EBF-1 is Essential for Normal B-cell Development

**Please see Mammalian Development homepage for more background information

Overview

Immune cells originate from hematopoietic stem cells. They differentiate into multipotent progenitors, which can then further differentiate into the common lymphoid progenitor. This simply means that it becomes more fated to becoming a B-cell. From this stage of the cell pathway, it can become a thymocyte, pre-NK cell or a pre-B cell.
The production and development of B lineage cells is a precise and controlled process (Zandi, et al. 2008). If the B-cell pathway is chosen, the cell will physically rearrange its genes in order to create antibodies that will be expressed on its surface. An immature B cell is one that has fully developed in the bone marrow and has traveled out to the periphery of the body. Once in the periphery, B-cells can become activated by certain proteins, which cause it to further develop to fight infections. B-cells are important in activating T-cells, providing the body with memory, and creating antibodies.

http://course1.winona.edu/kbates/Immunology/images/figure_06_25.jpg. The figure above shows the developmental stages of B-cells.

B-cells are antigen presenting cells, which means they can create small proteins or obtain them from foreign invaders and present them to T-cells. The roles of the B cell are to bind to T-helper cells. This complex of T-cell and B-cell will initiate B-cells to undergo clonal expansion and differentiation into plasma cells and memory B-cells. The plasma cells create antibodies that can neutralize or opsonize pathogens. As complex and diverse as a B-cell is, it is important to understand where the B-cells originate from and what factors cause normal stem cells to become fated towards the lymphoid progenitor lineage.

www.nature.com/nri/journal/v2/n1/fig_tab/nri706_F1.html. This figure shows the different fates of B-cells, which include plasma cells (short and long-lived) and memory cells.

EBF1 is an Important Transcription Factor for Normal B-cell Development

Transcription factors are proteins that aid in the transcription of genes. There are specific genes responsible for creating proteins that are essential for normal B-cell development. A few transcription factors responsible for the development of B-cells are E2A, EBF1, PAX5, and Ikaros. EBF1 is said to be required for B-cell commitment, pro–B cell development, and subsequent transition to the pre–B cell stage (Vilagos B., et al. 2012). According to Perez-Vera, et al., the loss of function of some of these genes can result in cancers in humans. One of the most common pediatric cancers, acute lymphoblastic leukemia (ALL), is characterized by impaired ability of these transcription factors from working properly.

It is known that if mutations are induced in EBF1 (early B-cell factor 1), it will synergize with STAT5 gene transcripts and lead to acute cases of ALL (Heltemes-Harris, Lynn, et al. 2011). The literature that will be analyzed is from Sasan Zandi and colleagues who study how EBF1 is essential for the B-lineage cells and the establishment of these transcription factors to form a network in common lymphoid progenitors.

http://en.wikipedia.org/wiki/EBF1. The image above shows the structural form of the EBF1 protein with its helix-loop-helix motif.

Structurally, the EBF1 protein has a helix-loop-helix motif which indicates that this portion of the protein binds to the DNA during transcription. If this DNA-binding region is disturbed or other structural defects occur in EBF1, it leads to a block in the formation of peripheral B-lymphoid cells and a large disturbance in immature B-cell lineage populations in bone marrow (Zandi, et al. 2008). EBF1 has a pleiotropic effect, meaning that it influences many B-cell genes and not just one. Studies have also shown that when transcription factor E2A (influences B-cell development) is absent, EBF1 can serve as a rescuing protein and still produce normal B-cells. During the investigation of the bone marrow B-cell compartments, there was a complete absence of pre and pro-B cells derived from EBF1 deficient cells.

One of the methods performed in the study was qRT-PCR of common lymphoid progenitor cells to test the presence of normal B-cell genes being expressed in the presence or absence of EBF1. In each instance of every gene, the wild type trial always contained more mRNA than the EBF1-knockout.

This figure shows the results of the transcription of genes that are known to be transcribed in normal B-cells. WT are the wild type samples and KO are the samples with the knocked out EBF1. (Zandi, et al. 2008)

To further prove their point about EBF1’s role in normal B-cell development, Zandi, et al. performed qRT-PCR on common lymphoid progenitor cells of wild type and knockout strains to measure the production of IgH, and D-J rearrangement. They also looked at Rag1 and Rag2 expression levels, examined the production of IgH and D-J rearrangement in CD3+ cells and studied the binding sites of EBF1. It is known that Rag1 and Rag2 are proteins involved in creating random double stranded breaks in the DNA of B-cells and T-cells. Without these proteins, B-cells cannot fully mature and will most likely not leave the bone marrow. Without D-J rearrangement, the cell cannot fully create functional antibodies.

The video above shows the general process of VDJ rearrangement and the influence that RAG1 and RAG2 have on this task. http://www.youtube.com/watch?v=AxIMmNByqtM

A This figure shows autoradiograms of blotted PCR products from D-J recombined IgH locus. Each lane represents one independant sort from different transplanted mice. B This image shows D-J PCR analysis of sorted thymic CD3+cells. C qRT-PCR analysis of Rag1, Rag2 and sterile IgH expression in wild type and EBF-knockout CLP populations. D Electrophoretic Mobility Shift Assay (EMSA) analysis using nuclear extract from pre-B cell lines and potential EBF1 binding site from the Cd79a promotor. (Zandi, et al. 2008)

Strengths and Weaknesses of Zandi, et al. Work

The studies showed that expression of EBF1 even in the absence of other B-cell lineage genes indicates that there is a defined pre-commitment stage of B-cell development represented by EBF1 expression (Zandi, et al. 2008). Through their methods of PCR and EMSA, there were able to find that EBF1 is needed for the production of certain gene products that are normal in B-cell development, like Pax5 and Foxo1. However, they could not determine a crucial target of EBF1. It may have been because of the fact that EBF1 is pleiotropic and may have many binding sites rather than one favored site. The experimenters did conclude that EBF1 is the key regulator of B-lineage transcriptional program and commitment because it activates B-lineage genes as well as proteins that are able to make the B-cell differentiation fate stronger (Zandi, et al. 2008).

With this wealth of information about a specific transcription factor’s role in differentiation of B-cells, the information can be pieced together to create a much broader picture of how cells are fated to a particular path and how exactly the processes occur.

http://i.imgur.com/RGpjh.png

References

Heltemes-Harris, Lynn M, Mark J L Willette, et al. “Ebf1 or Pax5 Haploinsufficiency Synergizes with STAT5 Activation to Initiate Acute Lymphoblastic Leukemia.” J Exp Med, 208.6 (2011): 1135-1149.

Perez-Vera, P, A Reyes-Leon, and EM Fuentes-Panana. “Signaling Proteins and Transcription Factors in Normal and Malignant Early B Cell Development.” Bone Marrow Research, 2011 (2011): 502751.

Vilagos, Bojan, Mareike Hoffmann, et al. “Essential Role of EBF1 in the Generation and Function of Distinct Mature B Cell Types.”J Exp Med, (2012).

Zandi, S, R Mansson, P Tsapogas, J Zetterblad, D Bryder, and M Sigvardsson. “EBF1 is Essential for B-lineage Priming and Establishment of a Transcription Factor Network in Common Lymphoid Progenitors.” Journal of Immunology (Baltimore, Md.: 1950), 181.5 (2008): 3364-3372.

*Note: Not all sources used in original T-square submission due to updated collaboration with group members and new topics chosen.

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