longer than wild-type adults. daf-16 which is required for the enhanced survival of arrested larvae, suggested that the increased longevity is a physiological response to the essential gene inactivation. These results suggest that insulin-signaling pathways play a role in regulation of aging at any stage in the life of C. elegans.

DNA repair is an important mechanism by which cells maintain genomic integrity. Long-lived C. elegans mutants have been identified and shown to be resistant to oxidizing agents and UV irradiation, suggesting a genetically determined correlation between DNA repair capacity and life span. Gene-specific DNA repair has been compared in wild-type C. elegans and stress-resistant C. elegans mutants for the first time. DNA repair capacity is higher in long-lived C. elegans mutants than in wild-type animals. In addition, RNAi knockdown of the nucleotide excision repair gene xpa-1 increased sensitivity to UV and reduced the life span of long-lived C. elegans mutants. These findings support that DNA repair capacity correlates with longevity in C. elegans.



Shona Krishna

X-inactivation in female human embryonic stem cells is in a nonrandom pattern and prone to epigenetic alterations

Yin Shen*, Youko Matsuno†, Shaun D. Fouse*, Nagesh Rao‡, Sierra Root§, Renhe Xu§, Matteo Pellegrini¶, Arthur D. Riggs†_, and Guoping Fan*_

There is a need for a common set of quality control guidelines in the use of female human embryonic stem cells (hESCs). Suboptimal culture conditions can have an effect on X Chromosome Inactivation (XCI); an example of which is the irreversible, epigenetic silencing of XIST, a gene involved in XCI.

In females, x-inactivation is a required mechanism for dosage compensation. Dosage compensation is important as it means that females, who have two x-chromosomes, only express x-linked genes in the same dosage as males. It is important to make sure that x-inactivation in hESC lines is quality controlled as they are seen to be the most useful stem cells for regenerative medicine, and if they are not controlled, they cannot be used.

Yin Shen and associates have proven the need for common quality control guidelines when they showed us that x-inactivation in hESCs is in fact in a nonrandom pattern and prone to epigenetic alterations. They found that stress and suboptimal culture conditions can lead to the irreversible, epigenetic silencing of XIST. Shen et. al. also reports that when polymorphic x-linked genes (genes that have 2 different alleles) undergo x-inactivation, the genes are monoallelically expressed (the same allele is always expressed). This suggests that x-inactivation is in a nonrandom pattern. It was also found that in cells where XIST was no longer expressed there was 100% methylation of the XIST promoter suggesting that DNA hypermethylation is coupled with the loss of XIST expression. Shen et.al. also report that in 12.2% of x-linked genes can be reactivated in the absence of XIST. This means that dosage compensation in disrupted in the absence of XIST expression.

What does all of this mean? Human embryonic stem cells, the cells that are set to be the basis for human regenerative medicine, when cultured in a lab, are not stable. Standards need to be put in place to make sure that should any of these cells be used in regenerative medicine, they will not have undergone irreversible epigenetic silencing of the XIST gene, DNA hypermethylation of the XIST promoter has not occurred, and x-linked genes haven’t been reactivated and disrupted dosage compensation.

XCI markers need to be monitored in established cell lines of hESC’s.

Steph Grehan

s4029138

Article taken from PNAS, March 12, 2008, vol 105, no. 12

Epigenetic variation mostly due to heritable factors than to age and environmental factors.

The researchers used two cohots of twins, adolescents and middle aged to study DNA methylation at the IGF2/H19 locus. Each cohot was half monozygote and half Dizygote. They observed that heritable factors influenced DNA methylation at the IGF2/H19 locus. Combined influence of environmental and stochastic factors on methylation of this locus did not increase from adolescence to middle age as has been hypothesized. This suggest that some loci are resistant to age related changes in methylation, and it is hyphothesized that when changes do occur in these loci, an epigenetic disease results. Studies have to be conducted to test this..


Source:

Heijmans BT, Kremer D, Tobi EW, Boomsma DI, Slagboom PE. Heritable rather than age related environmental and stochastic factors dominate variation in DNA methylation of the human IGF2/H19 locus. Human Mol Gen 2007;16(5)547-554

doi:10.1093/hmg/ddm010

G. Sebetso

Rice Epigenetics Helps Vindicate Lamarck

Epigenetic Inheritance in Rice Plants
KEIKO AKIMOTO1 , HATSUE KATAKAMI1 , HYUN-JUNG KIM1, EMIKO OGAWA1 ,
CECILE M. SANO2, YUKO WADA1 and HIROSHI SANO1,*
Annals of Botany 100: 205–217, 2007
doi:10.1093/aob/mcm110, available online at www.aob.oxfordjournals.org

Who would have thought that Jean Baptiste de Lamarck's (1744-1829) theory of evolution, based on inheritance of acquired characteristics would one day be supported by the study of epigenetics in rice plants?

The study conducted by Keiko Akimoto etal, in epigenetics of the rice plant Oryza sativa ssp. japonica has provided evidence that acquired traits were stably inherited by offspring. It would seem that changes to the structure of chromatin (DNA and Histone proteins that make up the chromosomes) but not to the DNA base sequence, causes a change in gene expression or traits. These changes are heritable and passed onto the next generation as a type of Lamarckian inheritance.

DNA methylation (adding a methyl group to the 5th carbon of the ctyosine base) is one way of modifying DNA without changing the original DNA sequence (epigenetics). Demethylation (removing the methyl group from cytosine) is also a way of modifying DNA epigenetically. In this study artificial demethylation of cytosine was achieved by treating japonica rice seeds with 5-azadeoxycytidine (methylation inhibitor). This process was lethal to most of the seedlings that germinated but a small percentage survived. Of these surviving seedlings a line that bred true for dwarfism over 9 successive generations (1998-2006) was selected for investigation. A wild form of the rice was the control. Seedlings of the control and the test group were inoculated with the rice blight pathogen Xanthomonas oryzae and various DNA molecular tests for methylation/demethylation sites in the genome were carried out. To direct the search researchers targeted the regions near the leaf blight resistant gene, Xa21.

Results of the molecular tests revealed that infected wild type rice displayed no transcripts (copies) of the Xa21 gene, while the test group rice showed heavy transcription of the Xa21 gene. Methylation analysis by DNA Blot Hybridisation also revealed that the promoter region upstream of the Xa21 gene was methylated in the wild type rice. The test group however were totally void of methylated cytosine. This indicated that DNA methylation had silenced the disease resistant gene Xa21 in the wild type rice and DNA demethylation had activated the gene. This acquired resistance was stably inherited by the progeny. Other studies have also revealed that demethylation can occur naturally when plants are placed under stress, be it environmental or pathogenic.

Lamarck's shouts of vindication can almost be heard from here, as this evidence supports his theory that acquired characteristics during an organism's lifetime are inherited by the next generation. There may also be some evolutionary biologists and X-teachers that will be able to change socks now that they have taken their foot out of their mouth. Well, at least long enough to be able to eat some Lamarckian humble pie!


Gerard Scalia