Abstract
We identified a distinct mouse pDC lineage derived exclusively from the common lymphoid progenitor (CLP) that is dependent on expression of Bcl11a. CLP-derived pDC have a unique gene expression profile that includes hallmark B cell genes not normally expressed in pDCs and therefore we refer to this lineage as “B-pDCs.” Unlike classical pDC, B-pDC express an inherent activation signature, localize preferentially to secondary lymphoid organs and expand more robustly and also induce increased T cell proliferation relative to classical pDCs following Toll-like receptor 9 (TLR9) engagement. B-pDCs lack IFN-α secretion but instead express a distinct cytokine profile and display high levels of the cell-surface receptor tyrosine kinase Axl. Murine B-pDCs represent a distinct CLP-derived DC lineage that is genetically, phenotypically, and functionally homologous to an AXL+ DC subtype recently discovered in human blood1-4
We identified a distinct mouse pDC lineage derived exclusively from the common lymphoid progenitor (CLP) that is dependent on expression of Bcl11a. CLP-derived pDC have a unique gene expression profile that includes hallmark B cell genes not normally expressed in pDCs and therefore we refer to this lineage as “B-pDCs.” Unlike classical pDC, B-pDC express an inherent activation signature, localize preferentially to secondary lymphoid organs and expand more robustly and also induce increased T cell proliferation relative to classical pDCs following Toll-like receptor 9 (TLR9) engagement. B-pDCs lack IFN-α secretion but instead express a distinct cytokine profile and display high levels of the cell-surface receptor tyrosine kinase Axl. Murine B-pDCs represent a distinct CLP-derived DC lineage that is genetically, phenotypically, and functionally homologous to an AXL+ DC subtype recently discovered in human blood1-4
Plasmacytoid dendritic cells (pDCs) specialize in the production of type I interferons (IFNs) and promote antiviral immune responses following engagement of pattern recognition receptors. They have been implicated in the pathogenesis of autoimmune diseases that are characterized by a type I IFN signature (notably, IFN-α), yet pDC also can induce tolerogenic immune responses5-8. Remarkably, considering the interest in pDC and their clinical significance, our understanding of pDC lineage derivation remains muddled. The transcription factor TCF4 is required for pDC development and for lineage identity9-11. TCF4 is a component of a multiprotein complex that includes both positive and negative regulators6,12. One of these components, the transcription factor Bcl11a, which is also essential for B cell development13-15, induces Tcf4 expression and initiates a positive feedback loop with Tcf4 to maintain pDC lineage commitment and function14-16.
Unlike their conventional dendritic cell (cDC) counterparts, pDCs express multiple other transcriptional regulators and markers associated with B-lymphocyte development in addition to Bcl11a (e.g. B220, SpiB)17,18. These features, along with established generation of pDC from myeloid restricted precursors, have made it difficult to define pDC lineage affiliation11,17,19,20 leading to the hypothesis that pDC may have distinct origins derived from either the Common Lymphoid Progenitor (CLP) or the Common Myeloid Progenitor (CMP)18. Beyond the enigmatic nature of their lineage derivation, pDCs with different functional attributes (e.g. variable IFN-α expression levels) or different surface markers (e.g. Cd19+ pDCs detected in tumor-draining lymph node) have also been identified17,21-23. Recently, a novel dendritic cell (DC) population with pDC-like properties which can be identified by expression of the receptor tyrosine kinase AXL (AXL+ DC) was discovered in human blood1-4. While AXL+ DCs express many “classical” pDC markers (CD123, BDCA2/CD303) they also express the Ig-like lectins SIGLEC1 and SIGLEC6, and the activation marker CD861. In a separate study2 AXL+ DCs were shown to be CD5+ and CD81+, two glycoproteins normally associated with the B cell receptor (BCR) signaling complex. The origin, exact nature and lineage of the AXL+ pDC-like population is currently unknown. Here, we report the identification of a murine, lymphoid-derived counterpart of the human AXL+ DC subset and demonstrate an in vivo requirement for Bcl11a in its transcriptional specification.
We previously demonstrated that conditional deletion of Bcl11a in the hematopoietic stem cell (HSC) compartment mediated by Vav-l-Cre or by inducible Mxl-Cre recombinase results in complete abolishment of pDC development14. Next, spurred by previous speculation of pDC origin from the CLP17,20, we selectively deleted floxed (F) Bcl11a alleles in the late CLP stage24,25 mediated by mbl-Cre in vivo. Expression of the mbl gene (Cd79a) begins at the Ly6d+ CLP stage, in B-cell-biased lymphoid progenitor (BLPs)26, downstream of Ly6d− CLPs (Fig. S1). Bcl11F/Fmbl-Cre mice (cKO) and littermate controls were examined for pDC frequencies among nucleated cells in the bone marrow (BM). B220+ Pdca1+pDCs were consistently and significantly reduced by an average of ~25% (24.8 ± 2.4%) in the cKO mice relative to littermates (Fig. 1a). A near complete loss of mature recirculating B cells (B220hiPdca1−) served as a gauge of mbl-Cre deletion efficiency. Additionally, immature B cell numbers were reduced by >50% in the KO mice (Fig. 1a, S2). Taken together, these data indicate that a significant proportion of pDC are derived from BLP progenitors, and are Bcl11a dependent.
To expand these in vivo observations, and to confirm that this defect is intrinsic to hematological progenitor cells, we transferred BM from either Bcl11a-sufficient reporter control mice (mbl-Cre-YFP) or Bcl11a-deficient cKO mice (Bcl11aF/Fmbl-Cre-YFP−) into lethally irradiated C57BL/6 recipients. After 8 weeks, <6% of B cells in the spleens of mbl-Cre-YFP recipients were YFP−, confirming elimination of recipient hematopoiesis (Fig. 1b). As expected, Bcl11aF/Fmbl-Cre-YFP BM resulted in significantly reduced B cell and pDC cellularity compared to mbl-Cre-YFP controls, whereas Bcl11a-sufficient (YFP) pDC persisted (Fig. 1b and c, representative plots Fig. S2). Approximately 1/3 of pDCs in the spleen of wild-type chimeras were YFP+ compared to only 1/5 in BM (Fig. 1d). This increased fraction of YFP+ pDCs in the spleen suggests that CLP-derived pDC preferentially home to that organ. Other hematopoietic lineages were capable of development in normal numbers and contain a paucity of YFP+ cells (Fig. 1e). Of note, Bcl11a-deficient progenitors yielded higher splenic T cell chimerism at the expense of B cells and pDCs, but T cells were <2% YFP+. This indicated that mbl-cre expression occurs subsequent to T-B lineage divergence, consonant with previous observations26 and our mbl-cre progenitor analysis in which YFP+ cells are confined to CLP stage (Fig. S1).
Collectively, these results provide in vivo evidence for Bcl11a-dependent and BLP-derived pDCs (generated downstream of CLP) which populate the BM and periphery of adult mice. Because of their exclusive lymphoid derivation post T-B bifurcation, we were prompted hereafter to refer to this pDC lineage as “B-pDC.” To further elucidate their phenotype at the genetic level, we performed RNA-seq analyses of purified B-pDC and compared them to classical, myeloid-derived pDC (Fig. 2a). Purity of each population was confirmed as pDC by RT-PCR of shared pDC markers (Pdcal and Bcl11a) (Fig. S3). While the overall gene expression patterns were highly similar across the two subsets, ~1% of transcripts (~220 genes) differed significantly (q value < 0.05, Fig. 2b). Differentially expressed transcripts generated Gene Ontology (GO)27 or Panther28 terms including “immune response”, “inflammatory response”, “cell activation”, and “regulation of immune response” (p=1.36×10−17, 2.15×10−12, 3.67×10−11, and 1.45×10−10, respectively; Fig. S4). Gene set enrichment analysis (GSEA) revealed elevation of each of these same GO/Panther terms within the B-pDC subset as compared to a pDC-related GSEA control dataset that showed no enrichment (Fig. 2c). Next we compared all genes expressed by both pDC populations to one another and to published29
RNA-seq of BM-derived mouse pre-B cells (B220+IgM-Ki-Cd25+)—a post-CLP B cell progenitor (pre-B) population (Fig. 2d and e, S5. pDC and B-pDC expression levels showed a strong correlation to each other relative to B cells (R2 values = 0.8959, 0.4145, and 0.404, respectively). Detailed analysis of these RNA expression data (Fig. 2e, S5, coupled with cell phenotyping by flow cytometry (Fig. 3a and b), showed that B-pDCs express genes critical to early B cell biology. Cdl9 was expressed at intermediate levels relative to B cells (Fig 2e, S5) and flow cytometry analysis revealed virtually all YFP+ B-pDC from mbl-Cre-YFP WT mice displayed Cd19 on their surface (Fig. 3a and b). In this same context, pre-B cell receptor components VpreBl and Igl11/lambda5 were transcriptionally upregulated in B-pDCs (Fig. 2e), analogous to human AXL+ pDC1; however, surface expression of neither protein could be detected by flow cytometry. Likewise, surface expression of BCR components IgM or Cd79a could not be detected by flow cytometry (data not shown). While several key B cell genes are highly expressed in B-pDC, other B-cell defining transcription factors are not (e.g. Pax5, Foxpl, Tcf3., Fig. 2e, S5).
A previously undefined DC subset captured in the conventional CD123+BDCA2+ pDC gate (AXL+ DC) was recently identified in humans by several groups1,2,4. This pDC subset is defined by high expression of AXL, SIGLEC1, SIGLEC6, CD2, CD22 CD81, CD86 and LYZ relative to classical pDC. We found that in mice, all of the homologous genes are also transcribed at elevated levels in the B-pDC compartment relative to classical pDC (Fig. 2e, S5), implying there is a conserved genetic program in common between mouse B-pDC and human AXL+ DC. Phenotypic analysis of total B220+Pdca1+ cells in the mouse confirmed that ~20% of pDCs were AXL+ Siglec1+, Cd86+, Cd19+, and YFP+ (Fig. 3b).
Relative to classical pDC, resting Axl+ B-pDC express higher levels of activation markers30 MHC Class II, Cd83, and Cd86 (Fig 2e, 3a and b) suggesting that they may be primed for immediate response to pro-inflammatory signals. To test this hypothesis, we delivered TLR9 ligand (CpG:ODN) into mbl-Cre-YFP mice via tail vein injection and splenic pDC were phenotyped 24 hours later (Fig 3c and d). While both pDC and B-pDC compartments expanded relative to controls, the YFP+ B-pDC fraction increased ~3-fold above that of the YFP- pDC fraction (~2.7x and ~7x expansion, respectively, Fig. 3d). Additionally B-pDC had markedly enhanced expression of MHC class II, Cd83, and Cd86 levels upon activation, (Fig. 3c and d). These results indicate that, relative to classical pDC, B-pDC are intrinsically activated and primed for rapid expansion upon TLR9 engagement.
Ccr7 is strongly expressed upon activation of pDC by TLR9 engagement following CpG stimulation31, and such upregulation has been shown to be critical for chemotactic migration of activated DC into lymphoid organs for interaction with T cells32-34. B-pDC showed 4-fold higher basal Ccr7 expression compared to classical pDC pDC; Fig. 2e, S5) consistent with their preferential residence in spleen (Fig. 1d). These data further suggest that B-pDC are prepositioned for immediate responses rather than summoned upon infection. Finally, coincident with the tenet that B-pDC may be primed and prepositioned for immediate response to pro-inflammatory signals, we note unexpected and extraordinarily high expression of complement (Clqa, Clqb, and Clqc) and lysozyme (Lyzl, Lyz2) genes in the B-pDC subset (Fig. S7)—rivaling only that of red pulp or peritoneal cavity resident macrophages35. Human AXL+ DC overexpress these genes too1,4.
To confirm the functional phenotype of B-pDC, we tested their ability to secrete cytokines known to be elicited by pDCs after Toll-like receptor (TLR) engagement. Specifically, we tested each pDC lineage for the production of IFN-α or IL-12p40 when activated by TLR9-binding CpG oligonucleotides. We sorted B-pDC and pDC, engaged TLR9 with CpG:ODNs for 24 hours and collected supernatant for cytokine specific ELISAs. IFN-α production was almost negligible in B-pDC (p<0.0001, Fig. 3e) yet IL-12p40 production was significantly augmented over pDC (p<0.0001, Fig. 3f), mirroring the functional phenotype of their human AXL+ phenotypic counterparts2. That classical pDCs originally were identified as professional type I interferon-producing cells (IPC)36 endowed with robust secretory machinery for the abundant secretion of cytokines, we speculate that B-pDC might instead utilize this attribute for copious secretion of alternative immune system modulators which they overexpress, such as C1q and Lyz (Fig. S7). We note that C1q can inhibit pDC production of IFN-α37.
To test their ability to expand T lymphocytes in culture, each were incubated with CpG:ODNs and co-cultured with freshly isolated CFSE-labeled lymphocytes. After 6 days, co-cultures were stained for CD3 and CFSE-negative cell percentages were recorded (Fig. 3g). B-pDCs were significantly better at expanding T cells in co-cultures (p=0.02, Fig. 3g and h), mirroring the superior ability of AXL+ DC to expand lymphocytes in human co-cultures2.
Both pDCs and B cells require BCL11A for their development and exhibit gene expression overlap between the two lineages14. This suggests that pDC and B cells may share transcriptional control mechanisms downstream of BCL11A. We employed ChIP-seq in model human cell lines to compare direct BCL11A chromatin-bound target genes and how these gene sets might differ between a pDC line (CAL1) and model B cell lines: (1) the pre-B cell line NALM6; (2) the germinal center Burkitt’s lymphoma B cell line RAJI; and (3) the lymphoblastoid B cell line, GM1287838. CAL1 expresses classical pDC markers9,39 as well as significantly upregulated AXL expression40 suggesting they are B-pDC-like. CAL1 was derived from a patient with blastic plasmacytoid dendritic cell neoplasm (BPDCN), a rare and deadly malignancy that uniformly expresses high levels of BCL11A and which, although its ontogeny is unclear, is widely believed to originate from pDCs9,41,42. Indeed, single-cell analysis has confirmed that whereas primary BPDCN malignant cells express several key B cell markers (IGLL1, IGLL5, SPIB) they are most closely related to pDCs and AXL+ pDCs1. The occupancy pattern of BCL11A in CAL1 pDC bore a striking resemblance to its binding distribution in the three human B cell types, with nearly a quarter of all CAL1 targets shared among all B cell lines, including, intriguingly, BCL11A occupancy on its own promoter region in each cell line (Fig 4a and b). In contrast, while B cells and pDC share numerous BCL11A targets, others are bound in a cell context-dependent fashion, including B cell only binding in PAX5, TCF3, and ID3 loci; CAL1 only binding in AXL, SIGLEC1, and IGLL1; and binding across cell types but with non-overlapping peaks in SPIB and ID2 (Fig. 4b).
Dendritic cell-subset biology and development, and the ensuing nomenclature, have long been unclear. Here, we provide definitive evidence in support of the long-suspected “lymphoid past” of pDCs by establishing their ability to arise in vivo from CLP progenitors with B/pDC bipotential lineage capacity17-19,43,44. Our data suggest that the murine pDC compartment is bipartite, being comprised of B-pDC—diverted from the CLP post T-B bifurcation—as well as myeloid-derived classical pDC. One possible mechanism by which post-CLP B cell to B-pDC diversion might be achieved is suggested by elevated B-pDC expression of transcription factors Id2 and SpiC (Fig. 2e, S5). Differentiation of the classical pDC is blocked by Id2 repression of Tcf4 activity45 and driven by SpiB46. During B cell development, SpiC, which is highly related to SpiB, acts to both reduce B cell numbers47 and to oppose SpiB-driven transcriptional regulation48. Once formed in the late CLP, mouse B-pDC exhibit additional canonical B cell genes; most notably Cd19, possibly displayed with help from high expression of the membrane tetraspanin Cd81, which is required for trafficking Cd19 to the surface in B cells49.
Recently, several groups have identified in human blood a new AXL+ DC subset with distinct functions1,2,4. Although defined differently (AXL+Siglec1+Siglec6+1 vs. AXL+Cd2hiCd81+2,4), both definitions encompass the mouse B-pDC described here. Our study provides compelling evidence that the murine B-pDC is the homologue of this evolutionarily conserved DC subset. As with its human counterpart3, the B-pDC is found at higher frequency in the spleen than in the BM, indicating the possibility they preferentially home to secondary lymphoid organs. The mouse B-pDC also intrinsically express activation markers at higher levels, divide more rapidly after TLR9 engagement, and expand T cells more rapidly in culture than conventional pDCs, similar to AXL+ DCs2,4. While further functional definition awaits discovery, our work provides a framework for the identification and segregation of the B-pDC lineage (comprising about 1/5 of the total pDC compartment) from its myeloid-derived counterpart(s). In particular, we have elucidated the genetic, phenotypic, and functional conservation in mouse of the human counterpart AXL+ DCs by using a lymphoid conditional knockout of the pDC transcription factor Bcl11a; and we have validated an experimental strategy for enriching Axl+ DCs in wild-type or mbl-driven reporter mice, thereby providing a useful tool for the continued study of this novel pDC lineage.
Our observations support the hypothesis that DC functionality derives primarily from ontogeny rather than from tissue environment50, exemplified by evolution of a specialized pDC lineage from a lymphoid progenitor. To date, the field of pDC biology has yet to identify a clonogenic progenitor restricted to the pDC compartment and perhaps that is because there is no single clonogenic progenitor for the present definition of pDC lineage, and that there are, at a minimum, two such progenitors for the pDC compartment. In the case of the B-pDC, our data suggest such a cell should exist within the late CLP, and may deviate from B cell commitment before or after Ly6d expression, thought to be the earliest marker of B cell specification26. Future experiments will address this question via refined cell sorting and expansion of CLP populations from mbl-driven reporter mice.
Plasmacytoid dendritic cells are thought to be involved in the pathogenesis of a variety of diseases, from autoimmune diseases to multiple treatment-resistant cancer types. For example, chronic activation of human pDC contributes to systemic lupus erythematosis, systemic sclerosis, and IFN-related autoimmune diseases51,52. At the same time pDC have been shown to play an important role in normal immune tolerance as well as induce unwarranted immune tolerance in human cancers51. The demarcation of mouse B-pDCs may help clarify the perceived plasticity of the pDC compartment in normal and disease contexts51, and provides a new cell for targeted study within the context of autoimmune disease, cancer, and infection models.
Online Methods
Mice
Generation of Bcl11a cKO Mice was performed as described14. Mbl-Cre deleter and Rosa26-YFP reporter strains were obtained from Jackson labs (catalog #’s 020505 and 006148, respectively).
Flow cytometry
Analytical cytometry was performed on a FACS Fortessa and sorting was performed on a FACS Aria (BD Biosciences) followed by analysis using FlowJo (Tree Star) software. For sorting prior to RNA collection, BM from 12 mice was prepared from femurs at 6 weeks of age, combined into three groups (4 mice/group), and single-cell suspensions were stained with antigen-specific monoclonal antibodies. Anti-mPDCA1-PE (Miltenyi Biotech), Anti-Cd45R/B220-V605, Anti-Cd19-Alexa Flour 700, Anti-Cd86-APC-Cy7, Anti-I-A/I-E-Pacific Blue, Anti-Cd179a-PE, Anti-IL7Ra-V421, Anti-cKit-PE-Cy7, Anti-Cd11b-PerCP-Cy5.5, Anti-Cd3e-PerCP-Cy5.5, Anti-Gr-1-PerCP-Cy5.5, Anti-Cd45R/ B220-PerCP-Cy5.5 Anti-Cd4-PerCP-Cy5.5, Anti-Cd8-PerCpCy5.5, Anti-NK1.1-PerCp-Cy5.5, Anti-Sca1-BV711, Anti-Cd135-APC, Anti-Cd150-BV605, Anti-Siglec1-PE-Cy7 (Biolegend), Anti-Cd34-Alexa Flour 700, Anti-Cd11c-PerCP-Cy5.5 (eBioscience), Anti-Cd179b-Biotin-V450 Streptavidin, Anti-AA4.1-PE (BD Biosciences), Anti-Axl-APC (R6D Systems) in D-PBS/2% (vol/vol) FBS FACS buffer.
Adoptive transfer
BM from 6-week-old Bcl11aF/F/mbl-Cre + cKO or Mb-l-Cre-YFP reporter mice (n=3 per group) were collected from femurs and transferred via retro-orbital injection into recipient immunocompetent C57/B6 mice lethally irradiated with two doses of 450 rad 1 hour apart. Mice were kept on an antibiotic diet for 3 weeks to allow for immune reconstitution. Eight weeks post transfer, mice were sacrificed and cells collected from BM and spleen to investigate reconstitution of cellular subsets.
RNA isolation and RNA-seq
Total RNA was extracted from FACS sorted Bcl11a cKO BM pDC using TRIzol reagent (Invitrogen). Oligo-dT-primed cDNA was prepared using SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). Taq polymerase (New England Biolabs) and a Perkin-Elmer 2700 thermocycler were used to amplify transcripts for the following mouse genes; Bcl11a (F: 5’-GTGGATAAGCCGCCTTCCCCTT-3’, R: 5’-GGGGACTTCCGTGTTCACTTTC-3’), Pdca1 (F; 5’-AGGCAAACTCCTGCAACCTG-3’, R; 5’-ACCTGCACTGTGCTAGAAGTC-3’), Spi-C (F; 5’-ATCCTCACGTCAGAGGCAAC-3’, R; 5’-TGTACGGATTGGTGGAAGCC-3’, Id2 (F; 5’-GGACATCAGCATCCTGTCCTTGC-3’ R; 5’-GTGTTCTCCTGGTGAAATGGCTGA-3’, and β-actin (F; 5’-CAAGGTGTGATGGTGGGAAT-3’, R; 5’-GGTGTAAAACGCAGCTCAGT-3’ For RNA-seq, twelve mice were pooled into three YFP+ groups, and total RNA was extracted as above. cDNA was prepared using the Ultra Low kit from Clontech (Mountain View, CA). Library preparation employed the Nextera DNA Library Preparation kit from Illumina (San Diego, CA). cDNA was analyzed by deep sequencing using Illumina sequencing technology. Data were analyzed using a high-throughput next-generation sequencing analysis pipeline; FASTQ files were aligned to the mouse genome (mm9, NCBI Build 37) using TopHat253. Gene expression profiles for the individual samples were calculated with Cufflinks54 as RPKM values. YFP+ pDC samples were normalized to each control YFP- pDC sample. GO terms identified as significantly different; GO:0006955, GO:0009611, GO:0048584, GO:0006954, GO:0002684, GO:0001775, GO:0009986. Panther terms: BP00148, BP00155, BP00102, BP00120. Gene Set Enrichment Analysis (GSEA) using ordered gene expression levels of Common Lymphoid Progenitor-derived B-pDC and Common Myeloid Progenitor-derived pDC were significantly enriched in both GO terms and Panther-derived gene sets. A randomly selected control GSEA curated set, GSE7831: UNSTIM_VS_ INFLUENZA_STIM_ PDC_4H_DN (defined as genes down-regulated in untreated pDC versus influenza virus infected pDC 55 showed insignificant enrichment. Normalized enrichment score (NES) and false discovery rate q-values (FDR); FDR ≤ 0.25 is considered significant56.
TLR9 engagement
Mb1-cre-YFP reporter mice were injected via tail veins with 100ul of PBS containing 50ug of CpG:ODN (n=4) or GpC:ODN control (n=4) (CpG-B no.1826, TCCAT GACG TTCCT GACGTT; control non-CpG-B no.2138, TCCATGAGCTTCCTGAGCTT, Invivogen, USA). At 24 hours, mice were sacrificed and bone marrow and spleens were collected for pDC phenotyping.
Cell co-cultures
B-pDC (B220+Pdca1+AXL+Siglec1+) and pDC (B220+Pdca1+Axl’Siglec?) subsets were sorted on a FACS Aria (BD Biosciences) by and cultured in 96-well round bottom plates with RPMI medium 1640 containing 10% (vol/vol) FBS, 2 mM L-glutamine, 100 units/mL penicillin and streptomycin, 1 mM sodium pyruvate, and 10 mM Hepes, and with or without class A CpGs (ODN 1585, InvivoGen) for 24 hours. pDCs were then washed three times to remove residual CpG. Preactivated pDCs (5 × 103) were cultured with CFSE-labeled lymphocytes (2.5 × 104) for 6d with 20 units/mL of IL-2.
Enzyme-linked immunosorbent assays (ELISA)
BM from 6-week-old wildtype C57/BL6 mice was collected from femurs and B-pDC and pDC populations were segregated with a FACS Aria (BD Biosciences). Cells were stimulated with CpG-A (ODN 1585, Invivogen) or CpG-C (ODN 2395, Invivogen) and ELISAs for IFN-α (Invitrogen) or IL-12p40 (BioLegend), respectively, were performed according to manufacturers recommendation.
ChIP followed by ChIP-seq
ChIP assays for BCL11A were performed and analyzed as described14. ChIP-seq data was analyzed by deep sequencing using Illumina sequencing technology as described above.
Data Deposition
RNA-seq; GSE105827; ChIP-seq; GSE99019
Accession numbers to previously published data sets; GSE52868 (pre-B RNA-seq); GSE55043 (CAL1 ChIP-seq); ENCODE BCL11A ChIP-seq in GM1287838.
Author contributions
J.D.D. and G.C.I. designed research; J.D.D., C.R., Z.H., and B-K.L., performed research; J.D.D., J.L. V.R.I, L.I.E., G.G., and G.C.I., contributed new reagents/analytic tools; J.D.D., C.R., B.-K.L., H.O.T. analyzed data; J.D.D., H.O.T., and G.C.I. wrote the paper.
The authors declare no conflict of interest.
Acknowledgements
We thank June V. Harriss for expert assistance in the generation of Bcl11a conditional knockout mice, and Chhaya Das and Maya Ghosh for help in ChIP experiments and cell culture. The CAL1 cell line was kindly provided by Drs. Takahiro Maeda and Boris Reizis. Library preparation and Illumina ChIP- and RNA-seq were performed at the NGS core of the MD Anderson Cancer Center. The Lymphoma Research Foundation Fellowship 300463 (to J.D.D.); NIH grant F32CA110624 and Owens Medical Research Foundation grant (to G.C.I.); NIH grant R01AI104870 (to L.I.R.E.); NIH grant R01CA130075 (to V.R.I.); NIH Grant R01CA31534; Cancer Prevention Research Institute of Texas (CPRIT) Grant RP120348 to the MD Anderson NGS core and CPRIT RP120459 and the Marie Betzner Morrow Centennial Endowment (to H.O.T.) provided support for this work.