Elucidating the role of MOZ and its implications for KAT6A Global Developmental Delay syndrome

The histone acetyltransferase Monocytic Leukaemia Zinc Finger Protein (MOZ) was originally identified at the breakpoint of a chromosomal translocation (inv8) associated with acute myeloid leukaemia (AML). MOZ, encoded by the KAT6A gene, belongs to the MYST family of histone acetyltransferases (HATs) and is expressed in a range of human tissues. It is required for the transcriptional regulation of HOX genes during development and is indispensable for the self-renewal ability and differentiation of haematopoietic stem cells. In mouse studies, KAT6A inactivation results in cardiac development abnormalities and impaired B lymphocyte maturation. Furthermore, this HAT is required for dentinogenesis and the inhibition of cell senescence. Initially this protein was primarily studied in the context of cancer, leading to advances in the understanding of the structure and function of this epigenetic regulator, for example the DPF domain which confers histone substrate specificity. MOZ functions as a component of a chromatin regulatory complex containing ING5, EAF6 and the bromodomain protein BRPF1, catalysing H3K9, H3K14 and H3K23 acetylation. Recently, pathogenic sequence variants in KAT6A, clustering in exons 17 & 18 have been implicated in global developmental delay (GDD) and intellectual disability syndromes. Most of these arise as de novo heterozygous mutations, resulting most commonly in premature termination codons (PTCs). Whether these variants result in nonsense-mediated mRNA decay (NMD), or the production of dominant-negative truncated MOZ proteins, is unknown. In this study, we used CRISPR-Cas9 nickase to generate PTCs in the KAT6A gene in erythroleukemia K562 cells, to resemble those found in individuals displaying KAT6A GDD. Clones were generated in which 2 or 3 alleles of KAT6A were targeted (K562 is triploid for this gene). We observed a reduction in spliced mRNA transcript levels of KAT6A harbouring PTCs in exon 17, indicating the action of transcript degradation by NMD, as no change in pre-mRNA levels were observed. Inhibitors of NMD were able to rescue the expression of KAT6A and MOZ target gene transcripts. In contrast, no evidence of NMD was observed in clones harbouring PTCs in KAT6A exon 18, which expressed truncated MOZ proteins. MTT assays indicated defects in all KAT6A PTC clones with regard to their rates of increase in the total number of viable, metabolically active cells over time, accompanied by changes in mRNA expression levels of selected MOZ target genes, as determined by qRT-PCR. Analysis of publically available RNA-seq data from two individuals with this syndrome indicated significant changes in HOX gene expression. Gene Ontology terms identified from differentially expressed gene lists indicates transcriptional changes in an enrichment of genes involved in cell differentiation and Wnt signalling. Taken together, our results offer a clearer insight into the molecular mechanisms behind the KAT6A GDD syndrome, demonstrating that C-terminal regions of the MOZ protein are indeed essential for some normal molecular functions of this HAT. This paves the way for potential therapies in the future

MIR137 is an androgen regulated repressor of an extended network of transcriptional coregulators

Androgens and the androgen receptor (AR) play crucial roles in male development and the pathogenesis and progression of prostate cancer (PCa). The AR functions as a ligand dependent transcription factor which recruits multiple enzymatically distinct epigenetic coregulators to facilitate transcriptional regulation in response to androgens. Over-expression of AR coregulators is implicated in cancer. We have shown that over-expression of KDM1A, an AR coregulator, contributes to PCa recurrence by promoting VEGFA expression. However the mechanism(s) whereby AR coregulators are increased in PCa remain poorly understood. In this study we show that the microRNA hsa-miR-137 (miR137) tumor suppressor regulates expression of an extended network of transcriptional coregulators including KDM1A/LSD1/AOF1, KDM2A/JHDM1A/FBXL11, KDM4A/JMJD2A, KDM5B JARID1B/PLU1, KDM7A/JHDM1D/PHF8, MED1/TRAP220/DRIP205 and NCoA2/SRC2/TIF2. We show that expression of miR137 is increased by androgen in LnCaP androgen PCa responsive cells and that the miR137 locus is epigenetically silenced in androgen LnCaP:C4-2 and PC3 independent PCa cells. In addition, we found that restoration of miR137 expression down-regulates expression of VEGFA, an AR target gene, which suggests a role of miR137 loss also in cancer angiogenesis. Finally we show functional inhibition of mIR137 function enhanced androgen induction of PSA/KLK3 expression. Our data indicate that miR137 functions as an androgen regulated suppressor of androgen signaling by modulating expression of an extended network of transcriptional coregulators. Therefore, we propose that epigenetic silencing of miR137 is an important event in promoting androgen signaling during prostate carcinogenesis and progression

MrkH, a Novel c-di-GMP-Dependent Transcriptional Activator, Controls Klebsiella pneumoniae Biofilm Formation by Regulating Type 3 Fimbriae Expression

Klebsiella pneumoniae causes significant morbidity and mortality worldwide, particularly amongst hospitalized individuals. The principle mechanism for pathogenesis in hospital environments involves the formation of biofilms, primarily on implanted medical devices. In this study, we constructed a transposon mutant library in a clinical isolate, K. pneumoniae AJ218, to identify the genes and pathways implicated in biofilm formation. Three mutants severely defective in biofilm formation contained insertions within the mrkABCDF genes encoding the main structural subunit and assembly machinery for type 3 fimbriae. Two other mutants carried insertions within the yfiN and mrkJ genes, which encode GGDEF domain- and EAL domain-containing c-di-GMP turnover enzymes, respectively. The remaining two isolates contained insertions that inactivated the mrkH and mrkI genes, which encode for novel proteins with a c-di-GMP-binding PilZ domain and a LuxR-type transcriptional regulator, respectively. Biochemical and functional assays indicated that the effects of these factors on biofilm formation accompany concomitant changes in type 3 fimbriae expression. We mapped the transcriptional start site of mrkA, demonstrated that MrkH directly activates transcription of the mrkA promoter and showed that MrkH binds strongly to the mrkA regulatory region only in the presence of c-di-GMP. Furthermore, a point mutation in the putative c-di-GMP-binding domain of MrkH completely abolished its function as a transcriptional activator. In vivo analysis of the yfiN and mrkJ genes strongly indicated their c-di-GMP-specific function as diguanylate cyclase and phosphodiesterase, respectively. In addition, in vitro assays showed that purified MrkJ protein has strong c-di-GMP phosphodiesterase activity. These results demonstrate for the first time that c-di-GMP can function as an effector to stimulate the activity of a transcriptional activator, and explain how type 3 fimbriae expression is coordinated with other gene expression programs in K. pneumoniae to promote biofilm formation to implanted medical devices

Elucidating the role of MOZ and its implications for KAT6A Global Developmental Delay syndrome

The histone acetyltransferase Monocytic Leukaemia Zinc Finger Protein (MOZ) was originally identified at the breakpoint of a chromosomal translocation (inv8) associated with acute myeloid leukaemia (AML). MOZ, encoded by the KAT6A gene, belongs to the MYST family of histone acetyltransferases (HATs) and is expressed in a range of human tissues. It is required for the transcriptional regulation of HOX genes during development and is indispensable for the self-renewal ability and differentiation of haematopoietic stem cells. In mouse studies, KAT6A inactivation results in cardiac development abnormalities and impaired B lymphocyte maturation. Furthermore, this HAT is required for dentinogenesis and the inhibition of cell senescence. Initially this protein was primarily studied in the context of cancer, leading to advances in the understanding of the structure and function of this epigenetic regulator, for example the DPF domain which confers histone substrate specificity. MOZ functions as a component of a chromatin regulatory complex containing ING5, EAF6 and the bromodomain protein BRPF1, catalysing H3K9, H3K14 and H3K23 acetylation. Recently, pathogenic sequence variants in KAT6A, clustering in exons 17 & 18 have been implicated in global developmental delay (GDD) and intellectual disability syndromes. Most of these arise as de novo heterozygous mutations, resulting most commonly in premature termination codons (PTCs). Whether these variants result in nonsense-mediated mRNA decay (NMD), or the production of dominant-negative truncated MOZ proteins, is unknown. In this study, we used CRISPR-Cas9 nickase to generate PTCs in the KAT6A gene in erythroleukemia K562 cells, to resemble those found in individuals displaying KAT6A GDD. Clones were generated in which 2 or 3 alleles of KAT6A were targeted (K562 is triploid for this gene). We observed a reduction in spliced mRNA transcript levels of KAT6A harbouring PTCs in exon 17, indicating the action of transcript degradation by NMD, as no change in pre-mRNA levels were observed. Inhibitors of NMD were able to rescue the expression of KAT6A and MOZ target gene transcripts. In contrast, no evidence of NMD was observed in clones harbouring PTCs in KAT6A exon 18, which expressed truncated MOZ proteins. MTT assays indicated defects in all KAT6A PTC clones with regard to their rates of increase in the total number of viable, metabolically active cells over time, accompanied by changes in mRNA expression levels of selected MOZ target genes, as determined by qRT-PCR. Analysis of publically available RNA-seq data from two individuals with this syndrome indicated significant changes in HOX gene expression. Gene Ontology terms identified from differentially expressed gene lists indicates transcriptional changes in an enrichment of genes involved in cell differentiation and Wnt signalling. Taken together, our results offer a clearer insight into the molecular mechanisms behind the KAT6A GDD syndrome, demonstrating that C-terminal regions of the MOZ protein are indeed essential for some normal molecular functions of this HAT. This paves the way for potential therapies in the future

Phosphorylation of the Pseudomonas aeruginosa Response Regulator AlgR Is Essential for Type IV Fimbria-Mediated Twitching Motility

The response regulator AlgR is required for both alginate biosynthesis and type IV fimbria-mediated twitching motility in Pseudomonas aeruginosa. In this study, the roles of AlgR signal transduction and phosphorylation in twitching motility and biofilm formation were examined. The predicted phosphorylation site of AlgR (aspartate 54) and a second aspartate (aspartate 85) in the receiver domain of AlgR were mutated to asparagine, and mutant algR alleles were introduced into the chromosome of P. aeruginosa strains PAK and PAO1. Assays of these mutants demonstrated that aspartate 54 but not aspartate 85 of AlgR is required for twitching motility and biofilm initiation. However, strains expressing AlgR D85N were found to be hyperfimbriate, indicating that both aspartate 54 and aspartate 85 are involved in fimbrial biogenesis and function. algD mutants were observed to have wild-type twitching motility, indicating that AlgR control of twitching motility is not mediated via its role in the control of alginate biosynthesis. In vitro phosphorylation assays showed that AlgR D54N is not phosphorylated by the enteric histidine kinase CheA. These findings indicate that phosphorylation of AlgR most likely occurs at aspartate 54 and that aspartate 54 and aspartate 85 of AlgR are required for the control of the molecular events governing fimbrial biogenesis, twitching motility, and biofilm formation in P. aeruginosa

Predicting puberty in partial androgen insensitivity syndrome: Use of clinical and functional androgen receptor indices.

BACKGROUND: PAIS exhibits a complex spectrum of phenotypes and pubertal outcomes. The paucity of reliable prognostic indicators can confound management decisions including sex-of-rearing. We assessed whether external masculinisation score (EMS) at birth or functional assays correlates with pubertal outcome in PAIS patients and whether the EMS is helpful in sex assignment. METHODS: We collected pubertal outcome data for 27 male-assigned PAIS patients, all with confirmed androgen receptor (AR) mutations, including two previously uncharacterized variants (I899F; Y916C). Patients were grouped as follows; EMS at birth <5 and ≥ 5 (EMS in normal males is 12; median EMS in PAIS is 4·7) and pubertal outcomes compared. FINDINGS: Only 6/9 patients (67%) with EMS <5 underwent spontaneous onset of puberty, versus all 18 patients with EMS ≥5 (p = .03). Only 1/6 patients (17%) with EMS <5 developed adult genitalia reaching Tanner stage 4 or 5, versus 11/13 (85%) with EMS ≥5 (p = 0·01). There was no significant difference between the two groups of patients in being prescribed androgen replacement, who reached adult testicular volume ≥ 15 ml, pubic hair Tanner stage 4 or 5, above average adult height, had gynaecomastia, and mastectomy. No correlation was observed between EMS and in vitro AR function. INTERPRETATION: In PAIS with AR mutation, birth EMS is a simple predictor of spontaneous pubertal onset and satisfactory adult genitalia. This provides useful information when discussing the likely options for management at puberty. FUND: European Commission Framework 7 Programme, NIHR Cambridge Biomedical Research Centre, BBSRC DTP

Cell morphology changes with high-MGO honey and high-hydrogen peroxide honey treatment^a.

a<p>Actual mean cell lengths and statistics are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055898#pone.0055898.s003" target="_blank">Table S1</a>.</p><p><b>↓</b>Statistically significant decrease compared to no-honey treated cells (<i>p&lt;0.05</i>).</p><p><b>↑</b>Statistically significant increase compared to no-honey treated cells (<i>p&lt;0.05</i>).</p><p>–No change.</p

Effect of sugar, MGO and catalase on growth of bacteria.

<p>Overnight cultures of <i>B. subtilis, E. coli, S. aureus</i> and <i>P. aeruginosa</i> were treated with various components, including catalase, MGO, sugar, and a combination of MGO and sugar at various concentrations equivalent to honeys at the corresponding concentrations shown on the x-axis. The MGO/sugar experiments were performed in the absence (left-hand graphs) and presence (right-hand graphs) of catalase as indicated. The MGO levels correspond to honeys M1 (651.4 mg/kg MGO), M2 (1004.3 mg/kg MGO) and M3 (1541.3 mg/kg MGO) at 1%–32% (w/v). Optical density was recorded at 595 nm every hour for 24 hours. For each component concentration, the time it takes for the culture to reach log phase (assessed as at least 10% of the final culture absorbance of the untreated culture) is plotted on the x-axis. The derivation of this value is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055898#s2" target="_blank">Materials and Methods</a>. A value of 24 hours on the y-axis denotes ‘no growth’. An untreated control was performed alongside each particular treatment, and the starting OD₅₉₅ (zero time-point on x-axis) is plotted for that particular honey experiment.</p

Cellular morphology of bacterial cells treated with a high-MGO honey and a high-hydrogen peroxide honey.

<p>The effects of 4% (w/v) of a high-MGO honey (M3) and a high-hydrogen peroxide honey (MK1) on bacterial cellular morphology were examined. Overnight cultures of <i>B. subtilis, E. coli, S. aureus</i> and <i>P. aeruginosa</i> were treated with these honeys, cells collected at both lag and log phases of growth as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055898#pone.0055898.s002" target="_blank">Figure S2</a>, fixed with glutaraldehyde, stained with DAPI and imaged using fluorescence microscopy. All images are overlays of the phase-contrast image and the DAPI-stained (red) fluorescence image. The two left-hand panels show the no-honey treated control cells, the two middle panels M3 honey-treated cells, and the two right-hand panels show the MK1 honey-treated cells. In all images, condensed DNA is shown by green arrows; and dispersed DNA in <i>B. subtilis</i> cells is shown by blue arrows. An asterisk indicates lysed cells for <i>B. subitlis</i> (MK1, lag-phase cells). The scale bar represents 2 µm, except for <i>S. aureus</i> images, where it represents 1 µm.</p

Floral source, MGO and H₂O₂ Levels of Honeys.

a<p>As reported in Stephens <i>et al.</i> (2010).</p>b<p>MGO (methylglyoxal) levels, reported in Stephens <i>et al.</i> (2010).</p>c<p>H₂O₂ (hydrogen peroxide) levels are expressed as mean H₂O₂ production rate in 1 mL of 10% w/v honey.</p>d<p>Samples collected from hive sites.</p>e<p>Aged samples from drums supplied by apiarists and purchased as designated type.</p>f<p>Obtained commercially.</p