hamlet: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References
Gene name - hamlet

Synonyms -

Cytological map position - 37A2--3

Function - transcription factor

Keywords - dendrite morphogenesis, peripheral nervous system

Symbol - ham

FlyBase ID: FBgn0045852

Genetic map position - 2L

Classification - C2H2 zinc finger protein

Cellular location - nuclear



NCBI links: Entrez Gene | UniGene |
BIOLOGICAL OVERVIEW

Recent literature
Nagy, V., Cole, T., Van Campenhout, C., Khoung, T. M., Leung, C., Vermeiren, S., Novatchkova, M., Wenzel, D., Cikes, D., Polyansky, A. A., Kozieradzki, I., Meixner, A., Bellefroid, E. J., Neely, G. G. and Penninger, J. M. (2015). The evolutionarily conserved transcription factor PRDM12 controls sensory neuron development and pain perception. Cell Cycle: [Epub ahead of print]. PubMed ID: 25891934
Summary:
PR homology domain-containing member 12 (PRDM12) belongs to a family of conserved transcription factors implicated in cell fate decisions. This study show that PRDM12 is a key regulator of sensory neuronal specification in Xenopus. Modeling of human PRDM12 mutations that cause hereditary sensory and autonomic neuropathy (HSAN) revealed remarkable conservation of the mutated residues in evolution. Expression of wild-type human PRDM12 in Xenopus induced the expression of sensory neuronal markers, which was reduced using various human PRDM12 mutants. In Drosophila, Hamlet as identified was the functional PRDM12 homologue that controls nociceptive behavior in sensory neurons. Furthermore, expression analysis of human patient fibroblasts with PRDM12 mutations uncovered possible downstream target genes. Knockdown of several of these target genes including thyrotropin-releasing hormone degrading enzyme (TRHDE) in Drosophila sensory neurons resulted in altered cellular morphology and impaired nociception. These data show that PRDM12 and its functional fly homologue Hamlet are evolutionary conserved master regulators of sensory neuronal specification and play a critical role in pain perception. These data also uncover novel pathways in multiple species that regulate evolutionary conserved nociception.

The dendritic morphology of neurons determines the number and type of inputs they receive. In the Drosophila peripheral nervous system (PNS), the external sensory (ES) neurons have a single nonbranched dendrite, whereas the lineally related multidendritic (MD) neurons have extensively branched dendritic arbors. hamlet, coding for a zinc finger transcription factor, is a binary genetic switch between these contrasting morphological types. In hamlet mutants, ES neurons are converted to an MD fate, whereas ectopic hamlet expression in MD precursors results in transformation of MD neurons into ES neurons. Moreover, hamlet expression induced in MD neurons undergoing dendrite outgrowth drastically reduces arbor branching (Moore, 2002).

In Drosophila embryos, a single external sensory organ precursor (ESOP) cell gives rise to an ES neuron and an MD neuron through a stereotypical series of asymmetric cell divisions. The IIA daughter of the ESOP divides once to produce a trichogen and a tormagen, the two external support cells of the ES neuron. The IIB daughter of the ESOP generates an MD neuron and the IIIB cell, which divides to form an ES neuron and a glia (Moore, 2002).

In a genetic screen designed to identify mutants in Drosophila embryonic dendrite development, a larval lethal mutant was isolated that appeared to affect determination of cells descended from the IIB precursor of the ESOP lineage. This mutation was named hamlet (ham) after the 'To be or not to be' soliloquy in the Shakespeare play of the same name (Moore, 2002).

In embryos homozygous for the ham1 mutation, supernumerary MD neurons are evident in each PNS cluster of the embryo, as well as a concomitant decrease in the number of ES neurons. For example, in the wild-type dorsal PNS cluster there are 13 neurons: five ES neurons, which express the pan-neural marker ELAV (Embryonic lethal, abnormal vision), and eight MD neurons, which express ELAV and the enhancer-trap E7-2-36, a pan-MD marker. In ham1 mutants, however, the number of MD neurons is increased up to 13 and the number of ES neurons decreases (Moore, 2002).

Do these extra MD neurons arise at the expense of their sibling ES neurons? To answer this question, focus was placed on the ventral pore sensory organs (vp1 to vp4a) because their lineage is fully described and each organ develops clearly spaced from those surrounding it. These organs were labeled with an antibody to Cut, which is expressed in all cells of the lineage. In the five differentiated cells of the lineage, Cut is expressed at low levels in the ES and MD neurons and at a much higher level in the trichogen, tormagen, and glia. In addition, labeling was performed with antibodies to the MD marker E7-2-36 and Prospero (Pros), which is expressed in the IIB cell and its descendants and remains expressed in the differentiated glia. In ham1 mutants the vp ESOP lineage division pattern appears normal and produces five cells; however, in the differentiated organ of these mutants, the ES neuron and glia are lost. A second MD neuron appears in the position normally occupied by the ES neuron, and a third external cell (trichogen) in the position normally occupied by the glia. Taken together, these observations indicate that the daughter cells of the IIIB cell are transformed to an MD neuron and a trichogen in ham1 mutants (Moore, 2002).

To test whether the supernumerary MD neurons can extend the characteristic complex branched dendritic arbor, ham1/ham1 MARCM (mosaic analysis with a repressible cell marker) clones were generated in the ESOP lineage. In this analysis, clones of positively marked neurons are derived from a single mitotic recombination event within the ESOP lineage. In wild-type clones of all PNS clusters, MD neurons either label alone, representing a clone derived from recombination within the IIB cell, or in association with an ES neuron, representing recombination at the level of the ESOP cell or its precursors. In ham1 homozygous clones of all PNS clusters, either one or two MD neurons are labeled. In the latter case, one of these neurons must represent the transformed ES cell. Both of these neurons have the arbors characteristic of MD neurons specific to the location at which they arise. Thus, in ham1 the MD neuron transformed from an ES neuron has a full MD arbor morphology. Additionally, this analysis also shows that ham functions in a cell-autonomous manner within the ESOP lineage (Moore, 2002).

To determine whether ectopic Ham expression can alter cell fate, the full-length ham cDNA was cloned into the pUAST vector and UAS-ham transgenic flies were created. Ectopic Ham expression in the IIB precursor of the MD neuron using pros-gal4 resulted in loss of labeling for the E7-2-36 MD marker in all embryonic PNS neurons, indicating an MD-to-ES marker transformation. To test whether the dendrite morphology of these neurons also reflects an MD-to-ES transformation, elav-gal4 driving UAS-gfp was used to visualize dendrites. In the wild-type dorsal cluster of stage 17 embryos, there are several neurons with multiply branched dendrites (i.e., MDs) and two dorsal ES neurons, each with a single dorsally projecting dendrite. In embryos where both pros-gal4 and elav-gal4 drive UAS-ham and UAS-mCD8-gfp in parallel, the multiple dendritic arbor of the MD neurons has clearly been transformed; the dendrites of all neurons in the cluster are unbranched and project dorsally (Moore, 2002).

Given that Ham is expressed in the postmitotic ES neuron during dendrite extension, whether postmitotic expression of Ham in an MD neuron can alter its dendrite morphology was investigated. elav-gal4 was used to drive UAS-ham and UAS-mCD8-gfp in parallel in the embryo. Indeed, postmitotic expression of Ham in the MD neurons drastically reduces dendritic branching, leading to arbors with a structure intermediate between that of MD and ES neurons. In addition, these neurons still express the MD marker E7-2-36 at high levels, indicating that these neurons have transformed dendrite morphology but not cell fate. To investigate this effect further, the MD-specific driver 109(2)80-gal4 was used to drive UAS-gfp and UAS-ham. In these embryos 109(2)80-gal4 remains active, implying that the neurons in which it is expressed remain MD; however, the branching of dendrites in these MD neurons is clearly reduced. These two lines of evidence illustrate that postmitotic expression of Ham in MD neurons does not switch the fate of these neurons to ES but does still act to suppress the formation of complex dendritic arbors (Moore, 2002).

The PR domain and bipartite multiple ZF structure define a small family of proteins, of which Ham is the sole member described in Drosophila . The sequence of the PR domain and ZFs as well as the overall domain structure are conserved between Ham and two other proteins, human MDS1/EVI1 (myelodysplasia syndrome 1/ectopic viral integration 1) and Caenorhabditis elegans Egl43 (egg laying defective 43). Both are implicated in neural development. MDS1/EVI1 was originally isolated because ectopic expression of EVI1 alone (leading to a protein still containing the ZFs but lacking the PR domain) can cause leukemia. A partial disruption of the Mds1/Evi1 locus in mouse, however, leads to mid-gestation lethality. Among multiple defects in these embryos are regions of hypocellularity in the neuroectoderm and a failure of peripheral nerve formation. Egl43 is required for hermaphrodite-specific neuron migration and phagemid sensory neuron development. The defect in phagemid neuron development is intriguing because these neurons fail to fill with dye through normally exposed sensilla, a phenotype that could be attributable to a failure of correct dendrite formation. Thus, it will be interesting to investigate whether MDS1/EVI1 or Egl43, like ham, have a role in dendrite specification (Moore, 2002).

As far as is known, ham represents the only binary genetic switch identified that acts to repress a multiple dendritic arbor and promote single-dendrite morphology. It is expected that Ham is a transcription factor; it has a nuclear subcellular distribution, and MDS1/EVI1 can bind DNA and regulate transcription. Ham must exert its effect in a short developmental window, because its expression is initiated in the IIIB neural precursor and continues only during the initial stages of ES neuron differentiation. Given this short period of activity, it is likely that Ham induces a cascade of events. Its transcriptional targets are likely to include key players in the genetic determination of dendrite morphology that act to repress dendritic branching and promote single- over multiple-dendrite morphology (Moore, 2002).

Chromatin modification of Notch targets in olfactory receptor neuron diversification

Neuronal-class diversification is central during neurogenesis. This requirement is exemplified in the olfactory system, which utilizes a large array of olfactory receptor neuron (ORN) classes. An epigenetic mechanism was discovered in which neuron diversity is maximized via locus-specific chromatin modifications that generate context-dependent responses from a single, generally used intracellular signal. Each ORN in Drosophila acquires one of three basic identities defined by the compound outcome of three iterated Notch signaling events during neurogenesis. Hamlet, the Drosophila Evi1 and Prdm16 proto-oncogene homolog, modifies cellular responses to these iteratively used Notch signals in a context-dependent manner, and controls odorant receptor gene choice and ORN axon targeting specificity. In nascent ORNs, Hamlet erases the Notch state inherited from the parental cell, enabling a modified response in a subsequent round of Notch signaling. Hamlet directs locus-specific modifications of histone methylation and histone density and controls accessibility of the DNA-binding protein Suppressor of Hairless at the Notch target promoter (Endo, 2011).

This study analyzed the ORN lineage history that gives rise to three primary ORN identities (Naa, Nab and Nba). These three identities arise in a sensillum via iterated rounds of Notch-mediated binary cell-fate decisions. Together with previous findings, these results suggest that diversification of Drosophila ORN classes is the result of the combined output of two predominantly hardwired mechanisms; spatially localized factors determine at least 21 types of sensilla, and Notch and Ham then act in each sensillum to maximize ORN class variety (Endo, 2011).

Biochemical and molecular analyses of Ham function indicate that it can repress Notch target enhancers. In the ORN lineage, Notch signaling is used in consecutive cell fate decisions, and it was found that Ham acts to turn off Notch targets before a subsequent a round of selective reactivation. Ham is expressed specifically in pNa, the neuronal-intermediate precursor with high Notch activity, and inherited by both of the pNa daughter cells. In addition to the current findings, studies in other contexts have observed that some Notch targets require a Notch signal for their transcriptional induction, but not for maintaining their expression. These Notch targets could aberrantly persist in both pNa progeny without the intervention of a mechanism to erase the effects of the preceding Notch signal (Endo, 2011).

ham mutants showed an unusual ORN fate switch. They not only transformed ORN fate with respect to Notch state, but also altered sublineage-specific identity (low-Notch Nab to high-Notch Nba identity). This phenotype suggests that, in addition to suppressing the previous round of Notch activation, Ham may delineate the selection of the next round of targets. As Ham activity resulted in altered chromatin modifications at Notch targets, this suggests that Ham could set an epigenetic context in which the terminal round of Notch signaling occurs (Endo, 2011).

Although this was demonstrated with respect to Ham, it is suggested that this approach to modifying the transcriptional outputs of a signaling pathway may have widespread importance in other lineages that utilize iterative signals. Notch signaling iteration is a widespread phenomenon. One important example is in the maintenance of neural and other stem cells, and it is now known that some chromatin-modifying factors promote stem cell self-renewal. Notably, several Prdm factors have regionalized expression in neural precursor domains of the embryonic mouse spinal cord and could modify and diversify stem cell identity during mammalian CNS development (Endo, 2011).

In Drosophila, Notch signaling and Ham expression are transient in nascent ORNs. Thus, Notch- and Ham-mediated fate choices must be perpetuated during the later selection of alternative axon guidance factors and odorant receptors. It is possible that chromatin methylation not only sets the context of immediate Notch signaling outcomes, but also maintains initial fate choice by priming or silencing promoters for readout during differentiation. The existence of such mechanisms in neural development is now beginning to emerge. It was recently shown that, in mouse cortical precursor cells, the trithorax factor Mixed-lineage leukemia1 (Mll1) prevents epigenetic silencing of the neural differentiation gene Distal-less homeobox 2 (Dlx2), enabling it to be properly upregulated during differentiation stages. In contrast, in the mammalian olfactory system, epigenetic repression is used during the transition from multipotent precursor to immature ORN to silence all ~2,800 odorant receptor genes before subsequent de-repression of a single odorant receptor per neuron (Endo, 2011).

To determine how chromatin modifications create a context-dependent outcome from signaling and how resultant cell-fate choices are perpetuated during Drosophila ORN differentiation, it will be necessary to elucidate the components and action of the chromatin-modification complex targeted by Ham. Furthermore, genome-wide identification of the promoters targeted by Su(H) and Ham will reveal the genes regulated by these factors to confer specific ORN fates (Endo, 2011).


GENE STRUCTURE

cDNA clone length - 2973bp (Entree Nucleotide)

Exons - 3

PROTEIN STRUCTURE

Amino Acids - 910

Structural Domains

To determine the identity of the ham gene, genetic complementation was used to map the ham1 mutation between the tup and msl1 genes on the second chromosome. Because the Gadfly database predicts 27 putative transcripts between tup and msl1, a polymerase chain reaction was used to amplify digoxigenin-labeled DNA probes representing each one of these transcripts from embryonic cDNA; these probes were then used for mRNA in situ hybridization of a stage 0 to stage 16 embryo collection. Of these 27 putative transcripts, only CG10568 is expressed in the developing PNS of the embryo. It was expected that ham expression might be largely PNS specific because ham1 mutants have no defects in muscle, gut, trachea, central nervous system, or cuticle formation, and because ham functions in a cell-autonomous manner within the PNS ESOP lineage. CG10568 was therefore persued as the candidate likely to be disrupted in these mutants (Moore, 2002).

Two lines of investigation have revealed that CG10568 corresponds to the ham transcript. In the first, probes derived from CG10568 were used to isolate from an embryonic library (LD) a full-length cDNA corresponding to eight exons spanning about 25 kb of genomic DNA. The predicted protein is 990 amino acids with a mass of 109 kD. It contains an NH2-terminal PRD1-BF1-RIZ1 homology (PR) domain followed by a group of six zinc fingers (ZF) and a group of three additional ZFs at the COOH-terminus. Sequencing of the genomic region encoding this transcript in ham1 mutants has revealed a single base pair loss at G2866, which leads to an altered amino acid composition for 41 residues after the mutation site and a truncation that removes the three COOH-terminal ZFs. This mutation thus gives rise to a protein predicted to be 88 kD. Polyclonal antibodies were raised in guinea pigs against a unique 135-amino acid portion of the predicted Ham protein. On a Western blot, the antibody detected a protein of about 110 kD in wild-type embryos. In ham1 heterozygotes the same band was visible in addition to a protein band at about 90 kD, most likely corresponding to the truncated protein produced by the ham1 allele (Moore, 2002).

Second, double-stranded RNA interference (dsRNAi) could phenocopy the ham1 mutation. dsRNA corresponding to the same sequence used in the CG10568 mRNA in situ was injected into the posterior pole of embryos carrying the MD marker 109(2)80-gal4 driving UAS-gfp or the MD marker E7-2-36. In embryos containing 109(2)80-gal4 driving UAS-gfp, CG10568 dsRNAi leads to a consistent and significant increase in the number of MD neurons in the dorsal cluster of these embryos (up to 11 neurons) compared with uninjected embryos (8 neurons). This increase is at the expense of ES neurons, as revealed by CG10568 dsRNAi in embryos labeled with antibodies to the MD marker E7-2-36 and the pan-neural marker ELAV (Moore, 2002).

These findings verify that CG10568 corresponds to the ham gene. The ham1 allele is either a genetic null or strong hypomorph, as embryos containing one copy of the ham1 mutation and one copy of a deficiency (Df(2L)OD15) deleting the gene have the same highly penetrant phenotype as ham1 homozygotes. Moreover, dsRNAi with ham RNA abolishes all Ham protein expression in the injected embryo and phenocopies the ham1 allele (Moore, 2002).


hamlet: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

date revised: 30 September 2003

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