Hematopoietic stem cell quiescence and DNA replication dynamics maintained by the resilient ß-catenin/Hoxa9/Prmt1 axis.

ABSTRACT: Maintenance of quiescence and DNA replication dynamics are 2 paradoxical requirements for the distinct states of dormant and active hematopoietic stem cells (HSCs), which are required to preserve the stem cell reservoir and replenish the blood cell system in response to hematopoietic stress, respectively. Here, we show that key self-renewal factors, ß-catenin or Hoxa9, largely dispensable for HSC integrity, in fact, have dual functions in maintaining quiescence and enabling efficient DNA replication fork dynamics to preserve the functionality of hematopoietic stem and progenitor cells (HSPCs). Although ß-catenin or Hoxa9 single knockout (KO) exhibited mostly normal hematopoiesis, their coinactivation led to severe hematopoietic defects stemmed from aberrant cell cycle, DNA replication, and damage in HSPCs. Mechanistically, ß-catenin and Hoxa9 function in a compensatory manner to sustain key transcriptional programs that converge on the pivotal downstream target and epigenetic modifying enzyme, Prmt1, which protects the quiescent state and ensures an adequate supply of DNA replication and repair factors to maintain robust replication fork dynamics. Inactivation of Prmt1 phenocopied both cellular and molecular phenotypes of ß-catenin/Hoxa9 combined KO, which at the same time could also be partially rescued by Prmt1 expression. The discovery of the highly resilient ß-catenin/Hoxa9/Prmt1 axis in protecting both quiescence and DNA replication dynamics essential for HSCs at different key states provides not only novel mechanistic insights into their intricate regulation but also a potential tractable target for therapeutic intervention.

Transcriptional memory of cells of origin overrides ß-catenin requirement of MLL cancer stem cells.

While ß-catenin has been demonstrated as an essential molecule and therapeutic target for various cancer stem cells (CSCs) including those driven by MLL fusions, here we show that transcriptional memory from cells of origin predicts AML patient survival and allows ß-catenin-independent transformation in MLL-CSCs derived from hematopoietic stem cell (HSC)-enriched LSK population but not myeloid-granulocyte progenitors. Mechanistically, ß-catenin regulates expression of downstream targets of a key transcriptional memory gene, Hoxa9 that is highly enriched in LSK-derived MLL-CSCs and helps sustain leukemic self-renewal. Suppression of Hoxa9 sensitizes LSK-derived MLL-CSCs to ß-catenin inhibition resulting in abolishment of CSC transcriptional program and transformation ability. In addition, further molecular and functional analyses identified Prmt1 as a key common downstream mediator for ß-catenin/Hoxa9 functions in LSK-derived MLL-CSCs. Together, these findings not only uncover an unexpectedly important role of cells of origin transcriptional memory in regulating CSC self-renewal, but also reveal a novel molecular network mediated by ß-catenin/Hoxa9/Prmt1 in governing leukemic self-renewal.

Elucidating combinatorial histone modifications and crosstalks by coupling histone-modifying enzyme with biotin ligase activity.

Histone post-translational modifications (PTMs) often form complex patterns of combinations and cooperate to specify downstream biological processes. In order to systemically analyse combinatorial PTMs and crosstalks among histone PTMs, we have developed a novel nucleosome purification method called Biotinylation-assisted Isolation of CO-modified Nucleosomes (BICON). This technique is based on physical coupling of the enzymatic activity of a histone-modifying enzyme with in vivo biotinylation by the biotin ligase BirA, and using streptavidin to purify the co-modified nucleosomes. Analysing the nucleosomes isolated by BICON allows the identification of PTM combinations that are enriched on the modified nucleosomes and function together within the nucleosome context. We used this new approach to study MSK1-mediated H3 phosphorylation and found that MSK1 not only directly phosphorylated H3, but also induced hyperacetylation of both histone H3 and H4 within the nucleosome. Moreover, we identified a novel crosstalk pathway between H3 phosphorylation and H4 acetylation on K12. Involvement of these acetyl marks in MSK1-mediated transcription was further confirmed by chromatin immunoprecipitation assays, thus validating the biological relevance of the BICON results. These studies serve as proof-of-principle for this new technical approach, and demonstrate that BICON can be further adapted to study PTMs and crosstalks associated with other histone-modifying enzymes.

Histone code pathway involving H3 S28 phosphorylation and K27 acetylation activates transcription and antagonizes polycomb silencing.

Histone H3 phosphorylation is a critical step that couples signal transduction pathways to gene regulation. To specifically assess the transcriptional regulatory functions of H3 phosphorylation, we developed an in vivo targeting approach and found that the H3 kinase MSK1 is a direct and potent transcriptional activator. Targeting of this H3 kinase to the endogenous c-fos promoter is sufficient to activate its expression without the need of upstream signaling. Moreover, targeting MSK1 to the α-globin promoter induces H3 S28 phosphorylation and reactivates expression of this polycomb-silenced gene. Importantly, we discovered a mechanism whereby H3 S28 phosphorylation not only displaces binding of the polycomb-repressive complexes, but it also induces a methyl-acetylation switch of the adjacent K27 residue. Our findings show that signal transduction activation can directly regulate polycomb silencing through a specific histone code-mediated mechanism.