Nuclear import of BCL11B is mediated by a classical nuclear localization signal and not the Krüppel-like zinc fingers.

The Krüppel-like transcription factor (KLF) BCL11B is characterized by a wide tissue distribution and crucial functions in key developmental and cellular processes, as well as in various pathologies including cancer and HIV infection. Although the basics of BCL11B activity and relevant interactions with other proteins have been uncovered, how this exclusively nuclear protein localizes to its compartment remained unclear. Here, we demonstrate that unlike other KLFs, BCL11B does not require the C-terminal DNA-binding domain to pass through the nuclear envelope but has an independent, previously unidentified, nuclear localization signal (NLS), which is located distantly from the zinc finger domains and fulfills the essential criteria of being an autonomous NLS. First, it can redirect a heterologous cytoplasmic protein to the nucleus. Second, its mutation causes aberrant localization of the protein of origin. Finally, we provide experimental and in silico evidences of the direct interaction with importin-α. The relative conservation of this motif allows formulating a consensus sequence (K/R)K-X13-14-KR+K++ ('+' indicates amino acids with similar chemical properties), which can be found in all BCL11B orthologs among vertebrates and in the closely related protein BCL11A.

Unveiling the N-Terminal Homodimerization of BCL11B by Hybrid Solvent Replica-Exchange Simulations.

Transcription factors play a crucial role in regulating biological processes such as cell growth, differentiation, organ development and cellular signaling. Within this group, proteins equipped with zinc finger motifs (ZFs) represent the largest family of sequence-specific DNA-binding transcription regulators. Numerous studies have proven the fundamental role of BCL11B for a variety of tissues and organs such as central nervous system, T cells, skin, teeth, and mammary glands. In a previous work we identified a novel atypical zinc finger domain (CCHC-ZF) which serves as a dimerization interface of BCL11B. This domain and formation of the dimer were shown to be critically important for efficient regulation of the BCL11B target genes and could therefore represent a promising target for novel drug therapies. Here, we report the structural basis for BCL11B-BCL11B interaction mediated by the N-terminal ZF domain. By combining structure prediction algorithms, enhanced sampling molecular dynamics and fluorescence resonance energy transfer (FRET) approaches, we identified amino acid residues indispensable for the formation of the single ZF domain and directly involved in forming the dimer interface. These findings not only provide deep insight into how BCL11B acquires its active structure but also represent an important step towards rational design or selection of potential inhibitors.

The N-Terminal CCHC Zinc Finger Motif Mediates Homodimerization of Transcription Factor BCL11B.

The BCL11B gene encodes a Krüppel-like, sequence-specific zinc finger (ZF) transcription factor that acts as either a repressor or an activator, depending on its posttranslational modifications. The importance of BCL11B in numerous biological processes in multiple organs has been well established in mouse knockout models. The phenotype of the first de novo monoallelic germ line missense mutation in the BCL11B gene (encoding N441K) strongly implies that the mutant protein acts in a dominant-negative manner by neutralizing the unaffected protein through the formation of a nonfunctional dimer. Using a Förster resonance energy transfer-assisted fluorescence-activated cell sorting (FACS-FRET) assay and affinity purification followed by mass spectrometry (AP-MS), we show that the N-terminal CCHC zinc finger motif is necessary and sufficient for the formation of the BCL11B dimer. Mutation of the CCHC ZF in BCL11B abolishes its transcription-regulatory activity. In addition, unlike wild-type BCL11B, this mutant is incapable of inducing cell cycle arrest and protecting against DNA damage-driven apoptosis. Our results confirm the BCL11B dimerization hypothesis and prove its importance for BCL11B function. By mapping the relevant regions to the CCHC domain, we describe a previously unidentified mechanism of transcription factor homodimerization.

Identification of multiple complex rearrangements associated with deletions in the 6q23-27 region in Sézary syndrome.

The 6q23-27 region, recurrently deleted in Sézary syndrome (SS), was characterized at the molecular level in 13 SS patients and SS cell line SeAx. Using fine-tiling comparative genomic hybridization, deletions within the 6q23-27 region were detected in half of the samples (six patients and SeAx). All samples with deletions were further analyzed by ligation-mediated PCR. In addition, in one patient sample and in SeAx, paired-end next-generation sequencing was performed on the HiSeq2000 Illumina platform. Using those techniques, 23 rearrangements associated with the deletions were identified. The majority of rearrangements showed enormous complexity and diversity, including eight inversions, three transpositions, and four translocations (with chromosomes 3, 17, 10, and 12). Fifteen genes were disrupted by those rearrangements, the MYB proto-oncogene three times and the interleukin-22 receptor subunit alpha-2 gene (IL22RA2) twice. All three patients with MYB alterations showed low MYB expression, whereas seven of the remaining patients showed overexpression. Most patients overexpressing MYB also presented increased expression of MYC, HSPA8, and BCL2. Five gene fusions were identified, of which two, CCDC28A-IL22RA2 and AIG1-GOSR1, both in SeAx, were in the same orientation and were expressed at the messenger RNA level.

Increased expression of bcl11b leads to chemoresistance accompanied by G1 accumulation.

BACKGROUND: The expression of BCL11B was reported in T-cells, neurons and keratinocytes. Aberrations of BCL11B locus leading to abnormal gene transcription were identified in human hematological disorders and corresponding animal models. Recently, the elevated levels of Bcl11b protein have been described in a subset of squameous cell carcinoma cases. Despite the rapidly accumulating knowledge concerning Bcl11b biology, the contribution of this protein to normal or transformed cell homeostasis remains open. METHODOLOGY/PRINCIPAL FINDINGS: Here, by employing an overexpression strategy we revealed formerly unidentified features of Bcl11b. Two different T-cell lines were forced to express BCL11B at levels similar to those observed in primary T-cell leukemias. This resulted in markedly increased resistance to radiomimetic drugs while no influence on death-receptor apoptotic pathway was observed. Apoptosis resistance triggered by BCL11B overexpression was accompanied by a cell cycle delay caused by accumulation of cells at G1. This cell cycle restriction was associated with upregulation of CDKN1C (p57) and CDKN2C (p18) cyclin dependent kinase inhibitors. Moreover, p27 and p130 proteins accumulated and the SKP2 gene encoding a protein of the ubiquitin-binding complex responsible for their degradation was repressed. Furthermore, the expression of the MYCN oncogene was silenced which resulted in significant depletion of the protein in cells expressing high BCL11B levels. Both cell cycle restriction and resistance to DNA-damage-induced apoptosis coincided and required the histone deacetylase binding N-terminal domain of Bcl11b. The sensitivity to genotoxic stress could be restored by the histone deacetylase inhibitor trichostatine A. CONCLUSIONS: The data presented here suggest a potential role of BCL11B in tumor survival and encourage developing Bcl11b-inhibitory approaches as a potential tool to specifically target chemoresistant tumor cells.