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.

Homozygous TAF8 mutation in a patient with intellectual disability results in undetectable TAF8 protein, but preserved RNA polymerase II transcription.

The human general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). In eukaryotic cells, TFIID is thought to nucleate RNA polymerase II (Pol II) preinitiation complex formation on all protein coding gene promoters and thus, be crucial for Pol II transcription. In a child with intellectual disability, mild microcephaly, corpus callosum agenesis and poor growth, we identified a homozygous splice-site mutation in TAF8 (NM_138572.2: c.781-1G > A). Our data indicate that the patient's mutation generates a frame shift and an unstable TAF8 mutant protein with an unrelated C-terminus. The mutant TAF8 protein could not be detected in extracts from the patient's fibroblasts, indicating a loss of TAF8 function and that the mutation is most likely causative. Moreover, our immunoprecipitation and proteomic analyses show that in patient cells only partial TAF complexes exist and that the formation of the canonical TFIID is impaired. In contrast, loss of TAF8 in mouse embryonic stem cells and blastocysts leads to cell death and to a global decrease in Pol II transcription. Astonishingly however, in human TAF8 patient cells, we could not detect any cellular phenotype, significant changes in genome-wide Pol II occupancy and pre-mRNA transcription. Thus, the disorganization of the essential holo-TFIID complex did not affect global Pol II transcription in the patient's fibroblasts. Our observations further suggest that partial TAF complexes, and/or an altered TFIID containing a mutated TAF8, could support human development and thus, the absence of holo-TFIID is less deleterious for transcription than originally predicted.

Querkopf is a key marker of self-renewal and multipotency of adult neural stem cells.

Adult neural stem cells (NSCs) reside in the subventricular zone (SVZ) and produce neurons throughout life. Although their regenerative potential has kindled much interest, few factors regulating NSCs in vivo are known. Among these is the histone acetyltransferase querkopf (QKF, also known as MYST4, MORF, KAT6B), which is strongly expressed in a small subset of cells in the neurogenic subventricular zone. However, the relationship between Qkf gene expression and the hierarchical levels within the neurogenic lineage is currently unknown. We show here that the 10% of SVZ cells with the highest Qkf expression possess the defining NSC characteristics of multipotency and self-renewal and express markers previously shown to enrich for NSCs. A fraction of cells expressing Qkf at medium to high levels is enriched for multipotent progenitor cells with limited self-renewal, followed by a population containing migrating neuroblasts. Cells low in Qkf promoter activity are predominantly ependymal cells. In addition, we show that mice deficient for Bmi1, a central regulator of NSC self-renewal, show an age-dependent decrease in the strongest Qkf-expressing cell population in the SVZ. Our results show a strong relationship between Qkf promoter activity and stem cell characteristics, and a progressive decrease in Qkf gene activity as lineage commitment and differentiation proceed in vivo.

Chromatin-Binding Protein PHF6 Regulates Activity-Dependent Transcriptional Networks to Promote Hunger Response.

Understanding the mechanisms of activity-dependent gene transcription underlying adaptive behaviors is challenging at neuronal-subtype resolution. Using cell-type specific molecular analysis in agouti-related peptide (AgRP) neurons, we reveal that the profound hunger-induced transcriptional changes greatly depend on plant homeodomain finger protein 6 (PHF6), a transcriptional repressor enriched in AgRP neurons. Loss of PHF6 in the satiated mice results in a hunger-state-shifting transcriptional profile, while hunger fails to further induce a rapid and robust activity-dependent gene transcription in PHF6-deficient AgRP neurons. We reveal that PHF6 binds to the promoters of a subset of immediate-early genes (IEGs) and that this chromatin binding is dynamically regulated by hunger state. Depletion of PHF6 decreases hunger-driven feeding motivation and makes the mice resistant to body weight gain under repetitive fasting-refeeding conditions. Our work identifies a neuronal subtype-specific transcriptional repressor that modulates transcriptional profiles in different nutritional states and enables adaptive eating behavior.

The transcriptional coactivator Querkopf controls adult neurogenesis.

The adult mammalian brain maintains populations of neural stem cells within discrete proliferative zones. Understanding of the molecular mechanisms regulating adult neural stem cell function is limited. Here, we show that MYST family histone acetyltransferase Querkopf (Qkf, Myst4, Morf)-deficient mice have cumulative defects in adult neurogenesis in vivo, resulting in declining numbers of olfactory bulb interneurons, a population of neurons produced in large numbers during adulthood. Qkf-deficient mice have fewer neural stem cells and fewer migrating neuroblasts in the rostral migratory stream. Qkf gene expression is strong in the neurogenic subventricular zone. A population enriched in multipotent cells can be isolated from this region on the basis of Qkf gene expression. Neural stem cells/progenitor cells isolated from Qkf mutant mice exhibited a reduced self-renewal capacity and a reduced ability to produce differentiated neurons. Together, our data show that Qkf is essential for normal adult neurogenesis.

Chromatin immunoprecipitation of mouse embryos.

During prenatal development, a large number of different cell types are formed, the vast majority of which contain identical genetic material. The basis of the great variety in cell phenotype and function is the differential expression of the approximately 25,000 genes in the mammalian genome. Transcriptional activity is regulated at many levels by proteins, including members of the basal transcriptional apparatus, DNA-binding transcription factors, and chromatin-binding proteins. Importantly, chromatin structure dictates the availability of a specific genomic locus for transcriptional activation as well as the efficiency, with which transcription can occur. Chromatin immunoprecipitation (ChIP) is a method to assess if chromatin modifications or proteins are present at a specific locus. ChIP involves the cross linking of DNA and associated proteins and immunoprecipitation using specific antibodies to DNA-associated proteins followed by examination of the co-precipitated DNA sequences or proteins. In the last few years, ChIP has become an essential technique for scientists studying transcriptional regulation and chromatin structure. Using ChIP on mouse embryos, we can document the presence or absence of specific proteins and chromatin modifications at genomic loci in vivo during mammalian development. Here, we describe a ChIP technique adapted for mouse embryos.

MOZ regulates the Tbx1 locus, and Moz mutation partially phenocopies DiGeorge syndrome.

DiGeorge syndrome, caused by a 22q11 microdeletion or mutation of the TBX1 gene, varies in severity greatly, even among monozygotic twins. Epigenetic phenomena have been invoked to explain phenotypic differences in individuals of identical genetic composition, although specific chromatin modifications relevant to DiGeorge syndrome are elusive. Here we show that lack of the histone acetyltransferase MOZ (MYST3/KAT6A) phenocopies DiGeorge syndrome, and the MOZ complex occupies the Tbx1 locus, promoting its expression and histone 3 lysine 9 acetylation. Importantly, DiGeorge syndrome-like anomalies are present in mice with homozygous mutation of Moz and in heterozygous Moz mutants when combined with Tbx1 haploinsufficiency or oversupply of retinoic acid. Conversely, a Tbx1 transgene rescues the heart phenotype in Moz mutants. Our data reveal a molecular mechanism for a specific chromatin modification of the Tbx1 locus intersecting with an environmental determinant, modeling variability in DiGeorge syndrome.

HBO1 is required for H3K14 acetylation and normal transcriptional activity during embryonic development.

We report here that the MYST histone acetyltransferase HBO1 (histone acetyltransferase bound to ORC; MYST2/KAT7) is essential for postgastrulation mammalian development. Lack of HBO1 led to a more than 90% reduction of histone 3 lysine 14 (H3K14) acetylation, whereas no reduction of acetylation was detected at other histone residues. The decrease in H3K14 acetylation was accompanied by a decrease in expression of the majority of genes studied. However, some genes, in particular genes regulating embryonic patterning, were more severely affected than "housekeeping" genes. Development of HBO1-deficient embryos was arrested at the 10-somite stage. Blood vessels, mesenchyme, and somites were disorganized. In contrast to previous studies that reported cell cycle arrest in HBO1-depleted cultured cells, no defects in DNA replication or cell proliferation were seen in Hbo1 mutant embryo primary fibroblasts or immortalized fibroblasts. Rather, a high rate of cell death and DNA fragmentation was observed in Hbo1 mutant embryos, resulting initially in the degeneration of mesenchymal tissues and ultimately in embryonic lethality. In conclusion, the primary role of HBO1 in development is that of a transcriptional activator, which is indispensable for H3K14 acetylation and for the normal expression of essential genes regulating embryonic development.

Moz and retinoic acid coordinately regulate H3K9 acetylation, Hox gene expression, and segment identity.

We report that embryos deficient in the histone acetyltransferase Moz (Myst3/Kat6a) show histone H3 lysine 9 (H3K9) hypoacetylation, corresponding H3K9 hypermethylation, and reduced transcription at Hox gene loci. Consistent with an observed caudal shift in Hox gene expression, segment identity is shifted anteriorly, such that Moz-deficient mice show a profound homeotic transformation of the axial skeleton and the nervous system. Intriguingly, histone acetylation defects are relatively specific to H3K9 at Hox loci, as neither Hox H3K14 acetylation nor bulk H3K9 acetylation levels throughout the genome are strongly affected; H4K16 acetylation actually increases in the absence of Moz. H3K9 hypoacetylation, Hox gene repression, and the homeotic transformation caused by lack of Moz are all reversed by treatment with retinoic acid (RA). In conclusion, our data show that Moz regulates H3K9 acetylation at Hox gene loci and that RA can act independently of Moz to establish specific Hox gene expression boundaries.

Mof (MYST1 or KAT8) is essential for progression of embryonic development past the blastocyst stage and required for normal chromatin architecture.

Acetylation of histone tails is a hallmark of transcriptionally active chromatin. Mof (males absent on the first; also called MYST1 or KAT8) is a member of the MYST family of histone acetyltransferases and was originally discovered as an essential component of the X chromosome dosage compensation system in Drosophila. In order to examine the role of Mof in mammals in vivo, we generated mice carrying a null mutation of the Mof gene. All Mof-deficient embryos fail to develop beyond the expanded blastocyst stage and die at implantation in vivo. Mof-deficient cell lines cannot be derived from Mof(-/-) embryos in vitro. Mof(-/-) embryos fail to acetylate histone 4 lysine 16 (H4K16) but have normal acetylation of other N-terminal histone lysine residues. Mof(-/-) cell nuclei exhibit abnormal chromatin aggregation preceding activation of caspase 3 and DNA fragmentation. We conclude that Mof is functionally nonredundant with the closely related MYST histone acetyltransferase Tip60. Our results show that Mof performs a different role in mammals from that in flies at the organism level, although the molecular function is conserved. We demonstrate that Mof is required specifically for the maintenance of H4K16 acetylation and normal chromatin architecture of all cells of early male and female embryos.

C3G regulates cortical neuron migration, preplate splitting and radial glial cell attachment.

Neuronal migration is integral to the development of the cerebral cortex and higher brain function. Cortical neuron migration defects lead to mental disorders such as lissencephaly and epilepsy. Interaction of neurons with their extracellular environment regulates cortical neuron migration through cell surface receptors. However, it is unclear how the signals from extracellular matrix proteins are transduced intracellularly. We report here that mouse embryos lacking the Ras family guanine nucleotide exchange factor, C3G (Rapgef1, Grf2), exhibit a cortical neuron migration defect resulting in a failure to split the preplate into marginal zone and subplate and a failure to form a cortical plate. C3G-deficient cortical neurons fail to migrate. Instead, they arrest in a multipolar state and accumulate below the preplate. The basement membrane is disrupted and radial glial processes are disorganised and lack attachment in C3G-deficient brains. C3G is activated in response to reelin in cortical neurons, which, in turn, leads to activation of the small GTPase Rap1. In C3G-deficient cells, Rap1 GTP loading in response to reelin stimulation is reduced. In conclusion, the Ras family regulator C3G is essential for two aspects of cortex development, namely radial glial attachment and neuronal migration.

Monocytic leukemia zinc finger protein is essential for the development of long-term reconstituting hematopoietic stem cells.

Monocytic leukemia zinc finger protein (MOZ), a transcriptional coactivator and member of the MYST family of histone acetyltransferases, is the target of recurrent translocations in acute myeloid leukemia. Since genes associated with translocations in leukemia are typically important regulators of blood formation, we investigated if Moz has a role in normal hematopoiesis. We generated mice carrying a mutation in the Moz gene. Homozygous Moz mutant mice died at birth. Moz mutant fetal liver hematopoietic cells were incapable of contributing to the hematopoietic system of recipients after transplantation. We observed profound defects in the stem cell compartment of Moz-deficient mice. Progenitors of all lineages were reduced in number. However, blood cell lineage commitment was unaffected. Together, these results show that Moz is essential for a fundamental property of hematopoietic stem cells, the ability to reconstitute the hematopoietic system of a recipient after transplantation and that Moz is specifically required in the stem cell compartment.

The central domain of Escherichia coli TyrR is responsible for hexamerization associated with tyrosine-mediated repression of gene expression.

TyrR from Escherichia coli regulates the expression of genes for aromatic amino acid uptake and biosynthesis. Its central ATP-hydrolyzing domain is similar to conserved domains of bacterial regulatory proteins that interact with RNA polymerase holoenzyme associated with the alternative sigma factor, sigma(54). It is also related to the common module of the AAA+ superfamily of proteins that is involved in a wide range of cellular activities. We expressed and purified two TyrR central domain polypeptides. The fragment comprising residues 188-467, called TyrR-(188-467), was soluble and stable, in contrast to that corresponding to the conserved core from residues 193 to 433. TyrR-(188-467) bound ATP and rhodamine-ATP with association constants 2- to 5-fold lower than TyrR and hydrolyzed ATP at five times the rate of TyrR. In contrast to TyrR, which is predominantly dimeric at protein concentrations less than 10 microm in the absence of ligands, or in the presence of ATP or tyrosine alone, TyrR-(188-467) is a monomer, even at high protein concentrations. Tyrosine in the presence of ATP or ATPgammaS promotes the oligomerization of TyrR-(188-467) to a hexamer. Tyrosine-dependent repression of gene transcription by TyrR therefore depends on ligand binding and hexamerization determinants located in the central domain polypeptide TyrR-(188-467).