Homeobox genes

Question 1

What is homeobox?

Answer 1

The sequence homology observed between genes resposible for different homeotic mutations.

• homeobox is translated into homeodomain
• homeodomain are transcription factors that are made of 3 alpha helices, the third of which binds to DNA in a sequence specific manner (TAAT) at promoters and enhancers.

Question 2

From Homeobox –> Hox genes

Answer 2

1. Homeobox gene is transcribed into homeodomain transcription factors
   
   1. Note: homeodomain transcription factors contain the homeodomain which has 3rd alpha helix that is TAAT sequence specific in DNA enhancers and promoters
   2. Homeodomain transcription factors also bind a variety of other proteins to form protein complexes that regulate DNA binding specificity.
2. Homeobox transcription factors come in two classes:
   
   1. HOX transcription factors encoded by HOX genes
      
      1. Shh transcribes HOX genes into HOX transcription factors
   2. Separate class of homeobox transcirption factors found in fly and mouse

Question 3

Hox genes in evolution

Answer 3

1. Four Hox clusters are present in the mouse and human which are commonly called Hoxa, Hoxb, Hoxc and Hoxd
2. Common theme in evolution - conservation and expansion in number
   
   1. Number of genes in a cluster may increase but in general the genes towards the 3’ end will be responsible for the anterior portion of the body while the 5’ end genes will be responsible for the posterior end of the body

Question 4

Gain of Function vs. Loss of Function Hox genes

Answer 4

1. Loss of function or knockout of Hox genes often lead to anterior transformations
2. Gain of function (transgenics: insertion of extra genes) have posterior transformation

Question 5

Hox a2 gene and Hoxb2 gene location and function

Answer 5

1. Hoxa2 and Hoxb2 are located in 3’ region of Hox cluster
2. Hoxa2 and Hoxb2 encode transcription factors with homeodomain that bind to TAAT region in genes needed to generate C2 vertebrae.

Question 6

Hox a3, Hox b3, and Hox d3 gene location and function

Answer 6

1. Hoxa3, Hoxb3, and Hoxd3 genes are located in region posterior to Hoxa2 and Hoxb2.
2. A combination of Hoxa2, Hoxb2,Hoxa3, Hoxb3, Hoxd3 encode transcription factors with homeodomain with 3rd alpha helix that bind to TAAT region in DNA promoters and enhancers of genes needed to generate C3 vertebrae.

KO of Hoxa3, Hoxb3, and Hoxd3 will result in a duplication of C2 vertebrate because only Hoxa2 and Hoxb genes are expressed and they create transcription factors that only transcribe genes needed for C2 in the posterior compartment.

Question 7

Spatial colinearity

Answer 7

Hox genes further 3’ have more anterior expression

Question 8

Temporal colinearity

Answer 8

3’ genes needed for expression of anterior structures are expressed earlier than 5’ genes that are needed for expression of posterior structures in development

Question 9

Hox d4 gene location and function

Answer 9

1. Hox d4 gene is located in posterior 5’ region of Hox cluster
2. Hox d4 gene encodes transcription factors containing a homeodomain with the 3rd alpha helix binding to TAAT sequence element in DNA enhancers and promoters of genes needed to transcribe C4 vertebrae.

*Misexpression of Hoxd4 earlier on will result in expression of C4 genes in more anterior compartments resulting in replacement of C2/C3 vertebrae with a C4 vertebrae duplication

Question 10

Formation of head structure

Answer 10

1. Drosophila head structure is controlled by homeobox transcription factor called Otd
2. Mice and humans have two Otd orthologs called Otx1 and Otx2

Question 11

Otd and Otx mutants

Answer 11

1. Otd -/- fly mutants do not have anterior brain regions
2. Otx1 -/- mouse mutants survive embryogenesis but different brain structures including the cortex are smaller
3. Otx2-/-mouse mutants die early in embryogenesis and are lethal. The mutant deletes the portion of the developing CNS that will generate forebrain structures.

*The Otx2 mutants resemble Otd fly mutants

Question 12

Functional conservation of Otd and Otx1

Answer 12

1. In mouse with Otx 1 -/- mutant phenotype, Otd knockin from fly was able to rescue phenotype
2. Threfore, fly Otd is functionally equivalent to Otx1 -/-

Question 13

Hox genes and implications in stem cell therapy for ALS and other neurodegenerative diseases

Answer 13

• Hedgehog initiates cascade of Hox tfs
• Researchers used this knowledge that there is code of tfs that can generate motor neurons by using these different codes of tfs.
• Can take stem cells, introduce these combinations.
• Stem cells can be turned into motor neurons/motor neuron progenitors (MNP), and use them to implant chicken/mouse
• MNPs will integrate into circuit and be functional.
• May be possible in humans someday.

Question 14

Ectopic expression of Otd and Otx

Answer 14

• Otd and Otx tfs are both sufficient and necessary to make brain structures in non-neuronal tissues
• When you don’t have them, don’t turn on these genes needed to make the part of the brain.
• Misexpress Otd in non-neuronal tissue, sufficient to turn on these downstream genes with that tf alone, makes bigger brain

Question 15

iPSCs made from differentiated cells

Answer 15

1. Two of the tfs (OCT4 and NANOG) are both sufficient.
2. Knock them out, and you don’t maintain pluoripotency.
3. Now, can take a blood cell, introduce those 4 tfs (OCT4, NOGGIN, SOX2, C-MYC, KLF-4), and turn them into induced pluripotent stem cells (IPSCs)

Question 16

Synpolydactyly

Answer 16

1. Mutations in HoxD13 gene in humans and mice that impact homeodomain (3rd helix) and therefore DNA binding results in hand or limb malformations.