Sub theme 2.1.5
Transcriptional control of erythroid differentiation

Goals of research: general outline
Scientific achievements
Future plans: special goals and approach
Running projects
Associated staff

Goals of research: general outline

The major cell type in the blood circulation is the red blood cell or erythrocyte. These cells are highly specialized to carry out their main functions: the transport of oxygen from the lungs to the other parts of the body, and the transport of carbon dioxide from these tissues back to the lungs. The oxygen-carrier hemoglobin comprises approximately 90% of the soluble protein in erythrocytes. Each hemoglobin molecule is composed of four subunits: two alpha-like and two beta-like globin proteins. It is essential for the function of the erythrocytes that the alpha- and beta-globins are present at high levels in a 1:1 ratio, so that sufficient amounts of hemoglobin can be formed. An adult has approximately 5 litres of blood, containing 5x106 erythrocytes per microlitre. Since the average life span of an erythrocyte is 120 days, a healthy person needs to produce 2x106 erythrocytes per second to maintain these numbers. Diseases that affect the function of erythrocytes, known as anemias, are very common in the human population. Anemias can have severe consequences since a constant oxygen supply is essential for the survival of all tissues in the body. Anemias can occur as acquired diseases for instance due to blood loss and as a secondary consequence of medical treatments such as chemotherapy of cancer patients. In addition, anemias occur as inherited diseases, most commonly due to impaired expression of alpha-globin (alpha-thalassemia) or beta-globin (beta-thalassemia), or changes in the globin proteins such as in Sickle Cell disease where a single amino acid of the beta-globin protein (Glu6Val) is altered. 6% of the world population is carrier of a disorder affecting red cell function. We use mice to study the role of transcription factors in the formation of erythrocytes. We have focused on members of the GATA- and Sp/KLF transcription factor families.

Scientific achievements

Sp/KLF transcription factors

Mutations in Sp/KLF binding sites have been implicated in human disorders such as osteoporosis, hypercholesterolemia and beta-thalassemia, but it is usually not known which Sp/KLF factors are involved in the regulation of the affected genes. To fully understand the role of Sp/KLF factors in development and physiology, it is therefore extremely important to detect their target genes and in vivo binding sites. Gene inactivation in mice has been a powerful first approach to elucidate the biological role of individual family members. Using transgenic, (conditional) knockout, and knockin approaches we have focused our studies on Sp1, Sp3 and EKLF (or KLF1), since these factors are expressed in the erythroid lineage. This work established that KLF1 is a direct activator of the beta-globin locus, and has a role in loop formation between distant regulatory elements and the beta-globin promoter. Furthermore, through genome-wide expression analysis we identified novel KLF1 target genes that have essential roles in hemoglobin metabolism and the erythroid cell membrane.

GATA transcription factors

Through erythroid overexpression of transgenic GATA1, we have shown that dynamic regulation of GATA1 activity is required for terminal erythroid differentiation to occur. We demonstrated that GATA1 is functionally interchangeable with other GATA factors, provided that the expression profiles follow that of endogenous GATA1. Acquired mutations in GATA1 are a hallmark of the transient myeloproliferative disorder (TMD) that occurs occasionally in newborn children with constitutional trisomy 21 (Down syndrome). In collaboration with Prof. Dr. Masayuki Yamamoto (Tsukuba, Japan) we have shown that mice with a knockdown allele of GATA1 develop leukemias with high frequency. These mouse models provide the basis to unravel the molecular details of the observed leukemogenic activities of GATA1. Finally, we discovered that GATA1 plays an important role in the development of dendritic cells.

Future plans: special goals and approach

We will integrate genomics and proteomics analyses to further understand the biological functions of the GATA- and Sp/KLF transcription factors. We will perform an RNAi screen aimed at alleviating the suppression of the fetal gamma-globin gene. Reactivation of gamma-globin would cure the majority of beta-thalassemia and sickle cell disease patients.

We will implement new genomics technologies, such as the lentiviral shRNA library and high throughput sequencing, and make this available to everybody at Erasmus MC.

Most recent publications
  1. Philipsen, S., Talbot, D., Fraser, P. and Grosveld, F. (1990) The beta-globin dominant control region: hypersensitive site 2. EMBO J, 9, 2159-2167.
  2. Marin, M., Karis, A., Visser, P., Grosveld, F. and Philipsen, S. (1997) Transcription factor Sp1 is essential for early embryonic development but dispensable for cell growth and differentiation. Cell, 89, 619-628.
  3. Gillemans, N., Tewari, R., Lindeboom, F., Rottier, R., de Wit, T., Wijgerde, M., Grosveld, F. and Philipsen, S. (1998) Altered DNA-binding specificity mutants of EKLF and Sp1 show that EKLF is an activator of the beta-globin locus control region in vivo. Genes Dev, 12, 2863-2873.
  4. Whyatt, D., Lindeboom, F., Karis, A., Ferreira, R., Milot, E., Hendriks, R., de Bruijn, M., Langeveld, A., Gribnau, J., Grosveld, F. and Philipsen, S. (2000) An intrinsic but cell-nonautonomous defect in GATA-1-overexpressing mouse erythroid cells. Nature, 406, 519-524.
  5. Gillemans, N., McMorrow, T., Tewari, R., Wai, A.W., Burgtorf, C., Drabek, D., Ventress, N., Langeveld, A., Higgs, D., Tan-Un, K., Grosveld, F. and Philipsen, S. (2003) Functional and comparative analysis of globin loci in pufferfish and humans. Blood, 101, 2842-2849.
  6. Drissen, R., Palstra, R.J., Gillemans, N., Splinter, E., Grosveld, F., Philipsen, S. and de Laat, W. (2004) The active spatial organization of the beta-globin locus requires the transcription factor EKLF. Genes Dev, 18, 2485-2490.
  7. Drissen, R., von Lindern, M., Kolbus, A., Driegen, S., Steinlein, P., Beug, H., Grosveld, F. and Philipsen, S. (2005) The erythroid phenotype of EKLF-null mice: defects in hemoglobin metabolism and membrane stability. Mol Cell Biol, 25, 5205-5214.
  8. Ferreira, R., Wai, A., Shimizu, R., Gillemans, N., Rottier, R., von Lindern, M., Ohneda, K., Grosveld, F., Yamamoto, M. and Philipsen, S. (2007) Dynamic regulation of Gata factor levels is more important than their identity. Blood, 109, 5481-5490.
  9. Gutierrez, L., Nikolic, T., van Dijk, T.B., Hammad, H., Vos, N., Willart, M., Grosveld, F., Philipsen, S. and Lambrecht, B.N. (2007) Gata1 regulates dendritic-cell development and survival. Blood, 110, 1933-1941.
  10. Gutierrez, L., Tsukamoto, S., Suzuki, M., Yamamoto-Mukai, H., Yamamoto, M., Philipsen, S. and Ohneda, K. (2008) Ablation of Gata1 in adult mice results in aplastic crisis, revealing its essential role in steady-state and stress erythropoiesis. Blood, 111, 4375-4385