GENE REGULATORY MECHANISMS FOR T-CELL DEVELOPMENT FROM STEM CELLS
The Rothenberg group studies the gene regulatory mechanisms that guide blood stem cells to ultimate fates as T lymphocytes. This developmental process is distinct from many of the developmental systems studied at Caltech, because hematopoietic stem cells provide a continuing source of new T cell precursors throughout life, and development of new T-cell cohorts is mobilized in fetal life, neonatal life, and on through adulthood. This system is also distinctive because it is particularly good for shedding light on the stepwise choices the cells need to make in order to complete their differentiation as T cells. Blood precursor cells need to migrate to the thymus and expose themselves to sustained Notch1-Delta-like 4 (DL4) interactions in order to be triggered to differentiate into T cells. All the steps from multipotent precursor to committed T-lineage cell occur in this thymic environment, where cells in each stage are relatively easy to isolate, characterize, and manipulate. Thus we have been able to learn that these cells pass through a hierarchical decision tree that involves: the choice not to become a red blood cell or a platelet, the choice not to become a B cell, the choice not to become a macrophage or granulocyte, the choice not to become an antigen-presenting dendritic cell, and finally the choice not to become a natural killer cell, which leaves only various T-cell fates as the last options. This last decision concludes the T-lineage commitment process. The goal of research in this lab is to understand not only how the cells acquire the properties they will need to work as T cells, but also why the options that remain open to the precursors still are open, and how the cells make the decisions they do at each branch point. The answers we are interested in provide explanations in terms of specific transcription factor actions in gene regulatory networks.
A convergence of cell biological and molecular biological studies has revealed that the main events in early T-cell development can be broken into two major phases, split by the conclusion of commitment. Although both phases are normally dependent on Notch1-DL4 signaling, they involve different "jobs" for the cells. The first phase seems to drive the precursors to proliferate, with only limited acquisition of T-cell characteristics. The cells then cross the boundary into the second phase, when they reduce their proliferation and activate the full T-cell differentiation program. The clean division between these two phases appears to be crucial to avoid derangement of T-cell development and progression toward lymphoma.
One of the regulators we have studied for many years, the Ets-family transcription factor PU.1, now emerges as a principal actor in the first phase. This factor can participate in gene regulatory networks pushing the cells to several different fates, but its early T-cell role is kept focused by interaction with Notch pathway signals. We have found evidence that in this context, PU.1 is a direct positive regulator of multiple genes involved in the self-renewal circuit operating in phase 1 pro-T cells, based on a convergence of data chromatin immune precipitation analyzed by deep sequencing (ChIP-seq) and on gain and loss of function perturbation experiments. PU.1 must then be repressed during commitment, and we have gained insight into new cis-elements and unexpected deployments of trans-acting factors that probably cause PU.1 to be repressed during the transition from phase 1 to phase 2.
We have also determined the identity of a factor that may be a major switch controller at the transition from phase 1 to phase 2, and this turns out to be the T-cell specific zinc finger factor Bcl11b. We have shown that if Bcl11b is deleted, phase 1 pro-T cells fail to undergo commitment, spawning non-T cells abnormally even in the presence of Notch ligands. Intriguingly, Bcl11b knockout pro-T cells uncouple proliferation from differentiation, gaining the ability to keep proliferating as long as growth factors are available without developmental progression. The cis- and trans-elements required to turn Bcl11b on can be equated with those that define T-lineage identity, and so they are a major focus of our current work. Further, the mechanism through which Bcl11b works to bring about commitment involves identifying its own direct target genes and interaction partners, and that has become another important focus. Bcl11b's action at the last major identity determination point for T-cell precursors may involve network interactions with competing phase 1 regulators, and the gene regulatory network aspects of its role are another important project.
The strong punctuation created by the phase 1—phase 2 transition machinery provides a new framework in which to view the roles of other essential T-lineage factors, like GATA-3, that have long appeared to have paradoxical roles. GATA-3 and TCF-1 (encoded by the Tcf7 gene) are the two factors that are initially induced by Notch signaling to distinguish the first T-cell developmental stages before commitment. GATA-3 especially has been difficult to study because its level needs to be very precisely regulated in developing T cells. The methodology we have developed to dissect stage-specific actions of PU.1 and Bcl11b has now given us more insight into the reasons why GATA-3 levels must be so tightly titrated for T cell development to proceed. Our ChIP-seq analyses of GATA-3 binding sites reveal that the phase 1—phase 2 split may not only alter the constellation of available regulatory factors in the nucleus but also alter the deployment of those factors that are present throughout the transition.
We proposed an initial gene regulatory network model to account for the T-cell development pathway three years ago, based on the effects of transcription factor perturbation on the expression of multiple developmentally regulated genes. The newest iteration of our network model has just been published. Network construction has illuminated the need for three additional kinds of information in order to complete and confirm the model. First, a more complete "parts list" for the T-cell specification process: we needed to know all the transcription factors and potential signaling systems that might be candidates for regulatory roles. Second, we needed a way to locate the candidate cis-regulatory sites at which these factors might work on their target genes. Third, we needed better tools for dissecting the roles of these factors via stage-specific loss or antagonism of function. To address the first and second needs, we have carried out a major survey of all the changes in both RNA expression and epigenetic histone marks throughout the genome as the cells progress from the earliest T-cell development stages to commitment and beyond. This enterprise, carried out through a collaboration with the Wold lab, has yielded a broad and detailed picture of the cis- and trans-regulatory changes at each stage of the T-cell specification process. Now, to verify direct functional effects of transcription factors on target genes in a stage-specific way, we have also developed a combination of inducible deletion and dominant negative strategies that resolve direct and indirect positive and negative regulation.
Another way we have sought to establish causality is by tracking the regulation of PU.1 and Bcl11b expression over time in individual cells by live imaging. This work, carried out in collaboration with the Elowitz lab, is based on following the expression of key regulatory genes under defined developmental conditions by tracking fluorescent protein transgenes inserted into the genome under the control of the PU.1 or Bcl11b cis-regulatory elements. We are able to track cells and their descendants across least three cell cycles as they select different developmental fates in real time, and thus transcription factor gene regulation changes can be directly coupled with the changes in developmental status of living cells.
The commitment process is not only a way for T-cell precursors to renounce other hematopoietic fates; it is also closely intertwined with poorly understood events that will go on to influence the subspecialization of T-cell fate that the cells will undertake, and even to determine whether or not they will be allowed to survive in the T-cell lineage. A long-standing project in the lab has been to study the variants of this program in genetically distinct mouse strains with potentially altered T-cell generation. Genome-wide transcriptome analysis now suggests that one genetic background associated with immunological defects also causes important defects in phase 1 to phase 2 progression of thymocytes. These early defects can undermine later developmental checkpoint control and lead to a high-penetrance preleukemic phenotype. At substantial frequency, these cells can then progress to malignancy, in which the persistent phase 1 gene expression serves as a hallmark for a specific early T-cell precursor type of acute lymphoblastic lymphoma related to a virulent form of T-ALL in humans. Thus the accurate regulation of the transition from phase 1 to phase 2 in the early stages of T-cell development not only works to regulate the size of the pro-T cell pool, but also may be a matter of life and death for the organism.
Current Rothenberg Lab Projects and Investigators
Precise definition of lineage commitment and developmental branch points
Hao Yuan Kueh, Mary Yui
GATA-3 roles in early T-cell development
Sagar Damle, Jonas Ungerbäck
PU.1 target genes and DNA binding related to function in early T lineage fate decisions
Ameya Champhekar, Sagar Damle, Jonas Ungerbäck
Bcl11b roles in early T-cell development
Satoshi Hirose, Hao Yuan Kueh, Mary A. Yui
Manipulation of the T-cell differentiation progression gene regulatory network
Shuyang Qin (Caltech undergraduate), Sagar Damle, George Freedman
Cell cycle kinetics as an integral component of gene regulatory network dynamics
Hao Yuan Kueh
Computational modeling and quantitative analysis of early T cell developmental kinetics
Hao Yuan Kueh, Xun Wang, Pawel Krupinski*, Erica Manesso*, Carsten Peterson*
Cis-regulatory elements of Bcl11b
Kenneth Ng, Hao Yuan Kueh
An approach for analyzing multiple cis-regulatory element roles in a dynamic developmental system
Single-cell and single-molecule imaging of regulatory states in early T cells
Mary Yui, Ahmet Coskun†, Long Cai†
A high-penetrance model for variant T-ALL linked to checkpoint violation
*University of Lund
†Long Cai Lab, Chemistry and Chemical Engineering, Caltech