MYB insufficiency disrupts proteostasis in hematopoietic stem cells, leading to age-related neoplasia.

MYB plays a key role in gene regulation throughout the hematopoietic hierarchy and is critical for the maintenance of normal hematopoietic stem cells (HSC). Acquired genetic dysregulation of MYB is involved in the etiology of a number of leukemias, although inherited noncoding variants of the MYB gene are a susceptibility factor for many hematological conditions, including myeloproliferative neoplasms (MPN). The mechanisms that connect variations in MYB levels to disease predisposition, especially concerning age dependency in disease initiation, are completely unknown. Here, we describe a model of Myb insufficiency in mice that leads to MPN, myelodysplasia, and leukemia in later life, mirroring the age profile of equivalent human diseases. We show that this age dependency is intrinsic to HSC, involving a combination of an initial defective cellular state resulting from small effects on the expression of multiple genes and a progressive accumulation of further subtle changes. Similar to previous studies showing the importance of proteostasis in HSC maintenance, we observed altered proteasomal activity and elevated proliferation indicators, followed by elevated ribosome activity in young Myb-insufficient mice. We propose that these alterations combine to cause an imbalance in proteostasis, potentially creating a cellular milieu favoring disease initiation.

Chromatin occupancy and target genes of the haematopoietic master transcription factor MYB.

The transcription factor MYB is a master regulator in haematopoietic progenitor cells and a pioneer factor affecting differentiation and proliferation of these cells. Leukaemic transformation may be promoted by high MYB levels. Despite much accumulated molecular knowledge of MYB, we still lack a comprehensive understanding of its target genes and its chromatin action. In the present work, we performed a ChIP-seq analysis of MYB in K562 cells accompanied by detailed bioinformatics analyses. We found that MYB occupies both promoters and enhancers. Five clusters (C1-C5) were found when we classified MYB peaks according to epigenetic profiles. C1 was enriched for promoters and C2 dominated by enhancers. C2-linked genes were connected to hematopoietic specific functions and had GATA factor motifs as second in frequency. C1 had in addition to MYB-motifs a significant frequency of ETS-related motifs. Combining ChIP-seq data with RNA-seq data allowed us to identify direct MYB target genes. We also compared ChIP-seq data with digital genomic footprinting. MYB is occupying nearly a third of the super-enhancers in K562. Finally, we concluded that MYB cooperates with a subset of the other highly expressed TFs in this cell line, as expected for a master regulator.

A c-Myb mutant causes deregulated differentiation due to impaired histone binding and abrogated pioneer factor function.

The transcription factor c-Myb is involved in early differentiation and proliferation of haematopoietic cells, where it operates as a regulator of self-renewal and multi-lineage differentiation. Deregulated c-Myb plays critical roles in leukaemias and other human cancers. Due to its role as a master regulator, we hypothesized it might function as a pioneer transcription factor. Our approach to test this was to analyse a mutant of c-Myb, D152V, previously reported to cause haematopoietic defects in mice by an unknown mechanism. Our transcriptome data from K562 cells indicates that this mutation specifically affects c-Myb's ability to regulate genes involved in differentiation, causing failure in c-Myb's ability to block differentiation. Furthermore, we see a major effect of this mutation in assays where chromatin opening is involved. We show that each repeat in the minimal DNA-binding domain of c-Myb binds to histones and that D152V disrupts histone binding of the third repeat. ATAC-seq data indicates this mutation impairs the ability of c-Myb to cause chromatin opening at specific sites. Taken together, our findings support that c-Myb acts as a pioneer factor and show that D152V impairs this function. The D152V mutant is the first mutant of a transcription factor specifically destroying pioneer factor function.