Gene Expression

Differential Gene Expression

some genes are expressed similarly across cell types, while some others are expressed in specific cell types

methods:
1. Chromatins Expression

2. Transcription initiation
   
   

Chromatin modification

Transcription initiation

• requires both general and regulatory transcription factors.

  • General transcription factors present in all cells.

  • Regulatory transcription factors only present in specific cells.

Three requirements of transcription initation for differential gene expression

requirements:

1. Transcriptional activators that bind to enhancer sequence on DNA.

2. a Mediator complex that bridge the communication between transcriptional activator and general transcription factors and RNA Pol II.

3. Chromatin modifying enzymes that remodels the chromatin.

Mediator complex

bridge the communication between transcriptional activator and general transcription factors and RNA Pol II.

Multiprotein complex (~2 M Da):

• Forms molecular bridge between an activator bound to an enhancer and RNA Pol II bound to the promoter.

• Facilitates assembly of RNA Pol II preinitiation complex

• Some subunits bind to RNA Pol II

• Some subunits bind activation domains of transcription factors

• One subunit has histone acetylase activity: maintain a promoter region in a hyperacetylated state.

Heterochromatin

• Tightly wound chromatin

• DNA is NOT accessible for transcription.

• Histone methylation results in condensed chromatin.

Euchromatin

Histone acetylation relaxes DNA, allowing genes to be transcribed

histone methylation

can activate or repress transcription depending on the site and degree of methylation

maintained during replication (at least for mitosis)

RNA sequencing

How to determine levels of RNA transcription in different cells?

ChIP (Chromatin ImmunoPrecipitation) -seq

How to examine histone modifications in the genome?

ATAC (Assay for Transposase-Accessible Chromatin) -seq

How to detect chromatin state?

regulatory transcription factors

all contain DNA binding domain and regulatory domain that interacts with the mediator

DNA binding domains of TFs

-- mostly interact with major groove of helical DNA

Transcription Activators

bound to enhancer sequences PROMOTE formation of a transcriptional complex.

facilitate interactions w promoter bound transcription machinery through DNA looping

Transcription Repressor

bound to silencer sequences INHIBIT formation of a transcriptional complex

enhanceosome

Transcription factors often function together

enhancer sequences can be upstream, downstream of the coding sequence, or within the intronic region of the gene.

DNA loops to bring all elements together.

Epigenetics

 the study of changes in gene expression that happen without altering the DNA sequence

Epigentic modifications

“Epi” (above, over) → modifications that do not change nucleotide sequence.

Ex. methylation/acetylation of histones and methylation of DNA nucleotides.

Reversible; potentially passed to offspring.

DNA methylation

• Can inhibit gene expression

• Prevents binding transcription

• recruits proteins that recognize 5-methyl cytosine:

  • recruits proteins with repressor activity

  • recruits chromatin modifiers that compress chromatin

Epigenetic inheritance

a cell memory that is not based on an inherited change in DNA sequence

RNA decay

Noncoding RNAs

RNA molecules that are not translated into proteins but instead perform structural, catalytic, or regulatory functions in the cell.

ex. tRNA, rRNA, miRNA

tRNA

Carries amino acids during translation.

rRNA

Makes up part of the ribosome and helps catalyze peptide bond formation.

miRNA

Regulates gene expression by inhibiting translation or promoting mRNA degradation

What distinguishes miRNA from siRNA?

Similarities

cleaved by dicer, assembled in RISC

Differences

miRNA → binds to 3'-UTR of targe mRNAs, translation inhibition

siRNA → RNA cleavage; binds to corresponding RNA

RISC (RNA induced Silencing Complex

 

Histone acetylation

increased transcriptional activity and chromatin accessibility

Histone deacetylation

decreased transcriptional activity, and less accessible chromatin accessibility

how does gene expression work in specialized cells

the transcription factor that binds to the enhancer of this gene

 

RNAseq

Addition of a protein X to cells leads to increased expression of multiple genes. A experiment was performed on samples with and without protein X added, which detected different expression levels of many RNAs at the same time. Which experiment will it be?

 

Histone lysine demethylase

these proteins lead to increased expression of multiple genes, but it doesn’t have a DNA binding domain