Evaluation of a Master's in Genomic Medicine framework: a national, multi-professional programme to educate healthcare professionals in NHS England.

PURPOSE: Genomic medicine is revolutionising healthcare but requires healthcare professionals to update their understanding of genomics and its application to clinical practice for successful implementation. To meet this need, Heath Education England developed the 'Master's in Genomic Medicine', a national multi-professional programme to increase genomic literacy in the National Health Service workforce. This study summarises an evaluation of the programme which will inform its future development. METHODS: Underpinned by Moore's evaluation framework, a mixed methods approach was used to characterise (i) learner demographics, (ii) perceptions of the programme, (iii) knowledge and/or qualifications achieved, and (iv) the outcome(s) for practice in the workplace. RESULTS: Learners were a diverse cohort of healthcare professionals, including doctors, healthcare scientists, nurses and midwives. Participant satisfaction was high for all elements of the programme, including the curriculum, learning environment(s), and multi-professional cohort(s), despite the challenges of engaging working professionals in part-time learning. Both learners and their managers reported enhanced genomic practice after completion of their studies. CONCLUSION: The Master's in Genomic Medicine programme is an effective approach to professional education in genomic medicine. This broad multi-professional learning complements training aimed at specific groups of healthcare professionals.

Protein kinase Msk1 physically and functionally interacts with the KMT2A/MLL1 methyltransferase complex and contributes to the regulation of multiple target genes.

BACKGROUND: The KMT2A/MLL1 lysine methyltransferase complex is an epigenetic regulator of selected developmental genes, in part through the SET domain-catalysed methylation of H3K4. It is essential for normal embryonic development and haematopoiesis and frequently mutated in cancer. The catalytic properties and targeting of KMT2A/MLL1 depend on the proteins with which it complexes and the post-translational protein modifications which some of these proteins put in place, though detailed mechanisms remain unclear. RESULTS: KMT2A/MLL1 (both native and FLAG-tagged) and Msk1 (RPS6KA5) co-immunoprecipitated in various cell types. KMT2A/MLL1 and Msk1 knockdown demonstrated that the great majority of genes whose activity changed on KTM2A/MLL1 knockdown, responded comparably to Msk1 knockdown, as did levels of H3K4 methylation and H3S10 phosphorylation at KTM2A target genes HoxA4, HoxA5. Knockdown experiments also showed that KMT2A/MLL1 is required for the genomic targeting of Msk1, but not vice versa. CONCLUSION: The KMT2A/MLL1 complex is associated with, and functionally dependent upon, the kinase Msk1, part of the MAP kinase signalling pathway. We propose that Msk1-catalysed phosphorylation at H3 serines 10 and 28, supports H3K4 methylation by the KMT2A/MLL1 complex both by making H3 a more attractive substrate for its SET domain, and improving target gene accessibility by prevention of HP1- and Polycomb-mediated chromatin condensation.

Podcasting in the STEM disciplines: the implications of supplementary lecture recording and 'lecture flipping'.

Lecture capture or 'podcasting' technology offers a new and engaging format of learning materials that can be used to increase the flexibility and interactivity of learning and teaching environments. Here we discuss different ways that these recordings can be incorporated into STEM discipline teaching, and the impact this can have on students' learning.

The histone deacetylase inhibitor sodium valproate causes limited transcriptional change in mouse embryonic stem cells but selectively overrides Polycomb-mediated Hoxb silencing.

BACKGROUND: Histone deacetylase inhibitors (HDACi) cause histone hyperacetylation and H3K4 hypermethylation in various cell types. They find clinical application as anti-epileptics and chemotherapeutic agents, but the pathways through which they operate remain unclear. Surprisingly, changes in gene expression caused by HDACi are often limited in extent and can be positive or negative. Here we have explored the ability of the clinically important HDACi valproic acid (VPA) to alter histone modification and gene expression, both globally and at specific genes, in mouse embryonic stem (ES) cells. RESULTS: Microarray expression analysis of ES cells exposed to VPA (1 mM, 8 h), showed that only 2.4% of genes showed a significant, >1.5-fold transcriptional change. Of these, 33% were down-regulated. There was no correlation between gene expression and VPA-induced changes in histone acetylation or H3K4 methylation at gene promoters, which were usually minimal. In contrast, all Hoxb genes showed increased levels of H3K9ac after exposure to VPA, but much less change in other modifications showing bulk increases. VPA-induced changes were lost within 24 h of inhibitor removal. VPA significantly increased the low transcription of Hoxb4 and Hoxb7, but not other Hoxb genes. Expression of Hoxb genes increased in ES cells lacking functional Polycomb silencing complexes PRC1 and PRC2. Surprisingly, VPA caused no further increase in Hoxb transcription in these cells, except for Hoxb1, whose expression increased several fold. Retinoic acid (RA) increased transcription of all Hoxb genes in differentiating ES cells within 24 h, but thereafter transcription remained the same, increased progressively or fell progressively in a locus-specific manner. CONCLUSIONS: Hoxb genes in ES cells are unusual in being sensitive to VPA, with effects on both cluster-wide and locus-specific processes. VPA increases H3K9ac at all Hoxb loci but significantly overrides PRC-mediated silencing only at Hoxb4 and Hoxb7. Hoxb1 is the only Hoxb gene that is further up-regulated by VPA in PRC-deficient cells. Our results demonstrate that VPA can exert both cluster-wide and locus-specific effects on Hoxb regulation.

Genes are often sheltered from the global histone hyperacetylation induced by HDAC inhibitors.

Histone deacetylase inhibitors (HDACi) are increasingly used as therapeutic agents, but the mechanisms by which they alter cell behaviour remain unclear. Here we use microarray expression analysis to show that only a small proportion of genes (∼9%) have altered transcript levels after treating HL60 cells with different HDACi (valproic acid, Trichostatin A, suberoylanilide hydroxamic acid). Different gene populations respond to each inhibitor, with as many genes down- as up-regulated. Surprisingly, HDACi rarely induced increased histone acetylation at gene promoters, with most genes examined showing minimal change, irrespective of whether genes were up- or down-regulated. Many genes seem to be sheltered from the global histone hyperacetyation induced by HDACi.

Elevated FOSB-expression; a potential marker of valproate sensitivity in AML.

Histone deacetylase inhibitors (HDIs) are emerging as valuable new agents in the treatment of acute myeloid leukaemia (AML). However, since response rates to these agents alone are low, we sought to identify markers associated with responsiveness. In a trial of 20 patients treated with the HDI sodium valproate (VPA) in combination with all trans retinoic acid and theophylline, three patients responded clinically with one complete remission (CR) and two partial remissions. The in vivo response of the CR patient was mirrored by high in vitro sensitivity of their blasts to VPA, indicating that similar factors determine both in vivo and in vitro sensitivity. Microarray analysis of the primary AMLs and a panel of haemato-lymphoid cell lines, with a similar range of VPA sensitivities as the primary leukaemic blasts, identified elevated FOSB-expression as a potential marker of VPA sensitivity. Quantitative polymerase chain reaction confirmed overexpression of FOSB in the CR patient blasts compared to patients failing to achieve CR, and in a subset of a larger panel of AML samples. Overexpression of FOSB in K562 myeloid cells significantly increased in vitro sensitivity to VPA. Thus, we propose that FOSB is a novel, potential marker of VPA sensitivity in AML.

Directed evolution of a histone acetyltransferase--enhancing thermostability, whilst maintaining catalytic activity and substrate specificity.

Histone acetylation plays an integral role in the epigenetic regulation of gene expression. Transcriptional activity reflects the recruitment of opposing classes of enzymes to promoter elements; histone acetyltransferases (EC 2.3.1.48) that deposit acetyl marks at a subset of histone residues and histone deacetylases that remove them. Many histone acetyltransferases are difficult to study in solution because of their limited stability once purified. We have developed a directed evolution protocol that allows the screening of hundreds of histone acetyltransferase mutants for histone acetylating activity, and used this to enhance the thermostability of the human P/CAF histone acetyltransferase. Two rounds of directed evolution significantly stabilized the enzyme without lowering the catalytic efficiency and substrate specificity of the enzyme. Twenty-four variants with higher thermostability were identified. Detailed analysis revealed twelve single amino acid mutants that were found to possess a higher thermostability. The residues affected are scattered over the entire protein structure, and are different from mutations predicted by sequence alignment approaches, suggesting that sequence comparison and directed evolution methods are complementary strategies in engineering increased protein thermostability. The stabilizing mutations are predominately located at surface of the enzyme, suggesting that the protein's surface is important for stability. The directed evolution approach described in the present study is easily adapted to other histone modifying enzymes, requiring only appropriate peptide substrates and antibodies, which are available from commercial suppliers.

Acetylation increases access of remodelling complexes to their nucleosome targets to enhance initiation of V(D)J recombination.

Targeted chromatin remodelling is essential for many nuclear processes, including the regulation of V(D)J recombination. ATP-dependent nucleosome remodelling complexes are important players in this process whose activity must be tightly regulated. We show here that histone acetylation regulates nucleosome remodelling complex activity to boost RAG cutting during the initiation of V(D)J recombination. RAG cutting requires nucleosome mobilization from recombination signal sequences. Histone acetylation does not stimulate nucleosome mobilization per se by CHRAC, ACF or their catalytic subunit, ISWI. Instead, we find the more open structure of acetylated chromatin regulates the ability of nucleosome remodelling complexes to access their nucleosome templates. We also find that bromodomain/acetylated histone tail interactions can contribute to this targeting at limited concentrations of remodelling complex. We therefore propose that the changes in higher order chromatin structure associated with histone acetylation contribute to the correct targeting of nucleosome remodelling complexes and this is a novel way in which histone acetylation can modulate remodelling complex activity.

Cross-talk between histone modifications in response to histone deacetylase inhibitors: MLL4 links histone H3 acetylation and histone H3K4 methylation.

Histones are subject to a wide variety of post-translational modifications that play a central role in gene activation and silencing. We have used histone modification-specific antibodies to demonstrate that two histone modifications involved in gene activation, histone H3 acetylation and H3 lysine 4 methylation, are functionally linked. This interaction, in which the extent of histone H3 acetylation determines both the abundance and the "degree" of H3K4 methylation, plays a major role in the epigenetic response to histone deacetylase inhibitors. A combination of in vivo knockdown experiments and in vitro methyltransferase assays shows that the abundance of H3K4 methylation is regulated by the activities of two opposing enzyme activities, the methyltransferase MLL4, which is stimulated by acetylated substrates, and a novel and as yet unidentified H3K4me3 demethylase.

Histone modifications: signalling receptors and potential elements of a heritable epigenetic code.

The genetic code epitomises simplicity, near universality and absolute predictive power. By contrast, epigenetic information, in the form of histone modifications, is characterised by complexity, diversity and an overall tendency to respond to changes in genomic function rather than to predict them. Perhaps the transient changes in histone modifications involved in intranuclear signalling and ongoing chromatin functions mask stable, predictive modifications that lie beneath. The current rapid progress in unravelling the diversity and complexity of epigenetic information might eventually reveal an underlying histone or epigenetic code. But whether it does or not, it will certainly provide unprecedented opportunities, both for understanding how the genome responds to environmental and metabolic change and for manipulating its activities for experimental and therapeutic benefit.

MAP kinase-mediated phosphorylation of distinct pools of histone H3 at S10 or S28 via mitogen- and stress-activated kinase 1/2.

ERK and p38 MAP kinases, acting through the downstream mitogen- and stress-activated kinase 1/2 (MSK1/2), elicit histone H3 phosphorylation on a subfraction of nucleosomes--including those at Fos and Jun--concomitant with gene induction. S10 and S28 on the H3 tail have both been shown to be phospho-acceptors in vivo. Both phospho-epitopes appear with similar time-courses and both occur on H3 tails that are highly sensitive to TSA-induced hyperacetylation, similarities which might suggest that MSK1/2 phosphorylates both sites on the same H3 tails. Indeed, on recombinant histone octamers in vitro, MSK1 efficiently phosphorylates both sites on the same H3 tail. However, sequential immunoprecipitation studies show that antibodies against phosphorylated S10-H3 recover virtually all this epitope without depletion of phosphorylated S28-H3, and vice versa, indicating that the two phospho-epitopes are not located on the same H3 tail in vivo. Confocal immunocytochemistry confirms the clear physical separation of the two phospho-epitopes in the intact mouse nucleus. Finally, we used transfection-based experiments to test models that might explain such differential targeting. Overexpression and delocalisation of MSK1 does not result in the breakdown of targeting in vivo despite the fact that the ectopic kinase is fully activated by external stimuli. These studies reveal a remarkable level of targeting of S10 and S28 phosphorylation to distinct H3 tails within chromatin in the interphase mouse nucleus. Possible models for such exquisite targeting are discussed.

Recruitment of the nucleolar remodeling complex NoRC establishes ribosomal DNA silencing in chromatin.

The rRNA gene cluster consists of multiple transcription units. Half of these are active, while the other half are transcriptionally inactive. Previously, in vivo studies have demonstrated that silencing of ribosomal DNA (rDNA) is mediated by the chromatin remodeling NoRC (nucleolar remodeling complex). To explore the mechanisms underlying NoRC-directed silencing of rDNA transcription, we investigated the effect of recombinant NoRC on RNA polymerase I transcription on reconstituted chromatin templates. We show that NoRC interacts with the transcription terminator factor (TTF-I), and this interaction is required both for the binding of TTF-I to its promoter-proximal target site and for the recruitment of NoRC to the promoter. After association with the rDNA promoter, NoRC alters the position of the promoter-bound nucleosome, thereby repressing RNA polymerase I transcription. This NoRC-directed rDNA repression requires the N terminus of histone H4. Repression is effective before preinitiation complex formation and as such is unable to exert an effect upon activated rDNA genes. Furthermore, the early steps of rDNA repression do not depend on DNA and histone modifications. These results reveal an important role for TTF-I in recruiting NoRC to rDNA and an active role for NoRC in the establishment of rDNA silencing.

A critical epitope for substrate recognition by the nucleosome remodeling ATPase ISWI.

The ATPase ISWI is the catalytic core of several nucleosome remodeling complexes, which are able to alter histone-DNA interactions within nucleosomes such that the sliding of histone octamers on DNA is facilitated. Dynamic nucleosome repositioning may be involved in the assembly of chromatin with regularly spaced nucleosomes and accessible regulatory sequence elements. The mechanism that underlies nucleosome sliding is largely unresolved. We recently discovered that the N-terminal 'tail' of histone H4 is critical for nucleosome remodeling by ISWI. If deleted, nucleosomes are no longer recognized as substrates and do not stimulate the ATPase activity of ISWI. We show here that the H4 tail is part of a more complex recognition epitope which is destroyed by grafting the H4 N-terminus onto other histones. We mapped the H4 tail requirement to a hydrophilic patch consisting of the amino acids R17H18R19 localized at the base of the tail. These residues have been shown earlier to contact nucleosomal DNA, suggesting that ISWI recognizes an 'epitope' consisting of the DNA-bound H4 tail. Consistent with this hypothesis, the ISWI ATPase is stimulated by isolated H4 tail peptides ISWI only in the presence of DNA. Acetylation of the adjacent K12 and K16 residues impairs substrate recognition by ISWI.

Methylation at arginine 17 of histone H3 is linked to gene activation.

The nuclear hormone receptor co-activator CARM1 has the potential to methylate histone H3 at arginine residues in vitro. The methyltransferase activity of CARM1 is necessary for its co-activator functions in transient transfection assays. However, the role of this methyltransferase in vivo is unclear, given that methylation of arginines is not easily detectable on histones. We have raised an antibody that specifically recognizes methylated arginine 17 (R17) of histone H3, the major site of methylation by CARM1. Using this antibody we show that methylated R17 exists in vivo. Chromatin immunoprecipitation analysis shows that R17 methylation on histone H3 is dramatically upregulated when the estrogen receptor-regulated pS2 gene is activated. Coincident with the appearance of methylated R17, CARM1 is found associated with the histones on the pS2 gene. Together these results demonstrate that CARM1 is recruited to an active promoter and that CARM1-mediated R17 methylation on histone H3 takes place in vivo during this active state.