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Molecular Biology Program
 

James Hagman, Professor

Ph.D. (1989), University of Washington in Seattle


 

 

 

 

Contact Info:

Molecular Biology
University of Colorado

James Hagman, Ph.D.  National Jewish Medical Research Center, Room K516-B hagmanj@njhealth.org Phone: 303-398-1398

 

Introduction
During hematopoiesis multipotent precursor cells with many possible fates undergo differentiation with the progressive loss of developmental potential. This process can be divided into successive steps of lineage determination and commitment. In activating a new program of transcribed genes (determination), cells must overcome barriers imposed by epigenetic mechanisms, including CpG methylation, repressive histone modifications and chromatin compaction. The repressed states of chromatin are maintained by negatively acting remodeling complexes and repressor proteins, which act as safeguards against inappropriate gene expression. To initiate lineage determination, the earliest activators of the gene program begin the process of changing the repressed, or 'closed' state of chromatin to active, or 'open' chromatin. In concert with programmatic activation, the limitation of potential fate choices (commitment) is the function of nuclear factors that repress programs of other lineage contexts. In this manner a single fate choice is established, while other fate choices are blocked.

My laboratory studies mechanisms that control lineage determination and commitment in the context of B lymphocyte development. As a post-doctoral fellow in the laboratory of Dr. Rudolf Grosschedl (UCSF), I was the first to identify and clone cDNAs encoding Early B cell Factor (EBF). Today, my laboratory continues our studies of EBF, which we have identified as a transcriptional 'pioneer': a protein that initiates early epigenetic changes necessary for the activation of target genes. We are also studying Pax5, the B lineage commitment factor. To understand how these proteins control B lineage development and function (Fig.1), my laboratory uses biochemical methods (including structural determinations) and genetic approaches, including the generation and use of knock-out, conditional knock-out and knock-in mice. Each of these areas of interest is highlighted below.

 

 

 

Fig.1-Stages of B cell development in normal bone marrow. B cell development in the bone marrow. At indicated stages, targeted deletion of transcription factor genes including runx1, spi-1 (encoding PU.1), ebf1 (EBF), tcfe2a (E2A) and pax5 (Pax5) resulted in arrested development. The onset of expression of various stage-specific markers is indicated above and rearrangements of Ig genes are indicated below.

Areas of Research Interest
How does EBF function as a 'pioneer' of B cell lineage determination? EBF initiates early events necessary for the activation of the B cell lineage program, including CpG demethylation of target gene promoters, nucleosome remodeling, recruitment of partner proteins and transcriptional activation. Recently, we determined that EBF functions, in part, by recruiting SWI/SNF nucleosome remodeling complexes. SWI/SNF counteracts the effects of Mi-2/NuRD, which maintains the closed inactive state of EBF target genes. We are dissecting this mechanism using novel cell lines and ex vivo cell systems developed in my laboratory, together with shRNA and inducible systems for expressing EBF and its co-factors. Other studies are addressing how EBF initiates DNA demethylation, which we are studying using inducible systems for transcription factor expression.

Which genes constitute the EBF-dependent transcriptome? EBF is of central importance in the hierarchy of B lineage-specific factors (Fig.2). To address the identities of EBF-regulated genes and B cell-specific transcriptional networks, we have developed ex vivo B cell progenitor cell lines from knock-out mice lacking EBF or EBF and Pax5. These cytokine-dependent cell lines can be transduced with retroviruses to restore all or part of the B cell program. Key experiments are using an EBF:ER fusion protein, which initiates chromatin remodeling and B lineage-specific transcription in response to tamoxifen in vitro. 

 

 

             Fig.2- EBF is of central importance in the hierarchy of B cell regulatory factors.

 

 

What is the molecular structure of EBF? EBF is a member of a novel family of DNA-binding proteins. We are carrying out mutagenesis and structural studies to understand how EBF binds DNA and activates transcription.

What is the significance of EBF interactions with Runx family proteins? EBF recruits Runx1 to target genes. To study these interactions in vivo, we have developed a mouse model of ebf1-runx1 haploinsufficiency. These mice exhibit severely depressed bone marrow B cell development suggesting defective lineage progression. We are also studying the effects of of ebf1-runx1 haploinsufficiency on peripheral B cell function, which is severely impaired in these mice. Together, our data suggest that EBF-Runx interactions are important for the humoral response to antigens.

How does Pax5 mediate B lineage commitment? Pax5 is the B lineage commitment factor. My laboratory previously demonstrated that Pax5 recruits Ets-1 to bind DNA by inducing allosteric changes in the Ets-1 inhibitory domains (Fig.3). To block recruitment of Ets proteins by Pax5 in vivo, we have 'knocked-in' point mutations into pax5 genes in ES cells for incorporation into the mouse germline. A second set of mutations will address how Pax5 is controlled by 'redox' mechanisms in vivo.

 
Fig.3- Pax5:Ets-1 complex binding to the mb-1 promoter. Left: X-ray crystallographic structure of Pax5 and Ets-1 DNA binding domains at 2.25Å. Right- Network of contacts between Pax5, Ets-1 and DNA.

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