A pioneer factor locally opens compacted chromatin to enable targeted ATP-dependent nucleosome remodeling.

To determine how different pioneer transcription factors form a targeted, accessible nucleosome within compacted chromatin and collaborate with an ATP-dependent chromatin remodeler, we generated nucleosome arrays in vitro with a central nucleosome containing binding sites for the hematopoietic E-Twenty Six (ETS) factor PU.1 and Basic Leucine Zipper (bZIP) factors C/EBPα and C/EBPß. Our long-read sequencing reveals that each factor can expose a targeted nucleosome on linker histone-compacted arrays, but with different nuclease sensitivity patterns. The DNA binding domain of PU.1 binds mononucleosomes, but requires an additional intrinsically disordered domain to bind and open compacted chromatin. The canonical mammalian SWI/SNF (cBAF) remodeler was unable to act upon two forms of locally open chromatin unless cBAF was enabled by a separate transactivation domain of PU.1. cBAF potentiates the PU.1 DNA binding domain to weakly open chromatin in the absence of the PU.1 disordered domain. Our findings reveal a hierarchy by which chromatin is opened and show that pioneer factors can provide specificity for action by nucleosome remodelers.

KAT6A and ENL Form an Epigenetic Transcriptional Control Module to Drive Critical Leukemogenic Gene-Expression Programs.

Epigenetic programs are dysregulated in acute myeloid leukemia (AML) and help enforce an oncogenic state of differentiation arrest. To identify key epigenetic regulators of AML cell fate, we performed a differentiation-focused CRISPR screen in AML cells. This screen identified the histone acetyltransferase KAT6A as a novel regulator of myeloid differentiation that drives critical leukemogenic gene-expression programs. We show that KAT6A is the initiator of a newly described transcriptional control module in which KAT6A-catalyzed promoter H3K9ac is bound by the acetyl-lysine reader ENL, which in turn cooperates with a network of chromatin factors to induce transcriptional elongation. Inhibition of KAT6A has strong anti-AML phenotypes in vitro and in vivo, suggesting that KAT6A small-molecule inhibitors could be of high therapeutic interest for mono-therapy or combinatorial differentiation-based treatment of AML. SIGNIFICANCE: AML is a poor-prognosis disease characterized by differentiation blockade. Through a cell-fate CRISPR screen, we identified KAT6A as a novel regulator of AML cell differentiation. Mechanistically, KAT6A cooperates with ENL in a "writer-reader" epigenetic transcriptional control module. These results uncover a new epigenetic dependency and therapeutic opportunity in AML. This article is highlighted in the In This Issue feature, p. 587.

Post-Translational Modifications in Polypyrimidine Tract Binding Proteins PTBP1 and PTBP2.

RNA binding proteins play an important role in regulating alternative pre-mRNA splicing and in turn cellular gene expression. Many of these RNA binding proteins occur as gene families with members sharing a high degree of primary structure identity and domain organization yet have tissue-specific expression patterns and regulate different sets of target exons. How highly similar members in a gene family can exert different splicing outcomes is not well understood. We conducted mass spectrometry analysis of post-translational phosphorylation and acetylation modifications for two paralogs of the polypyrimidine tract binding protein family, PTBP1 and PTBP2, to discover modifications that occur in splicing reaction mixtures and to identify discrete modifications that may direct their different splicing activities. We find that PTBP1 and PTBP2 have many distinct phosphate modifications located in the unstructured N-terminal, linker 1, and linker 2 regions. We find that the two proteins have many overlapping acetate modifications in the RNA recognition motifs (RRMs) with a few distinct sites in PTBP1 RRM2 and RRM3. Our data also reveal that lysine residues in the nuclear localization sequence of PTBP2 are acetylated. Collectively, our results highlight important differences in post-translational modifications between the paralogs and suggest a role for them in the differential splicing activity of PTBP1 and PTBP2.