cell differentiation 2

how is initiation of transcription regulated?

• RNA polymerase is directed to the gene transcription start sites by a series of helper proteins

  • TATA binding protein TFIID controls where on DNA transcription starts

what is the TATA box?

• a DNA signal sequence that binds binding protein TFIID

• its the same for all protein coding genes

  • it controls where on the DNA transcription starts, but not in which cells the gene is transcribed 

what are the proteins that recognise different signal sequences called?

transcription factors

how do transcription factors initate transcription?

• when a transcription factor is present, it binds with its DNA signal

• it can activate or repress RNA polymerase function

  • the gene can either be ‘switched on’ or ‘switched off’

what does a genes transcriptional activity depend on?

• what binding sites are in its DNA enhancenr sequence

• whether the corresponding transcription factors are present in the cell

• some cells require multiple transcription factors

how can a transcription factor act directly?

• it can directly recruit RNA polymerase to the TATA box

how can a transcription factor act indirectly?

• it can indirectly recruit RNA polymerase by altering chromatin structure

  • histone acetyl transferase (HAT) - acetylation loosens histone interactions with DNA which makes the gene more accessible

  • chromatin remodelling complex

  • the chromatin modifying enzymes promote RNA polymerase binding and function

how does muscle cell differentiation work?

• it starts as mesoderm and divides into separate parts to form somites

• it then forms mysotome which contains precursors for muscle

• myoblasts proliferate as the surrounding tissue releases fibroblast growth factor

• once theres enough GF, its stopped being released and so cells leave the cell cycle

• they align and there is cell fusion to form a muscle fiber

• myoblasts can be cultured with GF to form myosin

  • myosin 2 is then strongly shown under a microscope

what is the transcription factor in muscle cell differentiation?

MyoD

how does MyoD act as the transcription factor for muscle cell differentiation?

• MyoD is present in muscle cells

• it binds DNA as a protein dimer

• it comes under the basic-helix-loop-helix domain

  • the recognition sequence is known as the E box

what does bHLH stand for?

basic helix loop helix domain

how does MyoD coordinate activation of multiple muscle genes?

• MyoD switched on all the 100s of muscle specific genes required for muscle cell differentiation 

• e.g. muscle myosin 2, troponin

• they are known as target genes

• they all have the MyoD E box recognition sequence

• 1 transcription factor can regulate the co-ordination of many downstream target genes

• one gene can have large scale effects via its regulation of other genes

what is the effect of mutating the MyoD gene?

• myosin 2 is stained as a target gene

• if you remove and culture myoblasts, they form myotubules in culture

• if mutated:

  • tongue muscles form to a certain stage to form myoblasts, but not any further

  • if you culture myoblasts, they remain as undifferentiated myoblasts

  • stalled division between cell cycle exit and termination differentiation

what was the first experiment in a multicellular organism that showed that a singe transcription factor can coordinate gene activity?

• cultured cells of different types are transfected with a plasmid that triggers MyoD

• fiborblasts become myotubules expressing myosin 2

what is the genetic analysis of TF function?

• expression pattern

• loss of function

• gain of function

what are plant leaf trichomes?

• hair like structures on leaves that deter insects, break up wind currents and reduce sun exposure

• scientists can do a genetic screen to look at mutations and see what genes are present

• they took areabidopsis leaves and screaned them for mutations which leads to trichomes

• Glabra1 mutant is when leaves are formed but they arent hairy

• Myb- family DNA binding domain

how do transcription factors recognise their DNA binding sites?

• TF have a DNA binding domain and are forced into grooves in DNA

• amino acids bind to certain bases by hydrogen bonds

• base pairs can only bond to certain amino acids

how many TF are there in humans?

• between 2000 and 3000

• there are more TF functioning than we have cell types

• we can assume that there are as many TF as we have access to the human genome which allows us to see how many genes are present

• Tf are around 10% of the genome

• each TF belongs to a small number of protein families

how do transcription factors work in combinations?

• there are groups of Tf binding sites called enhancers

• enhancer regions may contain more than 1 site for TF

• either TF may be requierd or sometimes they are sufficient together

how do transcription factors act as repressors?

• if 2Tf bind, the gene can be prevented from functioning

• expression may only require 1

• the repressor may

  • prevent binding of activators

  • recruit proteins that tighten chromatin, making the gene less accessible

what does red blood cell differentiation require?

• needs coordination of synthesis of the 2 chains of haemoglobin : alpha and beta

• a TF is required for final differentiation to erythrocytes

what is the GATA 1 TF?

• has enhancers down stream and in the middle

• its a zinc finger transcription factor

• binds to the A/T GATA A/G DNA sequence

• mutations of the mouse GATA1 gene lead to anaemia due to the death of the erythroid precursor cellsw

what are examples of GATA 1 target genes?

• alpha and beta globin genes

• erythropoietin receptor

• haem bioysnthesis enzymes

• spectrin

what are the enahncers of the beta globin gene

• have enhancers in the middle and downstream

• several different TF bound to the 5’ control region

• the gene is activated if GATA1 and CP1 and NF1 are bound

• GATA1 cant be activated by itself

how does GATA1 regulate genes in several blood lineages?

• GATA1 is expressed in several blood lineages

• GATA1 only activates globin genes in erythroblasts

• GATA1 works in combination with different TF in each lineage

• human mutations lead to anaemia and blood clotting disorders