Chromatin looping defines expression of TAL1, its flanking genes, and regulation in T-ALL

The TAL1 gene encodes a basic helix loop helix transcription factor that is a key regulator during vertebrate hematopoiesis.^1,2  Although TAL1 is not normally expressed in lymphoid cells, ectopic T-cell expression of TAL1 occurs in 60% of cases of T-cell acute lymphoblastic leukemia (T-ALL).^3,4  In ∼6% to 26% of T-ALL cases, TAL1 is expressed via a fusion messenger RNA (mRNA) resulting from a microdeletion (∼74-82 kb) involving TAL1 and its neighboring STIL gene, bringing TAL1 under the control of STIL expression.^5,6  Of these deletions, the majority have common breakpoints within TAL1 promoter 1b and within STIL intron 1 (the latter known as the TAL^d breakpoint). The mechanisms which regulate TAL1 expression in T-ALL and predispose TAL1 and STIL to undergo structural rearrangements are not well understood.

The importance of TAL1 in normal hematopoiesis and in T-ALL has fueled systematic dissection of the TAL1 locus and led to the identification of multiple cis-acting regulatory elements capable of directing TAL1 expression.^7-10  In addition to TAL1 promoters 1a and 1b, these elements include enhancers and CTCF-bound elements (supplemental Figure 1, available on the Blood Web site). Collectively, these cis-regulatory elements span ∼88 kb in humans, a genomic region which contains the entire TAL1 “regulon” defined by CTCF-bound elements at either end.⁹  This regulon also contains the 3′ segment of STIL, and the entirety of PDZK1IP1, the latter being juxtaposed between TAL1 and its erythroid enhancer (supplemental Figure 1). PDZK1IP1 is expressed in all hematopoietic cell types where TAL1 is expressed¹¹  (albeit at low levels; supplemental Figure 2), suggesting that their transcription is coregulated. The basis for this coregulation is unclear, although others have suggested that TAL1 and PDZK1IP1 share cis-regulatory elements.¹²  However, this is unlikely to occur given that a CTCF-bound element with enhancer-blocking activity⁹  is positioned between the PDZK1IP1 promoter and the majority of the TAL1 cis-regulatory circuitry (supplemental Figure 1B). Similarly, the location of this CTCF-bound element makes it difficult to envisage how the TAL1 erythroid enhancer communicates with its promoters to regulate TAL1 expression in the erythroid lineage (supplemental Figure 1C). Finally, the presence of a CTCF-bound element within the 3′ portion of the transcribed region of the STIL gene presents an impediment to STIL transcription (supplemental Figure 1D).

Because of this genomic organization, we hypothesized that context-dependent chromatin looping would be required at the TAL1 locus to facilitate appropriate communication between genes and their cis-regulatory circuitry. We rationalized that such looping interactions may also predispose the TAL1 locus to be expressed and undergo structural rearrangement in T-ALL (the latter by bringing sequences which are prone to rearrange into close physical proximity). We report here transcription-associated looping models of the TAL1 locus as detected by 3C- and 4C-based methods. We discuss how these models could account for a range of biological and evolutionary features associated with the expression of TAL1 and its flanking genes and how they could predispose to, or facilitate, TAL1 expression in cases of T-ALL.

K562, HPB-ALL, Jurkat, MEL (C88), and BW5147 cell lines were cultured as described previously.⁹  Primary murine lymphocytes were isolated from spleens of wild-type ICR mice (Harlan-Olac Ltd.) by density centrifugation (lympholyte-mammal; VH-Bio). Nuclei from primary murine erythroblasts were obtained as described previously.¹³ 

K562 cells (5 × 10⁶) were transfected with 20nM double-stranded small interfering RNA (siRNA) oligonucleotides targeted to GATA1 (sense: 5′-GGAUGGUAUUCAGACUCGAdTdT-3′) or firefly luciferase (sense: 5′-CUUACGCUGAGUACUUCGAdTdT-3) using the Amaxa Nucleofector (T-016 program). Cells were monitored for growth arrest (trypan blue; Sigma-Aldrich), morphologic changes (Stain Quick-Staining kit; Lamb), and apoptosis (annexin V-phycoerythrin; BD Biosciences) by microscopy.

Total cellular RNA from cultured cells was purified (TRIzol; Invitrogen) and reverse-transcribed (Superscript II kit; Invitrogen). SyBr green quantitative polymerase chain reaction (PCR) was performed (Roche) (supplemental Table 1) on an Mx3000P QPCR Systems PCR machine (Agilent Technologies). Protein was extracted and quantified (Protein Assay Kit II; Bio-Rad). Western detection used the anti-GATA1 antibody (M-20; Santa Cruz Biotechnology).

Chromatin immunoprecipitation (ChIP) from multiple bioreplicate samples was performed as described elsewhere⁹  (supplemental Table 2). ChIP enrichment levels were determined by SyBr green PCR as noted in the previous paragraph (supplemental Table 3).

3C libraries were prepared as described previously.¹⁴  Bacterial artificial clones for the human TAL1 locus (RP1-18D14; Invitrogen) and the murine Tal1 locus (RP23-453H14; BPRC) were used to prepare mock 3C libraries. PCR conditions for each 3C assay (supplemental Table 4) are available on request. Quantification of PCR band intensities was performed with background correction (AIDA; Raytest). 3C-relative ligation frequencies were obtained by normalizing band intensities obtained from 3C libraries with those from mock 3C libraries. Statistical significance within or between samples was determined by the Student t test (P < .05) and via normalization strategies as described previously.¹⁵  4C libraries were prepared as described previously¹⁶  (supplemental Table 5). PCR cycling conditions are available on request. Microarray analysis of fluorescently labeled 4C and genomic DNAs, hybridized to a TAL1 tiling path microarray, were performed as described previously.⁹  3C and 4C data were derived from at least 2 independent biological replicates.

We performed 3C analysis^17,18  (supplemental Figure 3) in human and murine cell types to determine (1) whether cis-regulatory elements at the TAL1 locus showed enhanced frequencies of ligation between each other in 3C libraries, relative to appropriate controls, and (2) whether there were differences in looping frequencies of these elements between TAL1-expressing and nonexpressing cell types.

We first asked whether specific looping interactions occurred between the 2 human TAL1 promoters (1a and 1b) in combination with the erythroid enhancer (+51), the stem cell enhancer (+19/+20/+21), or a 5′ proximal enhancer (−10) (Figure 1A). In the TAL1-expressing erythroid¹⁹  K562 cell line, we detected looping between the TAL1 promoters and both the erythroid (+51) and stem cell enhancers (+19) (Figure 1B). These looping interactions were also observed in equivalent murine erythroid cell types at +40 and +18, respectively (Figure 1C; supplemental Figure 4). There did, however, appear to be differences in the levels of looping between the promoters and the 5′ proximal enhancer in humans and mice (−10 and −9, respectively) relative to their cognate control regions (−8 and −5, respectively). These differences may be explained by the complexity of multiple cis-regulatory elements upstream of TAL1.^7,9,20,21  This is supported by comparisons of ligation frequencies at the TAL1/Tal1 locus in both erythroid and lymphoid cells normalized against those from the ERCC3/Ercc3 control locus (supplemental Figure 5) which showed that all 3 enhancers, as well as the control regions upstream of TAL1, had significantly higher levels of looping with the TAL1/Tal1 promoters when the gene was transcribed (Figure 1D; supplemental Figure 4). Furthermore, additional control regions (eg, +15 in human; +30, +15 in mouse) showed higher interaction frequencies in erythroid cells than in lymphoid cells, suggesting that a number DNA segments of the downstream of the TAL1/Tal1 gene were also in close contact with its promoters when the gene was transcribed. Although we did see some evidence for elevated ligation frequencies between the TAL1/Tal1 promoters and the human +19 stem cell enhancer (mouse +18) in lymphoid cells (Figure 1B-C; supplemental Figure 4), these occurred at relatively low levels compared with those in TAL1/Tal1-expressing cell types. These data provided our first lines of evidence that there are transcription-associated looping interactions which occur between TAL1 cis-regulatory elements.

Our previous work demonstrated that the human +51 erythroid enhancer and TAL1 promoter 1a were both occupied with RNA polymerase II (Pol II) and components of the TAL1 erythroid complex (TEC) containing GATA1, TAL1, LMO2, TCF3, and LDB.⁹  We hypothesized that these proteins were involved in looping between these elements. We optimized an siRNA assay for a core component of the TEC, GATA1, in K562 cells (Figure 2A). At 48 hours after knockdown, GATA1 protein expression was reduced by 87% and was accompanied by a reduction in TAL1 mRNA expression to 60% of its wild-type level (Figure 2B). Maximum knockdown of GATA1 was achieved 96 hours after transfection (98% decrease in protein; Figure 2A) and resulted in a further decrease in TAL1 expression to 40% of its wild-type level (Figure 2B). Transient knockdown of GATA1 for 96 hours in K562 cells did not adversely affect cell viability and morphology compared with controls (supplemental Figure 6).

There was substantial loss of GATA1 from both TAL1 promoter 1a and the +51 erythroid enhancer after 48 hours of GATA1 knockdown (Figure 2C). Surprisingly, GATA1 occupancy showed the most significant decrease at the stem cell enhancer (+20); at 48 hours of knockdown, its level was indistinguishable from background. GATA1 was not known previously to bind to the stem cell enhancer in K562 cells. By 96 hours of knockdown, GATA1 occupancies at both the TAL1 promoter 1a and the +51 erythroid enhancer were also reduced to background levels. Occupancy of other members of the TEC (LDB1 and TCF3) also decreased in GATA1 knockdown cells from both the TAL1 promoter 1a and the +51 enhancer (Figure 2D-E). GATA1 knockdown also resulted in decreased transcription of LDB1, TCF3, and GATA2 (Figure 2B), all known to be downstream targets of GATA1 in hematopoietic cells.^19,22-25  However, decreased expression of LDB1 and TCF3 could not account for the much larger drop in occupancies for these transcription factors at both +51 and TAL1 promoter 1a, and supported that loss of GATA1 directly impaired recruitment of LDB1 and TCF3 to these elements. Impairment of Pol II recruitment at TAL1 cis-regulatory elements was also evident by 96 hours of GATA1 knockdown (Figure 2F). These data demonstrated a direct relationship between TAL1 transcription, the kinetics of GATA1 depletion from its cis-regulatory elements, and the dependence of other factors on GATA1 occupancy at these sites.

To determine whether GATA1 was required for looping between TAL1 regulatory elements, we performed 3C on material obtained from the GATA1 knockdown cells (Figure 3). After 48 hours of knockdown, we observed significant loss in the looping interaction between the +19 stem cell enhancer and the TAL1 promoters (Figure 3A), coinciding with the complete loss of GATA1 from +19 and a reduction in TAL1 transcription. Only modest decreases in looping between the +51 erythroid enhancer with either the TAL1 promoters or with the +19 enhancer were observed at 48 hours. However, at the 96-hour time point, we observed a substantial loss of the looping interactions between these elements (Figure 3B-C), coinciding with complete loss of GATA1 from TAL1 promoter 1a and +51 and a further drop in TAL1 transcription. All of these data supported a model where the TAL1 promoters and the stem cell and erythroid enhancers were in contact in a transcriptionally active erythroid “hub” (Figure 2D). The kinetics of looping interactions within this hub were in agreement with the kinetics of GATA1 depletion and decreases in TAL1 transcription, supporting a model whereby this hub structure was linked to GATA1-dependent TAL1 expression in erythroid cells. We could not, however, rule out the possibility that reductions in TCF3, LDB1, and GATA2 expression also impact upon these looping interactions and TAL1 expression.

We investigated whether the 4 CTCF-bound elements at the human TAL1 locus (+57, +53, +40, and −31) were involved in looping to facilitate the formation of the TAL1 erythroid hub. Each of these elements acts as enhancer-blocking insulators in reporter assays⁹  and is also occupied by RAD21 and SMC3, components of the cohesin complex (supplemental Figure 7). We considered all possible looping interactions between these elements (Figure 4A; supplemental Figure 8) using 3C. In TAL1-expressing K562 cells, looping interactions were identified between the +57 and −31 elements (Figure 4B) and between +53 and −31 (supplemental Figure 9) in a transcription-associated manner when compared with levels found in HPB-ALL (Figure 4C; supplemental Figure 10). We did not detect looping interactions between +40 and any of +53, +57, or −31 in either K562 or in HPB-ALL which we considered to be biologically relevant (supplemental Figures 9-10). These data would support a configuration where the TAL1 promoters and the stem cell and erythroid enhancers were compartmentalized in a single chromatin loop in TAL1-expressing erythroid cells, but not in TAL1-nonexpressing lymphoid cells (Figure 4D). Based on this, we predicted that the interaction between +51 and the TAL1 promoters would bring the +57/+53/−31 interaction into close proximity to the TAL1 promoters. Using 3C, we were able to demonstrate that the TAL1 promoters and −31 were in close proximity in the K562 cell line (Figure 4E). This proximity was significantly increased in K562 when compared with levels in HPB-ALL (Figure 4E-F), linking it to TAL1 transcription.

We asked whether disassembly of the TAL1 erythroid hub during GATA1 knockdown was accompanied by loss of CTCF-associated loops. Forty-eight hours after transfection, CTCF and RAD21 occupancies at +57, +40, and −31 remained unchanged in GATA1 knockdown cells when compared with controls (Figure 5A-B). However, there was considerable loss of both CTCF and RAD21 (≤47% remaining) from the −31 element at the 96-hour time point. This loss was accompanied by a significant decrease in looping between +57 and −31 (Figure 5C). Thus, while GATA1 loss from TAL1 regulatory elements began early in our perturbation time course and was accompanied by partial disassembly of the hub, loss of CTCF and RAD21 from −31 only occurred once the hub was completely disassembled, and GATA1 was completely depleted from cis-regulatory elements in the hub (Figure 5D). This demonstrated a hub-dependent recruitment of CTCF and RAD21 to the −31 insulator element when TAL1 is transcribed. Surprisingly, this recruitment is GATA1-mediated, despite GATA1 not being directly bound to −31. We identified several features of the −31 element in K562 cells which distinguished it from other CTCF/RAD21-bound elements at the TAL1 locus (supplemental Figures 7 and 11). We also showed that RAD21 recruitment to −31 is substantially reduced when the +57/+53/−31 loop is not formed in TAL1-nonexpressing HPB-ALL cells (supplemental Figure 12).

Based on our erythroid model (Figures 3D and 5D), we predicted that expression of genes flanking TAL1 may be dependent on the hub for transcriptional regulation because of their increased proximity to the TAL1 promoters. We used 4C²⁶  (supplemental Figure 13) in combination with a TAL1 genomic tiling microarray⁹  to determine whether the TAL1 hub showed transcription-associated proximities with its flanking genes.

In addition to detecting interactions between the TAL1 promoters, its enhancers, and various points along the gene body of TAL1, our 4C-microarray analysis revealed that both PDZK1IP1 and STIL showed increased frequencies of interactions with the TAL1 promoters in TAL1-expressing cells (K562), compatible with the idea that the hub was bringing these flanking genes into close contact with the TAL1 promoters (Figure 6Ai). These interactions were found to be distributed at discrete points throughout the PDZK1IP1 and STIL genes (shown in red in Figure 6A). Levels of these interactions were noticeably higher than those detected by 4C in TAL1-nonexpressing HPB-ALL cells (supplemental Figure 14), and this was confirmed for one of these interactions by 3C (supplemental Figure 15; referred to as −81/TAL^d in Figure 6A, see the next section). mRNA levels of PDZK1IP1, STIL, and CMPK1 in GATA1 knockdown cells, all showed statistically significant decreases after 96 hours of GATA1 knockdown (Figure 6B) correlating with the complete dissolution of the TAL1 looping hub and loss of contact between the TAL1 promoters and the −81/TAL^d looping interaction (Figure 6C). Furthermore, Pol II recruitment at the STIL promoter was reduced during GATA1 knockdown, although this was not the case for CMPK1 (Figure 6D) or PDZK1IPI (which did not have detectable levels of Pol II at its promoter⁹ ). All of these data correlate well with a model whereby the TAL1 flanking genes have some degree of dependence on the TAL1 hub in erythroid cells for their expression.

The −81/TAL^d interaction with the TAL1 promoters was of particular interest to us because it was associated with TAL1 transcription (in our K562 model), was also present in lymphoid cells, and was mapped to the sites of the common TAL1/STIL deletion breakpoints in patients with T-ALL (supplemental Figure 16). We hypothesized that this interaction, and possibly others at the TAL1 locus, could be related to ectopic expression of TAL1 in T-ALL either through (1) predisposing the TAL1 locus to TAL1/STIL deletions or (2) alterations in TAL1 looping patterns in the lymphoid lineage in T-ALL cases where TAL1/STIL deletions were absent. We tested the latter of these 2 hypotheses by performing 3C on a TAL1-expressing T-ALL cell line (Jurkat) and compared this to looping interactions in a TAL1-nonexpressing T-ALL cell line (HPB-ALL). Jurkat cells showed high-frequency interactions between the TAL1 promoters and sequences located at 8 and 10 kb upstream (supplemental Figure 16). One of these interactions (at −8) was located ∼500 bp from a site where the TAL1, RUNX1, and GATA3 proteins had previously been shown to bind and enhance TAL1 transcription in Jurkat cells (“the Jurkat enhancer”).²¹  The second of these was at the −10 enhancer known to be in contact with the TAL1 promoters in K562 cells (Figure 1). One or both of these upstream interactions was at significantly higher frequencies in Jurkat when compared with HPB-ALL (Figure 6E) or K562 (supplemental Figure 16). Furthermore, interaction of the TAL1 promoters with the TAL^d STIL breakpoint region (−81/TAL^d) was significantly higher in Jurkat cells than in HPB-ALL (Figure 6F). These data strongly suggest that TAL1 expression in Jurkat cells may be regulated by at least 3 transcription-associated looping interactions involving the TAL1 promoters and sequences upstream of TAL1 including the −81/TAL^d breakpoint region.

We describe here looping models of TAL1 transcriptional control (Figure 7). In our erythroid model, the transcriptionally active hub contains multiple loops which bring together the TAL1 promoters, several enhancers, and CTCF elements flanking the TAL1 regulon. In this model, contact between the erythroid enhancer and the TAL1 promoters reflects the activity of an enhancer known to be active in the erythroid lineage.^8,9  Central to this hub is the presence of Pol II and the TEC (GATA1, TAL1, TCF3, LDB1, and LMO2). In Jurkat cells, where TAL1 is inappropriately expressed in T-ALL, looping between the TAL1 promoters and elements upstream are linked to TAL1 expression. In our lymphoid model, the transcriptionally inactive hub contains the TAL1 promoters, the stem cell enhancer and the −31 element, but these interactions occur at a significantly reduced frequency than in either erythroid or Jurkat cells and may reflect residual looping activity when TAL1 was expressed at some point prior to commitment to the lymphoid lineage. In all 3 models, interaction of the TAL1 promoters with the TAL^d breakpoint region is shown, as our data support that this interaction is present in all 3 cell types, but is elevated when TAL1 is transcribed. These models are consistent for data obtained from human cell lines, murine cell lines, and murine primary cells, thus providing substantial proof that these looping structures are found in hematopoietic cells in vivo. Recently, 2 other studies have described looping models for TAL1 expression.^27,28  Both are highly compatible with our models and also account for the proximities of +51²⁸  and +53²⁷  to the TAL1 promoters. These studies also demonstrate requirements for Mediator²⁷  and hSET1²⁸  in TAL1 looping interactions, suggesting further that GATA1 is not the only requirement for loop formation at the TAL1 locus.

We demonstrated that GATA1 is important for both the transcription of TAL1 and the maintenance of the active erythroid hub. The kinetics of hub disassembly and decreases in TAL1 transcription correlate well with the loss of GATA1 or complexes containing GATA1 from TAL1 cis-regulatory elements during GATA1 knockdown experiments, although we cannot exclude the possibility that other factors such as Mediator, hSET1, and CTCF may influence the kinetics of hub disassembly during GATA1 knockdown. GATA1 could facilitate the formation of such a hub via bridging and/or recruitment of other factors, such as LDB1, which may directly mediate looping.^15,29,30  Alternatively, the transcription factory model of gene expression¹³  would predict that local concentrations of Pol II, GATA1, and other factors are sufficient to provide a loose scaffold that maintains the proximity between cis-regulatory elements. Furthermore, the ability of a single GATA factor to regulate the integrity of the TAL1 active hub has significant evolutionary implications for the TAL1 locus, which has undergone cis-regulatory remodeling over the last 360 million years while maintaining highly conserved GATA sites (supplemental Figure 17).

It is easy to envisage why the TAL1 erythroid enhancer is present in a transcriptionally active erythroid hub but it is less easy to understand why the stem cell enhancer, bound with GATA1, is also present, and why loss of this enhancer from the hub is accompanied by significant reductions in TAL1 transcription in K562 cells. The stem cell enhancer has previously been shown to be active only in hematopoietic progenitors by recruiting GATA2.³¹  However, exchange between GATA1 and GATA2 occupancy at the stem cell enhancer during lineage fate decisions could regulate its activity as has previously been described for other cis-regulatory elements.³²  It is also plausible that K562 is a cell line which has attributes of both stem cell and erythroid function although our data from primary murine erythroblasts would argue that the stem cell enhancer is in contact with the TAL1 promoters in the erythroid lineage. Our data suggest a possible new function for this element in hub formation and enhancing TAL1 transcription in the erythroid lineage.

The roles of CTCF-bound elements in transcription-dependent looping at the TAL1 locus were also studied here. While all 4 CTCF elements at the TAL1 locus are bound by both CTCF and RAD21 in both erythroid and lymphoid cells, looping between 3 of these elements (+57 and/or +53 in contact with −31) occurs only in erythroid cells where TAL1 is expressed. This looping configuration facilitates compartmentalization of the TAL1 regulon and assembly of the erythroid hub because all TAL1 cis-regulatory circuitry is located between +57 and −31. Furthermore, this looping pattern overcomes issues created by the juxtaposition of the +40 CTCF element located between +51 erythroid enhancer and the TAL1 promoters (Introduction and supplemental Figure 1) because +40 does not participate in local insulator-based looping.

The −31 element appears to have a crucial role in determining looping interactions at the TAL1 locus in erythroid cells because both RAD21 and CTCF are lost from this element upon disassembly of the transcriptional hub in GATA1 knockdown cells. Furthermore, −31 appears to be dependent on GATA1 binding at other TAL1 cis-regulatory elements for the recruitment of CTCF and RAD21 to −31. This relationship may reflect the cooperative nature of GATA1, bound at TAL1 promoters and enhancers, in facilitating looping between TAL1 insulators through the recruitment of CTCF and RAD21 to −31 (as all of these elements are in close proximity in the hub). This mechanism is reminiscent of GATA1 and CTCF function at the HBBa locus in erythroid cells where they bind to different cis-regulatory elements but act cooperatively to enable looping.³³ 

We demonstrated here that the TAL1 erythroid hub also coregulates, to some degree, the transcription of PDZK1IP1, STIL, and CMPK1 through their interaction with the hub via looping (supplemental Figure 11C). Thus, the TAL1 regulon definition which we previously proposed⁹  no longer adequately describes the regulatory influence that TAL1 imposes on its flanking genes because the majority of STIL and the entirety of CMPK1 reside outside of the predicted regulon boundaries. We propose that there are 2 models which could explain the coregulation of genes at the TAL1 locus, both of which rely on activity of the TAL1 hub and are consistent with our data (supplemental Figure 18). Importantly, in both models, expression of STIL relies on the dynamic assembly and disassembly of CTCF and RAD21 at −31. However, we cannot rule out the possibly that these, or other models, may account for transcription of genes flanking TAL1 in a coregulated way. Nevertheless, our data would suggest that conventional views of gene regulation may not apply to TAL1.

The occurrence of TAL1/STIL deletions (TAL^d deletions), in cases of T-ALL, is well documented.^5,6,34-36  How these deletions occur with such frequency in T-ALL is remarkable and somewhat of a paradox. Although recombination by aberrant immunoglobulin/T-cell receptor (Ig/TCR) recombinase activity could be involved in mediating these deletions, sequences at these breakpoints are relatively poor substrates for this recombinase.³⁴  Our 3C and 4C data show that regulatory hubs which control TAL1 transcription bring the TAL1/STIL common breakpoint regions (TAL^d) into close proximity in both TAL1-expressing cells (K562 and Jurkat) and TAL1-nonexpressing cells (HPB-ALL). Thus, physical proximity between these regions in early hematopoietic progenitors, or even in committed lymphoid cells, may predispose to TAL^d deletions by increasing the likelihood that they will recombine and give rise to an expressed TAL1/STIL fusion mRNA. Recent evidence suggests that chromosomal proximity may be a key determinant in double-stranded breakage and rejoining which lead to structural alterations in cancer genomes.³⁷ 

Moreover, in cases of T-ALL where TAL^d deletions are not found, our data also support that ectopic expression of TAL1 is linked to changes in TAL1 regulation, mediated by aberrant looping between TAL1 cis-regulatory elements upstream of TAL1. To this end, loops involving the TAL^d STIL breakpoint and the TAL1 promoters may also be relevant to TAL1 transcription in T-ALL, in addition to any possible roles these loops may have in mediating deletion events. Our view of the events leading to TAL1 expression in T-ALL demonstrates that local transcription-associated looping topology may be deterministic in pathobiology via multiple mechanisms. Further analysis into these events is ongoing in our laboratory.

This work was supported by a University of Glasgow studentship (Y.Z.).

Contribution: Y.Z. and R.S. performed 3C analysis; Y.Z. performed 4C analysis, ChIP, and siRNA studies; S.K. developed the 4C procedure in our laboratory; M.V. and A.M.M. provided primary murine lymphocytes; P.S. provided bioinformatic support; D.V. and A.G.W. planned the study; and Y.Z., A.G.W., and D.V. wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

The current affiliation for S.K. is Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, India.

The current affiliation for P.S. is lllumina United Kingdom, Chesterford Research Park, Little Chesterford, United Kingdom.

The current affiliation for R.S. is Department of Physiology Development and Neuroscience, University of Cambridge, Downing Site, Cambridge, United Kingdom.

Correspondence: David Vetrie, Epigenetics Unit, Institute of Cancer Sciences, University of Glasgow, Glasgow, United Kingdom G12 8QQ; e-mail: david.vetrie@glasgow.ac.uk.