Characterisation of the interaction interface of the MOZ/BRPF1 acetyltransferase complex and its role in gene regulation

Monocytic leukaemia zinc-finger protein (MOZ) is a histone lysine acetyltransferase, which plays important roles in haematopoiesis and tissue development. Alterations of the Lysine (K) acetyl-transferase 6A (KAT6A) gene encoding (MOZ) are associated with cancers and in particular, the expression of MOZ-fusion proteins in haematopoietic progenitors has been shown to be causative of acute myeloid leukaemia (AML). More recently, mutations in KAT6A have been associated with developmental syndromes, the clinical features of which are global developmental delay and intellectual disability. MOZ has been shown to associate with a protein complex that includes BRPF1 (bromodomain and PHD finger containing protein 1) and direct association of MOZ and BRPF1 stabilises this complex. Mutations in the BRPF1 gene are also associated with intellectual disability syndromes. Importantly, association with BRPF1 modulates the substrate specificity of MOZ and enhances its acetyltransferase activity for Histone H3 in nucleosomes. However, both MOZ and BRPF1 contain chromatin reader domains that allow them to associate with chromatin by their ability to recognise specific histone modifications. Thus normal MOZ function appears to be regulated by BRPF1, although they may also function independently in different tissues. In animal models, the induction of AML by the MOZ-Transcriptional Intermediary Factors 2 (TIF2) was shown to be dependent on its association with BRPF1. Therefore, in this study, I set out to investigate the molecular interface between human MOZ and BRPF1 proteins, to gain a better insight into how the complex is formed and to understand the functional cooperativity of these proteins. Firstly, to map the minimal sequences required for the interaction of the MOZ/BRPF1 complex, I performed yeast-two-hybrid mapping of protein-protein interactions. This was achieved by generating a series of deletion constructs of both proteins in yeast two hybrid expression vectors. This identified that the sequence BRPF1 59-190 is sufficient to bind the MOZ sequence 676-763, which encodes helix-turn-helix motif located C-terminus of the MOZ MYST acetyltransferase domain. Site-directed mutagenesis identified key residues in the MOZ 676-763 sequence that when altered/disrupted interaction with BRPF1.These results strongly suggest that MOZ 676-763 is a BRPF1 recruitment domain, consistent with a recent structure of the related HBO1/BRPF2 complex. A dual expression vector has been constructed in which the minimal interaction domains were successfully co-expressed in E.coli, which will be taken forward for large scale purification and crystallisation trials. To extend our study to full-length proteins, we performed co-localisation and co-immunoprecipitation studies with epitope-tagged proteins transiently expressed in mammalian HEK293 cells. Interestingly, while FLAG-MOZ localised to the nucleus, expression of HA-BRPF1 alone showed it to be located to the cytoplasmic compartment. In contrast co-expression of both wild type FLAG-MOZ and HA-BRPF1 resulted in their strong co-localisation to the nucleus, and co-immunoprecipitation as a complex. In contrast, a FLAG-MOZ mutant lacking a key region of the BRPF1 binding site, was unable to induce nuclear localisation of HA-BRPF1, suggesting that the helix turn helix region (676-767) is the major interface between these proteins. Surprisingly, both the wild type and mutant FLAG-MOZ proteins continued to co-precipitate together from cell extracts, but we show using a BRPF1 bromodomain-selective inhibitor that this may be in part explained by indirect mechanism involving co-localisation to chromatin sites involving the histone reader domains. To investigate the functional consequences of disrupting the MOZ/BRPF1 complex, rescue experiments were performed using CRISPR-CAS9 edited HEK293 cells developed in our laboratory in which MOZ expression was inactivated. Due to the repressive function of BRPF1, we focussed on genes that we upregulated after MOZ deletion. The RTqPCR data showed YFP-MOZ successfully repressed several of these upregulated genes, whereas a YFP-MOZ mutant deficient in BRPF1 binding was slightly less efficient. These results are consistent with a role for MOZ/BRPF1 complexes in suppression of gene expression, although further studies will be required to investigate this fully. In summary, the minimal sequences involved in the molecular interface between MOZ and BRPF1 have been defined and shown to be contained within a helix-turn helix domain conserved in MYST acetyltransferase domains. This interaction is essential for co-localisation of MOZ and BRPF1 to the nucleus, and for MOZ-mediated repression of several of its target genes, although histone modification reader domains also play a role in assembling these complexes on chromatin. Future work will focus on how specific MYST/BRPF1 complexes are assembled

Characterisation of the interaction interface of the MOZ/BRPF1 acetyltransferase complex and its role in gene regulation

Monocytic leukaemia zinc-finger protein (MOZ) is a histone lysine acetyltransferase, which plays important roles in haematopoiesis and tissue development. Alterations of the Lysine (K) acetyl-transferase 6A (KAT6A) gene encoding (MOZ) are associated with cancers and in particular, the expression of MOZ-fusion proteins in haematopoietic progenitors has been shown to be causative of acute myeloid leukaemia (AML). More recently, mutations in KAT6A have been associated with developmental syndromes, the clinical features of which are global developmental delay and intellectual disability. MOZ has been shown to associate with a protein complex that includes BRPF1 (bromodomain and PHD finger containing protein 1) and direct association of MOZ and BRPF1 stabilises this complex. Mutations in the BRPF1 gene are also associated with intellectual disability syndromes. Importantly, association with BRPF1 modulates the substrate specificity of MOZ and enhances its acetyltransferase activity for Histone H3 in nucleosomes. However, both MOZ and BRPF1 contain chromatin reader domains that allow them to associate with chromatin by their ability to recognise specific histone modifications. Thus normal MOZ function appears to be regulated by BRPF1, although they may also function independently in different tissues. In animal models, the induction of AML by the MOZ-Transcriptional Intermediary Factors 2 (TIF2) was shown to be dependent on its association with BRPF1. Therefore, in this study, I set out to investigate the molecular interface between human MOZ and BRPF1 proteins, to gain a better insight into how the complex is formed and to understand the functional cooperativity of these proteins. Firstly, to map the minimal sequences required for the interaction of the MOZ/BRPF1 complex, I performed yeast-two-hybrid mapping of protein-protein interactions. This was achieved by generating a series of deletion constructs of both proteins in yeast two hybrid expression vectors. This identified that the sequence BRPF1 59-190 is sufficient to bind the MOZ sequence 676-763, which encodes helix-turn-helix motif located C-terminus of the MOZ MYST acetyltransferase domain. Site-directed mutagenesis identified key residues in the MOZ 676-763 sequence that when altered/disrupted interaction with BRPF1.These results strongly suggest that MOZ 676-763 is a BRPF1 recruitment domain, consistent with a recent structure of the related HBO1/BRPF2 complex. A dual expression vector has been constructed in which the minimal interaction domains were successfully co-expressed in E.coli, which will be taken forward for large scale purification and crystallisation trials. To extend our study to full-length proteins, we performed co-localisation and co-immunoprecipitation studies with epitope-tagged proteins transiently expressed in mammalian HEK293 cells. Interestingly, while FLAG-MOZ localised to the nucleus, expression of HA-BRPF1 alone showed it to be located to the cytoplasmic compartment. In contrast co-expression of both wild type FLAG-MOZ and HA-BRPF1 resulted in their strong co-localisation to the nucleus, and co-immunoprecipitation as a complex. In contrast, a FLAG-MOZ mutant lacking a key region of the BRPF1 binding site, was unable to induce nuclear localisation of HA-BRPF1, suggesting that the helix turn helix region (676-767) is the major interface between these proteins. Surprisingly, both the wild type and mutant FLAG-MOZ proteins continued to co-precipitate together from cell extracts, but we show using a BRPF1 bromodomain-selective inhibitor that this may be in part explained by indirect mechanism involving co-localisation to chromatin sites involving the histone reader domains. To investigate the functional consequences of disrupting the MOZ/BRPF1 complex, rescue experiments were performed using CRISPR-CAS9 edited HEK293 cells developed in our laboratory in which MOZ expression was inactivated. Due to the repressive function of BRPF1, we focussed on genes that we upregulated after MOZ deletion. The RTqPCR data showed YFP-MOZ successfully repressed several of these upregulated genes, whereas a YFP-MOZ mutant deficient in BRPF1 binding was slightly less efficient. These results are consistent with a role for MOZ/BRPF1 complexes in suppression of gene expression, although further studies will be required to investigate this fully. In summary, the minimal sequences involved in the molecular interface between MOZ and BRPF1 have been defined and shown to be contained within a helix-turn helix domain conserved in MYST acetyltransferase domains. This interaction is essential for co-localisation of MOZ and BRPF1 to the nucleus, and for MOZ-mediated repression of several of its target genes, although histone modification reader domains also play a role in assembling these complexes on chromatin. Future work will focus on how specific MYST/BRPF1 complexes are assembled

Fast, multicolour optical sectioning over extended fields of view with patterned illumination and machine learning

Structured illumination can reject out-of-focus signal from a sample, enabling high-speed and high-contrast imaging over large areas with widefield detection optics. However, this optical-sectioning technique is currently limited by image reconstruction artifacts and poor performance at low signal-to-noise ratios. We combine multicolour interferometric pattern generation with machine learning to achieve high-contrast, real-time reconstruction of image data that is robust to background noise and sample motion. We validate the method in silico and demonstrate imaging of diverse specimens, from fixed and live biological samples to synthetic biosystems, reconstructing data live at 11 Hz across a 44 X 44 μm^2 field of view, and demonstrate image acquisition speeds exceeding 154 H

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

Supplementary document for Fast, multicolour optical sectioning over extended fields of view by combining interferometric SIM with machine learning - 6796904.pdf

Supplementary text and figure

Supplementary document for Fast, multicolour optical sectioning over extended fields of view by combining interferometric SIM with machine learning - 6834994.pdf

Supplementary materia

Feasibility of Coacervate-Like Nanostructure for Instant Drug Nanoformulation.

Despite the enormous advancements in nanomedicine research, a limited number of nanoformulations are available on the market, and few have been translated to clinics. An easily scalable, sustainable, and cost-effective manufacturing strategy and long-term stability for storage are crucial for successful translation. Here, we report a system and method to instantly formulate NF achieved with a nanoscale polyelectrolyte coacervate-like system, consisting of anionic pseudopeptide poly(l-lysine isophthalamide) derivatives, polyethylenimine, and doxorubicin (Dox) via simple "mix-and-go" addition of precursor solutions in seconds. The coacervate-like nanosystem shows enhanced intracellular delivery of Dox to patient-derived multidrug-resistant (MDR) cells in 3D tumor spheroids. The results demonstrate the feasibility of an instant drug formulation using a coacervate-like nanosystem. We envisage that this technique can be widely utilized in the nanomedicine field to bypass the special requirement of large-scale production and elongated shelf life of nanomaterials

Thyroid and androgen receptor signaling are antagonized by μ-Crystallin in prostate cancer.

Androgen deprivation therapy (ADT) remains a key approach in the treatment of prostate cancer (PCa). However, PCa inevitably relapses and becomes ADT resistant. Besides androgens, there is evidence that thyroid hormone thyroxine (T4) and its active form 3,5,3'-triiodo-L-thyronine (T3) are involved in the progression of PCa. Epidemiologic evidences show a higher incidence of PCa in men with elevated thyroid hormone levels. The thyroid hormone binding protein μ-Crystallin (CRYM) mediates intracellular thyroid hormone action by sequestering T3 and blocks its binding to cognate receptors (TRα/TRβ) in target tissues. We show in our study that low CRYM expression levels in PCa patients are associated with early biochemical recurrence and poor prognosis. Moreover, we found a disease stage-specific expression of CRYM in PCa. CRYM counteracted thyroid and androgen signaling and blocked intracellular choline uptake. CRYM inversely correlated with [18F]fluoromethylcholine (FMC) levels in positron emission tomography/magnetic resonance imaging of PCa patients. Our data suggest CRYM as a novel antagonist of T3- and androgen-mediated signaling in PCa. The role of CRYM could therefore be an essential control mechanism for the prevention of aggressive PCa growth