KDM5-mediated activation of genes required for mitochondrial biology is necessary for viability in Drosophila.

Histone-modifying proteins play important roles in the precise regulation of the transcriptional programs that coordinate development. KDM5 family proteins interact with chromatin through demethylation of H3K4me3 as well as demethylase-independent mechanisms that remain less understood. To gain fundamental insights into the transcriptional activities of KDM5 proteins, we examined the essential roles of the single Drosophila Kdm5 ortholog during development. KDM5 performs crucial functions in the larval neuroendocrine prothoracic gland, providing a model to study its role in regulating key gene expression programs. Integrating genome binding and transcriptomic data, we identify that KDM5 regulates the expression of genes required for the function and maintenance of mitochondria, and we find that loss of KDM5 causes morphological changes to mitochondria. This is key to the developmental functions of KDM5, as expression of the mitochondrial biogenesis transcription factor Ets97D, homolog of GABPα, is able to suppress the altered mitochondrial morphology as well as the lethality of Kdm5 null animals. Together, these data establish KDM5-mediated cellular functions that are important for normal development and could contribute to KDM5-linked disorders when dysregulated.

KDM5-mediated transcriptional activation of ribosomal protein genes alters translation efficiency to regulate mitochondrial metabolism in neurons.

Genes encoding the KDM5 family of transcriptional regulators are disrupted in individuals with intellectual disability (ID). To understand the link between KDM5 and ID, we characterized five Drosophila strains harboring missense alleles analogous to those observed in patients. These alleles disrupted neuroanatomical development, cognition and other behaviors, and displayed a transcriptional signature characterized by the downregulation of many ribosomal protein genes. A similar transcriptional profile was observed in KDM5C knockout iPSC-induced human glutamatergic neurons, suggesting an evolutionarily conserved role for KDM5 proteins in regulating this class of gene. In Drosophila, reducing KDM5 changed neuronal ribosome composition, lowered the translation efficiency of mRNAs required for mitochondrial function, and altered mitochondrial metabolism. These data highlight the cellular consequences of altered KDM5-regulated transcriptional programs that could contribute to cognitive and behavioral phenotypes. Moreover, they suggest that KDM5 may be part of a broader network of proteins that influence cognition by regulating protein synthesis.

The histone demethylase KDM5 controls developmental timing in Drosophila by promoting prothoracic gland endocycles.

In Drosophila, the larval prothoracic gland integrates nutritional status with developmental signals to regulate growth and maturation through the secretion of the steroid hormone ecdysone. While the nutritional signals and cellular pathways that regulate prothoracic gland function are relatively well studied, the transcriptional regulators that orchestrate the activity of this tissue remain less characterized. Here, we show that lysine demethylase 5 (KDM5) is essential for prothoracic gland function. Indeed, restoring kdm5 expression only in the prothoracic gland in an otherwise kdm5 null mutant animal is sufficient to rescue both the larval developmental delay and the pupal lethality caused by loss of KDM5. Our studies show that KDM5 functions by promoting the endoreplication of prothoracic gland cells, a process that increases ploidy and is rate limiting for the expression of ecdysone biosynthetic genes. Molecularly, we show that KDM5 activates the expression of the receptor tyrosine kinase torso, which then promotes polyploidization and growth through activation of the MAPK signaling pathway. Taken together, our studies provide key insights into the biological processes regulated by KDM5 and expand our understanding of the transcriptional regulators that coordinate animal development.

Caregiver-reported characteristics of children diagnosed with pathogenic variants in KDM5C.

Loss of function variants in the lysine demethylase 5C (KDM5C) gene account for approximately 0.7-2.8% of X-linked intellectual disability (ID) cases and pose significant burdens for patients and their caregivers. To date, 45 unique variants in KDM5C have been reported in individuals with ID. As a rare disorder, its etiology and natural history remain an area of active investigation, with treatment limited to symptom management. Previous studies have found that males present with moderate to severe ID with significant syndromic comorbidities such as epilepsy, short stature, and craniofacial abnormalities. Although not as well characterized, females have been reported to predominantly display mild to moderate ID with approximately half being asymptomatic. Here, we present caregiver-reported data for 37 unrelated individuals with pathogenic variants in KDM5C; the largest cohort reported to-date. We find that up to 70% of affected females were reported to display syndromic features including gastrointestinal dysfunction and hearing impairment. Additionally, more than half of individuals reported a diagnosis of autism spectrum disorder or described features consistent with this spectrum. Our data thus provide further evidence of sexually dimorphic heterogeneity in disease presentation and suggest that pathogenic variants in KDM5C may be more common than previously assumed.

KDM5 interacts with Foxo to modulate cellular levels of oxidative stress.

Increased cellular levels of oxidative stress are implicated in a large number of human diseases. Here we describe the transcription co-factor KDM5 (also known as Lid) as a new critical regulator of cellular redox state. Moreover, this occurs through a novel KDM5 activity whereby it alters the ability of the transcription factor Foxo to bind to DNA. Our microarray analyses of kdm5 mutants revealed a striking enrichment for genes required to regulate cellular levels of oxidative stress. Consistent with this, loss of kdm5 results in increased sensitivity to treatment with oxidizers, elevated levels of oxidized proteins, and increased mutation load. KDM5 activates oxidative stress resistance genes by interacting with Foxo to facilitate its recruitment to KDM5-Foxo co-regulated genes. Significantly, this occurs independently of KDM5's well-characterized demethylase activity. Instead, KDM5 interacts with the lysine deacetylase HDAC4 to promote Foxo deacetylation, which affects Foxo DNA binding.

The Drosophila ubiquitin-specific protease Puffyeye regulates dMyc-mediated growth.

The essential and highly conserved role of Myc in organismal growth and development is dependent on the control of Myc protein abundance. It is now well established that Myc levels are in part regulated by ubiquitin-dependent proteasomal degradation. Using a genetic screen for modifiers of Drosophila Myc (dMyc)-induced growth, we identified and characterized a ubiquitin-specific protease (USP), Puffyeye (Puf), as a novel regulator of dMyc levels and function in vivo. We show that puf genetically and physically interacts with dMyc and the ubiquitin ligase archipelago (ago) to modulate a dMyc-dependent cell growth phenotype, and that varying Puf levels in both the eye and wing phenocopies the effects of altered dMyc abundance. Puf containing point mutations within its USP enzymatic domain failed to alter dMyc levels and displayed no detectable phenotype, indicating the importance of deubiquitylating activity for Puf function. We find that dMyc induces Ago, indicating that dMyc triggers a negative-feedback pathway that is modulated by Puf. In addition to its effects on dMyc, Puf regulates both Ago and its cell cycle substrate Cyclin E. Therefore, Puf influences cell growth by controlling the stability of key regulatory proteins.

Myc in stem cell behaviour: insights from Drosophila.

The Myc family proteins are key regulators of animal growth and development, which have critical roles in modulating stem cell behaviour. Since the identification of the oncogenic potential of c-Myc in the early 1980s the mammalian Myc family, which is comprised of c-Myc, N-Myc, and L-Myc, has been studied extensively. dMyc, the only Drosophila member of the Myc gene family, is orthologous to the mammalian c-Myc oncoprotein. Here we discuss key studies addressing the function of the Myc family in stem cell behaviour in both Drosophila Models and mammalian systems.

Essential functions of the histone demethylase lid.

Drosophila Little imaginal discs (Lid) is a recently described member of the JmjC domain class of histone demethylases that specifically targets trimethylated histone H3 lysine 4 (H3K4me3). To understand its biological function, we have utilized a series of Lid deletions and point mutations to assess the role that each domain plays in histone demethylation, in animal viability, and in cell growth mediated by the transcription factor dMyc. Strikingly, we find that lid mutants are rescued to adulthood by either wildtype or enzymatically inactive Lid expressed under the control of its endogenous promoter, demonstrating that Lid's demethylase activity is not essential for development. In contrast, ubiquitous expression of UAS-Lid transgenes lacking its JmjN, C-terminal PHD domain, and C(5)HC(2) zinc finger were unable to rescue lid homozygous mutants, indicating that these domains carry out Lid's essential developmental functions. Although Lid-dependent demethylase activity is not essential, dynamic removal of H3K4me3 may still be an important component of development, as we have observed a genetic interaction between lid and another H3K4me3 demethylase, dKDM2. We also show that Lid's essential C-terminal PHD finger binds specifically to di- and trimethylated H3K4 and that this activity is required for Lid to function in dMyc-induced cell growth. Taken together, our findings highlight the importance of Lid function in the regulated removal and recognition of H3K4me3 during development.

Proximity labeling reveals a new in vivo network of interactors for the histone demethylase KDM5.

BACKGROUND: KDM5 family proteins are multi-domain regulators of transcription that when dysregulated contribute to cancer and intellectual disability. KDM5 proteins can regulate transcription through their histone demethylase activity in addition to demethylase-independent gene regulatory functions that remain less characterized. To expand our understanding of the mechanisms that contribute to KDM5-mediated transcription regulation, we used TurboID proximity labeling to identify KDM5-interacting proteins. RESULTS: Using Drosophila melanogaster, we enriched for biotinylated proteins from KDM5-TurboID-expressing adult heads using a newly generated control for DNA-adjacent background in the form of dCas9:TurboID. Mass spectrometry analyses of biotinylated proteins identified both known and novel candidate KDM5 interactors, including members of the SWI/SNF and NURF chromatin remodeling complexes, the NSL complex, Mediator, and several insulator proteins. CONCLUSIONS: Combined, our data shed new light on potential demethylase-independent activities of KDM5. In the context of KDM5 dysregulation, these interactions may play key roles in the alteration of evolutionarily conserved transcriptional programs implicated in human disorders.

DVT: a high-throughput analysis pipeline for locomotion and social behavior in adult Drosophila melanogaster.

BACKGROUND: Drosophila melanogaster is excellent animal model for understanding the molecular basis of human neurological and motor disorders. The experimental conditions and chamber design varied between studies. Moreover, most previously established paradigms focus on fly trace detection algorithm development. A comprehensive understanding on how fly behaves in the chamber is still lacking. RESULTS: In this report, we established 74 unique behavior metrics quantifying spatiotemporal characteristics of adult fly locomotion and social behaviors, of which 49 were newly proposed. By the aiding of the developed analysis pipeline, Drosophila video tracking (DVT), we identified siginificantly different patterns of fly behavior confronted with different chamber height, fly density, illumination and experimental time. Meanwhile, three fly strains which are widely used as control lines, Canton-S(CS), w1118 and Oregon-R (OR), were found to exhibit distinct motion explosiveness and exercise endurance. CONCLUSIONS: We believe the proposed behavior metrics set and pipeline should help identify subtle spatial and temporal differences of drosophila behavior confronted with different environmental factors or gene variants.

Expression of retrotransposons contributes to aging in Drosophila.

Retrotransposons are a class of transposable elements capable of self-replication and insertion into new genomic locations. Across species, the mobilization of retrotransposons in somatic cells has been suggested to contribute to the cell and tissue functional decline that occurs during aging. Retrotransposons are broadly expressed across cell types, and de novo insertions have been observed to correlate with tumorigenesis. However, the extent to which new retrotransposon insertions occur during normal aging and their effect on cellular and animal function remains understudied. Here, we use a single nucleus whole genome sequencing approach in Drosophila to directly test whether transposon insertions increase with age in somatic cells. Analyses of nuclei from thoraces and indirect flight muscles using a newly developed pipeline, Retrofind, revealed no significant increase in the number of transposon insertions with age. Despite this, reducing the expression of two different retrotransposons, 412 and Roo, extended lifespan, but did not alter indicators of health such as stress resistance. This suggests a key role for transposon expression and not insertion in regulating longevity. Transcriptomic analyses revealed similar changes to gene expression in 412 and Roo knockdown flies and highlighted changes to genes involved in proteolysis and immune function as potential contributors to the observed changes in longevity. Combined, our data show a clear link between retrotransposon expression and aging.

Molecular and cellular events linking variants in the histone demethylase KDM5C to the intellectual disability disorder Claes-Jensen syndrome.

The widespread availability of genetic testing for those with neurodevelopmental disorders has highlighted the importance of many genes necessary for the proper development and function of the nervous system. One gene found to be genetically altered in the X-linked intellectual disability disorder Claes-Jensen syndrome is KDM5C, which encodes a histone demethylase that regulates transcription by altering chromatin. While the genetic link between KDM5C and cognitive (dys)function is clear, how KDM5C functions to control transcriptional programs within neurons to impact their growth and activity remains the subject of ongoing research. Here, we review our current knowledge of Claes-Jensen syndrome and discuss important new data using model organisms that have revealed the importance of KDM5C in regulating aspects of neuronal development and function. Continued research into the molecular and cellular activities regulated by KDM5C is expected to provide critical etiological insights into Claes-Jensen syndrome and highlight potential targets for developing therapies to improve the quality of life of those affected.

A KDM5-Prospero transcriptional axis functions during early neurodevelopment to regulate mushroom body formation.

Mutations in the lysine demethylase 5 (KDM5) family of transcriptional regulators are associated with intellectual disability, yet little is known regarding their spatiotemporal requirements or neurodevelopmental contributions. Utilizing the mushroom body (MB), a major learning and memory center within the Drosophila brain, we demonstrate that KDM5 is required within ganglion mother cells and immature neurons for proper axogenesis. Moreover, the mechanism by which KDM5 functions in this context is independent of its canonical histone demethylase activity. Using in vivo transcriptional and binding analyses, we identify a network of genes directly regulated by KDM5 that are critical modulators of neurodevelopment. We find that KDM5 directly regulates the expression of prospero, a transcription factor that we demonstrate is essential for MB morphogenesis. Prospero functions downstream of KDM5 and binds to approximately half of KDM5-regulated genes. Together, our data provide evidence for a KDM5-Prospero transcriptional axis that is essential for proper MB development.

The histone demethylase KDM5 is required for synaptic structure and function at the Drosophila neuromuscular junction.

Mutations in the genes encoding the lysine demethylase 5 (KDM5) family of histone demethylases are observed in individuals with intellectual disability (ID). Despite clear evidence linking KDM5 function to neurodevelopmental pathways, how this family of proteins impacts transcriptional programs to mediate synaptic structure and activity remains unclear. Using the Drosophila larval neuromuscular junction (NMJ), we show that KDM5 is required presynaptically for neuroanatomical development and synaptic function. The Jumonji C (JmjC) domain-encoded histone demethylase activity of KDM5, which is expected to be diminished by many ID-associated alleles, is required for appropriate synaptic morphology and neurotransmission. The activity of the C5HC2 zinc finger is also required, as an ID-associated mutation in this motif reduces NMJ bouton number, increases bouton size, and alters microtubule dynamics. KDM5 therefore uses demethylase-dependent and independent mechanisms to regulate NMJ structure and activity, highlighting the complex nature by which this chromatin modifier carries out its neuronal gene-regulatory programs.

Drosophila Histone Demethylase KDM5 Regulates Social Behavior through Immune Control and Gut Microbiota Maintenance.

Loss-of-function mutations in the histone demethylases KDM5A, KDM5B, or KDM5C are found in intellectual disability (ID) and autism spectrum disorders (ASD) patients. Here, we use the model organism Drosophila melanogaster to delineate how KDM5 contributes to ID and ASD. We show that reducing KDM5 causes intestinal barrier dysfunction and changes in social behavior that correlates with compositional changes in the gut microbiota. Therapeutic alteration of the dysbiotic microbiota through antibiotic administration or feeding with a probiotic Lactobacillus strain partially rescues the behavioral, lifespan, and cellular phenotypes observed in kdm5-deficient flies. Mechanistically, KDM5 was found to transcriptionally regulate component genes of the immune deficiency (IMD) signaling pathway and subsequent maintenance of host-commensal bacteria homeostasis in a demethylase-dependent manner. Together, our study uses a genetic approach to dissect the role of KDM5 in the gut-microbiome-brain axis and suggests that modifying the gut microbiome may provide therapeutic benefits for ID and ASD patients.

Abnormalities in cell proliferation and apico-basal cell polarity are separable in Drosophila lgl mutant clones in the developing eye.

In homozygous mutants of Drosophila lethal-2-giant larvae (lgl), tissues lose apico-basal cell polarity and exhibit ectopic proliferation. Here, we use clonal analysis in the developing eye to investigate the effect of lgl null mutations in the context of surrounding wild-type tissue. lgl- clones in the larval eye disc exhibit ectopic expression of the G1-S regulator, Cyclin E, and ectopic proliferation, but do not lose apico-basal cell polarity. Decreasing the perdurance of Lgl protein in larval eye disc clones, by forcing extra proliferation of lgl- tissue (using a Minute background), leads to a loss in cell polarity and to more extreme ectopic cell proliferation. Later in development at the pupal stage, lgl mutant photoreceptor cells show aberrant apico-basal cell polarity, but this is not associated with ectopic proliferation, presumably because cells are differentiated. Thus in a clonal context, the ectopic proliferation and cell polarity defects of lgl- mutants are separable. Furthermore, lgl- mosaic eye discs have alterations in the normal patterns of apoptosis: in larval discs some lgl- and wild-type cells at the clonal boundary undergo apoptosis and are excluded from the epithelia, but apoptosis is decreased elsewhere in the disc, and in pupal retinas lgl- tissue shows less apoptosis.

The transcriptional repressor dMnt is a regulator of growth in Drosophila melanogaster.

The Myc-Max-Mad/Mnt network of transcription factors has been implicated in oncogenesis and the regulation of proliferation in vertebrate cells. The identification of Myc and Max homologs in Drosophila melanogaster has demonstrated a critical role for dMyc in cell growth control. In this report, we identify and characterize the third member of this network, dMnt, the sole fly homolog of the mammalian Mnt and Mad family of transcriptional repressors. dMnt possesses two regions characteristic of Mad and Mnt proteins: a basic helix-loop-helix-zipper domain, through which it dimerizes with dMax to form a sequence-specific DNA binding complex, and a Sin-interacting domain, which mediates interaction with the dSin3 corepressor. Using the upstream activation sequence/GAL4 system, we show that expression of dMnt results in an inhibition of cellular growth and proliferation. Furthermore, we have generated a dMnt null allele, which results in flies with larger cells, increased weight, and decreased life span compared to wild-type flies. Our results demonstrate that dMnt is a transcriptional repressor that regulates D. melanogaster body size.

The Histone Demethylase KDM5 Is Essential for Larval Growth in Drosophila.

Regulated gene expression is necessary for developmental and homeostatic processes. The KDM5 family of transcriptional regulators are histone H3 lysine 4 demethylases that can function through both demethylase-dependent and -independent mechanisms. While loss and overexpression of KDM5 proteins are linked to intellectual disability and cancer, respectively, their normal developmental functions remain less characterized. Drosophila melanogaster provides an ideal system to investigate KDM5 function, as it encodes a single ortholog in contrast to the four paralogs found in mammalian cells. To examine the consequences of complete loss of KDM5, we generated a null allele of Drosophila kdm5, also known as little imaginal discs (lid), and show that it is essential for viability. Animals lacking KDM5 show a dramatically delayed larval development that coincides with decreased proliferation and increased cell death in wing imaginal discs. Interestingly, this developmental delay is independent of the well-characterized Jumonji C (JmjC) domain-encoded histone demethylase activity of KDM5, suggesting key functions for less characterized domains. Consistent with the phenotypes observed, transcriptome analyses of kdm5 null mutant wing imaginal discs revealed the dysregulation of genes involved in several cellular processes, including cell cycle progression and DNA repair. Together, our analyses reveal KDM5 as a key regulator of larval growth and offer an invaluable tool for defining the biological activities of KDM5 family proteins.

A Drosophila Model of Intellectual Disability Caused by Mutations in the Histone Demethylase KDM5.

Mutations in KDM5 family histone demethylases cause intellectual disability in humans. However, the molecular mechanisms linking KDM5-regulated transcription and cognition remain unknown. Here, we establish Drosophila as a model to understand this connection by generating a fly strain harboring an allele analogous to a disease-causing missense mutation in human KDM5C (kdm5A512P). Transcriptome analysis of kdm5A512P flies revealed a striking downregulation of genes required for ribosomal assembly and function and a concomitant reduction in translation. kdm5A512P flies also showed impaired learning and/or memory. Significantly, the behavioral and transcriptional changes in kdm5A512P flies were similar to those specifically lacking demethylase activity. These data suggest that the primary defect of the KDM5A512P mutation is a loss of histone demethylase activity and reveal an unexpected role for this enzymatic function in gene activation. Because translation is critical for neuronal function, we propose that this defect contributes to the cognitive defects of kdm5A512P flies.

Early programming of the oocyte epigenome temporally controls late prophase I transcription and chromatin remodelling.

Oocytes are arrested for long periods of time in the prophase of the first meiotic division (prophase I). As chromosome condensation poses significant constraints to gene expression, the mechanisms regulating transcriptional activity in the prophase I-arrested oocyte are still not entirely understood. We hypothesized that gene expression during the prophase I arrest is primarily epigenetically regulated. Here we comprehensively define the Drosophila female germ line epigenome throughout oogenesis and show that the oocyte has a unique, dynamic and remarkably diversified epigenome characterized by the presence of both euchromatic and heterochromatic marks. We observed that the perturbation of the oocyte's epigenome in early oogenesis, through depletion of the dKDM5 histone demethylase, results in the temporal deregulation of meiotic transcription and affects female fertility. Taken together, our results indicate that the early programming of the oocyte epigenome primes meiotic chromatin for subsequent functions in late prophase I.