Analysis of Biological Development (K. Kalthoff)

Updates to Topic 16: Transcriptional Control

Answers to Questions in Text

Control of Embryonic hunchback Gene Expression by Maternal bicoid Protein in Drosophila (p. 415/416)

  1. Where would you expect the hunchback-lacZ fusion gene shown in Fig. 16.11 to be expressed if it were introduced into embryos derived from females lacking a functional bicoid gene? Answer: nowhere.
  2. Given that deletion of the A1 module from the enhancer element resulted in expression of the fusion gene in a smaller anterior domain, how could the gradient of bicoid protein in the Drosophila embryo (see Fig. 16.8b) control the expression of several natural target genes with staggered expression domains? Answer: If their enhancer activities depended on the concentration of bicoid protein.

Gene Activation by Histone Acetylation in Yeast (p. 421/422)

  1. How do you think the investigators assayed for the activation of GCN5 target genes? Answer: By Northern blotting or by using fusion genes combining a target gene's regulatory region with the transcribed region of lacZ.
  2. The N-terminus of yeast histone H4 contains four highly conserved lysine residues that are reversibly acetylated in H4 molecules located near the promoters of genes that are activated in the presence of a particular nutrient, such as galactose. How do you expect the activation of these genes to change in mutants in which the H4 lysine residues are changed to arginine – an amino acid that conserves the positive charge of lysine but cannot be acetylated? Answer: The mutant will show a sharply reduced response to the activating nutrient, e.g. galactose.

Drosophila Homeotic Gene Activity (p. 423-425)

  1. Using the in situ hybridization technique, where would you expect to find Antennapedia mRNA accumulation in embryos from an Antp gain-of-function mutant in which the adults show antennae replaced with legs? Answer: In addition to its normal expression domain, Antp should be expressed in other regions including the antennal segment.
  2. Based on the results with transfected cells shown in Fig. 16.22, Ubx+ gene expression feeds back positively on itself. Based on this information, where would you expect a transgene consisting of the Ubx+ regulatory region and the lacZ transcribed region to be expressed in wild-type and in Ubx- deficient embryos? Answer: The transgene should be expressed where the resident Ubx+ gene is expressed, that is, primarily in T3 of wild-type embryos. Little or no transgene expression should be expected in Ubx-deficient embryos, because there is no Ubx protein to feed back positively on the transgene's regulatory region.


Clarifications and Corrections

p. 409, Fig. 16.4: The core promoter region upstream of the TATA box should be bound by TFIIB rather than TFIIA. See review article by Butler and Kadonaga (2002), Fig.1 for update.

New Review Articles

Brivanlou AH and Darnell JE Jr. (2002) Signal transduction and the control of gene expression. Science 295: 813-818

Butler J.E.F. and Kadonaga J.T. (220) The RNA polymerase II core promoter: a key component in the regulation of gene expression. Genes & Devel. 16: 2583-2592

Courey AJ and Jia S. (2001) Transcriptional repression: the long and the short of it. Genes Dev. 15: 2786-2796.

Emerson B.M. (2002) Specificity of gene regulation. Cell 109: 267-270

Hochheimer A. and Tijan R. (2003) Diversified transcription initiation complexes expand promoter selectivity and tissue-specific gene expression. Genes & Devel. 17: 1309-1320

Jones P.A. and Takai D. (2001) The role of DNA methylation in mammalian epigenetics. Science 293: 1068-1070

Lemon B. and Tijan R. (2000) Orchestrated response: a symphony of transcription factors for gene control. Genes & Devel. 14: 2552-2569

Maniatis T and Reed R. (2002) An extensive network of coupling among gene expression machines. Nature 416: 499-506

Orlando V. (2003) Polycomb, epigenomes, and control of cell identity. Cell 112: 599-606

Orphanides G and Reinberg D. (2002) A unified theory of gene expression. Cell 108: 439-451

West AG, Gaszner M and Felsenfeld G. (2002) Insulators: many functions, many mechanisms. Genes Dev. 16: 271-288

Zhang Y. and Reinberg D. (2001) Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails. Genes & Devel. 15: 2343-2360

New Research Articles

Bell A.C. and Felsenfeld G. (2000) Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene. Nature 405: 482-485

Hark A.T., Schoenherr C.J., Katz D.J., Ingram R.S., Levorce J.M. and Tilghman S.M. (2000) CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus. Nature 405: 486-489

Srivastava M., Hsieh S., Grinberg A., Williams-Simons L., Huang S.P. and Pfeifer K. (2000) H19and Igf2 monoallelic expression is regulated in two distinct ways by a shared cis-acting regulatory region upstream of H19. Genes & Devel. 14: 1186-1195

These three studies show that DNA methylation may control gene expression by modulating the binding site for a boundary protein. The H19 and Igf2 genes in mice share an enhancer region located downstream of both genes and close to H19. The two genes are oppositely imprinted: On the maternal chromosome, H19 uses the enhancer and is expressed whereas Igf2 is silent. On the paternal chromosome, Igf2 uses the enhancer and is expressed whereas H19 is silent. The three papers describe a imprinting-control region (ICR), which is located between Igf2 and H19, but closer to H19. ICR is controlled by methylation. In the unmethylated state, ICR is bound by CTCF, a "boundary" protein preventing the interaction between enhancers and promoters. On the maternal chromosome, ICR is unmethylated, allowing CTCF to bind and to prevent the enhancer from interacting with Igf2, which is not expressed. However, the enhancer can interact with H19, which is also unmethylated, so that H19 is expressed. On the paternal chromosome, ICR is methylated so that CTCF does not bind. Since the H19 promoter is also methylated this gene is not expressed. However, in the absence of the CTCF boundary, the enhancer can interact with Igf2, so that this gene is expressed.


Evolutionary Explanation of Imprinting

Haig D. and Westoby M. (2006) An earlier formulation of the genetic conflict hypothesis of genomic imprinting. Nature Genet. 38: 271.

The sex-specific and reversible silencing of certain autosomal genes in mammals is known as genomic imprinting
•  Some genes (about 30-40) are imprinted during spermatogenesis while others are imprinted during oogenesis.
•  The imprinted genes remain silent in early embryonic and in all somatic cells, including the brain.   The homologous genes are then expressed uniparentally.
•  The imprinting is erased is erased in the primordial germ cells of each new generation, so that germ line cells express both alleles of previously imprinted genes.
•  During gametogenesis, each parent again imprints the sex-specific set of genes.
The ultimate cause of imprinting seems to be a conflict over the amount of maternal nutrients diverted to her offspring via placental circulation or nursing.   Fathers seem to imprint genes that would otherwise limit the amount of maternal nutrients received by the offspring, whereas mothers imprint genes that would otherwise have the opposite effect.

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