The epigenetic regulator Histone Deacetylase 1 promotes transcription of a core neurogenic programme in zebrafish embryos
© Harrison et al; licensee BioMed Central Ltd. 2011
Received: 13 August 2010
Accepted: 12 January 2011
Published: 12 January 2011
The epigenetic regulator Histone Deacetylase 1 (Hdac1) is required for specification and patterning of neurones and myelinating glia during development of the vertebrate central nervous system (CNS). This co-ordinating function for Hdac1 is evolutionarily conserved in zebrafish and mouse, but the mechanism of action of Hdac1 in the developing CNS is not well-understood.
A genome-wide comparative analysis of the transcriptomes of Hdac1-deficient and wild-type zebrafish embryos was performed, which identified an extensive programme of gene expression that is regulated by Hdac1 in the developing embryo. Using time-resolved expression profiling of embryos, we then identified a small subset of 54 genes within the Hdac1-regulated transcriptome that specifically exhibit robust and sustained Hdac1-dependent expression from early neurogenesis onwards. 18 of these 54 stringently Hdac1-regulated genes encode DNA-binding transcription factors that are implicated in promoting neuronal specification and CNS patterning, including the proneural bHLH proteins Ascl1a and Ascl1b, as well as Neurod4 and Neurod. Relatively few genes are strongly repressed by Hdac1 but expression of the Notch target gene her6 is attenuated by Hdac1 in specific sub-regions of the developing CNS, from early stages of neurogenesis onwards. Selected members of the stringently Hdac1-regulated group of genes were tested for Hdac1 binding to their promoter-proximal cis-regulatory elements. Surprisingly, we found that Hdac1 is specifically and stably associated with DNA sequences within the promoter region of ascl1b during neurogenesis, and that this Hdac1-ascl1b interaction is abolished in hdac1 mutant embryos.
We conclude that Hdac1 regulates histone acetylation and methylation in the developing zebrafish embryo and promotes the sustained, co-ordinate transcription of a small set of transcription factor genes that control expansion and diversification of cell fates within the developing CNS. Our in vivo chromatin immunoprecipitation results also suggest a specific function for Hdac1 in directly regulating transcription of a key member of this group of genes, ascl1b, from the beginning of neurogenesis onwards. Taken together, our observations indicate a novel role for Hdac1 as a positive regulator of gene transcription during development of the vertebrate CNS, in addition to its more well-established function in transcriptional repression.
Histone modifying enzymes are key catalytic components of the transcriptional control systems that programme multicellular development. Many different histone modifying enzymes contribute to the dynamic regulation of chromatin structure and function, with concomitant impacts on gene transcription. For example, the balance of Histone acetyltransferase (HAT) and Histone deacetylase (HDAC) activities that are associated with any given gene determines the distribution of histone acetylation marks in the chromatin domain encompassing that gene. Histone acetylation is a hallmark of transcriptionally active chromatin, whereas transcriptionally silent chromatin lacks this modification . Mechanistic analysis of protein complexes that establish and maintain transcriptional repression has revealed the presence of HDACs in these complexes [2, 3]. Whilst there is much evidence in support of functions for HDACs in transcription silencing, the roles of HDACs in facilitating transcription have been less well appreciated. Nevertheless, some genome-wide studies in yeast have demonstrated that HDACs are associated with transcriptionally active genes and that they promote gene transcription [4–6]. More recently, mammalian HDACs have been shown to be specifically enriched in chromatin encompassing the transcriptional start sites of transcriptionally active genes, as well as at transcriptionally silent genes that are poised for activation . Moreover, HDAC-containing protein complexes such as REST/CoREST have been demonstrated to poise transcriptionally silent genes in a specific configuration in neural progenitors, which facilitates their robust transcriptional activation when these cells are induced to differentiate into neurons .
In zebrafish, the Class I HDAC, Hdac1, is required for specification of neurones and glia during embryonic development [9–12]. In addition, prominent roles are known for this gene in the development of the gastrointestinal system and neural crest derivatives [13–15]. In the mouse, there are two murine orthologues of zebrafish hdac1, Hdac1 and Hdac2, which together promote the transformation of embryonic neural progenitors into neurones and glia [16, 17]. In both zebrafish and mouse, Hdac1 regulates neural progenitor differentiation by facilitating the integration of Hedgehog, Notch and Wnt signalling pathway activities into the mechanisms governing neuronal and glial specification. However, precisely how Hdac1 accomplishes this role is still not well understood.
The establishment of proneural gene expression patterns in early embryonic ectoderm delineates zones of active neurogenesis within the neural plate [reviewed in ]. Proneural gene expression is repressed by the Notch pathway as a result of negative feedback from Delta-expressing neuronal precursors, which activates Notch in proliferating neural progenitors, thus limiting the rate at which these cells are transformed into neuronal precursors [reviewed in ]. Whilst this negative feedback mechanism has been well-characterised, the mechanisms that positively regulate expansion and diversification of differentiated cell types within the nervous system are less well understood. Our previous studies demonstrated that Hdac1 promotes expression of proneural genes, represses Notch target gene expression and enables neural fate-determining responses to Hedgehog pathway activity [9, 12]. In view of the well-documented roles of HDACs as components of transcriptional repressor complexes, these observations suggested a role for Hdac1 as a potential direct repressor of her6 transcription, and thus as an indirect activator of proneural genes. Here, we describe the results of our recent work to elucidate further the role of Hdac1 in neurogenesis, using global gene expression profiling tools to define the Hdac1-regulated transcriptome in an unbiased way and to identify direct targets of Hdac1 using chromatin immunoprecipitation.
Identification of Hdac1-regulated genes by transcriptome analysis of hdac1 mutant zebrafish embryos
Hdac1-targeted morpholinos closely phenocopy the transcriptional defects exhibited by hdac1 mutant embryos
Comparison of hdac1 mutant and wild-type transcriptomes at 27 hpf identified large numbers of Hdac1-regulated genes, many of which were likely to be indirect, downstream effectors of the primary target genes for Hdac1. To identify candidate Hdac1 direct target genes, we sought to identify genes whose expression was regulated by Hdac1 during early developmental stages that preceded the emergence of a morphologically recognisable hdac1 mutant phenotype. Since morpholinos can be used to create defined batches of embryos that are all deficient in a specific gene function, we compared the phenotypes of embryos microinjected with a translation-blocking morpholino, Hdac1ATG1  or a splice-blocking morpholino Hdac1SPL1, with the phenotype of hdac1 mutant embryos. Microinjection of either Hdac1ATG1 or Hdac1SPL1 morpholinos into embryos caused morphological abnormalities that closely resembled the hdac1 mutant phenotype (data not shown), and which were first detectable at ~24 hpf. Moreover, both the Hdac1ATG1 and Hdac1SPL1 morpholinos significantly reduced Hdac1 protein levels in the embryo, although the Hdac1ATG1 morpholino induced a larger reduction in Hdac1 protein levels than did the Hdac1SPL1 morpholino (Figure 2C). By contrast, neither an Hdac1 Mismatch Control morpholino (Hdac1 Control), nor a Standard Control morpholino, exhibited any appreciable effect on embryo morphology, or on levels of Hdac1 protein (Figure 2C).
In order to begin to elucidate the effects of Hdac1ATG1 and Hdac1SPL1morpholinos on embryonic transcription, the transcriptomes of these morphant embryos were analysed at 27 hpf. Both the Hdac1ATG1 and Hdac1SPL1 morpholinos elicited major effects on the embryonic transcriptome (Additional file 1). A set of 6117 probes was identified, from within the group of probes whose expression is affected in the hdac1 mutant, that also exhibited statistically significant (p < 10-5) Hdac1-regulated gene expression in 27 hpf morphant samples. This set of 6117 probes was further analysed with the Explore Gene Ontology (eGOn) software, to identify Gene Ontology (GO) categories that were significantly enriched in the Hdac1-regulated probe set (Additional file 2). The Biological Process category was found to be considerably over-represented in the annotations of Hdac1-regulated genes, within which the sub-categories of GO terms related to Transcriptional Regulation (highlighted in purple, Additional file 2) or Developmental Processes occurred particularly frequently. Other prominent GO terms that are enriched in the Hdac1-regulated probe set include nervous system development, neuronal differentiation and photoreceptor differentiation (Additional file 2). Thus, both hdac1 mutant and morphant transcriptomes exhibit similar alterations to the expression of genes that encode transcription factors and developmental regulators, with an emphasis on development of the nervous system. These results implied that Hdac1 performs a central role in regulating expression of these classes of genes and further validated the morpholinos as tools for perturbing Hdac1 function during nervous system development.
In order to determine which of the two Hdac1 morpholinos most closely phenocopied the hdac1 mutation, hierarchical cluster analysis  was used to order the transcriptome datasets obtained from 27 hpf hdac1 mutant, Hdac1ATG1 and Hdac1SPL1 morphant embryos, according to the similarities in the patterns of gene expression changes observed in each of the three different manipulations (Additional file 1). This analysis showed that overall, the set of Hdac1-regulated genes that was identified using the Hdac1ATG1 morpholino, more closely resembled the group of genes identified by analysis of 27 hpf hdac1 mutant embryos than did those identified using the Hdac1SPL1 morpholino. The Hdac1ATG1 morpholino was therefore chosen to investigate Hdac1 function during early stages of CNS development before a morphologically distinct phenotype can be recognised. As a first step in this analysis, we determined whether this morpholino could reduce Hdac1 protein levels from early stages of CNS development onwards. Hdac1ATG1 morphant embryos and individually genotyped wild-type and hdac1 mutant embryos, aged 12 hpf, 18 hpf and 27 hpf (corresponding to early, mid- and late neurogenesis stages, respectively) were immunostained with an anti-Hdac1 antibody and analysed by confocal imaging (Figure 2D). Whilst Hdac1 protein was abundant in all cell nuclei of wild-type embryos at each of the stages analysed, both hdac1 mutant and Hdac1ATG1 morphant embryos exhibited a substantially lower level of Hdac1 protein, which was more pronounced at later developmental stages (Figure 2D). The results of the transcriptomic analysis, together with the experiments to compare the distribution of Hdac1 protein in hdac1 mutant and morphant embryos, revealed a close correspondence between the 27 hpf hdac1 mutant and morphant phenotypes and validated the Hdac1ATG1 morpholino as a precision tool with which to investigate Hdac1 function in the embryo between 12 hpf and 27 hpf.
Hdac1 promotes expression of a core set of neurogenic regulatory genes throughout the developing CNS
Pharmacological inhibition of HDAC function during early neurogenesis closely phenocopies the Hdac1ATG1 morphant phenotype
Hdac1 binds directly to the promoter of the proneural gene ascl1b in 12 hpf and 27 hpf zebrafish embryos
Hdac1 promotes sustained expression of a small subset of developmental regulatory genes from the beginning of neurogenesis onwards
In zebrafish and mouse embryos, Hdac1 promotes the transformation of neural progenitors into differentiated neurons and glia. The experiments reported here further elucidate the mechanism of Hdac1 function in zebrafish CNS development. We initially sought to identify all of the genes whose expression levels were robustly Hdac1-regulated in 27 hpf embryos. We found that approximately 20% of all the probes on the microarrays exhibited statistically significant differences in transcript abundance in 27 hpf hdac1 mutant and wild-type sibling embryos, although only 7% of these Hdac1-regulated probes exhibited more than a two-fold change in transcript abundance. However, as this comparative analysis was performed on morphologically distinct 27 hpf hdac1 mutants and wild-type siblings, it seemed likely that many of the genes identified in this way as being Hdac1-regulated are indirect downstream effectors of a smaller programme of gene expression that is directly regulated by Hdac1 from earlier stages of neurogenesis. Therefore, to identify transcriptional changes that were likely to be more direct consequences of loss of Hdac1 function, we used the Hdac1ATG1 morpholino to generate defined batches of Hdac1-deficient embryos, whose transcriptomes were then analysed well before any morphological differences appeared in these embryos. Gene Ontology analysis of Hdac1-regulated genes, in both hdac1 mutant and Hdac1ATG1 morphant embryos, revealed a consistent and robust enrichment of genes with known functions in both transcriptional control and CNS development. We then discovered within this group a set of 54 genes that exhibited consistently increased or decreased changes in transcript abundance in Hdac1-deficient embryos at 12 hpf, 18 hpf and 27 hpf. More than half of these genes are implicated in CNS development and down-regulated in Hdac1-deficient embryos. Within this set of co-regulated genes, we identified a core subset of 18 genes encoding sequence-specific DNA binding transcription factors, whose down-regulation could readily account for many of the neural specification defects that characterise the Hdac1-deficient CNS. Interestingly, the gene that exhibited the greatest-fold decrease in gene expression in hdac1-deficient embryos encodes a novel member of the methyltransferase-encoding NOL1/NOP2/Sun domain family of proteins, Nsun5, but nothing currently is known about the function of this protein.
Many of the 18 Hdac1-regulated CNS-specific transcription factor genes that were identified in our experiments play important roles in the specification of neuronal subtype identities in the spinal cord, brain and retina. Thus, dbx2, lhx9, lbx1b, gsx1, bhlhe22, ascl1a, ascl1b or their vertebrate orthologues have been implicated in neuronal specification within the spinal cord [22–26]. In the brain, neurod, neurod4 and bhlhe22 are are required for the production of cortical projection neurones [27, 28], whilst upper motoneurones of the corticospinal tract depend on fezf1 and specification of branchiomotor neurones of the mouse hindbrain requires the combined activities of ascl1 and neurod4 orthologues . In the vertebrate retina, formation of amacrine cells is positively regulated by orthologues of ascl1a/b, neurod, neurod4, foxn4, bhlhe22 and nr4a2[31–34]. Moreover, mammalian photoreceptor specification also requires neurod, crx and rx functions [35–37]. Taken together with these observations, our genome-wide expression analysis indicates that Hdac1 is deployed widely throughout the developing CNS to specify a wide variety of neuronal subtypes, by promoting the transcription of this core group of developmental regulatory genes. This conclusion was confirmed by our in situ hybridisation analysis of the expression patterns of proneural/bHLH genes ascl1b, neurod4, and neurod, along with lhx9 and the Notch ligand gene dlb. Transcription of these genes was considerably reduced throughout the Hdac1-deficient CNS, both at 12 hpf and at 27 hpf, and transient incubation of embryos in the HDAC inhibitor TSA between 10 hpf and 14 hpf almost completely extinguished their expression. By contrast, widespread expression of the neural transcription factor gene sox2 was not appreciably altered at 12 hpf, demonstrating that at this early stage, the reduced expression of ascl1b, neurod4, neurod, dlb and lhx9 in Hdac1-deficient embryos is not due to reduction in the pool of neural precursors, but rather the result of a failure to specify particular neuronal identities. Intriguingly, forced co-expression of the murine proneural gene Ascl1 (the orthologue of zebrafish ascl1a and ascl1b) with a small set of neuronal transcription factors in cultured murine fibroblasts, was recently shown to directly re-programme these cells to differentiate into distinct neuronal subtypes . Remarkably, this group of collaborating murine transcription factors, defined by Vierbuchen et al., includes close relatives of brn1 (Brn2), neurod (Neurod1), lhx9 (Lhx2) and nr4a2 (Nr2f1), all of which we have found to exhibit stringent Hdac1-dependent co-expression throughout zebrafish neurogenesis. Interestingly, the murine Ascl1 and Brn1 proteins co-regulate a neurogenic programme by co-operative binding to a conserved DNA sequence motif in the cis-regulatory regions of Delta genes . Thus, loss of brn1 and ascl1b expression in hdac1 mutant CNS could be responsible for the reduced expression of dlb that was also observed in hdac1 mutant zebrafish embryos. Overall, our analysis has uncovered a group of co-regulated neurogenic transcription factors that may function as a regulatory network driving neuronal patterning and differentiation in the CNS. It will now be of interest to investigate whether expressing particular combinations of these transcription factors in hdac1 mutant embryos can rescue defects of neuronal specification that are caused by loss of Hdac1 function.
Somewhat surprisingly, relatively few genes were identified in the time-resolved transcriptome analysis whose expression was robustly repressed by Hdac1 between 12 hpf and 27 hpf. Overall, 11 genes were identified, of mostly unknown functions, which exhibited a consistent, two-fold or greater increase in expression in Hdac1-deficient embryos across this period of development. Of these genes, 4 have been implicated previously in aspects of CNS development, raising the possibility that their increased transcription could contribute to the defects of neural development in Hdac1-deficient embryos. TRIM9, for example, encodes a brain-specific E3 ubiquitin ligase  and fjx1 encodes a Notch-regulated inhibitor of neuronal dendrite branching that is expressed in both the central and peripheral nervous systems . Our previous work demonstrated that Hdac1 repressed the Notch target her6 at 26hpf and 33hpf , but her6 was not initially identified as an Hdac1-regulated gene in the time-resolved transcriptome analysis because its transcript abundance was not statistically significantly changed in 12 hpf and 18 hpf morphant embryos. Nevertheless, whole mount in situ hybridisation to Hdac1ATG1 morphant embryos revealed that her6 expression was appreciably increased in the hindbrain and optic vesicles at 12 hpf, which was confirmed in TSA-treated embryos, and that the increased her6 expression in the hindbrain persisted in 18 hpf hdac1 morphants. Thus, we conclude that Hdac1 attenuates her6 expression in restricted regions of the CNS from early stages of neurogenesis onwards.
The role of Hdac1 in promoting transcription of genes required for CNS development
A recent microarray-based study of HDAC function in the differentiation of mouse retinal explants revealed that transcription of genes involved in promoting photoreceptor specification, such as Crx, Neurod4 and Neurod, as well as Otx2 and Nrl was rapidly suppressed within 3 hours of administering TSA to retinal explants . Moreover, attenuation of Crx and Nrl expression by TSA was independent of a requirement for protein synthesis, implying a direct role for HDAC enzymes in promoting the transcription of these genes. We observed a similarly rapid and near complete extinction of ascl1b, neurod4, dlb, neurod and lhx9 expression, when 10 hpf embryos were incubated in TSA. Interestingly, many HDACs, including HDAC1, are abundant at the promoters of active genes in mammalian T lymphocytes, and closely associated with both HATs and phosphorylated RNA Polymerase II in the transcribed regions of these genes . In yeast, HDACs biochemically oppose HAT functions by removing acetylation marks from chromatin at active genes, which facilitates their HAT-mediated re-acetylation in subsequent cycles of re-transcription. Accordingly, in yeast HDAC mutants, genes that are embedded within hyperacetylated chromatin are transcriptionally impaired [4–6], and moreover, similar observations have been made in mammalian T-cells . Thus, it seems possible that some of the hyperacetylated histones we observed in hdac1 mutant zebrafish embryos are associated with genes whose transcription has been impaired by loss of Hdac1 function. The finding that Hdac1 is stably and specifically associated with sequences within the promoter region and first exon of ascl1b in 12 hpf and 27 hpf zebrafish embryos, transcription of which is Hdac1-dependent throughout this period, identifies ascl1b as a novel in vivo direct target of Hdac1. To our knowledge, this is the first such direct in vivo target to be defined for Hdac1 in vertebrate embryos. Taken together, our results suggest that Hdac1 could promote transcription of this proneural gene directly, by removing acetyl modifications from transcription unit-associated histones to enable their transcription-coupled re-acetylation, and/or by maintaining the ascl1b promoter in a deacetylated, H3K4 methylated, transcriptionally poised state. Indeed, the fact that global H3K4 methylation levels were unchanged by loss of hdac1 function (Figure 1) suggests that Hdac1 may bind to promoters within H3K4 methylated chromatin of zebrafish embryos, thus poising them for activation . In view of the fact that our experiments were performed on whole embryos, it is possible that Hdac1 also binds to the ascl1b promoter in non-neuronal cells, although the significance of such possible interactions is unclear, as no ectopic expression of ascl1b was observed in Hdac1-deficient embryos. Future studies will aim to identify the DNA sequences within the ascl1b promoter, and the cognate DNA binding proteins, which recruit Hdac1 to this target gene, and determine whether these interactions are specific for the neuronal lineage in the developing CNS. We also intend to investigate how the distribution of epigenetic modifications across the genome is regulated by Hdac1.
Our in vivo chromatin immunoprecipitation studies detected no interactions between Hdac1 and the DNA sequences immediately upstream and downstream of the transcription start sites of neurod4, neurod, deltab and her6. Whilst it is possible that these genes are regulated by Hdac1 indirectly, it is also conceivable that Hdac1 binds directly to other cis-regulatory elements that are located further away from the transcription start sites of these genes. Another possibility is that the level of Hdac1 binding to these promoters in vivo may be below the threshold for detection in the chromatin immunoprecipitation assay. Despite the lack of Hdac1 binding to her6 promoter sequences, this gene remains a candidate direct target for direct repression by Hdac1, as Hdac1 repressed her6 expression in restricted regions of the CNS from 12 hpf onwards. Indeed, it remains possible that Hdac1-mediated repression of her6 facilitates the transcription of proneural genes such as ascl1b independently of the physical interaction between Hdac1 protein and the ascl1b promoter that we describe here. More extensive studies of Hdac1 binding to the chromatin in which these and other genes are embedded, and identification of proteins that recruit Hdac1 to its direct target genes, will allow the Hdac1-regulated genes identified by our transcriptome analysis to be evaluated further as candidate direct targets for Hdac1 binding. This information will help to define better the functional interrelationships between components of the Hdac1-regulated genetic network that promotes neurogenesis.
We demonstrate that Hdac1 is an epigenetic regulator that governs the global levels of histone acetylation and H3K9 methylation during zebrafish development. Using a sensitive, in-depth, time-resolved transcriptome analysis of the in vivo function of Hdac1 during embryogenesis, we defined a principal requirement for Hdac1 to positively regulate the co-ordinated expression of 18 sequence-specific DNA binding transcription factor genes with known roles in neural specification and patterning, including several proneural bHLH genes. Chromatin immunoprecipitation analysis of candidate Hdac1 direct target genes in developing zebrafish embryos identified stable and specific binding of Hdac1 protein to the promoter of the proneural gene ascl1b. Although it is possible that this binding of Hdac1 to ascl1b occurs in non-neuronal cells, our results show that transcription of ascl1b is exquisitely sensitive to loss of hdac1 function, implying a role for Hdac1 in promoting ascl1 transcription in the neuronal lineage. Taken together, our results suggest that in addition to its well-documented functions in transcriptional repression, Hdac1 may also facilitate the direct transcriptional activation of target genes during vertebrate embryogenesis. In the developing CNS, this role could underpin the transcriptional poising of Hdac1 target genes encoding neuronal fate determinants, where these genes are silent but competent for transcriptional activation in response to neural specification and patterning signals.
hdac1hi1618 mutant zebrafish were maintained at University of Sheffield. Individual embryos were genotyped using primers for hdac1 (Hdac1F: 5'-GGC AGG CGC AGG CTG TAA TT-3'; Hdac1 Intron1R: 5'-GGC TAA ACC CGG CTA ACA AT-3'); the 3' Long Terminal Repeat of the MLV vector (MLV 3LTR F: 5'- AAA GAC CCC ACC TGT AGG TTT G-3') . Animal care and use was in accordance with the UK Animals (Scientific Procedures) Act 1986.
Morpholino microinjection and drug treatment of embryos
Morpholino sequences were as follows: Hdac1ATG1: 5'-ttg ttc ctt gag aac tca gcg cca t-3'; Hdac1SPL1: 5'-ata ttc tta ccg tca taa taa tag c-3'; Standard Control (human β-globin): 5'-cct ctt acc tca gtt aca att tat a-3'; Hdac1 Mismatch control: 5'-ttg ctc gtt gag aac tct gca caa t-3'. 1-2nl of 0.3 mM morpholino solution in milli-Q water was microinjected into embryos at the 1-2-cell stage.
Trichostatin A (TSA) was dissolved in DMSO to 3 μM and added to E3 medium to a final concentration of 1 μM. 10 hpf wild-type embryos were incubated in E3 medium containing 1 mM TSA for four hours until 14 hpf, then samples were fixed and analysed by in situ hybridisation
Whole-mount in situ hybridization and immunostaining of embryos
Whole-mount in situ hybridisation was performed using standard procedures.
For immunostaining with anti-Hdac1 antibody (abcam ab41407), embryos were fixed overnight, dehydrated in methanol and stored at -20°C. After rehydrating and permeabilizing with acetone at -20°C for 7 minutes, embryos were blocked in PBS containing 0.5% Triton-X, 1% DMSO, 1% BSA and 2% sheep serum (PBDTss) for 2 hours at 4°C and incubated with Hdac1 antibody (ab41407, 1:100, abcam) overnight. The next day, embryos were rinsed in PBDTss and incubated with alexa488-conjugated rabbit IgG (1:500, Invitrogen) before mounting for confocal microscopy.
Western Blotting analysis of protein samples
200 μg of zebrafish embryo protein extract, corresponding to 5 whole 24hpf embryos, was separated by SDS-PAGE, transferred to Nitrocellulose (Amersham), and incubated with the following antibodies: anti-Hdac1 (ab41407, abcam), Pan-acetyl lysine (gift from C. Crane-Robinson, University of Portsmouth , H3acetylK9 (ab4441, abcam), H3dimethlyK9 (ab1220, abcam), H3trimethlyK4 (ab8580, abcam), H3 (ab1791, abcam). Signals were visualized using Horse Radish Peroxidase-conjugated secondary antibodies and the ECL system (GE Healthcare).
RNA extraction, microarray hybridisation and gene expression analysis
40-60 embryos were treated with RNAlater (Ambion) and RNA was extracted using TRIzol. Traces of DNA were removed with RNase-free DNase I and RNA was purified on RNeasy columns (QIAGEN). Cy-dye (Perkin Elmer)-labelled amino Allyl-Modified aRNA probes (Ambion Amino Allyl MessageAmp II) were synthesised and hybridized to a custom Agilent 4 × 44 K array. Hybridizations were scanned using the standard Two-Color Microarray-Based Gene Expression Analysis Protocol (publication number G4140-90050; Agilent Technologies). The custom 44 K design contained a 22 K Agilent probe set together with a 16 K Sigma-Compugen probe set and bespoke oligo list . In total the array contained probes corresponding to 18636 unique Unigene clusters and approximately 2000 additional Ensembl-annotated genes.
For the comparative analysis of 27 hpf wild-type and hdac1 mutant embryos, duplicate biological samples for each genotype were analysed in technical duplicate and with dye swaps at the probe labelling stage, giving total of 8 separate microarray datasets. For the analysis of morphant transcriptomes, biological duplicate batches of embryos injected with Hdac1ATG1, Hdac1SPL1, Hdac1Control and Standard Control morpholinos were all compared together in one microarray experiment, and for the time-resolved analysis of the Hdac1ATG1 transcriptome, biological triplicate batches of Hdac1ATG1 and Standard Control morphant embryos were compared at each time point.
To analyse the hybridisation data, the probe intensity values for each scanned Agilent chip was uploaded to the Rosetta Resolver system (Rosetta Biosoftware). Data from duplicates (technical, biological and dye-swap) were combined to give a combined fold-change and an associated p-value . In all experiments, a p-value threshold of p < or = 10-5 was used to define a probe as being significantly differentially expressed between two samples. The selection of this p-value as appropriate was determined by previous error-modelling and is an especially stringent criterion of significance . Datasets were compared using Rosetta Resolver tools and selected data were then exported. Unannotated probes were annotated using homology searches (nBLAT, Ensembl) against the zebrafish genome and associated cDNAs or GenScan sequences were interrogated with BLAST searches. The microrray datasets used in our analyses have been deposited into GEO with Accession Number GSE26710.
Gene Ontology analysis was carried out using eGOn (v2.0, NTNU Gene Tools). Probe lists of significantly Hdac1-regulated genes (p < 10-5) were compared to a master probe list comprising all probes on the array in a Master-Target analysis to identify particular Gene Ontology classes that were over- or under-represented in the Hdac1-regulated list .
Chromatin Immunoprecipitation and real-time quantitative PCR
For each batch of chromatin, three hundred wild-type (AB), hdac1 mutant, wild-type sibling, or morphant embryos were enzymatically dechorionated at 12 hpf or 27 hpf. Embryos were fixed immediately in 1.5 mM ethylene glycolbis[succinimidyl succinate] (EGS; Sigma-Aldrich) for 45 minutes, followed by 1.5% formaldehyde for 20 minutes . Glycine (0.125 M) was added to quench the formaldehyde and embryos were washed in ice cold PBS. Embryos were deyolked  and homogenised on ice in extraction buffer with protease inhibitors. Chromatin was sheared by sonication to give DNA fragments of approximately 300-700 bp in size. Sonicated samples were centrifuged at 12,000 × g at 4°C for 30 minutes, and insoluble material was discarded. The supernatant was incubated at 4°C for 3 hours with 6 μg of either anti-Hdac1 antibody (Abcam ab41407) or control IgG antibody (Abcam ab46540). 80 μl of protein G magnetic beads (Pierce) were added to each sample and the samples rotated at 4°C overnight. Beads were washed six times with RIPA buffer at 4°C and bound complexes were eluted from the beads in Elution Buffer at 65°C for 20 minutes with vortexing. Cross-links were reversed overnight at 65°C and DNA fragments were purified by treatment with RNase A, followed by proteinase K digestion, phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation. Quantitative real-time PCR (QPCR) of immuno-precipitated DNA was used to detect DNA sequences around the transcription start sites (+1) of ascl1b: between -785bp and +790 bp; dlb: between -964bp and +1018 bp; neurod4: between -803bp and +856 bp; her6: between -838bp and +825 bp. QPCR analyses were performed in triplicate and DNA abundance was normalised against Input values.
This research was funded by a Wellcome Trust Project Grant (WT081884MA) to V.T.C. M.R.M.H. was supported by an MRC postgraduate studentship. Confocal microscopy and zebrafish facilities were supported through MRC Centre awards to Professor P.W. Ingham (Grants G0400100 and G0700091), and also by a grant from The Wellcome Trust (GR077544AIA) to support the core BMS-MBB Light Microscopy Facility.
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