Transcription, or the process of RNA Polymerase copying DNA into RNA, is an important control point in development, because it is the first step of gene expression. “Thousands of proteins (transcription factors, cofactors, and chromatin regulators) control gene expression programs that start and maintain specific cell states.” (Lee and Young 2013)
In the early embryonic development of the fruit fly, Drosophila melanogaster, a hierarchy of genes control segmentation and determine the body plan. The gene cascade starts with the maternal genes, of which the mRNA is deposited by the mother in the egg, and expressed early in the development of the embryo. The protein Bicoid is expressed in a gradient that is most concentrated in the anterior of the embryo, and decreases toward the posterior of the embryo and levels off. Bicoid activates the transcription of the zygotic gap gene hunchback in the anterior of the embryo. Other gap genes are expressed along the gradient in broad overlapping domains, and regulate the pair rule genes and set their boundaries. Both the gap genes and pair-rule genes code for DNA-binding transcription factors. The pair rule genes regulate the segment polarity genes. These genes establish the development of the 14 segments of the fruit fly’s body. Segmentation is a conserved process in vertebrates.
A segmentation gene, Runt, which is important for the proper expression of other segmentation genes, is involved in three developmental pathways in flies; sex determination, segmentation and, neurogenesis. Runt is the founding member of the RUNX transcription factor family, which has homologues in all animals. In mice, Runx1 is necessary for life, because it is involved in the development of hematopoiesis and the loss of Runx1 in mice is embryonic lethal. In humans, some mutations in Runx1 or AML1 are associated with leukemia, for example, one mutation associated with acute myeloid leukemia is a chromosomal translocation (t8:21) resulting in a fusion protein of AML-ETO. (Reikvam et. al 2011) The mutant AML-ETO fusion protein has the N-terminal domain of AML1, and the C-terminal domains of ETO. Since it has the wrong C-terminal domain it misregulates transcription.
I have had experience studying transcription factors and enhancers using Drosophila genetics as a graduate student researcher in Dr. John Peter Gergen’s lab. Runt, Opa, Eve, and Ftz are pair-rule transcription factors that regulate the early expression of the gene sloppy-paired and this regulation is mediated by two enhancers, the distal early stripe element (DESE) and the proximal early stripe element (PESE). (Hang and Gergen 2017, Prazak et. al 2010) Enhancers are stretches of DNA, usually a few hundred basepairs, which contain clusters of transcription factor binding sites and mediate regulation by transcription factors.(Sakabe et. al 2012) They can be located upstream or downstream of the gene or in an intron of the gene, and regulate the gene from a relative distance. Enhancers are often essential for the expression of developmental genes. (Sakabe et. al 2012)
Protein complexes are involved in transcription. Some enhancers affect transcription activation through a protein complex called the Mediator.
Another protein complex, Negative Elongation Factor (NELF), cause RNA Polymerase II (Pol II) to pause and stop making RNA, and repress the gene. A cyclin dependent kinase, P-TEFb, phosphorylates NELF and the C-terminal domain of Pol II, allowing for transition from promoter proximal pausing to transcription elongation. I will write more on this topic in another blog post.
One question I asked in my research was if an enhancer is mediating repression by preventing Pol II release, does that prevent another enhancer near the same promoter from activating transcription? Runt and Ftz repress DESE by preventing Pol II release, and Eve also represses PESE by preventing Pol II release. When these enhancers were each combined with an enhancer from the short gastrulation gene (sog), Runt and Ftz repressed the DESE-sog-lacZ gene, and Eve repressed DESE-sog-lacZ gene. This demonstrated that repression by preventing paused Pol II release is dominant.
References
Lee, Tong Ihn and Richard A. Young. “Transcription regulation and its misregulation in disease” Cell. 2013 Mar 14; 152(6): 1237–1251.
Hang, S., & Gergen, J. P. (2017). Different modes of enhancer-specific regulation by Runt and Even-skipped during Drosophila segmentation. Molecular Biology of the Cell, 28(5), 681–691. http://doi.org/10.1091/mbc.E16-09-0630
Prazak L, Fujioka M, Gergen JP. Non-additive interactions involving two distinct elements mediate sloppy-paired regulation by pair-rule transcription factors. Developmental biology. 2010;344(2):1048-1059. doi:10.1016/j.ydbio.2010.04.026.
Reikvam et. al “Acute Myeloid Leukemia with the t(8;21) Translocation: Clinical Consequences and Biological Implications” Journal of Biomedicine and Biotechnology
Volume 2011 (2011), Article ID 104631, 23 pages http://dx.doi.org/10.1155/2011/104631
Sakabe et. al “Transcriptional enhancers in development and disease” Genome Biology https://genomebiology.biomedcentral.com/articles/10.1186/gb-2012-13-1-238