Zbtb11, an Evolutionarily Conserved Pu.1-Regulated Transcriptional Repressor of TP53, Is Required for Neutrophil Development

In response to infection and injury, the hematopoietic system must orchestrate rapid expansion of the neutrophil population. Maintaining this high number of short-lived cells and rapidly increasing their supply on demand requires tightly coordinated but flexible regulation. Neutrophils derive from a common myeloid progenitor that can also give rise to macrophages. Although transcriptional mechanisms have been identified that influence the balance of neutrophil or macrophage production from this common progenitor, the exact pathways governing the relative preponderance of neutrophils and macrophages in the output remain unknown. Here we identify the transcriptional repressor Zbtb11, describe its requirement for neutrophil but not macrophage development, and establish Zbtb11-dependent regulation of TP53 as a new functional pathway instructing neutrophil lineage development.

We identified the requirement for Zbtb11 in myeloid development by forward genetics and characterizing a resultant temperature-sensitive Zbtb11-deficient mutant, marsanne (mne). Zbtb11 is a little-studied transcriptional repressor of the BTBZF transcription factor family (which includes PLZF and BCL6). Zbtb11 shows enriched expression in myeloid precursors. Its deficiency leads to a pleiotropic phenotype including abnormally few neutrophils.

Demand-driven granulopoiesis is obtunded in mne whether stimulated by Csf3a (G-CSF) or microorganisms. Normalizing basal granulopoiesis in mne by temperature shift did not restore emergency granulopoietic capacity. Hence, there is also a strong genetic requirement for Zbtb11 in emergency granulopoiesis.

The genetic requirement in myelopoiesis for Zbtb11 is neutrophil lineage specific, since macrophage numbers are normal in mne and repopulation of macrophages following ablation occurs regardless of Zbtb11 sufficiency/deficiency.

In luciferase assays, the Zbtb11 promoter is regulated by master myeloid transcription factors, including Pu.1, C/ebp-a, and Gfi1, positioning Zbtb11 directly within myeloid transcriptional networks. Microarray analysis of mne embryos revealed enrichment of pro-apoptotic genes including tp53 and bbc3 (Puma), known for its importance in hematopoietic cells. Zbtb11 directly represses the TP53 promoter in a luciferase assay and targets the TP53 locus by chromatin immunoprecipitation in K562 cells, suggesting the high tp53 levels in mne are at least, in part, a functional consequence of tp53 derepression by loss of Zbtb11 repressor function.

The mne mutation is a T>A transversion causing a Cys116>Ser substitution in the N-terminal domain of Zbtb11. The region surrounding this mutation has been regarded as devoid of any known protein structural motif. However, cross-species homology analysis identified a conserved C2H2 configuration shared with a viral integrase that includes the Cys116 in the N-terminal domain that is mutated in mne. In structure/function studies using phenotypic rescue of mne as an in vivo functional bioassay, mRNAs with mutations in each His/Cys residue showed compromised Zbtb11 function. Ability to rescue was further compromised by mutation of all four conserved residues but not mutation of non-conserved Gln98. Furthermore, the C116S mutation disables Zbtb11 regulation of the TP53 promoter. We propose that the C116S mutation compromises Zbtb11 function by disrupting a novel zinc finger structure identified by modeling the N-terminal of the protein, in which Cys116 directly contacts the central metal cation.

Ongoing studies are exploring this pathway further. ChIPseq in human K562 cells is determining genome wide downstream targets of ZBTB11 in hematopoietic cells. RNAseq transcriptional profiling of Zbtb11-deficient compared to WT neutrophils is further delineating precisely where and how the block in mne granulopoiesis occurs and identifying the required Zbtb11-integrated genetic pathways. These studies are supported by ongoing analysis and characterization of a Zbtb11 conditional knockout mouse.

These data provide genetic, biochemical and functional evidence identifying a Pu.1-Zbtb11-tp53 pathway as a new mechanism regulating the output of myelopoiesis. Collectively, the in vivo zebrafish genetic and phenotypic data and the concordant mammalian and zebrafish biochemical data underscore the evolutionary conservation of Zbtb11 function in this pathway.