Lineage-specific Gene Expression Programs Research Articles

Gro/TLE enables embryonic stem cell differentiation by repressing pluripotent gene expression

Gro/TLE proteins (TLE1-4) are a family of transcriptional corepressors acting downstream of multiple signalling pathways. Several TLEs are expressed in a dynamic manner throughout embryonic development and at high levels in embryonic stem cells (ESCs). Here we find that Gro/TLE is not required in ESC for sustaining pluripotency and suppressing differentiation genes, but rather is important for the shutting down of the pluripotency network in differentiation. Consistent with this view, we found that one of the Gro/TLE family, TLE4 is expressed heterogeneously in ESCs in a population that corresponds to a Nanog low subset of ESC culture. TLE4 expression is also increased in response to LIF withdrawal and Fgf/Mek/Erk stimulation. To explore the role of Gro/TLE in more detail we generated an allelic series of knockout ESCs of two TLE genes expressed most dynamically in early differentiation, TLE3 and TLE4. Genetic reduction in TLE dose resulted in an increase in the expression of pluripotency markers and inhibition of ESC differentiation towards both epiblast and endoderm lineages. Overexpression of a drug inducible TLE4 could both rescue TLE3/TLE4 compound phenotypes and induce early expression of endoderm (Hhex-Venus) and neural (Sox1-GFP) reporter genes. Taken together, our results suggest that TLE activity is essential for early differentiation where it acts to suppress the pluripotency network, allowing for the initiation of lineage specific gene expression programs.

Transcriptional and epigenetic regulation of T-helper lineage specification.

Combined with TCR stimuli, extracellular cytokine signals initiate the differentiation of naive CD4+ T cells into specialized effector T-helper (Th) and regulatory T (Treg) cell subsets. The lineage specification and commitment process occurs through the combinatorial action of multiple transcription factors (TFs) and epigenetic mechanisms that drive lineage-specific gene expression programs. In this article, we review recent studies on the transcriptional and epigenetic regulation of distinct Th cell lineages. Moreover, we review current study linking immune disease-associated single-nucleotide polymorphisms with distal regulatory elements and their potential role in the disease etiology.

Initiation of Hippo signaling is linked to polarity rather than to cell position in the pre-implantation mouse embryo.

In the mouse embryo, asymmetric divisions during the 8-16 cell division generate two cell types, polar and apolar cells, that are allocated to outer and inner positions, respectively. This outer/inner configuration is the first sign of the formation of the first two cell lineages: trophectoderm (TE) and inner cell mass (ICM). Outer polar cells become TE and give rise to the placenta, whereas inner apolar cells become ICM and give rise to the embryo proper and yolk sac. Here, we analyze the frequency of asymmetric divisions during the 8-16 cell division and assess the relationships between cell polarity, cell and nuclear position, and Hippo signaling activation, the pathway that initiates lineage-specific gene expression in 16-cell embryos. Although the frequency of asymmetric divisions varied in each embryo, we found that more than six blastomeres divided asymmetrically in most embryos. Interestingly, many apolar cells in 16-cell embryos were located at outer positions, whereas only one or two apolar cells were located at inner positions. Live imaging analysis showed that outer apolar cells were eventually internalized by surrounding polar cells. Using isolated 8-cell blastomeres, we carefully analyzed the internalization process of apolar cells and found indications of higher cortical tension in apolar cells than in polar cells. Last, we found that apolar cells activate Hippo signaling prior to taking inner positions. Our results suggest that polar and apolar cells have intrinsic differences that establish outer/inner configuration and differentially regulate Hippo signaling to activate lineage-specific gene expression programs.

Gene Regulation: A Genomic Perspective

Regulatory elements control the anatomic and cellular contexts, timing, and magnitude of gene expression patterns. Under the ENCODE and Roadmap Epigenomics Projects, human regulatory DNA has been mapped using a variety of approaches in over 300 cell and tissue types and developmental states. Collectively, the human genome encodes several million regulatory elements, most of which are located at some distance from promoters. The vast majority of these elements exhibit exquisite cell-and lineage-selective activation patterns, providing novel insights into the coordination of gene expression patterns. Genomic footprinting is a new and powerful technology that enables simultaneous profiling of the occupancy of hundreds of sequence-specific transcription factors within regulatory regions. These profiles in turn enable construction of transcription factor regulatory networks that are providing new insights into how cell-and lineage-specific gene expression programs arise. Hundreds of genetic variants associated with a wide range of hematological traits and disorders localize within regulatory regions. Many such variants disrupt specific transcription factor-DNA interactions, exposing pathophysiologically relevant transcriptional regulatory pathways. Disclosures:No relevant conflicts of interest to declare.

Correlated Spatio-Temporal Fluctuations in Chromatin Compaction States Characterize Stem Cells

Stem cells integrate signals from the microenvironment to generate lineage-specific gene expression programs upon differentiation. Undifferentiated cell nuclei are easily deformable, with an active transcriptome, whereas differentiated cells have stiffer nuclei and condensed chromatin. Chromatin organization in the stem cell state is known to be highly dynamic but quantitative characterizations of its plasticity are lacking. Using fluorescence imaging, we study the spatio-temporal dynamics of nuclear architecture and chromatin compaction in mouse embryonic stem (ES) cells and differentiated states. Individual ES cells exhibit a relatively narrow variation in chromatin compaction, whereas primary mouse embryonic fibroblasts (PMEF) show broad distributions. However, spatial correlations in chromatin compaction exhibit an emergent length scale in PMEFs, although they are unstructured and longer ranged in ES cells. We provide evidence for correlated fluctuations with large amplitude and long intrinsic timescales, including an oscillatory component, in both chromatin compaction and nuclear area in ES cells. Such fluctuations are largely frozen in PMEF. The role of actin and Lamin A/C in modulating these fluctuations is described. A simple theoretical formulation reproduces the observed dynamics. Our results suggest that, in addition to nuclear plasticity, correlated spatio-temporal structural fluctuations of chromatin in undifferentiated cells characterize the stem cell state.

Coordinating Self-Renewal and Proliferation via MLL1-HOX Pathways and Beyond

Abstract SCI-28Epigenetic regulation of gene expression plays a central role in normal hematopoietic stem cell (HSC) maintenance and leukemogenesis. The histone methyltransferase, MLL1, is essential for the maintenance of HSCs and is a common target of chromosomal translocations that result in acute leukemia. To discover genetic networks regulated by MLL1 in HSCs, we identified genes that were acutely deregulated upon Mll1 loss in HSCs, using a conditional knockout approach and lineage-negative, c-Kit+, Sca-1+, CD48-negative (LSK/CD48neg) cells. The majority of genes that changed were proliferation-associated genes, upregulated in Mll1−/− LSK/CD48neg cells. This reflected the fact that Mll1-deficient HSCs exhibit increased proliferation in vivo, a phenotype previously documented using the Mx1-cre inducible model. To determine whether the increased proliferation was cell-intrinsic, we performed single cell proliferation studies in serum-free medium containing SCF, IL-11, and Flt3L. We found that Mll1−/− LSK/CD48neg single cells entered the cell cycle earlier and that each cell cycle was shorter than wild-type controls. Evidence for failure to suppress lineage-specific gene expression was also observed; specifically, five percent of the upregulated genes encoded erythroid-specific proteins. These included erythroid transcriptional regulators such as GATA1 and KLF1, but also structural proteins such as spectrin, KEL, and EpoR. The relationship between erythroid-lineage genes and Mll1 was unique, since no other lineage-specific programs were upregulated in Mll1−/− LSK/CD48neg cells. Among the genes downregulated upon Mll1 loss, the largest category was comprised of transcriptional regulators, including Mecom, Pbx1, and Prdm16, which are known to control HSC self-renewal and quiescence. As observed in many other tissues, Mll1−/− LSK/CD48neg cells also exhibited reduced Hoxa9 expression. Interestingly, not all identified MLL1 target genes required menin, a cofactor thought to participate in directing MLL1 to particular genomic loci in vivo, and not all targets were Mll1-dependent in nonhematopoietic tissues. Chromatin immunoprecipitation and functional studies suggest that the identified genes act within a series of parallel pathways as direct transcriptional targets of MLL1. Interestingly, reexpression of Prdm16 alone could rescue Mll1-deficient cells from rapid attrition in bone marrow chimeras. Furthermore, Prdm16 corrected the hyperproliferation phenotype of Mll1−/− LSK/CD48neg cells. These data demonstrate that MLL1 coordinately regulates proliferation, lineage-specific gene expression programs, and self-renewal. By elucidating the normal MLL1-dependent transcriptional network within HSCs, we show that this pathway is overlapping but distinguishable from the leukemogenic pathway, suggesting that targeted therapy with minimal side effects on hematopoiesis will be feasible. Disclosures:No relevant conflicts of interest to declare.

Scl/Tal1 Directly Activates Hematopoiesis and Represses Cardiogenesis During Mesodermal Diversification

AbstractAbstract 3446Understanding the mechanisms of mesoderm specification into the different lineages during embryogenesis holds a great potential to advance the development of cell-based regenerative therapies for cardiovascular and blood disorders. The divergence of the developmental fates is dictated by transcription factors that induce lineage-specific gene expression programs. The basic helix-loop-helix transcription factor Scl is known as the master regulator for the specification of the hematopoietic fate. We recently discovered that, in addition to positive effects of Scl in promoting the establishment of hemogenic endothelium and hematopoietic stem/progenitor cells development, it is also required to repress cardiogenesis in hematopoietic tissues during developmentally defined window (Van Handel, Montel-Hagen, et al, Cell, 2012). However, how Scl regulates hematopoiesis and cardiogenesis remains unknown.To identify Scl’s direct target genes during mesoderm diversification, we determined the genome-wide Scl binding sites in Flk+ mesoderm from embryoid bodies using ChIP-sequencing. This analysis identified ∼4600 Scl binding sites throughout the genome, with predominance in inter-genic regions. Comparison with previously published Scl ChIP-seq datasets during later stages of development (HPC7 hematopoietic progenitor cell-line, Wilson et al. 2010, and red blood cells from fetal liver, Kassouf et al. 2010) revealed that the majority of the binding sites are developmental stage specific. Using nearest gene approach to intersect ChIP-seq data with gene expression data showed that the regulating regions of about 35% of Scl activated and 20% of repressed genes in Flk+ mesoderm were bound by Scl. Similar to later stages of hematopoietic development, robust binding of Scl to key hematopoietic transcription factors downstream of Scl, such as Runx1, Gata1, Gata2, Lyl1, Eto2, Erg, Fli1, Hhex, Gfi1, Gfi1b and Myb was observed during mesoderm specification. Interestingly, genomic regions enrichment analysis of Scl binding sites unique to Flk+ mesoderm showed enrichment for genes implicated in mesoderm formation and heart development, such as Gata4, Gata6, Msx1, Myocd, Nkx2–5 and Tbx5 indicating Scl functions as a direct repressor for cardiogenic transcriptional program.We then went on to investigate the mechanism of how Scl distinguishes between cardiac and hematopoietic genes to repress or activate them. Previous studies have shown that Scl forms complex with Gata1/2 transcription factors to activate red cell transcriptional program in erythroid cells. To clarify whether Gata1/2 are required in Scl binding and functional distinction between activation and repression during mesoderm specification, we performed Scl ChIP sequencing on Flk+ mesoderm from embryoid bodies induced from Gata1/2 double KO ES cells. Unexpectedly, Scl still bound to most hematopoietic as well as cardiac sites. However, some specific binding sites around key hematopoietic genes were completely lost or significantly reduced, such as ∼+300kb Runx1, ∼+30kb Myb and ∼TSS of Pu.1, and the expression of these genes was also down regulated in agreement with the loss of CD41+ hematopoietic progenitors in Day4.5 embryonic bodies. This suggests that Gata1/2 are required for Scl binding to the regulatory regions of a subset of crucial hematopoietic genes.These studies show that Scl has a direct critical function both as an activator of hematopoietic fate and a repressor of cardiac fate during mesoderm diversification, and only a fraction of Scl binding sites are Gata1 and/or Gata2 dependent. Disclosures:No relevant conflicts of interest to declare.

Structural Transitions in Higher Order Chromatin Assembly and Nuclear Plasticity during Stem Cell Differentiation

Undifferentiated cells integrate physico-chemical cues from the local microenvironment to elicit lineage specific gene expression programs. However the underlying mechanisms of this physical plasticity and how it impinges on gene expression is still unclear. In this study, using high resolution live-cell fluorescence polarization imaging, we analyze the spatio-temporal aspects of nuclear organization and chromatin structure in mouse embryonic stem (ES) cells and contrast them to a primary embryonic fibroblasts (PMEF). Higher-order chromatin compaction states exhibit unique features in ES cells, marked by homogeneous chromatin compaction but heterogeneity at the population level, but PMEFs evidence an inverse correlation. In addition, the nuclear lamina and actin cytoskeleton is highly flexible in ES cells but are frozen in PMEFs. This transition in nuclear architecture resembles that of fluid-like to solid-like transitions. Further the temporal evolution of nuclear plasticity is studied by differentiating ES cells on gelatin coated dishes. Taken together these results suggest that ES cells exhibit a broad epigenetic free energy landscape transitioning into a frozen configuration in higher-order chromatin assembly as lineage specific gene expression programs emerge.

Genome-Wide Programming of Myeloid Cell Fates

Abstract SCI-29Hematopoiesis represents one of the most tractable models of adult stem cell development and differentiation. Transcription factor (TF) proteins have long been recognized as major regulators of blood stem cell development as well as the subsequent differentiation into the multiple mature hematopoietic lineages. Seminal studies in multiple vertebrate model systems have identified specific TFs that control cell fate choices during myeloid differentiation (1). It remains largely unknown, however, how individual TFs are integrated into wider transcriptional regulatory networks, and how combinatorial TF interactions within these networks drive lineage specific gene expression programs. We are addressing these issues using two complementary approaches. First, we use a combination of transgenic reporter assays and network modeling approaches to reconstruct core transcription factor networks operating in early myeloid differentiation. Second, we employ genome-scale analysis of transcription factor binding sites for key hematopoietic regulators in both stem/progenitor cells and mature lineages (2,3). Integrated analysis of genome-scale datasets reveals previously unrecognized combinatorial interactions within core hematopoietic regulatory networks, which can be validated using both biochemical and mouse knockout approaches. Moreover, our studies also pinpoint novel candidate hematopoietic regulators, several of which we have validated using high throughput loss-of-function assays in zebrafish. Disclosures:No relevant conflicts of interest to declare.

Pax5 control the B lineage specific gene expression program through association with the nuclear matrix (153.14)

Abstract Accumulating studies indicate that the nuclear matrix provides a dynamic structural support for various biological reactions inside of nuclei such as DNA replication, RNA transcription, and RNA splicing. Pax5 is an important regulator for B lineage cell development, which controls the B lineage specific gene expression program involving hundreds of positive and negative target genes. Here, we show that majority of the endogenous Pax5 proteins in human and murine B lineage cells are associated with the nuclear matrix, where they occupy the centers for transcription on the nuclear matrix as indicated by co-localization with the RNA polymerase II or the TATA box binding protein (TBP). Detailed analyses identified that two different domains of Pax5 are required for its association with the nuclear matrix. Interestingly, lysine 67, 87, and 89 residues within the PRD domain of Pax5 are essential for its association with the nuclear matrix. Microarray analysis showed that mutation of these lysine residues compromise Pax5 mediated activation and repression of hundreds target genes. Furthermore, chromatin immunoprecipitation (ChIP) and nuclear matrix foot printing assays indicated that Pax5 is responsible for recruitment of Cd19 loci to the nuclear matrix bound RNA polymerase complex. These results demonstrate that association with the nuclear matrix is essential for Pax5 to control the B lineage specific gene expression program.

Histone Acetyltransferase p300 Acetylates Pax5 and Strongly Enhances Pax5-mediated Transcriptional Activity

Pax5/B cell lineage specific activator protein (BSAP) is a B lineage-specific regulator that controls the B lineage-specific gene expression program and immunoglobulin gene V(H) to DJ(H) recombination. Despite extensive studies on its multiple functions, little is known about how the activity of Pax5 is regulated. Here, we show that co-expression of histone acetyltransferase E1A binding protein p300 dramatically enhances Pax5-mediated transcriptional activation. The p300-mediated enhancement is dependent on its intrinsic histone acetyltransferase activity. Moreover, p300 interacts with the C terminus of Pax5 and acetylates multiple lysine residues within the paired box DNA binding domain of Pax5. Mutations of lysine residues 67 and 87/89 to alanine within Pax5 abolish p300-mediated enhancement of Pax5-induced Luc-CD19 reporter expression in HEK293 cells and prevent Pax5 to activate endogenous Cd19 and Blnk expression in Pax5(-/-) murine pro B cells. These results uncover a novel level of regulation of Pax5 function by p300-mediated acetylation.

Continuous Expression of the Transcription Factor E2-2 Maintains the Cell Fate of Mature Plasmacytoid Dendritic Cells

The interferon-producing plasmacytoid dendritic cells (pDCs) share common progenitors with antigen-presenting classical dendritic cells (cDCs), yet they possess distinct morphology and molecular features resembling those of lymphocytes. It is unclear whether the unique cell fate of pDCs is actively maintained in the steady state. We report that the deletion of transcription factor E2-2 from mature peripheral pDCs caused their spontaneous differentiation into cells with cDC properties. This included the loss of pDC markers, increase in MHC class II expression and Tcell priming capacity, acquisition of dendritic morphology, and induction of cDC signature genes. Genome-wide chromatin immunoprecipitation revealed direct binding of E2-2 to key pDC-specific and lymphoid genes, as well as to certain genes enriched in cDCs. Thus, E2-2 actively maintains the cell fate of mature pDCs and opposes the "default" cDC fate, in part through direct regulation of lineage-specific gene expression programs.

Aberrant expression of CD19 in AML with t(8;21) involves a poised chromatin structure and PAX5

Correct hematopoietic differentiation requires the tightly regulated execution of lineage-specific and stage-restricted gene expression programs. This process is disturbed in hematological malignancies that typically show incomplete differentiation but often also display a mixed lineage phenotype. Co-expression of lymphoid and myeloid molecules is a well-known feature of acute myeloblastic leukemia (AML) with t(8;21). These cells consistently express the B-cell-specific transcription factor PAX5, and the B-cell-specific cell surface protein CD19. However, the functional consequences of PAX5 expression are unknown. To address this question, we studied the chromatin features of CD19, which is a direct target of PAX5 in cells with and without the t(8;21) chromosomal translocation. We show that CD19 chromatin exists in a poised configuration in myeloid progenitors and that this poised chromatin structure facilitates PAX5-dependent CD19 activation. Our results also show a positive correlation between PAX5 and CD19 expression in t(8;21)-positive AML cells and demonstrate that PAX5 binds to the promoter and enhancer of CD19 gene and remodels chromatin structure at the promoter. This study shows that expression of PAX5 in leukemic cells has functional consequences and points to an important role of a progenitor-specific chromatin configuration in myeloid leukemia.

Long noncoding RNAs in neuronal-glial fate specification and oligodendrocyte lineage maturation

BackgroundLong non-protein-coding RNAs (ncRNAs) are emerging as important regulators of cellular differentiation and are widely expressed in the brain.ResultsHere we show that many long ncRNAs exhibit dynamic expression patterns during neuronal and oligodendrocyte (OL) lineage specification, neuronal-glial fate transitions, and progressive stages of OL lineage elaboration including myelination. Consideration of the genomic context of these dynamically regulated ncRNAs showed they were part of complex transcriptional loci that encompass key neural developmental protein-coding genes, with which they exhibit concordant expression profiles as indicated by both microarray and in situ hybridization analyses. These included ncRNAs associated with differentiation-specific nuclear subdomains such as Gomafu and Neat1, and ncRNAs associated with developmental enhancers and genes encoding important transcription factors and homeotic proteins. We also observed changes in ncRNA expression profiles in response to treatment with trichostatin A, a histone deacetylase inhibitor that prevents the progression of OL progenitors into post-mitotic OLs by altering lineage-specific gene expression programs.ConclusionThis is the first report of long ncRNA expression in neuronal and glial cell differentiation and of the modulation of ncRNA expression by modification of chromatin architecture. These observations explicitly link ncRNA dynamics to neural stem cell fate decisions, specification and epigenetic reprogramming and may have important implications for understanding and treating neuropsychiatric diseases.

The Histone H3 Lysine 27-Specific Demethylase Jmjd3 Is Required for Neural Commitment

Patterns of methylation at lysine 4 and 27 of histone H3 have been associated with states of gene activation and repression that are developmentally regulated and are thought to underlie the establishment of lineage specific gene expression programs. Recent studies have provided fundamental insight into the problem of lineage specification by comparing global changes in chromatin and transcription between ES and neural stem (NS) cells, points respectively of departure and arrival for neural commitment. With these maps of the differentiated state in place, a central task is now to unravel the chromatin dynamics that enables these differentiation transitions. In particular, the observation that lineage-specific genes repressed in ES cells by Polycomb-mediated H3-K27 trimethylation (H3-K27me3) are demethylated and derepressed in differentiated cells posited the existence of a specific H3-K27 demethylase.In order to gain insight into the epigenetic transitions that enable lineage specification, we investigated the early stages of neural commitment using as model system the monolayer differentiation of mouse ES cells into neural stem (NS) cells. Starting from a comprehensive profiling of JmjC-domain genes, we report here that Jmjd3, recently identified as a H3-K27me3 specific demethylase, controls the expression of key regulators and markers of neurogenesis and is required for commitment to the neural lineage.Our results demonstrate the relevance of an enzymatic activity that antagonizes Polycomb regulation and highlight different modalities through which the dynamics of H3-K27me3 is related to transcriptional output. By showing that the H3-K27 demethylase Jmjd3 is required for commitment to the neural lineage and that it resolves the bivalent domain at the Nestin promoter, our work confirms the functional relevance of bivalent domain resolution that had been posited on the basis of the genome-wide correlation between their controlled resolution and differentiation. In addition, our data indicate that the regulation of H3-K27me3 is highly gene- and context- specific, suggesting that the interplay of methyltransferases and demethylases enables the fine-tuning more than the on/off alternation of methylation states.

Helix-loop-helix proteins and lymphocyte development

Helix-loop-helix (HLH) proteins are transcriptional regulators that control a wide variety of developmental pathways in both invertebrate and vertebrate organisms. Results obtained in the past decade have shown that HLH proteins also contribute to the development of lymphoid lineages. A subset of HLH proteins, the 'E proteins', seems to be particularly important for proper lymphoid development. Members of the E protein family include E12, E47, E2-2 and HEB. The E proteins contribute to B lineage- and T lineage-specific gene expression programs, regulate lymphocyte survival and cellular proliferation, activate the rearrangement of antigen receptor genes and control progression through critical developmental checkpoints. This review discusses HLH proteins in lymphocyte development and homeostasis.

T cell lineage choice and differentiation in the absence of the RNase III enzyme Dicer

The ribonuclease III enzyme Dicer is essential for the processing of micro-RNAs (miRNAs) and small interfering RNAs (siRNAs) from double-stranded RNA precursors. miRNAs and siRNAs regulate chromatin structure, gene transcription, mRNA stability, and translation in a wide range of organisms. To provide a model system to explore the role of Dicer-generated RNAs in the differentiation of mammalian cells in vivo, we have generated a conditional Dicer allele. Deletion of Dicer at an early stage of T cell development compromised the survival of αβ lineage cells, whereas the numbers of γδ-expressing thymocytes were not affected. In developing thymocytes, Dicer was not required for the maintenance of transcriptional silencing at pericentromeric satellite sequences (constitutive heterochromatin), the maintenance of DNA methylation and X chromosome inactivation in female cells (facultative heterochromatin), and the stable shutdown of a developmentally regulated gene (developmentally regulated gene silencing). Most remarkably, given that one third of mammalian mRNAs are putative miRNA targets, Dicer seems to be dispensable for CD4/8 lineage commitment, a process in which epigenetic regulation of lineage choice has been well documented. Thus, although Dicer seems to be critical for the development of the early embryo, it may have limited impact on the implementation of some lineage-specific gene expression programs.

Cross-lineage expression of Ig-beta (B29) in thymocytes: positive and negative gene regulation to establish T cell identity.

Developmental commitment involves activation of lineage-specific genes, stabilization of a lineage-specific gene expression program, and permanent inhibition of inappropriate characteristics. To determine how these processes are coordinated in early T cell development, the expression of T and B lineage-specific genes was assessed in staged subsets of immature thymocytes. T lineage characteristics are acquired sequentially, with germ-line T cell antigen receptor-beta transcripts detected very early, followed by CD3epsilon and terminal deoxynucleotidyl transferase, then pTalpha, and finally RAG1. Only RAG1 expression coincides with commitment. Thus, much T lineage gene expression precedes commitment and does not depend on it. Early in the course of commitment to the T lineage, thymocytes lose the ability to develop into B cells. To understand how this occurs, we also examined expression of well defined B lineage-specific genes. Although lambda5 and Ig-alpha are not expressed, the mu 0 and I mu transcripts from the unrearranged IgH locus are expressed early, in distinct patterns, then repressed just before RAG1 expression. By contrast, RNA encoding the B cell receptor component Ig-beta was found to be transcribed in all immature thymocyte subpopulations and throughout most thymocyte differentiation. Ig-beta expression is down-regulated only during positive selection of CD4(+)CD8(-) cells. Thus several key participants in the B cell developmental program are expressed in non-B lineage-committed cells, and one is maintained even through commitment to an alternative lineage, and repressed only after extensive T lineage differentiation. The results show that transcriptional activation of "lymphocyte-specific" genes can occur in uncommitted precursors, and that T lineage commitment is a composite of distinct positive and negative regulatory events.