Einat zalckvar, Christina Paulus, Desiree Tillo, Alexandra Absach-Nitzsche, Yaniv Lubling, Carla Winterling, Nicholas Strieder, Katrin Mucke, Felicia Goodrum, Eran Segal, Michael Nevels.

Nucleosome maps of the human

Cytomegalovirus genome reveal

A temporal switch in chromatin Organization linked to a major

IE protein

Sara Puche GómezAndrés Felipe Restrepo RamírezMedicine studentsIII SemesterMolecular BiologyUPB

INTRODUCTION

Associated diseases:• Infectious mononucleosis• Congenital Infection Cytomegalic inclusion disease• Pneumonitis• Gastrointestinal disease

INTRODUCTION

Chromatin

Complex between

eukaryotic DNA and proteins

Constitutes chromosom

e

Have histone and non-histone

proteins

Basic structure is

the nucleosome

INTRODUCTION

146 pairs of DNA coiled 1,65 times

Histones H2A, H2B,

H3 AND H4, and them

with H1 form chromatoso

me

Nucleosome

INTRODUCTION

Its the first set of viral gene products to be expressed

Not require the novo protein synthesis for their expression

IE 2: essential transcriptional transactivator. Genes activated and inhibits downstream IE gene

transcription through their specific binding to DNA

IE 1: promiscuous transactivator. Inactive

histone deacetylase (HDAC) to repress gene expression

can viral

IE Protein

INTROCUTION

Cytomegalovirus

Have IE protein

Which expresses

to help

The virus to enter the cell nucleus

And use proteins

To form nucleosom

es

And then chromatin

So then is incorporat

ed in

DNA

OBJECTIVE

To investigate the role of the human cytomegalovirus IE protein in the temporal

organization of the nucleosome and involvement in the function and structure of chromatin

MATERIALES Y METODOS

PCR

• No es una tecnica analìtica, es una metología• Desnaturalización del DNA para dar dar hebras sencillas• Hibridación específica de la hebra sencilla con un primer• Replicación de la hebra sencilla por una DNA polimerasa a partir del primer

anterior

PCRq

• Basada en el analisis digital de imagen mediante cualquier programa de analisis de imagen, permite realizar una precisa cuantificación de bandas de ácidos nucléicos desarrollados en gel de agarosa

PCR real time

• Mas seguro que la PCR, procesos como la amplificación y la detección se dan simultaneamente y por fluorescencia se puede detectar la producción de DNA en el momento

Materiales y metodos

Southern blot• Identificar

secuencias especificas de DNA

Microarreglos• Analizar la

expresión diferencial de genes, monitorizándose los niveles de miles de ellos de modo simultaneo

Digestión• Rompimiento

especifico y reproducible, de segmentos de DNA con enzimas de restricción, para luego recombinar ese DNA obtenido

RESULTS

FIGURA 1

cambios masivos en la organización del nucleosoma en todo el genoma hCMV durante la infección productiva.

FIGURA 1

FIGURA 2

Cambios temporales IE1-dependientes en la organización del nucleosoma en el genoma del hCMV

FIGURA 2

FIGURA 3

Efectos IE1-dependientes en la ocupación nucleosomal a través del hCMV genoma

FIGURA 3

FIGURA 4

Correlación negativa entre cambios IE1 dependientes en la organización y expresión del nucleosoma en el genoma del hCVM

DISCUSIÓN

REFERENCES WHAT HE OR SHE SAY? AGREE/DISAGREE

Nevels M et al

herpesvirus genomes are

commonly considered to

formregular

nucleosomal arrays resembling

bulk cellular chromatin in

Latently infected cells

¿Why?

This type of chromatin organization has not been formally shown for hCMV or any β-herpesvirus.

1

Herpesvirus histones inside virus particles

Nonnucleosomal structure of viral genomes

nuclei of cells infected with herpes viruses

2

REFERENCES WHAT HE OR SHE SAID?

AGREE / DISAGREE

Greaves RF et al

Disruption of IE1 delays

transcriptionduring

productive hCMV infection

AGREE

REFERENCESWHAT HE OR SHE

SAID?AGREE/DISAGREE

Sekinger et al

Genome-wide analyses in yeast have indicated

thatnucleosome occupancy

correlates poorly with

transcriptionIt was found significant negative correlations betweenchanges in nucleosome occupancy and changes in gene

expression between WT and dlIE1 strains 96 h postinfection.

REFERENCES WHAT HE OR SHE SAY?

AGREE/DISAGREE

Munch K et al

IE1 is notconsidered to bind DNA directly , the viral protein associates with chromatin and nucleosome modifying factorsIE1 on nucleosome occupancy

and organization might be mediated through direct interaction

between the viral protein and chromatin components

AGREE

conclusions

Increasingly more importance is been given to the complex role of proteins in the chromatin organization and gene expression

understand the mechanism during productive hCMV infection would help to develop new treatments for patients worldwide.

In order to properly understand and analyze a research article is necessary to study deeply the experimental models and the basis of the used techniques to understand the housed results, and the choice of appropriate techniques is essential to get accurate results consistent with the investigation purpose.

The Chip on chip technique is needed to confirm the results because it show the interaction between DNA and nuclear proteins as histones

MAPAS

Sara Puche GSara

GRACIAS