Inhibitors of histone acetyltransferases KAT6A/B induce senescence and arrest tumour growth.

Acetylation of histones by lysine acetyltransferases (KATs) is essential for chromatin organization and function1. Among the genes coding for the MYST family of KATs (KAT5-KAT8) are the oncogenes KAT6A (also known as MOZ) and KAT6B (also known as MORF and QKF)2,3. KAT6A has essential roles in normal haematopoietic stem cells4-6 and is the target of recurrent chromosomal translocations, causing acute myeloid leukaemia7,8. Similarly, chromosomal translocations in KAT6B have been identified in diverse cancers8. KAT6A suppresses cellular senescence through the regulation of suppressors of the CDKN2A locus9,10, a function that requires its KAT activity10. Loss of one allele of KAT6A extends the median survival of mice with MYC-induced lymphoma from 105 to 413 days11. These findings suggest that inhibition of KAT6A and KAT6B may provide a therapeutic benefit in cancer. Here we present highly potent, selective inhibitors of KAT6A and KAT6B, denoted WM-8014 and WM-1119. Biochemical and structural studies demonstrate that these compounds are reversible competitors of acetyl coenzyme A and inhibit MYST-catalysed histone acetylation. WM-8014 and WM-1119 induce cell cycle exit and cellular senescence without causing DNA damage. Senescence is INK4A/ARF-dependent and is accompanied by changes in gene expression that are typical of loss of KAT6A function. WM-8014 potentiates oncogene-induced senescence in vitro and in a zebrafish model of hepatocellular carcinoma. WM-1119, which has increased bioavailability, arrests the progression of lymphoma in mice. We anticipate that this class of inhibitors will help to accelerate the development of therapeutics that target gene transcription regulated by histone acetylation.

MOZ directs the distal-less homeobox gene expression program during craniofacial development.

Oral clefts are common birth defects. Individuals with oral clefts who have identical genetic mutations regularly present with variable penetrance and severity. Epigenetic or chromatin-mediated mechanisms are commonly invoked to explain variable penetrance. However, specific examples of these are rare. Two functional copies of the MOZ (KAT6A, MYST3) gene, encoding a MYST family lysine acetyltransferase chromatin regulator, are essential for human craniofacial development, but the molecular role of MOZ in this context is unclear. Using genetic interaction and genomic studies, we have investigated the effects of loss of MOZ on the gene expression program during mouse development. Among the more than 500 genes differentially expressed after loss of MOZ, 19 genes had previously been associated with cleft palates. These included four distal-less homeobox (DLX) transcription factor-encoding genes, Dlx1, Dlx2, Dlx3 and Dlx5 and DLX target genes (including Barx1, Gbx2, Osr2 and Sim2). MOZ occupied the Dlx5 locus and was required for normal levels of histone H3 lysine 9 acetylation. MOZ affected Dlx gene expression cell-autonomously within neural crest cells. Our study identifies a specific program by which the chromatin modifier MOZ regulates craniofacial development.

PHF6 regulates hematopoietic stem and progenitor cells and its loss synergizes with expression of TLX3 to cause leukemia.

Somatically acquired mutations in PHF6 (plant homeodomain finger 6) frequently occur in hematopoietic malignancies and often coincide with ectopic expression of TLX3. However, there is no functional evidence to demonstrate whether these mutations contribute to tumorigenesis. Similarly, the role of PHF6 in hematopoiesis is unknown. We report here that Phf6 deletion in mice resulted in a reduced number of hematopoietic stem cells (HSCs), an increased number of hematopoietic progenitor cells, and an increased proportion of cycling stem and progenitor cells. Loss of PHF6 caused increased and sustained hematopoietic reconstitution in serial transplantation experiments. Interferon-stimulated gene expression was upregulated in the absence of PHF6 in hematopoietic stem and progenitor cells. The numbers of hematopoietic progenitor cells and cycling hematopoietic stem and progenitor cells were restored to normal by combined loss of PHF6 and the interferon α and ß receptor subunit 1. Ectopic expression of TLX3 alone caused partially penetrant leukemia. TLX3 expression and loss of PHF6 combined caused fully penetrant early-onset leukemia. Our data suggest that PHF6 is a hematopoietic tumor suppressor and is important for fine-tuning hematopoietic stem and progenitor cell homeostasis.

Stem cell plasticity, acetylation of H3K14, and de novo gene activation rely on KAT7.

In the conventional model of transcriptional activation, transcription factors bind to response elements and recruit co-factors, including histone acetyltransferases. Contrary to this model, we show that the histone acetyltransferase KAT7 (HBO1/MYST2) is required genome wide for histone H3 lysine 14 acetylation (H3K14ac). Examining neural stem cells, we find that KAT7 and H3K14ac are present not only at transcribed genes but also at inactive genes, intergenic regions, and in heterochromatin. KAT7 and H3K14ac were not required for the continued transcription of genes that were actively transcribed at the time of loss of KAT7 but indispensable for the activation of repressed genes. The absence of KAT7 abrogates neural stem cell plasticity, diverse differentiation pathways, and cerebral cortex development. Re-expression of KAT7 restored stem cell developmental potential. Overexpression of KAT7 enhanced neuron and oligodendrocyte differentiation. Our data suggest that KAT7 prepares chromatin for transcriptional activation and is a prerequisite for gene activation.

Loss of TAF8 causes TFIID dysfunction and p53-mediated apoptotic neuronal cell death.

Mutations in genes encoding general transcription factors cause neurological disorders. Despite clinical prominence, the consequences of defects in the basal transcription machinery during brain development are unclear. We found that loss of the TATA-box binding protein-associated factor TAF8, a component of the general transcription factor TFIID, in the developing central nervous system affected the expression of many, but notably not all genes. Taf8 deletion caused apoptosis, unexpectedly restricted to forebrain regions. Nuclear levels of the transcription factor p53 were elevated in the absence of TAF8, as were the mRNAs of the pro-apoptotic p53 target genes Noxa, Puma and Bax. The cell death in Taf8 forebrain regions was completely rescued by additional loss of p53, but Taf8 and p53 brains failed to initiate a neuronal expression program. Taf8 deletion caused aberrant transcription of promoter regions and splicing anomalies. We propose that TAF8 supports the directionality of transcription and co-transcriptional splicing, and that failure of these processes causes p53-induced apoptosis of neuronal cells in the developing mouse embryo.

HBO1 (KAT7) Does Not Have an Essential Role in Cell Proliferation, DNA Replication, or Histone 4 Acetylation in Human Cells.

HBO1 (MYST2/KAT7) is essential for histone 3 lysine 14 acetylation (H3K14ac) but is dispensable for H4 acetylation and DNA replication in mouse tissues. In contrast, previous studies using small interfering RNA (siRNA) knockdown in human cell lines have suggested that HBO1 is essential for DNA replication. To determine if HBO1 has distinctly different roles in immortalized human cell lines and normal mouse cells, we performed siRNA knockdown of HBO1. In addition, we used CRISPR/Cas9 to generate 293T, MCF7, and HeLa cell lines lacking HBO1. Using both techniques, we show that HBO1 is essential for all H3K14ac in human cells and is unlikely to have a direct effect on H4 acetylation and only has minor effects on cell proliferation. Surprisingly, the loss of HBO1 and H3K14ac in HeLa cells led to the secondary loss of almost all H4 acetylation after 4 weeks. Thus, HBO1 is dispensable for DNA replication and cell proliferation in immortalized human cells. However, while cell proliferation proceeded without HBO1 and H3K14ac, HBO1 gene deletion led to profound changes in cell adhesion, particularly in 293T cells. Consistent with this phenotype, the loss of HBO1 in both 293T and HeLa principally affected genes mediating cell adhesion, with comparatively minor effects on other cellular processes.

Loss of p53 Causes Stochastic Aberrant X-Chromosome Inactivation and Female-Specific Neural Tube Defects.

Neural tube defects (NTDs) are common birth defects in humans and show an unexplained female bias. Female mice lacking the tumor suppressor p53 display NTDs with incomplete penetrance. We found that the combined loss of pro-apoptotic BIM and p53 caused 100% penetrant, female-exclusive NTDs, which allowed us to investigate the female-specific functions of p53. We report that female p53-/- embryonic neural tube samples show fewer cells with inactive X chromosome markers Xist and H3K27me3 and a concomitant increase in biallelic expression of the X-linked genes, Huwe1 and Usp9x. Decreased Xist and increased X-linked gene expression was confirmed by RNA sequencing. Moreover, we found that p53 directly bound response elements in the X chromosome inactivation center (XIC). Together, these findings suggest p53 directly activates XIC genes, without which there is stochastic failure in X chromosome inactivation, and that X chromosome inactivation failure may underlie the female bias in neural tube closure defects.