A-T: Ataxia-Telangiectasia; ATM: Ataxia-Telangiectasia Mutated Kinase; Cα: Tcra Constant Region; Cδ: Tcrd Constant Region; Chromosome Conformation Capture Experiments, 3C and 4C; CTCF: CTCCC-Binding Factor; D: Diversity; DN: Double Negative; DP: Double Positive; Eα: Tcra Enhancer; Eδ: Tcrd Enhancer; eDP: Early DP; ETP: Early T-cell Progenitor; HS: DNaseI Hypersensitivity Site; IL-7R: Interleukin-7 Receptor; ISP: Immature Single Positive; J: Joining; LCR: Locus Control Region; lDP: Late DP; Rag: Recombinase Activating Gene; SP: Single Positive; T-ALL: T-cell Acute Lymphoblastic Leukemia; TEAp; T Early α Promoter; TCR: T-cell Receptor; TCRα: T-cell Receptor α; Tcra : T-cell Receptor α Gene; TCRαβ: αβ T-cell Receptor; TCRβ: T-cell Receptor β chain; Tcrb : T-cell Receptor β Gene; TCRδ: T-cell Receptor δ; Tcrd: Tcell Receptor δ gene; TCRγ: T-cell Receptor γ; Tcrg: T-cell Receptor γ gene; TCRγδ: γδ T-cell Receptor; TF: Transcription Factor; Traj: T-cell Receptor α J; Trav : T-cell Receptor α V; Trdd : T-cell Receptor δ D; Trdj : T-cell Receptor δ J; Trdv : T-cell Receptor δ V; V: Variable.

During thymic T-cell development (Figure 1), early T-cell progenitors (ETP) arising from fetal liver or bone marrow enter to the thymus, where they mature progressively through different stages that can be distinguished based on the expression of the CD4 and CD8 surface markers: CD4-CD8- double-negative (DN) thymocytes, immature single-positive (ISP) CD8⁺ thymocytes, CD4+CD8+ doublepositive (DP) thymocytes, and CD4⁺ or CD8⁺ single-positive (SP) thymocytes [1]. Among the DN thymocyte population, four subpopulations can be further distinguished based on the expression of CD25 and CD44 surface markers: DN1 (CD44+CD25-), DN2 (CD44⁺CD25⁺), DN3 (CD44-CD25⁺), and DN4 (CD44-CD25-) thymocytes. In addition, two DN3 subpopulations can be distinguished based on the expression of CD27: DN3a (CD27^low) and DN3b (CD27high) thymocytes [2]. Furthermore, two DP thymocyte populations can be distinguished based on the expression of CD71: early DP (eDP) (CD71+) and late DP (lDP) (CD71-) thymocytes [3]. For αβ T-cell development, thymocytes transition from DN1 to SP thymocytes by maturing successively through the following populations: DN1, DN2, DN3a, DN3b, DN4, ISP, eDP, lDP, and SP thymocytes; whereas for γδ T-cell development, thymocytes transit only from DN1 to DN2 or DN3a before becoming mature cells [1].

Figure 1: Temporal control of T-cell gene rearrangements and thymocyte development. The thymus is represented as a light pink rectangle. Schematic representation of thymocyte maturation depicting the various developmental stages and the TCR gene rearrangements is shown. β-, γδ-, and positive selection, which depend on the expression of pre-TCR, TCRγδ or TCRαβ, respectively, are indicated in red. T cell maturation is indicated by the transition from yellow to red (maturation to γδ T lymphocytes) or green (maturation to αβ T lymphocytes).

The loci that encode for the TCR chains are composed of dispersed variable (V), diversity (D), and joining (J) gene segments that are rearranged during thymocyte development by a process known as V(D)J recombination to generate a gene configuration capable of expressing the functional receptors, TCRαβ or TCRγδ, on the cell membrane [4,5]. The V(D)J recombination is completed in DN2 and DN3a thymocytes at the Tcrg and Tcrd loci, in DN3a thymocytes at the Tcrb locus, and in DP thymocytes at the Tcra locus [4].

A successful Tcrg VJ and Tcrd VDJ recombination permits the expression of a TCRγδ, which drives cell differentiation to γδ T lymphocytes in a process known as γδ-selection [4]. A successful Tcrb VDJ recombination in DN3a thymocytes permits the expression of a functional TCRβ chain that assembles with the invariant pre-Tα chain to form a pre-TCR, which drives cell differentiation to DP thymocytes in a process known as β-selection [1,4]. A successful Tcra VJ recombination in eDP and lDP thymocytes permits the expression of a TCRα chain that associates with the previously expressed TCRβ chain to form a TCRαβ [1,4]. The antigen affinity of the TCRαβ in lDP thymocytes will determine the positive selection of a few DP thymocytes that will survive and differentiate into CD4+ or CD8+ SP thymocytes [1]. SP thymocytes migrate to the periphery as mature αβ T lymphocytes [1].

In addition to the essential roles for pre-TCR- and TCR-mediated signaling on thymocyte development, signals mediated by Notch and interleukin-7 receptor (IL-7R) are required for T-cell commitment, survival, and differentiation [6-10]. Each of these signals has a pivotal role in controlling the process of V(D)J recombination at the different TCR loci [1]. In DN2/3a thymocytes, signaling mediated by IL-7R is essential for the Tcrg germline transcription and VJ recombination, as well as for γδ T lymphocyte development [11], whereas signaling mediated by Notch is essential for Tcrb gene VDJ recombination and αβ T lymphocyte development [12]. During β-selection, pre-TCRmediated signaling triggers the Tcra germline transcription and VJ recombination and the development of αβ T lymphocytes, and inhibits the transcription of the Tcrg and Tcrd loci [13-15]. The molecular targets of those signaling pathways are genomic regulatory sequences capable of controlling chromatin structure of the loci, such as the enhancers associated with the Tcrg, Tcrd, and Tcra loci, and the silencer and promoters associated with the Tcrg and Tcrb loci [11,13,14,16-25].

The Tcra and Tcrd genes are linked in a single genetic locus, Tcra/ Tcrd , which spans 1.8-Mb with a conserved genomic structure and a location between the olfactory receptor genes and the Dad1 gene on chromosome 14 in humans and mice [4,5,26]. The 1.7-Mb 5´-locus region includes 132 Tcra and Tcrd V (Trav and Trdv ) gene segments, while the remaining 0.1-Mb 3´-locus region contains the Tcrd D and J (Trdd and Trdj ) gene segments, the Tcrd constant region (Cδ), the Trdv 5 gene segment, the Tcra J (Traj) gene segments, and the Tcra constant region (Cα)(Figure 2). Among the Trav/Trdv gene segments, some only rearrange with the Trdd gene segments, some only with the Traj gene segments, and some can rearrange with either Trdd or Traj gene segments, contributing to both the TCRδ and TCRα chain repertoires [5]. The nested organization of these genes prevents the occurrence of the Tcrd and Tcra gene segment rearrangements on the same chromosome, because the Tcra VJ recombination results in the deletion of the Tcrd locus in an extra-chromosomal circle (Figure 2) [4].

Figure 2: Representation of the different structure of the Tcra/Tcrd locus during T-lymphocyte development. The V, D, and J gene segments are represented by black narrow rectangles. The Cδ and Cα regions are represented by black large rectangles. The 1.6-Mb 5´-locus region includes the Trav/Trdv gene segments, and the 0.1- Mb 3´-locus region includes the Trdd and Trdj gene segments, the Cδ region, the Trdv5 gene segment, the Traj gene segments, and the Cα region. Red lines indicate the areas occupied by Trav/Trdv, Trdd and Trdj , and Traj gene segments. The position of the functionally relevant regulatory elements is indicated as follows: INT1/2 as green circles, Eδ as a blue circle, TEAp as a purple circle, E3´-Jα as an orange circle, LCR as a pink line, Eα as a red circle, HS-1´as a black rectangle, and HS2-6 as brown ovals. The dashed lines represent the possible different rearrangements along the Trav/Trdv gene segment cluster and the continuous lines represent the Tcrd DJ recombination in DN2/3a thymocytes and the primary and secondary Tcra VJ recombination in DP thymocytes. The arrows represent regions of active germline transcription. Arrows anchored to a particular V gene segment represent the transcripts originated from specific V promoters. The rearranged Tcrd VDJ and Tcra VJ transcripts are written in bold blue characters. Low transcription is represented by dashed lines (low transcription level) whereas the width of the continuous lines is proportional to the level of transcription (medium or high). The deleted Tcrd locus in DP and SP thymocytes, and in αβ T lymphocytes, is represented as a separate circularized DNA fragment containing the rearranged Tcrd and several unrearranged Trav/Trdv and Traj gene segments produced as a consequence of a Tcra VJ recombination.

Each Tcra and Tcrd locus is equipped with one transcriptional enhancer, Eα and Eδ, located at the 3´-end of Cα and at the 5´-end of Cδ, respectively, and the numerous promoters that associated with the V, D, and J gene segments along the locus, including the T early α promoter, TEAp, associated with the most 5´-Traj gene segment, Traj61 (Figure 2) [4]. TEAp orchestrates different chromatin loops at the 3´-end of the locus during T-cell development [5,27,28] (see below). Eα is part of a previously described locus control region (LCR) located between Cα and the ubiquitously expressed Dad1 gene [29]. The Tcra LCR spans approximately 7.4-kb, with seven DNase Ihypersensitivity sites (HS): HS1, HS1´, HS2, HS3, HS4, HS5, and HS6 [30]. The most 5´-1.4-kb LCR fragment contains HS1 and HS1´ [30,31]. HS1 contains Eα whereas the 3´-contiguous HS1´ contains two binding sites for the CTCCC-binding factor (CTCF) involved in the Tcra/Tcrd locus chromatin organization and Eα function during thymocyte development [27,30-33]. Eα is responsible for activating the endogenous locus germline transcription and the Tcra VJ recombination, as well as the generation of αβ T lymphocytes [34]. Eα can also activate transcription and the V(D)J recombination of transgenic reporter constructs in a temporally regulated manner during thymocyte development [29,35-38]. In addition, Eα is required for the normal expression of the rearranged Tcrd locus in γδ T lymphocytes [34]. CTCF binding to HS1´, TEAp, and proximal Trav/Trdv promoters is important to generate a functional chromatin hub among Eα, TEAp, and proximal Trav/Trdv promoters to promote the endogenous Tcra VJ recombination in eDP thymocytes [27] (see below). CTCF binding to HS1´ also collaborates with Eα for the expression of transgenic reporter constructs in thymocytes and splenocytes [30]. The 3´-6-kb LCR fragment contains HS2-6 [29]. HS2-6 is transcriptionally active in non T-lineage cells and collaborates with Eα to confer a high-level, position-independent and copy number-dependent transgene expression in T-lineage cells by acting as an insulator that blocks Eα activity to maintain the distinct regulatory programs of the neighboring Tcra/Tcrd and Dad1 genes [29,30,39]. The HS4-HS6 fragment contains the greatest enhancer blocking activity [39], with HS4 and HS6 being the major contributors that confer Eα-dependent high-level, position-independent and copy number-dependent transgene expression in T-lineage cells in a CTCFindependent manner [32,40,41]. In addition, two other Tcra/Tcrd regulatory elements have been recently described (Figure 2): 1) two binding sites for CTCF located upstream of the Trdv4 gene segment, INT1/2, that creates a functionally relevant chromatin loop with TEAp in DN2/3a thymocytes to increase the Tcrd and Tcra repertoires (see below), and 2) a new transcriptional enhancer located between the Traj3 gene segment and Cα, called E3´-Jα, that is active in thymic and peripheral αβ T cells as assessed using transgenic mice [28,42].

Eδ is essential for normal Tcrd V(D)J recombination and generation of γδ T lymphocytes [43]. Eδ functions as a local enhancer important to confer the accessibility of the Trdv5, Trdd , and Trdj gene segments to the recombinase machinery in a 10-20-kb region of adult DN3a thymocytes, while Eα influences a 500-kb region including the proximal 1/3 of the Trav/Trdv (3´-Trav/Trdv ) and Traj gene segments in DP thymocytes (Figure 2) [44,45]. Eδ and Eα are responsible for the specificity of the Tcrd and Tcra gene segment rearrangement, respectively; across the developmental stages by regulating the germline transcription and chromatin structure that mediates the accessibility of the recombinase machinery to each specific gene [46]. To permit the generation of functional Tcrd VDJ recombination and expression of the TCRδ chain in DN2/3a, Eδ is active whereas Eα remains inactive in these cells [14,46]. Tcrd VDJ recombination is accomplished through the activation of the Eδ-dependent promoters associated with the Trdd and Trdj gene segments, which opens up the chromatin structure to provide accessibility for the recombination machinery in DN2/3a thymocytes (Figure 2) [43,47]. During β- selection, Eα is activated to induce the Tcra VJ recombination in DP thymocytes whereas Eδ becomes inactivated [14,34,46]. During γδ- selection, Eα is also activated to contribute to the transcription of the rearranged Tcrd locus, being required for normal expression of the TCRδ chain in γδ T lymphocytes (34). Interestingly, Eδ is inactivated during β-selection but presumably not during γδ-selection [14,43]. Therefore, both Eδ and Eα are relevant enhancers to dictate the patterns of the Tcrd and Tcra gene germline transcription and V(D)J recombination during thymocyte development [14,34,43,46].

The Tcra VJ rearrangements are accomplished through activation of germline transcription, which is initiated at Eα-dependent promoters associated with the most 3´-Trav/Trdv and 5´-Traj gene segments and opens up the chromatin structure to provide accessibility for the recombination machinery in eDP thymocytes (Figure 2) [48-50]. These initial Tcra VJ gene segment rearrangements occur in eDP thymocytes and are known as primary Tcra VJ recombination (Figure 2) [50]. As a consequence of the nested organization of the Tcra/Tcrd locus, the primary Tcra VJ recombination results in the deletion of the rearranged Tcrd locus in an extra-chromosomal circle (Figure 2) [4]. These extra-chromosomal circles will remain present in all DP and SP thymocytes, as well as in naïve αβ T lymphocytes [4]. If the primary Tcra VJ recombination is not productive, then the secondary Tcra VJ recombination involving the more 5´-Trav/Trdv and 3´-Traj gene segments will occur in lDP thymocytes (Figure 2) [51]. This strategy of successive Tcra VJ gene segment rearrangements using the further 5´- Trav/Trdv and 3´-Traj gene segments permits multiple VJ gene segment rearrangements at each Tcra allele to assure the expression of a productive TCRα chain in all lDP thymocytes and to provide a greater probability that positive selection and further αβ T lymphocyte maturation can occur.

Eδ is formed by seven protein-bound elements known as δE1, δE2, δE3, δE4, δE5, δE6, and δE7, in a 380-bp DNA fragment [46]. Although this fragment is functional in activating transcription of reporter constructs in transient transfection experiments, it is not able to activate rearrangement of a reporter construct in single-copy transgenic mice requiring the presence of two flanking matrix attachment regions for such function [52]. Eδ activity depends critically on the binding of the transcription factors (TFs) Runx1 and c-Myb to δE3 [53-55]. These TFs are dissociated from Eδ in the transition from DN3a to DP thymocytes, which is concomitant with the inactivation of the enhancer [14].

Eα is formed by four protein-bound elements known as Tα1, Tα2, Tα3, and Tα4, in a 275-bp DNA fragment that constitutes the minimal Eα with the correct temporal regulation during thymocyte development [46]. The 116-bp Tα1-Tα2 fragment constitutes the core enhancer with essential binding sites for the constitutive TFs CREB/ ATF, TCF-1/LEF-1, Runx1, and Ets-1 that bind cooperatively in an allor- none fashion [36,56-58]. These TFs are bound to a primed Eα prior to its activation in DN3a thymocytes as well as when the enhancer is fully active in eDP and lDP thymocytes [14,46,59]. Although the Tα1- Tα2 fragment is efficient in activating transcription and gene segment recombination at short distances in the context of a transgenic recombination reporter construct, is not sufficient to activate endogenous Tcra VJ recombination at large distances [36,60]. In addition, it does not display the proper Eα developmental regulation because it is activated prematurely in DN3a thymocytes, being necessary additional Tα3-Tα4-binding TFs including Sp1, GATA-3, E2A, and/or HEB for proper temporal activation of the enhancer [36,37]. Pre-TCR signaling triggers the activation of Eα through the binding of the inducible TFs NFAT, AP-1, and Egr-1 in eDP thymocytes, which are recruited to a pre-assembled Eα enhanceosome formed by the Eα-bound constitutive TFs [18]. In lDP thymocytes, prior to positive selection, Eα remains fully active through the induction of strong binding of constitutive TFs such as E2A [18].

Three-dimensional fluorescence in situ hybridization experiments revealed the Tcra/Tcrd locus changes its configuration in DN3a and DP thymocytes [5,61]. In DN3a thymocytes, the Tcra/Tcrd locus adopts a fully contracted configuration [61]. In DP thymocytes, the locus adopts a contracted configuration across the most 3´-region of the locus, including the 3´ Trav/Trdv and the Traj gene segments, as well as the Cα region, in a region of approximately 0.5-Mb; whereas it adopts an extended configuration across the 5´-region of the locus, including the centrally and upstream position Trav/Trdv gene segments (5´-Trav/Trdv ) in a region of over 1-Mb [61]. The fully contracted locus configuration in DN3a thymocytes is thought to facilitate the Tcrd VDJ recombination using the disperse Trdv gene segments across the entire locus, whereas the Tcra/Tcrd configuration in DP thymocytes is believed to facilitate the sequentially ordered primary and secondary Tcra VJ recombination (Figure 2) [5,61]. The molecular mechanism involved in the regulation of the different configurations adopted by the Tcra/Tcrd locus during thymocyte development is currently unknown.

Although the 3´-end of the locus remains similarly contracted in DN3a and DP thymocytes, chromosome conformation capture experiments (3C and 4C) have distinguished two distinct functional chromatin interactions within this 0.5-Mb region of DN3a and DP thymocytes using recombinase activating gene-deficient (Rag^-/-) mice (Figure 3) [5,27,28]. In Rag^-/- DN3a thymocytes, a functionally relevant discrete chromatin loop mediated by CTCF-bound INT1/2 and CTCF-bound TEAp has been recently identified [28]. Active Eδ is present within this chromatin loop attached to the Trdd and Trdj gene segments constituting a recombination center capable of recruiting the distant Trdv gene segments in DN2/3a thymocytes [5,28]. In addition to CTCF, other factors are required for loop formation because it is not present in B lymphocytes where occupancy of the relevant CTCF sites remains intact [28]. Interestingly, the formation of this chromatin loop favors the use of the diverse Trdv gene segments for Tcrd VDJ recombination in DN2/3a thymocytes and indirectly increases the diverse use of the Trav/Trdv gene segments for Tcra VJ recombination in DP thymocytes [28]. In Rag^-/- eDP thymocytes, binding of the pre- TCR inducible TFs to Eα triggers the formation of a chromatin hub through the physical interactions of the Eα-bound TFs, TFs bound to the promoters associated with the most 3´-Trav/Trdv gene segments and TEAp, and the CTCF bound to HS-1´ and each Eα-dependent promoter (Figure 3) [27,31]. This chromatin hub creates an additional recombination center at the 3´-Trav/Trdv and 5´-Traj gene segments to activate the primary Tcra VJ recombination in DP thymocytes [5].

Figure 3: Different chromatin loops are formed at the 3´-Tcra/Tcrd locus in DN3a and DP thymocytes. The V, D, and J gene segments are represented by black narrow rectangles. The Cδ and Cα regions are represented by black large rectangles. The diagram indicates the 5´- and the 3´-Trav/Trdv gene segments, the Trdd and Trdj gene segments, the Cδ region, the Trdv5 gene segment, the Traj gene segments, and the Cα region. The red lines indicate the areas occupied by 5´-Trav/Trdv , 3´-Trav/Trdv, Trdd and Trdj, and Traj gene segments. The position of the functionally relevant described regulatory elements is indicated as follows: INT1/2 as green circles, Eδ as a blue circle, TEAp as a purple circle, E3´-Jα as an orange circle, LCR as a pink line, Eα as a red circle, HS-1´as a black rectangle, and HS2-6 as brown ovals. Curved arrows represent the looping interactions between the regulatory elements demonstrated by 3C and 4C experiments [27,28]. In Rag -/- DN3a thymocytes, high-frequency looping interactions occur between the INT1/2 elements and the TEAp CTCF sites. In Rag -/- DP thymocytes, high frequency looping interactions occur between the Eα-, TEAp-, and the 3´-Trav/Trdv promoters-associated CTCF sites and bound TFs. These interactions are thought to promote the nucleation of recombination centers that facilitate both the Tcrd VDJ recombination in DN2/3a thymocytes and the Tcra VJ recombination in DP thymocytes involving distant gene segments.

Once the TCRαβ or TCRγδ is assembled on the thymocyte surface, Eα becomes active in γδ T lymphocytes and is essential for normal transcription of the rearranged Tcrd locus in these cells, but surprisingly this enhancer is significantly inhibited in SP thymocytes and αβ T lymphocytes (Figure 4) [34,62]. Although Eδ is accepted to be active in γδ T lymphocytes, its contribution toward the transcription of the rearranged Tcrd locus is negligible due to the strong activity of the Eα enhancer in these cells (Figure 4) [43].

Figure 4: Regulation of transcription of the rearranged Tcrd and Tcra genes by distant enhancers in γδ and αβ T lymphocytes, respectively. The V, D, and J gene segments are represented by black narrow rectangles. The Cδ and Cα regions are represented by black large rectangles. Red lines indicate the areas occupied by Trav/Trdv and Traj gene segments. The rearranged Tcrd VDJ and Tcra VJ transcripts are written in bold blue characters. The position of the functionally relevant described regulatory elements is indicated as follows: Eδ as a blue circle, TEAp as a purple circle, E3´-Jα as an orange circle, LCR as a pink line, Eα as a red circle, HS-1´as a black rectangle, and HS2-6 as brown ovals. Curved arrows represent the predicted enhancer-promoter interactions based on the functional experiments [34,43,62]. In γδ T lymphocytes, Eα, and also presumably Eδ, functionally interact with the rearranged Trav/Trdv promoter. The black arrow represents the functionally relevant interaction between Eα and the rearranged Trav/Trdv promoter in comparison to the presumed interaction between Eδ and the rearranged Trav/Trdv promoter, which is represented in grey. In αβ T lymphocytes, Eα is strongly inhibited and its contribution to the transcription of the rearranged Tcra locus is uncertain. The contribution of the recently described E3´-Jα enhancer to the transcription of the rearranged Tcra locus is also unknown. The putatively weak or uncertain interactions between these enhancers and the rearranged Trav/Trdv promoters are represented as dashed light lines.

In support of inhibition of Eα activity in the transition from DP to SP thymocytes and in αβ T lymphocytes, Eα inhibition is evidenced not only when it is located in its natural location at the unrearranged Tcra locus and also when positioned at an ectopic location [62].

Furthermore, expression of reporter transgenes directed by the 7.4- kb LCR containing Eα is significantly inhibited in splenocytes and αβ T lymphocytes compared to thymocytes [30,63]. The transcriptional inhibition of the unrearranged Tcra locus by Eα in SP thymocytes and αβ T lymphocytes suggests this enhancer does not contribute to the transcription of the rearranged Tcra locus in these cells [62]. In support of this hypothesis, transgenic rearranged Tcra constructs containing the 3-kb region from the downstream Cα, including Eα and HS1´, are expressed at very low and variable levels, ranging from 1 to 20% in αβ T lymphocytes [29,64]. Two main questions rise from these findings: How is Eα inactivated and what is the molecular mechanism for transcribing the rearranged Tcra locus in SP thymocytes and αβ T cells?

Recent experiments using chromatin immunoprecipitation to compare the active and inactive Eα enhanceosomes assembled in DP thymocytes and αβ T lymphocytes, respectively, have revealed that the presence of the E2A and HEB TFs is highly diminished in αβ T lymphocytes (Figure 5) [62].

Figure 5: Different Eα enhanceosomes are assembled in lDP thymocytes and αβ T lymphocytes. The diagram depicts the TFs that are bound to Eα in the indicated cell stages based on genomic footprinting and chromatin immunoprecipitation experiments, as well as in vitro experiments [14,57-59,62,76]. The TFs are represented by colored ovals and their identity is indicated. The Eα enhanceosome assembled in αβ T lymphocytes lacks bound E2A and HEB TFs compared to those assembled in lDP thymocytes [62,76].

No differences in the binding of CTCF to HS-1´ were detected between DP thymocytes and αβ T lymphocytes, indicating the binding of this factor is not involved in the inhibition of Eα function in αβ T cells [31,33,62]. These results suggest strong binding of E2A and HEB might be essential for Eα activity. The forced expression of E2A in αβ T lymphocytes through retroviral transduction cannot recover the enhancer activity of Eα, neither alone nor in combination with the upregulation of other TFs in the context of T-cell activation or T helper differentiation [62]. Future experiments are necessary to reveal the molecular mechanism of enhancer inactivation in mature αβ T-cells by evaluating the simultaneous functional effect of E2A and HEB on Eα activity, the analysis of the molecular consequences of different signaling pathways mediated by the pre-TCR and TCRα on Eα function, and a detailed comparison of the Eα enhanceosomes assembled in αβ and γδ T lymphocytes.

The inhibition of Eα in SP thymocytes and αβ T lymphocytes does not preclude the enhanced transcription of the rearranged Tcra locus in these cells compared to the unrearranged Tcra locus in preselected DP thymocytes in the presence of a fully active Eα [62]. The molecular basis for the transcription of the rearranged Tcra locus is currently unknown. Although a possible contribution of Eα to the transcription of the rearranged Tcra locus cannot be totally rejected, the inhibition of its activity through the disruption of the functional long-range enhancer-promoter interactions, the loss of activating histone modifications, and the decreased transcription of the unrearranged Tcra locus in αβ T lymphocytes compared to lDP thymocytes suggests the existence of an Eα-independent mechanism to activate transcription of the rearranged Tcra locus in αβ T cells [62]. In support of this, Eα is not required for copy number-dependent transgenic expression in splenocytes [30]. It is possible that different conformations of the unrearranged and rearranged Tcra locus, due to the deletion of intergenic sequences, may reveal a novel enhancer or activate an enhancer-independent activity in the rearranged Tcra V promoters. The putative novel enhancer must be located upstream of Trav1 or downstream of Traj2 gene segments to ensure its retention upon Tcra VJ recombination. Interestingly, transcription of reporter transgenes controlled by the LCR is also significantly inhibited in splenocytes and αβ T lymphocytes compared to thymocytes, suggesting the additional sequences required for proper transcription the rearranged Tcra locus in αβ T lymphocytes are not contained within the 7.4-kb LCR fragment [30,31,63]. A new putative enhancer, E3´-Jα, located between the Traj3 gene segment and Cα region, and is active in both thymocytes and peripheral αβ T lymphocytes has been recently described (Figures 2-4) [42]. However, transgenic constructs containing a rearranged Tcra locus with an intact Traj2 to HS1´ are expressed at very low and variable levels in αβ T lymphocytes, suggesting the genomic region containing E3´-Jα, Eα, and HS1´ is not sufficient to allow for the strong and stable transcription of the endogenous rearranged Tcra locus [29,64]. It will be important to test the relevance of Eα and other putative relevant sequences in the transcription the rearranged Tcra locus by their conditional deletion in peripheral αβ T lymphocytes and in transgenic mice.

Although beneficial, V(D)J recombination is a dangerous process. Defects in this process at the TCR loci cause for immunodeficiencies and chromosomal translocations that lead to lethal leukemia [4,65,66]. The most common T-lymphocyte leukemia, T-cell acute lymphoblastic leukemia (T-ALL), is composed by a heterogeneous group of acute leukemias derived from the transformation of thymocytes that are arrested at various developmental stages. 35% of human T-ALLs carry chromosomal translocations involving TCR loci in thymocytes. These aberrant translocations frequently involve the juxtaposition of a strong promoter or enhancer from a TCR gene with a TF gene or a gene involved in cell signaling or differentiation. These illegitimate TCR gene translocations lead to the aberrant expression of their corresponding proteins, resulting in abnormal proliferation and differentiation processes. Among all the aberrant translocations of TCR genes during thymocyte development, those involving the Tcra/ Tcrd locus have been found in a high percent of human T-ALLs. For example, 5-10% of pediatric and 30% of adult T-ALLs show translocations of the TLX1 and TLX3 genes into the Tcra/Tcrd locus. These translocations result in the overexpression of the TFs TLX1 and TLX3 and the arrest of DP thymocyte maturation. This arrest is a direct consequence of the recruitment of these TFs to Eα [67]. Binding of TLX1/TLX3 to Eα interferes with the recruitment of Ets-1 and results in reduced enhancer activity as evidenced by decreased gene chromatin accessibility and a drastic inhibition of Tcra gene segment recombination. The expression of a functional TCRα chain is needed for the assembly of the TCRαβ and the maturation of DP to SP thymocytes [34,68]. Other important aberrant translocations involving the Tcra/Tcrd locus include those that result in ataxia-telangiectasia (A-T) syndrome, which is rare immunodeficiency disorder due to mutations in the A-T mutated kinase (ATM) that cause chromosome instability and defects in DNA repair [69]. An important percent of AT syndrome patients develop the disease due to translocations and inversions involving specific breakpoints at the Tcra/Tcrd locus and most of all ATM^-/- mice die due to thymic lymphomas derived from and incorrect repair of the breaks that result from V(D)J recombination and aberrant Tcra/Tcrd locus translocations [70-75]. The knowledge of the precise mechanisms by which the Tcra/Tcrd locus transcription and recombination are regulated is important to understand the defective control of these processes that results in disease.

This work was supported by the Spanish government (Grant BFU2013-44660R) and the Andalusian government (Grant CTS-6587), which is financed in part by the European Regional Development Fund (ERDF/FEDER).