1. Describing hematopoiesis in terms of global networks


One of the frontiers of genome biology is to identify the regulatory mechanisms that underpin global patterns of gene expression.  In our laboratory we have focused our efforts on describing B and T cell development in terms of global networks that involve critical transcriptional regulators, including E2A, EBF and FOXO1.  We have established close collaborations with other members of the UCSD community that allow us to approach this problem using novel experimental and computation approaches.  Key projects involve the genome-wide distribution of transcriptional regulators and their associated epigenetic marks through developmental progression of lymphocytes using advanced chromatin-mapping and computational strategies. 

2. Dissecting the mechanisms that underpin hematopoietic stem cell self-renewal

Hematopoiesis is initiated from a small pool of hematopoietic stem cells (HSC).  HSCs are pluripotent and have the ability to differentiate into multipotent progenitors.   We have recently demonstrated a critical role for helix-loop-helix proteins in hematopoietic stem cell self-renewal.  Our studies are now aimed to decipher the mechansism by which HSCs develop from pre-HSCs.  In the near future we hope to establish a global network of transcription factors that underpins the ability of HSCs to self-renew.  The ultimate goal would be to generate HSCs from a cell type that does not have the ability to self-renew.  These studies are in progress.  

 

3. Dissecting the roles of large intergenic RNAs in early B cell development


Recent genome-wide studies have identified that a large fraction of the genome transcribed in non-coding regions.  We are now faced with the question as to how these large non-coding RNAs relate to the control of gene expression.  We are exploring the possibility that a fraction of such large non-coding RNAs plays a critical role in lineage development.  Additionally, we are interested in the potential roles of such non-coding RNAs in modulating long-range chromatin structure and genomic interactions.  Both genome-wide and imaging approaches will be used.

 

4. Deciphering the function of helix-loop-helix proteins in lymphocyte development and aging

We have a long-standing interest in deciphering the role and regulation of helix-loop-helix proteins in lymphocyte development.  This class of factors plays key roles in essentially every step of hematopoiesis.  Currently our main interests are in the role of these factors in the control of hematopoietic stem cell homeostasis, B- and T-lineage specification and commitment and their roles in the periphery in the response to invading pathogens.  Our future studies will be mainly focused on their global activities.  Recent studies have suggested that in aging humans the expression of these factors within the hematopoietic system is substantially affected.  We are interested in exploring how aging affects the expression of helix-loop-helix activity and how such changes relate the immune response in aging mice as well as humans.

5. Generating long-term cultures of E2A-deficient multipotent progenitors from human embryonic stem cells

In previous studies we have established that long-term cultures of E2A-deficient hematopoietic progenitors are multipotent and have the ability to indefinitely self-renew in vitro.  We are now exploring ways to generate E2A-deficient multipotent progenitors from human embryonic stem cells.  The ultimately goal would be to generate long-term cultures of human progenitors that retain multipotency as demonstrated in the murine system.  Additionally, such cultures would be utilized to generate global networks of transcription factors that promote the development of the human lymphoid and myeloid cell lineages.

 

  

6. Resolving the 3D-structure of the immunoglobulin heavy chain locus at high resolution

The immunoglobulin heavy chain locus spans a vast region of genomic DNA (over 3 Mbp).  It is comprised of variable regions (VH) as well as diversity (DH) and joining (JH) segments. During B cell development VH regions have to find the DHJH elements with similar frequencies in order to establish antigen receptor diversity.  Our studies have recently provided insight into the mechanism by which this is established.  The data showed that the immunoglobulin locus is organized into clusters of loops that are separated by linkers.  Briefly, we found that the DHJH region is juxtaposed to rosette-like structures that contain the entire repertoire of VH regions.  One of the great challenges is now to determine how these rosettes are organized and to identify the anchors and their locations that promote the formation of rosettes.  We are using both state-of-the-art imaging and molecular biology tools to address these issues.

7. Unraveling the 3D-structures of lymphoid genomes

We have recently initiated studies that are aimed to assess how genomes are assembled in committed lymphoid cells. Our knowledge of chromatin structure and long-range genomic interactions is still rudimentary.  We aim to resolve this question using imaging, formaldehyde cross-linking strategies as well as computational approaches to derive a global view of chromatin topology in developing B cells.  How do interactions between regulatory elements relate to gene activation or repression?  How do aberrant interactions lead to the development of lymphoproliferative disorders?  Can we derive the spatial organization of the entire genome from interaction frequencies?  How does the three-dimensional organization of the B cell genome relates to its function?

 

8. Exploring the spatial organization and regulation of gene expression and DNA recombination in living cells

While our knowledge of the spatial organization of genome is limited, even less is known regarding the structure and dynamics of the genome in living cells.  We have recently generated experimental tools to address this issue.  With regard to chromatin dynamics our main focus will be on the immunoglobulin heavy chain locus and how epigenetics, spatial organization and diffusion are related to promote long-range genomic interactions.