BCL11B Is a Key Regulator of T-Lineage Differentiation during the Initial Stages of Human Thymopoiesis

Introduction

The initial stages of thymopoiesis are characterized by a progressive commitment to the T-cell lineage, which involves the loss of alternative (myelo-erythroid, NK, B) lineage potentials. Dysregulated differentiation during these stages can result in T-cell acute lymphoblastic leukemia (T-ALL). While the regulation of murine T-lineage commitment has been investigated through studies of the early stages of thymopoiesis, very few studies of early human thymopoiesis exist. Hence, our understanding of the mechanisms underlying the initial stages of human thymopoiesis is incomplete. The transcription factor BCL11B is recurrently mutated in T-ALL. BCL11B is repressed in Early T-cell precursor ALL (ETP-ALL), a T-ALL subtype with a differentiation arrest at an early stage of thymopoiesis. However, the function of BCL11B in human thymopoiesis has not been defined. Furthermore, the DNA binding targets of BCL11B during thymopoiesis, and thereby its role in the regulation of T-lineage transcription networks, are largely undefined. We investigated the role of BCL11B in the initial stages of human thymopoiesis.

Methods

BCL11B expression in human bone marrow (BM) progenitors and thymic populations was analyzed by RNA-Seq and qPCR. CD34+ cord blood (CB) progenitors transduced with BCL11B knockdown (KD) or scrambled control shRNA lentivirus were co-cultured with OP9DLL1 stroma to assess the effect of BCL11B loss of function on the initial stages of thymopoiesis. The earliest stages of thymopoiesis are recapitulated in the OP9DLL1 system through the sequential appearance of multilineage CD7+CD1a- and T-lineage restricted CD7+CD1a+ T-cell precursors. The effect of KD on alternative lineage potentials was assessed by isolating CD7+CD1a- and CD7+CD1a+ T-cell precursors from KD and control OP9DLL1 co-cultures and then replating them in NK, B, myeloid and/or erythroid assays. T-ALL cells transduced with a BCL11B overexpression or control lentivirus were analyzed by RNA-Seq to determine the effect of BCL11B gain of function on T-cell differentiation at a transcriptional level. ChIP-Seq and RNA-Seq of CD34+ and CD34- cells from the human thymus were performed to define differentiation stage specific BCL11B DNA binding targets and their expression.

Results

BCL11B was not expressed in BM cells and first became detectable in CD34+ thymic progenitors. A progressive increase in expression was seen when comparing thymic populations at successive stages of T-lineage differentiation. Similar percentages of total CD7+ cells (sum of CD7+CD1a- and CD7+CD1a+ cells) were seen in OP9DLL1 co-cultures initiated with BCL11B KD and control CD34+ CB progenitors. However, KD CD34+ cells showed reduced differentiation into CD7+CD1a+ T-cell precursors (mean %CD7+CD1a+ cells seen in control co-cultures =30.88%, vs 17.85% in KD co-cultures, p<0.05, n=7 independent experiments, 4 shRNA sequences), indicating a differentiation arrest at the CD7+CD1a- stage. KD T-cell precursors showed increased myeloid and NK potentials (p<0.05, n=2 independent experiments). BCL11B overexpression rapidly induced a T-lineage transcriptional program in T-ALL cells (n=3 experiments), including the upregulation of several critical T-lineage genes. ChIP-Seq revealed distinct BCL11B DNA binding profiles in CD34+ (5032 binding sites) and CD34- (12849 binding sites) human thymic cells with an overall gain in binding with differentiation. Most sites were in gene promoter regions. Combined genome wide analysis of gene expression and BCL11B DNA binding targets during thymopoiesis revealed previously unknown differentiation stage-specific binding at multiple sites, which were enriched for critical T-lineage differentiation genes, and binding was associated with upregulation of these target genes (n=2 biological replicates).

Conclusions

BCL11B is essential for T-lineage differentiation and commitment through the repression of NK and myeloid potentials, during the initial stages of human thymopoiesis. BCL11B binds to multiple T-lineage genes in a stage specific manner and induces T-lineage genes, a BCL11B transcriptional program that has not been previously described in murine or human thymopoiesis. Our data strongly support the notion that BCL11B repression at least partly contributes to the aberrant differentiation in human ETP-ALL and provide a framework for understanding human thymopoiesis and T-ALL.