Biomedical subjects

Michael S Krangel

Publications and source records attributed to Michael S Krangel.

At least 19 recordsLinked to original sources

PubMed2007

Role for rearranged variable gene segments in directing secondary T cell receptor alpha recombination.

During the recombination of variable (V) and joining (J) gene segments at the T cell receptor alpha locus, a ValphaJalpha joint resulting from primary rearrangement can be replaced by subsequent rounds of secondary rearrangement that use progressively more 5' Valpha segments and progressively more 3' Jalpha segments. To understand the mechanisms that target secondary T cell receptor alpha recombination, we studied the behavior of a T cell receptor alpha allele (HYalpha) engineered to mimic a natural primary rearrangement of TRAV17 to Jalpha57. The introduced ValphaJalpha segment was shown to provide chromatin accessibility to Jalpha segments situated within several kilobases downstream and to suppress germ-line Jalpha promoter activity and accessibility at greater distances. As a consequence, the ValphaJalpha segment directed secondary recombination events to a subset of Jalpha segments immediately downstream from the primary rearrangement. The data provide the mechanistic basis for a model of primary and secondary T cell receptor alpha recombination in which recombination events progress in multiple small steps down the Jalpha array.

Alleles↗

PubMed2006

T-B+NK+ severe combined immunodeficiency caused by complete deficiency of the CD3zeta subunit of the T-cell antigen receptor complex.

CD3zeta is a subunit of the T-cell antigen receptor (TCR) complex required for its assembly and surface expression that also plays an important role in TCR-mediated signal transduction. We report here a patient with T(-)B(+)NK(+) severe combined immunodeficiency (SCID) who was homozygous for a single C insertion following nucleotide 411 in exon 7 of the CD3zeta gene. The few T cells present contained no detectable CD3zeta protein, expressed low levels of cell surface CD3epsilon, and were nonfunctional. CD4(+)CD8(-)CD3epsilon(low), CD4(-)CD8(+)CD3epsilon(low), and CD4(-)CD8(-)CD3epsilon(low) cells were detected in the periphery, and the patient also exhibited an unusual population of CD56(-)CD16(+) NK cells with diminished cytolytic activity. Additional studies demonstrated that retrovirally transduced patient mutant CD3zeta cDNA failed to rescue assembly of nascent complete TCR complexes or surface TCR expression in CD3zeta-deficient MA5.8 murine T-cell hybridoma cells. Nascent transduced mutant CD3zeta protein was also not detected in metabolically labeled MA5.8 cells, suggesting that it was unstable and rapidly degraded. Taken together, these findings provide the first demonstration that complete CD3zeta deficiency in humans can cause SCID by preventing normal TCR assembly and surface expression.

B-Lymphocytes↗

PubMed2006

Regulation of T cell receptor-alpha gene recombination by transcription.

Despite the longstanding correlation between transcription and variable-(diversity)-joining (V(D)J) recombination, it is unknown whether transcription itself can direct recombinase targeting. Here we show that blockade of transcriptional elongation through the mouse T cell receptor-alpha (Tcra) locus suppressed V(alpha)-to-J(alpha) recombination and chromatin remodeling of J(alpha) segments. Transcriptional blockade also derepressed cryptic J(alpha) promoters. Our results demonstrate two key functions for transcription in Tcra locus regulation. Transcription increases the recombination of J(alpha) segments located within several kilobases of a promoter and prevents the activation of downstream promoters through transcriptional interference. These influences promote an ordered progression of Tcra locus recombination events and selection of a robust Tcra repertoire.

Animals↗

PubMed2006

A role for MAPK in feedback inhibition of Tcrb recombination.

The Tcrb locus is subject to a host of regulatory mechanisms that impart a strict cell and developmental stage-specific order to variable (V), diversity (D), and joining (J) gene segment recombination. The Tcrb locus is also regulated by allelic exclusion mechanisms, which restrict functional rearrangements to a single allele. The production of a functional rearrangement in CD4-CD8- double-negative (DN) thymocytes leads to the assembly of a pre-TCR and initiates signaling cascades that allow for DN to CD4+CD8+ double-positive (DP) differentiation, proliferation, and feedback inhibition of further Vbeta to DJbeta rearrangement. Feedback inhibition is believed to be controlled, in part, by the loss of Vbeta gene segment accessibility during the DN to DP transition. However, the pre-TCR signaling pathways that lead to the inactivation of Vbeta chromatin have not been determined. Because activation of the MAPK pathway is documented to promote DP differentiation in the absence of allelic exclusion, we characterized the properties of Vbeta chromatin within DP thymocytes generated by a constitutively active Raf1 (Raf-CAAX) transgene. Consistent with previous reports, we show that the Raf-CAAX transgene does not inhibit Tcrb recombination in DN thymocytes. Nevertheless, DP thymocytes generated by Raf-CAAX signals display normal down-regulation of Vbeta segment accessibility and normal feedback inhibition of the Vbeta to DJbeta rearrangement. Therefore, our results emphasize the distinct requirements for feedback inhibition in the DN and DP compartments. Although MAPK activation cannot impose feedback in DN thymocytes, it contributes to feedback inhibition through developmental changes that are tightly linked to DN to DP differentiation.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Some nuts are tougher to crack than others.

In this issue of Immunity, Oestreich et al. (2006) show that, during V(D)J recombination, RSSs may have distinct accessibility requirements. Some rely on an enhancer-intrinsic, general chromatin opening function, whereas others require enhancer-promoter interactions that direct local chromatin remodeling.

Animals↗

PubMed2006

Turning T-cell receptor beta recombination on and off: more questions than answers.

Successful V(D)J recombination at the T-cell receptor beta (Tcrb) locus is critical for early thymocyte development. The locus is subject to a host of regulatory mechanisms that impart a strict developmental order to Tcrb recombination events and that insure that Tcrb recombination occurs in an allelically excluded fashion. Progress has been made in the understanding of the cis-acting control of Tcrb locus chromatin structure and the extent to which such accessibility control can account for the developmental regulation of Tcrb recombination. However, recent studies in our laboratory and elsewhere have made it abundantly clear that accessibility control is only part of the story, and multiple additional mechanisms impact both the developmental activation and inactivation of locus recombination events. Here we evaluate our current understanding of developmental regulation at the Tcrb locus. We highlight the many unresolved issues and we discuss how recent concepts emerging from studies of other antigen receptor loci may (or may not) help to resolve these issues.

Alleles↗

PubMed2005

Allele-specific regulation of TCR beta variable gene segment chromatin structure.

Allelic exclusion of the murine Tcrb locus is imposed at the level of recombination and restricts each cell to produce one functional VDJbeta rearrangement. Allelic exclusion is achieved through asynchronous Vbeta to DJbeta recombination as well as feedback inhibition that terminates recombination once a functional rearrangement has occurred. Because the accessibility of Vbeta gene segment chromatin is diminished as thymocytes undergo allelic exclusion at the CD4(-)CD8(-) (double-negative) to CD4(+)CD8(+) (double-positive) transition, chromatin regulation was thought to be an important component of the feedback inhibition process. However, previous studies of chromatin regulation addressed the status of Tcrb alleles using genetic models in which both alleles remained in a germline configuration. Under physiological conditions, developing thymocytes would undergo Vbeta to DJbeta recombination on one or both alleles before the enforcement of feedback. On rearranged alleles, Vbeta gene segments that in germline configuration are regulated independently of the Tcrb enhancer are now brought into its proximity. We show in this study that in contrast to Vbeta segments on a nonrearranged allele, those situated upstream of a functionally rearranged Vbeta segment are contained in active chromatin as judged by histone H3 acetylation, histone H3 lysine 4 (K4) methylation, and germline transcription. Nevertheless, these Vbeta gene segments remain refractory to recombination in double-positive thymocytes. These results suggest that a unique feedback mechanism may operate independent of chromatin structure to inhibit Vbeta to DJbeta recombination after the double-negative stage of thymocyte development.

Alleles↗

PubMed2005

Regulation of TCR delta and alpha repertoires by local and long-distance control of variable gene segment chromatin structure.

Murine Tcrd and Tcra gene segments reside in a single genetic locus and undergo recombination in CD4- CD8- (double negative [DN]) and CD4+ CD8+ (double positive [DP]) thymocytes, respectively. TcraTcrd locus variable gene segments are subject to complex regulation. Only a small subset of approximately 100 variable gene segments contributes substantially to the adult TCRdelta repertoire. Moreover, although most contribute to the TCRalpha repertoire, variable gene segments that are Jalpha proximal are preferentially used during primary Tcra recombination. We investigate the role of local chromatin accessibility in determining the developmental pattern of TcraTcrd locus variable gene segment recombination. We find variable gene segments to be heterogeneous with respect to acetylation of histones H3 and H4. Those that dominate the adult TCRdelta repertoire are hyperacetylated in DN thymocytes, independent of their position in the locus. Moreover, proximal variable gene segments show dramatic increases in histone acetylation and germline transcription in DP thymocytes, a result of super long-distance regulation by the Tcra enhancer. Our results imply that differences in chromatin accessibility contribute to biases in TcraTcrd locus variable gene segment recombination in DN and DP thymocytes and extend the distance over which the Tcra enhancer can regulate chromatin structure to a remarkable 525 kb.

Acetylation↗

PubMed2005

Regulation of T cell receptor alpha gene assembly by a complex hierarchy of germline Jalpha promoters.

Assembly of the gene encoding T cell receptor alpha (Tcra) is characterized by an orderly progression of primary and secondary V(alpha)-to-J(alpha) recombination events across the J(alpha) array, but the targeting mechanisms responsible for this progression are mostly unknown. Studies have shown that the T early-alpha promoter is important in targeting primary Tcra rearrangements. We found that T early-alpha and a previously unknown promoter associated with J(alpha)49 targeted primary recombination to discrete sets of constant alpha region (C(alpha))-distal J(alpha) segments and together directed nearly all normal primary recombination events. Furthermore, deletion of the T early-alpha promoter activated previously suppressed downstream promoters and stimulated primary rearrangement to centrally located J(alpha) segments. Central promoter derepression also occurred after primary rearrangement, thereby providing a mechanism to target secondary recombination events.

Acetylation↗

PubMed2005

Regulation of the murine Ddelta2 promoter by upstream stimulatory factor 1, Runx1, and c-Myb.

Accessibility control of V(D)J recombination at Ag receptor loci depends on the coordinate activities of transcriptional enhancers and germline promoters. Recombination of murine Tcrd gene segments is known to be regulated, at least in part, by the Tcrd enhancer (Edelta) situated in the Jdelta2-Cdelta intron. However, there has been little characterization of promoters and other cis-acting elements that are activated by or collaborate with Edelta and that might function to regulate Tcrd gene recombination events. We now describe a strong promoter that is tightly associated with the murine Ddelta2 gene segment. EMSAs reveal that upstream stimulatory factor 1, Runx1, c-Myb, lymphoid enhancer binding factor 1, NF1, and E47 all interact with this promoter in vitro. Of these, upstream stimulatory factor 1, Runx1, and c-Myb appear necessary for full promoter activity in transiently transfected cells. Moreover, the same three factors were found to interact with the promoter in vivo by chromatin immunoprecipitation. We suggest that these factors play important roles as Edelta-dependent regulators of Ddelta2 accessibility in vivo. Consistent with the established roles of c-Myb and Runx factors in Edelta function, we detected low level, enhancer-independent activity of the Ddelta2 promoter in transient transfection experiments. We speculate that the Ddelta2 promoter may play a role as a weak, enhancer-independent regulator in vivo, and might contribute to residual Tcrd rearrangement in Edelta(-/-) mice.

Animals↗

PubMed2005

Regulation of T cell receptor beta allelic exclusion at a level beyond accessibility.

Allelic exclusion of V(beta)-to-DJ(beta) recombination depends on asynchronous rearrangement of alleles of the gene encoding T cell receptor beta in double-negative thymocytes and feedback inhibition that is maintained in double-positive thymocytes. Feedback is thought to be enforced through downregulation of V(beta) accessibility. In an attempt to override this negative regulation, we introduced the enhancer of the gene encoding T cell receptor alpha into the V(beta) gene cluster downstream of V(beta)12. In double-negative thymocytes, the introduced enhancer had no measurable effect on accessibility, but V(beta)12 rearrangement was stimulated and V(beta)12 allelic exclusion was partially subverted. In contrast, double-positive thymocytes showed increased V(beta) transcription and accessibility, but feedback inhibition of V(beta)-to-DJ(beta) recombination remained intact. Our results indicate additional regulatory constraints on V(beta)-to-DJ(beta) recombination that operate beyond the accessibility barrier.

Alleles↗

PubMed2004

Developmental activation of the TCR alpha enhancer requires functional collaboration among proteins bound inside and outside the core enhancer.

The TCR delta enhancer (Edelta) and TCR alpha enhancer (Ealpha) play critical roles in the temporal and lineage-specific control of V(D)J recombination and transcription at the TCR alphadelta locus, working as a developmental switch controlling a transition from TCR delta to TCR alpha activity during thymocyte development. Previous experiments using a transgenic reporter substrate revealed that substitution of the 116-bp minimal Ealpha, denoted Talpha1-Talpha2, for the entire 1.4-kb Ealpha led to a premature activation of V(D)J recombination. This suggested that binding sites outside of Talpha1-Talpha2 are critical for the strict developmental regulation of TCR alpha rearrangement. We have further analyzed Ealpha to better understand the mechanisms responsible for appropriate developmental regulation in vivo. We found that a 275-bp Ealpha fragment, denoted Talpha1-Talpha4, contains all binding sites required for proper developmental regulation in vivo. This suggests that developmentally appropriate enhancer activation results from a functional interaction between factors bound to Talpha1-Talpha2 and Talpha3-Talpha4. In support of this, EMSAs reveal the formation of a large enhanceosome complex that reflects the cooperative assembly of proteins bound to both Talpha1-Talpha2 and Talpha3-Talpha4. Our data suggest that enhanceosome assembly is critical for developmentally appropriate activation of Ealpha in vivo, and that transcription factors, Sp1 and pCREB, may play unique roles in this process.

Animals↗

PubMed2004

Targeted inhibition of V(D)J recombination by a histone methyltransferase.

The tissue- and stage-specific assembly of antigen receptor genes by V(D)J recombination is regulated by changes in the chromatin accessibility of target gene segments. This dynamic remodeling process is coordinated by cis-acting promoters and enhancers, which function as accessibility control elements. The basic epigenetic mechanisms that activate or repress chromatin accessibility to V(D)J recombinase remain unclear. We now demonstrate that a histone methyltransferase overrides accessibility control element function and cripples V(D)J recombination of chromosomal gene segments. The recruited histone methyltransferase induces extensive revisions in the local chromatin environment, including altered histone modifications and de novo methylation of DNA. These findings indicate a key function for histone methyltransferases in the tissue- and stage-specific suppression of antigen receptor gene assembly during lymphocyte development.

Animals↗

PubMed2004

Enforcing order within a complex locus: current perspectives on the control of V(D)J recombination at the murine T-cell receptor alpha/delta locus.

V(D)J recombination proceeds according to defined developmental programs at T-cell receptor (TCR) and immunoglobulin loci as a function of cell lineage and stage of differentiation. Although the molecular details are still lacking, such regulation is thought to occur at the level of accessibility of chromosomal recombination signal sequences to the recombinase. The unique and complex organization of the TCRalpha/delta locus poses intriguing regulatory challenges in this regard: embedded TCRalpha and TCRdelta gene segments rearrange at distinct stages of thymocyte development, there is a highly regulated progression of primary followed by secondary rearrangements involving Jalpha segments, and there are important developmental constraints on V gene segment usage. The locus therefore provides a fascinating laboratory in which to explore the basic mechanisms underlying developmental control. We provide here a current view of cis-acting mechanisms that enforce the TCRalpha/delta locus developmental program, and we emphasize the unresolved issues that command the attention of our and other laboratories.

Animals↗

PubMed2003

Gene segment selection in V(D)J recombination: accessibility and beyond.

V(D)J recombination assembles genes encoding antigen receptors according to defined developmental programs in immature B and T lymphocytes. The 'accessibility hypothesis' was initially invoked to explain how a single recombinase complex could control the locus and allele specificity of V(D)J recombination. It has been since shown that recombination signal sequences themselves influence recombination efficiency and specificity in ways that had not been previously appreciated. Recent developments have increased our understanding of how the chromatin barrier to V(D)J recombination is regulated, and how chromatin control and the properties of the underlying recombination signal sequences may cooperate to create diverse, lineage-restricted and allelically excluded repertoires of antigen receptors.

Alleles↗

PubMed2002

Distinct roles for c-Myb and core binding factor/polyoma enhancer-binding protein 2 in the assembly and function of a multiprotein complex on the TCR delta enhancer in vivo.

Enhancers and promoters within TCR loci functionally collaborate to modify chromatin structure and to confer accessibility to the transcription and V(D)J recombination machineries during T cell development in the thymus. Two enhancers at the TCRalphadelta locus, the TCR alpha enhancer and the TCR delta enhancer (Edelta), are responsible for orchestrating the distinct developmental programs for V(D)J recombination and transcription of the TCR alpha and delta genes, respectively. Edelta function depends critically on transcription factors core binding factor (CBF)/polyoma enhancer-binding protein 2 (PEBP2) and c-Myb as measured by transcriptional activation of transiently transfected substrates in Jurkat cells, and by activation of V(D)J recombination within chromatin-integrated substrates in transgenic mice. To understand the molecular mechanisms for synergy between these transcription factors in the context of chromatin, we used in vivo footprinting to study the requirements for protein binding to Edelta within wild-type and mutant versions of a human TCR delta minilocus in stably transfected Jurkat cells. Our data indicate that CBF/PEBP2 plays primarily a structural role as it induces a conformational change in the enhanceosome that is associated with augmented binding of c-Myb. In contrast, c-Myb has no apparent affect on CBF/PEBP2 binding, but is critical for transcriptional activation. Thus, our data reveal distinct functions for c-Myb and CBF/PEBP2 in the assembly and function of an Edelta enhanceosome in the context of chromatin in vivo.

Base Sequence↗

PubMed2002

Regulation of V(D)J recombination: a dominant role for promoter positioning in gene segment accessibility.

Antigen receptor gene assembly is regulated by transcriptional promoters and enhancers, which control the accessibility of gene segments to a lymphocyte-specific V(D)J recombinase. However, it remained unclear whether accessibility depends on the process of transcription itself or chromatin modifications that accompany transcription. By using T cell receptor beta substrates that integrate stably into nuclear chromatin, we show that promoter location, rather than germ-line transcription or histone acetylation, is a primary determinant of recombination efficiency. These spatial constraints on promoter positioning may reflect an RNA polymerase-independent mechanism to target adjacent gene segments for chromatin remodeling events that facilitate rearrangement.

Acetylation↗

PubMed2002

Regulation of the TCRalpha repertoire by the survival window of CD4(+)CD8(+) thymocytes.

T cell receptor (TCR) alpha alleles undergo primary and secondary rearrangement in double-positive (DP) thymocytes. By analyzing TCRalpha rearrangement in orphan nuclear receptor RORgamma-deficient mice, in which the DP lifespan is shorter, and in Bcl-x(L)-transgenic mice, in which the DP lifespan is extended, we show that the progression of secondary V(alpha) to J(alpha) rearrangements is controlled by DP thymocyte survival. In addition, because Bcl-x(L) induces a bias towards 3' J(alpha) usage in peripheral T cells, we conclude that the programmed cell death of DP thymocytes is not simply a consequence of failed positive selection. Rather, it limits the progression of rearrangement along the J(alpha) locus and the opportunities for positive selection, thereby regulating the TCRalpha repertoire.

Animals↗