Chromatin remodeling week 3

can we reverse long term repression of stem cell genes in somatic cells?

(stem cells) repression of cell lineage specific genes with potential of their activation (somatic cell) long term repression of non cell type specific genes through histone modifications and dna methylation

stem cells differ from somatic cells in the overall condensation and accessibility of chromatin

- more loose chromatin structure (euchromatin) allows for potential for activation of any lineage specific genes during differentiation to somatic cells (stem) - confined to its lineage specific gene expression pattern (somatic)

modification of tails provides a binding site for specific proteins

- regulatory proteins recognize and bind histone tails - proteins with bromodomains and chromodomains recruit higher-order chromatin remodelers - promotes euchromatin if bromodomain protein binds- less charge- looser- gene expression - promotes heterochromain if chromodomain protein binds- methylated- tight on dna- no expression

different chromatin remodelers can move, eject, or replace nucleosomes

... Remodelers use the energy of ATP hydrolysis to move, destabilize, eject, or restructure nucleosomes - ATP dependent: epigenetic factors which use the energy from ATP hydrolysis to move nucleosomes: they do all the work- bring about changes in chromatin compaction All chromatin remodeling complexes contain a conserved ATPase subunit and use the energy from ATP hydrolysis to disrupt the many contact between nucleosomes and DNA, allowing nucleosomes to be ejected or repositioned on the DNA What are two classes of proteins that alter nucleosome arrangements Chromatin Remodeling Complex: slide the nucleosome to a different location or eject it from the DNA or replace it with a new nucleosome that contains a variant histone subunit. Histone Modifying Enzymes: covalently modify the N-terminal tails of histones, by attaching chemical groups to specific amino acid residues o nucleosome subunits. - Histone modifications are inheritable Three types each with specific binding domains and ATPases https://www.youtube.com/watch?v=fMqaop7CLfc

the nucleosome

11nm fiber has four core histones: H2a. H2b, H3, H4 - a nucleosome consists of 2 of each of the 4 core histones (histone octamer) and about 160 bp of DNA which is wrapped around the protein core twice

known modifications

Acetylation - by HAT (histone acetyl transferase); deacetylation - by HDAC (histone deacetylase)

organization of eukaryotic chromatin

DNA-Nucleosome-linker histones- extended form of chromosome(scaffold proteins)- condensed section of chromosome (cohesins and other scaffold proteins)- mitotic chromosome

chromatin treated wit limiting micrococcal nuclease reveals a repeating unit: the nucleosome

MNase - because a limited amount is used you get fragments of various sizes (if each arrow is a cut point not every arrow gets a cut every time)

pluripotent stem cells

Stem cells that can become almost all types of tissues and cells in the body

euchromatin

active gene expression - lesser positive charge- more negative so bond with dna less strong -looser around dna -acetylated - proteins with bromodomains binds to acetylated lysines - condensed onlly during mitosis or meiosis- so in interphase it contains many genes that are being actively transcribed- contains many useful genes- not condensed during interphase -

epigenetic mechanisms

are affected by these factprs and processes: - development (in utero, childhood) -environmental chemicals - drugs/pharmaceuticals -aging -diet

Covalent histone-modifying complexes

catalyze addition or removal of various chemical elements on histones. These enzymatic modifications include acetylation, methylation, phosphorylation, and ubiquitination and primarily occur at N-terminal histone tails. Such modifications affect the binding affinity between histones and DNA, and thus loosening or tightening the condensed DNA wrapped around histones, e.g., Methylation of specific lysine residues in H3 and H4 causes further condensation of DNA around histones, and thereby preventing binding of transcription factors to the DNA leading to gene repression. On contrary, histone acetylation relaxes chromatin condensation and exposes DNA for TF binding, leading to increase gene expression.

teratomas

congenital tumors that contain embryonic elements of all three primary germ cell layers

chimeric

contain cells from two different mouse strains

the future: using induced pluripotent stem cells for medical research and novel therapies

differentiated cells can be obtained from a patient with a specific disease and reprogrammed to become iPS cells. The resulting iPS cells can then be in vitro differentiated to various specialized cell fates, such as neurons, cardiomyocytes or hepatocytes, and used to gain new insights into the disease process or as a cell based platform to try to develop new disease therapies

micrococcal nuclease

digests away exposed portions of genomic DNA that are tethers between nucleosomes

chromatin remodeling

he dynamic modification of chromatin architecture to allow access of condensed genomic DNA to the regulatory transcription machinery proteins, and thereby control gene expression. Such remodeling is principally carried out by 1) covalent histone modifications by specific enzymes, e.g., histone acetyltransferases (HATs), deacetylases, methyltransferases, and kinases, and 2) ATP-dependent chromatin remodeling complexes which either move, eject or restructure nucleosomes. Besides actively regulating gene expression, dynamic remodeling of chromatin imparts an epigenetic regulatory role in several key biological processes, egg cells DNA replication and repair; apoptosis; chromosome segregation as well as development and pluripotency

post translationsl modifications (PTMs)

help regulate chromatin compaction and transcriptional activity -histone tails are sites of post translational modification - modifications can change tail charge and how they interact with other proteins

The histone code

histone modifications regulate transcription and other cellular processes - hypothesis that the transcription of genetic information encoded in DNA is in part regulated by chemical modifications to histone proteins, primarily on their unstructured ends. Together with similar modifications such as DNA methylation it is part of the epigenetic code.

method 1: nuclear transfer to an oocyte to make mature cell reprogrammed to become pluripotent

john gurdon used UV light (1) to destroy the cell nucleus in a frog egg. He then replaced the egg nucleus with a cell nucleus from a differentiated intestinal epithelial cell from a tadpole (2). Many manipulated eggs did not develop but in several cases normal swimming tadpoles were generated (3). This showed that the genetic information required to generate the differentiated cells in a tadpole remained intact in the donor cell nucleus. Laster studies have shown that also mammals can be cloned by this technique

dna methylation

methyl group (an epigenetic factor found in some dietary sources) can tag dna and activate or repress genes

chromatin at a transcription start site

nucleosome free promoter region activation: HAT- hyperacetylated, accessible chromatin Repression: HDAC-hypoacetylated, condensed chromatin

scaffold proteins

play a role in folding and packing of DNA, help maintain the shape of the chromosome

protein coding genes represent a small part of the genome and the majority is made of repetitive sequences

protein-coding genes 1.5%

heterochromatin

repressed gene expression - no change in charge - methylated - proteins with chromodomains bind to methylated lysines - more compact during interphase- remember interphase is where the cell spends most of its life- doesn't allow for the genes in that region to be transcribed easily - has repeat sequences of dna called satellite dna- therefor heterochromatin has very few genes

method 2: forcing the expression of stem cell specific genes through transcription factors

starting from a collection of 24 different transcription factors (symbolised by the test tube); (1), Takahashi and Yamanaka (2006) demonstrated that a set of only four transcription factors (Myc, Oct3/4, Sox2 and Klf4) was sufficient to convert cultured mouse embryonic or adult fibroblasts (2) to become pluripotent cells capable of producing teratomas in vivo and contributing to chimeric mice (3). The pluripotent cells were called induced pluripotent stem cells (iPS cells)

histone modification

the binding of epigenetic factors to histone "tails" alters the extent to which DNA is wrapped around histones and the availability of genes in the DNA to be activated

histone code hypothesis

the histone modifications serve to recruit other proteins by specific recognition of the modified histone via protein domains specialized for such purposes, rather than through simply stabilizing or destabilizing the interaction between histone and the underlying DNA. These recruited proteins then act to alter chromatin structure actively or to promote transcription.

cohesins

the linking together of like molecules, often by hydrogen bonds. sister chromatids are initially joined by ___ all along their lengths Cohesin is a protein complex that regulates the separation of sister chromatids during cell division, either mitosis or meiosis. Cohesins hold sister chromatids together after DNA replication until anaphase when removal of cohesin leads to separation of sister chromatids

epigenetics

the study of changes in gene expression that are not due to the dna sequencce - includes histone modifications, dna methylation, rna mediated processes